Handbook of Neurochemistry and Molecular Neurobiology

Development and Aging Changes in the Nervous System

Abel Lajtha(Ed.)

Handbook of
Neurochemistry and
Molecular Neurobiology
Development and Aging Changes
in the Nervous System
Volume Editors: J. Regino Perez-Polo and Steffen Rossner

With 48 Figures and 12 Tables

Editor
Abel Lajtha
Director
Center for Neurochemistry
Nathan S. Kline Institute for Psychiatric Research
140 Old Orangeburg Road
Orangeburg
New York, 10962
USA

Volume Editors
J. Regino PerezPolo Steffen Rossner
I. H. Kempner Professor and Chair Universitat Leipzig
Dept. Biochemistry & Molecular Biology Medizinische Fakultat
Senior Scientist PaulFlechsigInstitut fur Hirnforschung
Sealy Center for Molecular Medicine Jahnallee 59
Staff Scientist Shriners Hospital for Children 04109 Leipzig
Department of Biochemistry & Molecular Biology Germany
University of Texas Medical Branch Steffen.Rossner@medizin.uni-leipzig.de
301 University Blvd.
Galveston
TX, 77555
USA
regino.perez-polo@utmb.edu

Library of Congress Control Number: 2006922553

ISBN: 9780387326702

Additionally, the whole set will be available upon completion under ISBN: 9780387354439
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Preface

Animals share the challenge of maintaining an internal environment that is restricted to fairly low ranges of
temperature, pH, and water content within a well-protected envelope while engaged in continuous
exchanges with the environment in terms of gases, liquids, energy, even as movement of body parts and
the entire organism itself is necessary for survival. This dynamic spectrum of changes is further amplified
during developmental events or more acutely during responses to pernicious environmental factors in due
to trauma and disease. In addition, persistent incidents associated with aging can result in irreversible
changes to the allostasis that characterizes the living condition.
In the nervous system, a very high metabolic turnover, fragile but steep ionic gradients, and morpho-
logical and structural constraints dictated by the necessity for prompt neuronal transmission of electrical
impulses and necessary plasticity result in a highly fragile organ system.
Here we address a small sampling of major constituents of neural function at the cellular and molecular
level that play important roles in development and aging, two endogenous processes that embody features
of allostasis or the dynamic shifts in set points for specific homeostatic mechanisms associated with
development and aging.
The opening chapters discuss the major players in the neurotrophic hypothesis, the neurotrophins.
These growth factors have been shown to play a significant role during development and in the maintenance
of the adult cholinergic system in CNS as well as in the development of the sensory and sympathetic
nervous system. That they are also involved in plasticity events associated with memory and behavior points
to the degenerate nature of signaling molecules that achieve specificity by acting in concert as part of
ensembles of molecules rather than solitary regulators.
It is widely known that oligodendroglia and myelination events are late arrivals in the developmental
scheme of the brain and are also prime targets in early development of ischemic insults. Thus, a chapter on
oligodendroglia and myelination in development and aging serves to introduce these nonneuronal partners
vital to proper neuronal transmission. Molecular participants in stress responses to both acute and chronic
stressors are discussed from different perspectives in following chapters with varying degrees of emphasis
on injury versus normal aging and neurodegenerative disease.
The study of neural responses to stress of various kinds has led to a realization of the importance of
plasticity and the complexity of the mechanism allowing plasticity in the nervous system. The chapters
addressing the topic are followed by an introduction to the amyloid hypothesis, and what may be its central
character the enzyme held mostly responsible for the generation of beta amyloid. This is followed by a
broader discussion of misfolding proteins in the nervous system and its possible interventions to counteract
aging-associated deficits.
Limited in scope but offering a broad sampling, these chapters stress the dynamic features of neuronal
responses to internal (developmental) cues or the more harmful external events (injury and disease) in a
modern perspective.
Contributors
K. Abid
George and Cynthia Mitchell Center for
Neurodegenerative Diseases, Departments of
Neurology, Neuroscience and Cell Biology and
Biochemistry and Molecular Biology, University of
Texas Medical Branch 301 University Blvd, Galveston,
TX 77555, USA
J. M. Angelastro
Department of Pathology and Center for Neurobiology
and Behavior, Columbia University College of Physicians
and Surgeons, New York NY 10032, USA
T. Arendt
Paul Flechsig Institute for Brain Research, Department
of Neuroanatomy, 04109 Leipzig, Germany
C. S. Atwood
School of Medicine, University of Wisconsin and
William S. Middleton Memorial Veterans Administration,
Madison, Wisconsin 53705, USA
R. L. Bowen
Voyager Pharmaceutical Corporation, Raleigh, North
Carolina 27615, USA
D. A. Butterfield
Department of Chemistry, University of Kentucky,
Lexington, KY 40506-0055, USA
V. Calabrese
Section of Biochemistry and Molecular Biology,
Department of Chemistry, Faculty of Medicine,
University of Catania, Catania, Italy
G. Casadesus
Institute of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106, USA
# 2008 Springer ScienceBusiness Media, LLC.
M. V. Chao
Molecular Neurobiology Program, Skirball Institute of
Biomolecular Medicine, Departments of Cell Biology;
Physiology and Neuroscience, New York University
School of Medicine, 540 First Avenue, New York,
New York 10016, USA
J. de Vellis
Department of Neurobiology, Mental Retardation
Research Center, Semel Institute for Neuroscience and
Human Behavior, David Geffen School of Medicine at
UCLA, USA
L. A. Greene
Department of Pathology and Center for Neurobiology
and Behavior, Columbia University College of Physicians
and Surgeons, New York NY 10032, USA
X. Hu
Department of Biolchemistry and Molecular Biology
and the Sealy Center for Molecular Science, University
of Texas Medical Branch at Galveston, Texas
77555-1072, USA
J. A. Joseph
Human Nutrition Research Center on Aging, Tufts
University, 711 Washington St., Boston, MA 02111, USA
I. Konig
AG Molecular Mechanisms of Plasticity, Department of
Neurochemistry & Molecular Biology, Leibniz Institute
for Neurobiology, 39118 Magdeburg, Germany
M. R. Kreutz
AG Molecular Mechanisms of Plasticity,
Department of Neurochemistry and Molecular Biology,
Leibniz Institute for Neurobiology,
39118 Magdeburg, Germany
x Contributors
S. F. Lichtenthaler
Adolf-Butenandt-Institut, Ludwig-Maximilians
University Munich, 80336 Munich, Germany
E. Meshorer
Department of Biological Chemistry, Institute of
Life Sciences, The Hebrew University of Jerusalem,
Israel 91904
M. Mikhaylova
AG Molecular Mechanisms of Plasticity, Department of
Neurochemistry & Molecular Biology, Leibniz Institute
for Neurobiology, 39118 Magdeburg, Germany
J. Neman
Department of Neurobiology, Mental Retardation
Research Center, Semel Institute for Neuroscience
and Human Behavior, David Geffen School of
Medicine at UCLA, USA
D. B. Pereira
Molecular Neurobiology Program, Skirball Institute of
Biomolecular Medicine, Departments of Cell Biology;
Physiology and Neuroscience, New York University
School of Medicine, 540 First Avenue, New York,
New York 10016, USA
J. R. Perez-Polo
Department of Biolchemistry and Molecular Biology
and the Sealy Center for Molecular Science, University
of Texas Medical Branch at Galveston, Texas
77555-1072, USA
G. Perry
Institute of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106, USA
D. K. Rassin
Department of Pediatrics, The University of Texas
Medical Branch at Galveston, Texas 77555-0344, USA
S. Roner
Paul Flechsig Institute for Brain Research, Department
of Neurochemistry, 04109 Leipzig, Germany
M. A. Smith
Institute of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106, USA
H. Soreq
Department of Biological Chemistry, Institute of
Life Sciences, The Hebrew University of Jerusalem,
Israel 91904
C. Soto
George and Cynthia Mitchell Center for
Neurodegenerative Diseases, Departments of
Neurology, Neuroscience and Cell Biology and
Biochemistry and Molecular Biology, University of Texas
Medical Branch, 301 University Blvd, Galveston,
TX 77555, USA
C. Spilker
AG Molecular Mechanisms of Plasticity,
Department of Neurochemistry & Molecular Biology,
Leibniz Institute for Neurobiology,
39118 Magdeburg, Germany
A. M. G. Stella
Section of Biochemistry and Molecular Biology,
Department of Chemistry, Faculty of Medicine,
University of Catania, Catania, Italy
L. Vergara
George and Cynthia Mitchell Center for
Neurodegenerative Diseases, Departments of
Neurology, Neuroscience and Cell Biology and
Biochemistry and Molecular Biology, University of Texas
Medical Branch, 301 University Blvd, Galveston, TX
77555, USA
K. M. Webber
Institute of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106, USA
X. Zhu
Institute of Pathology, Case Western Reserve University,
Cleveland, Ohio 44106, USA
W. Zuschratter
AG Molecular Mechanisms of Plasticity,
Department of Neurochemistry & Molecular Biology,
Leibniz Institute for Neurobiology,
39118 Magdeburg, Germany
Table of Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

1 Neurotrophins and Central Nervous System Development . . . . . . . . . . . . . . . . . 1

D. B. Pereira . M. V. Chao

2 Nerve Growth Factor Regulated Gene Expression . . . . . . . . . . . . . . . . . . . . . . . 21

L. A. Greene . J. M. Angelastro

3 Myelinating Cells in the Central Nervous SystemDevelopment,

Aging, and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
J. Neman . J. de Vellis

4 SulfurContaining Amino Acids in the CNS: Homocysteine . . . . . . . . . . . . . . . . . 77

D. K. Rassin

5 Stress Response Signal Transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Xiaoming Hu . J. R. PerezPolo

6 Aging and Oxidative Stress Response in the CNS . . . . . . . . . . . . . . . . . . . . . . . 103

V. Calabrese . D. A. Butterfield . A. M. Giuffrida Stella

7 Parallels Between Neurodevelopment and Neurodegeneration:

A Case Study of Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
X. Zhu . G. Casadesus . K. M. Webber . C. S. Atwood . R. L. Bowen .
G. Perry . M. A. Smith

8 Differentiation and DeDifferentiationNeuronal CellCycle Regulation

During Development and AgeRelated Neurodegenerative Disorders . . . . . . . 157
T. Arendt

9 mRNA Modulations in Stress and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

E. Meshorer . H. Soreq

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viii Table of Contents

10 Molecular Mechanisms of Dendritic Spine Plasticity in

Development and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
M. R. Kreutz . I. Konig . M. Mikhaylova . C. Spilker . W. Zuschratter

11 Alzheimers Disease BACE Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261

S. Roner . S. F. Lichtenthaler

12 Protein Misfolding, a Common Mechanism in the Pathogenesis of

Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
L. Vergara . K. Abid . C. Soto

13 AntiAging Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

J. A. Joseph . J. R. PerezPolo

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
1 Neurotrophins and Central Nervous
System Development
D. B. Pereira . M. V. Chao

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 Neurotrophins and CNS Neuronal Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.1 NGF and the Survival of Central Cholinergic Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2 Role of BDNF, NT-3 and NT-4 in Central Neuron Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.1 Motor Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.2 Mesencephalic Trigeminal Nucleus Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.3 Neurons of Other Regions of the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3 Neurotrophins and CNS Neuronal Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.1 Modulation of Neuronal Migration in the Cortex by BDNF and NT-4 . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.1 Radial Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1.2 Tangential Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.1.3 Neurotrophin Signaling Pathways Involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2 Modulation of Neuronal Migration in the Cerebellum by BDNF and NT-3 . . . . . . . . . . . . . . . . . . . . . 9

4 Neurotrophins and CNS Dendritic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4.1 Effects of Exogenous Neurotrophins on Pyramidal Neuron Dendrites . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.2 Effects of Exogenous Neurotrophins on the Dendrites of Non-Pyramidal
Interneurons and Other Neuronal Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.3 Role of Endogenous Neurotrophins in Dendritic Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.4 Neurotrophin Receptors and Downstream Signaling Pathways
Involved in Dendritic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4.5 Interplay Between Neurotrophins and Neuronal Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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2 1 Neurotrophins and central nervous system development

Abstract: The formation of the vertebrate nervous system is characterized by widespread programed cell
death, which determines cell number and appropriate target innervation during development. The neuro-
trophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neuro-
trophin-3 (NT-3) and NT-4, represent an important family of trophic factors that are essential for survival
of selective populations of neurons during different developmental periods. Neurotrophins exert their cellular
effects through the actions of two different receptors, the tropomyosin-related kinase (Trk) receptor tyrosine
kinase and the p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily. Much
attention has been given to the consequences of neurotrophin action in the peripheral nervous system (PNS);
however, neurotrophins are widely expressed in the brain and spinal cord. This chapter focuses on new views
concerning effects of neurotrophins in central nervous system (CNS) development.

List of Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BDNF, brain-

derived neurotrophic factor; CA1, cornu ammonis 1; CA3, cornu ammonis 3; CNS, central nervous system;
CP, cortical plate; EGL, external germinal layer; ERK, extracellular signal-regulated kinase; GEF, guanine
nucleotide-exchange factor; IgG, immunoglobulin G; IGL, internal germinal layer; MEK, mitogen activated
protein kinase/ERK kinase; MGE, medial ganglionic eminence; ML, molecular layer; MTN, mesencephalic
trigeminal nucleus; MZ, marginal zone; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; NPY,
neuropeptide Y; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PI3K, phosphatidylinositol 3-kinase; PLCg,
phospholipase Cg; PNS, peripheral nervous system; RGC, retinal ganglion cell; SNP, single nucleotide
polymorphism; Tiam1, T lymphoma invasion and metastasis; Trk, tropomyosin-related kinase; VTA,
ventral tegmental area

1 Introduction

The neurotrophic hypothesis postulates that during nervous system development, neurons approaching the
same final target vie for limited amounts of target-derived trophic factor (Levi-Montalcini and Angeletti,
1968; Thoenen and Barde, 1980). In this way, the nervous system moulds itself to maintain only the most
competitive and appropriate connections. Competition among neurons for limited amounts of neurotro-
phin molecules produced by target cells accounts for selective cell survival. Several predictions can be
gleaned from this hypothesis. First, the efficacy of neuronal survival depends on the amount of trophic
factors produced during development. Second, specific receptor expression in responsive cell population
dictates neuronal responsiveness.
The neurotrophins are initially synthesized as precursors or pro-neurotrophins that are cleaved to
release the mature, active proteins. The mature proteins form stable, noncovalent dimers and are normally
expressed at very low levels during development. Pro-neurotrophins are cleaved intracellularly by furin or
pro-convertases utilizing a highly conserved dibasic amino acid cleavage site to release carboxy-terminal
mature proteins of approximately 13 kD (Chao and Bothwell, 2002). The mature proteins mediate
neurotrophin actions by selectively binding to members of the Trk family of receptor tyrosine kinases to
regulate neuronal survival, differentiation, and synaptic plasticity. NGF binds most specifically to TrkA;
BDNF and NT-4 to TrkB; and NT-3 to TrkC receptors (> Figure 11). Neurotrophin binding to Trk
receptors results in the activation of the phospholipase Cg (PLCg) pathway, involved in synaptic transmis-
sion and plasticity (Matsumoto et al., 2001; Minichiello et al., 2002), and in the recruitment of a number of
proteins that are involved in the activation of extracellular signal-regulated kinase (ERK), phosphatidyli-
nositol 3-kinase (PI3K) and its downstream target Akt. While ERK1/2 mediate neurotrophin-induced
neuronal survival and differentiation (Huang and Reichardt, 2003), PI3K activity is responsible for
retrograde survival signaling (Kuruvilla et al., 2000).
The p75 receptor can bind to each neurotrophin but has the additional capability of regulating a
Trks affinity for its cognate ligand. In addition, pro-neurotrophins can also interact with p75 independently
(Lee et al., 2001).
Neurotrophin factors fit well with the neurotrophic hypothesis, as many peripheral neuronal subpo-
pulations exhibit a predominant dependence on a specific neurotrophin during the period of naturally
 Neurotrophins and central nervous system development 1 3

. Figure 11
Selectivity of neurotrophin binding to Trk receptors and p75. NGF binds most specifically to TrkA, BDNF and NT-
4 to TrkB and NT-3 to TrkC. NT-3 can also potentially interact with TrkB and TrkA receptors. In addition, all
neurotrophins are capable of binding to the p75 receptor with equal affinity

occurring cell death. However, in the CNS, there appears to be a much more complex situation. In the brain
and spinal cord, there is overlapping expression of multiple neurotrophin receptors and their cognate
ligands. This allows for the generation of diverse connectivity that extends well into adulthood. In addition
to promoting axonal and dendritic branching, neurotrophins also possess acute regulatory effects on
neurotransmitter release, synaptic strength and connectivity (Thoenen, 1995; Bonhoeffer, 1996; McAllister
et al., 1999). Many of these effects reflect activity-dependent events based upon competitive remodeling of
axon and dendritic terminals. Other complexities remain, such as the molecular mechanisms underlying
the retrograde signal, a pathway that must efficiently transmit information over long distances, at times over
a meter, as well as anterograde transport of neurotrophins.
The NGF family of trophic factors has provided a remarkable mechanism for controlling a wide gamut
of cellular activities, including target innervation, apoptosis, differentiation, synaptic plasticity, and axon
guidance. Defining the populations in the central nervous system (CNS) that are affected bidirectionally by
neurotrophins provides insights into the mechanisms responsible for regulating cell growth, survival, and
changes in complex circuits. This chapter considers some of the key decision-making events in different
populations of central neurons that lead to these diverse biological outcomes.

2 Neurotrophins and CNS Neuronal Survival

The broad distribution of TrkB, TrkC and their ligands throughout the CNS is suggestive of a putative role
for neurotrophins in CNS neuronal survival during development, in similarity to their action in the PNS
(peripheral nervous system). The result of initial studies showing that exogenously applied neurotrophins
can rescue specific populations of central neurons from naturally occurring and induced cell death, seemed
to support this idea. Among the neuronal populations that show increased survival when cultured in the
presence of neurotrophins are embryonic cerebellar granule cells (reviewed in Segal et al., 1992) and
substantia nigra dopaminergic neurons (Hyman et al., 1991), which are responsive to brain-derived
4 1 Neurotrophins and central nervous system development

neurotrophic factor (BDNF), cerebellar Purkinje cells (Cohen-Cory et al., 1991), which are sensitive to
NGF, and septal cholinergic neurons, which are responsive to both (Hartikka and Hefti, 1988; Alderson
et al., 1990). In vivo studies have also shown that exogenously applied NGF and BDNF or NT-3, can rescue
axotomized basal forebrain neurons (Williams et al., 1986) and motor neurons (Oppenheim et al., 1992;
Sendtner et al., 1992; Yan et al., 1992; Koliatsos et al., 1993), respectively. Despite what these pharmacologi-
cal studies have initially suggested, mouse mutants lacking neurotrophins or their receptors, which were
generated in the early-to-mid 1990s, have failed to confirm a significant neuroprotective role for neuro-
trophins in the CNS during embryonic development (reviewed in Snider, 1994).

2.1 NGF and the Survival of Central Cholinergic Neurons

The TrkA
/
and the NGF
/
mice, while exhibiting the expected loss of neurons in peripheral ganglia, do
not show reduced numbers of basal forebrain cholinergic neurons (Crowley et al., 1994; Smeyne et al.,
1994). These are the prototypical NGF-responsive neurons in the brain, which were thought to depend on
NGF secreted by their hippocampal and cortical target fields for survival and function (Dreyfus, 1989).
Striatal cholinergic neurons also do not seem to be affected in NGF-deficient mice (Crowley et al., 1994).
Although cholinergic cell numbers are normal, a substantial decrease in acetylcholinesterase staining in
fibers extending to the hippocampus and cerebral cortex was observed in the TrkA mutant mice (Smeyne
et al., 1994). A reduction in the size of basal forebrain cholinergic neurons is also apparent in both NGF
mutant homozygous and heterozygous animals (Crowley et al., 1994; Chen et al., 1997), arguing in favor of
a physiological role for endogenous NGF on the differentiation of these neurons. We should also bear in
mind, though, that both the TrkA and the NGF deficient mice die very early in their postnatal life, while the
development of their CNS is still ongoing. This precludes a definitive answer on the NGF-dependence of
brain cholinergic neurons.
Understanding the role of NGF in the survival of these neurons is further complicated by the
controversy surrounding the function of the p75 neurotrophin receptor in neuronal apoptosis in the
CNS. This issue is aggravated by another controversy regarding the nature of the two existing p75-deficient
mouse strains. The first p75 mutant mouse was generated by a targeted deletion of exon III and is
commonly referred to as the p75exonIII mutant (Lee et al., 1992). This mouse was later suggested to express
a shorter form of the p75 receptor, arising from alternative splicing of exon III, which lacks three of the
four cysteine-rich domains responsible for neurotrophin binding (von Schack et al., 2001). A second
p75-deficient mouse, the p75exonIV mutant, was then generated by deleting exon IV of the p75 locus, in
an attempt to create animals lacking any form of the p75 receptor (von Schack et al., 2001). However, these
mice were described to express a 26 kDa p75 gene product, containing the extracellular stalk region and the
entire transmembrane and intracellular domains of p75 (Paul et al., 2004). This truncated protein, which
was not observed in the wildtype or in the p75exonIII mutant animals, was able to activate p75 signaling
cascades when overexpressed in heterologous cells (Paul et al., 2004).
While this controversy remains unresolved, the study of the putative role of p75 in basal forebrain
cholinergic neurons has originated discrepant results even when the same p75 mutant mouse strain was
used. An initial report described an increase in the number of septum cholinergic neurons in the p75exonIII
mutant in agreement with the previously purposed pro-apoptotic function of p75 (Van der Zee et al., 1996).
Although subsequent studies reported a similar increase in basal forebrain cholinergic neuron numbers
(Yeo et al., 1997), others have found exactly the opposite. Adult p75exonIII mice were described to have
reduced septal region volume and cholinergic neuron numbers (Peterson et al., 1997, 1999; Greferath et al.,
2000), which suggests a pro-survival function for p75, possibly through a positive regulation of neuro-
trophin signaling through Trk receptors. A lack of any significant change in cholinergic neuron numbers
was also described in a reexamination of the p75exonIII mutant mice by the same group that first reported a
decrease in neuronal numbers in these mice (Ward and Hagg, 1999). Differences in genetic background
and age of the mice used in these studies and quantitative stereology methods may account for some of
the discrepancies observed. In fact, a more recent study shows that a moderate increase in basal fore-
brain cholinergic neurons was only apparent in p75exonIII mutant mice after backcrossing in a C57BL/6
 Neurotrophins and central nervous system development 1 5

background (Naumann et al., 2002). On the other hand, the same study reports a significant increase in the
cholinergic neuron number in the p75exonIV mutant mouse (Naumann et al., 2002).
The role of p75 in basal forebrain cholinergic neuron survival remains, therefore, unresolved. Moreover,
the involvement of neurotrophins and Trk signaling in the putative role of p75 in the survival of the
aforementioned neurons was not yet investigated. Clarifying the controversy regarding the two different
strains of p75 mutant mice will require conditional mice, together with crossing these animals with other
neurotrophin mutants. This will be crucial to the understanding of neurotrophin and p75 regulation of
central cholinergic neuron survival.

2.2 Role of BDNF, NT-3 and NT-4 in Central Neuron Survival

Studying the role of TrkB and TrkC in CNS development by genetic ablation of either one of these receptors
or their specific ligands was complicated, as in the case of TrkA and NGF, by the limited life span of the
animals. With the exception of the NT-4
/
mice that survive well into adulthood (Conover et al., 1995;
Liu et al., 1995), these mutant mice die either shortly after birth, as it is the case for the NT-3 and the TrkB
mutants (Klein et al., 1993; Farinas et al., 1994; Ernfors et al., 1994b) or within the second or third postnatal
weeks, as for the TrkC and BNDF-deficient animals (Ernfors et al., 1994a; Jones et al., 1994; Klein et al.,
1994). Analyzing the CNS of these animals did indicate, though, that neither TrkB nor TrkC activity is
essential on its own for CNS neuronal survival, at least during embryonic development. Nevertheless, there
are some exceptions and controversies that are worth mentioning.

2.2.1 Motor Neurons

The TrkB
/
mouse, which was the first neurotrophin receptor mutant to be reported, was initially
described to have a decrease of 35% in the number of spinal cord motor neurons (Klein et al., 1993).
This would be in agreement with the rescue of developmentally regulated or axotomy-induced motor
neuron death by BDNF (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992; Koliatsos et al.,
1993). However, two different strains of mice lacking BDNF failed to show any reduction in the survival of
several motor neuron populations (Ernfors et al., 1994a; Jones et al., 1994), including spinal cord neurons.
Mice deficient for the other two neurotrophins that can activate TrkB, NT-4 and NT-3, did not exhibit any
reduction in facial motor neurons numbers (Ernfors et al., 1994b; Farinas et al., 1994; Conover et al., 1995;
Liu et al., 1995). BDNF and NT-4 double mutants also have normal facial and lumbar cord motor neurons
pools (Conover et al., 1995; Liu et al., 1995). A reduction in motor neuron survival was only observed in
triple BDNF, NT-4 and NT-3 deficient mice, which show a modest 20% decrease in neuronal numbers in
several nuclei (Liu and Jaenisch, 2000). Finally, reexamination of the TrkB mouse mutants and analysis of
mice lacking both TrkB and TrkC showed no significant difference in the number of facial or spinal motor
neurons between these mutant mice and their wildtype littermates (Silos-Santiago et al., 1997). Therefore,
neurotrophins appear to play a redundant and modest role in the survival of motor neurons during
embryonic development. It is still possible, since not all motor neuron pools were examined in each animal,
that particular sets of motor neurons are dependent on specific neurotrophins for survival, as it is the case
for other trophic factors (Huang and Reichardt, 2001). As an example, the survival of g-motor neurons,
whose nerve fibers are present at the L4 ventral spinal root, is greatly reduced in the NT-3
/
mouse
(Kucera et al., 1995).

2.2.2 Mesencephalic Trigeminal Nucleus Neurons

The most dramatic reduction in neuronal survival in the CNS of neurotrophin or neurotrophin-receptor
mutant mice was observed in the mesencephalic trigeminal nucleus (MTN). The MTN, which is located in
the brainstem, contains the proprioceptive neurons of the trigeminal system that convey information from
6 1 Neurotrophins and central nervous system development

the head region. Both the BDNF
/
and the NT-3
/
mice show an approximate 50% reduction in the
number of neurons in this area (Ernfors et al., 1994a, b; Farinas et al., 1994; Jones et al., 1994). Double
BDNF/NT-3-deficient mice show a loss of 88% of these neurons and triple BDNF/NT-3/NT-4 mutants are
practically devoid of MTN neurons, with only about 5% of them surviving (Fan et al., 2000). Therefore,
unlike their PNS counterparts, which are completely dependent on NT-3 (reviewed in Snider, 1994; Huang
and Reichardt, 2001), different subsets of these CNS proprioceptive neurons seem to depend on different
neurotrophins for survival.

2.2.3 Neurons of Other Regions of the Brain

Most of the other regions of the brain were surprisingly normal in these mutant mice. No gross abnormality
was detected in the cytoarchitecture of the brain areas examined, including the cortex, hippocampus, and
thalamus, in TrkB and TrkC single mutants (Klein et al., 1993, 1994). TrkB/TrkC double mutants also
appeared to have normal brain anatomy (Silos-Santiago et al., 1997), ruling out redundancy as a possibility
for the lack of a phenotype. A more comprehensive examination was performed on the BDNF mutants.
With the exception of the granule cerebellar neurons, which exhibit increased cell death in these animals
(Schwartz et al., 1997), most of the neuronal populations known to be sensitive to this neurotrophin
showed no decrease in survival. This included substantia nigra and VTA dopaminergic neurons, basal
forebrain cholinergic neurons, cerebellar Purkinje neurons, and GABAergic and cholinergic neurons of the
hippocampus and striatum (Jones et al., 1994; Ernfors et al., 1994a). There was, however, a marked decrease
in the expression of certain neuronal markers, such as calbindin, parvalbumin, and neuropeptide Y (NPY;
Jones et al., 1994). This was interpreted as a problem in neuronal differentiation since GABAergic neurons,
where calbindin, parvalbumin, and NPY are expressed, are present in normal numbers in the BDNF-
deficient mice. Importantly, although their brain structure seems normal, BDNF
/
brains are smaller and
have less neuropile (Conover et al., 1995). A reduction in the thickness of all cortical layers is also evident
(Jones et al., 1994).
Subsequent reports looking specifically for neuronal apoptosis in the postnatal brain of TrkB and
double TrkB/TrkC mutants uncovered a need for these receptors in the survival of several populations of
CNS postmitotic neurons. TrkB deficient mice show a progressive increase in the number of apoptotic cells
from P10 to P18, in all cortical layers and hippocampal subfields (Alcantara et al., 1997). The dentate gyrus
is the most affected area. In other brain regions, such as the striatum and the reticular thalamic nucleus, the
increase in apoptotic cells in the TrkB mutant is apparent only during the first postnatal weeks (Alcantara
et al., 1997). The majority of dying cells were confirmed to be neurons by the use of specific neuronal
markers. Mice deficient in both TrkB and TrkC also exhibit increased cell death in the hippocampus, after
the first postnatal week (Minichiello and Klein, 1996). Cerebellar granule neurons, but not Purkinje cells,
are also reduced in these animals, in similarity to what was observed in the BDNF mouse mutants
(Minichiello and Klein, 1996; Schwartz et al., 1997).
Conditional mouse mutants lacking TrkB at specific developmental time points and in specific cell types
have been generated using the Cre/loxP recombination system (Minichiello et al., 1999; Xu et al., 2000;
Medina et al., 2004). These mice have been a valuable tool to study the role of TrkB in the CNS of adult
mice. Two of these strains, where depletion of TrkB occurs either embryonically or during the second to
third postnatal weeks, show a severe reduction in the thickness of their visual and somatosensory cortex
(but Medina et al., 2004; see also Minichiello et al., 1999; Xu et al., 2000). Although it is likely that this
compression of the cortical layers is associated with migration and differentiation problems (Xu et al., 2000;
Medina et al., 2004), a decrease in neuronal survival was also observed in 10-week-old animals (Xu et al.,
2000). Interestingly, the survival of different neuronal populations, identified by the expression of specific
transcription factors, were differentially affected by the lack of TrkB (Xu et al., 2000).
Taken together, all the work involving conventional and conditional neurotrophin and Trk receptor
mouse mutants has revealed a disappointing small role for these molecules in the survival of CNS neurons
during embryonic development, as compared with their PNS counterparts. With the exception of the MTN,
 Neurotrophins and central nervous system development 1 7

other brain regions seem to be dependent on neurotrophins for neuronal survival only throughout
postnatal development. As we will describe in the following sections, neurotrophins appear to have a
bigger contribution to CNS development by modulating migration and differentiation of CNS neurons.

3 Neurotrophins and CNS Neuronal Migration

Neuronal migration is an essential step in brain tissue formation during development, allowing the proper
positioning of neurons before the onset of neuritogenesis and synaptogenesis. To reach their appropriate
position, neurons undergo one or two different types of migration. In radial migration, neurons migrate
from their progenitor regions following the radial disposition of the neural tube (reviewed in Marin and
Rubenstein, 2003). This usually involves the use of radial glia processes, which extend from the ventricular
zone to the pial surface of the brain, as a guide on which neurons migrate until they reach their destination.
Radial glia-independent radial migration can also occur by a process called somal translocation, where
neurons themselves extend a process to the pial surface and then translocate their soma to the proper
position. The other type of migration, termed tangential migration, follows a path that is orthogonal to the
direction of radial migration (reviewed in Marin and Rubenstein, 2003). The physical substrates used by
cells migrating tangentially are other migrating neurons or growing axons. Alternatively, cells can just
disperse in an individual manner not following any cellular substrates.
Neurotrophins have been described to influence both types of neuronal migration by acting as
motogenic factors that stimulate the motility of migrating cells. In the following sections, we will focus
on studies performed on the cerebral cortex and the cerebellum, where the role of neurotrophins in
neuronal migration has been most intensively investigated.

3.1 Modulation of Neuronal Migration in the Cortex by BDNF and NT-4

The formation of the layered architecture of the cerebral cortex relies on the radial migration of postmitotic
neurons, originating from the ventricular zone that lines the wall of the cerebral ventricle, toward the
surface of the brain (Rakic, 1975). The first migrating neurons form a subpial layer known as the preplate.
This layer is then split into the marginal zone (MZ; Layer I, the outermost layer) and the subplate by
successive migrating neurons that form the cortical plate in between (Marin-Padilla, 1971). The latter is
generated by an inside-out migratory sequence that leads to the formation of layers IIVI (Angevine and
Sidman, 1961; Rakic et al., 1974; Caviness, 1982; Bayer et al., 1991) (> Figure 1-2). In addition, GABAergic
neurons generated mostly in the medial ganglionic eminence (MGE), with a smaller contribution of the
lateral and caudal eminences, migrate tangentially into the cortex where they reach the appropriate layer by
a final step of radial migration (Anderson et al., 2001; Nery et al., 2002; Ang et al., 2003).

3.1.1 Radial Migration

Messenger RNA for the TrkB and TrkC receptors is present in high levels during cortical development, when
neuronal migration occurs (Klein et al., 1990; Lamballe et al., 1994). On the other hand, their ligands,
BDNF and NT-3, have opposite patterns of expression during corticogenesis: NT-3 expression is high
during embryogenesis and decreases with maturation while BDNF expression is low during embryonic
development increasing during adulthood (Maisonpierre et al., 1990; Timmusk et al., 1994). Surprisingly,
in spite of these expression patterns, it is BDNF and not NT-3 that seems to play a role in neuronal
migration in the neocortex. Both BDNF and NT-4 can induce chemotaxis (directed migration) of
embryonic cortical neurons in vitro in a manner dependent on Ca2+ and Trk activation (Behar et al.,
1997). In the same assay, NT-3 can only elicit migration when used at high concentrations (100 ng/ml).
In vivo studies have shown that increasing the concentration of BDNF in the developing embryonic brain,
8 1 Neurotrophins and central nervous system development

. Figure 1-2
Cerebral cortex organization during development and adulthood. Postmitotic neurons, originating from the
ventricular zone (VZ), migrate radially towards the surface of the brain to form the preplate (PP). Most of these
neurons migrate on radial glial processes represented in the diagram by vertical lines. For simplicity, the cell
body of these glial cells was not represented. The PP is then split into the marginal zone (MZ) and the subplate
(SP) by successive migrating neurons that form the cortical plate (CP) in between. Layers II-VI are then formed at
the CP by an inside-out migratory sequence. IZ, intermediate zone

either by intraventricular injection of the neurotrophin or by creating transgenic mice that overexpress
BDNF, produces neuronal heterotopias (collections of cells) in the MZ (Brunstrom et al., 1997; Ringstedt
et al., 1998; Alcantara et al., 2006). Injection of NT-4 has an even greater effect in the production of
heterotopias, as compared with the injection of BDNF, while infusion of NT-3 has no effect (Brunstrom
et al., 1997). This accumulation of cells in the MZ of the developing cortex appears to be the result of
increased neuronal migration and it is at least partially due to a decrease in the expression of reelin
(Brunstrom et al., 1997; Ringstedt et al., 1998). Reelin, which is produced by the CajalRetzius cells of
the MZ, is required for normal lamination of the cortex during embryonic development and is down-
regulated postnatally as maturation occurs (reviewed in Marin and Rubenstein, 2003). This downregulation
is dependent on BDNF since it is delayed in mice lacking this neurotrophin (Ringstedt et al., 1998).
Therefore, BDNF appears to act as a cortical maturation factor that, by being expressed in high amounts
ahead of time, induces cortical lamination defects. Does this imply, however, that endogenous BDNF plays a
role in migration in the neocortex?
Preliminary analysis of the BDNF-deficient mouse brain revealed a reduction in the thickness of the
cortical layers (Jones et al., 1994). A similar phenotype was seen in TrkB conditional mouse mutants where
TrkB expression is reduced during embryogenesis being virtually eliminated following birth (Medina et al.,
2004). These mice show a delay in migration of newly born cortical neurons that assume altered positioning
in the newly formed cortical layers (Medina et al., 2004). However, it is not clear whether the reduction in
TrkB expression during embryogenesis and early postnatal life, achieved in these conditional mutants, is
enough to reveal the full plethora of defects that the lack of this receptor would cause in the context of
neuronal migration. It would be interesting to study in more detail the brain architecture of embryonic and
early postnatal TrkB and BDNF/NT-4 double mutant mice to clarify the role of these neurotrophins in
neuronal migration.
In support of a role for endogenous TrkB ligands in migration to the appropriate cortical layer TrkB
expression is seen in migratory neurons in the CP in vivo, during the period of maximal neuronal migration
(Behar et al., 1997). In addition, a gradient of BDNF transcripts is observed in the neocortex, with the CP
 Neurotrophins and central nervous system development 1 9

expressing higher amounts of this neurotrophin as compared to the ventricular zone from where cortical
neurons originate (Behar et al., 1997). This may be the reason why BDNF, and not the more highly
expressed NT-3, is important for neuronal migration. By being present in low amounts in discrete regions,
which allows the formation of a gradient, BDNF may direct the migration of TrkB-expressing neurons
toward regions of higher BDNF concentration.

3.1.2 Tangential Migration

Tangential migration of GABAergic interneurons into the cortex is also promoted by NT-4 and BDNF.
Exogenous application of these neurotrophins to brain slices increased the number of MGE neurons that
migrate into the cortex and the rate at which they migrate (Polleux et al., 2002). NT-3 was again without
effect on the migration of these interneurons. The role of endogenous neurotrophins in this process was
demonstrated by a reduction in the number of MGE neurons that migrate into the cortex in brain slices of
TrkB deficient mice and in slices treated with the Trk inhibitor K-252a (Polleux et al., 2002). Interestingly,
transactivation of Trk receptors by endocannabinoids has been reported to induce interneuron migration
in vitro (Berghuis et al., 2005).

3.1.3 Neurotrophin Signaling Pathways Involved

The Trk signaling pathways involved in the modulation of cortical neuronal migration by neurotrophins
have only recently been studied. The PI3K pathway was suggested to mediate the effects of TrkB ligands
on radial migration as well as interneuron tangential migration, by pharmacological approaches (Polleux
et al., 2002; Yoshizawa et al., 2005). However, both the Shc-binding site, which leads to the activation of
PI3K and ERK, and the PLCg-binding sites on the TrkB receptor need to be mutated to induce a delay in
radial migration similar to that observed in conditional TrkB mouse mutants (Medina et al., 2004). Mice
with single point mutations at either site show normal CNS development (Medina et al., 2004). The PI3K
pathway was also suggested to lead to the activation of P-Rex1, a Rac guanine nucleotide-exchange factor
(GEF) that plays an important role in BDNF-induced migration of cortical neurons (Yoshizawa et al.,
2005). A dominant negative form of P-Rex1 was shown to inhibit the migration of cortical neurons
toward higher concentrations of BDNF in vitro and to give rise to radial migration impairments in vivo
(Yoshizawa et al., 2005).
Other molecules such as the Src-family kinases, which have been involved in Trk signaling and function
(Alema et al., 1985; Kremer et al., 1991; Tsuruda et al., 2004; Rajagopal and Chao, 2006), were recently
shown to be important for neuronal migration. Double mutant mice lacking both Src and Fyn show a defect
in cortical lamination similar to that observed in Reelin
/
mice (Kuo et al., 2005). However, it is unknown
whether these molecules play a direct role in neuronal migration in the context of neurotrophin receptor
signaling. Future studies will be necessary to uncover the relative contribution of several signaling pathways
to neurotrophin-induced migration in the cortex.

3.2 Modulation of Neuronal Migration in the Cerebellum by BDNF and NT-3

The characteristic layered structure of the cerebellum is formed by the coordinated migration of two
principle classes of neurons: Purkinje cells and granule neurons. Purkinje cells, the major output neurons of
the cerebellar cortex, originate from the ventricular zone. Once they reach the roof of the developing
cerebellar anlage and become postmitotic they migrate inwards along the radial glial fibers and settle on a
broad area just above the deep cerebellar nuclei (Altman and Bayer, 1985; Hatten, 1999). The precursors of
cerebellar granule neurons, which originate from the rhombic lip (Alder et al., 1996), first migrate in a
dorsorostral pathway to cover the surface of the emerging anlage, forming the external germinal layer (EGL;
Hatten, 2002). As they become postmitotic, granule neurons migrate radially, past the Purkinje cell layer, to
10 1 Neurotrophins and central nervous system development

form the internal germinal layer (IGL; Hatten, 1999). This leads to the establishment of the three layers of
the cerebellum: the outer molecular layer (ML), composed of granule cell axons and Purkinje cell dendrites,
the Purkinje cell layer and the IGL.
Neurotrophins appear to be important for the proper migration of cerebellar granule neurons during
development. Both TrkB and TrkC are expressed, although to various degrees, by the principal neurons of the
developing cerebellum (Klein et al., 1990; Ernfors et al., 1992). NT-3 mRNA levels peak in the IGL at the time
of granule neuron migration and decrease afterwards (Rocamora et al., 1993). Although BDNF mRNA was
not detected in the IGL in the first two postnatal weeks (Rocamora et al., 1993), when granule neurons
migrate radially to form that layer, BDNF protein was detected at P7, both in the IGL and the ML (Borghesani
et al., 2002). While this apparent controversy remains unsolved, it is interesting to note that BDNF protein
levels are low in the EGL, the starting point of granule neuron radial migration, and high in the ML and IGL,
where these neurons migrate to. Additionally, TrkB immunoreactivity in the developing cerebellum is highest
in migratory granule neurons when they are passing through the ML (Gao et al., 1995). Moreover, BDNF is
acutely motogenic for granule cells in culture and when these cells are exposed to a gradient of this
neurotrophin they migrate toward higher concentrations of BDNF (Borghesani et al., 2002).
A role for BDNF and TrkB in the migration of cerebellar granule neurons was confirmed in vivo by
studying the development of the cerebellum from BDNF and TrkB mouse mutants. The BDNF-deficient
mice exhibit an increase in the thickness of their EGL, at P14, while their IGL is sparser and thinner
(Schwartz et al., 1997; Borghesani et al., 2002). The EGL abnormally persists until later in development, in
BDNF mutants, being still present at P17-P21 (Jones et al., 1994; Schwartz et al., 1997). These phenotypes
are believed to be the result of a delay rather than a deficit in migration since at P23 the EGL is already not
apparent in the BDNF-deficient animals (Jones et al., 1994). Indeed, BDNF-deficient granule neurons show
a decreased migratory index: the number of cells that migrate is lower although the speed of migration is
similar to wildtype (Borghesani et al., 2002). Therefore, BDNF appears to be important for the initiation of
cerebellar granule neuron migration (Borghesani et al., 2002).
TrkB conventional and conditional mouse mutants also displayed a delay in granule cell migration, in
similarity to the BDNF-deficient mice (Minichiello and Klein, 1996; Rico et al., 2002). However, TrkB/TrkC
double mutants (TrkB
/
; TrkC+/
or TrkB+/
; TrkC
/
) exhibit a more pronounced enlargement of
the EGL as compared with the single mutants (Minichiello and Klein, 1996). This suggests that both NT-3
and BDNF may contribute to the proper migration of cerebellar granule neurons. Analysis of NT-3
conditional mutant mice failed to detect a defect in the layering of the cerebellum (Bates et al., 1999).
Nevertheless, this study was performed at P8, when the layering of BDNF-deficient cerebellum is also not
yet different from the wildtype (Schwartz et al., 1997). On the other hand, the NT-3-deficient cerebellum
presents a similar foliation defect to that seen in BDNF mouse mutants, which may or may not be related
with a problem in migration (Schwartz et al., 1997; Bates et al., 1999).
In conclusion, neurotrophins appear to be important for the proper timing of neuronal migration in
both the cerebral cortex and the cerebellum. However, the overlapping effects of different neurotrophins
in these brain regions may be preventing the clarification of the exact role of these molecules in neuronal
migration. A more comprehensive analysis of conventional and conditional single and double neurotrophin
mutants could undoubtedly further clarify the promising role of neurotrophins in neuronal migration.

4 Neurotrophins and CNS Dendritic Development

Soon after neurons reach their appropriate location, or even during migration, they extend neurites that
eventually differentiate into dendrites and axons. This polarization sets the stage for the formation of an
elaborate network of synaptic connections, in which neurite outgrowth, axonal guidance, and synaptogen-
esis take part. Although no evidence was yet presented for a role of neurotrophins in neuronal polarization,
these molecules are established regulators of dendritic growth and complexity while a role in axonal
guidance and synaptogenesis is also emerging (McAllister, 2001; Gillespie, 2003). On the last section of
this chapter, we will focus on the most documented aspect of neurotrophin function on central neuron
differentiation: the regulation of dendritic growth and complexity.
 Neurotrophins and central nervous system development 1 11

Neurons in the CNS evolve from immature cells with just a few processes to bearers of highly complex
dendritic arbors, which integrate most of the afferent input received by these neurons. To achieve this level
of intricate dendritic morphology neurons appear to follow an intrinsic growth program, capable of
generating a basic dendritic arborization, and to respond to extrinsic signals that shape their dendritic
tree into a final mature form. Neuronal activity, in response to afferent neuronal connections, has been
regarded as a key factor in the regulation of dendritic morphology by controlling intracellular Ca2+
concentration. Alterations in the concentration of this ion can activate signaling pathways that may locally
induce cytoskeletal reorganization and direct the synthesis of new proteins needed for dendritic growth
(reviewed in Dijkhuizen and Ghosh, 2005b).
Neurotrophins and their receptors are highly expressed in the developing CNS during the time period
when dendritic growth is occurring (reviewed in McAllister et al., 1999). Moreover, neurotrophins are
secreted in response to neuronal activity and therefore appear as plausible candidates to mediate activity-
dependent dendrite plasticity (Ghosh et al., 1994; Blochl and Thoenen, 1995; Goodman et al., 1996;
Wang and Poo, 1997; Hartmann et al., 2001). Indeed, neurotrophins, and in particular BDNF, have
been extensively described as crucial regulators of dendrite number, length and branching (reviewed in
Dijkhuizen and Ghosh, 2005b; McAllister, 2001).

4.1 Effects of Exogenous Neurotrophins on Pyramidal Neuron Dendrites

Early studies using ferret visual cortex organotypic slice cultures showed that all the neurotrophins have the
potential of modulating dendritic morphology of pyramidal neurons (McAllister et al., 1995). These and
other types of organotypic slice culture systems have been extensively used in the study of dendritogenesis
since the basic cytoarchitecture of the tissue is conserved and dendritic development can be followed for
extended periods in culture. In this system, all neurotrophins, and more effectively BDNF, NT-3 and NT-4,
were shown to increase dendritic complexity of cortical pyramidal neurons by increasing dendritic length
and branching and the number of primary dendrites (McAllister et al., 1995). Basal dendrites appear to be
more responsive to neurotrophins than apical dendrites, and to be maximally stimulated by a given
neurotrophin according to the specific cortical layer at which the neuron is located (McAllister et al.,
1995). Moreover, BDNF and NT-3 oppose each others actions on dendritic growth. In layer 4, NT-3 blocks
BDNF-induced increase in dendritic growth while BDNF inhibits the effects of NT-3 on layer 6 pyramidal
neuron dendrites (McAllister et al., 1997). The positive effects of exogenous BDNF, NT-3 and NT-4 on
pyramidal neuron dendritic length and number were also demonstrated to occur in rat brain, cortical and
hippocampal organotypic cultures, with region-specific and basal versus apical variations (Baker et al.,
1998; Niblock et al., 2000; Schwyzer et al., 2002; Wirth et al., 2003).
The effects mentioned above on dendrite morphology were obtained by exposing young organotypic
slice cultures, where neurons are still elaborating their dendritic arbors, to neurotrophins, for 36 h to a few
days. BDNF treatment of older ferret visual cortex organotypic slices, where pyramidal neurons have
already established their mature morphology, increases the number of basal dendrites at the expense of
distal branches, in just 24 h (Horch et al., 1999). Dendrite dynamics and instability were increased by
BDNF, with new basal dendrites being rapidly added and lost, in contrast to control neurons that
rarely loose basal dendrites (Horch et al., 1999). The effect of BDNF on dendrite morphology was
demonstrated to be extremely localized, occurring within 4.5 mm from the neurotrophin source (Horch
and Katz, 2002).

4.2 Effects of Exogenous Neurotrophins on the Dendrites of Non-Pyramidal

Interneurons and Other Neuronal Types
Non-pyramidal interneuron dendrites are also responsive to neurotrophin treatment. BDNF was shown to
increase total dendritic length and the number of dendritic branch points both in organotypic cortical
cultures and in dissociated cortical neurons in culture (Jin et al., 2003; Kohara et al., 2003; Wirth et al.,
12 1 Neurotrophins and central nervous system development

2003). NT-4 was also able to increase the same dendritic parameters on non-pyramidal interneurons in
cortical organotypic slice cultures (Wirth et al., 2003), while NT-3 was without effect (Baker et al., 1998).
However, it is not certain whether the differential effects of these neurotrophins were not due to different
experimental conditions, such as differences in the developmental stage of the culture, duration of
neurotrophin treatment, and brain region analyzed.
Other types of central neurons that show increased dendritic growth in response to neurotrophins
include rat Purkinje cells and mouse parvalbumin-positive interneurons of the olfactory bulb, which are
responsive to BDNF (Hirai and Launey, 2000; Berghuis et al., 2006), rat dopaminergic mesencephalic
neurons, which are affected by NT-4 (DeFazio et al., 2000), and mouse cerebellar granule neurons and
basilar pontine nuclei neurons that are responsive to both neurotrophins (Gao et al., 1995; Rabacchi et al.,
1999). An interesting effect of exogenous BDNF was reported in the Xenopus visual system, where
microsphere-coupled BDNF increases retinal ganglion cell (RGC) dendritic complexity in vivo when
injected in the optic tectum, their target area, while having the opposite effect when injected in the retina,
where RGC dendrites are located (Lom and Cohen-Cory, 1999; Lom et al., 2002). These results indicate that
local effects of neurotrophins in different subcellular domains of central neurons may exert distinct
outcomes on dendrite differentiation.

4.3 Role of Endogenous Neurotrophins in Dendritic Differentiation

The role of endogenous neurotrophins on neuronal dendritic development has been addressed through
the study of transgenic animals or the use of neurotrophin-neutralizing antibodies and the Trk receptor
fusion proteins TrkA-IgG, TrkB-IgG, and TrkC-IgG. The later, which bind to the respective Trk ligands
preventing activation of the corresponding neurotrophin receptor, have been shown to inhibit dendritic
development in the ferret visual cortex (McAllister et al., 1997). While TrkA-IgG has minor effects on
dendritic growth, TrkB-IgG causes retraction of existing basal dendrites in layer 4 pyramidal neurons and
TrkC-IgG reduces dendritic complexity of layer 6 neurons (McAllister et al., 1997). However, in rat visual
cortex organotypic slices, neutralizing antibodies against NT-4, but not those against BDNF, decrease
dendritic length and complexity (Wirth et al., 2003). In addition, activity-deprived organotypic cultures,
which have decreased BDNF mRNA levels, show normal dendritic parameters (Wirth et al., 2003). These
results argue in favor of NT-4 being the most important TrkB ligand in early dendritic development of the
visual cortex pyramidal neurons. Interestingly, analysis of a forebrain-restricted early onset BDNF
/

mouse strain showed that a decrease in primary dendrite number and branching is only seen in animals
older than 35 weeks, after the peak of primary dendrite development (Gorski et al., 2003). Therefore,
endogenous BDNF appears to be important for the maintenance but not the initial development of cortical
pyramidal dendrites. The need for TrkB ligands in the maintenance of cortical neuron dendritic arbors
was confirmed in late onset TrkB mutant mice, which show fewer and shorter branches at 6 weeks of age
(Xu et al., 2000).
For other neuronal types, such as the cerebellar Purkinje cells, the role of endogenous neurotrophins in
dendritic development is still controversial. Although one report shows that BDNF increases Purkinje cell
dendritic differentiation, while TrkB-IgG has the opposite effect (Hirai and Launey, 2000), another study
shows no effect of either BDNF or TrkB-IgG on Purkinje cell dendritic parameters (Shimada et al., 1998).
On the other hand, of two different strains of BDNF-deficient mice, one shows altered Purkinje cell
dendritic arbors with smaller and more numerous primary dendrites (Schwartz et al., 1997), while the
other has normal Purkinje cell dendritic arbors (Jones et al., 1994). A conditional TrkB mutant mouse strain
that lacks TrkB in the cerebellum also shows normal Purkinje cell dendritic development (Rico et al., 2002).
Purkinje cell dendrites were also unaffected in organotypic cultures of BDNF-deficient mouse cerebella or
wildtype cultures treated with BDNF or K-252a (Adcock et al., 2004).
A transgenic mouse bearing a single nucleotide polymorphism (SNP) in the bdnf gene, which leads to
a valine to methionine substitution at position 66 of the BDNF prodomain, was recently described
to have abnormal dendritic morphology (Chen et al., 2006). This polymorphism, which reduces
depolarization-evoked BDNF secretion, leads to decreased dendritic complexity of dentate gyrus neurons
 Neurotrophins and central nervous system development 1 13

in 8-week-old mice. These mice also exhibit behavior abnormalities such as increased anxiety and decreased
contextual-dependent memory, in similarity to the BDNF heterozygous mice (Chen et al., 2006). These are
important findings since this SNP was found to decrease hippocampal-dependent memory in humans
(Egan et al., 2003), thus associating dendritic development deficits with a behavior-impairing human
polymorphism.

4.4 Neurotrophin Receptors and Downstream Signaling Pathways

Involved in Dendritic Development
An important issue in neurotrophin-mediated dendritic development is to identify the neurotrophin
receptors and downstream signaling molecules that are involved. Trk neurotrophin receptors have been
shown to mediate many of the effects of endogenous and exogenously added neurotrophins on dendritic
morphology, by the inhibitory actions of the Trk inhibitor K-252a (Morfini et al., 1994; Horch et al., 1999;
Jin et al., 2003; Wirth et al., 2003). Additionally, transfection of ferret visual cortex organotypic slice
cultures with full-length TrkB induces an increase in proximal dendritic complexity similar to what was
observed with neurotrophin treatment (Yacoubian and Lo, 2000). NT-4 and BDNF potentiate the increase
in dendritic complexity observed after full-length TrkB transfection. Interestingly, transfection with the
truncated TrkB receptor T1 increases distal dendritic mass by net elongation of preexisting dendrites, which
is similar to the effect of K-252a on its own (Yacoubian and Lo, 2000).
Analysis of two different strains of p75-deficient mice revealed an increase in the dendritic complexity
of hippocampal pyramidal neurons, while overexpression of p75 had the opposite effect (Zagrebelsky et al.,
2005). This suggests that the outcome of neurotrophin action on dendritic development may depend on a
balance of competing signals from Trk receptors, which promote dendritic growth, and p75 receptors,
which inhibit growth. However, a positive effect of p75 on dendritic growth and complexity in response to
BDNF, NT-3, and NGF was reported to occur in young cultures of subventricular zone cells that later
become responsive to BDNF only, through the activation of TrkB receptors (Gascon et al., 2005).
The identity of the Trk downstream pathways involved in dendritic development has only recently been
addressed by studies using pharmacological approaches. Inhibitors of the ERK and Akt pathways were shown
to prevent the BDNF-induced increase in primary dendrite number and dendritic complexity in dissociated
cortical neurons and cortical slices in culture (Dijkhuizen and Ghosh, 2005a), respectively. PLCg inhibition
had only a slight inhibitory effect. Expression of a constitutively active form of PI3K was able to mimic the
effect of BDNF on primary dendrite numbers, while constitutively active MEK (mitogen activated protein
kinase), the kinase that activates ERK, was without effect (Dijkhuizen and Ghosh, 2005a). This confirms an
involvement of the Akt pathway in dendritic development. Interestingly, the PI3K-Akt pathway controls
dendritic size in cultured rat hippocampal CA3/CA1 pyramidal neurons, while a coordinated activation of
this pathway and the ERK signaling cascade is necessary to regulate dendritic complexity (Kumar et al.,
2005). Also suggesting a role for the ERK pathway in dendritic development, the small GTPase Rap1, which
is known to lead to sustained ERK activation downstream of Trk receptors in PC12 cells (York et al., 1998),
is required for depolarization-induced dendritic growth and branching in rat cortical cultures (Chen et al.,
2005). In rat olfactory GABAergic neurons in culture, both PLCg and ERK inhibitors reduce the BDNF-
induced increase in neurite branching and elongation. Further studies will be necessary to clarify which Trk-
activated signaling pathway or pathways play the most relevant role in neurotrophin-regulated dendritic
development, and whether this is a common mechanism among different types of neurons and different
regions of the brain.
An interesting molecule downstream of TrkB is Tiam1, a guanine-nucleotide exchange factor that
activates the Rho family GTPases Rac1 and Cdc42. TrkB directly activates Tiam1 by phosphorylating a
specific tyrosine residue on this protein (Miyamoto et al., 2006). TrkB-phosphorylated Tiam1 is capable of
activating Rac1 in vitro. Treatment of cultured cortical neurons with BDNF induces the association of TrkB
with Tiam1, as well as Tiam1 tyrosine phosphorylation and activation. Moreover, BDNF-induced increase
in neurite number and length is inhibited by siRNA knockdown of Tiam1 and by transfection with mutant
Tiam1 carrying a single point mutation on the tyrosine residue that is phosphorylated by TrkB (Miyamoto
14 1 Neurotrophins and central nervous system development

et al., 2006). Tiam1 is therefore a promising new TrkB effector, suggesting the existence of a simple signaling
pathway with a few intermediaries that closely links TrkB activation and actin cytoskeleton dynamics, which
are crucial for dendritic remodeling.

4.5 Interplay Between Neurotrophins and Neuronal Activity

As we previously mentioned, neuronal activity is an established regulator of dendritic development. What is
the relationship between neurotrophins and activity in this context? Only a few reports address this
question and do not offer a consensual answer. One of the initial studies on the effects of neurotrophins
on dendritic differentiation, using the ferret visual cortex organotypic cultures, showed that blocking
activity, with NMDA or AMPA receptor antagonists or voltage-gated Na+ or Ca2+ channel inhibitors,
prevents most of BDNF-induced dendritic growth (McAllister et al., 1996). However, in rat brain organo-
typic slices, NMDA and AMPA receptor antagonists were not able to inhibit NT-3-induced increase in
pyramidal neuron dendritic length and number (Baker et al., 1998).
A lack of effect of activity blockade on BDNF-induced increase in GABAergic interneuron dendritic
complexity was also demonstrated in mouse organotypic cortical slices (Jin et al., 2003). On the other hand,
the effect of BDNF on interneuron dendrite morphology was mimicked by KCl depolarization, which was
blocked by anti-BDNF antibodies (Jin et al., 2003). This suggests that neuronal activity induces the release
of BDNF, which is responsible for at least some of the activity-induced effects on dendritic differentiation. A
similar notion was suggested in studies using cerebellar Purkinje cells and granule neuron co-cultures,
where activity-induced release of BDNF by granule neurons was proposed to mediate Purkinje cell dendrite
development (Hirai and Launey, 2000). This would be in agreement with previous reports that show that
BDNF is secreted in response to activity (Ghosh et al., 1994; Blochl and Thoenen, 1995; Goodman et al.,
1996; Wang and Poo, 1997; Hartmann et al., 2001).
Whether neurotrophins increase activity, act in conjunction with activity or are released by activity in
order to exert their effects on dendritic development is an extremely important issue that needs to be
clarified. Judging from the studies mentioned above, the three scenarios may co-exist and the comparative
weight of each one of them for neurotrophin-induced dendritic growth and complexity probably depends
on the type of neuron analyzed, among other factors. Understanding the relationship between neuronal
activity and neurotrophins, and the consequences on higher order function and behavior, is perhaps the
most exciting direction in the study of neurotrophins on CNS neuronal differentiation.

Acknowledgments

The authors are supported by grants from NIH (NS21072 and HD23315). D.B.P. is supported by a
postdoctoral fellowship from Fundacao Portuguesa para a Ciencia e a Tecnologia.

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2 Nerve Growth Factor Regulated
Gene Expression
L. A. Greene . J. M. Angelastro

1 Overall Aim and Content of This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

2 The Role of Regulated Gene Expression in the NGF MechanismWhat Genes Does NGF
Regulate and Why Do We Want to Know? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

3 Regulation of Gene Expression by NGFA View from the Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

4 LongTerm Regulation of Gene Expression by NGFResults of a SAGE

Study with PC12 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

5 Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

# 2008 Springer ScienceBusiness Media, LLC.

22
2 Nerve growth factor regulated gene expression

Abstract: This chapter provides a current accounting of the identities of genes and gene products that are
subject to longterm regulation by nerve growth factor (NGF). We provide tabular listings of (a) NGF
responsive genes and proteins that are reported in the literature and (b) transcripts found by comprehensive
serial analysis of gene expression (SAGE) to be differentially expressed in PC12 cells before and after longterm
NGF exposure. Transcriptional changes are key elements in the mechanisms by which NGF affects neuronal
differentiation, function, protection, and repair. The information provided here is thus meant to serve as a
resource for those interested in the molecular mechanisms and consequences of trophic factor actions.
List of Abbreviations: ATF5, activating transcription factor 5; EST, expressed sequence tag; NGF, nerve
growth factor; SAGE, serial analysis of gene expression

1 Overall Aim and Content of This Chapter

The aim of this chapter is to provide a current account of what we know about the identities of the genes and
gene products that are subject to longterm regulation by NGF. Our objective is like that of traditional
handbooks of chemistry and physics, that is, to list information without including extensive interpretive
rhetoric. We hope that the reader will find this information to be a useful resource that will inspire further
experimental and intellectual advances regarding the development, function, and repair of the nervous system.
There are two principal portions of this chapter. One is a tabular listing of the genes and gene products
that have been identified in the literature as being regulated by NGF. The second is a tabular listing of
identified NGF regulated transcripts that we have detected in a largescale SAGE comparison of the
transcriptomes of nave and longterm NGFtreated PC12 cells. In each case, the regulated genes have
been subdivided into categories based on the current information about their functional properties.
Part of the appeal of putting together such a chapter is the opportunity provided by current electronic
technology to easily and swiftly update and extend the information herein and to make such modifications
rapidly accessible to readers. We intend to continue to add to this chapter new findings in the literature as
well as the results of our continuous efforts to fully match the output of our SAGE study with known genes.
We will also continue to take into account new findings about the functional activities of the various genes
listed here. An additional aspect is that, such a format will permit us to correct errors and omissions that we
anticipate have not only occurred, but will be (and we encourage this) pointed out to us by readers. Finally, as
noted earlier, the present chapter includes genes and gene products subject to longterm regulation by NGF.
By long term, we mean to the exclusion of immediate early genes and potentially, to some genes that are only
transiently regulated by NGF. Should the opportunity arise, we will endeavor to add such genes in the future.

2 The Role of Regulated Gene Expression in the NGF MechanismWhat

Genes Does NGF Regulate and Why Do We Want to Know?

Though we now largely take for granted the notion that neurotrophic factors such as NGF act in part by
regulating gene expression, this was not the case always. The capacity of NGF to promote neuron survival as
well as differentiation led to an early debate over whether the factor was merely permissive for differentia-
tion or whether it truly possessed instructive actions. Moreover, the relative stability of NGFregulated
transcripts and proteins and the capacity of NGF to promote neurite outgrowth in explanted ganglia led to
the hypothesis that the factor acted by a mechanism that was independent of transcription (Partlow and
Larrabbee, 1971; Mizel and Bamburg, 1976). The advent of cell systems such as PC12 cells without a
requirement for NGF or without prior exposure to the factor (in contrast to dissected neurons) permitted
the first demonstrations that NGF does regulate genes (McGuire et al., 1978 ; Greene, 1981; Greenberg et al.,
1985) and that such effects play essential roles in neuronal differentiation and function (Burstein and
Greene, 1978; Greene et al., 1983). Moreover, the introduction of means to culture adult sensory neurons,
which respond to NGF, but do not require it, has extended our capacity to uncover effects of the factor on
gene expression (Lindsay, 1988; Lindsay and Harmar, 1989). Additional advances in neuron culture, in
 Nerve growth factor regulated gene expression
2 23

introducing NGF in vivo, and in generating animals that are null for key cell death genes and in which
neurons can consequently survive in absence of trophic support, have further widened the opportunities to
identify NGFregulated genes and gene products. Finally, molecular technologies have greatly improved the
sensitivity and efficiency with which NGFdependent changes in gene expression can be monitored.
If NGF acts in part by regulating genes, then the question naturally arises about their identities.
Knowing the identities of NGFregulated genes is important for several reasons. NGF participates in a
number of fundamental activities including promotion of differentiation (itself a multifaceted response),
survival, plasticity, repair, neuroprotection, and neurite outgrowth. Many of the genes regulated by NGF are
key regulators and participants in these major activities and their identification thus provides fundamental
insight into the molecular mechanisms by which they occur. Furthermore, understanding the mechanisms
by which NGF acts, must include a clear description of the genes subject to its regulation. In this context,
knowledge about the genes that NGF regulates not only permits construction of mechanistic pathways, but
also provides candidates for experimental extension of, and intercalation within, these pathways. Con-
fronting ourselves (and our colleagues) with the knowledge that there are large numbers of NGFregulated
genes for which we understand neither function nor potential roles, is not only humbling, but also spurs us
to understand how they contribute to the NGF mechanism of action. Finally, NGF is one of many
neurotrophic factors. If we can identify and understand the functions of the genes that NGF regulates,
we will have an enormous insight into the role of such genes in the actions of other trophic factors as well.

3 Regulation of Gene Expression by NGFA View from the Literature

> Table 21 is a listing based on a search of the literature (up to July 2004), of genes and gene products that
have been reported to be subject to regulation by NGF. Though we have attempted to be comprehensive,
there are likely to be unintentional omissions and readers are invited to point out these for future inclusion.
Multiple entries for the same gene/gene product are given in cases in which the findings have been made in
different experimental systems, in which observations regarding RNA and protein were in different papers,
or in cases in which different authors have reported contrasting results. We have not attempted to list all
authors or papers that report similar observations in the same experimental system or necessarily those that
were published first. Where sufficient information was given about the molecule in question, we have listed
an NCBI accession number. On the basis of information provided within the cited papers, the general
literature, and NCBI genome listings, we have subdivided the various genes/proteins into functional
categories. In some cases, the same gene is listed under multiple functional headings. We have not
quantified the reported changes (and in many cases, neither did the authors); hence, there is no threshold
for degree of regulation other than the authors interpretation that the reported changes were significant.
Finally, a word about the definition of gene regulation in the context of this chapter. By this we mean
changes in the levels of cellular proteins and transcripts. We are aware that such changes are not necessarily
due to altered gene transcription and may arise from changes in stability. However, few of the responses to
NGF have been studied at this level; hence, we follow this broader interpretation.

4 LongTerm Regulation of Gene Expression by NGFResults of a SAGE

Study with PC12 Cells

Although the list of NGFregulated genes in the literature is quite substantial, we felt that it was likely to be
far short of the numbers that are actually responsive to NGF. To address this, in 2000 we carried out a
comprehensive comparison, using SAGE technology, of transcripts expressed by rat PC12 pheochromocy-
toma cells before and after 9days of treatment with NGF (Angelastro et al., 2000). PC12 cells proliferate in
serumcontaining medium and do not require NGF for survival. In response to NGF under the same
conditions, PC12 cells exit the cell cycle and over a timecourse of days undergo differentiation into neurite
bearing, electrically excitable cells that resemble sympathetic neurons. Thus, they represent a convenient
system in which to identify NGFpromoted changes in gene expression. SAGE is an unbiased and highly
24
2 Nerve growth factor regulated gene expression

. Table 2-1
NGF regulated changes in gene expression
Up/
Gene name Access. # Down RNA/Prot System References
Antioxidant
Catalase (Cat) NM_012520 Up R PC12 Sampath et al.
(1994)
Cat NM_012520 Up R Striatum in vivo Frim et al. (1994)
Glutathione peroxidase Up R PC12 Sampath et al.
(1994)
GlutathioneStransferase mu Up P PC12 NurEKamal
et al. (2000)
GlutathioneStransferase pi Up P PC12 NurEKamal
et al. (2000)
Hypoxia inducible factor AJ277828 Down R, P PC12 NaranjoSuarez
2 alpha (Hif2a) et al. (2003)
Thioredoxin NM_053800 Up R, P PC12 Bai et al. (2003)

Calcium binding
Calbindin 1 (Calb1) NM_031984 Up P, R PC12 Iacopino et al.
(1992)
Calmodulin Up R PC12 Bai and Weiss
(1991)
Calpactin I heavy chain/ NM_019905 Up P, R PC12 Jacovina et al.
Annexin II (2001)
S100 related protein, clone NM_031114 Up R PC12 Masiakowski and
42C/calpactin I light chain Shooter (1988)

Cytoskeleton and neurite outgrowth

Alpha actinin Up P PC12 Sobue and
Kanda (1989)
Alphatubulin/T alpha 1 alpha NM_022298 Up R PC12 Miller et al.
tubulin (Tuba1) (1987)
Tuba1 NM_022298 Up R Sympathetic Mathew and
neurons in vivo Miller (1990)
Tuba1 NM_022298 Up R Adult DRG in Mohiuddin et al.
vitro (1995)
Adenomatous polyposis coli NM_012499 Up P PC12 Dobashi et al.
(APC) (1996)
Beta1 adducin AF130338 Up R Cultured DRGs Ghassemi et al.
(2001)
Gammaactin X52815 Up P PC12 Chang et al.
(1986)
Growth associated protein 43 NM_017195 Up P, R PC12 Costello et al.
(Gap43) (1990)
Gap43 NM_017195 Up R Adult sensory Mohiuddin et al.
neurons in vitro (1995)
Gelsolin Up P PC12 Furnish et al.
(2001)
Integrin alpha 1 (Itga1) NM_030994 Up R PC12 Danker et al.
(2001)
Microtubuleassociated AF079778 Up P PC12 Greene et al.
protein 1b (Map1b) (1983)
 Nerve growth factor regulated gene expression
2 25

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Microtubuleassociated NM_017212 Up P, R PC12 Drubin et al.
protein tau (Mapt) (1985)
Microtubuleassociated NM_013066 Up P, R PC12 Fischer et al.
protein 2 (Mtap2) (1991)
Neurofilament, light NM_031783 Up P, R PC12 Lindenbaum
polypeptide (Nfl) et al. (1987)
Nef3 neurofilament 3, NM_017029 Up P, R PC12 Lindenbaum
medium/Nfm et al. (1987)
Neuronal kinesin heavy chain Up P, R PC12, Vignali et al.
neuroblastoma (1996)
in vitro
Peripherin 1 (Prph1) NM_012633 Up P, R PC12 Aletta et al.
(1988)
Stathmin 1 (Stmn1) NM_017166 Up R PC12 Takekoshi et al.
(1998)
Stathminlike 2/SCG10 NM_053440 Up R PC12 Stein et al.
(Stmn2) (1988)
Thymosin beta4 NM_031136 Up R PC12 Leonard et al.
(1987)
Tropomyosin 1 (Tpm1), alpha/ M34135 Up R PC12 Weinberger
alphatropomyosin (TMBr1) et al. (1993)
Tropomyosin 1 (Tpm1), alpha/ M34136 Up R PC12 Weinberger
alphatropomyosin (TMBr3) et al. (1993)

Cell cycle
APC NM_012499 Up P PC12 Dobashi et al.
(1996)
Cdc2/Cdk1 Down P PC12 Buchkovich and
Ziff (1994)
Cyclin dependent kinase NM_199501 Down P PC12 Buchkovich and
2 (Cdk2) Ziff (1994)
Cyclindependent kinase 4 NM_053593 Down P PC12 Yan and Ziff
(CDK4) (1995)
Cdkn1a cyclindependent NM_080782 Up P, R PC12 Yan and Ziff
kinase inhibitor 1A/Waf1 (1997)
Cyclin B1 (Ccnb1) NM_171991 Down P PC12 Yan and Ziff
(1995)
Cyclin D1 (Ccnd1) NM_171992 Up P, R PC12 Yan and Ziff
(1995)
Cyclin D2 (Ccnd2) D16308 Down P PC12 Tamaru et al.
(1994)
Cyclin F (Ccnf) XM_340763 Down P, not R PC12 Movsesyan et al.
(1996)
Cyclin G Up P PC12 Gollapudi and
Neet (1997)
Deleted in colorectal U68725 Up R PC12 Lawlor and
carcinoma (Dcc) Narayanan
(1992)
E2F transcription factor 1 D63165 Down P PC12 Persengiev et al.
(E2f1) (1999)
26
2 Nerve growth factor regulated gene expression

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
E2F transcription factor Up P PC12 Persengiev et al.
2 (E2f2) (2001)
E2F transcription factor 3 Down P PC12 Persengiev et al.
(E2f3) (2001)
E2F transcription factor 4 Up P PC12 Persengiev et al.
(E2f4) (2001)
E2F transcription factor 5 XM_342209 Down P PC12 Persengiev et al.
(E2F5) (2001)
Growth arrest specific 7 NM_053484 Up P PC12 Chao et al.
(GAS7) (2003)
Retinoblastomalike 2 (Rbl2); NM_031094 Up P, R PC12 Paggi et al.
p130 (2001)
Proliferating cell nuclear NM_022381 Down P PC12 Yan and Ziff
antigen (Pcna) (1995)
Retinoblastoma 1 (Rb1) XM_344434 Down P PC12 Persengiev et al.
(2001)

Death and survival

Bcl2l1 Bcl2like 1/Bclxl NM_031535 Up R PC12 Rong et al.
(1999)
Bcl2l1 Bcl2like 1/Bclxs NM_031535 Up R PC12 Rong et al.
(1999)
Bcl2l11 BCL2like 11 (apoptosis NM_022612 Down P PC12 Biswas and
facilitator)/BIM Greene (2002)
Caspase 3 (Casp3) NM_012922 Up R PC12 Rong et al.
(1999)
Clusterin (Clu) NM_053021 Up R PC12 Gutacker et al.
(1999)
ADPribosyltransferase 1 U94340 Down R, P PC12 Taniguchi et al.
(Adprt) (1988)

Disease related
Amyloid beta (A4) precursor NM_019288 Up P, R PC12 Villa et al. (2001)
protein (App)
App NM_019288 Up R Developing Mobley et al.
brain (1988)
Prion protein (Prnp) NM_012631 Up R PC12 Wion et al.
(1988)
Prnp NM_012631 Up R Developing Mobley et al.
brain (1988)

Growth factors and receptors/signal transduction

Epidermal growth factor NM_031507 Down R PC12 Shibutani et al.
receptor (EGFR) (1998)
Fibroblast growth factor NM_024146 Up R PC12 Meisinger et al.
receptor 1 (FGFR1) (1996)
Interleukin 1 alpha (Il1a) NM_017019 Up R, P PC12 Alheim et al.
(1996)
Transforming growth factor, NM_021578 Up R, P PC12 Kim et al. (1994)
beta 1 (Tgfb1)
 Nerve growth factor regulated gene expression
2 27

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Tgfb1 NM_021578 Up R, P Chromaffin cells Forander et al.
in oculo (2001)
Vascular endothelial growth NM_031836 Down R PC12 NaranjoSuarez
factor (Vegf) et al. (2003)

Ion channels
Ca channel, voltage NM_147141 Up R PC12 Colston et al.
dependent, N type, alpha 1B (1998)
subunit
Cacna1c Ca channel, voltage NM_012517 Down R PC12 Colston et al.
dependent, Ltype alpha 1C (1998)
Kcnb1 K voltage gated NM_013186 Up P, not R PC12 Sharma et al.
channel, Shabrelated, (1993)
member 1
Cacna1d Ca channel, voltage NM_017298 Down R PC12 Colston et al.
dependent, Ltype alpha 1D (1998)
K small conductance Ca NM_019313 Up P DRG in vitro Boettger et al.
activated channel N1 (Kcnn1) (2002)
Proton gated cation channel NM_173135 Up R Sensory Mamet et al.
DRASIC neurons (2003)
Na channel, voltagegated, NM_030875 Up R DRG neurons in Zur et al. (1995)
type 1, a polypeptide (Scn1a) vitro
Na channel, voltagegated, NM_012647 Up R PC12 Mandel et al.
type 2, a1 polypeptide (1988)
(Scn2a1)
Na channel, voltagegated, NM_012647 Up R DRG neurons in Zur et al. (1995)
type 2, a1 polypeptide vitro
(Scn2a1)
Na channel, voltagegated, NM_013119 Down R Adult DRG in Black et al.
type III, a polypeptide (Scn3a) vitro (1997)
Na channel, voltagegated, NM_133289 Up R PC12 DArcangelo
type 9, a polypeptide (Scn9a) et al. (1993)
Na channel, voltagegated, NM_017247 Up P Adult DRG in Black et al.
type 10, a polypeptide vitro (1997)
(Scn10a)
Na channel, voltagegated, NM_017288 Up R DRG neurons in Zur et al. (1995)
type I, beta polypeptide vitro
(Scn1b)
Lectins
Lectin, galactose binding, NM_031832 Up P, R PC12 Kuklinski et al.
soluble 3 (Lgals3) (2003)
Lgals3 NM_031832 Up P DRG in vitro Pesheva et al.
(2000)

Miscellaneous

ATPase, Na K transporting, NM_012504 Up P PC12 Kurihara et al.
alpha 1 (1994)
Collagenase 1 Up R PC12 Vician et al.
(1997)
CTP: phosphocholine Up R PC12 Carter et al.
cytidylyltransferase beta2 (2003)
28
2 Nerve growth factor regulated gene expression

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Lactic Dehydrogenase type M Down R PC12 Calissano et al.
(LDHM) (1985)
Low density lipoprotein Up R PC12 Bu et al. (1998)
receptor related protein
LRP1B/LRPDIT
Prostaglandinendoperoxide NM_017043 Up P, R PC12 Kaplan et al.
synthase 1 (Ptgs1) (1997)
Nmyc downstream regulated NM_031967 Up P PC12 Nakada et al.
4 (Ndr4) (2002)
NeuroendocrineSpecific Up P PC12 Hens et al.
ProteinA (NSPA) (1998)
NeuroendocrineSpecific Up P PC12 Hens et al.
ProteinC (NSPC) (1998)
NID67 putative small NM_173126 Up R PC12 Vician et al.
membrane protein (2001)
Nin283/Znfr1 Up R PC12 Araki et al.
(2001)
Nnat neuronatin NM_053601 Down R PC12 Joseph et al.
(1996)
Ornithine decarboxylase 1 NM_012615 Up P, R PC12 Feinstein et al.
(Odc1) (1985)
Peripheral myelin protein 22 NM_017037 Up R PC12 De Leon et al.
(1994)
Plaur plasminogen activator, NM_017350 Up R PC12 FariasEisner
urokinase receptor et al. (2000)
Serine (or cysteine) proteinase NM_012620 Up R PC12 Vician et al.
inhibitor, member 1 (1997)
Similar to polypyrimidine tract Down R PC12 Ichikawa et al.
binding protein 2/PTBLPL (2002)
Similar to polypyrimidine tract Up R PC12 Ichikawa et al.
binding protein 2/PTBLPS (2002)
Similar to REN XM_343923 Up R PC12, TC1S Gallo et al.
cells (2002)
Tff3 trefoil factor 3/ITF NM_013042 Up P, R PC12 Probst et al.
(1997)

Neurotrophins and receptors

Brain derived neurotrophic NM_012513 Up R Adult DRGs in Apfel et al.
factor (Bdnf) vivo (1996)
NGF receptor (Ngfr) (TNFR NM_012610 Up P, R Cholinergic Cavicchioli et al.
superfamily, member neurons in vivo (1989)
16)/p75
Ngfr/p75 NM_012610 Up R DRG neurons in Lindsay et al.
vitro (1990)
Ngfr/p75 NM_012610 Up R Sympathetic Miller et al.
neurons, PC12 (1991)
Trk precursor NM_021589 Up R Adult DRG in Mearow (1998)
vivo
Trk precursor NM_021589 Up R Forebrain Kojima et al.
neurons in vitro (1994)
 Nerve growth factor regulated gene expression
2 29

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Neuropeptides
Adenylate cyclase activating NM_016989 Up R PC12 Hashimoto et al.
polypeptide 1 (Adcyap1)/ (2000)
PACAP
Adcyap1/PACAP NM_016989 Up R Adult DRG in Jongsma Wallin
vivo et al. (2001)
PACAP receptor 1 NM_133511 Up R PC12 Cavallaro et al.
(Adcyap1r1) (1995)
Adrenomedullin (Adm) NM_012715 Down P, R PC12 Kobayashi et al.
(2004)
Angiotensin II receptor, type NM_012494 Down R Cultured Huang et al.
2 (Agtr2) hypothalamus/ (1997)
brainstem
Bradykinin receptor b2 NM_173100 Up P, R Cultured adult Lee et al. (2002)
(Bdkrb2) DRG
Calcitonin/calcitoninrelated NM_017338 Up P, R Cultured adult Lindsay and
polypeptide, alpha (Calca) DRG Harmar (1989)
Galanin (Gal) NM_033237 Up R Basal forbrain in Planas et al.
vivo (1997)
Gal NM_033237 Down R DRG, in vitro and Corness and
in vivo Hokfelt (1998)
Neuropeptide Y (Npy) NM_012614 Up P, R PC12 Allen et al. (1987)
Neurotensin/neuromedin N M21187 Up P, R PC12 Caillaud et al.
gene (1995)
Tachykinin 1 (Tac1) NM_012666 Up P, R DRG, trigeminal Vedder et al.
neurons in vitro (1993)
Vanilloid receptor (Vr1)/Trpv1 AF327067 Up R Adult DRG Winston et al.
neurons in vitro (2001)
Neuropeptide Y receptor Y1 XM_344502 Up R PC12 Bournat et al.
(Npy1r) (2001)

Neurotransmission
Acetylcholinesterase (Ache) NM_172009 Up R PC12 DeschenesFurry
(stabilized) et al. (2003)
Adenosine A2a receptor NM_053294 Down P, R PC12 Arslan et al.
(Adora2a) (1997)
Agrin (Agrn) NM_175754 Up R PC12 Smith et al.
(1997)
Choline acetyltransferase Up R Basal forebrain in Higgins et al.
(ChAT) vivo (1989)
Cholinergic receptor, NM_080773 Down R Telencephalic Eva et al. (1992)
muscarinic 1 (Chrm1) neurons in vitro
Cholinergic receptor, NM_031016 Up R Striatal neurons Ebstein et al.
muscarinic 2 (Chrm2) in vitro (1993)
Cholinergic receptor, NM_012527 Down R Telencephalic Eva et al. (1992)
muscarinic 3 (Chrm3) neurons in vitro
Cholinergic receptor, XM_345403 Up R Telencephalic Eva et al. (1992)
muscarinic 4 (Chrm4) neurons in vitro
Chrm4 XM_345403 Up R PC12 Lee and Malek
(1998)
30
2 Nerve growth factor regulated gene expression

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Chromogranin A (Chga) NM_021655 Up R PC12 Mahata et al.
(1999)
Cholinergic receptor, nicotinic, NM_052805 Up R PC12 Henderson et al.
alpha polypeptide 3 (Chrna3) (1994)
Chrna3 NM_052805 Down R PC12 Rogers et al.
(1992)
Cholinergic receptor, nicotinic, NM_017078 Up R PC12 Takahashi et al.
alpha polypeptide 5 (Chrna5) (1999)
Chrna5 NM_017078 Up R PC12 Henderson et al.
(1994)
Chrna5 NM_017078 Down R PC12 Rogers et al.
(1992)
Cholinergic receptor, nicotinic, NM_012832 Up R PC12 Henderson et al.
alpha polypeptide 7 (Chrna7) (1994)
Cholinergic receptor, nicotinic, NM_019297 Up R PC12 Rogers et al.
beta polypeptide 2 (Chrnb2) (1992)
Cholinergic receptor, nicotinic, AY574259 Up R PC12 Takahashi et al.
beta polypeptide 3 (Chrnb3) (1999)
Chrnb3 AY574259 Down R PC12 Rogers et al.
(1992)
Cholinergic receptor, nicotinic, NM_052806 Up R PC12 Henderson et al.
beta polypeptide 4 (Chrnb4) (1994)
Chrnb4 NM_052806 Up R PC12 Avila et al. (2003)
Chrnb4 NM_052806 Down R PC12 Rogers et al.
(1992)
Dopamine beta hydroxylase NM_013158 Down R PC12 Badoyannis
(Dbh) et al. (1991)
Dopa decarboxylase (Ddc) NM_012545 Down R PC12 Li et al. (1997)
Dystrophin Dp71 Up P, R PC12 Cisneros et al.
(1996)
Glutamate receptor, NM_017011 Up P, R PC12 Kane et al.
metabotropic 1 (Grm1) (1998)
GTP cyclohydrolase 1 (Gch) NM_024356 Up R PC12 Anastasiadis
et al. (1996)
Gch NM_024356 Up R Sympathetic Hirayama and
neurons in vitro Kapatos (1995)
Nitric oxide synthase 1, NM_05279 Up P,R PC12 Sheehy et al.
neuronal (Nos1) (1997)
Rab3a: RAB3A, member RAS NM_013018 Up R PC12 Sano et al.
oncogene family (1989)
Nitric oxide synthase 1, NM_05279 Up R Cholinergic Holtzman et al.
neuronal (Nos1) neurons in vivo (1996)
Secretogranin 2 (Scg2) NM_022669 Up P, R PC12 Laslop and
Tschernitz (1992)
Synuclein, alpha (Snca) NM_019169 Up P, R PC12 Stefanis et al.
(2001)
Solute carrier family 6, member NM_031343 Down R PC12 Ikeda et al.
2/noradenalin transporter (2001)
 Nerve growth factor regulated gene expression
2 31

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Synapsin 1 (Syn1) NM_019133 Up P PC12 Romano et al.
(1987)
Synaptophysin (Syp) NM_012664 Down P PC12 Vetter and Betz
(1989)
Synaptotagmin 1 (Syt1) XM_343205 Up P PC12 Lah and Burry
(1993)
Tyrosine hydroxylase (Th) NM_012740 Up P Sympathetic Max et al. (1978)
ganglia in vitro
Th NM_012740 Up R Sympathetic Ma et al. (1992)
neurons in vitro
Th NM_012740 Up P PC12 Osaka and
Sabban (1997)
Solute carrier family 18, NM_031663 Up R Septal neurons Oosawa et al.
member 3 (Slc18a3)/VAChT in vitro (1999)
VGF nerve growth factor NM_030997 Up R PC12 Levi et al. (1985)
inducible (Vgf)
Signal transduction
Aldehyde dehydrogenase NM_053896 Up R Adult DRG in Corcoran and
family 1, subfamily A2 vitro Maden (1999)
(Aldh1a2)
Dual specificity phosphatase 6 NM_053883 Up R PC12 Mourey et al.
(Dusp6)/MKP3 (1996)
Guanine nucleotide binding NM_017327 Up P PC12 Zubiaur and
protein, alpha o (Gnao) Neer (1993)
G beta Up P PC12 Zubiaur and
Neer (1993)
Gnai2: GTPbinding protein (G NM_031035 Up P PC12 Zubiaur and
alphai2) Neer (1993)
Guanylate cyclase 1, soluble, NM_017090 Down P, R PC12 Liu et al. (1997)
alpha 3 (Gucy1a3)
Guanylate cyclase 1, soluble, NM_012769 Down P, R PC12 Liu et al. (1997)
beta 3 (Gucy1b3)
Neurofibromatosis type I Up R PC12 Metheny and
(NF1) Skuse (1996)
Opioid receptor, sigma 1 NM_030996 Up P PC12 Takebayashi
(Oprs1) et al. (2002)
Phospholipase D1 (Pld1) NM_030992 Up R PC12 Hayakawa et al.
(1999)
Protein kinase C, alpha (Prkca) XM_343975 Up P PC12 Min et al. (2001)
Protein kinase C, beta 1 NM_012713 Up P PC12 Min et al. (2001)
(Prkcb1)
Phospholipase D2 (Pld2) NM_033299 Up P, R PC12 Gibbs and Meier
(2000)
Protein tyrosine phosphatase, NM_053769 Up R Sympathetic PeinadoRamon
nonreceptor type 16/MKP1 neurons in vitro et al. (1998)
Protein tyrosine phosphatase, NM_053594 Transient R PC12 Sharma and
receptor type, R (Ptprr) Up Lombroso
(1995)
32
2 Nerve growth factor regulated gene expression

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Ptprr NM_053594 Down R PC12h Shiozuka et al.
(1995)
Protein tyrosine phosphatase, XM_341950 Transient R PC12 Mukouyama
receptor type, epsilon (Ptpre) Up et al. (1997)
Retinoic acid receptor beta2 Up P, R PC12 Cosgaya and
Aranda (2001)
Thioredoxin NM_053800 Up R, P PC12 Bai et al. (2003)

Substrate interactions
Integrin alpha 1 (Itga1) NM_030994 Up R PC12 Danker et al.
(2001)
Neural cell adhesion molecule NM_017345 Up P, R PC12 Grant et al.
L1 (NcamL1) (1996)
Neural cell adhesion molecule NM_031521 Up P, R PC12 Prentice et al.
1 (Ncam1) (1987)
Matrix metalloproteinase 3 NM_133523 Up R PC12 Machida et al.
(Mmp3)/stromelysin1 (1989)
Thymus cell antigen 1, theta NM_012673 Up P,W PC12 Doherty et al.
(Thy1) (1988)

Transcriptional regulators
Achaetescute complex NM_022384 Down R PC12 Grumolato et al.
homologlike 1 (Ascl1)/ (2003)
MASH1
AP2 Up P PC12 Paggi et al.
(2001)
Activating transcription factor NM_172336 Down P, R PC12 Angelastro et al.
5 (ATF5) (2003)
DNA (cytosine5) NM_053354 Down P, R PC12 Deng and Szyf
methyltransferase 1 dnmt1 (1999)
Estrogen receptor Up P, R PC12 Sohrabji et al.
(1994)
Fak1/fetal Alzheimer antigen/ Up R PC12 Rhodes et al.
falz (2003)
Foslike antigen 1/FRA1 NM_012953 Up P PC12 Cosgaya and
(Fosl1) Aranda (2001)
Foslike antigen 1/FRA NM_012954 Up P PC12 Cosgaya and
2 (Fosl2) Aranda (2001)
LIM homeobox protein 1 NM_145880 Up R DRG in vitro Jameson and
(Lhx1)/Rlim Lillycrop (2001)
Hypoxia inducible factor AJ277828 Down R, P PC12 NaranjoSuarez
2 alpha (Hif2a) et al. (2003)
LIM homeobox protein 3 AF370447 Up R DRG in vitro Jameson and
(Lhx3) Lillycrop (2001)
Math2/Nex1/Neurod6 XM_001059051 Up P PC12 Uittenbogaard
and Chiaramello
(2002)
Cmyc Y00396 Down R, P Human Chen et al.
neuroblastoma (1994)
lines
 Nerve growth factor regulated gene expression
2 33

. Table 2-1 (continued)

Up/
Gene name Access. # Down RNA/Prot System References
Mycn vmyc viral related XM_234025 Down R Neuroblastoma Woo et al. (2004)
oncogene, neuroblastoma
derived
P48ZnF (transcription factor) AY377983 Up R PC12 Heese et al.
(2004)
POU domain, class 2, XM_341802 Up R, P Sensory neurons Wood et al.
transcription factor 2/Oct2 in vitro (1992)
Sox21 Down P PC12 Ohba et al.
(2004)
Tumor protein p53 (Tp53) NM_030989 Up P PC12 Gollapudi and
Neet (1997)
Wilms tumor 1 (Wt1) NM_031534 Down R PC12 Liu et al. (2001)

Translational regulators
Eukaryotic translation Z48225 Up P PC12 Kleijn et al.
initiation factor 2B (eIF2B) (1998)

sensitive approach to defining cellular transcriptomes (Velculescu etal., 2000). Put briefly, each transcript
recovered from the cell of interest is converted into a defining SAGE tag, which includes the most 30 CATG
in the transcript followed by the next 11 bases. The relative number of tags representing a given transcript
that are recovered in the analysis is directly proportional to its relative abundance in the cell. If a large
number of SAGE tags are analyzed to compare the same cell in two different states (in this case
NGF
treatment), this provides a very comprehensive view of the changes that occur in gene regulation. In our
study, we analyzed large libraries of approximately 80,000 tags each (76,280 without NGF; 87,004 with
NGF). The results revealed that approximately 4% of the 22,000 transcripts detected in PC12 cells
responded to NGF by undergoing changes in expression of sixfold or greater. One of the challenges of
interpreting SAGE data is to match the various tags with known transcripts. Thus far, we have, by a variety
of informatic means (Angelastro etal., 2000), matched to known transcripts approximately half of the tags
representing genes that are regulated by sixfold or more in response to longterm NGF exposure.
> Table 2-2 lists the NGFresponsive transcripts thus far (up to September 2004) identified from our

SAGE study. The fold changes in expression are listed either as fold increase after NGF treatment (positive
numbers) or as fold decrease in expression after NGF treatment (negative numbers). When no tags were
detected, for the purposes of calculating a fold change, the tag number was set to 1 (to avoid a ratio of
infinity). Thus, some of the fold changes may be underestimated. The fold changes reported reflect
normalization for the differences in tag numbers for the two libraries. With the sixfold cutoff that we
have chosen, all reported changes are significant with a p value of 0.05 or less.
As in the previous table, we have subdivided the regulated genes into functional categories and in some
cases, we have listed the same gene under multiple categories. We have also included relevant accession
numbers. The present table updates and substantially expands the lists we published in Angelastro etal.
(2000) and such additions reflect the enormous increase in genome information available since that time.
We have done our very best to ensure the accuracy of the data presented in the table, but acknowledge that
errors are possible. Readers are encouraged to provide feedback in this respect.
We wish to note that because of the considerable delay between preparation of > Table 2-1 (September,
2004) and publication of this chapter, the Table does not reflect recent advances in annotation of the rat
genome. Nevertheless, the accession numbers given in the Table should permit the interested reader to find
the current annotation for each of the listed transcripts. It is our intention to provide updates for the Table
in future electronic versions of this chapter. Readers may also directly request the lastest version of this table
by email (lag3@columbia.edu).
34
2 Nerve growth factor regulated gene expression

. Table 2-2
NGFPROMOTED longterm changes in gene regulation in PC12 cells as detected by SAGE
() (;) Fold
Tag NGF NGF change Identity Accession #
Energetics
AAGGTTCACTC 19 1 17 Similar to Mtch1 protein (mitochondrial carrier XM_215358
homolog 1)
GCATACGGCGC 18 0 16 Atp5k: ATP synthase, H transporting, NM_080481
mitochondrial F1F0 complex, subunit e
AGCTTGATTAA 16 1 14 Similar to RIKEN cDNA 2410011G03O; XM_216880
0rtholog of human NADH: ubiquinone
oxidoreductase
GTGGCCCACTT 23 2 10 Cox17: cytochrome c oxidase, subunit XVII NM_053540
assembly protein homolog (yeast)
AATAAAAGTTC 15 2 7 Atp5a1: mitochondrial HATP synthase alpha NM_023093
subunit
TATCCAAACAG 7 1 6 Pdha1: pyruvate dehydrogenase E1 XM_343787
alpha 1
CCAAGGAAAAC 51 8 6 Ldha: lactate dehydrogenase A NM_017025
TACCATCTTTC 1 6 7 Similar to integral membrane protein CII3/ XM_2139361
Sdhc: succinate dehydrogenase complex,
subunit C
TACTAGAAAAG 1 7 8 Similar to NADH dehydrogenase (ubiquinone) XM_213940
FeS protein 2
ATCCAAGTCGC 1 8 9 Similar to Cytochrome oxidase biogenesis XM_214182
protein OXA1, mitochondrial precursor (OXA1
like protein) (OXA1Hs)
AGGTCGCTTGG 1 8 9 Similar to xylulokinase homolog; xylulokinase NW_000361
(H. influenzae) homolog
CCCGACTGGGT 5 66 15 Homolog of human LOC56901: ubiquinone NM020142
oxidoreductase MLRQ subunit homolog
Metabolism
GCTGGAATTGA 10 1 9 Farnesyl diphosphate synthase (Fdps) NM_031840
AAGGGTCCCCG 8 0 7 Prpsap1: phosphoribosylpyrophosphate NM_022545
synthetaseassociated protein (39kDa)
TCTGTCCTGCT 8 0 7 Degs: degenerative spermatocyte homolog XM_346454
(Drosophila); transmembrane protein involved
in meiosis in fly; lipid metabolism
ATTTGCTTCTT 8 0 7 Similar to ectonucleotide pyrophosphatase/ XM_236956
phosphodiesterase 5
TTTCAGCAGTG 7 0 6 Hprt: hypoxanthine guanine phosphoribosyl NM_012583
transferase
GGCCCCCAAGT 1 5 6 Ortholog of murine BC004012: cDNA sequence BC004012
BC004012; putative inorganic polyphosphate/
ATPNAD kinase
GCTTTAATGGA 6 31 6 Similar to 2310047E01Rik protein; encodes XM_343416
ortholog of murine Car12: carbonic
anyhydrase 12
GACAATGAAAA 2 12 7 Isocitrate dehydrogenase 3 (NAD), gamma X74125
(Idh3g)
GGAGGACCTCG 2 12 7 Dimethylarginine dimethylaminohydrolase XM_215315
2 (Ddah2)
 Nerve growth factor regulated gene expression
2 35

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TTGGGGGGTGA 2 12 7 Hadha: hydroxyacylCoenzyme A NM_130826
dehydrogenase/3ketoacylCoenzyme A
hiolase/enoylCoenzyme A hydratase
(trifunctional protein), a subunit
GACCCCTCAAA 1 7 8 Glutaredoxin 2 (Glrx2) (thioltransferase) XM_213890
TGTCCAGCTGG 1 7 8 Similar to thymidine kinase 2, mitochondrial; XM_226211
thymidine kinase 2
CTGGGTGGGGG 4 32 9 Similar to dehydrogenase/reductase (SDR XM_213723
family) X chromosome
AGACCTAGGAA 1 8 9 Similar to ecto ADPribosylhydrolase XM_342918
CTTGTGACAGG 0b 9 10 Monoglyceride lipase (Mgll) NM_138502
TTTACAGCTGC 0 10 11 Similar to cysteinetRNA ligase isoform b; XM_215134
cysteine translase; cysteinetRNA synthetase
ACCTACAGGAT 1 12 14 Branched chain alphaketoacid dehydrogenase J02827
subunit E1 alpha (Bckdha)
ATCCCTCCCCA 1 15 17 Ortholog of human COMMD1: copper NM_152516
metabolism (Murr1) domain containing 1

Signaling
AATGTGAGTCA 14 0 12 1433 protein gammasubtype NM_019376
TGCACAGTGCT 14 0 12 S100a4: S100 calciumbinding protein A4 NM_012618
AAATCCTTTCA 12 0 11 Pleiotrophin NM_017066
TGTAGCTCAAT 11 1 11 Guanine nucleotide binding protein, alpha o NM_017327
(Gnao)
ACTCGGAGCCA 10 0 9 Calmodulin 1 (Calm1) (phosphorylase kinase, NM_031969
delta)
AGACACTTCCT 10 1 9 Anxa2: calpactin I heavy chain/ANNEXIN 2 NM_019905
ATTCTGTGCTG 10 1 9 Cd9: CD9 antigen (p24) X76489
GGTGTGCCAGG 10 0 9 Similar to RIKEN cDNA 5730466P16; similar to NM_003876
protein PM1; putative receptor protein
AAGCCTTGCTG 9 0 8 Growth factor receptor bound NM_030846
protein 2 (Grb2)
TTTTGTGATGG 9 0 8 Similar to RIKEN cDNA 2010107K23 encoding XM_346291
protein with MAGE domain; Homolog to
human MAGEH1
TCAGGCATTTT 9 0 8 Similar to Rasrelated protein Rab1B X13905
CCCCCTGGATC 26 3 8 S100a6 calcium binding protein A6 (calcyclin) NM_0533485
CTGCCATCCCT 8 0 7 LOC294900: similar to PC1; Tpd52: tumor XM_215524
protein D52; possible roles Camediated
signaling and proliferation
TAGTCCAGGCT 8 0 7 LOC291675: similar to RIKEN cDNA AI028965
2010001M09encodes protein similar to
human PACAP
TCCTGTGTCCT 8 1 7 Inositol 1, 4, 5triphosphate receptor 3 (Itpr3) NM_013138
CTTCCAAATGT 15 2 7 Rtn4: NogoA NM_031831
AATGCCCCCAG 7 1 6 Presenilin2 (Psen2) NM_031087
CGCGCGCGCGC 7 0 6 Ptpns1: protein tyrosine phosphatase, non NM_013016
receptor type substrate (Bit, SHPS1)
CTGTTAGGTGG 7 0 6 Nicastrin Component of the gammasecretase NM_174864
complex (Ncstn)
36
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TAGGTGTCAAA 7 0 6 Mir16: membrane interacting protein of RGS16 NM_032615
GCGAATACAAG 7 0 6 Tspan2: Tspan2 protein; tetraspan protein NM_022589
GCTACAGGGAG 7 1 6 Similar to rod outer segment membrane XM_219564
protein 1; tetraspanin family
ACGTGCATCAT 1 5 6 RGD:628676: protein phosphatase 1G (formerly NM_147209
2C), magnesiumdependent, gamma isoform
GACTGGAAGCT 1 5 6 Ortholog of murine1700019B16Rik: RIKEN NM_028829
cDNA 1700019B16 gene putative G protein
receptor
AGTCCCTCCCG 1 5 6 Protein kinase LYK5; STRAD; activates tumor NM_182820
suppressor LKB1 involved in cell polarity
CGCGCGCTAGT 1 5 6 LOC292887: similar to lobe homologlike; XM_238103
mouse orthologAkt1s1, AKT1 substrate 1
(prolinerich), binds 1433
CCAGCCAGCGT 1 5 6 Crcp: calcitonin generelated peptidereceptor NM_053670
component protein
CTGCTAGCACC 1 5 6 Similar to Epidermal growth factor receptor XM_341958
pathway substrate 8like protein 2; Eps8l2; PTB
domain/Eps8 actin regulator
CCCCACACTGG 1 5 6 Ortholog of murine Ssh3: slingshot homolog 3 NM_198113
(Drosophila); Slingshot family phosphatases
that dephosphorylate cofilin
TGGAGGAGGCG 1 5 6 LOC308453: similar to aarF domain containing XM_218358
kinase 4 XM_218358
GGCACCTCCTA 2 11 6 Similar to Serine/threonineprotein kinase SNK XM_234920
(Serum inducible kinase)
CACACAAAAAA 1 5 6 Ortholog of murine Gmip: Geminteracting NM_198101
protein putative RhoGAP activity
CTGTTAGAACT 1 5 6 Protein phosphatase 4, regulatory subunit 1 NM_080907
(Ppp4r1)
CTGCTGTGTGG 2 10 6 Aip: arylhydrocarbon receptorinteracting NM_172327
protein/XAP2; may play positive role in AHR
mediated signaling
CTGTCAAGACC 0 5 6 LOC293783: similar to 4933402K05Rik protein; XM_215158
ortholog of murine Lpxn: leupaxin;
homologous to paxillin
TTTGTGGGAGG 1 5 6 Serine threonine kinase pim3 NM_022602
ACCACACCGGC 0 5 6 Inositol 1,4,5trisphosphate 3kinase C (Itpkc); NM_178094
conversion IP 1,4,5 to IP 1,3,4,5
CTTTCTGTAAT 0 5 6 Ortholog of human FGF11: fibroblast growth NM_004112
factor 11
GGTTGATTCTG 0 5 6 Ortholog of human PDE4D phosphodiesterase NM_006203
4D, cAMPspecific (phosphodiesterase E3
dunce homolog, Drosophila)
TCTCACCCACT 0 6 7 Haspp28: kinase substrate HASPP28 NM_022595
TAGAGTGTAAA 1 6 7 3Phosphoinositide dependent protein NM_031081
kinase1 (Pdpk1)/PDK1
 Nerve growth factor regulated gene expression
2 37

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TGCAATAGGGA 1 6 7 Ortholog of human PPP1R12C: protein NM_017607
phosphatase 1, regulatory (inhibitor) subunit
12C/myosin binding subunit 85
GCAAGGGGTGG 0 6 7 Ortholog of murine Efna2: ephrin A2 BC048697
GCGAGGAGTCC 0 6 7 Stk25: serine/threonine kinase 25 (STE20 NM_184049
homolog, yeast); (oxidant stress response
kinase 1
TGCCTCTTCCG 3 19 7 Similar to tetraspanin similiar to uroplakin 1 XM_230297
ATGGAGCAGTC 2 13 7 Similar to NAKAP95: neighbor of Akinase XM_216820
anchoring protein 95; binds regulatory subunit
(RII) of PKA and to DNA
GACCCAGCTCT 0 7 8 LOC303259 similar to Map4k6pending XM_239248
protein
TGCACACACTG 0 7 8 Similar to protein kinase BRPK/protein XM_216565
kinase BRPK /PINK1 (PTEN induced putative
kinase 1)
TACCTCGATGG 1 8 9 Acid nuclear phosphoprotein 32 (leucine rich) NM_012903
(Anp32); may be involved in signal
transduction
TTCTGCCTCCA 1 8 9 Serinethreonine kinase 16 (STK16)/F52/EDPK, D86220
Krct, PKL12
CTACAGTTCCT 1 13 15 CL1BA: CL1BA protein/Latrophilin/CIRL/CL1; 7 NM_022962
transmembrane domain receptor of secretin
family

Cytoskeleton
TGGAACCTTGC 16 1 14 Dncli2: LIC2 dynein light intermediate chain NM_031026
53/55
AGTTGATGCAA 32 2 14 Nfl: neurofilament, light polypeptide NM_031783
AGTTTGCTGAT 43 3 13 Ortholog of murine Tagln2: transgelin 2 BC009076
GAAAAATAGTC 11 0 10 Nfl: neurofilament, light polypeptide NM_031783
TATTTGTTTTG 10 1 9 Prnp: Prion protein, structural NM_012631
AGCTTTCCTGT 10 1 9 Cortactin isoform B (Cttnb) NM_021868
CCACACTGTCT 9 1 9 Ortholog of murine Capza1: capping protein NM_009797
(actin filament) muscle Zline, alpha 1
CAGTATCCCTA 9 1 8 Msn: Moesin NM_030863
GACTGTGCCAA 8 1 7 Pin: dynein, cytoplasmic, light chain 1 NM_053319
GAGGAGGGGGA 8 0 7 Ortholog of murine Tuba6: tubulin, alpha 6 NM_009448
CCCTTCCCTGC 7 0 6 Ctxn: cortexin L15011
GCAATAAATGG 7 0 6 Dbn1: drebrin 1; actin binding protein NM_031024
GCTTAGCCATT 13 2 6 Microtubuleassociated protein 4 XM_345984
CTGCTAGCACC 1 5 6 Similar to Epidermal growth factor receptor XM_341958
pathway substrate 8like protein 2; Eps8l2; PTB
domain/Eps8 actin regulator
ACCTTCTTGGT 2 11 6 Add1: adducin 1, alpha NM_016990
GAAGGAGAACT 0 6 7 Add3: Adducin 3, gamma; Actin capping NM_031552
protein
GCAGTGGGCTC 1 7 8 Coronin relative protein; actin binding protein NM_139115
38
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TGACTAGTGTC 0 10 11 Similar to hypothetical protein MGC37888; XM_215147
encoded proteinEml2: echinoderm
microtubule associated protein like 2
AGAGGTCTGAG 1 16 18 similar to coactosinlike 1; coactosinlike XM_341700
protein, actinbinding protein that may link
5lipoxygenase to actin

DNA binding proteins and transcription factors

GAGGCAGCTGG 10 0 9 Similar to PEA3: polyomavirus enhancer XM_340910
activator 3, transcription factor
GTTCTACCCCA 9 0 8 Ortholog of murine Rnf14: ring finger protein NM_020012
14/TRIAD2; transcriptional coactivator
CCTTTAATCCT 9 0 8 Similar to CyclicAMPdependent transcription XM_222871
factor ATF6 alpha (Activating transcription
factor 6 alpha)
CCCTTCACCTC 8 1 7 Similar to RIKEN cDNA 2310074H19; Homolog XM_215177
of human DRAP1: Dr1associated protein 1;
transcriptional corepressor
CGAAGTCAGGC 8 1 7 Ssrp1: Structure specific recognition protein 1; L08814
DNA binding protein
TTCCCCACACA 8 1 7 Tgfb1i4: Transforming growth factor beta NM_013043
stimulated clone 22; transcription factor
TGAATGGCCTA 7 0 6 LOC299113: similar to RIKEN cDNA XM_216721
2310022K15; Klhdc2 kelch domain containing
2; putative transcriptional regulator
CAAATAAGTTT 7 0 6 Ortholog of murine 2310042L19Rik: RIKEN XM_136134
cDNA 2310042L19 gene which encodes
protein highly homologous to human pirin, a
transcriptional activator
TGATCTTTTTG 2 11 6 Atf4: activating transcription factor ATF4 NM_024403
AGAAGCGCAAG 2 11 6 RGD:621323: aristaless (Drosophila) homeobox, NM_053869
Arix; Phox2a; Control noradrenergic phenotype
TGAGTGAAGAG 3 15 6 Ortholog of human SOX12: SRY (sex NM_006943
determining region Y)box 12
TTCCAGACGGA 1 5 6 LOC313666: similar to polyomavirus late XM_233676
initiator promoter binding protein; Zbtb17;
Lp1; Miz1; mZ13; Zfp100
TTGTGGTAACC 0 5 6 Ortholog of murine Zik1: zinc finger protein NM_009577
interacting with K protein 1; transcriptional
repressor
CTGAGCAGTGG 0 5 6 RGD:727889: vrel reticuloendotheliosis viral NM_199267
oncogene homolog A; Rela
CACCTTGAGTG 0 5 6 Nr1h2: nuclear receptor subfamily 1, group H, NM_031626
member 2 nuclear orphan receptor; may
interact with RXR
GAAAAATCCAC 0 5 6 AZF1: zinc finger protein 1; Putative DNA XM_342745
binding protein
GGGATGCTGCT 0 5 6 Ortholog of murine Zhx3: zinc fingers and NM_177263
homeoboxes 3
 Nerve growth factor regulated gene expression
2 39

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TCTCTTCTCGA 0 5 6 LOC305471: similar to transcription factor XM_223592
MAZR; ZNF278; ZSG; MAZR; PATZ; RIAZ;
ZBTB19; transcription repressor
CCACCTACATC 0 6 7 Similar to Transcription factor E3; Xlinked XM_228760
bHLH zip transcriptional activator
GAGAACATCAC 0 6 7 Similar to RIKEN cDNA 5730434I03 gene: XM_345561
homolog of human similar to RNA polymerase
B transcription factor 3
TCCCCAACGGC 0 6 7 Ascl1: achaetescute complex homologlike 1 NM_022384
(Drosophila)/Mash1
CTGCTGAGCCT 0 6 7 Similar to helixloophelix protein; nescient XM_222898
helix loop helix 1/Hen1, Nscl, Tal2; implicated in
neuronal differentiation.
TTGGCCAGAAT 1 6 7 Similar to transcription elongation regulator 1; XM_225983
transcription factor CA150; TATA box binding
proteinassociated factor
AAAAAGAATAA 0 6 7 Gata2: GATAbinding protein 2; Zn finger NM_033442
transcription factor
CGGAAATGATG 1 6 7 Usf2: transcription factor USF2 (UPSTREAM AB047556
STIMULATORY FACTOR 2) (HLH zipper
transcriptional activator via AP1 sites
CCCCAACCCTA 1 7 8 Orthololg of murine RIKEN cDNA C630022N07 NM_032711
gene; similar orto mafG
GAGAGAAGTGG 0 7 8 Similar to RIKEN cDNA 2400009B11 gene; NM_025886
encodes homolog to PUTATIVE
TRANSCRIPTIONAL REGULATORY PROTEIN
HRC1putative
CGGGAGTGCCT 1 8 9 Ortholog of murine Zfp61: zinc finger protein BC079015
61; putative DNA binding protein
TACTTGGGGGC 0 9 10 LOC293513 hypothetical LOC293513: encodes XM_238138
protein that matches murine Snf2related CBP
activator protein
TGACAGTCAGG 0 9 10 Similar to Meis3 (myeloid ecotropic viral XM_341796
integration siterelated gene 3); homeodomain
family member regulating gene expression
CCGGGAGTGTG 0 9 10 Similar to fork headrelated protein like A XM_343526
GACAGTGGAGA 1 9 10 Similar to methylCpG binding domain protein XM_214544
2; transcription regulator
AATAACTTTAA 1 10 11 Cnbp: cellular nucleic acid binding protein; NM_022598
Znfinger protein potentially regulating
transcription and translation
GCTGGGCAAGG 1 10 11 Similar to KIAA0138 gene product; encodes XM_238571
protein with RNA and DNA binding sites that
homologous to scaffold protein B.
TGCTCCGTGTA 1 10 11 Supt5h: suppressor of Ty 5 homolog XM_218382
(S. cerevisiae)
GGAGCAGGAAC 0 10 11 Lisch7: Liverspecific bHLHZip transcription NM_032616
factor
40
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
GTGTGTAGGGG 1 11 12 Nfat5: nuclear factor of activated Tcells 5, XM_226436
tonicityresponsive
TGATTGGTAGA 1 13 15 Ortholog of murine Tef: thrytroph embryonic NM_017376
factor; transcription factor member of PAR
family of bZip transcription factors
GCGGCCGGCTT 1 15 17 Cebpb: Liver activating protein; LAP, NFIL6, NM_024125
nuclear factorIL6, previously designated TCF5,
sfb ilencer factor B, CEB/PB
AGAACCTAGTC 5 130 30 Atf5: activating transcription factor 5 NM_172336

RNA binding, splicing, processing, and stability

TCTGGCTCCTT 11 0 10 Similar to RNA binding motif protein 5 XM_217263
AAGCTGGTTTA 9 0 8 Snrpb: small nuclear ribonucleoprotein M29295
polypeptides B and B1: splicing factor
ATGAGGAACTT 8 0 7 Similar to U1 snRNPspecific protein C XM_342101
GTACCAGGACA 8 1 7 Similar to hypothetical protein MGC14151; XM_213347
encodes snRNP Sm related protein
AGGATGCTTTG 7 1 6 Similar to RIKEN cDNA 2610528E23; member XM_213638
DEAD box helicase family
TTCTGTCCTAT 1 5 6 Ortholog of murine Rpo13: RNA NM_009087
polymerase 13
CCATCGAGGAG 3 17 6 LOC290660: similar to R27090_2; DEADc; XM_214290
DEADbox helicase
AATAAAGTTGT 2 10 6 Ortholog of human PABPN1: poly(A) binding NM_004643
protein, nuclear 1; role poly adenylation
TCGGTGCAGGC 0 5 6 Similar to murine Tdrd9: tudor domain XM_127120
containing 9 HELICc, Helicase superfamily
cterminal domain; Tudor domain
GAAATGTAAGA 2 12 7 Ortholog of murine Pcbp2: poly(rC) binding XM_128023
protein 2; RNA binding protein
CGGAAGGAATT 2 13 7 Similar to UPF3 regulator of nonsense XM_341460
transcripts homolog A isoform hUpf3p; mRNA
surveillance
GTAGGAACATA 0 6 7 Similar to splicing factor U2AF homolog XM_218195
mouse
TTGATCGAAGT 0 6 7 Similar to poly(A) polymerase V XM_234508
AGACAAGCTGG 2 13 7 Sfrs5: splicing factor, arginine/serinerich 5 L13635
(SRp40, HRS)
GCCCGAAAGAT 1 6 7 Ortholog of murine Sfrs16: splicing factor, NM_016680
arginine/serinerich 16 (suppressorofwhite
apricot homolog, Drosophila)
TCCAGGGCCTT 0 7 8 Homolog of murine Tia1: cytotoxic granule AK033792
associated RNA binding protein 1
CACAACTGTGA 0 7 8 Similar to splicing factor, arginine/serinerich 2, XM_231361
interacting protein; SC35interacting protein 1;
RNA splicing
CTTGGAGAACG 0 7 8 Similar to MADP1 protein; HAS RNA XM_343320
RECOGNITION MOTIFS;
 Nerve growth factor regulated gene expression
2 41

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
ACAGAGGCATT 1 8 9 Ortholog of murine Sfrs6: splicing factor, NM_026499
arginine/serinerich 6
TTGCTGGCTTT 1 8 9 Ortholog of human RBM9: RNA binding motif XM_086858
protein 9; transcription
GAAGAGTGTAA 2 15 9 Ptb: pyrimidine tract binding protein; NM_022516
premRNA splicing
AGCAAACCCCC 1 8 9 Similar to DAZ associated protein 1 isoform b; XM_343164
deleted in azoospermia associated protein 1
GTCGCTTCTGA 1 9 10 Similar to U2 auxiliary factor 26; RNA binding XM_341828
protein
GATCGAGCAAG 1 9 10 Ddx24: DEAD (AspGluAlaAsp) box NM_199119
polypeptide 24
GAAGGATGGCT 0 10 11 Cirbp: cold inducible RNAbinding protein NM_031147
ATGACTTGGGT 0 10 11 Ortholog of murine Rbm18: RNA binding motif NM_026434.2|
protein 18
GCTGGAGAGCC 0 10 11 Similar to RNAbinding protein Raly XM_215880
hnRNPassociated with lethal yellow
CCTGCCCTGTG 0 11 12 Similar to polymerase (RNA) III (DNA directed) XM_341388
(155kD)
GGCTTCACGGG 1 14 16 LOC363134: similar to U3 snoRNPassociated NXM_343469
protein; nucleolar protein
Proliferation
ACTGAGTGCTT 12 1 11 Similar to putative oral cancer suppressor; XM_341076
(DOC1)/CDK2associated protein 1
GTCCAGGAAAA 9 1 8 Ccnd1: Cyclin D1 D14014
CAGTGCTGGGT 9 1 8 Similar to NimArelated protein kinase; NEK9: XM_216755
NIMA (never in mitosis gene a) ortholog;
regulates mitotic progression
CTTTGGGTACA 8 0 7 Cyclin G1 NM_012923
GAGTGAAGAAT 1 5 6 Phb: prohibitin; suppresses proliferation and NM_031851
activates p53
TCCTATCTGCA 1 6 7 Similar to Cyclin K; CCNK: member of the cyclin XM_234516
family and may regulate PolII
TTTCCAAAGTT 0 6 7 Similar to ringbox 1; ringbox protein 1; XM_216991
ubiquitin ligases componentpossible cell cycle
protein
GACAGGGAGGT 0 6 7 Similar to PISSLRE; ortholog of murine Cdk10: XM_341712
cyclindependent kinase (CDC2like) 1; cell
cycle progression
ACCAGAGCAAC 2 14 8 Similar to prostate tumor over expressed gene XM_214944
1 (Ptov1); Promotes S phase entry
CCATCAGTGGG 2 15 9 Similar to centromere autoantigen B; CENPB: XM_342521
Centromeric protein B; centrosome assembly
protein
CGCAAGAAGGT 1 9 10 Pold1: DNA polymerase delta, catalytic subunit; NM_021662
DNA replication and repair
CAAACTGCATT 0 9 10 Cdk4: cyclindependent kinase 4 NM_053593
GGGCAGACAGG 1 9 10 Rpa2: p32subunit of replication protein A; X98490
DNA replication and repair
42
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TCGGAGAAGAG 0 10 11 Ptms: parathymosin/Zn binding protein; NM_031975
nuclear Zn binding protein possibly involved in
proliferation

Protein synthesis
CTCAGACAGTG 14 1 12 Similar to 40S ribosomal protein S27 X59375
CTCAAACACCA 1 6 7 LOC363023: similar to mitochondrial ribosomal XM_343354
protein L4 isoform a; Mrpl4
TGTATGAAGCA 1 7 8 Metap2: methionine aminopeptidase NM_022539
2/Initiation factor 2 associated 67 kDa protein
(Amp2); protein synthesis
TTCGTGGTCAA 2 16 9 Eef2: eukaryotic translation elongation factor 2 NM_017245
ATCCTCGCTGA 1 9 10 LOC287126: similar to TCE2; Ortholog of XM_213234
murine Mrps34 mitochondrial ribosomal
protein S34
ACAGAAAGTGG 1 9 10 Similar to mitochondrial ribosomal XM_214751
protein L18

Proteasomal pathway
GTGCTGGACCT 10 1 9 Psme2: protease (prosome, macropain) 28 NM_017257
subunit, beta; activator of proteasome
AGAGGAAGTGG 10 1 9 Similar to Fbox protein FBL2; related to skp2 XM_217519
so putative involvement in proteasomal
targeting
AGACGCCTGTG 10 0 9 Similar to 26S proteasomeassociated pad1 XM_215745
homolog
CCTTACACTTG 9 1 8 Similar to tetratricopeptide repeat domain 3; XM_340973
Ortholog of murine Ttc3; putative ubiquitin
ligase
AAAACACCTTG 7 1 6 Nedd4a: neural precursor cell expressed, XM_343427
developmentally downregulated gene 4; E3
UBIQUITIN PROTEIN LIGASE
AAGTAGCTGGA 7 0 6 Similar to RIKEN cDNA 1300013G12; encoded XM_222627
protein very similar to murine Ubxd2: UBX
domain containing 2
TGATGTCTCTC 0 6 7 Ortholog of human COP1: constitutive NM_022457
photomorphogenic protein; E3 ligase that can
target p53
CAGGGCGAGAT 1 6 7 Similar to RIKEN cDNA 6330414O09; ortholog XM_218405
of Irf2bp1: interferon regulatory factor2
binding protein1; E3 ligase domain
CTCCTCCTGAT 1 6 7 Similar to Ubiquitin carboxylterminal XM_341033
hydrolase 12/USP12 ubiquitin specific
protease 12
GACCTTGGAGT 0 11 12 Ortholog of murine Rnf26: ring finger NM_153762
protein 26; putative ubiquitinprotein ligase

Lysosomal function
CTGATCCCCAT 3 17 6 Lamp1: Lysosomal associated membrane NM_012857
protein 1 (120 kDa); membrane glycoprotein
 Nerve growth factor regulated gene expression
2 43

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
GGTAAGTCATC 1 6 7 Ortholog of murine Cln2: ceroidlipofuscinosis, NM_009906
neuronal 2; lysosomal serine protease
AATCGGAACAA 0 7 8 Dnase2: deoxyribonuclease II; lysosomal DNAse NM_138539
GTTCACCGACG 0 8 9 Man2b1: alphaDmannosidase; lysosomal NM_010764
enzyme

ER, molecular chaperones

AGCCTCCCTTG 12 1 11 Similar to carboxy ter of Hsp70interacting XM_213270
protein
GATTGTCTTGA 12 0 11 Similar to 25 kDa FK506binding protein XM_216717
GGTTTGATTCC 9 1 8 Ortholog of murine Canx: calnexin AK017254
AGTTCTGCTTG 17 2 7 Plp2: proteolipid protein 2 XM_217597
TGTGCAGTGAA 17 2 7 Similar to signal peptidase 12kDa XM_214276
TGGGTTAGACC 8 1 7 Similar to prefoldin 1 XM_341596
GCGCGCGTTTA 8 1 7 Ortholog of human VBP1: von HippelLindau NM_003372
binding protein 1/PREFOLDIN SUBUNIT 3 /
VHLBINDING PROTEIN1) (VBP1); Chaperone
GTATTGGCCAG 0 5 6 Similar to SREBP cleavage activating protein: XM_217279
SCAP; Required for sterolregulated transport
of SREBPs from ER to Golgi
CTCTCACCCCT 0 5 6 Ortholog of murine Ca activated AK081118
nucleotidase 1 (Cant1);Ca2dependent ER
nucleoside diphosphatase/apyrase 1
CCTGGTTATAC 0 5 6 Ortholog of murine Dnaja4: DnaJ (Hsp40) NM_021422
homolog, subfamily A, member 4; heat shock
protein, DNAJlike 4
GGCCTAAGGCA 1 5 6 LOC291671: similar to grp75 XM_214583
ATTGGGAAGCT 1 5 6 Ortholog of murine 1110021N07Rik: RIKEN NM_024207
cDNA 1110021N07 gene; encodes protein
derlin1 involved in ER transport
GTATTGGCCAG 0 5 6 Similar to TRAM1: translocating chain XM_232596
associating membrane protein
ACCTTGCCCTC 3 18 7 Similar to signal sequence receptor, beta; XM_215619
translocates newly synthesized polypeptides
across ER membrane
ACGAGCTTTAA 1 6 7 Similar to FK506binding protein: molecular XM_215758
chaperone
TCTTACTGGCA 1 7 8 Similar to HRD1 protein; synoviolin 1; involved XM_341999
in ERassociated degradation
TCATCTTTAAC 1 7 8 Calr: calreticulin; ER chaperone NM_022399

Trafficking, vesicular transport

TGTTGTTGATC 11 1 10 Similar to evectin2 (postGolgi vesicular XM_217372
membrane protein)
TGTGAAGTAGC 17 2 7 Arf1: ADPribosylation factor 1; vesicular NM_022518
transport
TGGTGACTAAG 7 1 6 Similar to ADPribosylation factor binding XM_215045
protein GGA2 (Golgilocalized, gamma ear
containing, ARFbinding protein 2)
44
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
GACTTCTGTCA 1 5 6 Similar to leptin receptor overlapping BC058504
transcriptlike 1
AAGATCATCGA 1 5 6 Ortholog of murine Ap1m2: adaptor protein NM_009678
complex AP1, mu 2 subunit
TCTCTGGGCCA 1 5 6 RGD:621591: tumor specific antigen 70 kDa. XM_220167
Ortholog of human and mouse Coronin 7;
vesicular trafficking
TCAGCTGACCA 1 5 6 Ortholog of murine Aspscr1: alveolar soft part NM_026877
sarcoma chromosome region, candidate 1 /
TUG; traps intracellular GLUT4
TTAATTCATTT 1 5 6 Timm23: translocase of inner mitochondrial NM_019352
membrane 23; intracellular protein
transporter
GTCTTTTCAGA 1 6 7 Rab10: rasrelated protein rab10; putative role NM_017359
in protein transport/neurotransmitter release
GAAGGCAGTTT 1 7 8 Ortholog of human SCFD2: sec1 family domain NM_152540
containing 2; vesicle dependent protein
transport
AACTGGGTCTG 4 27 8 Arl3: ADPribosylation factorlike 3; binds X76921
GTP and may regulate intracellular
transport
CCCCATTCCCA 0 7 8 Similar to zinedin; A calmodulinbinding, WD XM_218432
repeat protein with putative role in membrane
trafficking
TCAGCTGAATA 1 8 9 Gosr2: golgi SNAP receptor complex member NM_031685
2; vesicle transport from the cis/medial to the
transGolgi/TGN

Carbohydrate binding and metabolism

GCGGCGGATGG 215 15 13 Galectin 1 M19036
TTCAGAGGGGC 14 0 12 Similar to 106 kDa OGlcNAc transferase XM_236715
interacting protein
TGCTCCTGTGA 14 0 12 Hexa: hexosaminidase A XM_217144
ATCTAAGCCAG 10 1 9 Lgals3: lectin, galactose binding, soluble 3/ NM_031832
galectin3
GTTCCCCTCAC 8 0 7 Similar to glucuronosyltransferase I XM_238155
ATTTTCCCCCG 7 0 6 Similar to Stromal cellderived factor XM_213377
2 precursor (SDF2)
GAGACCTCTGG 1 7 8 Ortholog of murine Fn3k: fructosamine 3 NM_022014
kinase
GAGACGGCATC 1 10 11 Similar to Vesicular integralmembrane protein XM_214428
VIP36 precursor; lectin family
Endocytosis

ACTCCTGTCAG 8 0 7 Cltb: clathrin, light polypeptide (Lcb) NM_053835

TACAGAAGGAG 1 7 8 Ortholog of human ITSN2: intersectin XM_039680
2, involved in clathrin mediated
endocytosis
 Nerve growth factor regulated gene expression
2 45

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
CTGTCTGACTC 0 12 14 Ap2b1: adaptorrelated protein complex 2, NM_08083
beta 1 subunit/betaadaptin; betachain
clathrin associated protein complex AP2;
component of complex linking clathrin to
receptors in coated pits and vesicles,
intracellular protein transport

Transporters
TTCTAGCATAT 11 0 10 Atp1b1: ATPase Na/K transporting beta 1 NM_013113
polypeptide
TGCTACCACAC 7 1 6 HesB protein; contains Hesb domain involved NM_181626
in Inorganic ion transport and metabolism
GTGGGCCAAAC 2 10 6 Similar to solute carrier family 39 (zinc XM_342286
transporter), member 1; zinciron regulated
transporterlike gene;
TAGAAAAATGG 1 5 6 Slc16a1: solute carrier family 16, member 1 NM_012716
GTGGGCCAAAC 1 5 6 Ortholog of murine Slc39a1: solute carrier NM_013901
family 39 (zinc transporter), member 1/Zip1
GGGCAGACACA 1 5 6 Timm44: translocator of inner mitochondrial NM_017267
membrane 44 NM_017267
GACATAGCCCA 2 10 6 CHOT1: choline transporter NM_017348 NM_017348
GTGGCCAATCA 0 6 7 LOC291840: amino acid transporter NM_1003705
TTGTATAATAG 1 6 7 Ant2: Adenine nucleotide translocator 2, D12771
fibroblast isoform (ATPADP carrier protein)
CAGTGGGTGGG 5 33 8 LOC287642: galactose transporter NM_199081
ATTCTCTGGAT 1 7 8 Atp2a2: ATPase, Ca transporting, cardiac NM_017290
muscle, slow twitch 2
GGCTTGCTCCT 1 8 9 LOC290673: similar to cationtransporting XM_214310
atpase
CTGGAGCTGGG 1 9 10 Slc25a10: solute carrier family 25 NM_133418
(mitochondrial carrier; dicarboxylate
transporter), member 10
ACAGTGAAGGG 3 26 10 Slc3a2: solute carrier family 3 (activators of NM_019283
dibasic and neutral amino acid transport),
member 2
AACGCTGACCA 1 13 15 LOC288919: similar to arsenic resistance XM_213848
ATPase
TTGGTGAGGTA 1 16 18 Slc7a8: solute carrier family 7 (cationic amino NM_053442
acid transporter, Y system), member B/ LAT4

Neurotransmission/Synapses
CTGGAGGTGTG 22 2 10 Homolog of murine Syn2: synapsin II NM_013681
CCGCTATAACA 8 0 7 Syn2: synapsin II NM_919159
CTAGACACCTG 8 1 7 Scg3: secretogranin III NM_053856
AGTAATTTTAG 7 1 6 Acp1: acid phosphatase 1, soluble; possible NM_021262
role synaptic transmission
GCACACTGTGT 7 0 6 Sh3d2c1: SH3 domain protein 2 C1; implicated NM_031238
in vesicle recycling
ACATTTCAATT 1 5 6 Similar to gammaaminobutyric acid (GABA(A)) XM_216288
receptorassociated proteinlike 1
46
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TCCAGACCAGG 1 5 6 RGD: 621786: myc box dependent interacting NM_053959
protein 1; Bin1; may play role in synaptic
vesicle endocytosis
TCTCACTGCAG 1 5 6 RGD: 63135: shankinteracting protein; sharpin; NM_031153
interacts with the ankyrin Shank; at PSDs
GCCACAAGCTT 0 5 6 Ortholog of murine Unc13b: unc13 homolog B NM_021468
(C. elegans); Munc131; Involved in exocytosis
and synaptic transmission.
GTGTAAGGGAG 2 11 6 Syt4: synaptotagmin; vesicular trafficking and NM_03169
exocytosis
TGATGGAACCA 1 10 11 Ortholog of murine Nptxr: neuronal pentraxin XM_128199
receptor; synaptic protein uptake

Ion channels
GCTCAGATCCA 0 5 6 Kcnh2: potassium voltagegated channel, NM_053949
subfamily H (eagrelated), member 2
GAGAGCTAACA 1 6 7 Nnat: neuronatin; possibly regulates ion U08290
channels during brain development
GAAATGTCTGA 0 11 13 Similar to hypothetical protein MGC27385; XM_223921
ortholog of murine Kctd6: K channel
tetramerization domain containing 6
TTGGGCTGGTT 0 12 14 Asic4: SPASIC protein: Noninactivating NM022234
protongated ion channel in brain

Antioxidant action
GTGATAGACAA 0 6 7 Peroxiredoxin 6 (Prdx6); antioxidant enzyme NM_053610
AGCTGGGACTT 0 7 8 Sod2: Superoxide dismutase 2, mitochondria X566001

Cell death related

GACAGCACAAG 1 5 6 Similar to Apoptosis regulatory protein Siva XM_343117
(CD27binding protein) (CD27BP)
CTGCCGCCTCA 1 5 6 Adprt: ADPribosyltransferase 1; poly(ADP NM_013063
ribose) polymerase PARP1
TTTGTTAAAAC 0 5 6 Ddit4: DNAdamageinducible transcript 4; NM_080906
HypoxiaInducible Factor 1Responsive Gene,
RTP801
ACATCCACCCA 0 6 7 Similar to direct IAP binding protein with low PI XM_213814
(DIABLO); SMAC
GCTGAGGGAGA 1 12 14 Ortholog of human paternally expressed 3 XM_042345
(PEG3) protein with KRUPPEL ZINC FINGER
PROTEIN/PW1;
TGCCCAATAAA 0 15 17 LOC246273: kinase; novel kinase induced NM_144755
during PC12 cell death

Neurodegenerative disease associated

GAAGTCAGCCA 1 5 6 SMN1: survival of motor neuron 1, telomeric NM_022509
CACGCACAGTC 0 15 17 Drpla: dentatorubral pallidoluysian atrophy NM_017228
(atrophin1)
AGTGGAGGGAA 1 26 30 LOC361649: similar to ataxin 2 related protein XM_341928
isoform A; ataxin2 domain protein; ATXN2L:
ataxin 2like
 Nerve growth factor regulated gene expression
2 47

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
Extracellular matrix effects
CTCTGACTTTA 27 4 6 Bsg: Basignin/Ox47 antigen/CE9/EMMPRIN; NM_012783
extracellular matrix metalloproteinase inducer
TTAAGACCAAG 7 0 6 Serpinb6: Serine (or cysteine) proteinase NM_199085
inhibitor, clade B (ovalbumin), member 6
ACCCAGCTCAG 0 5 6 A disintegrin and metalloproteinase domain 15 AJ251198
(Adam15) (metargidin).
CCTCCGCCTCC 1 5 6 Lu: Lutheran blood group (Auberger b antigen NM_031752
included) Sequence; may act as an adhesion
molecule and receptor for laminin [RGD]

Peroxisomal function
TGCTTGCCACA 0 7 8 Similar to peroxisomal acylCoA thioesterase XM_234398
2B; likely ortholog of mouse peroxisomal acyl
CoA thioesterase 2B

DNA repair/damage response

CTGAGGAGGGG 10 1 9 DNAdamage inducible transcript 3 (Ddit3)/ U36994
GADD153/Chop10
TATGCACAGGC 8 1 7 Xray repair crosscomplementing group 1 NM_053435
protein (Xrcc1)
CTGCCGCCTCA 1 5 6 Adprt: ADPribosyltransferase 1; poly(ADP NM_013063
ribose) polymerase PARP1
TGATCTGCCTG 0 5 6 MGC5178: hypothetical protein MGC5178; BC000803
encoded protein has nuclease domain
putatively involved in DNA repair

Miscellaneous
TAGAGCGTGCT 11 0 10 Similar to nucleosome assembly protein 1like XM_341967
4; nucleosome assembly and gene expression
GTTTTGCTACC 8 0 7 Similar to NIPSNAP1 protein: XM_341249
4nitrophenylphosphatase domain and non
neuronal SNAP25like protein homolog 1
(C. elegans)
GAACGCACACC 7 0 6 Similar to erthyrocyte band 7 integral XM_216045
membrane protein, protein 7.2B, stomatin
GTCTAGGTCAC 7 0 6 Similar to hepatitis B virus xinteracting XM_215674
protein; HBxinteracting protein; HBixP
ACACGGAGGAG 0 5 6 Haptoglobin (Hp); has serinetype NM_012582
endopeptidase activity
CTCAGCAAAAC 0 5 6 LOC317431: similar to RIKEN cDNA 2610028I09; XM_228840
ortholog of murine Ribc1: RIB43A domain with
coiledcoils 1
GTGCGGTACCT 0 5 6 LOC300222: similar to microspherule protein 1 XM_217048
P78; MCRS1; MSP58; ICP22BP; putative cell
cycle/transcription regulator
GGAAGCTGCAA 1 5 6 LOC361425: similar to hypothetical protein XM_341703
COX4AL ortholog of murine Noc4: neighbor
of Cox4
GTAGCAGCCAG 1 5 6 LOC296731: similar to NEDD8 ultimate buster XM_231280
1; NUB1; binds NEDD8 a ubiquitinlike protein
48
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
CAAATTACTAA 0 6 7 Ortholog of murine RIKEN cDNA A530089I17 NM_133999
gene; encodes SAC3: Sac domaincontaining
inositol phosphatase 3 (Lipid transport and
metabolism)
CGCTGCAGAAA 1 6 7 Nurim (Nrm) (nuclear envelope membrane NM _212508
protein)
GTGTGTGGTGC 1 6 7 LOC362571: similar to prion protein interacting XM_342890
1 like (30.8kDa) (4B10); appears to be in
exonuclease family; prnpip
GCCATTTGGTG 0 6 7 LOC361035 similar to methyltransferase like 3; XM_341310
putative methyltransferase; m6a
methyltransferase
CAAATTACTAA 0 6 7 Ortholog of murine RIKEN cDNA A530089I17 NM_133999
gene; encodes SAC3: Sac domaincontaining
inositol phosphatase 3 (Lipid transport and
metabolism)
GCAATATTGGC 0 6 7 Similar to RIKEN cDNA 1200009I24 gene XM_213963
(LOC289323); Nuclear valosincontaining
proteinlike; AAA ATPase family
GGATGATGGTC 1 8 9 LOC292306: similar to Ribonuclease 6 XM_214769
precursor; Rnaset2; extracellular ribonuclease
AAACATTGGGG 2 16 9 LOC309475: similar to transmembrane protein XM_220013
TM9SF3
ACCTTGTTGAT 3 30 11 Ddopachrome tautomerase (Ddt); converts D NM_024131
dopachrome to 5,6dihydroxyindole; melanin
synthesis
CAGGTTCTCCT 1 15 17 Stellate cell activationassociated protein NM_130744
(Staap); cytoglobin with peroxidase activity
GACTGAAAAAG 0 16 18 LOC309161: similar to CG17265PA; Ortholog XM_219510
of human DIPA: hepatitis delta antigen
interacting protein A
GCAGCAAGAAG 0 19 22 LOC361682: similar to tumorsuppressing XM_341965
subchromosomal transferable fragment 4;
TSSc4

Transcripts encoding novel proteins of unknown function

TGCTCTGCATA 11 0 10 Ortholog of murine Fin14 fibroblast growth AK002917
factor inducible 14
GTGACCGGCCC 10 0 9 Hypothetical LOC294291 encoding XM_215355
hypothetical protein of unknown function
CTCTGTGGGTT 9 1 8 LOC294362: similar to DNA segment, Chr 10, XM_215378
ERATO Doi 214, expressed; has Zn finger
domain
GTGCCCACTGG 9 1 8 Ortholog of murine DNA sequence BC020184 XM_128892
encoding novel protein member of TB2/DP1,
HVA22 family.
ATGCTTCCTGT 9 0 8 Similar to chromosome 11 open reading frame; XM_235640
ortholog of murine Tmem16f: transmembrane
protein 16F
 Nerve growth factor regulated gene expression
2 49

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
CGTTCCACCAG 9 0 8 Similar to Protein C22orf5 XM_343289
TAGGGGTGGAG 9 1 8 Ortholog of murine 2610017J04Rik: RIKEN AI463119
cDNA 2610017J04 gene of unknown function
TGCACAGATGT 68 8 7 Growth and transformationdependent M17412
protein; Encodes novel gene of unknown
function
AAGAAAGTCGC 8 0 7 LOC287828: similar to HN1; hematological and XM_213527
neurological expressed sequence 1
GTTTCCTGCTT 8 1 7 LOC361797: LOC361797 XM_347003
TGGTACACGGA 8 1 7 Similar to DNA segment, Chr 5, Brigham & XM_222140
Womens Genetics 0834 expressed
TATACAGAGCG 8 0 7 Ortholog of human DKFZP564D166: putative XM_044366
ankyrinrepeat containing protein
TCTCAAAGAAC 8 0 7 Similar to hypothetical protein MGC45400 XM_346349
GAGCAGCCACC 8 1 7 Similar to RIKEN cDNA E030034P13 XM_340760
GTTCCGACAGT 8 0 7 Similar to CDV3B (carnitine deficiency XM_236579
associated gene expressed in ventricle 3)
CACCACTGGAT 8 1 7 Transcribed sequence with weak similarity to AI228578
protein pir:T43483 (H.sapiens) translation
initiation factor IF2 homolog
GCTATATTCCA 8 0 7 Ortholog of murine 1110061A14Rik: RIKEN AK004333
cDNA 1110061A14 gene of unknown function
AGCCGTGTATA 7 1 6 Ortholog of human C9orf25: chromosome 9 AK022819
open reading frame 25
TTTAGTGACGT 7 1 6 Ortholog of murine 9430029L20Rik RIKEN AK079127
cDNA 9430029L20 gene
ATTGTCTTTTC 7 0 6 Ortholog of murine Tub: tubby candidate XM_207983
gene
CCTGGCCAGCC 7 0 6 Ortholog of murine 2010004M13Rik: RIKEN AK008102
cDNA 2010004M13 gene encoding product of
unknown function
CTACTTCTGTA 7 0 6 Similar to 0610010K06Rik protein XM_223020
GGCCTGGCTTA 7 0 6 Similar to hypothetical protein FLJ20627 XM_214758
CCCAGACTGAA 7 0 6 Similar to RIKEN cDNA 2010315L10 XM_214304
CCCCTTCACCC 13 2 6 Similar to hypothetical protein 4933417N17 XM_214392
GGATGTGAACT 7 0 6 Similar to Ser/Thrrich protein T10 in DGCR XM_341010
region
TGGGGAGAAAT 3 16 6 Putative ISG12(b) protein; interferon inducible XM_238467
TTAAGTCCTTG 1 5 6 LOC309763: similar to DKFZP586B0923 XM_228159
protein
TATGCAACCTG 2 10 6 LOC296565: similar to hypothetical protein XM_216002
MGC36831
GTGTTCCTCCG 0 5 6 LOC288557: similar to RIKEN cDNA XM_213738
1190005J19; MOSPD3 motile sperm domain
containing 3
GCAGCCCCATA 0 5 6 LOC304649: similar to CG2662PA XM_222452
TGCAGAATCCA 0 5 6 LOC313668: similar to RIKEN cDNA XM_233612
1700027M01
50
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TGCAGGCGACA 0 5 6 MGC94549 similar to cDNA sequence XM_216853
BC005632
CGTATTCAGAA 1 5 6 Clone UIRFJ0cqbf240UI unknown mRNA. AY724538
CGCCCTTGAGC 1 5 6 Ortholog of murine 2610528K11Rik: RIKEN NM_175184
cDNA 2610528K11 gene
CGGTGGAGATA 0 5 6 7530403E16Rik: RIKEN cDNA 7530403E16 gene; BC051225
mRNA Sequence NM_175184
AGCAGAGAATG 0 5 6 Ortholog of murine 1700020I14Rik: RIKEN XM_488956
cDNA 1700020I14 gene
ATGCCAAACAC 0 5 6 LOC363077: similar to expressed sequence XM_343408
AV340375
GTGCTCAAACC 1 5 6 Ortholog of human JMJD3: jumonji domain XM_043272
containing 3
AGCCTCCAGGG 0 5 6 Ortholog of murine 1110002E23Rik: RIKEN NM_025365
cDNA 1110002E23 gene
ACTCTAGCCAG 0 5 6 LOC296751: similar to KIAA1897 protein; XM_216060
ATGCCAAACAC 0 5 6 LOC362914: similar to hypothetical protein XM_343244
FLJ10204
GCCAGCCAGCA 0 5 6 Similar to putative zinc finger protein XM_217318
(LOC301122)
GCCTTCCCTCA 1 5 6 Similar to RIKEN cDNA 5730469M10 XM_341406
GTATTTGCAAA 1 5 6 Ortholog of murine 1810006K23Rik: RIKEN AI835402
cDNA 1810006K23 gene; similar to CaMKII
inhibitor protein alpha
GTGGGTTTCTG 1 5 6 Ortholog of murine D130072O21Rik: RIKEN NM_175322
cDNA D130072O21 gene
GGGGTACCTGG 3 17 6 Putative ISG12(b) protein; interferon inducible XM_213523
TACTGGAGTAT 0 6 7 LOC308795: similar to MESDC1: mesoderm XM_218853
development candidate 1
ACTGTAGCTTC 1 6 7 Ortholog of murine 2300002D11Rik: RIKEN XM_130600
cDNA 2300002D11 gene; encodes novel
protein of unknown function
AGAGACCCTGC 0 6 7 Ortholog of murine RIKEN cDNA 1600010O03 BC002221
gene
ATGTGAAACTG 0 6 7 Similar to mKIAA0236 protein (LOC316524) XM_237291
CTCCTAAACCT 1 6 7 Similar to leucine zipper domain protein XM_213213
GAAGCCTGTAG 1 6 7 Similar to hypothetical protein MGC2494 XM_213267
GACCAGCAGGG 1 6 7 Ortholog of murine RIKEN cDNA 2610510D13 AK012090
CCTGGCCCTTT 0 6 7 Ortholog of murine clone C230062K19; full AK048777
insert sequence
GCCAGCACAGC 1 6 7 Similar to mKIAA1930 protein XM_214666
GAGTCACGGAG 0 6 7 Similar to KIAA1193 protein XM_343172
TGGAGCCTCAA 1 6 7 Ortholog of murine LOC209064: RIKEN cDNA XM_124946
1810055D05 encoding novel protein with a
DNAJ domain
GGGGTGGGGGG 1 6 7 Otholog of murine expressed sequence NM_134096
AW049604; encodes Tafa5, a secreted brain
specific protein
 Nerve growth factor regulated gene expression
2 51

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
CCTGGAATCTC 1 6 7 Brainenriched SH3domain protein Besh3 NM_139334
(Besh3)
TCCAACTCTAG 1 6 7 Ortholog of murine RIKEN cDNA 2610510H01 C019606
gene
GTGTCAGCAAG 1 6 8 Similar to CG31635PA XM_218397
TCCCTATAGTC 0 7 8 LOC291609: similar to Nedd4 WW domain XM_214564
binding protein 5
TCCTTTTTCAC 1 7 8 Ortholog of murine Mea1: male enhanced NM_010787
antigen 1
TCAGAGCCTCA 0 7 8 LOC314598: similar to wizL; Widelyinterspaced XM_234841
zinc finger motifs
AGGAGAAGGTG 0 7 8 Similar to oriLyt TDelement binding protein 7 XM_345879
GAGAGACTTTC 0 7 8 Similar to RIKEN cDNA 4921536I21 XM_342358
(LOC362056); encodes protein similar to
spermatogenesis associated protein 1
GCACGAACATC 1 7 8 Similar to CG14977PA XM_341044
GGCCAGGACAG 1 8 9 Ortholog of murine RIKEN cDNA 1600002K03 AK005397
gene
GTTGGAACACC 1 8 9 Ortholog of murine LOC328644: hypothetical NM_198629
gene supported by AK045595 mRNA
TAGACTGTGCA 1 8 9 Bladder cancer associated protein (Blcap); NM_133582
novel protein of unknown function
TTCGTGTGTCT 0 9 10 Similar to hypothetical protein MGC10120 XM_215245
TGGCCAGTAAC 1 10 11 Wmp1: Fertility related protein WMP1 NM_138862
GCACTCCTCCT 1 10 11 Ortholog of murine 1110021J02Rik: RIKEN XM_128573
cDNA 1110021J02 gene encoding protein of
unknown function
GTGAAAAAGGA 1 11 13 Ortholog of murine similar to hypothetical NM_145008
protein FLJ30213; encoded protein has
putative YIPPEE Zn binding domain
AGCCTGGAGAG 0 11 13 Ortholog of murine PERQ amino acid rich, with NM_031408
GYF domain 1 (Perq1)
TGCTGGTGGGT 1 11 13 Similar to KIAA0540 protein; Beige/BEACH XM_236649
domain
CACACCTCAGG 1 13 15 LOC361550 similar to RIKEN cDNA AY171575
1300003M23
GCCGGCCGGAC 1 19 22 LOC293514: similar to BCL7C; unknown XM_215076
function

ESTs
GTATTAAATAG 11 1 10 ESTs BQ192965
AGCTAGAGCTG 9 1 8 ESTs BF392924
TAAAGTACTCA 9 1 8 ESTs BQ202150
TGTAATGAGAT 8 0 7 ESTs BE117509
GCTCCAGCTAC 8 1 7 EST AA858996
ATTGTCTTTTC 7 0 6 ESTs AI548927
TGGGCACTGGG 7 0 6 EST AA818499
TGTTCTATAGG 7 1 6 ESTs AI060317
52
2 Nerve growth factor regulated gene expression

. Table 2-2 (continued)

() () Fold
Tag NGF NGF change Identity Accession #
TGTTCACTTGT 7 1 6 ESTS CB715527
GAATACAGCCT 2 11 6 ESTs BQ194973
CATTTTAGAAT 0 5 6 ESTs AW527014
ACCACAGGCCT 0 5 6 ESTs AW528651
GCTGCCACACA 0 5 6 ESTs BQ195365
GATAGCCATAG 0 5 6 ESTs BF398152
TAGCCCAACCC 0 5 6 ESTs BQ209689
TTGTGGTAACC 0 5 6 ESTs AW532812
TTAAATAATTG 1 5 6 ESTs CK843774
ATGGTGGTGAT 0 6 7 ESTs BE117794
TCTTTAACCCC 0 6 7 EST AI576247
TTTGGTAACTG 0 6 7 ESTs AI711568
AACGTGTACAC 1 6 7 ESTs BI287446
GGCCACATTAG 0 6 7 ESTs CR474415
CACGCACACAC 3 20 8 EST BF412923
CAAGGAGGAAC 1 7 8 ESTs AI070392
CAGGCAAACCC 0 7 8 EST BE097768
CAAGCAAAACA 1 7 8 EST XM_214182
GAGGCAGAGAA 3 30 11 ESTs BG378614
TCTCGTCCTAG 0 10 11 ESTs AI385367
AGGGGAGGGGA 0 11 12 EST BF407565
GCCCCACAGCA 0 12 14 EST BE105351

Though our aim here is not to interpret or speculate the significance of the various regulated genes, a
few generalizations seem warranted. One is that although the findings in > Tables 2-1 and > 2-2 do overlap
to some extent, they also include many genes that do not. In many cases, this reflects the difference in fold
change required to reach significance in that changes of only greater than sixfold are reported for the SAGE
study. In other cases, due to the absence of 30 sequence, it may not be presently possible to verify the SAGE
tag corresponding to the genes given in > Table 2-1.
Another point of interest is that the SAGE study has uncovered many regulated transcripts that encode
novel proteins of unknown function. Likewise, a number of additional regulated transcripts encode
proteins of at least partially defined function, but that have no obvious or previously known roles in
neuronal differentiation or function. This suggests that we have a very long way to go in our quest to fully
understand how NGF works and that there are many opportunities to fit new proteins into the NGF
mechanism. One example of this is our initial work on ATF5, a transcription factor that is 30fold
downregulated by NGF and that previously had no known role in the nervous system. Functional studies
inspired by our SAGE findings revealed that ATF5 is highly expressed in neuroprogenitor cells but not
mature neurons and glia and that it must be downregulated by trophic factors such as neurotrophins to
permit neuroprogenitor cells to exit the cell cycle and to differentiate (Angelastro etal., 2003; Angelastro
et al., 2005; Mason et al., 2005).-

5 Closing Remarks

In summary, we provide here information about the identities of genes and gene products that are subject
to regulation by NGF. We anticipate that many such genes will be regulated by other neurotrophic factors in
multiple cellular settings. In contrast, others will show both cell and factorspecific regulation. We hope that
 Nerve growth factor regulated gene expression
perusal of the information here will permit the reader to formulate overarching ideas about how NGF and
other trophic factors influence the properties of their target cells and will perhaps inspire them to choose
among the regulated genes their own favorites for further study.
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3 Myelinating Cells in the Central
Nervous SystemDevelopment,
Aging, and Disease
J. Neman . J. de Vellis

1 Oligodendrocyte Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
1.1 General Developmental Stages/Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
1.2 Embryonic Origins of Oligodendrocyte in the Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1.2.1 Motor NeuronOligodendrocyte Precursor (MNOP) Cell Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1.2.2 Dorsal Spinal Cord Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
1.2.3 Glial-Restricted Precursor Cell Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
1.3 Oligodendrocyte Development in the Forebrain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
1.4 Oligodendrocyte Development from the SVZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
1.5 Myelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

2 AxonMyelin Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

3 Aging and Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

# 2008 Springer ScienceBusiness Media, LLC.

62 3 Myelinating cells in the central nervous systemdevelopment, aging, and disease

Abstract: Many neurological diseases are caused by myelin deficiencies, which can be the result of both
genetic and environmental factors. The myelin forming cell, oligodendrocyte (OL), is a major target for the
known causes of white matter diseases. During development, oligodendrocytes pass through a series of cell
phenotypes from undifferentiated stem cells to mature myelin-forming cells. The general idea that there
might be different subclasses of oligodendrocyte derived from different precursor subtypes is an area of
active debate. Can cells that are born of progenitors in the different parts of the embryo, under the influence
of different positional signals and expressing different sets of patterning genes, ever converge on precisely
the same phenotypic endpoint? The following sections will review literature about controversy over
oligodendrocyte origins and degenerative process resulting in demyelination in the postnatal aging brain
and Alzheimers disease.

List of Abbreviations: AD, Alzheimers Disease; AEP, anterior entopeduncular; APP, amyloid precursor
protein; bFGF, basic fibroblast growth factor; CGE, caudal ganglionic eminence; CNS, central nervous
system; Gsh2, genomic screened homeobox 2; LGE, lateral ganglionic eminence; MBP, myelin basic protein;
MGE, medial ganglionic eminence; MNOP, motor neuronoligodendrocyte precursor; NEP, neuroepithe-
lial cells; NFT, neurofibrillary tangles; Nrcam, neuronal-adhesion molecules; NRG, neuregulin; NRP,
neuron-restricted precursor; OL, oligodendrocyte; OLP, oligodendrocyte progenitor; PDGF, platelet-
derived growth factor; PDGFR/, platelet-derived growth factor receptor-/; PLP, proteolipid protein;
PSA-NCAM, polysialylated form of the neural cell adhesion molecule; SHH, sonic hedgehog; SVZ,
subventricular zone; VZ, ventricular zone

1 Oligodendrocyte Development

1.1 General Developmental Stages/Scheme

The oligodendrocyte (OL) is the myelin-forming cell of the central nervous system (CNS). Myelin, which is
essential for the normal functioning of the mammalian CNS, is a fatty insulation composed of modified
plasma membrane that surrounds axons and promotes the rapid and efficient conduction of electrical
impulses. Many neurological diseases are caused by myelin deficiencies, which can be the result of both
genetic and environmental factors. These white matter disorders occurring during the development of the
CNS generally display hypomyelination or dysmyelination. Deficiencies of myelin may also occur after its
formation in the postnatal brain through a degenerative process resulting in demyelination.
Oligodendrocytes pass through a series of cell phenotypes from undifferentiated stem cells to
mature myelin-forming cells (> Figure 31). This sequential process of maturation of oligodendrocytes
can be reproduced in culture. The oligodendrocyte cell lineage culture is an excellent system to study the
influence of growth factors on cell lineage development. Each stage of oligodendrocyte lineage progression
can be identified by its characteristic markers, and through the use of growth factors, experiments can be
designed to increase or decrease the degree of proliferation and/or block, delay, or accelerate maturation of
developing precursor cells. Treatment of oligodendrocyte progenitor (OLP) cells with growth factors
known to be present in the developing CNS yields a complex set of ligand-dependent, phenotypic
responses.
Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) are two of the key
molecular signals controlling oligodendrocyte cell development. In the presence of PDGF, OLP cells are
stimulated to divide with a short cell cycle length of 18 h; are highly motile and bipolar; and differentiate in
a synchronous, symmetrical, clonal fashion with a time course similar to that in vivo. In the presence of
bFGF, however, progenitor cells are stimulated to divide with a longer cell cycle length of 45 h; become
nonmotile preoligodendrocytes with a multipolar shape; and are inhibited from expressing galactocerebro-
side glycolipid, proteolipid protein (PLP), or myelin basic protein (MBP). Progenitor cells treated with
both bFGF and PDGF exhibit a third phenotypic response, remaining motile, bipolar progenitor cells that
divide indefinitely and do not differentiate. This ligand-dependent, conditional immortalization can
 Myelinating cells in the central nervous systemdevelopment, aging, and disease 3 63

. Figure 31
Model for oligodendrocyte development. The figure illustrates the characteristics that accompany the sequen-
tial differentiation within the oligodendrocyte lineage beginning from very early stem cells to the mature
oligodendrocyte. Abbreviation for cell marker: SSEA1, stage specific embryonic antigen-1; A2B5, polysialic acid-
NCAM, ganglioside; CNP, 2,3-cyclic nucleotide-3-phosphohydrolase; GD3, ganglioside; GPDH, glycerolpho-
sphate dehydrogenase; GC, galactocerebroside glycolipid; O4, O1, ganglioside; PLP, proteolipid protein; MBP,
myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; Tf, transferrin; GSTpi, glutothione S-trans-
ferases pi; PLP, proteolipid protein; GPDH, glycerol 3-phosphate dehydrogenase

greatly expand the OLP cell population over extended periods of time. Upon removal of bFGF or both
mitogens, the progenitor cells differentiate along the oligodendrocyte lineage.
In addition, insulin-like growth factor 1 has been implicated in OLP proliferation and differentiation.
While transforming, growth factor-beta 1 is also associated with differentiation because it inhibits the
mitogen-induced proliferation of OLP through exit from the cell cycle and thus might have a role in
triggering differentiation (Franklin, 2002).
The morphology of oligodendrocytes varies according to the axons that they myelinate. Those that
ensheath large-diameter axons have a large cell body that lies close to the axon and they may synthesize only
a single internodes worth of myelin, while other oligodendrocytes make as many as 30 internodes small
axons. There are also molecular differences between oligodendrocytes on large- versus small-bore axons
for example, in their gap junction proteins (connexins). It is not known whether these are intrinsic
differences or phenotypic variations of a single, plastic cell type. The general idea that there might be
different subclasses of oligodendrocyte is derived from different precursor subtypes is an area of active
64 3 Myelinating cells in the central nervous systemdevelopment, aging, and disease

debate. Can cells that are born of progenitors in different parts of the embryounder the influence of
different positional signals and expressing different sets of patterning genesever converge on precisely
the same phenotypic endpoint? The following sections will review literature about controversy over
oligodendrocyte origins (Richardson et al., 2006).

1.2 Embryonic Origins of Oligodendrocyte in the Spinal Cord

The fundamental question in developmental neurobiology is how a relatively simple and undifferentiated
neuroepithelium in the embryo can give rise to the remarkable cellular diversity and specialization of the
mature CNS. Extrinsic signaling molecules and cell-intrinsic factors instruct multipotent progenitor cells,
thereby restricting their potential to become specialized neurons, oligodendrocytes, or astrocytes.
It is now clear that the initial specification of spinal cord oligodendrocytes takes place in the embryo.
This process requires precise interplay between cell-intrinsic and regionally restricted extrinsic factors,
which has much in common with the mechanisms that underlie the development of neuron. A substantial
progress in understanding neural cell fate specification has resulted from a focus on the roles of transcrip-
tion factors in the acquisition of progenitor subtype identity in the developing neural tube. An extensive
body of work has shown how restricted patterns of transcription factor-encoding genes are crucial for the
organization and initial fate choices of restricted sets of progenitor cells along the anteroposterior and
dorsoventral axes of the developing neural tube. In this section, we will discuss how neuronal and glial
progenitor domains are established in the spinal cord.

1.2.1 Motor NeuronOligodendrocyte Precursor (MNOP) Cell Hypothesis

The MNOP hypothesis is based on observations that both motor neurons and oligodendrocytes arise in
similar zone of the ventral spinal cord with similar concentrations of Sonic Hedgehog (SHH) required for
the induction of both cell types. In addition, in vitro studies show that the induction of oligodendrocytes is
frequently accompanied by the induction of motor neurons (Pringle et al., 1996; Orentas et al., 1999).
Therefore, it seems logical to have motor neurons and oligodendrocytes developmentally related to each
other since ensheating the axons of these neurons with myelin would increase the conduction velocity.
Interestingly, in annelids and crustaceans, myelin-like membranes are associated with axons required for
rapid escape responses. Therefore, it would be intuitive to ensure that motor neurons and the cells that
ensheath them arise at the same place would be to derive both cells from the same precursor.
In 2002, it was discovered by three separate groups that the Olig gene family of basic HLH transcription
factors is involved in the developmental production of both motor neurons and oligodendrocytes (Lu et al.,
2002; Takebayashi et al., 2002; Zhou and Anderson, 2002). Specifically, Olig1 and Olig2 genes are
expressed in the developing mouse spinal cord within the specific region that appears to give rise to both
oligodendrocytes and motor neurons. Forced expression of Olig1 or Olig2 in neuroepithelial stem cells
induces expression of early markers of the oligodendrocyte lineage. Expression of Olig2 in conjunction with
neurogenin2 appears to be critical for the generation of motor neurons. Moreover, the targeted disruption
of Olig2 prevents oligodendrocyte and motor neuron specification in the spinal cord. Disruption of Olig1,
in contrast, disrupted normal maturation of oligodendrocytes (Noble et al., 2004).

1.2.2 Dorsal Spinal Cord Development

Initially, it was believed that oligodendrocytes in the spinal cord are only derived from ventral sources.
However, recent new evidence shows that oligodendrocytes in the spinal cord are derived from both ventral
and dorsal sources (Cai et al., 2005; Fogarty et al., 2005; Vallstedt et al., 2005) (> Figure 32).
The Nkx6 transcription factors are normally expressed in the ventral part of the embryonic spinal cord
in progenitor domains p3, pMN, p2, and p1, and are activated by SHH signaling from the notochord and
 Myelinating cells in the central nervous systemdevelopment, aging, and disease 3 65

. Figure 32
Oligodendrocyte development in the spinal cord. Neurons are formed before glia (astrocytes and oligodendro-
cyte precursors (OLPs)). In general, OLPs are formed before astrocytes and ventral cell types before dorsal. The
figure shows the expression domains of several transcription factors. Dashed bars indicate that the expression
domain boundaries shift during development, in the direction of the small arrowsfor example, expression of
the transcription factor Nkx2.2 expands dorsally, and expression of the developing brain homeobox gene Dbx1
contracts. Approximately 85% of all spinal cord oligodendrocytes are generated from pMN and the remainder
from other progenitor domains. It is not known whether astrocytes are also generated from pMN but, if so, they
are probably produced in small numbers relative to oligodendrocytes. Abbreviations: dP1dP6, dorsal progen-
itor domains; Msx3, a homeobox gene; Olig2, oligodendrocyte lineage gene 2; Pax7, paired box gene 7; pMN
and p0p3, ventral progenitor domains. (Reproduced with permission from Richardson et al. (2006).)

floor plate at the ventral midline. Once activated, they in turn activate Olig2, which as stated before, is
required for the generation of both motor neurons and oligodendrocyte precursors from progenitors in the
ventral progenitor domain pMN. Therefore, in spinal cords of Nkx6-null mice (specifically Nkx6.1 and
Nkx6.2) there is a loss of Olig2 expression in pMN leading to developmental blockage of both motor
neurons and oligodendrocytes.
However, oligodendrocyte precursors that express normal markers of platelet-derived growth factor
receptor-/ (PDGFR/) and Olig2 continue to be produced in the dorsal spinal cord of Nkx6-null mice. The
dorsal precursors coexpress paired box gene 7 (Pax7), confirming their dorsal origin. In wild-type mice,
some oligodendrocyte precursors in the dorsal part of the cord are also found to express Pax7, which
indicates that dorsal production is a normal phenomenon. These precursors were missed in previous
studies presumably because they are generated after their ventrally derived counterparts and mingle with
them unnoticed. There are fewer Pax7-expressing oligodendrocyte precursors in wild-type spinal cord than
in Nkx6 mutant cord, which suggests that ventrally produced oligodendrocyte precursors normally
suppress their dorsal counterparts, perhaps because they compete more effectively for essential proliferation
and/or survival signals such as PDGF alpha. Generation of the ventral precursors starts a couple of days
earlier than that of the dorsal ones (embryonic day (E) 12.5 compared with
E15), so that they have plenty
of time to get preestablished (Richardson et al., 2006) (> Figure 33).
66 3 Myelinating cells in the central nervous systemdevelopment, aging, and disease

. Figure 33
Oligodendrocyte developmental sequence in developing spinal cord and forebrain. (a) In the mouse spinal
cord,
85% of oligodendrocyte precursors are generated from pMN in the ventral ventricular zones (1), starting
at about embryonic day (E)12.5. At about E15, generation of a secondary wave of precursors starts in more
dorsal regions by trans-differentiation of radial glia (2). (b) In the telencephalon, the ventral-most precursors in
the medial ganglionic eminence are produced from about E12.5 (1), production of the lateral ganglionic
eminence-derived precursors starts a few days later (2), and production of the cortex-derived precursors occurs
mainly after birth (3). Diagram not to scale. (Reproduced with permission from Richardson et al. (2006).)

Further evidence for dorsally derived oligodendrocytes has been provided by Cre-lox fate-mapping
experiments in transgenic mice under the control of regulatory elements surrounding the Dbx1 (developing
brain) homeobox gene. In these mice, Cre expression mirrors the normal pattern of Dbx1 expression, which
is restricted to neuroepithelial precursors in p1, p0, dP6, and dP5that is, four progenitor domains
centered on the dorsalventral midline. The Dbx1-derived oligodendrocytes comprised
3% of all oligo-
dendrocytes in the spinal cord and were less widely spread than the majoritybeing mainly located in the
lateral white matter radially opposite to their site of origin in the ventricular zone. Some Dbx1-derived,
Olig2-positive cells retained a radial process and transiently coexpressed the radial glial cell marker RC2,
which indicates that they are formed by direct interconversion from radial glia.
In more recent fate-mapping studies with Msx3Cre transgenic mice it appeared that 1015% of all
oligodendrocytes in the cervical spinal cord originate in the dorsal half of the cord. Many of these are
concentrated in the dorsal funiculus where they contribute up to 50% of the oligodendrocytes (Richardson
et al., 2006).

1.2.3 Glial-Restricted Precursor Cell Hypothesis

At the same time that studies have been ongoing on origin and relation of motor neurons and oligo-
dendrocytes, a separate line of investigation has raised questions about whether oligodendrocytes are
developmentally more closely related to astrocytes rather than motor neurons.
This latter investigation has led to the idea that during spinal cord development, the progression from
pluripotent neuroepithelial stem cell to differentiated cell types requires prior generation of lineage-
restricted precursor cells. Furthermore, it is thought that neurons and glial cells come from different
lineage-restricted precursor cells, NRP and GRP, respectively. GRP cells, in response to local environmental
signals, can generate either astrocytes or oligodendrocytes, with oligodendrocyte generation involving the
intermediate generation of OLPs (Noble et al., 2004).
Both GRP cells and neuron-restricted precursor (NRP) cells can be directly isolated from the devel-
oping rat spinal cord and grown as purified populations. Freshly isolated cells exhibit the same lineage
restrictions as those cells derived from neuroepithelial stem cells in vitro. Clonal studies have demonstrated
that GRP cells retain their tripotential nature even after weeks of in vitro expansion and several serial
 Myelinating cells in the central nervous systemdevelopment, aging, and disease 3 67

reclonings and also exhibit these same restrictions following transplantation in vivo. GRP cells generate
both oligodendrocytes and astrocytes following transplantation into the brain or spinal cord, and do not
generate neurons even when they migrate into such neurogenic zones as the rostral migratory stream and
olfactory bulb. NRP cells, in contrast, generate only neurons (including motor neurons), even upon
transplantation into such CNS regions as the adult spinal cord (Noble et al., 2004).
GRP cells exist throughout the rostra caudal axis and were initially isolated from the developing
rat caudal neural tube around E12 to E14 and can be distinguished from neuroepithelial cells (NEP) by
the acquisition of A2B5 immunoreactivity and their initial lack of NG2 and PDGFRa expression, and
their limited adhesion to laminin and preference for fibronectin. GRP cells therefore represent an early
intermediate progenitor committed to glial lineage between NEP cells and fully differentiated glia and
may be one of the earliest born glial precursors (Liu and Rao, 2004).
NRP cells, in contrast, expressed the polysialylated form of the neural cell adhesion molecule
(PSA-NCAM) and were shown to give rise to multiple different kinds of neurons and not to glia (Noble
et al., 2004).

1.3 Oligodendrocyte Development in the Forebrain

However, just like the spinal cord, there are still ambiguities as to ventral and dorsal sources for oligoden-
drocytes in the forebrain as well. The controversy about the origins of oligodendrocytes extends to the
forebrain as well. Here, too, there is evidence for a ventral source from the ventricular zone of the basal
forebrain. Cells that express oligodendrocyte lineage markers such as Olig1, Olig2, Sox10, and PdgfR/ first
appear in the neuroepithelium of the medial ganglionic eminence (MGE), and appear to migrate laterally
and dorsally from there to all parts of the developing forebrain, including the cerebral cortex, before birth
(Tekki-Kessaris et al., 2001).
Recent fate mapping by William Richardson and colleagues has helped resolve some of the confusion.
Using an Nkx2.1-Cre transgenic mouse line that marks neural progenitors in the basal forebrain (including
the MGE, anterior entopeduncular (AEP), and pre-optic area), it was found that the first oligodendro-
cyte precursors migrate into the cortex about E16 from ventral territories. By E18, a second wave of
oligodendrocyte precursors migration from the lateral ganglionic eminence (LGE) and/or caudal ganglionic
eminence (CGE) that are genomic screened homeobox 2 (Gsh2) positive occurs. Therefore, at E18, all
oligodendrocyte lineage cells in the cortex are ventral in origin. After E18, however, the contribution of
ventral cells starts to decrease as they are joined by yet another wave of oligodendrocyte precursors that
originate in the cortex itself (Emx1-positive neuroepithelium) (> Figure 34). Therefore, similar to the
spinal cord there are both ventral and dorsal sources depending on the stage of development studied.
Furthermore, the original population of MGE/AEP-derived precursors disappears after birth, being rapidly
eliminated from the cortex and more gradually from all other parts of the brain. Almost no trace can be
found of the initial Nkx2.1-derived oligodendrocyte population anywhere in adults (Richardson et al.,
2006).
In addition, a subpopulation of OLPs exists in the telencelphelon, characterized by the expression of
PLP/DM20 and does not depend on PDGFRa signaling for survival and proliferation (Spassky et al., 2001).
These PDGF-AA-independent OLPs expressing PLP/DM-20 are detected in several regions of the embry-
onic brain prior to the emergence of PDGFRa-expressing cells. In addition, after birth PLP/DM-20 OLPs
are also distinct from the population of PDGFRa cells in the subventricular zone (SVZ) of the cerebral
cortex (Ivanova et al., 2003).

1.4 Oligodendrocyte Development from the SVZ

As mentioned previously, forebrain oligodendrocytes originate first from progenitors in the embryonic
ventral telencephalon. A second wave of oligodendrocytes originates later in development from the dorsal
telencephalon. In addition, in the developing telencephalon, the neuroepithelieum gives rise to the SVZ,
68 3 Myelinating cells in the central nervous systemdevelopment, aging, and disease

. Figure 34
Sources of oligodendrocytes in the developing forebrain. (a) Three waves of oligodendrocytes generated from
different lineages in the telencephalic ventricular zone. Cells from the Nkx2.1 population begin to migrate
around E12.5, those from the Gsh2 population around E15.5, and those from the Emx1 population around P0.
Nkx2.1 and Gsh2 expressions overlap in the ventricular zone overlying the medial ganglionic eminence. Also
shown are presumed migratory pathways of oligodendrocyte precursors from each of these areas. (b) Sources
of oligodendrocytes in the corpus callosum (square) at three time points. At P0, most of the cells arise from the
ventral, Nkx2.1 and Gsh2 domains. By P10, cells from the Nkx2.1 lineage have mostly disappeared, replaced by
a mix of Emx1 and Gsh2 cells. In the adult, the Emx1 and Gsh2 lineages make up most of the oligodendrocytes,
but an unlabeled population is also present. (Reproduced with permission from Ventura and Goldman (2006).)

a secondary proliferative population of progenitors that simultaneously generates both neurons and glia.
Progenitor cells specified to become glia migrate radially from the SVZ into the subcortical white matter,
corpus collosum, striatum, and cerebral cortex, where they differentiate into astrocytes and oligodendro-
cytes (Marshall et al., 2003). In addition, oligodendrocytes continue to be produced in the adult brain and
participate in myelin repair (Chari and Blakemore, 2002).
Furthermore, work from James Goldmans laboratory show that in the postnatal forebrain, oligo-
dendrocytes are generated from progenitor cells that reside near the tips of the lateral ventricles (Levison
et al., 1999). Yet, the primary progenitors for new oligodendrocytes born in the adult SVZ have not
been identified.
Recent work from Alvarex-Buylla and colleagues suggest that GFAP-positive type B astrocytes and a
subpopulation of actively dividing type C cells in the SVZ express the transcription factor Olig2. They
also show that type B cells generate a small number of nonmyelinating NG2-positive OLPs and mature
myelinating oligodendrocytes. Olig2-positive, polysialylated neural cell adhesion molecule-positive, PDGFRa,
and beta-tubulin-negative cells originating in the SVZ migrated into corpus callosum, striatum, and fimbria
fornix to differentiate into the NG2-positive nonmyelinating and mature myelinating oligodendrocytes.
Furthermore, primary clonal cultures of type B cells gave rise to oligodendrocytes alone or oligodendrocytes
and neurons. Importantly, the number of oligodendrocytes derived from type B cells in vivo increased fourfold
after a demyelinating lesion in corpus callosum, indicating that SVZ-derived oligodendrocytes participate
in myelin repair in the adult brain (Ligon et al., 2006; Menn et al., 2006) (> Figure 35).
 Myelinating cells in the central nervous systemdevelopment, aging, and disease 3 69

. Figure 35
Olig2 contributes to ongoing oligodendrogenesis in progenitor cells of the adult SVZ. The cartoon depicts cells
surrounding the lateral ventricle (V). Type B cells give rise to rapidly cycling type C progenitor cells, which about
95% of the time produce type A neuroblasts that enter the rostral migratory stream and become olfactory
bulb interneurons (OBN). A small subpopulation (
5%) of type C cells express Olig2 and give rise to NG2+ OLP
and mature oligodendrocytes. Abbreviation: E, ependymal layer. Reproduced with permission from Ligon
et al. (2006).)

It would be interesting to know what the relationship between the embryonic and postnatal germinal
zones is with respect to oligodendrocyte development. As suggested by Richardson and colleagues, when
neurogenesis comes to an end during late embryogenesis, the forebrain ventricular zone (VZ) regresses
until only a remnant remains at the cortico-striatal boundary, which remains active and continues to
generate new oligodendrocytes after birth and into adulthood. The postnatal VZ and its neighboring SVZ is
derived mainly from the embryonic LGE and lateral cortex, with no contribution from more ventral
regions. Therefore, the most ventral, MGE/AEP-derived progenitors leave no descendants in the postnatal
SVZ. This might contribute to the gradual loss of MGE/AEP-derived oligodendrocytes during postnatal life
(Richardson et al., 2006).

1.5 Myelination
Myelin, a multilamellar membrane, is formed by the spiral wrapping of glial (oligodendrocytes in the CNS
and by Schwann cells in the peripheral nervous system (PNS)) plasma membrane extensions around the
axon. The evolutionary need for the rapid and efficient conduction of action potentials in vertebrate
neurons has resulted in the development of the myelin sheath. Neuron-derived signaling molecules can and
do regulate the proliferation, differentiation, and survival of oligodendrocytes.
Myelin is a lipid-rich membrane (lipids constitute 70% of dry myelin weight) that is highly enriched in
glycosphingolipids and cholesterol. The major glycosphingolipids in myelin are galactosylceramide and its
sulfated derivative sulfatide (20% of lipid dry weight). The two major CNS myelin proteins are MBP and
the proteolipid proteins (PLP/DM20). During the active phase of myelination, each oligodendrocyte must
produce as much as
550  103 mm2 of myelin membrane surface area per day (Pfeiffer et al., 1993).
The timing of myelination is crucial because the ensheathment of axons must not occur before
neurons signal to oligodendrocytes. In turn, signals from oligodendrocytes to neurons are necessary to cluster
70 3 Myelinating cells in the central nervous systemdevelopment, aging, and disease

multiprotein complexes in the axonal membrane into distinct subdomains at the nodes of Ranvierthe
gaps between myelinated segments of neurons. Therefore, the next event after differentiation of OLPs
is the formation of myelin. Myelination is a multistep process requiring precise coordination of several
different signals.
Oligodendrocytes wrap their plasma membrane around axons that have a minimum axon caliber of
2.0 mm diameter. The physiological rationale for this requirement could be that saltatory conduction would
not be significantly enhanced on smaller diameter fibers. The g-ratio is the ratio of the axonal diameter
divided by the diameter of the axons and its myelin sheath. Most myelinated axons have an approximate
g-ratio of 0.6. Therefore, larger axons have thicker myelin, while smaller ones have thinner myelin. The
exception to the constant g-ratio is seen in axons that have been remyelinated and are therefore typically
thinner than expected (Sherman and Brophy, 2005).
The neuregulin (NRG)-ErbB receptor system has been shown to have considerable influence on the
thickness of the myelin sheath. Recent studies show that NRG-1 type III on the axonal surface is required
for the myelination by Schwann cells in the PNS (Taveggia et al., 2005). Transgenic mice with reduced NRG-
1 expression display hypomyelination, whereas over expression of NRG-1 induces increased myelin
thickness (Nave and Salzer, 2006). Myelin-forming Schwann cells thus appear to use NRG-1 signals to
know whether and to what extent axons require myelination. The signaling pathways involved in the CNS
are not known. Whether this signaling system operates in CNS myelination remains an open question of
major importance for human demyelinating diseases (Simons and Trajkovic, 2006).
After oligodendrocytes have established proper contact with the axonal membrane, the process of
myelination begins. One signal that seems to be required to trigger myelination is the electrical activity from
the neuron that leads to secretion of promyelinating factors of adenosine from neurons and/or leukemia
inhibitory factor from astrocytes (Demerens et al., 1996; Stevens et al., 2002; Ishibashi et al., 2006). Once the
coordinated differentiation of the axon and myelination has occurred, the axonal membrane is differen-
tiated into distinct molecular, structural, and functional domains. These domains include the nodes of
Ranvier, the paranodal junction, the juxtaparanodes, and the internodal regions.

2 AxonMyelin Membrane

The Nodes of Ranvier are periodical interruptions in the myelin sheath that are spaced at intervals that are
about 100 times the axonal diameter. These gaps are required for efficient and rapid propagation of action
potentials. The nodes are flanked by paranodal loops that form septate-like junctions with the axonal
membrane. Adjacent to them are the juxtaparanodal domains that lie under the compact myelin sheath.
There are some structural differences between PNS and CNS. In peripheral nerves, the entire myelin unit is
covered by a basal lamina, and the outermost layer of the Schwann cell extends microvilli that cover the
nodes. The perinodal space that lies between the axolemma and the basal lamina contains the microvilli and
is also filled with a filamentous matrix. In addition, in the CNS there are no basal lamina and the nodes are
contacted by perinodal astrocytes (synantocytes) (Butt et al., 2005).
The nodes are characterized by a high density of Na+ channels that are essential for the generation of the
action potential during saltatory conduction. Nodes of Ranvier in the adult CNS and PNS mostly contain
Nav1.6 (Yu et al., 2003). During development, both PNS and CNS nodes express Nav1.2, which is later
replaced by Nav1.6 that may allow neurons to adapt to high-frequency firing (Kaplan et al., 2001). In
addition, there exist two K+ channels at the nodes. Kv3.1 is mainly found in large axons in the CNS and only
in few nodes in the PNS, whereas Kcnq2 is located in all PNS nodes and most CNS nodes (Devaux et al.,
2003). Several transmembrane and cytoskeleton proteins have been identified at the node as well. They
include the neuronal-adhesion molecules (Nrcam), neurofascin-186, and their ligand gliomedin (Eshed
et al., 2005), the cytoskeleton adaptor/scaffolding protein ankyrin G, and the actin-binding protein spectrin
Beta-IV (Garrido et al., 2003). In general, it is believed that interaction with neurofascin, NrCAM, and
gliodemin recruits ankyrin G that contains binding sites for Na+ channels. Recruitment of stabilizing
components such as bIV spectrin further enhances the clustering of Na+ channels by anchoring the complex
more firmly to the axonal cytoskeleton (Simons and Trajkovic, 2006).
 Myelinating cells in the central nervous systemdevelopment, aging, and disease 3 71

The paranodal junction attaches the myelin sheath and serves as a fence that limits lateral diffusion of
axolemmal proteins and to separate the electrical activity at the node of Ranvier from the internodal region
under the compact myelin sheath. Both in the PNS and the CNS, the formation of the paranodes is
dependent on axonglia interactions. The interaction of the loops that form septate-like junctions with the
axonal membrane is mediated by neurofascin-155 and contactin (Sherman and Brophy, 2005). The
interaction of neurofascin-155 in glia to a complex of contactin and contactin-associated protein (Caspr)
in axons initiates the assembly of the paranodal complex, which is then stabilized by interactions with a
specialized axonal cytoskeleton consisting of proteins such as the membrane skeleton component protein
4.1B, ankyrin B, and a/bII spectrin (Ogawa et al., 2006).
The juxtaparanode is located in a short zone just beyond the innermost paranodal junction. Caspr2, the
second member of the Caspr family, interacts with heteromultimers of the delayed rectifier K+ channels of
the Shaker family, Kv1.1, Kv1.2, and Kv2. In addition, Kv1.6 is present at this site, predominantly in
small axons. These K+ channels may act as an active damper of reexcitation and to help in maintaining
the internodal resting potential Two other proteins that are found at the juxtaparanodes are transient
axonal glycoprotein-1 (Tag1), a GPI-anchored CAM that is related to contactin118, and connexin 29
(Cx29), which is found at the glial membrane. Two recent studies showed that Caspr2 and Tag1 form a
juxtaparanodal complex, consisting of a glial Tag1 molecule and an axonal Caspr2/Tag1 heterodimer. This
complex is essential for the accumulation of K+ channels in the juxtaparanodes, as targeted disruption of
Caspr2 or Tag1 results in a striking reduction in the juxtaparanodal accumulation of these channels in both
PNS and CNS axons (Poliak and Peles, 2003) (> Figure 36 and > 37).

3 Aging and Alzheimers Disease

Several functions of the myelin-producing cells, the oligodendrocytes, make it an integral cell of the CNS
and make it pertinent issue when discussing aging and pathology of Alzheimers Disease (AD). Most studies
on the effects of aging on the nervous system have focused on the changes that occur as a consequence of
AD. The age risk factor is present in nongenetic and genetic forms of AD; with increased amyloid beta
peptides (Ab), deposition in the latter form. Myelin breakdown may be a contributing factor to the
pathology of both aging and AD. In addition, these breakdowns have been observed early in disease in
spite of infarction, Wallerian degeneration, or white matter amyloid angiopathy (Bartzokis et al., 2004). An
attribute to this white matter breakdown maybe due to the accumulation Ab, reduced cholesterol, and
myelin proteins (Roher et al., 2002).
The dependence of the brain on oligodendrocyte-produced cholesterol has implications for CNS
development and its continual functional plasticity. All brain cholesterol is synthesized de novo by
oligodendrocytes. The human brain, which is approximately 2% of the body by weight, contains approxi-
mately 25% of the bodys membrane cholesterol. It is thus not surprising that myelin membrane changes
are found to drive brain lipid changes with age as well as species differences in membrane composition.
Cholesterol enrichment may contribute to the free exchange of cholesterol from oligodendrocytes to
neurons and astrocytes with the aid of apolipoproteins. The low water binding produced by high
cholesterol levels in myelin breakdown may promote the hydrophobic ends of Ab aggregates to preferen-
tially interact with and damage myelin.
As mentioned earlier, myelin is essential for normal brain function. Myelination results in salutatory
conduction that results in increases signal transmission. Once myelin function in salutatory conduction is
compromised, there is a decrease in transmission velocity and refractory period of the axon. These changes
and disruption due to these delays can have impact on synchronization of impulses. The effects of this
desynchronization would be most apparent on brain functions that involve encoding and retrieval of
memories. Aging reduces 50% number of fast-conducting CNS axons in cats. Human stereological studies
estimate that the total length of myelinated axons is reduced by 2745% in old age, primarily through loss
of fibers with small diameter that myelinate later in development and are most susceptible to Ab pathology.
This aging-related myelin breakdown negatively impacts cognitive performance in primates and humans.
The age-related loss of myelin function may also explain the conduction delays observed in aging animals,
72 3 Myelinating cells in the central nervous systemdevelopment, aging, and disease

. Figure 36
Myelinated axons. (a) Myelinating glial cells, oligodendrocytes in the central nervous system (CNS), or Schwann
cells in the PNS form the myelin sheath by enwrapping their membrane several times around the axon. Myelin
covers the axon at intervals (internodes), leaving bare gapsthe nodes of Ranvier. Oligodendrocytes can
myelinate different axons and several internodes per axon, whereas Schwann cells myelinate a single internode
in a single axon. (b) Schematic longitudinal cut of a myelinated fiber around the node of Ranvier showing a
heminode. The node, paranode, juxtaparanode (JXP), and internode are labeled. The node is contacted by
Schwann cell microvilli in the PNS or by processes from perinodal astrocytes in the CNS. Myelinated fibers in the
PNS are covered by a basal lamina. The paranodal loops form a septate-like junction (SpJ) with the axon. The
juxtaparanodal region resides beneath the compact myelin next to the paranode (PN). The internode extends
from the juxtaparanodes and lies under the compact myelin. (c) Schematic cross-section of a myelinated nerve
depicting the inner and outer mesaxons (IMA and OMA, respectively). (d) Drawing of the specializations found
along the internodes. A strand composed of paranodal molecules (Caspr, Contactin; inner line) flanked by
juxtaparanodal proteins (Caspr2, K+ channels and TAG-1; outer lines) extends along the internodal region (the
juxtamesaxon) and below the SchmidtLanterman incisures (the juxtaincisure). In addition, Nf155 as well as
connexins 29 and 32 are found at the glial side, opposite to these axonal strands. (Reproduced with permission
from Poliak and Peles (2003).)
 Myelinating cells in the central nervous systemdevelopment, aging, and disease 3 73

. Figure 37
Molecular composition of domains at the Nodes of Ranvier. Components at the nodes include neurofascin-186,
NrCAM, and voltage-gated Na+ and K+ channels (Nav1.2, Nav1.6, Kv3.1, and Kcnq2), which are tethered to a
complex containing ankyrin G and bIV-spectrin. The paranodes contain a complex of Caspr, contactin, and 4.1B
at the axonal membrane, which binds to neurofascin-155 (NF155) on the septate junction of a paranodal loop.
The multiprotein complex in the juxtaparanode contains a cis complex of Caspr2 and TAG-1, which interact with
4.1B and a PDZ domain-containing protein associated with the two shaker-type K+ channels (Kv1.1 and 1.2).
This complex is linked through a trans interaction with TAG-1 to the glial membrane

humans, and patients with AD. Myelin loss may also underlie the reduced myelin staining in postmortem
studies of aging and the aging-related loss of brain volume (Bartzokis, 2004).
Since cholesterol- and lipid-synthesizing enzymes require iron to function, oligodendrocytes have the
highest iron content of all and as much as 70% of brain iron is associated with myelin. Age-related increases
in iron levels may contribute to the increased intracellular oxidation necessary to trigger oligodendrocyte
precursors to differentiate. Inadequate iron levels result in poor myelination and mental deficiencies in
children. Normal ferritin, a spherical protein in which more than of 90% of tissue nonheme iron is stored,
can sequester and store iron and other transition metals. Many normal as well as pathological processes that
have been shown to damage oligodendrocytes can also release iron from ferritin. Oligodendrocytes may be
more vulnerable than other cells to such iron releases since in addition to containing the highest iron stores,
their particular ferritin subunit composition makes iron available with greater ease than in other cells.
Recent evidence suggests that elevated iron levels increase the production of amyloid precursor protein
(APP) and that the soluble Ab can act as an iron chelator. However, iron and other transition metals such as
copper and zinc can also promote Ab oligomerization. Oligomerization makes Ab toxic, thus making
the homeostasis of iron and Ab critically important. Therefore, late-myelinating oligodendrocytes and
their precursors are present at the cortical site of amyloid beta deposits observed in aging and AD
(Xu et al., 2001). In addition, iron and Ab to promote the formation of reactive oxygen species (Butterfield,
2003). This could explain as to why myelin breakdown from older and AD patients has been attributed to
increased levels of lipid peroxidation.
Since myelination markedly reduces neuronal energy expenditure, the loss of axonal myelin would
require an estimated increase of up to 5000-fold in neuronal energy expenditure in order to maintain
neurotransmission levels. Approximately 23% of the oxygen consumed in normal mitochondrial
respiration is obligatorily transformed into free radicals, and with aging, an increasing percentage of oxygen
74 3 Myelinating cells in the central nervous systemdevelopment, aging, and disease

is converted to superoxide. The aging-related loss/dysfunction of myelin would result in a further increase
in the production of damaging free radicals. Both neurons and especially oligodendrocytes are very
susceptible to damage from free radicals. Free-radical (oxidative) damage has been shown to be strongly
aging related and has been implicated in the pathophysiology of AD. An increase in neuronal free-radical
production has also been postulated to contribute to AD tangle-related neuropathology. Oxidation of tau
induces its dimerization and polymerization into insoluble filaments, the precursor to the intraneuronal
neurofibrillary tangles (NFT), the second pathognomonic lesion observed in AD brain. These damaging
oxidative processes are also observed in other neurodegenerative diseases and it is thus not surprising
that many other neurodegenerative disorders manifest NFTs while normal individuals rarely do so
(Bartzokis, 2004).
Oligodendrocytes are markedly heterogeneous based on when in the protracted process of human brain
development they differentiated into myelin-producing cells. Oligodendrocytes that differentiated late in
life ensheath 3050 smaller diameter axons as opposed to 1 oligodendrocyte per myelin segment of large
CNS motor and primary sensory area axons. These late-differentiating cells cannot produce the same
myelin thickness per axon segment as earlier-myelinating oligodendrocytes. The thinner, later-myelinating
sheaths are more susceptible to functional impairment and destruction. In addition, later-differentiating
oligodendrocytes have different lipid properties, may have a slower rate of myelin turnover, and reduced
ability for myelin repair than earlier-differentiating cells. This development-dependent oligodendrocyte
heterogeneity could contribute to reason as to why the neocortical regions of the brain are the most
vulnerable to developing AD lesions that consist of extracellular amyloid neuritic plaques and intraneuronal
NFT. Furthermore, the hippocampal and primary sensory and motor areas develop lesions only later on
in the disease process. Interestingly, it is the late-myelinating neocortical regions that are the most
vulnerable to AD lesions first, whereas the early-myelinating primary sensory and motor areas are affected
last (Braak et al., 2006).

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4 SulfurContaining Amino Acids in
the CNS: Homocysteine
D. K. Rassin

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

2 Overview of Methionine Metabolism in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

3 Homocysteine and Metabolic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

4 Homocysteine as a CNS Risk Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

5 Homocysteine: Potential Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

6 Methodological Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

# 2008 Springer ScienceBusiness Media, LLC.

78 4 Sulfurcontaining amino acids in the CNS: Homocysteine

Abstract: The sulfur amino acid metabolic pathway plays an important role in supporting a number of
functions within the CNS. The metabolic intermediate, homocysteine, sits at an important branch point in
this pathway and disturbances related to this compound are related to detrimental outcomes for the CNS.
Homocysteine first came to notice when biological dysfunction related to its excess was reported in
individuals with the inherited metabolic defect homocystinuria. Subsequently, a number of biological
dysfunctions have been associated with this amino acid unrelated to metabolic diseases. Increased homo-
cysteine has been identified as an epidemiologic risk factor for neural tube defects, cardiovascular disease,
cerebrovascular disease, Alzheimers disease and, potentially, several dysfunctions that occur in the new-
born. The mechanisms by which homocysteine may exert detrimental effects on the CNS include oxidative
damage, apoptotic induction, synthesis of the excitotoxic metabolite homocysteic acid, and inhibition of
energy-related enzymes. Methodologic issues, related to its disulfide/sulfhydryl structure and to the small
absolute increases that are associated with increased risk, make it difficult to define homocysteines role in
the CNS. The potential importance of modulating the amount of this amino acid is tempered by the
numerous unanswered questions about the mechanism of its functions.
List of Abbreviations: CNS, central nervous system; GABA, gaminobutyric acid; MTHFR, N5,N10
methylenetetrahydrofolate reductase; NMDA, NmethylDaspartate; PARP, polyADPribose polymerase;
SAH, Sadenosylhomocysteine; SAM, Sadenosylmethionine

1 Introduction

The methionine metabolic pathway supports numerous functions within the central nervous system (CNS)
beyond providing an essential amino acid precursor for protein synthesis. These functions include
neurotransmitter synthesis, methyl group donation, polyamine precursor, osmotic protection, antioxidant
synthesis, and DNA salvage synthesis. In previous editions of this Handbook the inherited genetic defects
related to this pathway have been described (Gaull, 1973), and the general metabolism of methionine,
especially as related to nutrition and development, has been reviewed (Tallan et al., 1983).
In this chapter a general overview of the various constituents of this pathway and their role in the CNS
are presented followed by a discussion of the role of homocysteine, as an agent that may have implications
for potential damage to the brain. In the years since the first edition of this Handbook, there has been an
evolution of our understanding of the role that homocysteine plays in biology. (Homocysteine exists in a
variety of forms, see > Sect. 6, throughout this chapter the compound is presented with this spelling and
discussed as if it represents the total of all forms unless stated otherwise.) Our appreciation of the functions
of this amino acid has changed as investigations have progressed from viewing homocysteine as the
potential toxic agent in a very rare inherited metabolic disease (homocystinuria) to a risk factor in several
common human conditions, such as cardiovascular disease, cerebrovascular disease, and Alzheimers
disease.

2 Overview of Methionine Metabolism in the CNS

The importance that the pathway of sulfur amino acid metabolism has in the CNS was first stimulated by
the findings of large concentrations of cystathionine in the brain (Tallan et al., 1958) and the association of
mental retardation with homocystinuria (Carson and Neill, 1962; Gerritsen et al., 1962). These latter
patients were found not to have cystathionine in the brain (Gerritsen and Waisman, 1964; Brenton et al.,
1965). This latter finding stimulated the discussion for this, as for many other inherited metabolic diseases,
of the role of toxic precursor metabolites versus deficient metabolic products in causing the mental
retardation observed in inherited diseases of amino acid metabolism (Gaull et al., 1975). In addition, the
finding that a number of the sulfur amino acids have neurotransmitter properties (cystathionine, cystei-
nesulfinic acid, cysteic acid, hypotaurine, and taurine) stimulated interest in this pathway (Curtis and
Watkins, 1960; Werman et al., 1966).
 Sulfurcontaining amino acids in the CNS: Homocysteine 4 79

Methionine is the essential amino acid precursor for the sulfur amino acid pathway (> Figure 4-1) and
is primarily metabolized via homocysteine either to taurine or is remethylated to methionine (a good
example of the fact that a nutritionally essential amino acid may be synthesized in vivo). Methionine is
supplied in the diet and enters the brain from the peripheral circulation via the large neutral amino acid
transporter (Sershen and Lajtha, 1979). Methionine itself has not been shown to have neurotransmitter
activity in the CNS; however, behavioral effects have been noted after its administration to schizophrenics.
Individuals with schizophrenia consistently worsened after methionine loading (Pollin et al., 1961; Brune

. Figure 4-1
Enzymes of the methionine metabolic pathway. 1 methionine adenosyltransferase, 2 numerous methylating
reactions, 3 Sadenosylhomocysteine hydrolase, 4 cystathionine bsynthase, 5 gcystathionase, 6
cysteine oxidase, 7 cysteinesulfinic acid decarboxylase, 8 hypotaurine oxidase, 9 chemical oxidation,
10 cysteic acid decarboxylase, 11 N5methyltetrahydrofolate: homocysteine methyltransferase, 12 serine
hydroxymethyltransferase, 13 N5,N10methylene tetrahydrofolate reductase. THFA, tetrahydrofolic acid; B6,
pyridoxal50 phosphate cofactor; B12, methylcobalamin cofactor

and Himwich, 1962), an effect that appeared to be associated with an increase in the synthesis of methylated
products of biogenic amines (Spaide et al., 1968). This response reflects the first step in the metabolism of
methionine to Sadenosylmethionine (SAM), catalyzed by the enzyme methionine adenosyltransferase.
SAM serves as the primary methyl donor in the brain (and elsewhere in the body) for biogenic amines,
RNA, DNA, and phosphatidylcholine metabolism and formation. SAM is also the precursor for the
polyamines (notably spermine and spermidine), metabolizing putrescine in the process catalyzed by the
enzyme Sadenosylmethionine decarboxylase. The polyamines have been proposed as regulators of DNA
and RNA metabolism, but of particular interest relative to the CNS is their potential role in the regulation of
neurogenesis (Malaterre et al., 2004). SAM has also been proposed as a nutritional supplement that may
help you feel good by the dietary supplement industry; however, this advice ignores the fact that increased
SAM may increase homocysteine resulting in greater health risks.
80 4 Sulfurcontaining amino acids in the CNS: Homocysteine

The demethylated product of reactions that utilize SAM as a methyl donor is Sadenosylhomocysteine
(SAH). SAH is catabolized to homocysteine and adenosine, catalyzed by the enzyme Sadenosylhomocys-
teine hydrolase. SAdenosylhomocysteine hydrolase appears to favor the reverse direction of synthesis of
SAH (Schatz et al., 1981), which may be one explanation for the low to absent amounts of homocysteine
usually observed in physiologic fluids and tissues.
Homocysteine is the major branch point of the methionine metabolic pathway; approximately half the
homocysteine that reaches this point is remethylated to homocysteine via the folic acid metabolic pathway
and the remainder is metabolized via the transsulfuration pathway (Mudd and Poole, 1975). The transsul-
furation pathway begins with the synthesis of cystathionine from homocysteine and serine catalyzed by the
enzyme cystathionine bsynthase. Cystathionine is an unusual sulfur ether compound that is found
primarily in the brain (Tallan et al., 1958). Although this compound has been suggested to be a neuro-
transmitter (Werman et al., 1966), proof of this function has not been forthcoming. Cystathionine
primarily serves as an intermediary in the transfer of a sulfur moiety from homocysteine to serine. This
conversion is completed by the catabolism of cystathionine to cysteine, catalyzed by the enzyme
gcystathionase. There is little measurable activity of gcystathionase in the brain (Shimizu et al., 1966;
Brown and Gordon, 1971), which probably accounts for the accumulation of cystathionine in this organ.
Cysteine, an amino acid required for protein synthesis, is a precursor for the major intracellular
antioxidant glutathione (gglutamylcysteinylglycine) and is further metabolized to cysteinesulfinic acid
catalyzed by cysteine oxidase. Cysteinesulfinic acid is decarboxylated to hypotaurine in a reaction catalyzed
by cysteinesulfinic acid decarboxylase. Cysteinesulfinic acid may also be oxidized to cysteic acid, which may
be decarboxylated to taurine, and hypotaurine also may be oxidized to taurine. Taurine may function as an
inhibitory neurotransmitter; it is a structural analogue of gaminobutyric acid (GABA) but is more likely to
be an important osmoregulator in the brain (PasantesMorales and Schousboe, 1988). Cysteinesulfinic acid
and cysteic acid are structural analogues of glutamic acid and have similar excitatory properties in the CNS;
however, it is not clear whether or not they are excitatory neurotransmitters in their own right. Taurine
appears to be the end product of the methionine metabolic pathway and is either excreted from the body via
the urine or as one of the bile salt conjugates, for example, taurocholic acid.
The enzymatic activities of gcystathionase and cysteinesulfinic acid decarboxylase are absent or low in
the brain during early development, thus both cysteine and taurine must be supplied from exogenous
sources (Sturman et al., 1970; Pascal et al., 1972; Gaull et al., 1977; Rassin, 1982).
The remethylation of homocysteine to methionine is catalyzed via a remethylation reaction in the CNS
by N5methyl tetrahydrofolate: homocysteine methyltransferase, an enzyme dependent upon a form of the
cofactor of vitamin B12, methylcobalamin. N5Methyltetrahydrofolate (the methyl donor for homocysteine)
is regenerated from the product of the remethylation reaction, tetrahydrofolic acid, in a sequence of two
reactions. The first, catalyzed by serine hydroxymethyltransferase, adds a methylene group from the amino
acid serine to tetrahydrofolic acid, subsequently producing the inhibitory neurotransmitter glycine and N5
N10methylenetetrahydrofolic acid. N5 N10Methylenetetrahydrofolic acid is converted to N5methyltetra-
hydrofolic acid in a reaction catalyzed by N5,N10methylenetetrahydrofolate reductase (MTHFR). Homo-
cysteine may also be remethylated to methionine by a reaction catalyzed by betaine/homocysteine
methyltransferase, utilizing betaine as the methyl donor. However, this reaction only appears to take
place in the liver and the kidney (Sunden et al., 1997).
Thus, methionine metabolic pathway is responsible for the synthesis of several putative neurotransmit-
ters (cystathionine, cysteinesulfinic acid, cysteic acid, hypotaurine, taurine, and glycine), the major intracel-
lular antioxidant (glutathione), the primary CNS methyl donor (SAM), the polyamines, and an important
osmoregulator (taurine), in addition to providing the protein precursors, methionine and cysteine. The
pivotal role of homocysteine in this pathway appears to be important for the health of the CNS.

3 Homocysteine and Metabolic Diseases

As noted above, original interest in homocysteine began when the enzymatic defect in cystathionine
bsynthase was identified and shown to be associated with a number of clinical signs and symptoms,
 Sulfurcontaining amino acids in the CNS: Homocysteine 4 81

including mental retardation, and biochemical signs, including increased plasma methionine and homo-
cysteine and decreased cysteine (Mudd et al., 2001). Subsequent to this discovery, it was found that
increased plasma homocysteine occurred associated with defects in the enzymes N5methyltetrahydrofolate:
homocysteine methyl transferase, N5,N10methylenetetrohydrofolate reductase, and in cobalamin transport
and metabolism (Rosenblatt and Fenton, 2001). These diseases all are associated with CNS deficits, with the
common biochemical feature being increased homocysteine in the blood and/or urine.
It is interesting to note that two additional metabolic defects in the methionine metabolic pathway have
been identified, one associated with methionine adenosyltransferase and the other with gcystathionase
(Mudd et al., 2001). Neither of these latter defects are associated with increased homocysteine, though
increased plasma methionine is a hallmark of methionine adenosyltransferase deficiency. Increased
cystathionine in the plasma and urine is the primary biochemical sign observed in cystathioninuria.
These two latter defects are not generally associated with functional CNS deficits, though demyelination
has been observed in some patients with methionine adenosyltransferase deficiency (Surtees et al., 1991).
Thus, the primary association with poor cognitive function in these inherited metabolic diseases is the
presence of increased homocysteine concentrations in the blood and/or urine. The possibility that increased
methionine, decreased cysteine, or a general imbalance of the various potential neurotransmitters in this
pathway are responsible for the CNS deficits appear to be unlikely explanations.

4 Homocysteine as a CNS Risk Factor

In searching for mechanisms to explain the neurologic deficits associated with homocysteine, the first
finding that seemed to be plausible was related to the thromboembolic episodes seen in a large proportion
of patients with cystathionine bsynthaserelated homocystinuria. McCully (1969) suggested that homo-
cysteine was responsible for initiating these events and developed a rabbit model that supported this
hypothesis (McCully and Ragsdale, 1970). Although not all experimental results supported the finding by
McCully and Ragsdale (1970) of increased atherosclerotic plaques in homocysteinetreated rabbits
(Donahue et al., 1974), these investigations stimulated further examination of the role of homocysteine
in cardiovascular disease.
Lead by the study of Wilcken and Wilcken (1976), a number of investigations identified mildly
increased plasma homocysteine as a risk factor for cardiovascular disease (Wilcken et al., 1983; Reis et al.,
1995; Graham et al., 1997). The finding by Mudd et al. (1981) that obligate heterozygotes for cystathionine
bsynthase deficiency were not at increased risk for thromboembolic episodes and the increased interest in
the role of folic acid in prevention of neural tube defects (MRC Vitamin Study Group, 1991; Czeizel et al.,
1992) suggested that the remethylation pathway of homocysteine might be of particular importance in
understanding the pathophysiologic effects of this compound.
The potential role of the remethylation pathway in the observed increased homocysteine concentrations
was further supported by the finding that a common thermolabile variant of MTHFR existed (MTHFR 677
C!T) that was associated with an increased risk for cardiovascular disease (Kang et al., 1991). However, the
association of the variant form of the enzyme itself with vascular disease has been inconsistent (Deloughery
et al., 1996). The variant is associated with increased homocysteine and reduced folate (Deloughery et al.,
1996). In another study that compared patients with acute ischemic stroke, high risk for atherosclerosis
related stroke, and elderly controls, no differences were observed amongst the groups related to either
MTHFR variants or to increased homocysteine (Press et al., 1999). However, this elderly population did
appear to have generally increased plasma homocysteine concentrations.
The combination of findings regarding neural tube defects and the benefits of folic acid, and the possible
role of homocysteine remethylation failure in cardiovascular disease, led to investigations of folate supple-
mentation on homocysteine. The grain supply of the United States was fortified with folic acid starting in
1998 (Mills and England, 2001) and this fortification appears to have reduced the incidence of neural
tube defects by 19% (Honein et al., 2001). Cereal fortification appears to result in a reduction in homocysteine
concentrations (Malinow et al., 1998; Jacques et al., 1999). However, this food fortification has raised the issue
that additional folic acid supplementation may not be effective in reducing homocysteine concentrations and
82 4 Sulfurcontaining amino acids in the CNS: Homocysteine

their further role as a risk factor (Bostom et al., 2001, 2002). In addition, lifestyles (smoking and overweight)
and aging are associated with increased homocysteine (Nurk et al., 2004). Increased homocysteine with age
has been associated with increased risk of hip fractures (McLean et al., 2004). Patients with renal disease may
not clear homocysteine well, so have increased homocysteine (Tamura et al., 1996). Given the results of these
studies, it is not surprising that increased homocysteine has been associated with an increased risk for
cerebrovascular disease, estimated to be between 2.0 to 2.5fold increases in risk (Boushey et al., 1995).
In considering the above studies, it might be concluded that only vascularrelated diseases of the CNS
would be associated with increased homocysteine. However, several studies have indicated that homocys-
teine is a risk factor for cognitive function in the normal aging process (Riggs et al., 1996), in geriatric
patients with various dementias (Nilsson et al., 1996), and in patients with depression (Bell et al., 1992; Fava
et al., 1997).
Intense interest arose in the role of homocysteine in CNS dysfunction with publication of the study by
Clarke and coworkers (1998) in which increased homocysteine was clearly associated with Alzheimers
disease. This study has been considered particularly important because a subset of the patients had
pathologically proven Alzheimers disease, and these patients had the highest plasma concentrations of
homocysteine (DiazArrastia, 1998; Miller, 1999). The study by Clarke and coworkers (1998) also indicated
that the progression of Alzheimers disease was accompanied by an increase in homocysteine concentra-
tions. Patients with Alzheimers disease and also those with vascular dementia have increased plasma
homocysteine and folate deficiency (Quadri et al., 2004). These findings suggested that early increased
homocysteine may signal cognitive decline in the elderly.
Data to date implicate homocysteine as a risk factor in both vascular and nonvascular diseases during
the aging process (Parnetti et al., 1997), and suggests that homocysteine is associated with a cognitive
decline in normal aging populations (Ravaglia et al., 2003; Lewerin et al., 2005). The unanswered questions
related to these studies are whether or not supplementing the aged population with B vitamins and folic
acid (cofactors at a number of steps in the methionine metabolic pathway) (see > Figure 4-1) will delay the
cognitive decline (even if they reduce homocysteine) and what are the exact biological mechanisms that
may explain the effects of homocysteine on the CNS (Vollset and Ueland, 2005). Further, few studies have
actually measured homocysteine in the CNS, none was detected in the brain of homocystinuric patients
(Brenton et al., 1965) or in the brain of a fetus carried by a mother homozygous for homocystinuria (Rassin
et al., 1979). However, methodologic issues, especially protein binding as discussed below, may have
influenced these results.
While most of the data described above reflect homocysteine as a risk factor to the brain during aging or
in disease processes usually found in the geriatric population, there is some evidence for similar risk
associations during early development. Homocysteine plasma concentrations increase with age in child-
hood and this increase appears to be associated with a decrease in folate (van Beynum et al., 2005). The
increased homocysteine was not strongly associated with the MTHFR 677 C!T polymorphism in this
study. Increased homocysteine has been associated with poor pregnancy outcomes (Vollset et al., 2000;
Hague, 2003), neural tube defects (Mills et al., 1995; Knott et al., 2003), and fetal death (Burke et al., 1992).
A number of investigators have associated increased plasma homocysteine in children with ischemic stroke
(Cardo et al., 1999; van Beynum et al., 1999) and venous thrombosis (Koch et al., 1999). Given these risk
factors, it is of particular interest that mild hypoxia in neonatal rats may result in increased homocysteine
when the animals are vitamin deficient (Blaise et al., 2005). This increased homocysteine may be a signal in
the cascade that translates oxidative stress in neonates to cognitive dysfunction.

5 Homocysteine: Potential Mechanisms of Action

A number of potential mechanisms have been proposed via which homocysteine may exert its toxic effects
upon the CNS. These mechanisms include the direct toxic effect of homocysteine, interaction with
the NmethylDaspartate (NMDA)glutamate receptor, stimulation of the production of oxidative
products, the production of neurotoxic products, inhibition of functional enzymes, and the induction
of apoptosis or programmed cell death.
 Sulfurcontaining amino acids in the CNS: Homocysteine 4 83

Homocysteine administered to rodents induces convulsions in a manner analogous to those observed

in animals treated with excitatory amino acids (Freed et al., 1980). Local application of homocysteine to rat
CNS neurons resulted in an excitatory response similar to that observed with the administration of
glutamate (Wuerthele et al., 1982). These investigations surmised that insufficient time or oxidation
occurred in their experimental paradigm, for the effect to be explained by metabolism or chemical
oxidation to the glutamate analogue, homocysteic acid (Wuerthele et al., 1982). The effect of homocysteine
was blocked by the methyl donor, betaine (permitting an alternative methylation pathway), further
suggesting the important role of remethylation in protecting against the adverse effects of homocysteine.
The effects of homocysteine were blocked by the glutamate antagonist, glutamate diethylester, which
suggested that homocysteine may act at the NMDA receptor. The potential direct interaction of homocys-
teine with the NMDA receptor was demonstrated in cultured cortical neurons. Homocysteine appeared to
directly act as an agonist at the NMDA site, and possible metabolism to other excitotoxic analogues was
ruled out in these experiments (Lipton et al., 1997).
A number of experiments have implicated homocysteine in the induction of oxidative stress as a
mechanism of action. Studies of transgenic mice expressing a human Cu/Zn superoxide dismutase
mutation as a model of amyotrophic lateral sclerosis documented increased homocysteine immunoreac-
tivity in the brain, especially the hippocampus (Chung et al., 2003). While the actual role of superoxide
dismutase mutants in familial amyotrophic lateral sclerosis is unknown, its association with this disease
(AlChalabi and Leigh, 2000) implicates oxidative mechanisms and, thus, implies that homocysteine may
be a component of the oxidative damage. Homocysteine also potentiates toxicity in primary neuronal
cultures induced by copper and amyloid b peptide (potential causative agents in the development of
Alzheimers disease) (White et al., 2001). In these latter experiments the toxicity of the combination of
amyloidb, copper, and homocysteine was particularly notable but could be blocked by the presence
of catalase. These investigators also found that homocysteine was associated with H2O2 production,
which would explain why catalase could ameliorate this oxidative effect as this enzyme catalyzes the
catabolism of H2O2 after its formation from oxygen radicals catalyzed by superoxide dismutase (White
et al., 2001). In vitro experiments using homogenates of rat hippocampus have shown that homocysteine
can increase thiobarbituricreactive substances (markers of oxidative stress) and reduce the radicaltrapping
potential of this tissue, although antioxidant protective enzymes were not affected (Streck et al., 2003).
Thus, homocysteine does appear to increase oxidative stress in the CNS.
The role of the excitatory amino acid product, homocysteic acid, in insults observed in the CNS related
to the presence of homocysteine is unclear. Data that indicate homocysteine exerts its neurotoxic effect via
the NMDAglutamate receptor (Kim and Pae, 1996) would suggest that perhaps conversion of homocyste-
ine to homocysteic acid may be important. Homocysteic acid has been identified in the rat CNS, in
amounts roughly a 1000fold less than glutamate (Kilpatrick and Mozley, 1986). However, as noted above,
the effects associated with homocysteine may occur more quickly than could be accounted for by
conversion to its acidic metabolite (Wuerthele et al., 1982). In addition, the formation of homocysteic
acid in vivo has remained somewhat controversial (Waller et al., 1991) and its possible association with
increased homocysteine unclear (FritzerSzekeres et al., 1998).
Several investigations by the same laboratory have presented evidence that homocysteine may exert its
effects via inhibition of the Na/KATPase in brain, an enzyme important for the generation and
maintenance of neuronal membrane potentials in the CNS (Streck et al., 2002a, b; Wyse et al., 2002).
These studies demonstrated an in vitro inhibition of Na/KATPase but not Mg2ATPase by homocyste-
ine and methionine but not by cysteine (Streck et al., 2002a). In vivo studies had similar results and also
implicated homocysteine in antioxidant effects (Streck et al., 2002b; Wyse et al., 2002).
Homocysteine may also exert its toxic effect via induction of programmed cell death. Studies in
cultured human umbilical vein endothelial cells found that homocysteine induced an apoptotic type of
cell death (marked by decreased cell viability, nuclear condensation, and caspase3dependent cell death)
(Lee et al., 2005). The action of homocysteine appeared to be via induction of reactive oxygen species
(it could be inhibited by superoxide dismutase plus catalase) and regulated by nitric oxide (Lee et al., 2005).
An investigation in rat embryonic primary hippocampal cell cultures also documented the promotion of
apoptosis by homocysteine (Kruman et al., 2000). These investigators suggested that the sequence of events
84 4 Sulfurcontaining amino acids in the CNS: Homocysteine

initiated by homocysteine are: DNA damage, polyADPribose polymerase (PARP) activation, caspase
activation, and activation of the tumor suppressor protein p53, followed by a decline in mitochondrial
potential and finally nuclear disintegration (Kruman et al., 2000). These investigators also concluded that
homocysteine may sensitize hippocampal neurons to the neurotoxic effects of excitatory amino acids such
as glutamate (Kruman et al., 2000). Thus, there are a number of different possible pathways by which
homocysteine may exert its toxic effects, with oxidation and interaction with the NMDA receptor being
particularly interesting potential mechanisms.

6 Methodological Issues

The measurement of homocysteine has been an issue because of its ability to bind to both proteins and
other sulfurcontaining moieties via disulfide bonds. The proteinbound pool of homocysteine was first
described by Kang and coworkers (1979) and further characterized by Malloy and coworkers (1981a, b).
Homocysteine (in a manner similar to that of both cysteine and glutathione) exists as the sulfhydryl, the
homogeneous disulfidehomocystine, the mixed disulfide (with cysteine or glutathione), or as the protein
homocysteine disulfide (Rassin, 1996) (> Table 4-1). Indeed, the complexity of representing the multiple
forms of homocysteine has been addressed in a paper specific to the problem (Mudd et al., 2000). Most of

. Table 4-1
The forms of homocysteine
Form Name Abbreviation/Brief description
Reduced (thiol, sulfhydryl) Homocysteine HcySH
Oxidized (disulfide) Homocystine HcySSHcy
Mixed disulfide Mixed disulfide RSSHcy
(R cysteine, glutathione, protein)
Protein bound (via disulfide bond) Protein bound ProteinSSHcy
Sum of all but protein bound Free homocysteine (Free) f Hcy
Sum of all forms Total homocysteine (Total) t Hcy

The various forms of homocysteine found in biology. Adapted from Rassin (1996) and Mudd et al. (2000)

the published literature addressing the role of homocysteine as a risk factor has presented the data in terms
of total homocysteine (usually measured by treatment of samples with reducing agents and then determin-
ing the total amount of the sulfhydryl form produced). However, it is not unreasonable to suggest that
in vivo the free homocysteine is most available for transfer into the CNS and that pool, in equilibrium with
that bound to proteins and other sulfhydryls, may determine the degree of damage that results from
increased homocysteine. Also, homocysteine in blood and urine usually is present in the disulfide form,
homocystine, which may alter its transport into tissues. Thus, homocysteine availability may be modified
by its chemical form and by changes in the amounts of plasma proteins (particularly albumin), cysteine
(with which homocysteine competes for protein binding), and other sulfhydrylcontaining compounds
such as glutathione (Malloy et al., 1981a, b).
A second methodological issue relates to the amount of homocysteine that circulates, and the changes
that have been associated with its role as an increased risk factor. In five typical patients with cystathionine
bsynthaserelated homocystinuria, their total plasma homocysteines (the sum of free homocysteine,
proteinbound homocysteine, and mixed disulfide homocysteine) ranged from 101186 mmole/L (Malloy
et al., 1981a). In most of the epidemiologic studies of homocysteine as a risk factor, plasma homocysteine is
in the range of 920 mmole/L, and often a statistically significant result will be obtained when a 10%
reduction or increase is observedin the order of 1 mmol/L. For example a reduction from 11.4
3.4
(mean
SD) to 9.7
2.3 mmol/L, an 11.0% change was very statistically significant (p < 0.001) in a study
 Sulfurcontaining amino acids in the CNS: Homocysteine 4 85

of the effect of folic acid supplementation in cereal on blood homocysteine (Malinow et al., 1998). The
amounts of homocysteine observed in patients with Alzheimers disease were 13.2
4.0 mmole/L in
controls, 15.3
8.4 mmole/L in clinically diagnosed and 16.3
7.4 mmole/L in histologically confirmed
patients (Clarke et al., 1998). Some investigators have addressed the difficulty of assessing these quantita-
tively small changes by analyzing their data in terms of quartiles (McLean et al., 2004) or setting a possible
abnormal limit, for example, total plasma homocysteine >13 mmole/L (Jacques et al., 1999). From a
population perspective, monitoring plasma total homocysteine may be very useful but determining an
individuals risk factor in the light of these data may be considerably more troublesome; for example, how
would one interpret a blood total homocysteine of 10.0 or 11.0 or 12.0 mmole/Ldo any of these
concentrations represent a specific risk?

7 Conclusion

The sulfur amino acid metabolic pathway plays an important role in supporting a variety of functions
within the CNS. Homocysteine sits at an important branch point in this pathway and disturbances of its
equilibrium appear to be related to detrimental outcomes for the CNS. A number of questions remain to be
answered regarding the role of this amino acid in the CNS.
1. What is the amount of plasma homocysteine that represents safety for the CNS?
2. How should homocysteine be best determined and expressed?
3. Does increased plasma homocysteine reflect increased brain homocysteine?
4. What is the most appropriate milieu relative to the availability of vitamins B6, B12, and folic acid?
5. What is the role of homocysteic acid in the toxicity of homocysteine?
6. Does the MTHFR677C ! T polymorphism determine the homocysteine risk factor?
7. Does homocysteine play a common role in the pathway leading to the degenerative processes observed
in diseases such as Alzheimers, amyotrophic lateral sclerosis, atherosclerosis, and in the natural aging
process?

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5 Stress Response Signal
Transduction
Xiaoming Hu . J. R. PerezPolo

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

2 Homeostasis: Stress, Inflammation, and Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

3 Inflammatory Responses: Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

4 Cytokine Responses and Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

5 Transcription Factors: NFkB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

6 Inflammatory Gene Expression: COX2 and iNOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

7 Decoy Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

# 2008 Springer ScienceBusiness Media, LLC.

90 5 Stress response signal transduction

Abstract: Trauma evokes a common response in all organ systems that includes early energy depletion
followed by dysfunction of ionic gradients and triggering of stress response mechanisms with outcomes
ranging from recovery of function to delayed cell death and further dysfunction. The specific outcome to
trauma in terms of this spectrum is determined to some extent by the specific signaling mechanisms
triggered. In the central nervous system (CNS), stress response signaling includes changes in transcription
of cytokines and chemokines and also inflammatory enzymes. A common regulatory component in all of
these is the nuclear factor kappa B (NF-B) transcription factor. Interventions interfering with NF-B
activation can affect the outcome to CNS trauma.
List of Abbreviations: CNS, central nervous system; COX2, cyclooxygenase2; iNOS, inducible nitric
oxide synthase; ICE, IL1converting enzyme; IL1, interleukin1; IL1R, IL1 receptor; IL1Ra, IL1-
Rantagonist; IL1RAacP, IL1R accessory protein; LPS, lipopolysaccharides; MCAO, middle cerebral arterial
occlusion; MnSOD, manganese superoxide dismutase; RPA, ribonuclease protection assay; RTPCR, real
time polymerase chain reaction; TNF, tumor necrosis factor

1 Introduction
Classical stress response studies dealt with hormonal changes in the face of abrupt environmental stimuli
and their effects on physiological processes: circulation, temperature regulation, motor activity, and so on.
This was followed by inclusion of immune responses as part of the physiological stress responses, typically
involving assessments of cytokine expression changes in the central nervous system (CNS) and associated
growth factors. At about this time, quantitative molecular approaches began to take the place of cellular
assays based on migration and inflammatory events at the organismic level. In addition, the role of cytokine
expression changes in the CNS in response to more traumatic and acute events, typically related to abrupt
physical trauma and ischemia, became more common even as various experimental and animal models of
head trauma, stroke, perinatal ischemia, and spinal cord injury were developed and characterized. Here, we
focus on some of the more common molecular components of stress in the CNS in the acute and chronic
situation. By necessity, this is not a definitive treatment but rather an attempt to integrate some mechan-
isms of responses to trauma in CNS into a cohesive, albeit complex, conceptual framework.

2 Homeostasis: Stress, Inflammation, and Recovery

Cellular stress responses are best characterized by an early stage of energy depletion inactivating membrane
bound pumps responsible for the maintenance of transmembrane gradients of an assortment of ions and
signaling molecules. This departure from cellular homeostasis triggers an inflammatory response that, if
sufficient energy stores are available, triggers active secondary cell death with apoptotic features. Cellular
apoptosis processes include chromatin condensation and subsequent margination against the nuclear
envelope, cytoplasmic and nuclear condensation, overall cell shrinkage, cytoplasmic vacuolization, and
convolution of the nuclear and the cytoplasmic membranes followed by the formation of apoptotic bodies.
These apoptotic bodies then undergo phagocytosis. Under conditions in which the initial disruption of
cellular processes is sufficiently robust, the cell death process displays necrotic features, characterized by
overall cell swelling, chromatin clumping, and organelle disruption, with early loss of membrane integrity
(Hockenbery, 1995; Majno and Joris, 1995).
For example, necrotic cell death is typically an early event at the core of the traumatic insult to the CNS
in the case of hypoxia ischemia (HI) (Ray et al., 2003). Delayed cell death with apoptotic features usually
starts between 12 and 24 h later at the core site and spreads over time to sites away from the impacted area
and includes apoptosis of neurons and glia (Northington et al., 2001). In adult animal models, both
apoptosis and necrosis have been reported following focal cerebral ischemia in rats and mice (Charriaut
Marlangue et al., 1996; Dux et al., 1996; Chen et al., 1997; Matsushita et al., 1998; Guegan and Sola, 2000)
and after global cerebral ischemia (Nitatori et al., 1995; Ni et al., 1998), often emphasizing delayed neuronal
death in the selectively vulnerable regions of the hippocampus.
 Stress response signal transduction 5 91

3 Inflammatory Responses: Cytokines

Inflammatory responses have also been implicated in chronic glial activation in the aged rodent brain, in
rodent models of adult spinal cord injury and stroke, head injury, as well as hypoxia ischemia (Yang et al.,
1995; Hagberg et al., 1996; Bona et al., 1999; Nesic et al., 2001; Rossner et al., 2001; Chu et al., 2002; Hedtjarn
et al., 2002; Nesic et al., 2002; ToliverKinsky et al., 2002; Cole and PerezPolo, 2004; Hu et al., 2005; Rossner
et al., 2005). Typically, CNS trauma increases glial expression of the proinflammatory cytokines: interleukin
1 (IL1)b, tumor necrosis factora/b (TNFa/b), and IL6 (FosterBarber et al., 2001; Lau and Yu, 2001; Nesic
et al., 2001, 2002; Saliba and Henrot, 2001; Hu et al., 2005).
The bestcharacterized early response inflammatory cytokine is IL1 (Szaflarski et al., 1995). The IL1
family includes the agonists IL1a and IL1b, the endogenous receptor antagonist IL1 receptor antagonist
(IL1Ra), and IL18/IL1g (Shapiro et al., 1998). The role of IL1b in CNS trauma is probably the best
understood (Rothwell et al., 1997; Nesic et al., 2001; Hu et al., 2005). There are reports showing that IL1b
mRNA and protein levels increase as early as 1 h after brain injury and persist during the development
of infarction in various adult animal models (Minami et al., 1992; Zhang et al., 1998a, b; Davies et al.,
1999). Sources of IL1 in CNS are endothelial cells, microglia, and macrophages or monocytes (Buttini
et al., 1994) although neurons, astrocytes, and oligodendrocytes can also secrete IL1b under traumatic
conditions (Sairanen et al., 1997). Levels of IL1a in CNS have been shown to also increase promptly and
robustly after temporary or permanent occlusion of the middle cerebral artery in mice (Hill et al., 1999;
Touzani et al., 1999).
Following synthesis, proIL1b (31 KD) remains primarily in the cytoplasm until cleaved by the IL1b
converting enzyme (ICE, caspase1), producing mature and active IL1b (17 KD). Caspase1 also cleaves
and activates IL18 but not IL1a (Dinarello, 1999). There is evidence showing correlation between
increased IL1b levels and subsequent neurodegeneration. Thus, administration of exogenous IL1b
markedly exacerbates neuronal or glial damage in rodents exposed to focal cerebral ischemia or excitotoxin
administration (Lawrence et al., 1998; Stroemer and Rothwell, 1998). Decreasing effective IL1b levels using
antiIL1b antibodies (Touzani et al., 1999) or inhibitors of ICE (Loddick and Rothwell, 1996; Hara et al.,
1997) decreases neuronal losses after cerebral ischemia. IL1Ra is widely used to study the function of IL1
because it is a selective endogenous receptor antagonist that blocks the actions of IL1a and IL1b
(Dinarello et al., 1998). Injection or overexpression of IL1Ra significantly inhibits cell death after hypoxia
ischemia although the mechanisms involved were not determined (Betz et al., 1995; Stroemer and Rothwell,
1997, 1998; Yang et al., 1998).

4 Cytokine Responses and Cell Survival

The responses to IL1a and IL1b are initiated by binding to IL1 typeI receptor (IL1RI), which then
associates with an IL1 receptor accessory protein (IL1RAcP) (Wesche et al., 1997), leading to signal
transduction (Cao et al., 1996; Muzio et al., 1997). IL1RIs are expressed on both glia and neurons (French
et al., 1999; Pinteaux et al., 2002). Ligand binding of IL1, in situ hybridization, and immunohistochemical
studies have shown the expression of IL1RI receptors in specific brain regions in rats, particularly, the
hippocampus, the dentate gyrus, the choroid plexus, and the cerebellum. A second receptor IL1 typeII
receptor (IL1RII) has a much shorter cytoplasmic domain and is thought to act as a decoy (nonsignaling)
receptor (McMahan et al., 1991). The activation of IL1RI stimulates ubiquitous transcription factors
(NFkB, AP1, AFP) and some key intracellular signaling molecules (JNK, IP3, or PKC kinases; Auron,
1998; ONeill and Greene, 1998). Based on in vitro studies, it has been suggested that activation of the
NFkB pathway through IL1RI binding after ischemia results in gene transcription with deleterious
outcomes (Dunn et al., 2002).
For hypoxia ischemia, it has been shown that IL1 mRNA and protein levels increase as early as 1 h after
the injury and persist during the development of infarction (Minami et al., 1992; Szaflarski et al., 1995;
Hagberg et al., 1996; Zhang et al., 1998a, b; Davies et al., 1999; Qiu et al., 2004). Regional differences in IL
1b distribution patterns have been observed in normal rodent brain (Ilyin et al., 1998; Vitkovic et al., 2000);
92 5 Stress response signal transduction

for example, there are lower levels of IL1b in hippocampus compared with cortex in shamtreated animals
(Hu et al., 2005). This variation might reflect endogenous neuromodulatory activities in an untreated rat.
It is reported that the hippocampus is more sensitive to hypoxia ischemia injury than other brain
structures (CervosNavarro and Diemer, 1991; Grafe et al., 1994; Yue et al., 1997). The time course of IL1b
protein levels correlates well with the time course of cell death level and caspase3 activity observed after
injury. Thus, it would be reasonable to speculate that the cell deathinducing threshold of IL1 is lower in
hippocampus than that in the cortex, which explains the lower level of IL1b after hypoxia ischemia in
hippocampus. This is consistent with there being temporal and spatial differences in IL1b protein
expression after HI, not surprising given similar reports of spatial temporal differences between hippocam-
pus and basal forebrain cell death mechanisms in an adult rodent hypoxia model (Qiu et al., 2001). Thus, it is
likely that there are different mechanisms of stress response signal transduction at the level of IL1 signaling
in different brain regions. This could also explain some of the particular aspects of longterm outcomes in the
clinical context and the reported fragility of hippocampus, for example, over other brain regions.
Using the techniques of in situ hybridization, ribonuclease protection assay (RPA), and realtime
polymerase chain reaction (RTPCR), it has been reported that IL1RI mRNA was expressed in high levels
in cortex (Gayle et al., 1997; Wang et al., 1997) and in low to moderate levels in hippocampus (Wong and
Licinio, 1994). At the cellular level, IL1RI mRNA has been localized in cerebrovascular endothelium,
astrocytic and oligodendroglial (Wong and Licinio, 1994; Hu et al., 2005), as well as neuronal components
(Takao et al., 1990; Wong and Licinio, 1994; Hu et al., 2005).
Significantly protective effects of IL1Ra have been documented in an adult stroke rat model, where cell
death after stroke was significantly reduced when IL1Ra was administered as a single injection at the time
of middle cerebral artery occlusion and hypoxia ischemia (Mulcahy et al., 2003; Hu et al., 2005). The maximal
cortical protection occurred after intracerebroventricular injection of IL1Ra 2 h post middle cerebral
arterial occlusion (MCAO) (Mulcahy et al., 2003). Protective effects of IL1Ra can last for at least 7 days
after injury (Garcia et al., 1995; Liu et al., 1999; Loddick and Rothwell, 1996). Injections of L1Ra 2 h after
hypoxia ischemia injury markedly decreased hippocampal cell death levels and caspase3 activity when
measured 24 h after injury, in support of the hypothesis that IL1 contributes to hypoxia ischemiainduced
cell death.

5 Transcription Factors: NFkB

The transcription factor NFkB consists of homo or heterodimers of subunits, which constitute a family of
related proteins, including p50, p52 (also called p49), p65 (also called RelA), CRel, and RelB (> Figure 51).
All of them share a highly conserved
300 residue NH2terminal domain for DNA binding or dimeriza-
tion (Rel homology domain), which enables them to form dimers and bind to an array of homologous
decanucleotide sequences with varying affinities. NFkB/Rel proteins can be divided into two classes based
on their Cterminal domain. One class includes p65, CRel, and RelB proteins. All of them contain C
terminal transactivation domain. Another class includes p50 and p52, which have no transactivation
domain at the C terminus (Bours et al., 1990, 1992; Ghosh and Baltimore, 1990; Nolan et al., 1991;
Ruben et al., 1991; Schmid et al., 1991; Ryseck et al., 1992). p50 and p52 are first synthesized as precursors
p105 and p100, respectively, both of which have ankyrinlike repeats with close homology to that of IkB
family in their C terminus. After degradation of the Cterminal region, mature p50 and p52 are released.
Dimerization of NFkB subunits produces species with various intrinsic DNAbinding specificities, trans-
activation properties, and subcellular localization (Baeuerle, 1991; Siebenlist et al., 1994). NFkB dimeric
combinations must contain at least one of the transactivating members (p65, CRel, or RelB) to have the
ability to activate DNA transcription (Baeuerle, 1991; Lenardo and Siebenlist, 1994; Siebenlist et al., 1994;
Chen et al., 1998). The most commonly described active NFkB subunit combinations are the p65/p50 and
CRel/p50 heterodimer. Homodimeric combinations of p50 or p52 have no transactivation activity and
often behave in vivo as transcriptional inhibitors at high affinitybinding sites (Franzoso et al., 1992, 1993;
Kang et al., 1992).
 Stress response signal transduction 5 93

. Figure 51
Schematic diagram illustrating possible effects of traumainduced IL1 increases on NFkB activation. IL1
stimulates degradation of IkBa and increased nuclear levels of Bcl3. IkBa degradation enables nuclear
translocation of p65/p50. Nuclear Bcl3 binds and dissociates p50 homodimers from cognate NFkBbinding
sites enabling p65/p50 dimers to bind and activate target genes

A mechanism underlying IL1induced cell death involves the activation of the transcription factor
NFkB and the subsequent induction of multiple genes. NFkB is constitutively expressed in CNS at low
levels (Kaltschmidt et al., 1994), but is stimulated above basal levels by stimuli associated with ischemic
insults. In the rodent brain, NFkB activation is stimulated in the forebrain and the hippocampus after
hypoxia ischemia (Koong et al., 1994; Schmidt et al., 1995; Yang et al., 1995; Gabriel et al., 1999; Nurmi
et al., 2004). In most of these studies, the expression and activity of p65/p50 was measured and used as a
representative for NFkB activity, which overlooks changes in other NFkB proteins. For example, NFkB
CRel/p50 selectively binds the Bclx gene promoter CS4 sequence while p65/p50 preferentially binds the
IgGkB promoter sequence (Hu et al., 2005).
NFkB regulates the expression of antiapoptotic genes such as BclxL, Bcl2, Manganese superoxide
dismutase (MnSOD), and inhibitorofapoptosis proteins, and proinflammatory genes such as IL1, TNFa,
matrix metalloproteinase9, cyclooxygenase2 (COX2), and inducible nitric oxide synthase (iNOS), thereby
playing a dual role in neuronal survival (Mattson and Camandola, 2001). Given that the p65/p50 dimer
activates genes coding for proteins with proinflammatory properties, while CRel/p50 dimer activates genes
coding for BclxL protein that prevent cell death (Qiu et al., 2001; Pizzi et al., 2002), we believe that some of
the present confusions in the field as to the proapoptotic or antiapoptotic nature of NFkB activities in in
vivo lesion paradigms result from a lack of data addressing activation of specific promoters by different
94 5 Stress response signal transduction

NFkB dimer species. The activation of different NFkB dimers under different stimuli and at different time
points may be responsible for the differential beneficial or detrimental outcomes in cell survival.
NFkB is expressed in the nervous system and shows a low level of constitutive activity in neurons
(Kaltschmidt et al., 1994). NFkB is induced above constitutive levels in the brain by various stimuli, most
of which are stressful or neurotoxic. For example, rodent NFkB is induced in the forebrain and the
hippocampus following hypoxia ischemia (Koong et al., 1994; Schmidt et al., 1995; Yang et al., 1995; Qiu
et al., 2001), in the cortex after traumatic brain injury (Yang et al., 1995), and in the limbic structures
following seizures (Rong and Baudry, 1996). NFkB is reported to regulate cell death by modulating a
diverse array of genes, known to be important for cell death or survival, such as Bcl2, BclxL, MnSOD,
iNOS, COX2, and some inflammatory cytokines (IL1a/b, TNFa/b) (FosterBarber et al., 2001; Lau and
Yu, 2001; Saliba and Henrot, 2001).
The extent to which NFkB activation contributes to neuropathology versus neuroprotection and
recovery remains unresolved. In a number of experimental models, including cell lines and tissues under
different stimuli, NFkB activation appears to result in both apoptotic and antiapoptotic outcomes
(Abbadie et al., 1993; Barger et al., 1995; Jung et al., 1995; Barger and Mattson, 1996; Grilli et al., 1996;
Grimm et al., 1996; Baichwal and Baeuerle, 1997; Lipton, 1997; Grilli and Memo, 1999). It is possible that
the different compositions of NFkB protein dimers determine the outcomes of their activation. For
example, the p65/p50 dimer may activate genes coding for proteins with proapoptotic properties whereas
CRel/p50 dimer may activate genes coding for proteins that prevent cell death (Qiu et al., 2001; Pizzi et al.,
2002; Hu et al., 2005). Therefore, different stimuli might activate different NFkB dimers, resulting in
beneficial or detrimental outcomes.
There is a generic NFkB DNA sequence consisting of a 10bp consensus sequence that encompasses 128
different decameric sequences, 50 GGGRNNTYCC30 (Gguanine, Rpurine, Nany nucleotide, Y
pyrimidine, Ccytosine, Tthymine). Distinct NFkB subunit combinations exhibit different binding
affinities for the multiple consensus sequence combinations, and this complex variation likely determines
the specificity of NFkB transcriptional regulation (Perkins et al., 1992; Qiu et al., 2001). For example,
p65/p50 heterodimers bind preferentially to the decameric sequence present on the IgGkB promoter
(50 GGGACTTTCC30 ) and the CRel/p50 heterodimer binds preferentially to the decameric sequence 50
GGGGTCTCC30 present on the Bclx promoter (Qiu et al., 2001). Specific combinations of NFkB proteins
can thus distinguish among the various kB sites to selectively regulate gene expression.
Nadjar et al. (2003) documented the activation of NFkB by the visualization of p65 translocation in the
cells of adult rat brain after intraperitoneal or i.c.v. IL1b injection. IL1RIdeficient mice demonstrated
impaired NFkB activation upon the stimulation of IL1b. Huang et al. (2003) documented that decreased
NFkB activity was observed in ischemic ICE null adult mice and suggested that IL1 signaling contributes
to NFkB activation and subsequent ischemic damage.
One of the most important features of NFkB proteins is that the function of NFkB is strictly regulated
by its subcellular localization. Its activation depends on the translocation from the cytoplasm to the
nucleus. The movement of NFkB proteins into the nucleus and binding to DNA is controlled by a family
of inhibitor proteins called IkB (Zabel and Baeuerle, 1990; Haskill et al., 1991; Thompson et al., 1995).
There are several inhibitory IkB proteins (IkBa, IkBb, IkBg, and Bcl3, > Figure 51) that regulate NFkB
activity. These proteins have several homologous amino acid stretches known as ankyrin repeats that
specifically interact with NFkB/Rel proteins (Bours et al., 1992; Grilli et al., 1993). IkBa and Bcl3 are the
two most important proteins in this family. They contribute to the regulation of NFkB activity via different
mechanisms.
IkBa takes effect in the cytoplasm, where they bind to NFkB heterodimers and inhibit their action. In
response to stimuli, including IL1, TNFa, and bacterial lipopolysaccharides (LPS), IkB is phosphorylated
by an IkB kinase, ubiquinated and degraded by proteasomes (Ghosh and Baltimore, 1990; Liu et al., 1993;
Israel, 1995), uncovering masked nuclear localization signals on the NFkB dimers, which are then
translocated to the nucleus, bind to DNA consensus sequences on gene promoters, and activate transcrip-
tion (Ghosh et al., 1998). In particular, it has been shown that IkBa must be phosphorylated on Ser32
and Ser36 and ubiquitinated at Lys21 and Lys22 so that it can be targeted for subsequent degradation by
the ubiquitin and proteasome system (Traenckner et al., 1995; Baldwin, 1996). Interestingly, only the NFkB
 Stress response signal transduction 5 95

dimers that contain at least one transactivation member (p65, CRel, or RelB) are effectively regulated by
IkBa. Thus, the p52 and p50 homodimers are not usually retained in the cytoplasm by IkBa. Their activity is
regulated within the nucleus by Bcl3.
Although structurally homologous, Bcl3 and IkBs act differently in regulating NFkB activity. Whereas
the IkBs are primarily cytoplasmic, Bcl3 is predominantly nuclear bound and has a binding specificity for
p52 or p50 homodimer (Bours et al., 1993; Franzoso et al., 1993; Fujita et al., 1993; Nolan et al., 1993;
Bundy and McKeithan, 1997). There are two mechanisms by which Bcl3 can regulate the NFkB activity.
First, Bcl3 can successfully compete with promoter DNAbound p50 or p52 homodimers, removing the
NFkB homodimers away from cognatebinding sites on target promoter DNA sites (Franzoso et al., 1993;
Nolan et al., 1993; Siebenlist et al., 1994; Zhang et al., 1998a, b), allowing p65/p50 or CRel/p65 to bind and
activate gene transcription. Second, Bcl3, together with p52 or p50 homodimer, can bind to DNA kB site
and form a ternary complex.
The tethering of Bcl3 to DNA via p52 or p50 homodimer allows the activation of gene transcription,
whereas Bcl3 and p50/p52 homodimers alone cannot since Bcl3 has only the transactivation domain and
p50/p52 has only the DNAbinding domain (Bours et al., 1993; Fujita et al., 1993; Rocha et al., 2003). The
different regulatory effects of Bcl3 may depend on its phosphorylation states and concentration in the
nucleus (Fujita et al., 1993; Nolan et al., 1993; Bundy and McKeithan, 1997; Cogswell et al., 2000).
NFkB transactivation involves IkBa and Bcl3 proteins. IkBa phosphorylation at Ser32 and Ser36
(Traenckner et al., 1995) is a requirement for its ubiquitination and subsequent degradation by the 26 S
proteasome, an important step in the translocation of NFkB from the cytoplasm to the nucleus. An in vitro
study of IL1bchallenged C6 (astrocytoma) cells showed newly synthesized IkBa to be phosphorylated at
Ser32 (Uehara et al., 1999), which may explain the increase of phosphorylated IkBa 24 h after hypoxia
ischemia (Hu et al., 2005). Increased nuclear Bcl3 levels also stimulate NFkB activation. Western blot
analyses have shown an increase in translocation of Bcl3 from cytoplasm to nucleus 24 h after hypoxia
ischemia in hippocampus and cortex (Hu et al., 2005). A report based on DNA microarray analyses and RT
PCR by Elliott et al. (2002) using cultured SW1353 cells also shows Bcl3 transcription to be stimulated by
IL1b signaling. It is reported that NFkB is stimulated by hypoxia via increased IkBa degradation and NFk
B nuclear translocation. A small number of researches indicated that an independent pathway involving
Bcl3 stimulation also might contribute to the NFkB activation after hypoxia (Gozal et al., 1998; Zhang
et al., 1998a, b; Qiu et al., 2001).

6 Inflammatory Gene Expression: COX2 and iNOS

There is evidence that nitric oxide (NO) is an important mediator of ischemic and neurodegenerative
pathology in the CNS (Hara et al., 1996; Mizushima et al., 2002; Brown and BalPrice, 2003). NO is
endogenously produced as a byproduct of arginine metabolism by different nitric oxide synthase (NOS)
isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Both nNOS and
eNOS are constitutively expressed, whereas iNOS is expressed in response to a variety of stimuli. There are
reports documenting the relationship between iNOS induction and ischemic lesions in the brain (Hara
et al., 1996; Mizushima et al., 2002). In addition, the administration of IL1b to cultured brain endothelial cells
and hippocampal neurons induces the expression of iNOS mRNA and increases NO release (Bonmann et al.,
1997; Serou et al., 1999). These observations, together with evidence showing that induction of iNOS is
regulated by NFkB p65/p50 (Teng et al., 2002), also support the hypothesis that activation of p65/p50 by IL1
after CNS trauma may further stimulate iNOS synthesis and NO formation, which in turn triggers cell death.
COX is an important enzyme in the inflammatory process. COX catalyzes the ratelimiting step in the
conversion of arachidonic acid to prostaglandins. There are two isoforms of COX, designated COX1 and
COX2. COX1 is expressed constitutively and appears to be responsible for ongoing physiological function,
whereas COX2 is present only in certain tissues where it is transiently induced by growth factors,
inflammatory cytokines, tumor promoters, and bacterial toxins (Chun et al., 2004). There is evidence to
support an involvement of COX2 in brain damage (Hara et al., 1998; Nagayama et al., 1999; Sugimoto and
Iadecola, 2003). IL1b transcriptional control of COX2 by NFkB p65/p50 has been demonstrated in cell
96 5 Stress response signal transduction

culture (Newton et al., 1997). There is a significant increase in iNOS and COX2 expression in the
hippocampus and the cortex 24 h after hypoxia ischemia (Hu et al., 2003). Treatment with IL1Ra reversed
the HIinduced increases in iNOS and COX2, a novel finding. Interestingly, sequence analyses of the iNOS
and COX2 promoters have shown the presence of DNAbinding consensus sequences specific to the p65/
p50 binding (Hu et al., 2005).

7 Decoy Treatments

Sequencespecific inhibition of NFkB can be accomplished with synthetic doublestranded (ds) phos-
phothiorate oligonucleotides containing a NFkB consensus sequence, which acts in vivo as a decoy cis
element to bind transcription factors and block the activation of cognate genes (Morishita et al., 1997; Qiu
et al., 2004; Tomita et al., 1998, 2000, 2001). The principle of the transcription factor decoy approach is
simply the inhibition of promoter activity due to the binding of endogenous transcription factors to
saturating amounts of exogenous doublestranded oligonucleotides displaying specific DNA sequences that
are present in the promoter region of the gene of interest. For example, an IgGkB decoy, which contains the
NFkB consensus sequence in the IgGkB promoter, has been used to inhibit p65/p50 activity (Blondeau
et al., 2001). However, there is no information about decoys targeted to other NFkB dimers.
The pharmacokinetic profiles of phosphothiorated oligonucleotides of varying base composition in rat
are similar (Agrawal and Zhao, 1998). The stereotaxic injection of fluorescent IgGkB decoys into brain
results in the cellular incorporation of decoys in hippocampi as early as 2 h after injection, with dissipation
taking place by 7 h. The ability to selectively intervene in the binding of different NFkBbinding consensus
sequences to cognate NFkB protein dimers during a fairly short time period should allow for the
determination of the role of NFkB in the regulation of specific genes at specific times after a traumatic
insult to the CNS. It has been proposed that transcriptional injury responses are biphasic and that there are
different consequences to delayed transcriptional activation or to persistent versus transient transcriptional
factor activation of the NFkB transcription factor. The ability to abolish NFkB binding to selective
promoter sites in a transient fashion should provide a useful tool in establishing the role of different
traumainduced pathways in cellular commitment to cell death and survival.
Since IgGkB decoy can specifically inhibit p65/p50binding activity, it is not surprising that it can
inhibit the transcription of inflammatory cytokines, which are the target genes for NFkB p65/p50 (Hu
et al., 2005). These results are consistent with recent reports showing antiapoptotic features to the
stimulation of CRel and proapoptotic features to the stimulation of p65 DNA binding (Qiu et al., 2001;
Pizzi et al., 2002). These results are also consistent with the CRelmediated regulation of Bclx gene
transcription in response to hypoxia ischemia (Qiu et al., 2001, 2004). Genes that were significantly
downregulated by IgGkB decoys were not affected by Bclx decoys, and vice versa.

8 Conclusion
Following injury to the CNS, there is a cascade of molecular and cellular responses that determine outcome
in terms of cell survival and function. One important pathway activated by CNS trauma is via induction of
IL1, which binds to cognate receptors and activates selective gene expression of genes such as COX2 and
iNOS via the transcription factor NFkB. The aim here was to describe the activation mechanisms by which
IL1 contributes to cell death.
We also discuss the effects of selective interventions in (i) IL1 receptor binding or (ii) injuryinduced
p65/p50 NFkB activation. The absence of side effects of IL1Ra and its safety in clinical trials in rheumatoid
arthritis would suggest that this intervention may be applicable to perinatal ischemia. Decoys to NFkB
binding DNA consensus sequences are useful to determine regulatory features affecting individual genes.
Use of decoy treatments to alter physiological and pathological outcomes to trauma requires concerted
pulsed treatments with cocktail of different decoy sequences that take into account the large number of
genes displaying NFkBbinding sites in their promoters.

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6 Aging and Oxidative Stress
Response in the CNS
V. Calabrese . D. A. Butterfield . A. M. Giuffrida Stella

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

2 The FreeRadical Hypothesis of Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

2.1 The Mitochondrial Theory of Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
2.2 Mitochondrial Damage, Reactive Nitrogen Species, and Neurodegenerative Disorders . . . . . . . . 110

3 Oxidative Stress and Brain Stress Tolerance: Role of Vitagenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

3.1 Neurogasobiology of Nitric Oxide and Carbon Monoxide: Two Molecules That Promote
Adaptive Responses in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
3.2 Nitric Oxide Synthase and Its Isoforms in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
3.3 Nitric Oxide as a Neurotransmitter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
3.4 Redox Activities Elicited by NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.5 Regulation of Gene Expression by Oxidative and Nitrosative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
3.6 Carbon Monoxide: A Signaling Molecule Endowed with Antiinflammatory Properties . . . . . . . 113
3.7 The HeatShock Pathway of Brain Stress Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
3.8 HO System: A Putative Vitagene Target for Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
3.9 Regulation of HO Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3.10 Glutathione, Thiol Redox State, and RNS: Intracellular Modulators of HO1 Expression . . . . . 119
3.11 Heme Oxygenase in Brain Function and Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
3.12 Bilirubin and Biliverdin: An Endogenous Antioxidant System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

4 Caloric Restriction and Endogenous Oxidative Stress: Relevance to Aging and

Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.1 Therapeutic Potential of Nutritional Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

5 Major CNS Disorders Associated with ROS/RNSMediated Damage . . . . . . . . . . . . . . . . . . . . . . . . . . 123

5.1 Amyotrophic Lateral Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
5.2 Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
5.3 Parkinsons Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5.4 Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
5.5 Friedreichs Ataxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5.6 Huntingtons Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.7 Downs Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.8 Ischemia/Reperfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

6 Genetics of Human Longevity: Role of Vitagenes in Prolongation of Healthy Life Span . . . . . 133

7 HO1 and Hsp70 as a Therapeutic Funnel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

# 2008 Springer ScienceBusiness Media, LLC.

104 6 Aging and oxidative stress response in the CNS

Abstract: Cellular oxidant/antioxidant balance has become the subject of intense study, particularly
focused on brain aging and neurodegenerative disorders. There is now evidence to suggest that reduction
of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative stress are
fundamental causes for both the aging processes and neurodegenerative diseases. However, to survive
different types of injuries, brain cells have evolved networks of different responses, which detect and control
diverse forms of stress. Efficient functioning of maintenance and repair process seems to be crucial for both
survival and physical quality of life. This is accomplished by a complex network of the socalled longevity
assurance processes, which are composed of several genes termed vitagenes. Among these, heatshock
proteins (Hsps), proteasome, and mitochondrial uncoupling protein systems are highly conserved mechan-
isms responsible for the preservation and repair of the correct conformation of cellular macromolecules,
such as proteins, RNA, and DNA.
Recent studies have shown that the heatshock response contributes to establishing a cytoprotective state
in a wide variety of human diseases, including ischemia and reperfusion damage, inflammation, metabolic
disorders, cancer, infection, trauma, and aging. Among the various Hsps, Hsp32 also known as heme
oxygenase I (HO1), has received considerable attention, as it has been recently demonstrated that HO1
induction, by generating the vasoactive molecule carbon monoxide (CO) and the potent antioxidant
bilirubin, could represent a protective system potentially active against brain oxidative injury. The major
neurodegenerative diseases, Alzheimers disease (AD), Parkinsons disease (PD), amyotrophic lateral sclero-
sis (ALS), Huntingtons disease (HD), and Friedreichs ataxia (FA), are all associated with the presence of
abnormal proteins. Given the broad cytoprotective properties of the heatshock response, there is now strong
interest in discovering and developing pharmacological agents capable of inducing the heatshock response.
These findings have opened up new perspectives in medicine and pharmacology, as molecules inducing this
defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly,
modulation of endogenous cellular defense mechanisms such as the heatshock response, and the protea-
somal system, through nutritional antioxidants or pharmacological compounds may represent an innova-
tive approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.
Moreover, by maintaining or recovering the activity of vitagenes, it would be possible to delay the aging
process and decrease the occurrence of agerelated diseases with resulting prolongation of a healthy life span.
List of Abbreviations: AD, Alzheimers disease; AP1, activator protein1; Ab, amyloid betapeptide; CNS,
central nervous system; GSH, reduced glutathione; GSSG, oxidized glutathione; Hsp, heatshock protein;
JAK, janus kinase; JNK, cjun Nterminal kinase; MAPK, mitogenactivated protein kinase; NFkB, nuclear
factor kappaB; NFT, intraneuronal fibrillary tangles; NOS, nitric oxide synthase; PD, Parkinsons disease;
PLA2, phospholipase A2; RNS, reactive nitrogen species; ROS, reactive oxygen species; SAPK, stress
activated protein kinase; STAT, signal transducer and transcription activator; TNF, tumor necrosis factor

1 Introduction

Reduction of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative
stress are fundamental causes in the aging processes and neurodegenerative diseases (Mattson et al., 2002).
Numerous theories have been suggested to explain the aging process (> Table 61). However, current
thoughts generally propose that senescence results from various extrinsic events that lead progressively to
cell damage and death and/or characteristic intrinsic events related to the genomebased theory. These
general theories have been presented in various ways. It is agreed that aging is a combination of several
theories, the free radicals and mitochondrial theories presumably being the most important, while others
may play a definite, although less critical, role. However, it should be emphasized that no single theory is
entirely satisfactory.
Although several lines of evidence suggest that accumulation of oxidative molecular damage is a
primary causal factor in senescence, it is increasingly evident that the mitochondrial genome may play a
key role in aging and neurodegenerative diseases. Mitochondrial dysfunction is characteristic of several
neurodegenerative disorders, and evidence for mitochondria being a site of damage in neurodegenerative
 Aging and oxidative stress response in the CNS 6 105

. Table 61
Theories of aging
A. Stochastic (random event)
1. Somatic mutation
2. Error catastrophe
3. Protein glycosylation
B. Developmental
1. Immune
2. Neuroendocrine
C. Genomebased
1. Intrinsic mutagenesis
2. Programmed
D. Free radical and mitochondrial dysfunction

disorders is partially based on decreases in respiratory chain complex activities in Parkinsons disease (PD),
Alzheimers disease (AD), and Huntingtons disease (HD) (Calabrese et al., 2001a). Such defects in
respiratory complex activities, possibly associated with oxidant/antioxidant balance perturbation, are
thought to underlie defects in energy metabolism and induce cellular degeneration. Among these, chaper-
ones are highly conserved proteins responsible for the preservation and repair of the correct conformation
of cellular macromolecules, such as proteins, RNA, and DNA. Chaperonebuffered silent mutations may be
activated during the aging process and lead to the phenotypic exposure of previously hidden features and
contribute to the onset of polygenic diseases, such as agerelated disorders, atherosclerosis, and cancer
(Soti and Csermely, 2002). Recently, the involvement of the heme oxygenase (HO) pathway in antidegen-
erative mechanisms operating in AD has received considerable attention, as it has been demonstrated
that the expression of HO is closely correlated to that of amyloid precursor protein (APP) (Dore, 2002;
Perry et al., 2003). HO induction, which occurs together with the induction of other Hsps during various
physiopathological conditions, by generating the vasoactive molecule carbon monoxide (CO) and
the potent antioxidant bilirubin, represents a protective system potentially active against brain oxidative
injury. HO1 gene is redox regulated and this is supported by the fact that HO1 gene has a heatshock
consensus sequence as well as activator proteins (AP)1, 2, and nuclear factor kappaB (NFkB) binding
sites in its promoter region. In addition, HO1 expression is rapidly upregulated by oxidative and
nitrosative stresses, as well as by glutathione depletion. Given the broad cytoprotective properties of the
heatshock response, there is now strong interest in discovering and developing pharmacological agents
capable of inducing the heatshock response (Butterfield et al., 2002a). In the present chapter, we discuss the
role of free radicals and mitochondria in brain aging and neurodegenerative disorders, then review the role
of NO and CO gases and that of the heatshock system in brain stress tolerance and their relevance to
mechanisms of longevity.

2 The FreeRadical Hypothesis of Aging

As one of the most prominent current theories of aging, the freeradical theory postulates that free radicals
generated through mitochondrial metabolism can act as a causative factor of abnormal function and cell death.
Various toxins in the environment can injure mitochondrial enzymes, leading to increased generation
of free radicals that over the life span would eventually play a major role in aging (Knight, 2000; Fries, 2002;
Anisimov et al., 2003; Sastre et al., 2003). During the last few years, cellular oxidant/antioxidant balance has
become the subject of intense study, particularly by those interested in brain aging and in neurodegenerative
mechanisms.
Several lines of evidence suggest that accumulation of oxidative molecular damage is a causal factor
in senescence. The direct evidence for this hypothesis is that overexpression of antioxidative genes for
106 6 Aging and oxidative stress response in the CNS

Cu/Znsuperoxide dismutase (Cu/ZnSOD) and catalase in transgenic Drosophila melanogaster prolongs

the life span, retarding the ageassociated accumulation of oxidative damage (Biesalski, 2002). Among the
correlative evidence supporting the involvement of oxidative stress are the following: (1) oxidative damage
to DNA and proteins increases exponentially with age, and concomitantly, the rates of mitochondrial
O2
 and H2O2 generation as well as the susceptibility of tissues to experimentally induced oxidative stress
are increased; (2) experimental regimens that extend life span, such as caloric restriction in mammals
and reduction of metabolic rate in insects, decrease the accumulation rates of oxidative damage; and
(3) mitochondria make two rather contradictory contributions to cell survival. The classically recognized
function is the synthesis of ATP for energizing endergonic reactions and the other is generation of reactive
oxygen species (ROS) that may compromise the longterm survival of cells and constitute a major
underlying cause of the aging process. Indeed, these two rather conflicting functions are part of the same
process, namely mitochondrial respiration.
More than 95% of the O2 taken up by the human body is used by mitochondrial cytochrome oxidase,
which adds four electrons to oxygen to generate a molecule of water. Cytochrome oxidase normally does
not release ROS into its surroundings. However, a number of investigations have indicated that brain
mitochondria undergo oxidative stress damage and a decrease of cytochrome c oxidase activity during aging
(Harris et al., 2003). It has been postulated that this complex may act as a bottleneck, creating a situation of
electron traffic jam upstream, which would alter the redox state of oxidoreductases in the electron transfer
chain and increase their autoxidizability and rate of superoxide generation. A finding that lends credibility
to this hypothesis is that cytochrome c oxidase activity is directly correlated with the average life span in
different species (Sharman and Bondy, 2001).
Oxidative damage to key intracellular targets such as DNA or proteins is an important feature of the
normal cellular aging process in the brain, and several studies have shown that oxidative damage to DNA or
protein extracted from brain tissue increases with age (Kalyuzhny, 2002). Oxidative damage to DNA has
been shown to be extensive and could be a major cause of the degenerative diseases related to aging such as
cancer. With respect to this, it has been proposed that DNA damage is a major factor underlying neuronal
degeneration in normal aging and that accelerated damage to DNA may be the basis of neurodegenerative
conditions such as AD, and it has been demonstrated that DNA damage distribution in the human brain, as
shown by in situ end labeling, shows areaspecific differences in aging and in AD (Mecocci et al., 1998).
Levels of the oxidized nucleotide 8hydroxydeoxyguanosine (8OHdG), a biomarker of DNA damage,
have also been shown to accumulate with aging. In several tissues, including brain and muscle, levels of
8OHdG in mitochondrial DNA (mtDNA) exceed that of nuclear DNA (nDNA) some 16fold, although as
yet there have been no studies performed using absolutely pure mtDNA (Halliwell, 1999). It has been
demonstrated that 8OHdG most frequently base pairs with cytosine, but also mispairs with adenine
approximately 1% of the time, causing misreading of adjacent residues. Mecocci and coworkers (1997)
found that 8OHdG significantly correlates with increases in levels of a 7.4kb deletion in the human brain.
The major DNA product formed by methylating agents in vitro and in vivo is 7methylguanine, and it
has been shown that in nDNA of normal mouse brains steadystate levels of 7methylguanine increased
approximately twofold between 11 and 28 months of age and that following treatment in vivo with
methylnitrosourea, a fraction of DNA damage in brain tissue was refractory to repair and was lost from
DNA much more slowly (Gaubatz and Tan, 1993). This repairresistant fraction of damage was greater in
DNA from old tissues, and it was suggested that although DNA repair enzymes are present and active
in senescent postmitotic tissues such as the brain, changes in the structure and function of old chromatin
somehow decrease the capacity of the DNA repair enzymes present in the nucleus to repair oxidatively
damaged DNA (Gaubatz and Tan, 1994). In addition, singlestrand and doublestrand breaks in DNA
accumulate in the aging brain, and on exposure of neurons isolated from young and aged rats to an excitotoxic
insult, more extensive DNA breaks were measured in neurons isolated from older rats (Mandavilli and Rao,
1996). During the course of normal metabolism in the brain there is production of ROS such as superoxide
and hydroxyl radicals, as well as the production of reactive nitrogen species (RNS) such as nitric oxide (NO)
and peroxynitrite. Therefore, the ability of DNA repair mechanisms within the nuclei of brain cells to repair
damage caused by a diverse range of oxidizing species is important to maintain normal brain functions.
 Aging and oxidative stress response in the CNS 6 107

There are several enzymes systems that have been found to repair damage to DNA caused by oxidizing species
(Demple and Harrison, 1994). These include endonucleases, exonucleases, thymine glycol glycolases, and
DNA polymerases. DNA polymerases so far detected in the mammalian brain (a, b, d, and E) undergo age
dependent changes in activity, but it is not known which cell types contain which polymerases, and the ability
of nuclei from different brain regions to repair specific types of oxidative DNA damage is unknown. In
addition, recent evidence indicates that genetic instability, such as telomere loss and somatic and mtDNA
mutations, increases with age (Aviv et al., 2003). Levels of oxidative damage in mtDNA isolated from various
brain regions appear to be at least tenfold higher than those of nDNA (Bohr and Dianov, 1999), although
owing to technical difficulties there has as yet been no definitive study of oxidative damage to mtDNA
(Beckman and Ames, 1998). This increase correlates with the 17fold higher evolutionary mutation rate in
mtDNA compared with nDNA. These higher levels of oxidative damage and mutations in mtDNA recognize
different causal factors, including location of the DNA near the inner mitochondrial membrane sites (where
oxidants are generated), lack of protective histones, mitochondrial polymerase errors, and activations of
genes involved in errorprone DNA repair (Schapira, 1998). The ageassociated accumulation of oxidative
damage to mtDNA correlates with the level of mtDNA deletions found in various tissues composed of
postmitotic cells (Floyd and Hensley, 2002). It is possible that this damage leads to mutations that result in
mitochondrial dysfunction, which has been suggested to be involved in the pathogenesis of neurodegenera-
tive disorders (Calabrese et al., 2003a).
An increase in protein oxidative damage, as indicated by the loss of protein sulfhydryl groups and by a
decline in the activity of enzymes such as glutamine synthetase (GS) and glucose6phosphate dehydroge-
nase (G6PDH), has been demonstrated to occur in the brain during aging (Stadtman, 2001). A number of
experimental evidence indicates that increased rate of freeradical generation and decreased efficiency of the
reparative/degradative mechanisms, such as proteolysis, are the two factors that primarily contribute to age
related elevation in the level of oxidative stress and brain damage. With respect to this, it has been suggested
that decreases in levels of enzymes which ordinarily protect neuronal cells against oxidative stress with age
may be responsible for increased levels of freeradical damage in the brain, or that these enzymes themselves
are susceptible to inactivation by freeradical molecules which increase with age in the brain (Calabrese
et al., 2000a). During aging a number of enzymes accumulate as catalytically inactive or less active forms.
The agerelated changes in catalytic activity are due in part to reactions of proteins with oxygen and/or
nitrogen freeradical species produced during exposure to ionizing radiation or to metal ioncatalyzed
oxidation systems. The levels of oxidized proteins in brain extracts of rats of different ages increase
progressively with age, and in old rats can represent 30%50% of the total cellular protein (Calabrese
et al., 2001a). The agerelated increase in oxidized protein is accompanied by a loss of GS and G6PDH
activities, and by a decrease in the level of cytosolic neutral protease activity, which is responsible for the
degradation of oxidized (denatured) protein. Of particular significance are the results of experiments
showing that similar agerelated changes occur in the gerbil brain and that these changes are accompanied
by a loss of shortterm memory. Chronic treatment of old animals with the freeradical spintrap reagent
Ntertbutylaphenylnitrone (PBN) resulted in normalization of the several biochemical parameters to
those characteristic of the young animals; coincidentally, the shortterm memory index was restored to the
values seen in young animals (Carney et al., 1991). These results provide strong evidence that there is a
linkage between the agedependent accumulation of oxidized proteins and the loss in brain physiological
functions. It has recently been proposed that a primary mechanism leading to neuronal cell death in aging
and common neurodegenerative disorders is interference with proteasome function (Hyun et al., 2003).
Proteasomal dysfunction can involve genetic defects, direct inactivation of proteasome by reactive
oxygen and nitrogen species, or overloading with proteins. The latter can be caused by excessive production
of normal proteins or by the formation of poorly degradable proteins as a result of genetic mutations, faulty
posttranslational modification, or protein modification by freeradical damage. The major neurodegenera-
tive diseases, Alzheimers disease (AD), Parkinsons disease (PD), amyotrophic lateral sclerosis (ALS),
Huntingtons disease (HD) and Friedreichs ataxia (FA), are all associated with the presence of abnormal
proteins (Hyun et al., 2002). The origin of HD and FA involve specific genetic defects that lead to
production of abnormal proteins, whereas AD, ALS, and PD have been described mostly as sporadic,
108 6 Aging and oxidative stress response in the CNS

although familial types of AD, ALS, and PD are recognized. Examples are the rare mutations in synuclein or
parkin that cause familial PD and the 2% of patients with ALS associated with mutation in the gene
encoding Cu/ZnSOD (Halliwell, 2002). Even in the more common sporadic version of PD, ALS, and AD
abnormal proteins are present to a significant extent. Thus, the senile plaques typical of AD contain not
only bamyloid but also a wide range of other proteins. Most of them are oxidized and nitrated. Similar
damage has been described for proteins in Lewy bodies in sporadic PD. Oxidative as well as nitrosative
protein damage are also elevated in ALS. In nondividing cells, such as the great majority of neurons in the
adult brain, the protein content of cells is approximately constant. Since protein synthesis is continuous,
there must be an equilibrium between synthesis and degradation. Cellular protein can be degraded by the
lysosomal system, but a system of equal or greater importance is the proteasome. The 20S proteasome
(according to its sedimentation coefficient) is a cylindrical structure comprised of multiple protein subunits
and containing a narrow channel where proteolysis occurs. The 20S proteasome can degrade a wide range of
proteins including oxidatively damaged proteins, but most or all of the 20S proteasome in the cell is
associated with a 19S cap complex, which binds in an ATPdependent manner and confers specificity for
the degradation of polyubiquitinated proteins (Halliwell, 2001). Several other proteins are known that can
associate with proteasomes and increase (or in some case decrease) rates of protein clearance. Although it is
usually assumed that increased levels of oxidative damage are due to the increased generation of freeradical
species, however, increased levels of oxidative damage can equally ensue a decreased clearance of oxidatively
modified biomolecules. Oxidized protein levels in the central nervous system (CNS) tend to increase with
age (Friguet, 2002), consistent with several reports that proteasome activity decreases with age and in
neurodegenerative disorders (Mc Naught et al., 2001).
The agedependent accumulation of oxidized dysfunctional proteins with reactive carbonyl groups
leads to interand intramolecular crosslinks with protein amino groups, thus altering the efficiency of the
electron transport. Imbalances in the stoichiometry of functional electron transport proteins is proposed
to lead to a leakage in the flow of electrons to the terminal electron acceptor, cytochrome oxidase (Ojaimi
et al., 1999), and increased likelihood of superoxide generation. Studies on isoprostanes, the end product
of lipid peroxidation that can be measured in the CSF and urine in various neurodegenerative disorders,
suggest that lipid peroxidation is an early stage in these disease processes. Similarly, another end product
of lipid peroxidation, the aldehyde 4hydroxytransnonenal (HNE), which is highly neurotoxic, avidly
binds to proteins, and HNEprotein adducts are demonstrable in senile plaques and tangles in AD, tissues
from ALS patients, and Lewy bodies in PD. Protein carbonyls can be generated by direct oxidative damage
to proteins, by binding of cytotoxic aldehyde such as HNE to proteins, and by glycosidation of proteins
(Drake et al., 2003a). The content of protein carbonyls in Alzheimers brain samples is greater than in age
matched controls (Butterfield, 2002), and this provides the clearest indication of greater accumulation of
oxidized proteins in this disease. Brain regions show specific changes in this regard, and carbonyl levels
correlate well with tangles (Butterfield, 2002). Importantly, the accumulation of oxidation products in
particular regions of the brain seems to be related to specific cognitive defects (Forster et al., 1996). In support
of this, administration of the spintrap PBN to gerbils retards both protein oxidation and such neurological
defects (Carney et al., 1991).

2.1 The Mitochondrial Theory of Aging

Harman in 1972 first proposed that mitochondria may have a central role in the process of aging (Harman,
1981). According to this theory, free radicals generated through mitochondrial metabolism can act as a
causative factor of abnormal function and cell death. Mitochondria are the cells most significant source of
oxidants and in vitro studies have indicated that approximately 1%2% of electron flow through the
electron transport chain (ETC) results in the univalent generation of superoxide (Calabrese et al., 2000a).
Moreover, various toxins in the environment can injure mitochondrial enzymes, leading to increased
generation of free radicals that over the life span would eventually play a major role in aging (Calabrese
et al., 2004a). Ultrastructural changes have been also reported to occur in mitochondria with age. They
become larger and less numerous with vacuolization, cristae rupture, and accumulation of paracrystalline
 Aging and oxidative stress response in the CNS 6 109

inclusions. Cardiolipin, an acidic phospholipid that occurs only in mitochondria, has been shown to
decrease with age (Paradies et al., 2002). This inner membrane lipid is known to have optimal electrical
insulating properties, thereby contributing significantly to the transmembrane potential that drives the
formation of ATP via ATP synthase. Indeed, a decrease in membrane potential in mitochondria from older
animals has been demonstrated (Calabrese et al., 2001a).
It has been proposed that accumulation of mtDNA during life is a major cause of agerelated disease,
and this is because of its high mutagenic propensity. As discussed before, the lack of introns and protective
histones, limited nucleotide excision and recombination DNA repair mechanisms, and location in proxim-
ity of the inner mitochondrial membrane which expose it to an enriched freeradical milieu are all factors
contributing to a tenfold higher mutation rate occurring in the mtDNA than in the nDNA. Moreover, a
large body of evidence indicates that mtDNA mutations increase as a function of age, reaching the highest
levels in the brain and muscle. More than 20 different types of deletions have been documented to
accumulate in aging human tissues. The first report on an agerelated increase in mtDNA deletion was
found in brains from elderly subjects and in PD (Calabrese et al., 2001a). This deletion has been described
to occur between 13bp sequence repeats beginning at nucleotides 8470 and 13447, removing almost a 5kb
region of mtDNA between the ATPase 8 and the ND5 genes. The deletion is thought to occur during
replication of the mtDNA, the absent sequence encoding for six essential polypeptides of the respiratory
chain and 5 tRNAs. It has been associated with several clinical diseases, such as chronic progressive external
ophthalmoplegia and Kearns Sayre syndrome. Several agerelated disorders have been shown to be linked to
higher levels of mtDNA mutations than agematched controls. In the CNS, 17 times higher levels of the
common deletion in the striatum of patients with PD have been demonstrated, compared with age
matched controls. Evidence also exists indicating higher levels of this deletion in patients with AD, which
parallel increased levels in the oxidized nucleotide 8OHdG (Bohr and Dianov, 1999).
A major feature of mtDNA disease in humans is the presence of cells with low cytochrome c oxidase
activity, and evidence exists that indicates that the mechanism for these changes is likely to be clonal
expansion of individual mtDNA deletions within single cells (Schapira, 1998). Complex IVdeficient cells,
which occurred only sporadically earlier than the sixth decade of life, were present regularly after this age,
with the loss of enzyme activity being always confined to single, randomly distributed cells. Similarly,
cytochrome c oxidasenegative neurons have been demonstrated to exist in abundance in the CNS of
patients with mitochondrial disorders (Cottrell et al., 2001). These findings establish the relationship
between ageassociated accumulation of mtDNA mutations and bioenergy dysfunction as a key feature of
the aging process, at least in tissues predominantly composed of postmitotic cells, such as the CNS and
skeletal muscle. Relevant to mitochondrial bioenergetics, in fact, is the finding of a significant decrease in
state 3/state 4 ratio, which has been observed to occur in brain during aging (Calabrese et al., 2001a). Since
this ratio relates to the coupling efficiency between electron flux through the ETC and ATP production, an
increase in state 4 would result in a more reductive state of mitochondrial complexes and, consequently, to
an increase in freeradical species production. A decrease in state 3/state 4 respiration during aging has been
found associated with a significant decrease in cardiolipin content in brain mitochondria (Cottrell and
Turnbull, 2000). This loss could play a critically important role in the agerelated decrements in mitochon-
drial function and appears to be associated with both quantitative and qualitative regionspecific protein
changes, which are parallel to structural changes, such as decrease of the inner membrane surface, smaller as
well as sparser cristae, decreased fluidity, and increased fragility. Modifications in cardiolipin composition is
recognized to accompany functional changes in brain mitochondria, which include all proteins of the inner
mitochondrial membrane that generally require interaction with cardiolipin for optimal catalytic activity
(PorteroOtin et al., 2001; Quiles et al., 2002). Acetylcarnitine fed to old rats increased cardiolipin levels to
that of young rats and also restored protein synthesis in the inner mitochondrial membrane, as well as
cellular oxidant/antioxidant balance (Paradies et al., 1999), suggesting that administration of this com-
pound may improve cellular bioenergetics in aged rats (Hagen et al., 1998a, b). Interestingly, caloric
restriction, a dietary regimen that extends life span in rodents, maintains the levels of 18:2 acyl side chains
and inhibits the cardiolipin composition changes (Selman et al., 2003). In addition, caloric restriction was
shown to retard the agingassociated changes in oxidative damage, mitochondrial oxidant generation, and
antioxidant defenses (Mattson, 2003).
110 6 Aging and oxidative stress response in the CNS

2.2 Mitochondrial Damage, Reactive Nitrogen Species,

and Neurodegenerative Disorders
Increasing evidence sustains the hypothesis that mitochondrial energy metabolism underlies the pathogen-
esis of neurodegenerative diseases (Papa and Skulachev, 1997; Genova et al., 2004). Decreased complex I
activity is reported in the substantia nigra of postmortem samples obtained from patients with PD (Beal,
1998). Similarly, impaired complex IV activity has been demonstrated in AD (Heales et al., 1999). Increased
freeradicalinduced oxidative stress has been associated with the development of such disorders and a large
body of evidence suggests that NO
plays a central role (Stamler and Hausladen, 1998). Cytokines (INFg)
that are present in the normal brain are elevated in numerous pathological states, including PD (Mayer,
2003), AD (Moore et al., 2003), multiple sclerosis (MS) (Calabrese et al., 1994, 1998, 2002a, 2003b; Bagasra
et al., 1995), ischemia, encephalitis, and viral infections of the CNS (Calabrese et al., 2001a). Accordingly, as
cytokines promote the induction of nitric oxide synthase (NOS) in the brain, a possible role for a glial
derived NO
in the pathogenesis of these diseases has been suggested (Stamler and Hausladen, 1998).
Excessive formation of NO
from glial origin has been evidenced in some study in which NADPH
diaphorase (a cytochemical marker of NOS activity)positive glial cells have been identified in the substantia
nigra of postmortem brains obtained from individuals with PD (Hyun et al., 2003). Loss of nigral GSH is
considered an early and crucial event in the pathogenesis of PD (Beal, 1998, 2003) and as a consequence
decreased peroxynitrite scavenging may also occur. Therefore, such perturbations in thiol homeostasis may
constitute the starting point for a vicious cycle leading to excessive ONOO generation in PD. In support of
this it has been reported that the selective inhibition of neuronal NOS (nNOS) prevents 1methyl4phenyl
1,2,3,6tetrahydropyridine (MPTP)induced parkinsonism in experimental animals (Dawson and Dawson,
2002; Moore et al., 2003).

3 Oxidative Stress and Brain Stress Tolerance: Role of Vitagenes

There is now evidence to suggest that reduction of cellular expression and activity of antioxidant proteins
and the resulting increase of oxidative stress are fundamental causes for both aging process and neurode-
generative diseases (Ferri et al., 2003; Genova et al., 2003). However, to survive different types of injuries,
brain cells have evolved networks of different responses, which detect and control diverse forms of stress.
Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical
quality of life. This is accomplished by a complex network of the socalled longevity assurance processes,
which are composed of several genes termed vitagenes. Signaling mechanisms adopted by regulatory
proteins to control gene expression in response to alterations in the intracellular redox status are very
common in prokaryotes. Gene activation by oxidative stress was first described in bacteria where
regulatory proteins such as OxyR were discovered as an activator of antioxidantand stressresponsive
genes. As the cytoprotective mechanism triggered by SoxR in E. coli includes the expression of critical
antioxidant defensive proteins, such as superoxide dismutase, the emerging concept is that an analogous
system might operate in mammalian cells (Calabrese et al., 2001a, 2003a, 2004a). In eukaryotes, typical
examples are the HO and Hsp genes, thioredoxin, detoxificant enzymes (MnSOD, glutathioneStransferase,
NADPH/quinone reductase), cytokines, immunoreceptors and growth factors (Calabrese et al., 2003c;
Colombrita et al., 2003).

3.1 Neurogasobiology of Nitric Oxide and Carbon Monoxide: Two Molecules

That Promote Adaptive Responses in the CNS
CO is the second gas discovered in the last 25 years to have salutary effects, the first being NO. Certain
findings raise the conceivable possibility that HO1 and/or CO and NOS2 and/or NO are functionally
interrelated in mediating their protective effects. In some situations, CO can activate the expression of
 Aging and oxidative stress response in the CNS 6 111

NOS2 and, in others, inhibits the expression of NOS2 and consequently of NO (Motterlini et al., 2002a).
NO upregulates HO1 with production of CO (Maines, 1997). We have recently found evidence for a
functional relationship between CO and NO. In endotoxic shock, the salutary action of CO in the rat brain
appears to depend sequentially on the activation of NFkB, which triggers transcription of NOS2 with
production of NO, and subsequently in the upregulation of HO1. In the absence of any of these steps, the
beneficial effect of CO is lost (Scapagnini et al., 2002a). This has been also demonstrated in mice treated for
hepatitis induced by TNFa and Dgalactosamine (Otterbein et al., 2003a). To what extent CO and NO act
interdependently in other physiopathological conditions that are responsive to CO and/or NO is unknown.

3.2 Nitric Oxide Synthase and Its Isoforms in the CNS

The enzyme responsible for NO synthesis is the NOS family of enzymes, which catalyze the conversion of
arginine to citrulline and NO
. NOS, localized in the CNS and in the periphery (Calabrese et al., 2000a), is
present in three wellcharacterized isoforms: (1) neuronal NOS (nNOS; type I), (2) endothelial NOS
(eNOS; type III), and (3) inducible NOS (iNOS; type II). Activation of different isoforms of NOS requires
various factors and cofactors. In addition to a supply of arginine and oxygen, an increase in intracellular
calcium leads to activation of eNOS and nNOS, and formation of calcium/calmodulin complexes is a
prerequisite before the functionally active dimer exhibits NOS activity, which depends also on cofactors
such as tetrahydrobiopterin (BH4), FAD, FMN, and NADPH (Dawson and Dawson, 1995). nNOS has a
predominant cytosolic localization whereas the eNOS is bound to the plasma membrane by Nterminal
myristaglation (Calabrese et al., 2000a). In contrast to nNOS and eNOS, iNOS can bind to calmodulin even
at very low concentrations of intracellular calcium; thus, iNOS can exert its activity in a calciumindependent
manner. iNOS, usually present only in the cytosol, also requires NADPH, FAD, FMN, and BH4 for full
activity. eNOS, expressed in cerebral endothelial cells, critically regulates cerebral blood flow. However, a
small population of neurons in the pyramidal cells of CA1, CA2, and CA3 subfields of the hippocampus and
granule cells of the dentate gyrus express eNOS. nNOS, which is expressed in neurons, is critically involved
in synaptic plasticity, neuronal signaling, and neurotoxicity. Activation of nNOS forms part of the cascade
pathway triggered by glutamate receptor activation that leads to intracellular cGMP elevation. The levels of
iNOS in the CNS are generally fairly low. However, an increased expression of iNOS in astrocytes and
microglia occurs following viral infection and trauma (Bredt, 1999). Activation of iNOS requires gene
transcription, and the induction can be influenced by endotoxin and cytokines (Calabrese et al., 2000a)
(IL1, IL2, lipopolysaccharide, IFNg, TNF). This activation can be blocked by antiinflammatory drugs
(dexamethasone), inhibitory cytokines (IL4, IL10), prostaglandins (PGA2), and tissue growth factors or
inhibitors of protein synthesis, e.g., cycloheximide.

3.3 Nitric Oxide as a Neurotransmitter

The discovery of the role of NO as a messenger molecule has revolutionized the concept of neuronal
communication in the CNS. NO is a gas freely permeable to the plasma membrane, thus, NO does not need
a biological receptor to influence the intracellular communication or signaling transduction mechanisms
(Stamler and Hausladen, 1998). Once generated, the cell cannot regulate the local concentration of NO,
therefore the other way to influence NO activity is to control its synthesis. The activity of NO also
terminates when it chemically reacts with a target substrate. NO when produced in small quantities can
regulate cerebral blood flow, local brain metabolism (Calabrese et al., 2001a), and neurotransmitter release
and gene expression, and play a key role in morphogenesis and synaptic plasticity. It is also generally
accepted that NO is a major component in signaling transduction pathways controlling smooth muscle
tone, platelet aggregation, host response to infection, and a wide array of other physiological and patho-
physiological processes. Under conditions of excessive formation, NO is emerging as an important media-
tor of neurotoxicity in a variety of disorders of the nervous system (Heales et al., 1999).
112 6 Aging and oxidative stress response in the CNS

3.4 Redox Activities Elicited by NO

In the last several years, a number of studies have shown a protective effect of NO in a variety of paradigms
of cell injury and cell death. These include (1) direct scavenging of free radicals, such as superoxide with
effects on intracellular iron metabolism, including interaction with iron to prevent, through formation
of nitrosyliron complexes, release of iron from ferritin (Sergent et al., 1977); (2) interaction of NO

(through its congener NO) with thiol group on the NMDA receptor with consequent downregulation
and inhibition of calcium influx (Ignarro, 2002); (3) inactivation of caspases (Ignarro, 2002); (4) activation
of a cGMPdependent survival pathway, as demonstrated in PC12 cells (Motterlini et al., 2002a); (5) induc-
ing expression of cytoprotective proteins, such as heatshock proteins (Hsps) (Motterlini et al., 2000a); and
(6) inhibition of NFkB activation or GADPH, whose activity appears to be required in one paradigm of
neuronal apoptosis (Piantadosi et al., 1997). In general, the current opinion holds that the intracellular
redox state is the critical factor determining whether in brain cells NO is toxic or protective (Rosenberg
et al., 1999). In addition, it has been proposed that NO might inhibit Tcell activation and cell trafficking
across the bloodbrain barrier, and hence limiting the setting of the autoimmune cascade associated with
degenerative damage (Dawson and Dawson, 1995). The difficulty in delineating a mechanistic involvement
of NO as proinflammatory or antiinflammatory agent and the controversy arising on whether excessive NO
elicits cytoprotective or cytotoxic actions are better appreciated by recognizing the complexity of NO
chemistry when applied to biological systems (Motterlini et al., 2002a). As minutely detailed by Stamler and
colleagues, the reactivity of the NO groups is dictated by the oxidation state of the nitrogen atom, which
enables the molecule to exist in different redoxactivated forms (Stamler and Hausladen, 1998). In contrast
to NO, which contains one unpaired electron in the outer orbital, nitrosonium cation (NO) and nitroxyl
anion (NO) are charged molecules being, respectively, the oneelectron oxidation and reduction products
of NO. Whereas NO can be transferred reversibly between cysteine residues (transnitrosation), NO can
be formed by hemoglobin, nNOS, and Snitrosothiols (RSNO). A fundamental aspect of NO biochemistry
is the attachment of NO groups to sulfhydryl centers to form Snitrosyl derivatives or RSNO (Ignarro,
2002). This chemical process, known as Snitrosation, has been suggested to represent a refined endogenous
tool to stabilize and preserve NO biological activity (Rosenberg et al., 1999; Motterlini et al., 2002a). It has
been speculated that lowmolecular weight RSNO, such as Snitrosoglutathione or nitrosocysteine, may
also represent a mechanism for storage of NO in vivo (Stamler et al., 1992; Rosenberg et al., 1999). In this
regard, glutathione becomes an important determinant of the reactivity and fate of NO because
this cysteinecontaining tripeptide is very abundant in most tissues and biological fluids. In addition,
Snitrosation is also an important process in modulating the activity and function of several enzymes and
proteins. However, deleterious and oxidative modification in protein structure and function may occur
when RNS reach a critical threshold, and hence nitrosative stress may ensue (Hausladen et al., 1996). At the
cellular level, nitrosative stress has been linked to inhibition of cell growth and apoptosis, and implicated in
NO pathogenesis (Sergent et al., 1977). The intriguing aspect in the parallelism between the effects
mediated by increased RNS and ROS is the ability of cells to respond to these two types of stress and,
depending on the severity of the nitrosative/oxidative insult, this response may result in both adaptation
and resistance to toxicity (Calabrese et al., 2002b).

3.5 Regulation of Gene Expression by Oxidative and Nitrosative Stress

Signaling mechanisms adopted by regulatory proteins to control gene expression in response to alterations
in the intracellular redox status are very common in prokaryotes. Gene activation by oxidative stress was first
described in bacteria where regulatory proteins such as OxyR was discovered as an activator of antioxidant
and stressresponsive genes. OxyR is a homotetramer that is activated by hydrogen peroxide and
Snitrosothiols. The protein contains six cysteine residues, one of each is absolutely necessary for activity
and two are required for maximal activation. Recent studies suggested that oxidation of a single thiol to a
sulfenic acid may represent a sensor mechanism, whereas the activation mechanism can be ascribed to
formation of an intramolecular disulfide, or alternatively to Snitrosylation of a single cysteine residue, with
 Aging and oxidative stress response in the CNS 6 113

Cys 199 being a likely candidate site of posttranslational modification (Rosenberg et al., 1999; Motterlini
et al., 2002a). The expression of these protective genes renders bacteria more resistant to oxidant
damage (Motterlini et al., 2002b). As the cytoprotective mechanism triggered by SoxR in E. coli includes
the expression of critical antioxidant defensive proteins, such as superoxide dismutase (Motterlini
et al., 2003), the emerging concept is that analogous systems might operate in mammalian cells. In
eukaryotes, typical examples are genes such as the HO gene, thioredoxin and detoxificant enzymes
(MnSOD, glutathioneStransferase, NADPH/quinone reductase), cytokines, immunoreceptors, and
growth factors. That the antioxidant protein HO could sense NO, and thus protecting against ROS and
RNS insults, is supported by the following findings: (1) NO and NOrelated species induce HO1 expression
and increase HO activity in human glioblastoma cells, hepatocytes, and aortic vascular cells; (2) cells
pretreated with various NOreleasing molecules acquire increased resistance to H2O2mediated cytotoxicity
when HO is maximally activated; and (3) bilirubin, one of the end products of heme degradation by HO,
protects against the cytotoxic effects caused by strong oxidants such as H2O2 and ONOO (Rosenberg et al.,
1999; Motterlini et al., 2002a). The conception that NO and RNS can be directly involved in the modulation
of HO1 expression in eukaryotes is based on the evidence that different NOreleasing agents can markedly
increase HO1 mRNA and protein, as well as HO activity, in a variety of tissues, including brain cells
(Scapagnini et al., 2002a). In rat glial cells, treatment with lipopolysaccharide (LPS) and interferong
(IFNg) results in a rapid increase in both iNOS expression and nitrite levels followed by enhancement of
HO1 protein (Calabrese et al., 2000b). In the same study, the presence of NOS inhibitors suppressed both
nitrite accumulation and HO1 mRNA expression. Modulation of HO1 mRNA expression by iNOS
derived NO following stimulation with LPS has also been reported in different brain regions, particularly in
the hippocampus and substantia nigra in an in vivo rat model of septic shock (Scapagnini et al., 2002a).
Moreover, the early increase in iNOS protein levels observed in endothelial cells exposed to low oxygen
tension seems to precede the stimulation of HO1 expression and activity, an effect that appears to be finely
regulated by redox reactions involving glutathione (Motterlini et al., 2000a, 2002a). Taken together, these
findings point to the central role of NO as a signaling molecule which, by triggering expression of
cytoprotective genes such as HO1, may lead to adaptation and resistance of brain cells to subsequent,
eventually more severe, nitrosative and oxidative stress insults (Butterfield et al., 2002b). Thus, a direct
interaction of NO groups with selective chemical sites localized in transcription proteins that can be
activated through nitrosative reactions could effectively contribute to the enhancement of both HO1
gene expression and stress tolerance. Recent knowledge concerning the modulation by thiol redox state of
the activity of several transcription factors that recognize specific binding sites within the promoter and
distal enhancer regions of the HO1 gene include Fos/Jun (AP1), NFkB, and the more recently identified
Nrf2 proteins (Balogun et al., 2003; Poon et al., 2004a). Importantly, both AP1 and NFkB contain cysteine
residues whose interaction with oxidant or nitrosant species might be crucial for determining the DNA
binding activity (Rosenberg et al., 1999; Motterlini et al., 2002a). Data in the literature show that NO can
either activate or inhibit these transcription factors, and that in many circumstances activation depends on
the reversibility of the posttranslational modification elicited by the various RNS (Butterfield et al., 2002a;
Poon et al., 2004b).
We have demonstrated in astroglial cell cultures that cytokineinduced nitrosative stress is associated
with an increased synthesis of Hsp70 stress proteins, which was also found after treatment of cells with the
NOgenerating compound sodium nitroprusside (SNP), thus suggesting a role for NO in inducing Hsp70
protein expression (Calabrese et al., 2000b). In vivo experiments performed in our laboratory have also
demonstrated that the redox glutathione status is a critical factor for induction of cytoprotective Hsp70
(Calabrese et al., 2000c, 2002c, 2004a; Balogun et al., 2003).

3.6 Carbon Monoxide: A Signaling Molecule Endowed with

Antiinflammatory Properties
Grehan first detected a combustible gas in the blood in 1894 (Grehan, 1894). This gas was supposed by de
Saint Martin and Nicloux to be CO. However, it was not until 1949 that Siorstrand discovered that
114 6 Aging and oxidative stress response in the CNS

endogenously produced CO arose from the degradation of hemoglobin released from senescing erythro-
cytes (Siorstrand, 1949). Greater than 75% of CO produced in humans arises from erythrocyte turnover
generated as a byproduct of heme metabolism. In 1969, the source of endogenous CO was discovered, as
Tenhunen and collaborators (1969) described and characterized HO as the enzyme responsible for breaking
down heme in the body, demonstrating that heme catalysis resulted in the subsequent release of CO and free
iron as byproducts (Tenhunen et al., 1969). Since then, supported by a large body of experimental
evidence, CO is proving to be an extraordinary signaling molecule generated by the cell, which is vital in
the regulation of cellular homeostasis. In the brain, CO is emerging as a chemical messenger molecule
which can influence physiological and pathological processes in the central and peripheral nervous systems.
This gaseous molecule is now considered a putative neurotransmitter, owing to its capability to diffuse
freely from one cell to another, thereby influencing intracellular signal transduction mechanisms. However,
unlike conventional neurotransmitters, CO is not stored in synaptic vesicles and is not released by
membrane depolarization and exocytosis. It seems likely that CO is involved in the mechanism of cell
injury (Turcanu et al., 1998). This is evidenced by the fact that CO binds to iron of heme of the enzyme
guanylyl cyclase to activate cGMP (Piantadosi et al., 1997). Indeed, it has been found that CO is responsible
for maintaining endogenous levels of cGMP. This effect is blocked by potent HO inhibitors but not by NO
inhibitors (Maines, 1997). On the basis of endogenous distribution of HO in the CNS, it has been suggested
that CO can influence neurotransmission like NO (Verma et al., 1993). CO appears to be involved as a
retrograde messenger in LTP and also in mediating glutamate action at metabotropic receptors (Graser
et al., 1990). This is evident from the fact that metabotropic receptor activation in the brain regulates the
conductance of specific ions channels via a cGMPdependent mechanism, which is blocked by HO
inhibitors (Glaum and Miller, 1993). Experimental evidence suggests that CO plays a similar role like
NO in the signal transduction mechanism in regulating cell function and celltocell communication
(Maines, 1997). HO resembles NOS in that the electrons for CO synthesis are donated by cytochrome
P450 reductase, which is 60% homologous at the amino acid level to the half carboxyterminal of NOS
(Calabrese et al., 2001a). CO like NO binds to iron in the heme moiety of guanylyl cyclase. However, there
are some differences in function between CO and NO. Thus, NO mainly mediates glutamate effect at
NMDA receptors while CO is primarily responsible for glutamate action at metabotropic receptors. Taken
together, it appears that CO and NO play an important role in the regulation of CNS function, therefore
impairment of CO and NO metabolism results in abnormal brain function (Calabrese et al., 2000a). A
number of evidence suggest a possible role of CO in regulating nitrergic transmission. Endogenous CO has
been suggested to control constitutive NOS activity. Moreover, CO may interfere with NO binding to
guanylyl cyclase, and this in addition to the important role of HO in regulating NO generation, owing to its
function in the control of heme intracellular levels, as part of the normal protein turnover (Calabrese et al.,
2003a). This hypothesis is sustained by recent findings showing that HO inhibition increases NO produc-
tion in mouse macrophages exposed to endotoxin (Turcanu et al., 1998). CO may also act as a signaling
effector molecule, by interacting with targets other than guanylate cyclase. Notably, it has been recently
demonstrated that K(Ca) channels are activated by CO in a cGMPindependent manner (Wang and Wu,
2003) and also that COinduced vascular relaxation results from the inhibition of the synthesis of the
vasoconstrictor endothelin1 (Coceani et al., 1997). Little, however, is known about how CO is sensed on a
biological ground. Interestingly, the photosynthetic bacterium Rhodospirillum rubrum has the ability to
respond to CO through the heme protein CooA that, upon exposure to CO, acquires DNAbinding
transcriptional activity for the CO dehydrogenase gene, thereby encoding for CO dehydrogenase, which
is the key enzyme involved in the oxidative conversion of CO to CO2. Remarkably, heart cytochrome c
oxidase possesses COoxygenase activity, thus metabolizing CO to CO2 (Calabrese et al., 2001a). Whether
this occurs also in brain mitochondria remains to be elucidated. Aside from the CNS, the protective effects
of CO were initially demonstrated in a model of acute lung injury and endotoxic shock, and subsequently in
a mouse cardiac xenotransplantation model (Otterbein et al., 2003a). Mouse heart transplanted to
immunosuppressed rats survive indefinitely. However, if HO1 activity cannot be expressed in the mouse
heart, either as a consequence of absent phenotypical expression of the HO1 gene (mice hmox/) or as a
consequence of HO1 activity being inhibited with a selective inhibitor Tin protoporphyrin (SnPPIX), the
hearts are rejected rapidly. HO1 expression in the transplanted heart is essential to prevent rejection in this
 Aging and oxidative stress response in the CNS 6 115

model. Surprisingly, if the donor and recipient were both treated with 250 ppm CO, even a heart that
cannot express HO1 activity still survives indefinitely (Otterbein, 2002). In this scenario CO appears to be
able to substitute for HO1 in suppressing the proinflammatory response, which is the leading cause of graft
rejection. CO emerges as a powerful antiinflammatory promoting agent acting at the level of the macro-
phage cell line, a cell that probably controls the balance of inflammation in many conditions. Macrophages
stimulated with bacterial LPS produce proinflammatory cytokines such as TNFa and an inflammatory
cytokine interleukin10 (IL10) is also produced (Otterbein et al., 2003b). If macrophages overexpress HO1
or are exposed to CO in vitro before stimulation with LPS, the proinflammatory response, and consequently
TNFa, is markedly diminished, whereas the antiinflammatory response, characterized by IL10 production,
is enhanced. At least, three important actions of CO contribute to its antiinflammatory effects: (1) CO
prevents platelet aggregation and the consequent thrombosis (Otterbein et al., 2003a); (2) CO down-
modulates the expression of plasminogen activator inhibitor type 1 (PAI1); and (3) CO prevents apoptosis
in several cell types, including endothelial cells, fibroblasts, hepatocytes, and pancreatic bcells (Otterbein
et al., 2003a). In addition, CO suppresses the proliferative response of smooth muscle cells, which contribute
to neointimal proliferation associated with inflammatory lesions in vivo. Many of the observed effects of CO
have been obtained by exposing cells or animals to gaseous CO and its subsequent inhalation. Interestingly,
the recently discovered COreleasing molecules (CORMS) appear to afford similar protective action,
thereby providing an alternative therapeutic approach for those pathophysiological conditions where CO
administration is warranted (Motterlini et al., 2002b, 2003).

3.7 The HeatShock Pathway of Brain Stress Tolerance

It is well known that living cells are continually challenged by conditions that cause acute or chronic stress.
To adapt to environmental changes and survive different types of injuries, eukaryotic cells have evolved
networks of different responses, which detect and control diverse forms of stress. One of these responses,
known as the heatshock response, has attracted a great deal of attention as a universal fundamental
mechanism necessary for cell survival under a wide variety of toxic conditions. In mammalian cells, Hsp
synthesis is induced not only after hyperthermia but also following alterations in the intracellular redox
environment and exposure to heavy metals, amino acid analogs, or cytotoxic drugs. While prolonged
exposure to conditions of extreme stress is harmful and can lead to cell death, induction of Hsp synthesis
can result in stress tolerance and cytoprotection against stressinduced molecular damage. Furthermore,
transient exposure to elevated temperatures has a crossprotective effect against sustained, normally lethal
exposures to other pathogenic stimuli. Hence, the heatshock response contributes to establish a cytopro-
tective state in a variety of metabolic disturbances and injuries, including stroke, epilepsy, cell and tissue
trauma, neurodegenerative disease, and aging (Calabrese et al., 2001a; Mattson et al., 2002). This has
opened new perspectives in medicine and pharmacology, as molecules activating this defense mechanism
appear as possible candidates for novel cytoprotective strategies (Scapagnini et al., 2002b; Soti and
Csermely, 2002; Colombrita et al., 2003). In mammalian cells, the induction of the heatshock response
requires the activation and translocation to the nucleus of one or more heatshock transcription factors,
which control the expression of a specific set of genes encoding cytoprotective Hsps. Some of the known
Hsps include ubiquitin, Hsp10, Hsp27, Hsp32 (or HO1), Hsp47, Hsp60, Hsc70, Hsp70 (or Hsp72),
Hsp90, and Hsp100/105. Most of the proteins are named according to their molecular weight.
HSP70. The 70kDa family of stress proteins is one of the most extensively studied. Included in this
family are Hsc70 (heatshock cognate, the constitutive form), Hsp70 (the inducible form, also referred to as
Hsp72), and GRP75 (a constitutively expressed glucoseregulated protein found in the endoplasmic
reticulum). After a variety of CNS insults, Hsp70 is synthesized at high levels and is present in the cytosol,
nucleus, and endoplasmic reticulum. Denatured proteins are thought to serve as stimuli for induction.
These denatured proteins activate heatshock factors (HSFs) within the cytosol by dissociating other Hsps
that are normally bound to HSF (Balogun et al., 2003; Calabrese et al., 2003a; Poon et al., 2004a, b). Freed
HSF is phosphorylated and forms trimers, which enter the nucleus and bind to heatshock elements (HSEs)
within the promoters of different heatshock genes, leading to transcription and synthesis of Hsps.
116 6 Aging and oxidative stress response in the CNS

After heat shock, for instance the synthesis of Hsp70 increases to a point where it becomes the most
abundant single protein in a cell. Once synthesized, Hsp70 binds to denatured proteins in an ATP
dependent manner. The Nterminal end contains an ATPbinding domain, whereas the Cterminal region
contains a substratebinding domain. Hsps serve as chaperones that bind to other proteins and regulate
their conformation, regulate protein movement across membranes or through organelles, or regulate the
availability of a receptor or activity of an enzyme.
In the nervous system, Hsps are induced in a variety of pathological conditions, including cerebral
ischemia, neurodegenerative disorders, epilepsy, and trauma. Expression of the gene encoding Hsps has
been found in various cell populations within the nervous system, including neurons, glia, and endothelial
cells (Kelly et al., 2002). Hsps consist of both stressinducible and constitutive family members. Whether
stress proteins are neuroprotective has been the subject of much debate, as it has been speculated that these
proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, however, with the
availability of transgenic animals and gene transfer, it has become possible to overexpress the gene encoding
Hsp70 to test directly the hypothesis that stress proteins protect cells from injury, and it has been
demonstrated that overproduction of Hsp70 leads to protection in several different models of nervous
system injury (Wang and Wu, 2003). Following focal cerebral ischemia, mRNA encoding Hsp70 is
synthesized in most ischemic cells except in areas of very low blood flow, because of limited ATP levels.
Hsp70 proteins are produced mainly in endothelial cells, in the core of infarcts in cells that are most
resistant to ischemia, in glial cells at the edges of infarcts, and in neurons outside the areas of infarction. It
has been suggested that this neuronal expression of Hsp70 outside an infarct can be used to define the
ischemic penumbras, which is the zone of protein denaturation in the ischemic areas (Balogun et al., 2003).
A number of in vitro studies show that both heat shock and Hsp overproduction protect CNS cells against
both necrosis and apoptosis. Mild heat shock protects neurons against glutamatemediated toxicity and
protects astrocytes against injury produced by lethal acidosis (Narasimhan et al., 1996). Transfection of
cultured astrocytes with Hsp70 protects them from ischemia or glucose deprivation (Fink et al., 1997).
Hsp70 has been demonstrated to inhibit caspase3 activation caused by ceramide, and also affects JUN
kinase and p38kinase activation (Mosser et al., 1997). In addition, Hsp70 binds to and modulates the
function of BAG1, the bcl2 binding protein (McLaughlin et al., 2003), thus modulating some type of
apoptosisrelated cell death.
A large body of evidence now suggests a correlation between mechanisms of oxidative and/or nitrosa-
tive stress and Hsp induction. Current opinion holds also the possibility that the heatshock response can
exert its protective effects through inhibition of NFkBsignaling pathway (Calabrese et al., 2001a). We have
demonstrated in astroglial cell cultures that cytokineinduced nitrosative stress is associated with an
increased synthesis of Hsp70. Increase in Hsp70 protein expression was also found after treatment of
cells with the NOgenerating compound SNP, thus suggesting a role for NO in inducing Hsp70 proteins.
The molecular mechanisms regulating the NOinduced activation of the heatshock signal seems to involve
cellular oxidant/antioxidant balance, mainly represented by the glutathione status and the antioxidant
enzymes (Calabrese et al., 2000a, 2003a; Motterlini et al., 2000a).
Ubiquitin. Ubiquitin is one of the smallest Hsps and is expressed throughout brain in response to
ischemia. It is involved in targeting and chaperoning of proteins degraded in proteasomes, which include
NFkB, cyclins, HSFs, hypoxiainducible factor, some apoptosisrelated proteins, tumor necrosis factor, and
erythropoietin receptors (Mayer, 2003).
Hsp27. Hsp 27 is synthesized mainly in astrocytes in response to ischemic situations or to kainic acid
administration. It chaperones cytoskeletal proteins such as intermediate filaments, actin, or glial fibrillary
acidic protein following stress in astrocytes. It also protects against FasApo1, staurosporine, TNF, and
etoposideinduced apoptotic cell death as well as H2O2induced necrosis (Bechtold and Brown, 2003).
Hsp47. Hsp47 is synthesized mainly in microglia following cerebral ischemia and subarachnoid
hemorrhage (Valentim et al., 2003).
Hsp60, glucoseregulated protein 75 (GRP75), and Hsp10. Hsp60, glucoseregulated protein 75 (GRP75),
and Hsp10 chaperone proteins within mitochondria. GRP75 and GRP78, also called oxygenregulated
proteins (ORPs), are produced by low levels of oxygen and glucose. These protect brain cells against
ischemia and seizures in vivo, after viralinduced overexpression (Turner et al., 1999). Hsp60 is encoded in
 Aging and oxidative stress response in the CNS 6 117

the nucleus and resides mainly in the mitochondria (Calabrese et al., 2003c, 2004a, 2005). Hsp60 forms
the chaperonin complex, which is implicated in protein folding and assembly within the mitochondria
under normal conditions (Izaki et al., 2001). Most mitochondrial proteins are synthesized in the cytosol
and must be imported into the organelles in an unfolded state (Izaki et al., 2001). During translocation, the
proteins interact with Hsp70. ATPdependent binding and release of Hsp70 provide the major driving force
for complete transport of polypeptides into the matrix. Most imported polypeptides are released from
soluble Hsp70; however, a subset of aggregationsensitive polypeptides must be transferred from Hsp70 to
Hsp60 for folding (Okubo et al., 2000). Owing to the close functional interaction between this chaperonin
system and the Hsp70 system, it is likely that upregulation of Hsp60 may be a fundamental mechanism
targeted by nutritional interventions leading to restoration of mitochondrial respiratory complex function
compromised by oxidative stress (Calabrese et al., 2004a, 2005; Perluigi et al., 2005; Poon et al., 2005;
Sultana et al., 2005).
Hsp32. Hsp32 or HO is the ratelimiting enzyme in the production of bilirubin. There are three
isoforms of HO, HO1 or inducible isoform, HO2 or constitutive isoform, and the recently discovered
HO3 (Scapagnini et al., 2002c).

3.8 HO System: A Putative Vitagene Target for Neuroprotection

In the last decade the HO system has been strongly highlighted for its potential significance in
maintaining cellular homeostasis. It is found in the endoplasmic reticulum in a complex with NADPH
cytochrome c P450 reductase. It catalyzes the degradation of heme in a multistep, energyrequiring system.
The reaction catalyzed by HO is the aspecific oxidative cleavage of the heme molecule to form equimolar
amounts of biliverdin and CO. Iron is reduced to its ferrous state through the action of NADPH
cytochrome c P450 reductase. CO is released by elimination of the amethene bridge of the porphyrin
ring. Further degradation of biliverdin to bilirubin occurs through the action of biliverdin reductase.
Biliverdin complexes with iron until its final release (Calabrese et al., 2003a). HO is present in various
tissues with the highest activity in the brain, liver, spleen, and testes. There are three isoforms of HO, HO1
or inducible isoform (Calabrese et al., 2004b), HO2 or constitutive isoform (Ewing and Maines, 1992;
Hon et al., 2000), and the recently discovered HO3, cloned only in rat to date (Scapagnini et al., 2002c).
They are all products of different genes and, unlike HO3, which is a poor heme degrading catalyst,
both HO1 and HO2 catalyze the same reaction (i.e., degradation of heme) but differ in many respects
and are regulated under separate mechanisms. The similarity between HO1 and HO2 consists of a
common 24 amino acid domain (differing in just one residue) called the HO signature, which renders
both proteins extremely active in their ability to catabolize heme (Calabrese et al., 2004b). They have
different localization, similar substrate and cofactor requirements, while presenting different molecular
weights. They also display different antigenicity, electrophoretic mobility, inducibility, as well as suscepti-
bility to degradation. The proteins for HO1 and HO2 are immunologically distinct and, in humans, the
two genes are located on different chromosome arms i.e., 22q12 for HO1 and 16q13.3 for HO2 (Ewing
and Maines, 1992).
Various tissues have different amounts of HO1 and HO2. Brain and testes have a predominance of
HO2, whereas HO1 predominates in the spleen. In the lung not subjected to oxidative stress more than
70% of HO activity is accounted for by HO2, whereas in the testes the pattern of HO isoenzyme expression
differs according to the cell type, although HO1 expression predominates after heat shock. This also occurs
in the brain tissue, where HO isoforms appear to be distributed in a cellspecific manner, and HO1
distribution is widely apparent after heat shock or oxidative stress. Although previous reports from us and
other groups have not found detectable levels of HO1 protein in the normal brain (Ewing and Maines,
1992), we have recently demonstrated that HO1 mRNA expression is physiologically detectable in the brain
and shows a characteristic regional distribution, with high level of expression in the hippocampus and
cerebellum (Scapagnini et al., 2002c). This evidence may suggest the possible existence of a cellular reserve
of HO1 transcripts quickly available for protein synthesis and a posttranscriptional regulation of its
expression.
118 6 Aging and oxidative stress response in the CNS

HO isoenzymes are also seen to colocalize with different enzymes depending on the cell type. In the
kidney, HO1 colocalizes with erythropoietin, whereas in smooth muscle cells HO1 colocalizes with NOS.
In neurons, HO2 colocalizes with NOS, whereas the endothelium exhibits the same isoform that coloca-
lizes with NOS III. The cellular specificity of this pattern of colocalization lends further support to the
concept that CO may serve a function similar to that of NO. Furthermore, the brain expression pattern
shown by HO2 protein and HO1 mRNA overlaps with the distribution of guanylate cyclase, the main CO
functional target (Coceani et al., 1997).
HO3, the third isoform of HO, shares a high homology with HO2, both at the nucleotide (88%) and
protein (81%) levels. Both HO2 and HO3, but not HO1, are endowed with two CysPro residues
considered the core of the hemeresponsive motif (HRM), a domain critical for heme binding but not
for its catalysis (Hon et al., 2000).
Although the biological properties of this isoenzyme still remains to be elucidated, the presence of two
HRM motifs in its amino acidic sequence might suggest a role in cellular heme regulation (Maines, 2000).
Studying the HO3 mRNA sequence (GenBank accession no.: AF058787), we have observed that its 50
portion corresponds to an L1 retrotransposon sequence, a member of a family of retrotransposons recently
found to be involved in evolutionary mechanisms (Kazazian, 2000). On the basis of the close similarity to a
paralogous gene (HO2) and on the preliminary data from our group demonstrating the absence of introns
in the HO3 gene (Scapagnini et al., 2002c), it is possible that this 50 -portion could have originated from the
retrotransposition of the HO2 gene. In addition, this genetic mutation in the rat may represent a species
specific event since no other sequence in the public databases match that of the rat HO3.
Induction of HO1 gene could be used for clinical diagnosis. However, the length of the GT polymor-
phism in the promoter of the gene encoding HO1 that regulates the magnitude of the HO1 response to a
given stress signal can render this approach difficult for those individuals with long GT repeats, which are
associated with low HO1 responsiveness. This polymorphism appears to be of functional significance in
the short repeats, which are associated with high responsiveness and seem to be also associated with lesser
likelihood of restenosis after angioplasty (Otterbein et al., 2003a).

3.9 Regulation of HO Genes

Coupling of metabolic activity and gene expression is fundamental to maintain homeostasis. Heme is an
essential molecule that plays a central role as the prosthetic group of many heme proteins in reactions
involving molecular oxygen, electron transfer, and diatomic gases. Although heme is integral to life, it is
toxic because of its ability to catalyze the formation of ROS, and consequently oxidative damage to cellular
macromolecules. In higher eukaryotes, toxic effects of heme are counteracted by the inducible HO1 system
(Maines, 2000). As in the classic theory of metabolic control, expression of HO1 is induced by the substrate
heme (Kanakiriya et al., 2003). In addition, expression of HO1 is robustly induced in mammalian cells by
various proinflammatory stimuli, such as cytokines, heavy metals, heat shock, and oxidants that induce
inflammatory damage (Keyse and Tyrrell, 1989). Thus, HO1 is an essential antioxidant defense enzyme
that converts toxic heme into antioxidants and is fundamental for coping with various aspects of cellular
stress and for regulating iron metabolism (Poon et al., 2004a). In clinical conditions, HO1 expression has
been associated with increased resistance to tissue injury, thus leading to a gene therapy approach employ-
ing HO1 (Otterbein, 2002).
HO2 gene consists of five exons and four introns. HO2 has a molecular weight of 34 kDa and exhibits
40% homology in amino acid sequence with HO1. It is generally considered a constitutive isoenzyme;
however, in situ hybridization studies have shown increases in HO2 mRNA synthesis, associated with
increased HO2 protein and enzyme activity in the neonatal rat brain after treatment with corticosterone
(Raju et al., 1997). The organization of the HO2 gene needs to be fully elucidated, although a consensus
sequence of the glucocorticoid response element (GRE) has been demonstrated in the promoter region of
the HO2 gene (Liu et al., 2000). In addition, endothelial cells treated with the NOS inhibitor LNAME and
HO inhibitor zinc mesoporphyrin exhibited a significant upregulation of HO2 mRNA.
 Aging and oxidative stress response in the CNS 6 119

HO1 gene is induced by a variety of factors, including metalloporphyrins and hemin, as well as
ultraviolet A (UVA) irradiation, hydrogen peroxide, prooxidant states, or inflammation (Tyrrell, 1999).
This characteristic inducibility of HO1 gene strictly relies on its configuration: the 6.8kb gene is organized
into four introns and five exons. A promoter sequence is located approximately 28 base pairs upstream from
the transcriptional site of initiation. In addition, different transcriptional enhancer elements such as the
HSE and metal regulatory element reside in the flanking 50 region. Also, inducerresponsive sequences have
been identified in the proximal enhancer located upstream the promoter and, more distally, in two
enhancers located 4 kb and 10 kb upstream the initiation site (HillKapturczak et al., 2003). The molecular
mechanism that confers inducible expression of HO1 in response to numerous and diverse conditions has
remained elusive. One important clue has recently emerged from a detailed analysis of the transcriptional
regulatory mechanisms controlling the mouse and human HO1 genes. The induction of HO1 is regulated
principally by two upstream enhancers, E1 and E2 (Sun et al., 2002). Both enhancer regions contain multiple
stress (or antioxidant)responsive elements (StRE, also called ARE) that also conform to the sequence of the
Maf recognition element (MARE) (Martin et al., 2004) with a consensus sequence (GCnnnGTA) similar to
that of other antioxidant enzymes (Balogun et al., 2003). There is now evidence to suggest that heterodimers
of NFE2related factors 2 (Nrf2) and one or another of the small Maf proteins (i.e., MafK, MafF and MafG)
are directly involved in induction of HO1 through these MAREs (Gong et al., 2002). A possible model,
centered on Nrf2 activity, suggests that the HO1 locus is situated in a chromatin environment that is
permissive for activation. Since the MARE can be bound by various heterodimeric basic leucine zipper
(bZip) factors including NFE2, as well as several other NFE2related factors (Nrf1, Nrf2, and Nrf3), Bach,
Maf, and AP1 families (Sun et al., 2002), random interaction of activators with the HO1 enhancers
would be expected to cause spurious expression. This raises a paradox as to how cells reduce transcriptional
noise from the HO1 locus in the absence of metabolic or environmental stimulation. This problem could
be reconciled by the activity of repressors that prevent nonspecific activation. One possible candidate is the
heme protein Bach1, a transcriptional repressor endowed with DNAbinding activity, which is negatively
regulated upon binding with heme. Bach1heme interaction is mediated by evolutionarily conserved heme
regulatory motifs (HRM), including the cysteineproline dipeptide sequence in Bach1. Hence, a plausible
model accounting for the regulation of HO1 expression by Bach1 and heme is that expression of HO1
gene is regulated through antagonism between transcription activators and the repressor Bach1. While
under normal physiological conditions expression of HO1 is repressed by Bach1/Maf complex, increased
levels of heme displace Bach1 from the enhancers and allow activators, such as heterodimer of Maf with
Nrf2, to promote the transcription of HO1 gene (Sun et al., 2002). To our knowledge, the Bach1/HO1
system is the first example in higher eukaryotes that involves a direct regulation of a transcription factor for
an enzyme gene by its substrate. Thus, regulation of HO1 involves a direct sensing of heme levels by Bach1
(by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate
effects repressoractivator antagonism.
The promoter region also contains two metalresponsive elements, similar to those found in the
metallothionein1 gene, which respond to heavy metals (cadmium and zinc) only after recruitment of
another fragment located upstream, between 3.5 and 12 kbp (CdRE). In addition, a 163bp fragment
containing two binding sites for HSF1, which mediates HO1 transcription, are located 9.5 kb upstream of
the initiation site (Balogun et al., 2003). The distal enhancer regions are important in regulating HO1 in
inflammation, as they have been demonstrated to be responsive to endotoxin. In the promoter region, there
also resides a 56 bp fragment that responds to the STAT3 acutephase response factor, involved in the
downregulation of HO1 gene induced by glucocorticoid (Raju et al., 1997).

3.10 Glutathione, Thiol Redox State, and RNS: Intracellular Modulators of

HO1 Expression
The major regulator of intracellular redox state is glutathione, a cysteinecontaining tripeptide with
reducing and nucleophilic properties. This tripeptide (GSH) is essential for the cellular detoxification of
120 6 Aging and oxidative stress response in the CNS

ROS in brain cells (Butterfield et al., 2002b). A compromised GSH system in the brain has been associated
with the oxidative stress occurring in neurological diseases (Butterfield et al., 2002c). Recent data demon-
strate that, besides intracellular functions, GSH has also important extracellular functions in the brain. In
this respect astrocytes appear to play a key role in the GSH metabolism of the brain, since astroglial
GSH export is essential for providing GSH precursors to neurons (Dringen and Hirrlinger, 2003). Of
the different brain cell types studied in vitro only astrocytes release substantial amounts of GSH. In
addition, during oxidative stress astrocytes efficiently export glutathione disulfide (GSSG). The multi-
drugresistance protein 1 participates in both the export of GSH and GSSG from astrocytes (Dringen and
Hirrlinger, 2003). Glutathione exists in either a reduced (GSH) or oxidized (GSSG) form and participates in
redox reactions through the reversible oxidation of its active thiol. In addition, GSH acts as a coenzyme of
numerous enzymes involved in cell defense. In unstressed cells the majority (99%) of this redox regulator is
in the reduced form, and its intracellular concentration is between 0.5 and 10 mM depending on the cell
type (Drake et al., 2003b). Depletion of glutathione has been shown to occur in conditions of moderate or
severe oxidative stress and has been associated with increased susceptibility to cell damage (Calabrese et al.,
2000b; Balogun et al., 2003; Catania et al., 2003). There is now evidence to suggest that a direct link between
a decrease in glutathione levels by oxidant stress and rapid upregulation of HO1 mRNA and protein exist
in a variety of cells, including the rat brain, human fibroblasts, endothelial cells, and rat cardiomyocytes
(Foresti and Motterlini, 1999). This finding is supported by the fact that Nacetylcysteine (a precursor of
glutathione) abolishes oxidative stressmediated induction of HO1 gene (Foresti et al., 1997, 2001). In
addition, increased production of NO and RSNO can also lead to changes in intracellular glutathione.
In astroglial cell cultures, stimulation of iNOS by exposure to LPS and IFNg decreases total glutathione, while
increasing GSSG, and this effect was abolished by pretreatment of glial cells with NOS inhibitors (Calabrese
et al., 2000b). Moreover, elevation of intracellular glutathione prior to exposure of endothelial cells to NO
donors almost completely abolishes activation of the HO pathway, which suggests that thiols can antagonize
the effect of NO and NOrelated species on HO1 induction (Calabrese et al., 2000b). We have recently
demonstrated in endothelial cells subjected to hypoxia that induction of HO1 is associated with a decrease in
the GSH/GSSG ratio and with an increase in RSNO levels resulting from early induction of iNOS (Motterlini
et al., 2000a). This implies that in conditions of low oxygen availability both oxidative and nitrosative reactions
may serve as a trigger for induction of the HO1 gene (Motterlini et al., 2000b). All these evidence corroborate
the notion that generation of ROS and RNS are important signal transduction mechanisms linking HO1
activation to cell stress tolerance (Mancuso et al., 2003; Pocernich et al., 2005).

3.11 Heme Oxygenase in Brain Function and Dysfunction

In the brain, the HO system has been reported to be very active and its modulation seems to play a crucial
role in the pathogenesis of neurodegenerative disorders. The HO pathway, in fact, has been shown to act as
a fundamental defensive mechanism for neurons exposed to an oxidant challenge (Chen et al., 2000).
Induction of HO occurs together with the induction of other Hsps in the brain during various experimental
conditions including ischemia (Dore, 2002). Injection of blood or hemoglobin results in increased
expression of the gene encoding HO1, which has been shown to occur mainly in microglia throughout
the brain (Calabrese et al., 2001a). This suggests that microglia take up extracellular heme protein following
cell lysis or hemorrhage. Once in the microglia, heme induces the transcription of HO1. In human
brains following traumatic brain injury, accumulation of HO1 microglia/macrophages at the hemor-
rhagic lesion was detected as early as 6 h post trauma and was still pronounced after 6 months (Beschorner
et al., 2000).
There is now evidence that oxidative stress contributes to secondary injury after spinal cord trauma.
Induction of HO1 in the hemisected spinal cord, a model that results in reproducible degeneration in the
ipsilateral white matter, was found in microglia and macrophages from 24 h to at least 42 days after injury.
Within the first week after injury, HO1 was induced in both the grey and the white matter. Thereafter, HO1
expression was limited to degenerating fiber tracts. Interestingly, Hsp70 was consistently colocalized with
HO1 in the microglia and macrophages, indicating that longterm induction of HO1 and Hsp70 in
 Aging and oxidative stress response in the CNS 6 121

microglia and macrophages occur long after traumatic injury and are correlated with Wallerian degeneration
and remodeling of surviving tissue (Mautes et al., 2000).
Since the expression of Hsps is closely related to that of APP, Hsps proteins have been studied in the
brain of patients with AD. Significant increases in the levels of HO1 have been observed in AD brains in
association with neurofibrillary tangles (NFTs) (Takeda et al., 2000), and also HO1 mRNA was found
increased in AD neocortex and cerebral vessels (Premkumar et al., 1995). HO1 increase was not only in
association with NFTs but also colocalized with senile plaques and glial fibrillary acidic proteinpositive
astrocytes in AD brains (Schipper et al., 2000). It is conceivable that the dramatic increase in HO1 in AD
may be a direct response to increased free heme associated with neurodegeneration and an attempt to
convert the highly damaging heme into the antioxidants biliverdin and bilirubin (Calabrese et al., 2004b).
Upregulation of HO1 in the substantia nigra of patients with PD has been demonstrated. In these
patients, nigral neurons containing cytoplasmic Lewy bodies exhibited in their proximity maximum HO1
immunoreactivity (Ewing and Maines, 1992). New evidence showed a specific upregulation of HO1 by
oxidative stress in the nigral dopaminergic neurons (Poon et al., 2004a).
Multiple sclerosis (MS) is a common, often disabling disease of the CNS. It has been suggested that
inappropriate stress response within the CNS could influence both the permeability of the bloodbrain
barrier and the expression of Hsps, thereby initiating the MS lesion (Aquino et al., 1977; Maines, 2000).
However, cytokines, immunoglobulins, and complement complexes may elicit a survival response in the
oligodendrocytes, involving the induction of endogenous Hsps and other protective molecules, which
indicates that redox systems and therefore the oxidant/antioxidant balance in these cells are of great
importance in MS (Calabrese et al., 1994, 1998, 2002a, 2003b; Bagasra et al., 1995). The expression of
HO1 is increased in the CNS of mice and rats with experimental allergic encephalomyelitis (EAE), an
animal model of MS (Catania et al., 2003). To investigate the role of HO1 in EAE, SnPPIX was
administered to SJL mice during active disease. SnPPIX (200 mmol/kg) attenuated clinical scores, weight
loss, and some signs of pathology in comparison to vehicle treatment. Glutathione levels were greater in
treated EAE mice than in those receiving vehicle, indicating lower oxidative stress in the former group.
These data suggest that inhibition of HO1 attenuated disease and suppressed freeradical production
(Chakrabarty et al., 2003). On the contrary, in another study, high expression of HO1 in lesions of EAE was
enhanced by hemin treatment, a procedure that resulted in the attenuation of clinical signs of pathology,
whereas tin mesoporphyrin, an inhibitor of HO1, markedly exacerbated EAE (Liu et al., 2001). These
results strongly suggest that endogenous HO1 plays an important protective role in EAE, and that targeted
induction of HO1 overexpression may represent a new therapy for the treatment of MS. We have recently
shown that thiol disruption and nitrosative stress are associated in active MS with induction of Hsp70 and
HO1 in central and peripheral tissues of MS patients and that acetylcarnitine was able to counteract nitrosative
stressmediated damage, an effect associated with enhancement of Hsp stress signaling (Poon et al., 2004a).
All these findings can open up new therapeutic perspectives, as molecules activating these defense mechanisms
appear to be possible candidates for novel neuroprotective strategies (Calabrese et al., 2000c).

3.12 Bilirubin and Biliverdin: An Endogenous Antioxidant System

Supraphysiological levels (>300 mM) of nonconjugated bilirubin, as in the case of neonatal jaundice,
are associated with severe brain damage. This is a plausible reason whereby bilirubin has generally
been recognized as a cytotoxic waste product. However, only in recent years its emerging role as a
powerful antioxidant has received wide sustain. The specific role of endogenously derived bilirubin as
a potent antioxidant has been demonstrated in hippocampal and cortical neurons, where accumulation of
this metabolite owing to phosphorylativedependent enhancement of HO2 activity protected against
hydrogen peroxideinduced cytotoxicity (Stocker et al., 1987; Mancuso et al., 2003). Moreover, nanomolar
concentrations of bilirubin resulted in a significant protection against hydrogen peroxideinduced toxicity
in cultured neurons as well as in glial cells following experimental subarachnoid hemorrhage. In addition,
neuronal damage following middle cerebral artery occlusion was substantially worsened in HO2 lacking
mice (Dore et al., 2000). Bilirubin can become particularly important as a cytoprotective agent for tissues
122 6 Aging and oxidative stress response in the CNS

with relatively weak endogenous antioxidant defenses such as the CNS and the myocardium. Interestingly,
increased levels of bilirubin have been found in the cerebrospinal fluid in AD, which may reflect the increase
of degraded bilirubin metabolites in the AD brain, derived from the scavenging reaction against chronic
oxidative stress (Kimpara et al., 2000). Similarly, a decreased risk for coronary artery disease is associated
with mildly elevated serum bilirubin, with a protective effect comparable to that of HDL cholesterol (Dore
et al., 2000). The most likely explanation for the potent neuroprotective effect of bilirubin is that a redox
cycle exists between bilirubin and biliverdin, the major oxidation product of bilirubin. In mediating the
antioxidant actions, bilirubin would be transformed to biliverdin, then rapidly converted back to bilirubin
by biliverdin reductase, which in the brain is present in large functional excess, suggesting a mechanism to
amplify the antioxidant effect (Poon et al., 2004b). Remarkably, the rapid activation of HO2 by protein
kinase C (PKC) phosphorylation parallels the availability of nNOS. Both are constitutive enzymes localized
in neurons, and nNOS is activated by calcium entry into cells binding to calmodulin. Similarly, PKC
phosphorylation of HO2 and the transient increase in intracellular bilirubin would provide a way for a
rapid response to calcium entry, this being a major activator of PKC. Recent evidence has demonstrated that
bilirubin and biliverdin possess strong antioxidant activities toward peroxyl radical, hydroxyl radical, and
hydrogen peroxide. Exposure of bilirubin and biliverdin to agents that release NO or nitroxyl resulted in a
concentrationand timedependent loss of bilirubin and biliverdin. Increasing concentrations of thiols
prevented bilirubin and biliverdin consumption by nitroxyl, indicating that bile pigments and thiol groups
can compete and/or synergize the cellular defence against NOrelated species. In view of the high inducibil-
ity of heme oxygenase1 by NOreleasing agents in different cell types, these findings highlight novel
antinitrosative characteristics of bilirubin and biliverdin, suggesting a potential function for bile pigments
against the damaging effects of uncontrolled NO production (Kaur et al., 2003).

4 Caloric Restriction and Endogenous Oxidative Stress: Relevance to

Aging and Cell Survival

Caloric restriction in mammals has been recognized as the best characterized and most reproducible
strategy for extending maximum life span, retarding physiological aging, and delaying the onset of age
related pathological situations. The overwhelming majority of studies using caloric restriction have used
shortlived rodent species, although current work using monkeys should reveal whether this paradigm is
also relevant for manipulating the rate of primate aging. The mechanisms by which restricted calorie intake
modifies the rate of aging and cellular pathology have been the subject of much controversy, although an
attenuation of accumulating oxidative damage appears to be a central feature (Hursting et al., 2003). A
major effect of calorierestricted feeding now appears to be on the rate of production or leak of free radicals
from mitochondrial sites, although the details of the adaptation and the signaling pathway that induce this
effect are currently unknown. General consensus, however, has been achieved that caloric restriction feeding
regimes reduce the rate of accrual of oxidative damage as measured by lipid peroxidation, nuclear and
mtDNA damage, and protein carbonyl formation. An analysis of published studies that used a degree of
food restriction in the range of 40%50% ad libitum intake revealed a significant positive correlation
between survival parameters, such as mean, maximum, and average survival time, and duration of caloric
restriction. The longer the animals are maintained on low calorie intake during the postweaning period of
the life span, the greater is the survival (Mattson et al., 2002). It is unclear whether caloric restriction
protects against random oxidative damage per se or is protective for those vulnerable proteins of key
pathways, such as those containing ironsulfur centers of the ETC or DNAbinding signaling proteins. This
is directly related to the question whether oxidative damage in genomic and mtDNA is primarily random as
a function of age or whether there is a specific pattern of distribution of ROS which may vary depending on
the tissue or the state of the cell cycle within any particular cell. It is generally accepted that agerelated
accrual of ROSinduced damage represents a balance between generation and defences, such as antioxidant
enzymes, repair systems, and turnover. It has been demonstrated that caloric restriction reduces cellular
injury and improves heat tolerance of old animals by lowering radical production and preserving cellular
ability to adapt to stress through antioxidant enzyme induction and translocation of these proteins to the
 Aging and oxidative stress response in the CNS 6 123

nucleus (Calabrese et al., 2001a). It has been also demonstrated that mitochondria from calorierestricted
animals produce less ROS per nanomole of O2 during state 4 respiration, and recent work on ETC
complexes suggests a modification in the Km for complex III associated with a retention of highaffinity
binding sites for complex IV as a possible mechanism operating in reducing superoxide generation
(Mattson et al., 2003). It is conceivable that low calorieinduced changes in unsaturated fatty acid
composition of the mitochondrial membranes not only may protect against ROSinduced lipid peroxida-
tion but also may influence the binding properties of ETC proteins embedded in the membrane and the
related transport processes. However, several questions need to be addressed such as the signaling pathway
underlying the adaptive responses triggered by caloric restriction, or the effect of chronic caloric restriction
on either the bioenergetics of individual mitochondria or the mitochondrial number and turnover rate.
Highdensity oligonucleotide array studies have recently provided compelling evidence that aging results in
a differential gene expression pattern indicative of a marked stress response associated with lower expression
of metabolic and biosynthetic genes, and also, these alterations are either completely or partially prevented
by caloric restriction. In addition, the transcriptional patterns of calorierestricted animals suggest that
caloric restriction retards the aging process by causing a metabolic shift toward increased protein turnover
and decreased macromolecular damage (Martin et al., 2003; Strauss, 2003; Calabrese et al., 2004b).

4.1 Therapeutic Potential of Nutritional Antioxidants

Recently, considerable attention has been focused on identifying dietary and medicinal phytochemicals
that can inhibit, retard, or reverse the multistage pathophysiological events underlying AD pathology
(Butterfield et al., 2002a). Spices and herbs contain phenolic substances with potent antioxidative and
chemopreventive properties (Scapagnini et al., 2002d). The active antioxidant principle in Curcuma longa, a
coloring agent and food additive used in Indian culinary preparations, has been identified as curcumin
(diferuloylmethane). Because of the presence in its structure of two electrophilic a, bunsaturated carbonyl
groups which, by virtue of the Michael reaction, can react with nucleophiles such as glutathione, curcumin
has the potential to inhibit lipid peroxidation and effectively to intercept and neutralize reactive oxygen and
NObased free radicals (Poon et al., 2004a). This agent is a potent inhibitor of tumor initiation in vivo and
possesses antiproliferative activities against tumor cells in vitro (Butterfield et al., 2002b). Recent epidemi-
ological studies (Ganguli et al., 2000) have raised the possibility that this molecule, as one of the most
prevalent nutritional and medicinal compounds used by the Indian population, is responsible for the
significantly reduced (4.4fold) prevalence of AD in India compared with the United States. On the basis of
these findings, compelling evidence has been provided that dietary curcumin given to an Alzheimer
transgenic APPSw mouse model (Tg2576) for 6 months resulted in a suppression of indices of inflamma-
tion and oxidative damage in the brain of these mice (Lim et al., 2001). Furthermore, in a human
neuroblastoma cell line it has recently been shown that curcumin inhibits NFkB activation, effectively
preventing neuronal cell death (Poon et al., 2004a). Remarkably, recent evidence has demonstrated that
curcumin is a potent inducer of HO1 in vascular endothelial cells (Balogun et al., 2003). We have also
recently demonstrated in astroglial cells the role of caffeic acid phenethyl ester (CAPE), an active compo-
nent of propolis, as a novel HO1 inducer (Scapagnini et al., 2002d). The similarity of CAPE to curcumin is
striking because CAPE is also a Michael reaction acceptor, endowed with antiinflammatory, antioxidant,
and anticancer effects (Butterfield et al., 2002a). These agents all appear capable of transcriptionally
activating a gene battery that includes antioxidant enzymes and HO (DinkovaKostova et al., 2001).
Gene induction occurs through the antioxidantresponsive element (ARE) (Alam, 2002; Alam and Cook,
2003). Thus, increased expression of genes regulated by the ARE in cells of the CNS may provide protection
against oxidative stress.

5 Major CNS Disorders Associated with ROS/RNSMediated Damage

The major CNS disorders associated with ROS/RNSmediated damage are listed in > Table 62.
124 6 Aging and oxidative stress response in the CNS

. Table 62
Neurodegenerative disorders associated with freeradical damage
1. Amyotrophic lateral sclerosis
2. Alzheimers disease
3. Parkinsons disease
4. Multiple sclerosis
5. Friedreichs ataxia
6. Downs syndrome
7. Huntingtons disease
8. Ischemia/reperfusion

5.1 Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a remarkably debilitating disease with inevitable lethal consequences.
It typically affects adults in midlife with progressive paralysis and causes death generally within 5 years. It is
characterized by degeneration of the motor neurons. These neural components include the anterior horn
cells of the spinal cord, motor nuclei of the brain stem, particularly the hypoglossal nuclei, and the upper
motor neurons of the cerebral cortex. ALS is currently untreatable and the pathogenesis is unknown,
although numerous possible etiologies have been studied including viral, immunologic, and metabolic.
However, none of these is considered a serious etiological candidate. On the other hand, the pathogenetic
role of oxidative stress has emerged as a distinct possibility. Recent data from a multicenter study indicate
that some but not all cases of inherited ALS arise because of mutations in the gene encoding the cytosolic
form of Cu/ZnSOD (Calabrese et al., 2004b). Familial ALS patients heterozygous for SOD mutations have
less than 50% of normal SOD activity in their erythrocytes and brains. This defective SOD gene is on
chromosome 21 (the gene for mtSOD is on chromosome 6). Thus, the implication is that the degeneration
of motoneurons in ALS may be initiated by oxidative freeradical damage. An alternative hypothesis is that
the mutation might impart harmful properties to the enzyme. This gainoffunction theory is based
primarily on the fact that familial ALS is dominantly inherited, and thus only one copy of the enzyme is
required to cause the disease, which arise from the toxic influence exerted by the abnormal protein. In this
regard, transgenic mice containing extra copies of human Cu/ZnSOD and ALS mutations showed a
disorder closely resembling human ALS, whereas overexpression of normal Cu/ZnSOD alone did not
produce the disorder (Brwon, 1994). These studies suggest that the enzyme is endowed with neurotoxic
properties. What the neurotoxic function might be remains to be elucidated. With regard to the familial
form of the disease, one line of evidence suggests that interaction between NO and superoxide, by yielding
the powerful oxidant ONOO, might constitute the primary pathogenic event leading to protein nitration,
which slowly injures the motor neurons (Beckman et al., 1993). It is possible that the active site of SOD is
altered, allowing greater access of ONOO to the copper center, and so favoring the subsequent formation
of a nitroniumlike species which nitrosylates tyrosine residues (Heales et al., 1999). Immunocytochemistry
studies have also revealed, in the neurofilament aggregates associated with ALS, a close association between
SOD1 and NOS activity (Chou et al., 1996). Since light neurofilaments are rich in tyrosine, it is proposed
that nitrotyrosine formation occurs, which impairs neurofilament assembly and ultimately leads to
motoneuron death. Recently, increased nitrotyrosine immunoreactivity has been demonstrated in motor
neurons of both sporadic and familial ALS, suggesting that ONOOmediated oxidative damage may play a
role in the pathogenesis of both forms of the disease (Beal et al., 1997). Some evidence is now available to
suggest that mitochondrial dysfunction is a central event in the disease process. Thus, a significant decrease
in complex IV activity is reported in the spinal cord (ventral, lateral, and dorsal regions) of patients with
sporadic ALS (Fujita et al., 1996). In addition, studies with a transgenic mouse model of ALS also suggest
that axonal transport of organelles, in particular mitochondrial transport, is impaired and may be an
important factor in ALS (Collard et al., 1995).
 Aging and oxidative stress response in the CNS 6 125

5.2 Alzheimers Disease

AD affects over two million Americans and is the major cause of admission to nursing homes. AD, which
rarely occurs before the age of 50 years, usually becomes clinically apparent as a subtly impaired cognitive
function or as affectivity disturbance. With time there is a progressive memory loss and disorientation,
which eventually progresses into dementia. Although most cases are sporadic, 5%10% or more are
familial. Gross examination of the brain in AD shows a variable degree of cortical atrophy with narrowed
gyri and widened sulci most apparent in the frontal, parietal, and temporal lobes. Microscopically, the
features include NFTs, neurite (senile) plaques, amyloid angiopathy, granulovacuolar degeneration, and
Hirano bodies. Importantly, all of these changes are present in the brains of nondemented older individuals
but to a much lesser extent. The finding that choline acetyltransferase is decreased by 40%90% in the
cerebral cortex and hippocampus of patients with AD has led to the hypothesis that AD is consequence of a
deficit in the cholinergic system (Calabrese et al., 2001a). Several lines of evidence now support an
important role for freeradicalmediated event in the pathogenesis of the disease. Advanced glycosylation
end products (AGEs) are a family of complex posttranslationally modified proteins that are initiated by
condensation of reducing sugars with proteins amino groups via the Maillard reaction. It has become
evident that glycation of proteins occurs in vivo in aged individuals (Christen, 2000). Oxidative stress
increases the frequency of hydroxyl radicalinduced autoxidation of unsaturated membrane lipids. Reactive
aldehydes, resulting by metal ionmediated fragmentation of lipid hydroperoxides, can modify proteins
through alteration of proteinprotein interactions and intermolecular crosslinking. Age modifications and
oxidative stress mechanisms can synergistically accelerate protein damage (Butterfield, 2004). Several
potential sources of oxidative stress should be considered in the pathogenesis of AD. First, the concentration
of iron, a potent catalyst of oxyradical generation, is increased in NFTbearing neurons (Calabrese et al.,
2000a). Second, increased concentrations of iron would result in increased protein modifications, which are
catalyzed by metal ions and reducing sugars (Calabrese et al., 2000a). Third, microglia are activated and
increased in number in AD and represent a major source of free radicals (El Khoury et al., 1998). Fourth, the
increased lipid peroxidation and the resulting membrane disturbances, which are observed in degenerating
neurons and neurites, are expected to lead to an influx of calcium, which causes destabilization of the
cytoskeleton and activation of specific degradative enzymes (Markesbery, 1997; Drake et al., 2004). A
decrease of complex IV activity has been reported in the cerebral cortex of individuals who died of AD (Kish
et al., 1992). While the exact mechanism for this loss of activity is not clear, it is known that this enzyme
complex is particularly susceptible to oxidative damage (Butterfield, 2002). In addition, there is now
evidence to suggest that NO metabolism is affected in AD. The glialderived factor, S100b, which is
overexpressed in this condition, causes induction of iNOS in astrocytes associated with NOmediated
neuronal cell death in a coculture system (Hu et al., 1997). Furthermore, bamyloid is reported to activate
NOS in a substantia nigra/neuroblastoma hybrid cell line (Heales et al., 1999). Analysis of postmortem
material has revealed in AD brain the presence of tyrosine, as result of the reaction of ONOO and
nitrotyrosine residues in protein, which was not detectable in agematched control brains (Smith et al.,
1997). In addition, using antibodies specifically directed against iNOS, the presence of this isoform in
neurofibrillary tanglebearing neurons was demonstrated (Sayre et al., 2000). Despite evidence for activa-
tion of NO metabolism in AD, analysis of the CSF nitrite nitrate (stable end products of ONOO
degradation) concentration revealed levels in AD patients comparable to controls (Corregidor and De
Pasamonte, 1996). While this observation does not dismiss a role for NO/ONOO in the etiology of AD, it
implies that formation of RNS occurs at a level that not necessarily leads to a rise in CSF RNS concentration.
Amyloid betapeptide (Ab), the principal component of senile plaques and the major neuropathologi-
cal hallmark of AD, is considered to be central to the pathogenesis of AD. Ab is a 4042 amino acid peptide
that accumulates in the neuritic plaques in AD. The AD brain is under extensive oxidative stress (Butterfield
et al., 2002a). These two observations were joined by a model to potentially account for neurodegeneration
in AD brain: the Abassociated freeradical oxidative stress hypothesis of brain cell death in AD (Castegna
et al., 2004; Drake et al., 2004). In this model, Abassociated free radicals initiate lipid peroxidation, protein
oxidation, ROS formation, intracellular and mitochondrial Ca2 accumulation, and eventual death of
126 6 Aging and oxidative stress response in the CNS

neurons. A prediction of this model is that the antioxidant vitamin E should prevent or modulate these Ab
induced effects to neurons (Butterfield et al., 1997). Consistent with this model, this freeradical scavenger
was shown to block Abinitiated lipid peroxidation in cortical synaptosomes (Butterfield et al., 1999).
Further, protein oxidation induced by Ab in astrocyte cultures and assessed by increased protein carbonyl
content was abrogated by the more soluble form of vitamin E, trolox (Koppal et al., 1998). Vitamin E also
blocked Abinduced inhibition of transmembrane protein function, including ionmotive ATPases, glucose
and glutamate transporters, Gprotein coupled signal transduction, and the energyrelated enzyme creatine
kinase, and the methionine residue 35 of Ab142 and Ab140 was shown to be critical to the oxidative stress
properties of these peptides (Butterfield and Kanski, 2002). Human Ab142, expressed in vivo in transgenic
Caenorhabditis elegans nematodes, led to protein oxidation in the living animal, and methionine was
important in this process as well (Yatin et al., 1999).
A risk factor for AD is the presence of allele 4 of apolipoprotein E (apoE) (Roses, 1996). Synaptosomes
from apoEknockout mice, containing no gene for apoE, show increased susceptibility to oxidative stress
induced by Ab, (Lauderback et al., 2001), while synaptosomes from knockout mice containing human
apoE4 with no mouse background show significantly increased Abinduced oxidative stress compared with
synaptosomes from human apoE2 or apoE3 knockin mice (Lauderback et al., 2002). Thus, apoE may serve
an antioxidant function, but apoE4 may be less able than apoE2 or apoE3 to do so (Lauderback et al., 2002).
This notion was tested using 1monthold control and apoEdeficient mice. Both received dietary vitamin E
for 12 months. Vitamin Efed animals had better behavioral outcomes of spatial motor activity and
decreased levels of lipid peroxidation relative to apoEdeficient mice fed a normal diet (Veinbergs et al.,
2000). The sum of these studies suggests a decreased risk for and diminished oxidative stress in AD in
persons taking high dose dietary, or perhaps supplemental, vitamin E (and vitamin C to regenerate vitamin
E from the tocopherol radical).
Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation,
classically associated with the heatshock response, which is one form of stress response. Hsps are proteins
serving as molecular chaperones involved in the protection of cells from various forms of stress. Increasing
interest has focused on identifying dietary compounds that can inhibit, retard, or reverse the multistage
pathophysiological events underlying AD pathology. AD, in fact, involves a chronic inflammatory response
associated with both brain injury and Abassociated pathology. All of the above evidence suggests that
stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various
ageassociated alterations in cells, tissues, and organisms. Spice and herbs contain phenolic substances with
potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is
responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry
spice turmeric, has emerged as a strong inducer of the heatshock response. In light of this finding, curcumin
supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative
damage and amyloid pathology associated with AD (Butterfield et al., 2002a, c; Calabrese et al., 2004b).
Conceivably, dietary supplementation with vitamin E or with polyphenolic agents, such as curcumin
and its derivatives, can forestall the development of AD, consistent with a major metabolic component to
this disorder. Nutritional biochemical research is providing optimism that this devastating brain disorder of
aging may be significantly delayed and/or modulated.

5.3 Parkinsons Disease

PD is a progressive neurodegenerative disorder that increases in frequency after the age of about 50 years. The
major clinical disturbances in PD result from dopamine depletion in the striatum, because of nigral neuronal
loss. Although, a number of hypothesis, including defective DNA repair mechanisms, specific genetic defects,
viral disorder, lack of a neurotrophic hormones, or toxic compounds present in the environment, have been
proposed, none completely explains the cascade of events responsible for the cause and the course of the
disease. A large body of evidence supports the role of free radicals in the pathogenesis of the disease (Hyun et al.,
2003). Levels of lipid hydroperoxides are increased tenfold in the substantia nigra in PD (Hyun et al., 2002).
 Aging and oxidative stress response in the CNS 6 127

Decreased glutathione peroxidase and catalase activities associated to increased SOD activity leads to increased
levels of hydrogen peroxide (Dexter et al., 1991). This, in dopaminergic cells, is primarily produced by MAO
via deamination of dopamine and also nonenzymatically by autoxidation of dopamine. Hydrogen peroxide, by
reacting with reduced forms of transition metals, e.g., iron (II) or copper (I), gives rise to the powerful oxidant
hydroxyl radical, and oxidative damage to nigral membrane lipids, proteins, and DNA ensues. The role of iron
in brain oxidative injury has been extensively considered (Mattson et al., 2002). Dexter and coworkers (1991)
reported a 31%35% increase in the total iron content in parkinsonian SN compared with control tissue,
which was associated with decreased levels of the iron storage protein ferritin, contrasting with a signifi-
cant decrease of the levels of ironbinding protein in the CSF. A shift of iron II/iron III ratio in the
substantia nigra from almost 2:1 in the normal brain to 1:2 in the parkinsonian brain is also served (Li
and Dryhurst, 1997). Hence, a distinct possibility exists that excessive freeradical generation occurs in this
region, leading to the death of nigral neurons. In addition, substantia nigra is a dopaminerich brain area,
and catechols, including DOPA and dopamine, have been demonstrated to be cytotoxic in vitro, presum-
ably by formation of covalent bonds between their quinone forms and macromolecules of vital impor-
tance, primarily represented by thiol groups (Spencer et al., 2002). In fact, an intermediate in the
autoxidation of catechols to quinone is the free radical semiquinone. Both autoxidation steps generate
reduced forms of molecular oxygen such as superoxide anion and hydrogen peroxide which, in addition to
hydrogen peroxide produced by the MAOdependent catabolism of dopamine, contribute to maintain
considerable levels of the highly reactive hydroxyl radical, which on reacting with free thiol groups may
contribute to the decreased levels of GSH and a corresponding increase in GSSG found in the SN
(Calabrese et al., 2002b, 2004a). This is of special importance considering that nigral cells also contain
neuromelanin, a pigmented substance related to lipofuscin and derived from dopamine. Neuromelanin has
been demonstrated to have high affinity for iron III, and this ironmelanin interaction might have
pathogenetic implications. In fact, the synthesis of neuromelanins from dopamine is known to produce
more oxidative damage than the synthesis from other catecholamines (Spencer et al., 1994) and, in
addition, neuromelanins polymerize from pheomelanin in a process that requires cysteine for synthesis,
thus competing with gglutamyl cysteine synthetase which utilizes cysteine for GSH synthesis. Under these
circumstances the GSH system in the substantia nigra could result in a position of increased demand and
decreased synthetic capability, and hence contribute to the high vulnerability of this region to peroxidative
injury (Calabrese et al., 2001a, 2002b, 2004a). This is confirmed by the study of Perry and coworkers (1982),
which showed that GSH levels in the SN were significantly lower than in other brain regions. Moreover, a 40%
decrease in GSH in the SN of PD, associated with significant increase in oxidized glutathione, has been also
reported (Sian et al., 1994). Recently, it has been demonstrated in PD patients that the proportion of
dopaminergic neurons with immunoreactive NFkB in their nuclei was more than 70fold than that in control
subjects (Hunot et al., 1997). A possible relationship between the nuclear localization of NFkB in mesence-
phalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary
cultures of rat mesencephalon, where translocation of NFkB is preceded by a transient production of free
radicals during apoptosis induced by activation of the sphingomyelindependent signaling pathway with C2
ceramide (FranceLanord et al., 1997). Data suggest that this oxidantmediated apoptogenic transduction
pathway may play a role in the mechanism of neuronal death in PD (Schapira et al., 1990; Mc Naught et al.,
2001; Dawson and Dawson, 2002; Moore et al., 2003). Moreover, a potential role for excitotoxic processes in PD
has been strengthened by the observation that there appears to be a mitochondrially encoded defect in complex
I activity of the ETC (Schapira et al., 1990). An impairment of oxidative phosphorylation will enhance
vulnerability to excitotoxicity (Xin et al., 2000). Substantia nigra neurons possess NmethylDaspartate
receptors, and there are glutamatergic inputs into the substantia nigra from both the cerebral cortex and
the subthalamic nucleus. After activation of excitatory amino acid receptors, it has been suggested that there
is an influx of calcium followed by activation of nNOS, which can then lead to the generation of
peroxynitrite (Bechtold and Brown, 2003). Consistent with such a mechanism, studies of MPTP neurotox-
icity in both mice and primates have shown that inhibition of nNOS exerts neuroprotective effects, raising
the prospect that excitatory amino acid antagonists for nNOS inhibitors might be useful in the treatment of
PD (Dawson and Dawson, 1995, 2002).
128 6 Aging and oxidative stress response in the CNS

5.4 Multiple Sclerosis

MS is a common often disabling disease of the CNS. Although evidence indicates that MS is a complex trait
caused by interaction of genetic and environmental factors, little is known about its cause or the factors that
contribute to its unpredictable course (Risch and Merikangas, 1996). It is generally accepted that vascular
factors, metabolic alterations, virus infections of the CNS, or disturbed immune mechanisms are responsi-
ble for the cause and course of MS. The clinical symptoms of MS result from inflammatory damage to the
insulating myelin sheath of axons in the CNS and at later stages to axons themselves. A local autoimmune
process involving activation of TH cells against CNS protein components is likely to be crucial for this
development. Once triggered, the immune system attacks and destroys myelin and the myelinforming cells
(Calabrese et al., 1998). Evidence exists which indicate that oligodendrocytes and their secreted products
respond to the attack by immune cells through modulation of its metabolism and gene expression (Lindsey
et al., 1997). It has been also suggested that inappropriate stress response within the CNS could influence
both the permeability of the bloodbrain barrier and the expression of Hsps, thereby initiating the MS
lesion (Calabrese et al., 2002a). In addition, cytokines, immunoglobulins, and complement complexes may
elicit a survival response in the oligodendrocytes, involving the induction of endogenous Hsps and other
protective molecules, which indicates that redox systems and therefore the oxidant/antioxidant balance in
these cells are of great importance in MS (Calabrese et al., 2003b). A variety of studies support a role for
oxidative stress in MS. These include studies on increased serum peroxide levels in MS relative to control
(Toshniwal and Zarling, 1992; Calabrese et al., 1994). Patients with MS in acute exacerbation exhibit
significantly higher levels of pentane and exane (products of lipid peroxidation) in expired breath
compared with either MS patients in remission or control subjects (Toshniwal and Zarling, 1992).
Moreover, recent clinical and animal studies suggest that NO and its reactive derivative peroxynitrite are
implicated in the pathogenesis of MS (Bagasra et al., 1995). Patients dying with MS demonstrate increased
astrocytic iNOS activity as well as increased levels of iNOS mRNA and nitrotyrosine residues (Bagasra et al.,
1995; Cross et al., 1998). In EAE, both astrocytes and microglia express iNOS (Tran et al., 1997). All this is
consistent with the demonstration that NOderivative species are cytotoxic to oligodendrocytes and
neurons by inhibiting the mitochondrial respiratory chain (complex IIIII and IV) and certain key
intracellular enzymes (Stamler and Hausladen, 1998; Heales et al., 1999), thereby representing a critical
determinant in the etiology of the disease.
MS is a relatively common disease of the CNS, the course of which is often of a progressive but
relapsing/remitting nature. The clinical symptoms of MS during relapse (numbness, paralysis, blindness,
and a variety of others) are mainly due to conduction block of axonal electrical impulses, caused by a variety
of different molecular pathologies, including inflammation and demyelination (Calabrese et al., 2001a). A
local autoimmune process involving activation of glia is likely to be crucial in the development of this
damage (Tran et al., 1997).
Activated glia secrete RNS products of NO metabolism with superoxide radicals (O2
) to form
peroxynitrite anion (ONOO). At physiological pH, it protonates to its conjugate acid peroxynitrous
acid, which decomposes with a t1/2 of less than 1 s. One of the fastest reactions of ONOO is with
(3 to 5.8  104 M1 s1 at 37 C). Together with the high concentrations of CO2 (1.3 mM) and HCO 3
(25 mM) this reaction is the most probable pathway of ONOO decomposition in vivo (Calabrese
et al., 2003b).
RNS can cause nitrosative stress, which results in the destruction of myelin and (myelinforming)
oligodendrocyte cells (Calabrese et al., 2002a). A direct link between NO and the conduction block that
occurs in MS has been suggested, as NO donors cause reversible conduction block in both normal and
demyelinated axons of the central and peripheral nervous systems (Calabrese et al., 2002a). In addition,
conduction in demyelinated and early remyelinated axons is particularly sensitive to block by NO (Heales
et al., 1999). This may be due to the direct effects of NO on glutamatergic neurotransmission, as it has been
shown that NMDA receptor is inactivated by nitrosylation (Heales et al., 1999). Furthermore, the formation
of Snitrosoglutathione (GSNO) can cause GSH depletion, and hence trigger redoxdependent changes in
cellular signaling as well as modification of key intracellular enzymes, such as chain respiratory complex
activities (Gegg et al., 2003).
 Aging and oxidative stress response in the CNS 6 129

Recent clinical and animal studies also indicate that NO and ONOO play a central role in
the pathogenesis of MS (reviewed in Calabrese et al., 2001a). It has also been shown that in CSF and
plasma nitrite nitrate (stable end products of NO metabolism) levels are elevated in patients with MS
(Giovannoni, 1998).
ROS and RNS have a major role in the mediation of cell damage, and free sulfhydryl groups are vital in
cellular defence against endogenous or exogenous oxidants (Calabrese et al., 2000a, 2001b). The possible
links between MS and oxidant/antioxidant balance in cell perturbation may be suggested by several factors,
including increased incidence of MS in populations consuming high proportions of animal fat, (Calabrese
et al., 1994), increased malonaldehyde levels in blood, and decreased glutathione peroxidase activity in MS
erythrocytes, lymphocytes and granulocytes (Calabrese et al., 1998) and, in addition, an inappropriate
expression of Hsps on oligodendrocytes (Calabrese et al., 2002a). This last event could represent a possible
initiating factor at the level of MS lesions, capable of modulating the subsequent susceptibility or resistance
of cells to oxidative stress. Moreover, a decrease in sulfhydryl groups and increased amounts of lipid
peroxidation products have also been measured in the CSF and plasma of MS patients (Calabrese et al.,
2002a). Nitrosative stress in isolated astrocytes in vitro causes modifications in the endogenous thiol pool
associated with induction of Hsp32 or HO1, which is prevented by antioxidants, suggesting a biochemical
link between nitrosative stress, sulfhydryl function, and the heatshock pathway (Scapagnini et al., 2002d;
Calabrese et al., 2004b). In addition, this evidence suggests that redoxactive compounds such as glutathi-
one and the overall oxidant/antioxidant balance in the CNS are potentially of great importance in MS,
although as yet there has been few studies addressing the relationships between NO, ONOO, and
glutathione in MS. The chemical composition of human CSF is considered to reflect brain metabolism
(Thompson, 1988), and we have recently demonstrated in MS patients decreased levels of protein sulfhydryl
groups associated with an increase in RNS and peroxidative products (Calabrese et al., 2002a, 2003b). More
recently, we have provided experimental evidence that increased levels of RNS are present in the CSF of MS
patients, and this is associated with increased nitrosylation of sulfhydryl moieties. Our results are consistent
with evidence indicating increased protein nitrosylation in MS patients (Cross et al., 1997) and pose
intriguing implications regarding clinical manifestations in MS, which are potentially linked to a failure
of action potentials to propagate along damaged axons and involve inflammatory processes as primary
causative factors in addition to demyelination. In favor of this possibility is the evidence that NO donors are
capable of blocking conduction in rat demyelinated axons (Garthwaite et al., 2002). All this would suggest a
broader potential role for NO in the symptomatic manifestations of MS. Whether or not NO is central to
the pathogenesis of MS remains to be clarified, owing to its role of being a doubleedged sword..
Consistently, a recent study (Sellebjerg et al., 2002) has demonstrated an association between high CSF
levels of NO metabolites with severe disease activity in relapsing/remitting MS, and high concentrations of
NO metabolites were associated with more pronounced treatment responses after methylprednisolone
treatment. However, other studies have shown no significant correlation between NO metabolites and
disability score, disease progression index, MRI activity, and development of cortical atrophy on MRI.
(Yuceyar et al., 2001). We have also demonstrated in MS patients an increase in nitrosative stress, which was
associated with a significant decrease of both protein SH groups and GSH, with increased levels of GSSG
and nitrosothiols (Calabrese et al., 2002a). Interestingly, treatment of MS patients with acetylcarnitine
resulted in decreased CSF levels of NOreactive metabolites and protein nitration and in a significantly
higher content of both GSH and GSH/GSSG ratio. In addition, urinary nitrites, which were higher in MS
patients than in controls, decreased significantly after treatment with acetylcarnitine. Several studies have
shown the capability of carnitines to interfere with changes in oxidant/antioxidant balance and metabolism
induced by oxidants (Hagen et al., 1998a, b). Although, so far, the exact mechanisms of action of
acetylcarnitine are still unknown, current research points to its ability to enhance neuronal mitochondrial
bioenergetics (Calabrese and Rizza, 1999), which in turn may influence cellular oxidant/antioxidant balance
(Calabrese et al., 2002d). We have recently shown in astrocytes exposed to LPS and INFginduced
nitrosative stress that acetylcarnitine protects against cytokinemediated mitochondrial chain respiratory
complex impairment and the associated increase in protein and lipid peroxidation. The increase in
astroglial antioxidative potential observed after acetylcarnitine treatment involves a secondary line of
antioxidant defenses, represented by stressresponsive genes, such as HO1 and the mitochondrial Hsp60
130 6 Aging and oxidative stress response in the CNS

and SOD (Calabrese et al., 2005). Furthermore, as a brain energy enhancer, acetylcarnitine could improve
survival of damaged neurons (Scapagnini et al., 2002b), and metabolic studies conducted noninvasively in
humans with NMR indicate that acetylcarnitine helps the brain to maintain the constant supply of energy
needed for effective homeostasis.
MS is a progressive inflammatory neurodegenerative disease. However, despite increasing research
efforts and although several explanations have been proposed for destruction of myelin and oligodendro-
cytes in MS, there is still no proven mechanism of injury. The possibility of manipulating these complex
glial cell functions and controlling their pathologic interactions with immune cells probably will illuminate
how myelin damage can be contained and how the injured tissue can be repaired.

5.5 Friedreichs Ataxia

FA is an autosomal recessive neurodegenerative disorder involving both the central and peripheral nervous
systems. Patients also show a systemic clinical picture presenting heart disease and diabetes mellitus or
glucose intolerance. The disease is caused by mutations in the FA gene mapped on chromosome arm 9q13.
The product of the gene is frataxin, an 18kDa soluble mitochondrial protein with 210 amino acids. Crystal
structure suggests a new, not previously reported, protein fold (Durr et al., 1996). The most frequent
mutation is the expansion of a GAA trinucleotide repeat located within the first intron of the gene, and
represents 98% of the mutations. This triplet motif can adopt a triplehelical DNA structure that inhibits
transcription (Harding, 1981). The severity of the disease correlates directly with the number of triplet
units and consequent decrease in protein levels, with patients having frataxin levels ranging from 6% to
30% of that of normal subjects (Campuzano et al., 1996). The primary tissues affected in the disease include
the large sensory neurons in the dorsal root ganglia and the nucleus dentatus, as well as cardiac and
pancreatic cells. The progressive gait and limb ataxia, hypertrophic cardiomyopathy, and diabetes mellitus
found in FA patients are attributed to low levels of ATP produced in these energyintensive tissues (Durr
et al., 1996). Point mutations are described in compound heterozygous subjects with one expanded allele.
A twostep model of GAA normal alleles toward premutation alleles, which might generate further full
expanded mutations in the population with IndoEuropean ancestry, has been postulated. Clinical pheno-
type is variable and an inverse correlation with the GAA expansion size has been observed. Analysis of the
GAA triplet is a strong molecular tool for clinical diagnosis, genetic counseling, and prenatal diagnosis.
Many approaches have been undertaken to understand FA, but the heterogeneity of the etiologic factors
makes it difficult to define the clinically most important factor determining the onset and progression
of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed
protein metabolism and their interaction in a vicious cycle are central to FA pathogenesis. Brains of
FA patients undergo many changes, such as disruption of protein synthesis and degradation, classically
associated with the heatshock response, which is the most important form of stress response. The precise
sequence of events in FA pathogenesis is uncertain. The impaired intramitochondrial metabolism with
increased free iron levels and a defective mitochondrial respiratory chain will result in increased freeradical
generation, causing oxidative damage, which may be considered a possible mechanism that compromises
cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione
peroxidase and elevation of thiols, and in addition, decreased expression of frataxin protein is associated
with FA.
Recent studies have shown that frataxin acts as a chaperone for Fe(II) and a storage compartment
for excess iron (Babcock et al., 1997). This is consistent with the roles played by frataxin in iron export, FeS
cluster assembly, heme biosynthesis, and prevention of oxidative stress. Also, frataxin plays a direct role in the
mitochondrial energy activation and oxidative phosphorylation. Several model systems have been developed
in an effort to understand the disease. In mouse models, deletion of the frataxin gene results in embryonic
lethality (Radisky et al., 1999) while its selective inactivation in neuronal and cardiac tissues leads to
neurological symptoms and cardiomyopathy associated with mitochondrial ironsulfur clustercontaining
 Aging and oxidative stress response in the CNS 6 131

enzyme deficiencies and timedependent mitochondrial iron accumulation. In contrast, a model expressing
25%35% of wildtype frataxin levels, by virtue of a (GAA)230 expansion inserted in the first intron of the
mouse gene, has no obvious phenotype (Bradley et al., 2000).
Over the last 5 years, it has become clear that mitochondrial iron accumulation generates oxidative
stress and results in damage to critical biological molecules.
Studies using the budding yeast Saccharomyces cerevisiae have provided a further understanding of the
consequences of frataxin loss (Radisky et al., 1999). Deletion of the yeast frataxin homolog YFH1 results in a
tenfold increase in iron within the mitochondria along with increased ROS production (Lodi et al., 2002).
This leads to loss of mitochondrial function and the appearance of a petite phenotype in nearly all strains
that have been examined (Radisky et al., 1999). Bradley and colleagues (2000) demonstrated an impaired
oxidative phosphorylation system with severe and significant deficiencies of mitochondrial respiratory chain
complexes I and II/III and aconitase activity in cardiac muscle from patients with FA; mtDNA levels were
reduced in FA heart and skeletal muscle and increased iron deposition was present in FA heart, liver, and spleen
in a pattern consistent with a mitochondrial location. In addition, there is the appearance of nDNA damage
(Bradley et al., 2000). Moreover, aconitase deficiency is suggestive that oxidative stress may induce a self
amplifying cycle of oxidative damage associated with mitochondrial dysfunction, which may also contribute to
cellular toxicity. Iron deposition and enzyme deficiencies have been reported in postmortem heart and brain
tissues (Foury and Talibi, 2000) of FA patients. The role of oxidative damage in the pathogenesis of FA is also
supported by the finding that idebenone, an antioxidant similar to ubiquinone, can reduce myocardial
hypertrophy and also decrease markers of oxidative stress in FA patients (Lodi et al., 2002).
Upregulation of protein manganese superoxide dismutase (MnSOD) fails to occur in FA fibroblasts
exposed to iron. This finding, together with the absence of activation of the redoxsensitive factor NFkB,
suggests that NFkBindependent pathway which may not require freeradical signaling is responsible for
the reduced induction of MnSOD. This impairment could constitute both a novel defense mechanism
against ironmediated oxidative stress in cells with mitochondrial iron overload and, conversely, an
alternative source of free radicals that could contribute to the disease pathology. Iron chelator drugs and
antioxidant drugs have therefore been proposed for the treatment of FA. Drugs that reduce oxidative stress
have a limited effect on the progression of the disease pathology, probably because these cannot properly
remove iron accumulation. The potential role of iron chelator analogues (e.g., 2pyridylcarboxaldehyde
isonicotinoyl hydrazone (PCIH)) as agents to remove mitochondrial iron deposits have been recently under
investigation (Jauslin et al., 2003). These ligands have been specifically designed to enter and target
mitochondrial iron pools, which is a property lacking in desferrioxamine, the only chelator in widespread
clinical use. This latter drug may not have any beneficial effect in FA patients, probably because of its
hydrophilicity that prevents mitochondrial access. Indeed, standard chelation regimens will probably not
work in FA, as these patients do not exhibit gross iron loading. Considering that there is no effective
treatment for FA, it is essential that the therapeutic potential of iron chelators focusses on the mitochon-
drial iron pools as their primary target. Remarkably, in an in vitro model of regulated human frataxin
overexpression, it was shown that downregulation of the expression of mitogenactivated protein kinase
kinase 4 was associated with a decreased phosphorylation of cJun Nterminal kinase. In addition, to
understand whether this alteration might result in cell death, the caspase pathway was investigated in FA
cells, revealing in FA patients a significantly higher activation of caspase9 after serum withdrawal compared
with controls. These findings suggest the presence, in FA patient cells, of a hyperactive stresssignaling
pathway. The role of frataxin in FA pathogenesis could be explained, at least in part, by this hyperactivity.
Pilot studies have shown the potential effect of antioxidant therapy using idebenone or coenzyme Q10 with
vitamin E administration and provide a strong rationale for designing larger randomized clinical trials
(Lodi et al., 2003). There is now strong evidence to suggest that mitochondrially localized antioxidant
ameliorates cardiomyopathy in FA patients, as well other lipophilic antioxidants can protect FA cells from
cell death, indicating novel treatment strategies for FA and presumably for other neurodegenerative diseases
with mitochondrial impairment. Antioxidants targeted to mitochondria appear a promising approach to
effectively slow disease progression.
132 6 Aging and oxidative stress response in the CNS

5.6 Huntingtons Disease

HD is an autosomal dominant, completely penetrant inherited neurodegenerative disorder, characterized
by the insidious progression of severe neuropsychological and motor disturbances. The clinical manifesta-
tions of HD primarily involve psychiatric abnormalities, most commonly mood disturbances, followed by
the development of involuntary choreiform movements and dementia. Onset of the disease is typically
apparent in the fourth or fifth decade of life, and its long duration of up to 1520 years results in death as
consequence of complicating immobility. The principal neuropathological features of the disease are
marked atrophy, neuronal loss, and astrogliosis in the neostriatum. The genetic defect in HD has been
recognized in an abnormal expanded trinucleotide (CAG) repeat in a gene located on the short arm of
chromosome 4 that encodes a protein termed huntingtin, whose function, so far, remains to be
elucidated. Several lines of evidence indicate that a defect in mitochondrial energy metabolism might
underlie the pathogenesis of the selective neuronal death occurring in HD. Evidence of bioenergetic defects
in HD comes from in vivo imaging studies showing a marked hypometabolism, as revealed by PET analysis
of [18F]fluorodeoxyglucose (FDG) utilization, in the caudate and putamen of symptomatic HD patients
(Calabrese et al., 2004b). Recent studies have also identified cortical hypometabolism in symptomatic
HD. Alterations in cerebral glucose utilization predominantly reflect changes in neuronal terminal activity,
the principal site of energy consumption. Most of the ATP produced in the brain is used by energy
dependent pumps to restore transmembrane potential following synaptic transmission (Calabrese et al.,
2004b). Consequently, the marked hypometabolism observed in specific brain regions in HD can be related
to loss of synaptic density due to the marked atrophy occurring in these regions. This hypothesis has
gained further sustain from NMR studies indicating increased lactate concentrations in the basal ganglia of
HD patients (Jenkins et al., 1993). This increase well correlates with the duration of the disease, implying
that normal energy metabolism is progressively impaired by the disease process. This might arise by the fact
that when oxidative phosphorylation is no longer sufficient in supplying cellular energy demands, cells
resort to reducing pyruvate by NADH in order to recycle NAD for ATP production via the glycolytic
pathway. A pathogenic role for mitochondrial dysfunction in HD arises from in vivo biochemical studies in
postmortem brain tissue, which have evidenced defects in succinate dehydrogenase as well as pyruvate
dehydrogenase activities in the striatum of HD patients, and also these defects have been found as a
function of illness duration. Further evidence supporting a mitochondrial defect in HD has been provided
by an NMR spectroscopy study demonstrating 60% increase in pyruvate levels in the CSF, and 60%
reduction in the activities of complex II and III in the caudate of HD patients, compared with controls
(Browne, 1997).

5.7 Downs Syndrome

The most important pathological features of Downs syndrome (DS) are mental retardation and accelerated
aging. Numerous studies link both of these disturbances to freeradicalinduced damage (Calabrese et al.,
2000a). DS patients have an extra chromosome 21 (trisomy 21) and the recent assignment to chromosome
21 of the gene for Cu/ZnSOD together with the observation of increased SOD activity in red blood cells in
DS patients has directed the interest on the role played by freeradical species in the pathogenesis of the
disease (Calabrese et al., 2000a). DS patients have a very high predisposition to develop the characteristics of
AD. Therefore, DS patients provide a genetic model for investigating the role of oxidative stress in AD. In this
regard, transgenic mice expressing the human SOD gene preferentially localize SOD in the hippocampus,
which is the most vulnerable region in AD. However, these changes are not compensated by corresponding
increase in catalase or glutathione peroxidase activities. This provides one possible explanation why increase
in SOD activity might be detrimental. In addition, increased SOD activity results in decreased steadystate
levels of superoxide anion, which also plays a role in terminating the chain reactions of lipid peroxidation.
All these evidence highlight the importance of oxidant/antioxidant balance as a critical determinant, and
with this the conceivable possibility that the use of exogenous antioxidants can slow the progression of the
disease (Halliwell, 2002).
 Aging and oxidative stress response in the CNS 6 133

5.8 Ischemia/Reperfusion
Ischemic brain damage is accompanied by an energy deficiency state and selective neuronal loss (Heales
et al., 1999). Under such conditions, there is an increase in the extracellular concentration of glutamate,
which may be neurotoxic due to activation of nNOS. Excess NO generation, causing impairment of energy
metabolism and other metabolic processes, may also downregulate glutamate (NMDA) receptors, thereby
minimizing the effect of glutamate. In addition, NO can cause vasodilation, and hence increase cerebral
blood flow to the infarcted area (Heales et al., 1999). These effects may provide an explanation for the
contradictory results that have been obtained when nonspecific NOS inhibitors have been evaluated in
various models of ischemia. Reperfusion, following ischemia, may exacerbate the generation of oxidizing
species, particularly superoxide. In a model of graded ischemia, loss of brain mitochondrial function, at the
levels of complexes I, II, IIIII, and ATP synthetase, has been reported (Powell and Jackson, 2003).
Reperfusion was associated with restoration of activity of these mitochondrial components, followed
after 2 h by a dramatic loss of complex IV activity (Brooks et al., 2002). The exact mechanism for this
loss of complex IV activity is not known, but could involve the oxidative and/or nitrosative stressmediated
reactions (Brooks et al., 2002). In fact, ischemia is accompanied by the formation of gliotic scar, principally
comprised of reactive astrocytes, which in large amount express iNOS (Heales et al., 1999). Thus, excessive
generation of glialderived ONOO may constitute an important contributing factor to the mitochondrial
damage associated with ischemia. Loss of brain ATP levels and mitochondrial complex IIIII and IV activity
has been demonstrated in a rat model of perinatal asphyxia (Bolanos et al., 1998); in addition, administra-
tion of an NOS inhibitor to the mothers prevented impairment of brain energy metabolism in the hypoxic
pups (Bolanos et al., 1998). Notably, ischemic preconditioning, which has been demonstrated to increase
Hsp expression, preserves brain mitochondrial functions during middle cerebral artery occlusion (Zhang
et al., 2003).

6 Genetics of Human Longevity: Role of Vitagenes in Prolongation of

Healthy Life Span

The first half of the twentieth century has seen a rapid increase in the life expectancy of individuals in
industrialized nations because of improved sanitation, public health, housing, nutrition, medical technol-
ogy, and pharmaceuticals. The second half of this century has been characterized by a growing concern with
the challenge produced by the increasing prevalence of old people in the society. Aging is a very common
feature in living organisms and can be described as the total effect of those intrinsic changes in an organism
that adversely affect its vitality and render it more susceptible to the many factors that can cause death.
Typically, mortality rate accelerates with time, but it is not clear whether this effect is the result of external
or internal causes of death. The full extent of aging in a population becomes apparent when most important
external hazards are removed, such as captive or laboratory conditions, and average longevity is usually
greatly extended (Calabrese et al., 2001a). Even if an organism is immortal it has nonzero probability of
dying because of extrinsic causes such as starvation, predation, and accidents. The probability of survival
decreases in the course of life and, since natural selection is effective only through the reproductive output
of individuals, the strength of natural selection decreases with age (Calabrese et al., 2001a).
The first genetic theories on the evolution of aging were proposed in 1957 by Medawar and Williams
almost simultaneously to the mechanistic theories of aging, such as the freeradical and the somatic
mutation theory, suggested by Harman (1956) and Szilard (1977), respectively. A synthesis of evolutionary
and mechanistic theories occurred in 1977 within the framework of the soma theory of aging postulated by
Kirkwood (1977). This theory provides a direct connection between evolutionary and physiological aspects
of aging, by recognizing the primary importance of the allocation of metabolic energy resources between
growth, somatic maintenance, and reproduction. It is suggested that longevity is determined through the
setting of longevity assurance mechanisms so as to provide an optimal compromise between investments in
somatic maintenance (including stress resistance) and in reproduction. As a corollary, increasing mainte-
nance promotes the survival and longevity of the organism only at the expense of significant metabolic
134 6 Aging and oxidative stress response in the CNS

investments that could otherwise be used to accelerate processes such as growth and reproduction. The
disposable soma theory of the evolution of aging also proposes that a high level of accuracy is maintained
in immortal germ line cells, or alternatively, defective germ cells, if any, are eliminated. The evolution of an
increase in longevity in mammals may be due to a concomitant reduction in the rates of growth and
reproduction, the socalled essential life and an increase in the accuracy of synthesis of macromolecules.
The theory can be tested by measuring accuracy in germ line and somatic cells and also by comparing
somatic cells from mammals with different longevities. Notably, the HO gene is evolutionarily different in
birds and mammals, with the biliverdin reductasebilirubin step present in the latter but absent in the
former.. Consistently, the organism sacrifices the potential for indefinite survival in favor of earlier and
more prolific fecundity. From an evolutionary perspective, aging is a nonadaptive phenomenon, since it
limits the reproductive potential of an individual. For this reason aging should be opposed by natural
selection, and hence the argument that it evolved to provide offspring with living space is now receiving
little credence. A clear prediction is that the actual mechanisms of senescence are stochastic, involving most
likely processes such as random accumulation of somatic mutations or oxidative damage to macromole-
cules. In the words of an anonymous poet, we are born as copies, but we die as originals.
It is becoming increasingly clear that genetic factors are prominently involved in aging, the major
lines of empirical evidence being: (1) the life span which in human populations shows significant
heritability; (2) different species have different intrinsic life spans due to genomic differences; (3) human
populations possess inherited progeroid disorders, such as Werners syndrome, a disease characterized by
premature agerelated disorders, including atherosclerosis, type II diabetes, osteoporosis, and cancers;
and (4) clear evidence of genetic effects on life span have been demonstrated in invertebrate model systems,
such as D. melanogaster and C. elegans. In this organism, five different genomic regions appear to be
associated with longevity, as assessed by quantitative genetic analysis (Rothschild and Jazwinski, 1988).
Also, in S. cerevisiae 13 longevity genes have been identified and cloned. Of these 13 genes, 11 have human
homologues (Rothschild and Jazwinski, 1988). At least, three categories of genes are predicted to affect
aging and longevity. They are: (1) genes that regulate levels of somatic maintenance and repair; (2)
pleiotropic genes, whose expression involves tradeoffs between earlylife fitness benefits and latelife fitness
disadvantages, which do not encompass somatic maintenance; and (3) lateacting deleterious mutations
that have escaped elimination as consequence of the decline in the force of natural selection at old ages
(Calabrese et al., 2001a). Efficient functioning of maintenance and repair process seems to be crucial for
both survival and physical quality of life. This complex network of the socalled longevity assurance
processes is composed of several genes, termed vitagenes (> Table 62). The homeodynamic property of
living systems is a function of such a vitagene network. Because aging is characterized by the failure of
homeodynamics, a decreased efficiency and accuracy of the vitagene network can influence gerontogenic
processes. It is not clear how various components of the vitagene network operate and influence each other
in a concordant or a discordant manner. Since aging is characterized by a progressive failure of maintenance
and repair, it is reasoned that genes involved in homeodynamic repair pathways, such as the HO1 or Hsp70
genes, are the most likely candidate vitagenes.

7 HO1 and Hsp70 as a Therapeutic Funnel

A promising approach for the identification of critical vitagenerelated processes is represented by the
hormesislike positive effect of stress, including regular muscle exercise (Butterfield et al., 2002a, b;
Calabrese et al., 2004b) caloric restriction, which can result in activation of the Hsp signal pathway and,
consequently, in stress tolerance. In particular, there is strong evidence that the HO/CO and biliverdin
bilirubin redox system might work critically as a therapeutic funnel in a number of physiopathological
situations where the sensing of redoxactive events is coupled to acquiring major resistance to the effects of
stressful and pathogenic conditions (> Figure 61). HO1 activity seems to be required for the action of
several other therapeutic molecules. In each case, the expression of HO1 or administration of one of its
metabolic products substitutes for the actions of the other protective molecule (Otterbein et al., 2003a).
 Aging and oxidative stress response in the CNS 6 135

. Figure 61
Redox regulation of gene expression involving the vitagene system. Proposed role for the vitagene member
heatshock proteins (Hsps) in modulating cellular redox state and cell stress tolerance. Various proteotoxic (or
genotoxic) conditions cause depletion of free Hsps that lead to activation of stress kinase and proinflammatory
and apoptotic signaling pathways. Hsp70 prevents stressinduced apoptosis by interfering with the SAPK/JNK
signaling and by blocking caspase proteolytic cascade. Nitrosativedependent thiol depletion triggers HO1
induction, and increased HO1 activity is translated into augmented production of carbon monoxide (CO) and
the antioxidant bilirubin. These molecules may counteract increased NOS activity and NOmediated cytotoxici-
ty. In addition, HO1 may directly decrease NO synthase protein levels by degrading the cofactor heme.
Abbreviations: PLA2, phospholipase A2; IL6, interleukin6; AP1, activator protein1; SAPK, stressactivated
protein kinase; JNK, cjun Nterminal kinase; NFkB, nuclear factor kappaB; GSNO: Snitrosoglutathione; HO1,
heme oxygenase1

In many inflammatory situations, the ability of IL10 to suppress TNFa expression in macrophages
requires the presence of HO1 and the generation of CO; HO1 expression or CO administration has the
same effects as IL10 (Lee and Chau, 2002; Soares et al., 2004). In concert with this conceivable possibility,
the protective effect of IL10 in a lethal endotoxic shock mice model is strongly dependent on the expression
of HO1 and the generation of CO (Lee and Chau, 2002; Soares et al., 2004). Moreover, rapamycin appears
not to exert its antiproliferative effects on smooth muscle cells unless HO1 is present (Lee and Chau, 2002;
Akamatsu et al., 2004), and it has been proven that, in order for NO to protect mice livers from hepatitis
induced by TNFa and galactosamine, upregulation of HO1 seems to be essential (Otterbein et al., 2003b).
Also, alcohol has antiinflammatory effects as TNFa is suppressed and IL10 is increased (Otterbein et al.,
2003b; Yamashita et al., 2004). However, protection is lost when HO1 is blocked (Foresti et al., 1997). In
addition, the antiinflammatory effect of 15deoxyD12,14prostaglandin J2 has been shown to require the
activity of HO1 (Lee and Chau, 2002; Otterbein et al., 2003b). Notably, during heat shock, which leads to
upregulation of several Hsps endowed with cytoprotective actions, entire cytoprotection is lost if HO1 is
blocked with SnPPIX. Last, relevant to brain physiopathology, dietary and medicinal phytochemicals that
can inhibit, retard, or reverse the multistage pathogenic events associated with degenerative damage,
136 6 Aging and oxidative stress response in the CNS

particularly polyphenols such as curcumin, caffeic acid, and ferulic acid, all capable of exerting powerful
antiinflammatory actions, have been shown to function by upregulating HO1 (Scapagnini et al., 2002d,
2004; Poon et al., 2004a). The fact that in all these situations specific molecules or biological phenomena
appear to lose most, if not all, of their effects when HO1 is absent represents a compelling evidence that the
HO1 system may represent a final common mediator of many biological events associated to cell stress
response and, as such, working as a critical vitagene, which links redoxdependent pathways of stress
tolerance to a versatile biological program of cell life.

8 Conclusion

Modulation of endogenous cellular defense mechanisms via the stress response signaling represents an
innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegenera-
tion (Calabrese et al., 2005; Sultana et al., 2005). Efficient functioning of maintenance and repair processes
seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network
of the socalled longevity assurance processes, which are composed of several genes termed vitagenes.
Consistently, by maintaining or recovering the activity of vitagenes it can be possible to delay the aging
process and decrease the occurrence of agerelated diseases with resulting prolongation of a healthy life span.
As one of the most important neurodegenerative disorders, AD is a progressive disorder with cognitive and
memory decline, speech loss, personality changes, and synapse loss. With the increasingly aging population
of the United States, the number of AD patients is predicted to reach 14 million in the midtwentfirst
century in the absence of effective interventions (Butterfield et al., 2002a). This will pose an immense
economic and personal burden on the people of this country. Similar considerations apply worldwide, except
in subSaharan Africa where HIV infection rates seem to be leading to decreased incidence of AD (Butterfield
et al., 2002b). There is now strong evidence to suggest that factors such as oxidative stress and disturbed
protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis. Brainaccessible
antioxidants, potentially, may provide the means of implementing this therapeutic strategy of delaying the
onset of AD, and more in general all degenerative diseases associated with oxidative stress. As one potentially
successful approach, potentiation of endogenous secondary antioxidants systems can be achieved by
interventions which target the HO1/CO and/or Hsp70 systems. In this chapter, the importance of the stress
response signaling and, in particular, the central role of HO1 together with the redoxdependent mechan-
isms involved in cytoprotection are outlined. The beneficial effects of HO1 induction result from heme
degradation and cytoprotective regulatory functions of biliverdin/bilirubin redox cycling. Thus, HO1 can
amplify intracellular cytoprotective mechanisms against a variety of insults. Consequently, induction of HO
1 by increasing CO and/or biliverdin availability can be of clinical relevance.
CO has been studied for >100 years and, until the last few years, has been touted as a molecule to avoid,
owing to its toxic effects exerted mostly on hemoglobin and cytochrome oxidase functions (Otterbein,
2002). However, these toxic effects are seen at concentrations of CO well above concentrations used
experimentally. Beneficial effects are obtained with relative low doses of CO (250 ppm for one to few
hours) in rodents (Otterbein et al., 2003b). Carboxyhemoglobin levels generated in such a model are not
too high from those of heavy smokers. If this beneficial effect is confirmed also in humans, limited exposure
of patients to CO might be considered as therapy for various syndromes, particularly to prevent restenosis
after angioplasty or treatment of an organ donor and/or the organ to suppress ischemiareperfusion injury
and to prolong allograft survival. Very importantly, HO1 and CO can suppress the development of
atherosclerotic lesions associated with chronic rejection of transplanted organs (Otterbein et al., 2003b;
Akamatsu et al., 2004). Interestingly, the recently discovered CORMS appears to afford similar protective
action, thereby providing an alternative therapeutic approach for those pathophysiological conditions
where CO administration may prove to be beneficial (Motterlini et al., 2002b, 2003). Furthermore,
administration of biliverdin or bilirubin after the first few weeks of life is proven not to have toxicity and
doses as much as 2.5 mg/dl used in experimental paradigms are only slightly above normal levels, yet
endowed with cytoprotective effects (Yamashita et al., 2004). Although clinical application of the HO
 Aging and oxidative stress response in the CNS 6 137

system should be fully considered, a better understanding of how HO mediates its action will guide
therapeutic strategies to enhance or suppress HO effects. Remarkably, the recent envisioned role of
Hsp70 as a vehicle for intracytoplasmic and intranuclear delivery of fusion proteins or DNA to modulate
gene expression (Wheeler et al., 2003), along with the evidence that binding of HO protein to HO1 DNA
modifies HO expression via nonenzymatic signaling events (Weng et al., 2003) associated to CO and P38
dependent induction of Hsp70, opens intriguing perspectives, as it is possible to speculate that synergy
between these two systems might impact cell proliferation and apoptotic processes during oxidative stress,
hence contributing to programmed cell life or programmed cell death (> Figure 61), depending on the
relative extent of activation.
Presented here is strong evidence that a cross talk between stress response genes is critical for cell stress
tolerance, highlighting a compelling reason for a renewed effort to understand the central role of this most
extraordinary defense system in biology and medicine. All of the above evidence supports also the notion
that stimulation of various maintenance and repair pathways through exogenous intervention, such as mild
stress or nutritional compounds targeting the heatshock signal pathway, may have biological significance as
a novel approach to delay the onset of various ageassociated alterations in cells, tissues, and organisms
(Poon et al., 2004a, b). Hence, by maintaining or recovering the activity of vitagenes (Calabrese et al., 2001a,
2004b, 2005) it can be possible to delay the aging process and decrease the occurrence of agerelated diseases
with resulting prolongation of a healthy life span.

Acknowledgments

This work was supported in part by grants from the Wellcome Trust, MIURCluster Biomedicine, and FIRB
RBNE01ZK8F, and by grants from the National Institute of Health (D.A.B.). The authors acknowledge
helpful discussions with John Clark (Institute of Neurology, UCL, London, UK) and with Roberto
Motterlini (Northwick Park Institute for Medical Research, Harrow, UK); Enrico Rizzarelli and Eduardo
Puleo (Department of Chemistry, University of Catania).

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7 Parallels Between
Neurodevelopment and
Neurodegeneration: A Case Study
of Alzheimers Disease
X. Zhu . G. Casadesus . K. M. Webber . C. S. Atwood . R. L. Bowen . G. Perry . M. A. Smith

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
1.1 Review Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

2 Alzheimers DiseaseAssociated Proteins: Critical Roles in Both Neurodevelopment

and Neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
2.1 Genetic Associations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
2.1.1 Amyloidb Protein Precursor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
2.1.2 Presenilins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
2.1.3 Apolipoprotein E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

3 Mitotic Alterations in Alzheimers Disease: Similarities to Neurogenesis . . . . . . . . . . . . . . . . . . . . 149

3.1 CellCycle Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3.2 Cyclins and CyclinDependent Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3.3 A Mitotic Catastrophe in Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
3.4 An Early, Not a Late, Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

4 Possible Mitotic Factors: Clues Provided by Gender Difference in Alzheimers Disease

and Hints on Therapeutic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.1 Cell Cycle and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.2 Mitogenic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.3 Gender Differences and Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.3.1 The HPG Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.3.2 Luteinizing Hormone/Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

# 2008 Springer ScienceBusiness Media, LLC.

148
7 Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimers disease

Abstract: An accumulating body of evidence shows significant similarities between cellular processes
involved in neurodegeneration and those involved in neurodevelopment. Most striking, in Alzheimers
disease, cell cycle re-entry appears to be a pathological signature of the disease that also occur in brain
development. Such inappropriate reactivation of a fetal program is likely to play a key role in both the
etiology and pathogenesis of disease.
List of Abbreviations: AD, Alzheimers disease; ApoE, apolipoprotein E; APP, amyloid- protein precur-
sor; A, amyloid-; Cdk, cyclin-dependent kinase; HPG axis, hypothalamic-pituitary-gonadal axis; HRT,
hormone replacement therapy; LH, luteinizing hormone; PS, presenilin; ROS, reactive oxygen species

1 Introduction

1.1 Review Summary

Alzheimers disease (AD) is the most common senile dementia in the elderly, which is characterized by three
main structural changes in the brain: neurofibrillary tangles, senile plaques, and extensive neuronal
degeneration. Quite surprisingly, a number of recent findings demonstrate similarities between neurogen-
esis during development and neurodegeneration during AD in that neuronal populations that are known to
degenerate in AD display phenotypic changes characteristic of dividing cells (Zhu et al., 2004a). Such a
notion of a direct relation between neurodevelopment and neurodegeneration is further strengthened by
studies of ADassociated proteins (Selkoe and Kopan, 2003). In this chapter, we highlight the parallels between
early neural development and latelife neurodegeneration and, thereafter, discuss possible therapeutic strategies
that this might afford.

2 Alzheimers DiseaseAssociated Proteins: Critical Roles in Both

Neurodevelopment and Neurodegeneration

2.1 Genetic Associations

Linkage studies show that mutations in at least three genes, the amyloidb protein precursor (AbPP)
gene located on chromosome 21, and the two homologous genes, presenilin 1 and 2 (PS1 and PS2)
located on chromosome 14 and 1, respectively, are associated with earlyonset AD (Hardy, 1997). AbPP
is a type 1 membrane protein with a large extracellular domain and a short cytoplasmic domain and
belongs to a larger AbPP family that also includes APLP1 and APLP2 (Coulson et al., 2000).
Presenilins are polytopic proteins with six to eight transmembrane domains that play a central role
in intramembrane proteolysis (gcleavage) of a number of cell surface proteins including AbPP, which
gives rise to amyloidb (Ab) peptide, the major component of senile plaques (Selkoe and Kopan, 2003).
The penetrance of the mutations is almost 100%, suggesting that these proteins play critical roles in the
neurodegeneration of AD. Unfortunately, however, their mechanistic roles in the pathogenesis of AD
are still elusive.

2.1.1 Amyloidb Protein Precursor

Interestingly, it appears that these ADassociated proteins also play central roles in early neural
development (Bothwell and Giniger, 2000). For example, the overexpression of human AbPP in
Drosophila leads to development of blisteredwing phenotype (Fossgreen et al., 1998). Mice genetically
deficient in AbPP, APLP1, or APLP2 are viable with only subtle neurological deficits, suggesting
redundancy in function among the AbPP family members (Heber et al., 2000). More recently, mutant
mice lacking all three AbPP genes displayed early lethality as well as high incidence of cortical dysplasia
 Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimers disease
7 149

suggesting a critical role in neural development (Koo, 2002). However, the actual function of AbPP
remains unresolved. Interestingly, in vitro studies suggest that AbPP may function as a cellcycle
regulator because AbPP and its proteolytic fragments (i.e., Ab peptide and AbPP) are mitogenic
(Schubert et al., 1989; Milward et al., 1992; Copani et al., 1999; Hoffmann et al., 2000; Schmitz
et al., 2002). Further, its adaptor proteins including Fe65 and AbPPBP1 have been shown to function
as regulators of the cell cycle. AbPPBP1 is a cellcycle protein that normally negatively regulates the
progression of cells into the S phase and positively regulates the progression into mitosis (Chen et al.,
2000), whereas Fe65 is a nuclear protein that negatively regulates G1 to Sphase progression by
inhibiting the key Sphase enzymes (Bruni et al., 2002). Therefore, it is likely that AbPP may act as
a cell surface receptor to relay cell cyclerelated signals.

2.1.2 Presenilins

The role of PS1 in neurodevelopment is more firmly established at least partially because of its involvement
in the gcleavage of the Notch receptor, which plays critical roles in determining cell fate during early
development (Selkoe and Kopan, 2003). It is shown that the PS1deficient mouse displays profound
developmental abnormalities, including failed somite formation and altered neurogenesis (Shen et al.,
1997; Wong et al., 1997). Additionally, the loop domain of PS1 serves as a scaffold for bcatenin degradation
(Willert and Nusse, 1998; Kang et al., 2002). It is known that bcatenin can migrate to the nucleus and
interact with transcription factors like TCF/LEF1 to induce the expression of genes such as Cyclin D1 and
c-myc, which are crucial in the G1/Sphase transition (Willert and Nusse, 1998). In fact, transgenic mice
that express a constitutively active bcatenin in neuroepithelial precursor cells develop enlarged brains
because a greater proportion of neural precursors reenter the cell cycle after mitosis (Chenn and Walsh,
2002). Therefore, PS1 may also be involved in cellcycle regulation. Indeed, the association of presenilins
with centrosome and centromeres suggests that they play a role in cell division and segregation of
chromosomes (Li et al., 1997). In addition, it has been shown that the overexpression of both PS1 and
PS2 proteins resulted in G1phase arrest of the cell cycle (Janicki and Monteiro, 1999; Janicki et al., 2000),
whereas PS1 deficiency results in accelerated entry into (Soriano et al., 2001) and significantly prolonged
length (Yuasa et al., 2002) of the S phase of the cell cycle. This evidence suggests that PS1 and PS2 play
important roles in cellcycle control.

2.1.3 Apolipoprotein E

Finally, the apolipoprotein E4 (ApoE4) alleles, risk factor of lateonset AD, which confer increased
susceptibility both to AD and prostate cancer, implicate an association between the ApoE4 allele and a
propensity toward developing a dysregulated cell cycle (Yuasa et al., 2002). In support of such a notion,
ApoE is itself associated with the amyloid deposits found in pituitary adenomas.

3 Mitotic Alterations in Alzheimers Disease: Similarities to Neurogenesis

3.1 CellCycle Control

The cell cycle is a highly regulated process and it is the sequential expression and activation of cyclin/Cdk
complexes, the main regulators of cellcycle progression, which orchestrate the orderly transition from one
phase to another. Cyclin dependent kinases (Cdks) are controlled through sophisticated mechanisms,
including binding to their appropriate cyclins, phosphorylation modifications, and binding to their specific
inhibitors. Adult neurons are generally thought of as being postmitotic and terminally differentiated and
therefore, it is somewhat surprising that vulnerable neurons in AD have certain phenotypic markers that
resemble a cyclin rather than a quiescent nondividing, cell, which highlights the similarities between
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7 Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimers disease

neurogenesis during development and neurodegeneration during AD. Indeed, this may well reflect the fact
that ADassociated proteins are involved in cellcycle regulation, which may be disrupted in the pathogen-
esis or progression of the disease.

3.2 Cyclins and CyclinDependent Kinases

Neurogenesis involves proliferation of the precursor cells and Cdks are crucial regulators of proliferation in
the precursor stage. For example, Cdk2 and Cdk4/6 are both critical in cellcycle regulation in neural
precursor cells (Ferguson et al., 2000). The activities of Cdks are blocked once the neural cells start
differentiation and these Cdks are normally absent in differentiated cells. However, various cellcycle
markers including Cdks are found in susceptible neurons in AD reminiscent of proliferating precursor
cells during neurogenesis. Indeed, the upregulation of Cdk7 and reappearance of Cyclin D, Cdk4, and/or
Ki67 suggest that the susceptible neurons are no longer quiescent (Smith and Lippa, 1995; McShea et al.,
1997; Nagy et al., 1997a, b; Zhu et al., 2000). The expression of Cdk2/Cyclin E complex reflects that
neurons may have emerged from phase G1 (Nagy et al., 1997a), whereas the aberrant expression of Cdc2/
Cyclin B1 complex and CARB indicates that the degenerating neurons may have reached phase G2 at
least in some cases (Nagy et al., 1997b; Vincent et al., 1997; Zhu et al., 2004b). The reported presence
of coordinated DNA replication suggests that the susceptible neurons may complete a nearly full
S phase (Yang et al., 2001), and this is supported by the fact that mitochondrial DNA is also increased
(Hirai et al., 2001).

3.3 A Mitotic Catastrophe in Alzheimers Disease

Despite the increasing evidence of reactivation of cellcycle machinery in the susceptible neurons in AD,
there is, as yet, no evidence suggesting a successful nuclear division or chromosome condensation in AD.
This implies that neurons do not complete mitosis, leading us to speculate that there is a mitotic
catastrophe. Indeed, the highly unorganized nature of the cellcycle reentry in AD neurons as evidenced
by the concurrent expression and aberrant localization of Cdk4 and p16 (McShea et al., 1997), and the
presence of Cyclin E and B1 but absence of Cyclin D and A (Nagy et al., 1997b), point to an inadequate or a
failed control of cell cycle in these neurons which may be due to the inherent incompetence of terminally
differentiated neurons to complete cell cycle. Therefore, it is very likely that an unscheduled activation of a
mitotic division cycle in postmitotic neurons leads to an abortive reactivation of cellcycle machinery and
the eventual demise of the cell.

3.4 An Early, Not a Late, Change

However, this does not necessarily mean that mitotic proteins are exclusively associated with end stage of
neuropathology. Rather, it was shown that cellcycle markers are associated with the earliest neuronal
change to occur in the disease (Nagy et al., 1997a; Busser et al., 1998; Yang et al., 2003). For example, cell
cycle markers have been shown in patients with mild cognitive impairment (MCI) (Yang et al., 2003). It
has also been shown that they occur prior to the appearance of gross cytopathological changes (Vincent
et al., 1998), which suggests that mitotic alterations may lead to those cytopathological changes. For
example, the expression, phosphorylation, and metabolism of AbPP, have been shown to be cell cycle
related, thus any perturbation of cell cycle, as apparently seen in AD, could lead to misregulated
metabolism and expression of AbPP, which may give rise to Ab production and amyloid pathology.
Further, increased phosphorylation and altered microtubule stability are coincident during progression
through the cell cycle. Therefore, it is not surprising that microtubule abnormities and tau phosphoryla-
tion are also associated with AD, because cellcycle reentrance is an early feature in AD and candidate tau
kinases such as Cdk2, Cdk5, Cdc2, and MAPK that are involved in cellcycle control are all increased in
 Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimers disease
7 151

AD in a topographical manner that overlaps with phosphotau. Moreover, free radicals produced during
oxidative stress are speculated to be pathologically important in AD and other neurodegenerative
diseases (Cross et al., 1987; Perry et al., 1998). Dysregulated cellcycle control may also lead to
increased cytoplasmic oxidative damage observed in susceptible neurons in AD, as it may adversely
affect mitochondria function. It is reported that mitochondrial DNA and protein are increased (Hirai
et al., 2001), which may be due to the same mitogenic signal that triggers cellcycle reentry and nuclear
DNA duplication. However, the incompetence of postmitotic neurons to complete the celldivision
cycle may result in the failure of organelle kinesis after mitochondrial DNA duplication that probably
leads to mitochondrial abnormalities. It is clear that dysregulated mitochondria are major sources of
ROS for surrounding cytoplasm.

4 Possible Mitotic Factors: Clues Provided by Gender Difference in

Alzheimers Disease and Hints on Therapeutic Considerations

4.1 Cell Cycle and Pathology

As discussed earlier, such mitotic alterations are not only some of the earliest neuronal abnormalities in the
disease (McShea et al., 1997; Vincent et al., 1998), but would also lead to all of the other pathological
changes reported in the disease, including tau phosphorylation, mitochondria dysfunction, and cytoplas-
mic oxidative damage, alterations in Ab and the ultimate neuronal dysfunction and death (Raina et al.,
2000). This strongly suggests that therapeutic interventions targeted toward normalizing these abnormal
mitotic changes may result in the slowing or prevention of disease progression.

4.2 Mitogenic Factors

In order to study this possibility, one must first examine possible candidates that can stimulate these
neurons to reenter the cell cycle. Among these, probable candidates are growth or cellular differentiation
factors. In this regard, aberrant and elevated levels of a variety of molecules with potential neurotrophic and
mitogenic activities are found in AD, including nerve growth factor (Allen et al., 1991; Crutcher et al., 1993;
Connor et al., 1996), luteinizing hormone (LH) (Bowen et al., 2002), epithelial growth factor (Birecree
et al., 1988; Styren et al., 1990), transforming growth factorb1, plateletderived growth factor (Masliah et al.,
1995), hepatocyte growth factor/SF (Fenton et al., 1998), insulinlike growth factorI (Connor et al.,
1997), insulinlike growth factorII (Tham et al., 1993), and basic fibroblast growth factor (Gomez
Pinilla et al., 1990; Stopa et al., 1990). However, although the precise effects of aberrant release of
growth factors in AD remains elusive, the gender bias present in AD has provided valuable information
in our attempt to determine the identity of this unknown mitogen.

4.3 Gender Differences and Hormones

Extensive epidemiological data suggest a genderbased predisposition that is specific to AD such that higher
prevalence (Jorm et al., 1987; Breitner et al., 1988; Rocca et al., 1991; McGonigal et al., 1993) and incidence
(Jorm and Jolley, 1998) of AD is present in women. This has led investigators to focus on the roles of the sex
steroids, estrogen and to a lesser extent testosterone, in the pathogenesis of the disease resulting in several
lines of evidence suggesting that estrogen deficiency following menopause may contribute to the etiology of
AD in women. In this regard, a positive correlation has been shown to exist between AD and decreased
estrogen levels following menopause (Manly et al., 2000) and earlier studies also demonstrate a decreased
incidence (Henderson et al., 1994) and a delay in the onset (Tang et al., 1996) of AD among women on
hormone replacement therapy (HRT) following menopause (Kawas et al., 1997). Nevertheless, HRT
protection against AD is restricted to administration during a critical period that constitutes the
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7 Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimers disease

climacteric years and is ineffective or even detrimental when administered after such time (Rapp et al.,
2003) or during the latent preclinical stage of AD, which usually occurs much later in life (Resnick and
Henderson, 2002).

4.3.1 The HPG Axis

Although falling levels of estrogen might explain the higher incidence of AD in females than in males, a
decline in sex steroids does not account for why males with Downs syndrome have a higher risk of
developing ADtype changes than females, as the levels of sex steroids in both males and females with
Downs syndrome are comparable to those found in the general population (Schupf et al., 1998). This
reversal in genderbased predisposition of AD combined with questions regarding a critical period of
HRTbased protection against dementia has prompted us to investigate the potential role of other
hypothalamicpituitarygonadal (HPG) axis hormones in the etiology of AD (Bowen et al., 2000, 2002;
Bowen 2001; Smith et al., 2003), which, along with sex steroids, regulate reproductive function, and also
show expression in many nonreproductive tissues including, most notably, the brain. In this regard,
previous reports demonstrate a twofold increase in the gonadotropin LH in AD patients when compared
to agematched control subjects (Bowen et al., 2000; Short et al., 2001). More important, the fact that the
highest density of LH receptors in the brain is found within the hippocampus (Lei et al., 1993; AlHader
et al., 1997a, b), the most vulnerable region in AD, and gonadotropins are known to cross the bloodbrain
barrier (Lukacs et al., 1995), we speculate that elevated LH levels may be a driving pathogenic force in
mitogenic dysfunction seen in AD (Bowen et al., 2002).

4.3.2 Luteinizing Hormone/Therapeutics

As LH is a powerful mitogen (Harris et al., 2002), and given the temporal and spatial overlap with mitotic
changes in AD (Bowen et al., 2002; unpublished observations), it is plausible that elevations in LH seen in
AD patients are responsible for the inappropriate cellcycle reentry in ADvulnerable neurons. In support of
such a notion, we found significant elevations of LH in ADvulnerable neuronal populations when
compared to agedmatched control cases (Bowen et al., 2002). More important, such increases appear to
be a very early change in disease history and parallel the ectopic expression of cellcycle and oxidative
markers that represent one of the initiating pathological changes preceding neuronal degeneration by
decades (Nunomura et al., 2001; Ogawa et al., 2003). Therefore, therapeutic strategies that are targeted
toward the release of LH may prove successful in treatment of AD. In this regard, it is likely that the
ineffectiveness of estrogen replacement therapy in the treatment of AD (Mulnard, 2000; Mulnard et al.,
2000) may be due to the incapacity of this hormone to restore the HPG axis to its premenopausal state.
Therefore, treatments such as leuprolide acetatea gonadotropinreleasing hormone agonist, which has
been shown to suppress LH to undetectable levels by downregulating pituitary gonadotropinreleasing
hormone receptorsfor mature patients (i.e., postmenopausal women) might be a more effective
method for AD. The results of phase III clinical trials of leuprolide acetate for the treatment of AD are
anticipated in late 2007.

5 Conclusions

As highlighted earlier, factors such as luteinizing hormone, which drive neuronal proliferation during
development, may also play a key role in neuronal degeneration during AD. What irony, that factors used
to build the brain are the same that deconstruct the brain after a lifetime of use. We hope that the
recognition of this fact will enable efforts to forestall deconstruction and preserve cognitive integrity to
the grave.
 Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimers disease
Acknowledgements
Work in the authors laboratories is supported by the National Institutes of Health and the Alzheimers
Association. CSA, GP, and MAS are consultants for and own equity in Voyager Pharmaceutical Corporation.
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8 Differentiation and
DeDifferentiationNeuronal
CellCycle Regulation During
Development and AgeRelated
Neurodegenerative Disorders
T. Arendt

1 The Eukaryotic Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

1.1 CyclinDependent KinasesKey Regulators of the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
1.2 Regulation of CyclinDependent Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
1.2.1 Cyclins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
1.2.2 Protein Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
1.2.3 Cdk Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
1.2.4 Subcellular Sorting and Degradation of CellCycle Regulating Proteins . . . . . . . . . . . . . . . . . . . . . . . . 165
1.2.5 Regulation of NoncellCycle Cdks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
1.3 Effects of Cdk ActivationCdk SubstratesThe Retinoblastoma Protein . . . . . . . . . . . . . . . . . . . . . 166
1.4 Quality Control of CellCycle Progression by Stop and Go Signals: Restriction
Point and Checkpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
1.5 Upstream Control of Cdk Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
1.5.1 Growth Factor SignalingThe RasMAPK Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
1.5.2 Signaling Induced by CellCell and CellMatrix InteractionsExternal Positional Cues . . . 170
1.5.3 Convergence of Ras and IntegrinDependent Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

2 CellCycle Regulators During Development and in Postmitotic Neurons . . . . . . . . . . . . . . . . . . . . . 172

2.1 Stem Cell Proliferation and Neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
2.2 Do CellCycle Regulators Have Alternative Functions Unrelated to CellCycle Regulation? . . . 174
2.3 Coupling Cell Cycle to Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
2.3.1 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
2.3.2 The Bcl2 Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
2.3.3 P53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
2.3.4 Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
2.3.5 Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

3 CellCycle Regulators in Neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

3.1 The Link Between Neurodegeneration and Neuroplasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
3.1.1 Coherent Hierarchical Pattern of Selective Neuronal Vulnerability and Neuroplasticity . . . . . . . 182
3.1.2 Potential Risks of Dynamic Stabilization of Neuronal Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
3.1.3 Aberrant Plasticity in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3.2 CellCycle Regulators Alternatively Execute Neuroplasticity or Cell Death . . . . . . . . . . . . . . . . . . . . . 185
3.2.1 Elevated Mitogenic Force and Activated Mitogenic Signaling in Alzheimers Disease . . . . . . . . . 185

# 2008 Springer ScienceBusiness Media, LLC.

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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

3.2.2 Oxidative Stress and the Autocrine Loop of SelfPerpetuating Mitogenic Activation . . . . . . . . . . 186
3.2.3 The Replay of Developmental Programs in Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
3.2.4 CellCycle Activation in Alzheimers Disease and Other Neurodegenerative Disorders . . . . . . . . . . . . 190

4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 159

Abstract: The eukaryotic cell cycle is an evolutionary highly conserved process that results in cell division
into two daughter cells. Nondividing cells rest outside the cell cycle. The cell cycle consists of an ordered set
of events that are tightly regulated by defined temporal and spatial expression, subcellular localization, and
degradation of cell cycle regulators. The transition through the cell cycle is controlled by stop and go
signals at defined checkpoints. These checkpoints allow alternate decisions between further progression
through the cell cycle and growth arrest, in order to provide potentials for DNA repair or induction of
apoptosis. Postmitotic neurons in the CNS are generated from neuroepithelial stem cells, which are
multipotent cells that differentiate into progenitor cells of neurons and glial cells. Differentiated neurons
are postmitotic cells that have permanently withdrawn from the cell cycle. Currently accumulating
evidence, however, shows that differentiated neurons express a number of cell cycle regulators which
suggest a role of these proteins beyond cell cycle regulation. Under conditions of neurodegeneration in
Alzheimers disease, neurons leave their differentiated stage and re-enter the cell cycle. This process which is
driven by an abnormal activation of mitogenic pathways eventually results in cell death. Alzheimers disease
should, thus, be added to the list of proliferative disorders such as cancer, cardiovascular disease, infections
and autoimmune diseases, where cell cycle regulators are recognized targets for treatment. Here we attempt
to give an overview on the current state of knowledge on the regulation of the cell cycle in neurons under
physiological and pathophysiological conditions.
List of Abbreviations: AD, Alzheimers disease; ATP, adenosine triphosphate; APC, anaphase-promoting
complex; APP, amyloid precorsor protein; Akt/PKB, phosphoinositide 3-kinase/protein kinase B; ApoE,
apoliporotein E; Abl, v-abl Abelson murine leukemia viral oncogene homolog 1 [Homo sapiens]; A,
amyloid-beta protein; ATM, ataxia telangiectasia mutated kinase; ATR, ATM-Rad-3 related kinase; Bad,
Bcl-2-associated death protein; Bak, Bcl-2 agonist killer 1; Bax, Bcl-2-interactive cell death susceptibility
regulator; Bcl-2, B-cell leukemia/lymphoma-2; Bcl-x, B-cell lymphoma 2-associated protein X; bFGF, basic
fibroblast growth factor; Bid, BH3 interacting domain death agonist; Bim, Bcl-2-interacting mediator of cell
death; BH, Bcl-2 homology; Bmf, Bcl-2 modifying factor; Bok, Bcl-2 related ovarian killer protein; CAK,
cdk-activating kinase; Cdc, cell-division-control; Cdk, Cyclin dependent kinase; C/EBP, CCAAT/enhancer-
binding protein transcription factor; Cip/Kip family, Cdk interacting protein/kinase inhibitory protein
(Cip/Kip) family; c-myc, avian virus inducing Myelocytomatosis, c = cellular; CNS, central nervous system;
DA, Dalton; Dab, Drosophila disabled homolog; DNA, deoxyribonucleic acid; DP5/Hrk, death protein5/
harakiri; ECM, extracellular matrix; E2F, transcription factor; EGF, epidermal growth factor; EGL, external
germinal layer; ELISA, enzyme linked immunosorbent assay; Elk-1, member of ETS oncogene family (ETS
erythroblast transformation specific domain); ERK, extracellular signal-regulated kinase; ERK1, Mitogen-
activated protein kinase p44 extracellular signal-regulated kinase; ERK2, Mitogen-activated protein kinase
p42 extracellular signal-regulated kinase; Ets-2, v-ets erythroblastosis virus E26 oncogene homolog
2 (avian); FAK, focal adhesion kinase; FKBP12, FK506-binding protein; FTD, Fronto-temporal dementia-
17 (FTD linked to 17q21); FAS, tumor necrosis factor ligand family, apoptose related molecule (CD95 or
APO-1), encodes region of fatty acid synthase activity; FAS; multifunctional protein; GAP-43, growth-
associated protein 43; GAPs, GTPase-activating proteins; G0, G1, G2, gap phase 0, 1, 2; GDP, guanine
diphosphate; GEFs, guanine nucleotide exchange factors; GSK3, glycogen synthase kinase 3-beta; GTP,
guanine triphosphate; HCMV, human cytomegalovirus; HGF/SF, hepatocyte growth factor/scatter factor;
HIV, human immunodeficiency virus; H3, histone 3; HP1, heterochromatin protein 1; HPV, human
papilloma virus; HSV, herpes simplex virus; IL, interleukin; IGF-1, IGF-2, insulin-like growth factor-1, -2;
INK4, inhibitors of cyclin-dependent kinase 4; JNK, c-Jun NH2-terminal kinase; Ki-67, cell-cycle related
nuclear protein; LEF/TCF, lymphoid enhancer factor/T-cell factor; LTP, long term potentiation; M, mitosis;
MAPK, mitogen activated protein kinase; MARCKS, myristoylated alanine-rich C-kinase substrate; MCI,
Mild Cognitive Impairment; Myt1, myelin transcription factor 1; MEK, Map kinase extracellular signal-
regulated kinase; mTOR/FRAP, mammalian target of rapamycin/FKBP12 rapamycin associated protein;
Mcl-1, myeloid cell leukemia sequence 1, antiapoptotic protein (Bcl-2 homologue); NCAM, neural cell
adhesion molecule; NMDA receptor, N-methyl-D-aspartate receptor; NR2A, subunit of NMDA receptor;
NO, nitric oxide; NOS, NO synthase; nNOS, Type I or neuronal isoform of NOS; iNOS, Type II or inducible
isoform of NOS; eNOS, Type III or endothelial isoform of NOS; NGF, nerve growth factor; NF-kB, nuclear
160
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

factor kappa-B, subunit 1; OA, okadaic acid; PCNA, proliferating cell nuclear antigen; p53, tumor protein
p53; PHFs, paired helical filaments; pRb, phospho-retinoblastoma protein; PI3K, phosphatidylinositol
3-kinase; PKC, protein kinase C; PP1, protein phosphatase 1; PHAS-1, Phosphorylated heat- and acid-
stable protein 1; PLA-2, phospholipase A2; p70S6k, p70 ribosomal protein S6 kinase; PAK, p21-activated
kinase; p75, p75 neurotrophin receptor; PC12, pheochromocytoma cell line; Puma, p53-upregulated
modulator of apoptosis; PMAIP1/Noxa, phorbol-12-myristate-13-acetate-induced protein1/BH3-containing
mitochondrial protein; PDGF, platelet derived growth factor; R, restriction point of the cell cycle; rhoA, ras-
related protein A; Ras, rat sarcoma virus; Raf, proto-oncogene serine/threonine-protein kinase; Rsk,
ribosomal protein S6 kinase; S, DNA synthesis phase of cell cycle; SAA, serum amyloid; SSeCKS, Src-
suppressed protein kinase C substrate with metastasis suppressor activity; Skp2, S-phase kinase-associated
protein 2 (p45); SUV39H1, Histone methyltransferase Suv39h1; SOS, son of sevenless protein; SMADs,
Mothers against decapentaplegic homolog (vertebrates) (derived from MAD [Mothers against dpp
CG12399-PA in Drosophila melanogaster] and SMA [in C. elegans]; STAT3, signal transducers and
activators of transcription; SOD1G37R, Superoxide dismutase 1 (transgenic mice overexpressing mutant);
Spike, small protein with inherent killing effect; Src, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene
homolog (avian) [Homo sapiens]; TGF, transforming growth factor; t-TGase, tissue-transglutaminase; TNF,
tumor necrosis factor; TUNEL, TdT-mediated dUTP-biotin nick end labeling (TdT = terminal deoxynu-
cleotidyl transferase); TrkA, tropomyosin receptor kinase A; Wnt, Wnt oncogene analog, wingless type

1 The Eukaryotic Cell Cycle

The cell cycle is an evolutionary, conserved, and ordered set of events resulting in the cell dividing into two
daughter cells. Nondividing cells rest outside the cell cycle in G0. They account for the major part of
nonproliferating cells. The cell cycle consists of two major consecutive events, basically characterized by
DNA replication and segregation of replicated chromosomes into two separate cells. Corresponding phases
of the cell cycle are the synthesis (S) phase in which DNA replication occurs and the mitosis (M) phase
in which the cell undergoes division into two daughter cells. G0 and S are separated by gap1 (G1) phase in
which cells increase in size and produce RNA and protein to get ready for replication, whereas S and M are
separated by gap2 (G2) phase in which cells continue to grow and produce new protein for subsequent cell
division (> Figure 8-1).
The transition from one phase to another and, thus, the progression of the cell cycle is tightly
regulated by defined temporal and spatial expression, subcellular localization, and degradation of cell
cycle regulators.

1.1 CyclinDependent KinasesKey Regulators of the Cell Cycle

Key regulators of the cell cycle are the cyclindependent kinases (Cdks), a family of small, serine/threonine
protein kinases (3035 kDa), whose members share greater than 40% identity. With the exception of Cdk3
and Cdk5, they are activated by a cyclin regulatory subunit (Morgan, 1997; Pavletich, 1999; Dhavan and
Tsai, 2001; Knockaert et al., 2002). Cdks are numbered in order of their discovery. In dividing cells, Cdks
regulate proliferation, differentiation, senescence, and apoptosis. A critical role in cellcycle regulation has
been demonstrated for Cdk1, Cdk2, Cdk4, Cdk6, and Cdk7, whereas other members of the Cdk family
subserve functions such as transcription (Cdk2, Cdk7, Cdk8, Cdk9, Cdk11), neurite outgrowth, neuron
migration, neurotransmitter signaling (Cdk4, Cdk5, Cdk11), differentiation (Cdk2, Cdk5, Cdk6, Cdk9),
and cell death (Cdk1, Cdk2, Cdk4, Cdk5, Cdk6, Cdk11) (Knockaert et al., 2002) (> Table 8-1).
The orderly progression through the G1, S, G2, and M phases of the cell division cycle is driven by the
sequential activation of Cdks (> Figure 8-2), which is controlled at multiple levels (1) by the accumulation
of cyclins as positive regulators, (2) at the assembly level into a cyclinCdk complex, (3) by phosphoryla-
tion, and (4) by their association with inhibitory proteins, the cyclindependent kinase inhibitors that can
either block activation or block substrate/ATP access (Evans et al., 1983; Sherr, 1993, 1994; Heichman and
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 161

. Figure 8-1
The phases of the cell cycle. The site of action of regulatory CDK/cyclin complexes is indicated

Roberts, 1994; Hunter and Pines, 1994; King et al., 1994; Sherr and Roberts, 1995; Poon et al., 1995; Rivard
et al., 1996; Coats et al., 1996; Morgan, 1997; Pavletich, 1999).

1.2 Regulation of CyclinDependent Kinases

1.2.1 Cyclins

The initial segment of the cell cycle, the first gap (G1) phase, is the site of integration of mitotic signals that result
from liganddependent activation of both growth factor receptors and integrins (Miyamoto et al., 1996; Assoian,
1997; Giancotti, 1997; Lin et al., 1997; Renshaw et al., 1997; Moro et al., 1998; Roovers et al., 1999; Assoian and
Schwartz, 2001; Danen and Yamada, 2001), which converge on the activation of G1cyclindependent kinases
(Cdks) Cdk4/6 and Cdk2 (Sherr 1993, 1994). The resultant Cdk activities determine whether mitogenic signals
are propagated downstream inducing phosphorylation of key substrates required for progression through G1 and
entry into Sphase. The major function of G1phase kinases is to partially phosphorylate the retinoblastoma
protein (pRb), thereby contributing to the inactivation of its repressive function. Consequently, the E2F
transcription factor can initiate transcription of cyclin E. Second, the cyclin D family sequesters the Cdk
inhibitor p27Kip1 from the previously inactive cyclinE/Cdk2 complex (> Figure 8-3).
. Table 8-1 162
Functional implications of cyclindependent kinases and associated cyclins
8
Cell cycle Cell differentiation Transcription Specific neuronal functions Cell death Other functions
CDK1 cyclinB prophase to metaphase amyloid
transition regulation of induced
topoisomerase 2 cytotoxicity
CDK2 cyclinA G1/S transition S phase regulation of Sp1 apoptosis in
cyclinE G2 phase centrosome mediated thymocytes,
duplication transcription mesangial cells
DNA damage
induced
apoptosis
CDK3 cyclinE G1/S transition
CDK4 cyclin D G1phase excitotoxin
induced neuronal
death
CDK5 p35, lens cell neurite outgrowth apoptosis Golgi membrane
p25 differentiation neurone migration traffic Insulin
P39, myogenesis induction of acetylcholine exocytosis by
p29 receptors dopamine and cells
glutamate signaling phototransduction
neurotransmitter release
CDK6 cyclin D G1phase neuronal cell
death
CDK7 cyclin H Cdk1, Cdk2 activation basal
transcription
CDK8 cyclin C regulation of basal
CDK7cyclin H transcription
CDK9 cyclinK MyoDmediated RNA signal
cyclinT myocyte transcription transduction
differentiation
CDK11 cyclin L RNA processing dopamine and glutamate apoptosis
or transcription signaling
Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

Modified after Knockaert et al. (2002)

 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 163

. Figure 8-2
In proliferating cells, the temporal association of Cdks to cyclins is tightly regulated to control the progression
of the cell cycle (modified after Nguyen et al., 2002)

When quiescent cells enter the G1 phase, genes encoding Dtype cyclins (D1, D2, and D3) are induced
in response to mitogenic signals in conjunction with the appropriate external stimuli such as cell adhesion.
During progression through G1, the level of Dtype cyclins increases, and, in a mitogenregulated manner,
these proteins associate with, and activate their catalytic partners, Cdk4 or Cdk6 (Sherr, 1993; Matsushime
et al., 1994; Vidal and Koff, 2000). The major function of Dtype cyclins is to provide a link between the
mitogenic stimulation and the autonomously progressing cellcycle machinery. Unlike the other cyclins,
cyclin D is not expressed periodically, but is synthesized as long as mitogenic stimulation persists (Assoian and
Zhu, 1997) and, thus, is usually absent from the cell cycle that progresses independent of the presence of
mitogenic signals. Conversely, constitutive activation of cyclin D can contribute to oncogenic cell transforma-
tion. Cyclin D1 mRNA translation can be inhibited by rapamycin, a mTOR/FRAP (target of rapamycin/FkBP12
rapamycinassociatedprotein) inhibitor. In contact inhibited cells, cyclin D is sequestered to the cytoplasm by
binding to SSeCKS/gravin protein, a major PKC substrate with tumor suppressor activity (Lin et al., 2000).
The first wave of cyclin Ddependent kinase activity is followed in late G1 by an increase in cyclin E,
which associates with Cdk2 (Dulic et al., 1992; Koff et al., 1992). The Cdk2/cyclin E complex is not only
responsible for the G1/S transition but also regulates centrosome duplication. Unlike the Dtype cyclin
dependent kinases, assembly of cyclin E and Cdk2 into active kinase is not mitogendependent. Cdk2
reinforces Cdk4 to complete Rb phosphorylation and induces the degradation of p27Kip1. The cell cycle is
now irreversibly committed to enter the S phase. CyclinE/Cdk2 phosphorylates a variety of substrates,
thereby contributing to relieve the Rbmediated repression. Cyclin E targets components necessary for DNA
synthesis, contributing to the assembly of the DNA replication complexes. Cyclin E, like cyclin D, is targeted
for degradation by phosphorylation. The active cyclin ECdk2 complex itself phosphorylates cyclin E,
which is subsequently recognized by the ubiquitin ligase SCF (Skp2) and targeted for degradation.
Proteolysis of cyclin E marks the transition into the S phase (Won and Reed, 1996).
Assembly of cyclin A with Cdk2 is required during the S phase. The Cdk2/cyclin A complex phosphor-
ylates various substrates allowing DNA replication. At the S/G2 transition, cyclin A associates with Cdk1.
In late G2 phase, cyclin B appears, complexes with Cdk1, and triggers the G2/M transition, where cyclin
A is degraded (Peters, 1998). This resets the system and reestablishes the requirement for mitogenic cues to
induce Dtype cyclins for the next cycle.
Taken together, cyclins can influence when and where the Cdks are active. They are more than a
conformational on/off switch for Cdk activity and influence Cdk activity by mediating critical interactions
between cyclin/Cdk complexes and cellular proteins such as substrates, inhibitors, and activators.
164
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

. Figure 8-3
The cell division cycle and its major regulatory elements

1.2.2 Protein Phosphorylation

In addition to cyclin binding, activity of Cdks is regulated by phosphorylation. Phosphorylation/dephos-

phorylation events prime Cdks for activation of regulatory subunits. Inhibitory phosphorylation on
Nterminal threonine and tyrosine residues of Cdk maintains kinase complexes in an inactive state. Late
in S phase, the dual specific kinases Wee 1 and Myt 1 phosphorylate Cdk1 on Thr14 and Tyr15,
thereby inactivating the kinase. Positive regulation of Cdk activity occurs in the following two steps:
dephosphorylation of the threonine and tyrosine residues by the Cdc25 phosphatase and phosphorylation
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 165

by the Cdk7cyclin H complex, also called Cdkactivating kinase (CAK) (Lew and Kornbluth, 1996). These
phosphorylations induce conformational changes and further enhance binding of cyclins (Jeffrey et al.,
1995).

1.2.3 Cdk Inhibitors

Activity of Cdks is negatively regulated by Cdkinhibitors, which bind directly to Cdks or to Cdk/cyclin
complexes (Biggs and Kraft, 1995; CordonCardo, 1995; Poon et al., 1995; Sherr and Roberts, 1995; Boice
and Fairman, 1996; Coats et al., 1996; Guan et al., 1996; Rivard et al., 1996). Based on structural features,
Cdk inhibitors are grouped into two families, the inhibitors of Cdk4 (INK4) family and the Cip/Kip family.
The INK4 family currently consists of four members, p16INK4a, p15INK4b, p18INK4c, and p19INK4d, which
commonly share the presence of ankyrin repeats (Serrano et al., 1993; Hannon and Beach, 1994; Guan et al.,
1994; Chan et al., 1995; Hirai et al., 1995). The INK4A locus encodes two independent but overlapping
genes, p16INK4a and p19ARF, the mouse homolog of p14ARF, which act in partly overlapping pathways
(Carnero et al., 2000). All these proteins can inhibit cyclin D associated kinases, Cdk4 and Cdk6. INK4
proteins compete with Dtype cyclins for binding to the Cdk subunit (McConnell et al., 1999; Parry et al.,
1999). The inhibition of cyclin binding is mediated by a conformational change in the kinase that prevents
association of the cyclins and distorts the ATP binding site (Russo et al., 1998). The INK4family of cyclin
dependent kinase inhibitors might be involved in the regulation of pathways that control cell growth and
proliferation as well as cell death. Deregulation of these Cdki proteins results in either uncontrolled
proliferation and neoplastic transformation or activation of apoptosis (Freeman et al., 1994; Kranenburg
et al., 1996; Liu et al., 1996a). The inhibitory action of the INK4 proteins is largely dependent on the
presence of pRb in the cell.
Three proteins currently comprise the following Cdk interacting protein/kinase inhibitory protein (Cip/
Kip) family: p21Cip1, p27Kip1, and p57Kip2 (Gu et al., 1993; Harper et al., 1993; Polyak et al., 1994a, b; Lee
et al., 1995a). These proteins share a homologous inhibitory domain, which is necessary for binding to
preformed complexes of cyclinCdks, thereby directly inhibiting the kinases. Compared to the INK4family,
they have a wider inhibiting specificity, affecting the activities of cyclin D, E, and Adependent kinases. In vivo
they preferentially act on Cdk2 complexes. Through binding to cyclin D/Cdk4 complexes, p27Kip1 and p21Cip1
can be sequestered without inhibiting the activity of the complex (Sherr and Roberts, 1995). Kip/Cip
proteins might even be able to promote activation of cyclin D/Cdk complexes by stabilizing the complexes
and directing their nuclear translocation (LaBaer et al., 1997; Cheng et al., 1999). When mitogens are
withdrawn and cyclin D is downregulated, this pool of Kip/Cip proteins is released, thereby inducing G1
phase arrest through inhibition of cyclin E/Cdk2. P21cip1 also associates with proliferating cell nuclear
antigen (PCNA), an essential binding partner of DNApolymerase delta, which might be an additional
mechanism through which p21Cip1 can inhibit DNA synthesis (Zhang et al., 1993; Luo et al., 1995).
In addition to binding to Cdks, p21Cip1 and p57Kip2 also bind to the PCNA (Gulbis et al., 1996). PCNA
is involved in DNA replication and binding of p21Cip1 or p57Kip2, prevents PCNAmediated DNA synthesis,
and arrests the cell cycle (Watanabe et al., 1998).
Cdkis are regulated by both external and internal signals. The expression of p21Cip1 is under the
transcriptional control of the p53 tumor suppressor gene (el Deiry et al., 1993). Expression and activation
of p15INK4b and p27Kip1 is increased by TGF, contributing to growth arrest (Reynisdottir et al., 1995).
Both p21Cip1 and p27Kip1 undergo phosphorylation, which determines their subcellular location. Recogni-
tion of the phosphorylated form of p27Kip1 by the ubiquitin ligase SCF (Skp2) targets p27Kip1 for
degradation.

1.2.4 Subcellular Sorting and Degradation of CellCycle Regulating Proteins

Correct cellcycle progression is also regulated by the subcellular sorting of cellcycle regulating proteins.
Cyclin B contains a nuclear exclusion signal and is actively exported from the nucleus until the beginning of
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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

the prophase. The Cdk inactivating kinases Wee1 and Myt1 are located in the nucleus and Golgi complex
and protect the cell from premature mitosis (Liu et al., 1997). Subcellular sorting of different proteins is also
regulated through sequestration by binding to the 1433 protein (Peng et al., 1997; Yang et al., 1999).
Many regulating proteins are subject to rapid degradation during the cell cycle. Proteins are irrevers-
ibly destroyed, which permits the orderly transition through the cycle, based on the completion of one
phase before entry into the next phase. The specific degradation of many proteins, including Cdks, cyclins,
and Cdk inhibitors, is achieved by targeted ubiquitination (Murray, 1995). Critical for the initiation of
mitosis and cellcycle exit is a specific E3type ubiquitin ligase, referred to as the anaphasepromoting
complex (APC) or the cyclosome (Peters et al., 1996). APC selectively targets proteins for ubiquitination
that contain a specific consensus sequence, which is found in cyclins and other mitotic proteins (Glotzer
et al., 1991).

1.2.5 Regulation of NoncellCycle Cdks

Regulation of a Cdk that is not related to cellcycle progression, such as Cdk5, differs markedly from that of
cellcycle Cdks (Nguyen et al., 2002). The association of Cdk5 with one of its neuronspecific coactivators,
p35 or p39, is required for neurite outgrowth, axonal migration, cortical lamination, control of cell
adhesion, axonal transport, synaptic activity, neuronal adaptive changes, and motor functions (Dhavan
and Tsai, 2001). Inhibition of Cdk5 or expression of a dominant negative form of the kinase in cultured
neurons prevents neurite outgrowth, whereas overexpression of p35/Cdk5 induces the formation of longer
neurites (Xiong et al., 1997; Nikolic et al., 1998). P35null mice display a loss of stratified cellular
organization (Chae et al., 1997; Kwon and Tsai, 1998), and an even more severe disruption of embryonic
cortical layering is observed in mice lacking Cdk5 (Ohshima et al., 1996). The cortical phenotypes of Cdk5,
p35, or p39null mice resemble a severe developmental disorder in humans, referred to as type 1
lissencephaly (Reiner, 2000). P35/Cdk5 is also involved in associative learning (Fischer et al., 2002). The
role of Cdk5 in plastic neuronal changes involves control of presynaptic function by phosphorylation of
Munc18, amphysin, and synapsin I (Dhavan and Tsai, 2001). Cdk5 also regulates neurotransmission and
longterm potentiation by phosphorylating the NR2A subunit of NMDA receptors (Li et al., 2001). The
involvement of Cdk5 in neurodegeneration has been suggested by reports of Cdk5 expression during
apoptosis and by the presence of the kinase in neurofibrillary tangles in AD and frontotemporal dementia
17 (FTD) (Yamaguchi et al., 1996; Pei et al., 1998). An abnormal localization of Cdk5 is also found in canine
motor neuron disease, Parkinsons disease, and amyotrophic lateral sclerosis (Nakamura et al., 1997; Bajaj
et al., 1998; Green et al., 1998).

1.3 Effects of Cdk ActivationCdk SubstratesThe Retinoblastoma Protein

The activities of G1 Cdk determine whether mitogenic signals are propagated downstream resulting in
phosphorylation of key substrates required for progression through G1. One of the key substrates is the
retinoblastoma protein (Rb), an inhibitor of progression through G1. Activation of cyclin Ddependent
Cdks initiate Rb phosphorylation in midG1 phase when cyclin E/Cdk2 becomes active and completes this
process by phosphorylating Rb on additional sites (Sherr, 1993, 1994, 1996; Weinberg, 1995; Taya, 1997;
Lundberg and Weinberg, 1998; Cheng et al., 1999). Cyclin A and Bdependent Cdks, which are activated
later during the cell cycle, maintain Rb in a hyperphosphorylated form (Ludlow et al., 1993). Resetting the
pRbE2F complex occurs in Mphase by dephosphorylation, probably by PP1 (Durfee et al., 1993; Ludlow
et al., 1993).
In quiescent cells, Rb is hypophosphorylated, and transcription factors such as E2F and DP bind
specifically to this hypophosphorylated form of pRb. RbE2F complexes bind to E2Fbinding sites in E2F
responsive genes and repress transcription (Kovesdi et al., 1986; Weintraub et al., 1992, 1995; Sherr, 1993,
1994; Weinberg, 1995; Luo et al., 1998; Zhang et al., 1999). Two other proteins, p130 and p107, are
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 167

Rbrelated members of the pRb pocket protein gene family. They are also substrates of the G1phase
Cdks, share structural and biochemical properties, and, like pRb, bind and modulate the activity of E2F
transcription factors (Ewen, 1998; Ferreira et al., 1998; Mulligan and Jacks, 1998). P107 and p130 are
structurally more closely related to each other than to Rb, and are thought to function redundantly in G0
and G1 to repress E2Fresponsive genes that are distinct from those regulated by Rb.
The E2F transcription factor consists of a heterodimer between E2F and DP family proteins. Six
members of the E2F family (E2F1 to E2F6) and three DPs (1, 2, 3) have been identified so far (Nervins,
1998). E2F1, E2F2, and E2F3 are structurally similar and form complexes exclusively with the retinoblas-
toma protein, whereas E2F4 and E2F5 interact primarily with p107 and p130. E2F1, E2F2, and E2F3 but
not E2F4 and E2F5 contain cyclinbinding motifs, which promote stable binding of cyclin A/Cdk2. E2F6
lacks both pocket proteinbinding and cyclinbinding sequences and acts as a transcriptional repressor for
E2Fresponsive genes (Morkel et al., 1997).
Rbmediated repression occurs through at least three mechanisms (Brehm et al., 1998; MagnaghiJaulin
et al., 1998; Luo et al., 1998; Swanton, 2004). First, Rb induces modification of histones in the nucleosome
by recruiting the histone deacetylase to E2F sites. Histone deacetylation causes tighter association between
DNA and nucleosomes and restricts access of the transcriptional apparatus to DNA, thereby repressing
transcription. Second, Rb regulates nucleosome structure by recruiting ATPdependent remodeling com-
plexes to promoters. Third, Rb influences chromatine structure by recruiting the SUV39H1 methylase and
HP1 to the cyclin E promoter leading to histone H3 methylation and silencing of cyclin E.
Cdkdependent phosphorylation of Rb (pRb) disrupts its association with E2F family members, relieves
pRbmediated repression, and allows E2Fdependent transcription of several genes involved in cell cycle
and growth regulation. Promoters of many genes have been found to contain consensus E2F binding sites,
including enzymes of DNA metabolism, protooncogenes, and cellcycle regulatory proteins such as cyclins
E and A both of which are required to catalyze the G1/S transition (Sherr, 1994; DeGregori et al., 1995;
Schulze et al., 1995; Weinberg, 1995; Dyson, 1998; Nevins, 1998; Black et al., 1999; Cheng et al., 1999).
Hypophosphorylated pRb also binds to other proteins, such as members of the Abl family, which seem to be
closely implicated in adhesiondependent growth control. The E2Fdependent induction of cyclin E, which
in turn stimulates Rb phosphorylation through Cdk2, provides a feedback loop that contributes to the G1/S
transition. This event of pRb phosphorylation and release of E2F corresponds to progression through the
restriction point R (Pardee, 1974).
In addition to regulation of E2F activity through phosphorylation of pocket proteins, direct phosphor-
ylation of E2F also influences its activity. Both cyclin A/Cdk2 and cyclin A/Cdc2 can phosphorylate E2F1
on multiple sites (Mudryj et al., 1991; Kitagawa et al., 1995; Adams et al., 1996; Dynlacht et al., 1997), which
might contribute to its downregulation in late S/G2 phase. Besides its involvement in cell growth and cell
cycle regulation, E2F also plays a role in apoptosis. Similar to growth regulation, E2Fmediated apoptosis is
regulated by pocket proteins (Shan et al., 1996).
Other Cdk substrates include histone H1 (Bradbury et al., 1974), DNA polymerase alpha primase
(Voitenleitner et al., 1997), their own regulators Wee1 and Cdc25, and cytoskeletal proteins such as lamins,
microtubules, and vimentin, which are required for correct mitosis (Hoffmann et al., 1993; Blangy et al., 1995).

1.4 Quality Control of CellCycle Progression by Stop and Go Signals:

Restriction Point and Checkpoints
Although initiation of the cell cycle depends on the presence of external cues, progression beyond G1 is
largely regulated by internal control. The restriction point, R (Pardee, 1974, 1989), marks the point of no
return, where cellcycle progression becomes independent of extracellular signals and the cells become
irreversibly committed to continue cellcycle progression.
The orderly sequence of cellcycle events resulting in successive waves of Cdk activation and inactivation
is controlled further, depending on the completion of previous steps, through the socalled checkpoint
controls (Lew, 2000). DNA damage checkpoints and spindle checkpoints have been identified. This
surveillance system is highly conserved from yeast to mammals, and its failure, especially in mammals,
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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

leads to genomic instability. These checkpoints allow to alternate decisions between further progression
through the cycle and growth arrest, in order to provide potentials for DNA repair or induction of
apoptosis. Apoptosis as an alternative to cellcycle progression is thus, aimed at preventing cell prolifera-
tion. In proliferating cells, this might be a protective mechanism against transmission of nonreparable
DNA. Depending on the chemical nature of the damage on DNA, different types of repair programs and
cellular responses are evoked (Laiho and Latonen, 2003). In highly differentiated cells, such as neurons, this
mechanism might be preserved to prevent inappropriate generation of new cells that cannot easily be
integrated into functional circuitry (Copani et al., 2001; Liu and Greene, 2001). Thus, an apoptotic
response might be inherent to an inappropriate activation of the cell cycle (Evan et al., 1995; Evan and
Littlewood, 1998).
DNA quality controls, i.e., damage checkpoints, are positioned before DNA replication, at the G1S
transition, and prior to mitosis, at the G2M transition. Further checkpoints are positioned during the S
and M phase. Cellcycle arrest induced by DNA damage at the G1S checkpoint is p53dependent. DNA
damage activates the product of the ataxiateleangectasia gene (ATM/ATR) and DNAdependent protein
kinase family, which leads to posttranslational stabilization of a normally labile p53 protein (Giaccia and
Kastan, 1998) (> Section 2.3.3).
Sphase checkpoints control initiation and elongation phase of DNA replication (Paulovich and Hartwell,
1995). Progression of the mammalian cell cycle requires the activity of Cdc25 family of phosphatases,
which dephosphorylate the inhibitory phosphorylations of Cdks and, thus, enable Cdk activities
required for S phase and mitosis. A G2 checkpoint prevents entry into mitosis by Cdk1 inhibition
through phosphorylation or sequestration outside the nucleus. The G2M checkpoint also involves p53,
which induces the Cdk inhibitor p21Cip1 as well as a member of the 1433 family, which sequesters cyclin
B outside the nucleus (Hermeking et al., 1997). In mitosis, the errorfree segregation of sister chromatides is
essential for division. In response to unreplicated or damaged DNA, the cell arrests at a DNA structure
checkpoint in mitosis. The spindle assembly checkpoint induces mitotic arrest in case of improper
alignments of chromosomes or defects in microtubule attachment (Amon, 1999). A further checkpoint,
dependent on both p53 and pRb, acts subsequent to mitotic errors to block proliferation of cells that have
entered G1 with tetraploid status (Borel et al., 2002).

1.5 Upstream Control of Cdk Activation

1.5.1 Growth Factor SignalingThe RasMAPK Pathway

Extracellular physiological signals affect the decision to transit the restriction point and this decision is
mediated through phosphorylation of pRb. This means, extracellular signals directly control phosphoryla-
tion of pRb. Mitogendependent induction of these mechanisms is often mediated through the RasMAP
kinase pathway (Filmus et al., 1994; Liu et al., 1995).
Small GTPases of the Ras family, which act as molecular switches in intracellular signaling (Bourne
et al., 1990), regulate cellular proliferation and mediate the mitogenic response of a variety of growth factors
(Mulcahy et al., 1985). In mammals, the Ras superfamily of GTPases contains more than 65 members.
According to their function and structure, this superfamily has been subdivided into six families repre-
sented by Ras, Rho, Rab, Ran, Rad, and Arf (Barbacid, 1987; Valencia, 1991). In mammals, three genes
encode the ras family: Hras, Kras, and Nras.
The characteristic of Ras function is its regulated transit between an inactive state, where it is bound to
guanine dinucleotides (GDPs), and an active state, bound to guanine trinucleotides (GTPs). The GDP/GTP
exchange is regulated by guanine nucleotide exchange factors (GEFs), which catalyze the GDP/GTP
exchange, and GTPaseactivating proteins (GAPs), which enhance the intrinsic capacity of Ras to hydrolyze
GTP into GDP, thereby returning Ras to the inactive state (Lowy and Willumsem, 1993; Schlessinger, 2000).
To be functional, Ras proteins must be recruited to the inner side of the plasma membrane. Upon ligand
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 169

binding, growth factor receptors dimerize, autophosphorylate tyrosine residues in their own cytoplasmic
domain, and recruit intracellular signaling molecules to the membrane. Among these recruited molecules
are GEFs for Ras protein, which promote binding to GTP (Schlessinger, 2000). Through conformational
changes that occur upon the exchange of GDP for GTP, the GTPbound form of Ras interacts with Raf,
phosphatidylinositol 3kinase (PI3K), and RalGdS, driving intracellular signaling cascades (> Figure 8-4).
Activated Raf stimulates MAPkinase extracellular signalregulated kinase (MEK) (Kyriakis et al., 1992),
which in turn activates mitogenactivated protein kinases p44 extracellular signalregulated kinase (ERK1)
and p42 (ERK2) (Gomez and Cohen, 1991). ERKs have a wide spectrum of substrates including cytoplas-
mic proteins such as SOS, MEK, Rsk, PHAS1, or PLA2, and nuclear proteins that are phosphorylated upon
ERK translocation to the nucleus, mainly transcription factors such as Elk1, Ets2, C/EBP, and SMADs
(Whitmarsh and Davis, 1996). The main substrates of PI3K are Akt/PKB and p70 ribosomal protein S6
kinase (p70S6K) (Downward, 1998). Activation of Akt generates an antiapoptotic signal and regulates
glycogen synthase kinase 3b (GSK3) among other substrates.
MAPkinase and PI3K pathways activate transcription factors to induce numerous genes, including
those of cyclin D1 (Hill and Treisman, 1999). The promoter of the cyclin D1 gene contains mitogen
responsive Ets and AP1 binding sites (Albanese et al., 1995), known to respond to Ras signaling (Galang
and Hauser, 1994). Ras signaling promotes nuclear location of cyclin D1 by negatively regulating GSK3,
which phosphorylates cyclin D1. This modification redistributes cyclin D1 to the cytoplasmic compartment
and labels cyclin D1 for degradation by the ubiquitinproteosome (Diehl et al., 1998). Ras also regulates the
cyclin D assembly. In quiescent cells, stimulated with growth factors, Ras signaling promotes transition
from G0 to G1 and from G1 to S. Proliferating cells make an Rasdependent commitment for completion of
the next cycle while they are in G2 phase of the preceding cycle (Hitomi and Stacey, 2001). Thus, Ras
activity during G2 phase induces cyclin D1 expression, which apparently persists independently of Ras until
the beginning of the next S phase (Sa et al., 2002).
Ras also controls cellcycle progression through control of the expression of other cyclins such as D3, A,
and E, and members of the E2F family of transcription factors (Fan and Bertino, 1997) as well as through
downregulation of p27Kip1 (Aktas et al., 1997).
Mutations conferring constitutive Ras activation are found in nearly 30% of human tumors (Bos,
1989). Oncogenic Ras promotes uncontrolled mitogenesis independently of the presence of growth factors
(Feramisco et al., 1984) but is unable to transform cells. Constitutively activated Ras, moreover, not only
regulates cellular proliferation, but also renders cells susceptible to apoptosis (Chen and Faller, 1995;
Gulbins et al., 1995; Mayo et al., 1997; Serrano et al., 1997; Downward, 1998; Joneson and BarSagi,
1999; Frame and Balmain, 2000; Liou et al., 2000; Khokhlatchev et al., 2002).
Depending on the activation of other signaling pathways, activation of Ras signaling pathways can also
arrest the cell cycle rather than activate proliferation (Kohl and Ruley, 1987; Ridley et al., 1988; Marshall,
1995; Serrano et al., 1997; Sewing et al., 1997; Woods et al., 1997). As a result, Ras suppresses oncogenic
transformation and induces a condition phenotypically identical to premature cellular senescence (Serrano
et al., 1997; Lin et al., 1998; Hahn et al., 1999). It selectively inhibits genes involved in mitosis, DNA
replication, segregation, and repair, and at the same time upregulates genes associated with cell death and
potentially involved in neurodegenerative disorders and tumorigenesis such as APP, serum amyloid (SAA),
and tissue transglutaminase (tTGase) (Chang et al., 2000). This process involves the upregulation of the
cyclindependentkinase inhibitors p15INK4b, p16INK4a, p21Waf1/Cip1, p19ARF, and p53 (Serrano et al., 1997;
Olson et al., 1998; Palmero et al., 1998; Ferbeyre et al., 2000; Fogal et al., 2000; Malumbres et al., 2000; Guo
et al., 2000a; Pearson et al., 2000; Zhong et al., 2000a, b; Gottifredi and Prives, 2001) and, thus, resembles
changes seen in early AD (Gartner et al., 1995, 1999; Arendt et al., 1996, 1998d, e). Activation of the p21Ras
cascade, thus, plays an essential role in oncogenesis and cellular senescence as well as AD (> Section 3.2.1).
Cellular effects of Ras activation are strictly cell type dependent and show pleiotropic features,
depending on the activity level of Ras signaling (Crespo and Leon, 2000). Whereas high levels of Raf
activity are able to induce cellular senescence, less intense Raf activation induces cyclin D1 expression and
drives cells into proliferation (Woods et al., 1997).
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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

1.5.2 Signaling Induced by CellCell and CellMatrix InteractionsExternal

Positional Cues

Activation of the cell cycle through growth factors also requires signals by the extracellular matrix. Entry
into G1 phase and further progression toward the S phase is a major downstream event of synergistic
signaling by mitogenic compounds and integrindependent cell adhesion. All of the important mitogenic
signaling cascades downstream of the Ras and Rho family small GTPases and the PI3kinasePKB/Akt
pathway are regulated by integrin mediated cell adhesion (Danen and Yamada, 2001) (> Figure 8-4). They
control critical molecular switches such as induction of cyclin D1 resulting in activation of Cdk4/6, the
suppression of p21Cip1 and p27Kip1 inducing Cdk2 activity (Fang et al., 1996; Schulze et al., 1996; Stromblad
et al., 1996; Zhu et al., 1996; Assoian, 1997; Resnitzky, 1997; Wu and Schonthal, 1997) and the subsequent
phosphorylation of Rb (Weinberg, 1995; Sherr and Roberts, 1999). Some of these matrixrelated effects
involve matrixdependent organization of the cytoskeleton. Adhesiondependent cellcycle progression
involves both integrin binding to the ECM and integrinmediated cytoskeletal organization (Hansen
et al., 1994; Assoian and Zhu, 1997; Chen et al., 1997). An organized cytoskeleton is, thus, a requirement
throughout the mitogendependent portion of the G1phase; and the adhesiondependent expression of
cyclin D and the phosphorylation of pRb are mediated by the cytoskeleton (Bohmer et al., 1996; Assoian
and Zhu, 1997). Cell adhesion to extracellular matrix (ECM) is mediated by binding to the cell surface
integrin receptors, which activate intracellular signaling cascades and mediate tensiondependent changes
in cell shape and cytoskeletal structure. Although growth control has focused on integrin and growth factor
signaling, the cell shape might play an equally critical role in cellcycle progression, which acts by
subjugating the molecular machinery that regulates the G1/S transition (Huang et al., 1998).
Adhesion to substratum has two separate effects on cells both of which could underlie the anchorage
requirement for cellcycle progression, i.e., the initial adhesion event and the subsequent clustering of
occupied integrins. Integrins might act as mechanoreceptors, transmitting mechanical information from
the extracellular matrix to the cytoskeleton (Wang et al., 1993; Hansen et al., 1994). Alternatively, an
organized cytoskeleton might be required to force integrins to remain clustered at focal contacts, and
integrin clustering seems to be a prerequisite for integrin signaling.

1.5.3 Convergence of Ras and IntegrinDependent Signaling

Signaling through Ras and integrin regulates cyclin D/Cdk4 and cylin E/Cdk2 at different levels. (1) Expression
of cyclin D1, often the rate/limiting step in the activation of cylin D/Cdk4/6, critically depends on synergistic

. Figure 8-4
Synopsis of current models of information flow (light gray) from cell surface events (dark gray) involved in
intercellular communication to gene expression. These pathways mediate structural plasticity and dynamic
regulation of intercellular connectivity as well as cellcycle activation and cellular proliferation (speckled). One
of the major downstream targets is cyclin D1, which is under positive control of bcatenin/LEF/TCF, RhoA, PAK,
JNK, and ERK1/2. RhoA also represses p21Cip1 and p27Kip1, inhibitors of Cdk2. Thus cyclin D/Cdk4/6 and cyclin E/
Cdk2 become active and phosphorylate E2Fpocket protein complexes (E2FpRb and E2Fp107), allowing for
E2Fdependent expression of cyclin A. This phosphorylation of pRb correlates with cellcycle progression out of
the growth factor dependent portion of the G1 phase. At multiple levels, these pathways are also potentially
involved in t phosphorylation (Cdk5, GSK3, JNK, ERK1/2) (hatched). (The individual regulatory relationships
depicted in this cartoon have been demonstrated in various published reports using different types of cells. These
signaling pathways, however, have neither been demonstrated in their entirety within a single experimental
system, nor is their action firmly established for neurons; for reference see: Assoian and Zhu, 1977; BenZeev, 1977;
Giancotti, 1997; Yamada and Geiger, 1997; Howe et al., 1998; Schlaepfer and Hunter, 1998; Schlaepfer et al., 1999;
Schoenwaelder and Burridge, 1999; Schwartz and Baron, 1999; Novak and Dedhar, 1999; Orford et al., 1999; Aplin
et al., 1999; Dedhar et al., 1999; Patapoutian and Reichert, 2000; Petit and Thiery, 2000; Dedhar, 2000; Welsh and
Assioan, 2000; Assoian and Schwartz, 2001; Baki et al., 2001) Modified after Arendt (2003)
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
. Figure 8-4 (continued)
8 171
172
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

signaling through the Ras/MAPK cascade, integrin, cadherin, and Wnt dependent signaling (Albanese et al.,
1995; Lavoie et al., 1996; Miyamoto et al., 1996; Sherr, 1996; Winston et al., 1996; Zhu et al., 1996; Aktas et al.,
1997; Kerkhoff and Rapp, 1997; Lin et al., 1997; Renshaw et al., 1997; Weber et al., 1997; Cheng et al., 1998;
Huang et al., 1998; Short et al., 1998; Aplin et al., 1999; Gille and Downward, 1999; Roovers et al., 1999; Roovers
and Assoian, 2000) (> Figure 8-4). (2) Assembly of newly synthesized cyclin D1 with Cdk4 similarly
depends on the Ras/MAPK cascade (Peeper et al., 1997; Cheng et al., 1998). (3) Turnover of Dtype cyclins,
moreover, is Rasdependent and mediated by the PI3kinaseAktpathway, which regulates the phosphory-
lation of cyclin D1 by GSK3b (Diehl et al., 1998). Inhibition of this pathway leads to cyclin D phosphory-
lation, enhancing its nuclear export and the ubiquitindependent proteasomal degradation. (4) Cell
adhesion is a prerequisite for efficiently downregulating the steadystate levels of p21Cip1 and p27Kip1
(Fang et al., 1996; Schulze et al., 1996; Zhu et al., 1996; Welsh and Assoian, 2000; Welsh et al., 2001).
P27Kip1 was first identified as a Cdk2inhibitory factor detected in contactinhibited cells (Koff et al., 1993;
Polyak et al., 1994a, b) and many factors, including cellcell contact, induce accumulation of p27Kip1
(Hengst and Reed, 1998). Under these conditions, p19INK4d and p27Kip1 cooperate to maintain differen-
tiated neurons in a quiescent state (Zindy et al., 1999). Adhesion lowers the levels of p21Cip1 and p27Kip1,
thus permitting activation of cyclin E/Cdk2 (Fang et al., 1996; Zhu et al., 1996) whereas high levels of
p27Kip1 persist in cells that were prevented from spreading by restriction of the size of the substratum,
despite normal MAPK activation (Huang et al., 1998).
Key intermediates of signaling of integrindependent cell adhesion upon cellcycle regulation are the
integrinlinked kinase (Radeva et al., 1997), which, through inhibition of GSK3 (Delcommenne et al.,
1998; Troussard et al., 1999), can also regulate both the expression and degradation of cyclin D1 (Diehl
et al., 1998) and Focal adhesion kinase (FAK), which controls cellcycle progression through G1 via JNK
(Oktay et al., 1999) and through RasERK1/2 (Chan et al., 1994; Cobb et al., 1994; Schaller et al., 1994;
Schlaepfer et al., 1994; Xing et al., 1994; Schlaepfer and Hunter, 1996; Zhao et al., 1998; Sastry et al., 1999;
Barberis et al., 2000; Assoian and Schwartz, 2001) (> Figure 8-4).
Downstream G1 events, including phosphorylation of pRb leading to release of E2Ftranscription
factors from their complex with Rb and cyclin A expression also require both soluble mitogens and cell
interaction with the extracellular matrix (Kang and Krauss, 1996; Bohmer et al., 1996; Koyama et al., 1996;
Schulze et al., 1996; Zhu et al., 1996; Assoian, 1997; Aplin et al., 1999).

2 CellCycle Regulators During Development and in Postmitotic Neurons

2.1 Stem Cell Proliferation and Neurogenesis

Postmitotic neurons in the CNS are generated from neuroepithelial stem cells, which are multipotent cells
that differentiate into progenitor cells of neurons and glial cells. Stem cells are undifferentiated cells capable
of selfrenewal and asymmetric divisions to generate both differentiated and undifferentiated cells. The
vertebrate CNS develops from the neuronal tube where the positions of the nuclei of stem cells are
correlated with the cellcycle phases (Sauer, 1935; Fujita, 1960) (> Figure 8-5). Cellular nuclei are located
in the outer part of the ventricular zone during S phase, where DNA replication occurs and move toward
the ventricular surface in G2 phase, where they complete mitosis. Daughter cells generated by this
symmetric division retain the characteristics of stem cells and enter into the G1 phase with their nuclei
in the inner part of the ventricular zone, which subsequently move further outward for a new round of
DNA replication. During neurogenesis, asymmetric division generates two daughter cells with one of them
differentiating into a neuroblast, which is incapable of replication and begins to migrate to the upper
marginal zone whereas the other daughter cell reenters the cell cycle as a stem cell. Symmetric divisions of
neuronal stem cells, which produce two identical daughter cells that reenter the cell cycle, are characterized
by vertically oriented cleavage, whereas asymmetric division generating two daughter cells with different
fates is characterized by horizontally oriented cleavage.
During neurogenesis, up to 50% of cells in both the ventricular zone and the postmitotic cortical plate
are estimated to undergo apoptosis (Raff et al., 1993). Apoptosis in the early stages of neurogenesis may
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 173

. Figure 8-5
Proliferation of stem cells during early stages of neuronal tube development (symmetric) and during neuro-
genesis (asymmetric). Modified after Yoshikawa (2000)

occur as a result of erroneous DNA replication. In later stages, neuronal apoptosis might be due to limited
availability of trophic support or lack of synaptic input from target cells (Oppenheim, 1991). Neurons of
particular types arise from cells that divide at particular times. Different neuronal fates, thus, appear to be
determined at specific developmental stages or after a certain number of cell divisions. Some fates are
determined independently of active cycling, and the mature phenotype of a neuron is often determined even
postmitotically. Other fates, however, are determined when a cell is actively proliferating, and the cellcycle
status might have a critical influence over the determination process. The balance between proliferation and
differentiation in the nervous system is, thus, critical for histogenesis, and there might indeed exist
bidirectional mechanisms for coordinating the cell cycle with the process of neuronal determination and
differentiation (Edlund and Jessell, 1999; Ohnuma et al., 2001).
A variety of molecules such as Cdc2, Cdk1, Cdk5, Dtype cyclins, p27Kip1, p57Kip2, or Rb may have
complementary but separable roles in regulating the cell cycle and promoting neuronal determination/
174
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

differentiation (Hamel et al., 1992; Okano et al., 1993; Ohnuma et al., 1999; Dyer and Cepko, 2000; Levine
et al., 2000) (see also > Table 8-1). In addition, various determination/differentiation factors such as
TGFa, TGFb, EGF, or Notch might regulate cellcycle factors through transcriptional or posttranscrip-
tional mechanisms (Dorsky et al., 1997; Hanahan and Weinberg, 2000).

2.2 Do CellCycle Regulators Have Alternative Functions Unrelated

to CellCycle Regulation?
Differentiated neurons are postmitotic cells that have permanently withdrawn from the cell cycle. Most
mammalian CNS neurons reach their postmitotic state during the embryonic period when they enter into
an extended G0phase. At this stage, neurons are incapable of dividing and they are also not able to
regenerate (Arendt, 2003). However, they express a number of cellcycle regulators (Yoshikawa, 2000;
Schmetsdorf et al., 2005). Why these genes that potentially promote cellcycle progression are expressed
in permanent mitotic quiescence remains elusive. From a phylogenetic point of view, the evolution of the
cyclin-Cdkbased engine to control the cell cycle from other, older protein kinases had been suggested
(Murray, 2004). It might, thus, be speculated that control of cell cycle is a phylogenetically newly acquired
role of these regulators that still might subserve a wider range of cellular functions, such as coordinating
tissue remodeling and neuronal plasticity (Nabel, 2002; Arendt, 2003; Schmetsdorf et al., 2005).
Functions unrelated to Cdk binding have recently been suggested for some cyclins and Cdk inhibitors
(Tannoch et al., 2000; Coqueret, 2003; Schmetsdorf et al., 2005). These additional functions of cellcycle
regulators rely on different cellular localizations and different targets, present either in the cytoplasm, in the
nucleus, and probably also on DNA (Coqueret, 2003). Cip/Kip proteins, for example, might act as
assemblypromoting factors of cyclin D1/Cdk4 complexes, and besides their nuclear inhibitory function,
also activate Dtype cyclin complexes through enhanced association and nuclear accumulation. Both p21Cip1
and p27Kip1 might also have antiapoptotic prosurvival effects, which are associated with its relocation to
specific cytoplasmic compartments and, in addition, might be required to maintain the differentiated state
of certain cells (Bunz et al., 1998 ; Chang et al., 2000; Levine et al., 2000; Blagosklonny, 2002).
Further, both cyclin D1 and Cdk inhibitors of the Cip/Kip family such as p21Cip1 and p27Kip1 might act
as transcriptional cofactors, thereby controlling the transcriptional activation of various genes in a Cdk
independent manner (Zwijsen et al., 1997; McMahon et al., 1999; Bienvenu et al., 2005). p21Cip1, for
example, regulates activity of NFkB, STAT3, Myc, C/EBP, and E2F (Coqueret and Gascan, 2000; Kitaura
et al., 2000; Harris et al., 2001), and it inhibits expression of several genes involved in cellcycle progression
such as those encoding for DNA polymerase a, topoisomerase II, cyclin B1, and Cdk1 (Chang et al., 2000).

2.3 Coupling Cell Cycle to Cell Death

2.3.1 Apoptosis

Apoptosis is a highly conserved mechanism by which eukaryotic cells actively commit suicide. Apoptosis in
the nervous system has been recognized as an essential process during development where it plays an
essential role in the control of the final number of neurons (Oppenheim, 1991). Apoptosis might also be
responsible for the loss of neurons during normal aging (Tagliatella et al., 1996; Kaufmann et al., 2001).
Apoptosis involves a series of stereotyped, morphologically welldefined phases. Its morphological
manifestations include nuclear and cytoplasmic condensation, intranucleosomal DNA cleavage, nuclear
membrane breakdown and plasma membrane blebbing, and the formation of apoptotic bodies. In
proliferating cells, apoptosis acts to prevent transmission of nonrepairable DNA modifications to the
progeny. Apoptosis, thus, prevents cell proliferation if it is inappropriate. This role is potentially maintained
in postmitotic cells such as differentiated neurons in which unscheduled proliferation would otherwise
generate neurons that cannot be easily integrated into functional circuitry. In proliferating cells, it is mostly
DNA damage that triggers cellcycle arrest, and if DNA damage is too extensive to be repaired, apoptosis.
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 175

During development, death is the default (Benn and Woolf, 2004). Immature neurons are compe-
tent to die (Deshmukh and Johnson, 1998) by an active apoptotic pathway unless it is shut off by sufficient
trophic support (Oppenheim, 1991). Thus, apoptosis during development results from the absence of
enough trophic factor support or can actively be triggered by signaling through the lowaffinity neuro-
trophin receptor p75NTR (Carter and Lewin, 1997; Miller and Kaplan, 2001) or the FAS receptor (Raoul
et al., 1999, 2000).
In mature neurons, two main pathways lead to apoptosis, an extrinsic or death receptorinitiated
pathway, and an intrinsic or mitochondrial pathway (Green, 1998; Strasser et al., 2000; Benn and Woolf,
2004). Both pathways converge at the mitochondrion on the release of apoptogenic proteins (Shimizu et al.,
2001; Rostovtseva et al., 2004; Sharpe et al., 2004). Extrinsic apoptosis is initiated by the activation of
plasma membrane death receptors of the tumor necrosis factor (TNF) receptor superfamily. The extrinsic
death receptor pathway either directly activates effector caspases through JNK or converges with the
intrinsic pathway at the mitochondrion. This intrinsic pathway results from mitochondrial damage that
leads to the release of cytochrome c from the mitochondria, which triggers the formation of the apoptotic
complex and the subsequent activation of effector caspases. Death pathways differ between neurons. The
specific deathsignaling pathway that leads to degeneration depends on the neuronal population that is
affected, as well on the nature, stage, cause, and extent of the deathinducing insult.
Apoptosis, proliferation, and differentiation are coupled through potentially multifunctional up-
stream signaling pathways. According to the cellular context, activation of identical pathways is translated
into such diverse cellular responses as growth arrest, cycling, cell death, or survival (Blagosklonny, 2003).
The cell cycle and apoptosis share both identical molecular regulators such as Rb, E2F, and p53 and cellular
phenomena such as detachment from the matrix, condensation of chromatin, disassembly of nuclear
lamina, and membrane blebbing (Ucker et al., 1991; Raff et al., 1993; Evan et al., 1995; Liu and Green,
2001). Deregulation of the cell cycle can either directly induce apoptosis or can increase the sensitivity to
apoptotic inducers (Evan et al., 1995; Evan and Littlewood, 1998). Successful proliferation, as an alternative
to apoptosis, requires active suppression of the apoptotic program.
The activation of various components of the cell cycle in postmitotic neurons might contribute to
apoptosis (King and Cidlowski, 1995). The induction of proteins that control the G1/S phase transition in
proliferating cells such as cyclin D1 occurs in dying neurons (Freeman et al., 1994; Kranenburg et al., 1996),
and G1 cyclindependent kinases can trigger neuronal death (Park et al., 1997a, b, 1998a). Similarly, proteins
that control the G2/M phase transition such as Cdc2 might be involved in apoptosis (Shi et al., 1994; Vincent
et al., 1997; Yu et al., 1998). Neuronal activity deprivation in cerebellar granule neurons can stimulate the
activity of Cdc2, which triggers apoptosis (Konishi et al., 2002). This process involves phosphorylation of the
BH3only protein Bad, thereby activating Badmediated apoptosis by inhibiting its sequestration through
1433 protein.
In neuronal apoptosis, induced in a variety of experimental in vitro paradigms, changes in Cdk
activities, levels of cyclins and Cdkis, and deregulation of Rb/E2F activity have been observed (Park et al.,
1997a, b, 1998a, b; Padmanabhan et al., 1999; OHare et al., 2000) (> Table 8-2). Induction of Cdks, for
example, occurs in vivo in mature adult neurons in mouse, rat, or rabbit during ischemia, excitotoxic cell
death, spinal cord injury, or various mutant mouse models associated with neuronal death such as mutant
SOD1G37R, weaver mouse, harlequin mouse, or staggerer mouse. It is also seen in vitro in neuroblastoma
cells or primary neuronal cultures deprived of trophic factor support or treated with DNA damaging agents
or amyloid (Giovanni et al., 1999, 2000; Park et al., 2000). The induction of Cdks in neurons is associated
with their dedifferentiation and triggers neuronal death that can be blocked by Cdk inhibitors or dominant
negative Cdks (Park et al., 2000; Liu and Greene, 2001).
NGF deprivation, for example, leads to increased Cdc2 activity and cyclin B expression in PC12 cells
(Gao and Zelenka, 1995), as well as elevated cyclin D1 levels in sympathetic neurons (Freeman et al., 1994)
whereas pharmacological inhibitors of Cdks, upregulation of Cdk inhibitors, or dominant negative Cdk4 or
Cdk6 promote survival of NGFdeprived sympathetic neurons (Park et al., 1997a, b, 1998a, b). Apoptosis,
induced by DNA damage, requires Cdk4 and Cdk6 activity and subsequent phosphorylation of pRb and
activation of the pRb/E2F/DP pathway (Park et al., 2000; Liu and Greene, 2001) and is suppressed by the G1/S
transition blockers deferoxamine and mimosine as well as by the Cdk inhibitors flavopiridol and olomoucine
176
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

. Table 8-2
Evidence of cellcycle activation under experimental conditions of in vivo and in vitro cell death
In Vivo Paradigms of Cell Death
Ischemia (transient focal or global ischemia in mouse, rat, or rabbit)
increased neuronal expression of cellcycle regulators/proliferation Guegan et al. (1997)
associated proteins Hayashi et al. (2000)
Katchanov et al. (2001)
Cdk4 Li et al. (1997a, b)
Cdk2 Osuga et al. (2000)
cyclin D1 Sakurai et al. (2000)
cyclin B1 Timsit et al. (1999)
cyclin G1 Tomasevic et al. (1998)
p21Cip1 van Lookeren Campagne
MPM2 and Gill (1998)
PCNA Gill (1998)
increased phosphorylation of Rb Wang et al. (2002)
neuroprotection through post/periischemic administration of Cdk inhibitors Wen et al. (2004)
Combined ischemia/hypoxia
increased neuronal expression of cellcycle regulators/proliferation Kuan et al. (2004)
associated proteins
Cdk2
Ki67
increased phosphorylation of Rb
DNAsynthesis (BrdU incorporation)
Kainateinduced seizures
increased neuronal expression of cellcycle regulators Ino and Chiba (2001)
Cdk4 Park et al. (2000)
cyclin D1 Timsit et al. (1999)
increased phosphorylation of Rb
Spinal cord injury (rat)
increased neuronal expression of cellcycle regulators/proliferation Di Giovanni et al. (2003)
associated proteins
Cdk4
cyclin D1
cyclin G
Gadd45a
cmyc
PCNA
increased phosphorylation Rb, E2F5
Amyotrophic lateral sclerosis caused by mutant SOD1G37R mice
increased neuronal expression of cellcycle regulators/proliferation Nguyen et al. (2003)
associated proteins
Cdk4
cyclin D1
increased phosphorylation of Rb
Weaver mouse (apoptosis in the external germinal layer (EGL)
of the cerebellum)
increased neuronal expression of cellcycle regulators/proliferation Migheli et al. (1999)
associated proteins
Cdk4
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 177

. Table 8-2 (continued)

cclin D
cyclin A
PCNA
harlequin mouse mutation (progressive degeneration of terminally
differentiated cerebellar and retinal neurons)
increased neuronal expression of cellcycle regulators/proliferation Klein et al. (2002)
associated proteins
PCNA
Targetrelated neuronal death during development (apoptotic staggerer
granule cells)
increased neuronal expression of cellcycle regulators/proliferation Herrup and Busser (1995)
associated proteins
cyclin D
PCNA

In Vitro Paradigms of Cell Death

Apoptotic cell death induced by:
Trophic withdrawal/serum starvation Freeman et al. (1994)
KCl withdrawal Gao and Zelenka (1995)
DNA damage induced by camptothecin or UV radiation Giovanni et al. (1999)
Cisplatin Gill and Windebank (1998)
amyloid
(murine Balb/c3T3 cells, neuroblastoma cells, PC12 cells, sympathetic Kranenburg et al. (1996)
neurons, and rat cortical neurons) Padmanabhan et al. (1999)
increased neuronal expression of cellcycle regulators/proliferation Pandey and Wang (1995)
associated proteins Park et al. (1998, 2000)
Cdk1
Cdc2
Cdk4
cyclin B
cyclin D1
PCNA
increased phosphorylation of Rb
BrdU incorporation

Paradigms of experimental neuroprotection

in vitro Ino and Chiba (2001)
CDK inhibitor flavopiridol and olomoucine Park et al. (1996, 1998a, b, 2000)
suppress pRb and p107 phosphorylation in paradigms, DNA damage
induced by camptothecin
suppress death of PC12 cells and sympathetic neurons after trophic factor
withdrawal
Ectopic expression of p16Ink4a, p21Waf/Cip1, and p27Kip1, as well as DNCdk4
and 6
protect rat sympathetic and cortical neurons against DNA damage and
apoptosis induced by camptothecin or UV radiation
in vivo
intraventricular infusion of Cdk4 or cyclin D1 antisense oligonucleotides
suppresses excitotoxin
induced neuronal cell death
178
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

(Park et al., 1997). Activation of the cyclin D/Cdk4/6RbE2F pathway might, thus, be a critical component of
the apoptotic mechanism induced by DNAdamage or trophic factor deprivation in postmitotic neurons (Liu
and Greene, 2001; Kruman et al., 2004).

2.3.1.1 Caspases Caspases are the executioners of the apoptotic program (Thornberry and Lazebnik,
1998). Caspases stands for cysteine aspartate proteases. They cause cell death by degrading critical structural
elements such as lamins and gelsolin, and by activating latent enzymes, such as DNAses, through proteolysis
of their inhibitors. Caspases are present in healthy cells as inactive precursors (zymogens) that can be
activated by caspasemediated cleavage. Proteolysis releases two fragments of about 10 kDa and 20 kDa that
are assembled into the active tetrameric enzyme (OConnor et al., 2000). There are 14 members in the
mammalian family of caspases, divided into three subfamilies on the basis of the peptidesequence
preferences of their substrates. Certain initiator caspases, such as caspase8 and caspase9 have low
enzymatic activity in their zymogen form and contain regulatory aminoterminal prodomains that can
bind to specific adapter proteins (Salvesen and Dixit, 1997; Thornberry and Lazebnik, 1998). Caspase8 is
associated with apoptosis involving death receptors, and loss of adhesion, whereas caspase9 is involved in
stressinduced apoptosis (Earnshaw et al., 1999). Stressed cells might receive conflicting signals for cell
division and growth arrest, which might trigger apoptosis (Arsura and Sonenshein, 1996; OConnor et al.,
2000). In response to apoptotic stimuli, adapter proteins bind these initiator caspases and form oligomeric
aggregates, which facilitate autocatalytic processing of the cymogens (Martin et al., 1998; Srinivasula et al.,
1998). Initiator caspases subsequently process effector caspases, creating a proteolytic cascade that
executes cell death. In addition, there are noncaspase proteases such as cathepsins, calpains, and
granzyme B, involved in apoptosis.

2.3.1.2 Anoikis The adhesionrelated form of apoptosis is called anoikis (Frisch and Francis, 1994). It is
triggered by inappropriate or inadequate contacts between the cell and the extracellular matrix
and essentially displays all the features of apoptosis such as membrane blebbing, nuclear fragmentation,
and DNA degradation.
Integrinmediated cell anchorage, furthermore, has a vital role in the control of apoptosis (Frisch and
Ruoslahti, 1997; Aplin et al., 1998). They regulate cell viability through their interaction with the extracel-
lular matrix and can convert cellmatrix interactions into intracellular signals. These mechanisms involve
FAK (Frisch et al., 1996b) and Rasdependent signaling (Chen and Guan, 1994; Datta et al., 1997; King et al.,
1997; Khwaja et al., 1997) as well as the Wnt1/catenin pathway (Frisch and Ruoslahti, 1997; Kuhl and
Wedlich, 1997). FAK when activated can suppress anoikis. Phosphatidylinositol 3kinase/Akt and mitogen
activated protein kinase may mediate the anoikissuppressing effects. Activation of TrkB, the major receptor
for brainderived neurotrophic factor, can also suppress anoikis through stimulation of PI3 kinase signaling
(Douma et al., 2004). Conversely, the stressactivated protein kinase/Jun aminoterminal kinase pathway
promotes anoikis (Zhan et al., 2004). Control of anoikis may vary substantially from one cell lineage to
another and Ras may fulfill both positive and negative regulatory functions depending on the cell type
(Meredith et al., 1993; Frisch and Francis, 1994; Hall et al., 1994; Polakowska et al., 1994; Ruoslahti and
Reed, 1994; Coucouvanis and Martin, 1995; Boudreau et al., 1995; Hungerford et al., 1996; Vachon et al.,
1996, Assoian, 1997; Frisch and Ruoslahti, 1997; Khwaja et al., 1997; Meredith and Schwartz, 1997; McGill
et al., 1997; Eckert et al., 2004).
Anoikis may also involve phosphorylation of FAK and p53dependent mechanisms (Zhang et al., 2004).
The proapoptotic protein Bim, a member of the Bcl2family, is a critical mediator of anoikis in epithelial
cells (Reginato et al., 2003). Detachmentinduced expression of Bim requires a lack of 1integrin engage-
ment, downregulation of EGFreceptor expression, and inhibition of ERK signaling (Reginato et al., 2003).

2.3.2 The Bcl2 Family

The Bcell leukemia/lymphoma2 (Bcl2) families of proteins are major gatekeepers of apoptosis (Cory
and Adams, 2002). They control cell survival through both caspasedependent and caspaseindependent
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 179

pathways and can act via regulation of permeabilization of the outer mitochondrial membrane (Cory and
Adams, 2002; Akhtar et al., 2004). The Bcl2 family consists of three main subgroups, which are categorized
according to their anti or proapoptotic function, and the presence or absence of the Bcl2 homology (BH)
domains (Korsmeyer, 1999).
Antiapoptotic members, such as Bcl2, BclxL, Bclw, and myeloid cell leukemia1 (Mcl1) contain four
Bcl2homology domains. They can also delay entry into the cell cycle (OReilly et al., 1996). The
proapoptotic members of this family fall into two categories: Baxlike proteins, such as Bcell lymphoma
2associated protein X (Bax), Bcl2 agonist killer 1 (Bak), Bcl2related ovarian killer protein (Bok), and BclxS
that contain multiple BH domains, or BH3only members, such as Bcl2associated death protein (Bad),
BH3 interacting domain death agonist (Bid), Bcl2interacting mediator of cell death (Bim), Bcl2 mod-
ifying factor (Bmf) death protein 5/harakiri (DP5/Hrk), Puma (p53upregulated modulator of apoptosis),
small protein with inherent killing effect (Spike), and phorbol12myristate13acetateinduced protein
(PMAIP1/Noxa) (Puthalakath and Strasser, 2002; Akhtar et al., 2004). Although dimerization among the pro
and antiapoptotic members of the Bcl2 family plays a major role in the regulation of apoptosis, the expression
levels of Bcl2 determine the cells fate. Bcl2 family members are involved in neuronal programmed cell death and
play a significant role in diseases of the nervous system such as hypoxicischemic cell death, seizureinduced
neurodegeneration, motor neuron disease, and neurotoxic and mechanical neuronal injury (for review see
Akhtar et al., 2004).

2.3.3 P53

Another major element in the regulation of apoptosis is p53. Its activation prevents cell proliferation by
inducing cellcycle arrest at the G1S checkpoint or apoptosis through activation of proapoptotic genes
such as bax.
Although the constitutive level of p53 is low, it can rapidly be induced through DNA damage (Levine,
1997), which stimulates the transcription of regulators such as p21Cip1, mdm2, and bax (Agarwal et al.,
1998). P21Cip1 inhibits Cdk activation and results in cellcycle arrest, thus, preventing replication of
damaged DNA (Ko and Prives, 1996). This function of p53 in shutting down the proliferation in response
to DNA damage reflects its role as guardian of the genome (Lane, 1992). Mdm2 provides a feedback loop
regulating degradation of p53 by ubiquitination (Oren, 1999). In severely damaged cells unable to repair
DNA, p53 induces bax, fas, and other genes involved in apoptotic signaling (Miyashita and Reed, 1995; Yin
et al., 1997; Gottlieb and Oren, 1998).

2.3.4 Nitric Oxide

Nitric oxide (NO) is a signaling free radical, which displays antiadhesive properties (Radomski et al., 1990)
and can be considered as a cause for anoikis (Monteiro et al., 2004). It has the capacity to signal and trigger
proapoptotic events in a variety of cell types. NO can inhibit cell adhesion, interfere with the assembly of
focal adhesion complexes, and disrupt the cellextracellular matrix interactions. NO is enzymatically
derived from Larginine and O2. For the enzymes NO synthases (NOS), three isoforms can be distin-
guished, Type I or neuronal isoform (nNOS), Type II or inducible isoform (iNOS), and Type III or
endothelial isoform (eNOS). Lipopolysacharides, cytokines, and toxins can induce the expression of the
iNOS with the production of large amounts of NO whereas the neuronal and endothelial isoforms are
Ca2/calmodulindependent, constitutively expressed enzymes that produce lower amounts of NO
(Nathan, 1992).
NO might have pleiotropic effects on the induction of apoptosis and cell survival, depending on factors
such as levels of NO production, coactivation of other signaling pathways, and other cell typespecific
features. In a variety of cell types such as lymphocytes, hepatocytes, and endothelial cells, NO, at tissue
concentrations, can effectively block lipid peroxidation, and protect against oxidative damage mediated cell
death and apoptosis (Padmaja and Huie, 1993; Liu and Stamler, 1999). Among other mechanisms, these
180
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

effects involve the activation of p21Ras signaling through both MAPkinase and PI3 pathway (Lander et al.,
1995; Deora et al., 1998; McFall et al., 2001). As opposed to the protective role of NO when produced at low
fluxes, NO generated at high fluxes through iNOS is cytotoxic (Brune et al., 1999). NO has the capacity to
trigger proapoptotic events. It can inhibit cell adhesion, and it interferes with the assembly of focal adhesion
complexes, thereby disrupting cellextracellular matrix interactions leading to anoikis. NO can potentially
uncouple cooperative signaling by growth factors and matrix receptors, which ultimately leads to apoptosis
(Frisch and Screaton, 2001; Monteiro et al., 2004). This underlies the importance of coupling integrin and
growth factorsignaling pathways for regulating cellular survival.

2.3.5 Transcription Factors

2.3.5.1 CMyc CMyc is a nuclear phosphoprotein that acts as a transcription factor stimulating both cellcycle
progression and apoptosis (Penn et al., 1990; Facchini and Penn, 1998). Activity of cMyc is regulated through
phosphorylation and interaction with other cellular proteins such as Max (Lutterbach and Hann, 1994).
It can exert its effects on the cell cycle by controlling transcription of genes coding for Cdc25A, cyclin
D1, cyclin D2, cyclin E, cyclin A, Cdk1, Cdk2, Cdk4, and E2F (Born et al., 1994; Kim et al., 1994; Beier et al.,
2000). It can also suppress transcription of the genes for Cdk inhibitors p15INK4b, p21Cip1, and p27Kip1
(Grandori et al., 2000). As cMyc responds directly to mitogenic signals, it might have a critical
role especially in the transition from G0 to S phase (Spencer and Groudine, 1991). CMyc expression,
however, is maintained throughout the cell cycle and might have a role in G2 (Hann et al., 1985; Mateyak
et al., 1997).
CMyc also plays a key role in apoptosis, which might involve p53dependent and independent path-
ways. CMyc can transactivate the p53 gene promoter and increase the halflife of p53 (Hermeking and Eick,
1994). CMycinduced apoptosis might also involve activation of Fas receptor (Wang et al., 1998) and
inhibition of Bcl2 and Mcl1 (Reynolds et al., 1994). Phosphorylated cMyc can be detected immunocyto-
chemically in dystrophic neurites and neurons with neurofibrillary degeneration in AD, and in neurons and
glial cells with abnormal t deposition in Picks disease, progressive supranuclear palsy, and corticobasal
degeneration (Ferrer et al., 2001).

2.3.5.2 E2F A number of cellcycle molecules that have been associated with neuronal death converge on
regulation of the E2F family of transcription factors. E2F acts as a gene silencer in neurons and that repression is
required for neuronal survival (Liu and Greene, 2001). Induction of apoptosis induced by DNA damaging
agents or trophic factor withdrawal is characterized by depression of E2F responsive genes (Liu and Greene,
2001). Among those genes that are derepressed in the process of cell death are the transcription factors B and
Cmyb. E2F can also induce the expression of the proapoptotic factor Apaf1 (Moroni et al., 2001). E2F1
deficient mice are more resistant to focal ischemia (MacManus et al., 1999). PRb/E2F and p53
pathways may be directly linked in the cell cycle and apoptosis. Activated p53 induces p21Cip1,
thereby preventing Cdk activation and subsequent phosphorylation of pRb. In contrast, free E2F induces
p53 transactivation, connecting the pRb/E2F directly to p53dependent apoptosis (Hiebert et al., 1995).

2.3.5.3 STATs Many cytokine receptors transduce signals that promote cell division, block apoptosis, and
induce differentiation. These pathways involve distinct members of the JAK family of kinases and the STAT
family of transcriptional regulators (Ihle, 1996). Depending on the specific stimulus or cell type, STATs can
mediate either proapoptotic or antiapoptotic signals (Battle and Frank, 2002). Regulation of apoptotic
pathway by STATs is largely due to transcriptional activation of genes, which encode proteins that are
involved in cell death processes, such as BclxL, caspases, Fas, and TRAIL as well as those that regulate cell
cycle progression such as p21Waf1.

2.3.5.4 NFkB The transcription factor NFkB is activated by a broad range of signals including cytokines,
mitogens, free radicals, and other stress signals. NFkB upregulates several survival factors, and might also directly
promote antiapoptotic mechanisms, such as upregulation of Bcl2 family members (Baichwal and Baeuerle, 1997;
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 181

Hinz et al., 1999; Glasgow et al., 2001). In contrast, there is also evidence for apoptosispromoting activity of
NFkB, depending on the nature of the apoptosisinducing stimulus (Kaltschmidt et al., 2000).

3 CellCycle Regulators in Neurodegeneration

Cellcycle regulators are recognized targets for the treatment of proliferative disorders such as cancer,
cardiovascular disease, infections, or autoimmune diseases (Brooks and La Thangue, 1999; Nabel, 2002;
Vermeulen et al., 2003a) (> Table 8-3). In cancer, cellcycle deregulation occurs through mutations affecting
proteins critically involved in different levels of the cell cycle such as Cdk, cyclins, Cdkactivating enzymes,
Cdk inhibitors, Cdk substrates, and checkpoint proteins (Sherr, 1996; McDonald and el Deiry, 2000).

. Table 8-3
Disorders with involvement of cellcycle regulators that are potential targets for treatment with cellcycle
inhibitors
Cancer
Cardiovascular disease
atherosclerosis
cardiac hypertrophy
Nervous system
Alzheimers disease
amyotrophic lateral sclerosis
stroke
Viral infections
human cytomegalovirus (HCMV)
human papillomavirus (HPV)
human immunodeficiency virus (HIV)
herpes simplex virus (HSV)
Fungal infections
Protozoan disease
malaria
leishmaniosis
trypanosomes
Psoriasis
Glomerulonephritis

Recent evidence indicates that the control of proliferation and differentiation, i.e., cellcycle control, is also
involved in degeneration of nondividing cells, such as mature neurons that might be of critical relevance to
the pathomechanism of AD and related neurodegenerative disorders (Arendt, 1993; Heintz, 1993; Nagy,
2000; Copani, 2001).
The control of cell division and cellular differentiation has repeatedly been linked in the past to
neurodegeneration in AD, and, based on this potential relationship, a variety of hypotheses have been
formulated such as the concept of hyperdifferentiation (Bouman, 1934), retrodifferentiation (Scheibel
and Scheibel, 1975), dysdifferentiation (Woolf and Butcher, 1990), or dedifferentiation (Arendt, 1993;
Heintz, 1993). About 10 years ago, it became increasingly clear that in AD, the mitogenic force is elevated
and neuronal mitogenic pathways are aberrantly activated in the early stages of the disease (Gartner et al.,
1995). From that time on, further insight into the pathomechanism progressed further downstream,
suggesting a potential involvement of cellcycle control as a result of abnormal mitogenic signaling (Arendt
et al., 1996; Vincent et al., 1996; Nagy et al., 1997).
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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

3.1 The Link Between Neurodegeneration and Neuroplasticity

3.1.1 Coherent Hierarchical Pattern of Selective Neuronal Vulnerability
and Neuroplasticity

Cell death in the nervous system, irrespective of whether caused by inflammation, ischemia, or primary
degeneration, selectively affects special types of neurons whereas the others are spared. In AD, this selective
neuronal vulnerability has systemic character. Neurodegeneration, initially very constantly affects circum-
scribed brain areas such as cholinergic basal forebrain projection neurons and the entorhinal cortex and
progresses from there throughout the brain, subsequently involving other areas in a defined sequence. The
reasons for this selective neuronal vulnerability are unknown, and several explanations have been given.
Paul Flechsig (1896) stressed the ontogenetic aspect of selective neuronal vulnerability when he advanced
the idea that variations in vulnerability of different groups of neurons are to be traced back in large part to
developmental conditions, a concept later defined by Cecile and Oscar Vogt (1951) as the principle of
pathoclisis. More recently, the phylogenetic dimension of selective neuronal vulnerability was recognized
(Ashford and Jarvik, 1985; Rapoport, 1989; DiPatre, 1991; Braak and Braak, 1996; Arendt et al., 1998;
Arendt, 2003). Brain areas and neuronal types highly vulnerable against neurofibrillary degeneration in AD
not only mature rather late during ontogenic development, but also have been acquired late during
phylogenetic development (or have completely been reorganized lately). Accordingly, these brain structures
typically subserve higher brain functions such as learning, memory, selfawareness, and others and as such
exhibit a particularly high degree of synaptic plasticity (> Figure 8-6).

3.1.2 Potential Risks of Dynamic Stabilization of Neuronal Connectivity

During development, after proliferation, migration and differentiation are completed, neurons become
integrated into a neuronal network. This integration is based on intercellular communication, which is
largely regulated through external cues. Connectivity and attachment of a cell are mechanisms that, during
evolution from single cellular systems to multicellular systems, have been acquired to assess signals from the
environment and respond appropriately by proliferation, differentiation, and cell death. In a multicellular
organism, external cues are thus, used for morphoregulation, i.e., the assembly of individual cells into
highly ordered tissues. Terminally differentiated neurons that have permanently withdrawn from the cell
cycle, however, make use of these mechanisms primarily developed to control connectivity for their genuine
function, i.e., for information processing in a multicellular network. Thus, cellcell interactions in the
nervous system subserve a phylogenetically newly acquired purpose, the formation of a dynamic network.
Once the network has been set up, the cell cycle is shut down and information from neighboring cells is
translated into continuous changes of synaptic strength and morphology, a process referred to as synaptic
plasticity. This means that, at least partially, identical signaling mechanisms are used to control and regulate
such divergent effectors as cellcycle control and synaptic plasticity.
From the phylogenetic perspective, it might be a great achievement for a neuron to control its synaptic
plasticity; to do this on the potential expense of differentiation control, however, might put the neuron on a
permanent risk. Neurons acquired particularly late during evolution of the human brain (e.g., cortical
associative circuits), which subserve typical human higher cortical functions such as learning, memory,
reasoning, consciousness, selfawareness, etc., need to display a particularly high degree of synaptic
plasticity, which might explain the particularly high sensitivity toward the loss of differentiation control
and cell death (> Figure 8-6). What makes the human brain so special, a large association cortex
characterized by a high degree of plasticitya prerequisite for selfconsciousness, i.e., the development
of a belief of itselfat the same time creates a vulnerability unique to humans (> Figure 8-2). Identical
signaling pathways might be used in the first stage of synaptic change in both development and maturity,
and higher cognitive functions such as learning and memory might be based on adaptive modifications of
an ancient mechanism initially evolved to wire the brain. The activitydependent modification of synapses
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 183

. Figure 8-6
Hierarchy of cortical vulnerability to neurofibrillary degeneration in the cerebral cortex. a: Neuropathological
staging of AD according to Braak and Braak (1991). b: This progression of neurofibrillary degeneration throughout
different cortical areas in AD follows a certain sequence (stage I to VI) that represents systematic differences in the
vulnerability (hierarchy of vulnerability), which matches inversely the hierarchic pattern of complexity of cortical
information processing, structural plasticity, and phylogenetic and ontogenetic age. Those neuronal systems that
play a crucial role in higher brain functions and therefore become increasingly predominant as the evolutionary
process of encephalization progresses, such as the hippocampus and the neocortical association areas, retain a
high degree of structural plasticity throughout their life. These areas of the brain take the longest to mature
during childhood and adolescence. Exactly the same brain structures display the highest degree of vulnerability
during aging and in AD. Modified after, Arendt et al. (1998); Braak and Braak (1991, 1996)

is, thus, a powerful mechanism for shaping and modifying the response properties of neurons, but it is also
dangerous (Abbott and Nelson, 2000) as it still hides its beasty potency, the risk of phylogenetic regression
into cellcycle activation, which ultimately results in cell death (Arendt, 2003). Synaptic plasticity might
thus, not only be beneficial providing the basis for information processing and memory formation in the
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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

brain, it might also be crucially involved in a variety of pathological conditions, including amnesia,
dementia, epilepsy, and psychosis (BenAri and Represa, 1990; McEachern and Shaw, 1996; Jeffery and
Reid, 1997).

3.1.3 Aberrant Plasticity in AD

Alterations of neuroplasticity are also a constant feature of AD (Ashford and Jarvik, 1985; Cotman and
Anderson, 1988; Butcher and Woolf, 1989; Phelps, 1990; Flood and Coleman, 1990; DiPatre, 1991; Geddes
and Cotman, 1991; Masliah et al., 1991c; Swaab, 1991; Walsh and Opello, 1992; Roberts et al., 1993; Neill,
1995; DeWitt and Silver, 1996, Mirmiran et al., 1996; Foster, 1999; Kondo et al., 1999; Mesulam, 1999; Teter
and Ashford, 2002).
Neurodegeneration in AD is associated with aberrant neuritic growth (Arendt et al., 1986). Abnormal
growth profiles preferentially affect neurons that are potentially vulnerable to neurodegeneration, such as
cholinergic basal forebrain neurons and cortical pyramidal cells (Arendt et al., 1995a, b, e) (> Figure 8-7).

. Figure 8-7
Aberrant morphological growth profiles in AD on neurons of the cholinergic basal nucleus of Meynert. Growth
profiles are localized on dendritic endings (d) or cover the soma (b, c). The appearance of these growth profiles
is associated with an upregulation of the NGF receptors TrkA (a) and p75 (b). (a: TrkA in situ hybridization;
b: p75 immunocytochemistry; c, d: Golgi impregnation). Scale bar: 30mm. Modified after Arendt and Bruckner
(1992); Arendt et al. (1995a)
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 185

Aberrant sprouts can be detected in early stages of the disease (Ihara, 1988) and precede the formation of
paired helical filaments or occur even without massive neuronal loss (Arendt and Bruckner, 1992b). They
might thus, represent a very early event of primary significance, inherent to the pathomechanism rather
than a secondary event triggered by ongoing degeneration.
As opposed to the continuous growth of dendritic elements during aging (Buell and Coleman, 1979),
growth in AD is aberrant with respect to its localization, morphological appearance, and composition of
cytoskeletal elements (Arendt et al., 1986, 1995a, b, d; McKee et al., 1989). Dystrophic neurites, mainly
dendritic but occasionally also axonal in origin, form a constant component of AD pathology. The original
identification of these neurites, in the early years of research on Alzheimers disease by Fischer (1907), has
been corroborated more recently by Golgi studies, ultrastructural evidence, and the identification of
elevated growthassociated proteins (for review see Arendt, 2003). Growth profiles on cholinergic basal
forebrain neurons, for example, are typically associated with receptors for NGF, indicating the potential
ability to respond to an increased trophic force (Arendt and Bruckner, 1992a, b) (> Figure 8-7).

3.2 CellCycle Regulators Alternatively Execute Neuroplasticity or Cell Death

3.2.1 Elevated Mitogenic Force and Activated Mitogenic Signaling in
Alzheimers Disease

The presence in the AD brain of growthassociated and growthpromoting proteins, as well as their receptors,
might be an indication of an increased mitogenic force particularly pronounced within the microenvironment
of plaques. The growing list of compounds contains GAP43, MARCKS, spectrin, heparansulfate, laminin,
NCAM, various cytokines, and neurotrophic factors such as NGF, bFGF, EGF, IL1, IL2, IL6, IGF1, IGF2,
PDGF, vascular endothelial growth factor, and HGF/SF (for review see Arendt, 2003).

3.2.1.1 Ras A number of these neurotrophic and potentially mitogenic compounds that are elevated early
in the course of the disease mediate their cellular effects through activation of receptor tyrosine kinases,
which recruit p21Ras, a GTPase that belongs to the Ras superfamily of smallGproteins and lead to the
sequential activation of Raf, MEK, and ERK (Seger and Krebs, 1995) (> Section 1.5.1). Ras proteins are
involved in the central coordination of a variety of functions including cytoskeletal organization, gene
expression, cellcycle progression, membrane trafficking, cell adhesion, migration and polarity, and
synaptic plasticity (Heumann et al., 2000; Arendt et al., 2004). During brain development, p21Ras
participates in the regulation of the G0/G1 transition of the cell cycle and might thus, be a critical
regulator for cellular proliferation and differentiation (Borasio et al., 1989). In the adult nervous system,
it plays a role in reactive dendritic proliferation and neosynaptogenesis (Phillips and Belardo, 1994; Holzer
et al., 2001a, b). Thus, Ras is a central regulator of synaptic plasticity in the adult brain (Arendt et al., 2004).
The RasERK/MAPK pathway is an evolutionary, conserved signaling mechanism that plays a fundamental
role in the regulation of cellular proliferation and differentiation and control of neuroplasticity, and is also
involved in modulating the expression and posttranslational processing of APP and t protein (Greenberg
et al., 1994; Mills et al., 1997; Sadot et al., 1998a), two proteins critically involved in AD pathomechanism.
In AD, p21Ras is highly expressed in vulnerable brain areas prior to its affection by neurofibrillary
degeneration (Gartner et al., 1999), which indicates an activation of the RasMAPK pathway in the early
stages of the disease (Arendt et al., 1995c) (> Figure 8-8).

3.2.1.2 MAPK The activation of MAPK that is associated by its nuclear translocation plays an essential role
in the expression of many immediate, early, and late response genes (> Section 1.5.1). MAPK activation and
nuclear translocation are required for longterm facilitation in Aplysia (Martin et al., 1997b) and LTP in
vertebrates (Impey et al., 1998). MAPK signaling might thus be a critical regulator for both shortterm
synaptic function and transcription of genes required for longterm plasticity. Ras/MAPK signaling
components are highly enriched in the adult CNS, and expression of many MAPK regulators is largely
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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

restricted to the CNS, where they are highly abundant in association areas implicating a role in memory
consolidation and synaptic plasticity.
ERK1/2 and other members of the MAPK family (Drewes et al., 1992; Goedert et al., 1992) are able to
phosphorylate the microtubuleassociated protein t on threonine and serine residues found phosphory-
lated in PHFs. In AD, both MAPKK and MAPK are activated in the early disease stages (Arendt et al., 1995c)
(> Figure 8-8). This activation of the MAPK pathway can be modeled in transgenic mice expressing
permanently activated ras (> Figure 8-9).

3.2.2 Oxidative Stress and the Autocrine Loop of SelfPerpetuating

Mitogenic Activation

Neuronal nitric oxide synthase (nNOS) was originally thought to be a constitutively expressed enzyme. It
becomes increasingly clear now, however, that its levels are dynamically regulated in response to neuronal
development, plasticity, and injury (Dawson et al., 1998). The transcriptional induction of nNOS, which is
controlled by neurotrophins and other growth factors, is in turn involved in regulating the expression of
immediate early genes in neurons, thereby controlling neuronal growth and differentiation (Peunova and
Enikolopov, 1993, 1995). NO may play a major role in nervous system morphogenesis and plasticity and
may be involved in activitydependent establishment of connections in both developing and regenerating
neurons (Dawson et al., 1998; Luth and Arendt, 1998a; Bogdan, 2001). Under developmental conditions,
NO may trigger growth arrest, a process that at least in certain cell types might involve inhibition of Cdk2,
a key regulator of the G1 and S phases of the cell cycle (see later). These antiproliferative effects of NO
involve the repression of cyclin A reexpression as well as an induction of the cyclindependent kinase
inhibitor p21Cip1 (Gansauge et al., 1998). The high degree of coexpression of nNOS with p16INK4a (Luth
et al., 2000) indicates that further regulators of the G1S transition might be involved in the NOinduced
cellcycle arrest or that additional mechanisms of proliferation and differentiationregulating mechanisms
are activated in parallel in the course of neurodegeneration in AD. Thus NO serves as an inducer of cell
cycle arrest, initiating the switch to cytostasis during differentiation (Peunova and Enikolopov, 1995;
Dawson et al., 1998), a process that can alternatively lead to apoptosis (Gansauge et al., 1998).
Although the molecular mechanism for the control of NO in proliferation, differentiation, cellular
survival, and death is not understood in detail, recent evidence indicates that activation of p21Ras is
critically involved in downstream signaling as a potential endogenous NOredoxsensitive effector molecule
(Lander et al., 1997b; Yun et al., 1998). Endogenous NO and intermediates generated through oxidative
stress can drive the Ras/MAPK cascade directly by direct activation of RasGTPase activity (Lander et al.,
1997; Bogdan 2001). NO might thus be a key mediator linking cellular activity to gene expression and long
lasting neuronal responses through the activation of p21Ras by redox sensitive modulation (Dawson et al.,
1998), a process with potential implications both in development and degeneration.
In AD, nNOS is aberrantly expressed in potentially vulnerable neurons of the isocortex and entorhinal
cortex. Since these neurons express nNOS before they are affected by neurofibrillary degeneration (Luth
and Arendt 1998b; Luth et al., 2000), transcriptional induction of nNOS might be an early event in the
process of neurodegeneration. As expression of nNOS in AD is highly colocalized with p21Ras (Luth et al.,
2000) (> Figure 8-10), an autocrine loop may exist within cells, whereby NO activates p21Ras, which in
turn leads to cellular activation and stimulation of NOS expression (Lander et al., 1997). In parallel,

. Figure 8-8
Activation of p21Rasdependent MAPkinase pathway in AD. Expression of p21Ras, bRaf, MAP kinase kinase
(MEK1), and the MAP kinases ERK1/2 quantified by ELISA and detected by immunohistochemistry in Brodmann
area 10. All four markers are elevated early in the course of the disease. P21Ras is highly enriched in plaques,
plaqueassociated astrocytes, as well as in tanglebearing neurons. BRaf and the associated protein 1433 are
also found in tangles. The subcellular translocation of MEK1 and ERK1/2 into neuronal nuclei indicate enzyme
activation. Modified after Arendt et al. (1995d); Arendt (2003); Gartner et al. (1995)
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 187

. Figure 8-8 (continued)

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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

. Figure 8-9
Changes in the subcellular distribution of MAP kinase (ERK1/2) in AD, and in a transgenic mice model
expressing permanently activated p21Ras under control of the synapsin promoter (synRas mouse). Both in
AD and transgenic mice, MAPK is translocated from the cytoplasm (asterisk) to the nucleus (arrows) indicating
an activation of the enzyme. Scale bar: 10 mm. Modified after Arendt et al. (1995c); Holzer et al. (2001)

. Figure 8-10
Double immunofluorescence for nNOS/p21Ras and nNOS/p16INK4a. The aberrant expression of nNOS in pyra-
midal neurons is highly colocalized with p21Ras (top row) and p16INK4a (bottom row), respectively. Interneurons
expressing nNOS constitutively (arrow), on the contrary, neither express p21Ras nor p16INK4a, modified after
Luth et al. (2000)
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 189

downstream mechanisms such as the cell cycle might be activated as indicated by the coexpression of
cellcycle regulators (> Figure 8-10). The coexpression of NOS and p21Ras in neurons vulnerable to
neurofibrillary degeneration early in the course of AD might thus provide the basis for a feedback
mechanism that might exacerbate the progression of neurodegeneration in a selfpropagating manner
(> Figure 8-11). This selfperpetuation of a process likely to be associated with limited prospects of
physiological control and termination might be the critical switch converting two potentially neuropro-
tective mechanisms such as NO (Farinelli et al., 1996) and p21Ras (Heumann et al., 2000) dependent
signaling into a disease process, leading to slowly but continuously progressing neuronal death.

3.2.3 The Replay of Developmental Programs in Alzheimers Disease

Several genes and proteins and their posttranslational modification associated with AD have recently been
found to play critical roles in neuronal development, particularly neuronal migration, and axon extension
(Arendt, 2000, 2001a, b; Bothwell and Giniger, 2000; Mehler and Gokhan, 2001). Neurodegeneration,

. Figure 8-11
Schematic illustration of the intracellular signaling events triggered by Morphodysregulation in AD that involve
an aberrant activation of p21Ras/MAPkinase signaling, a loss of differentiation control, the subsequent reentry
and partial completion of the cell cycle, and eventually result in cell death. Modified after Arendt et al. (1998),
J Neural Transm (Suppl 54), 147158; Arendt (2003)
190
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

furthermore, is associated with a complex structural reorganization, which involves a replay of develop-
mental programs. The aberrant neuritic growth in AD, as a likely indication of defect synapse turnover, is
accompanied by microtubular reorganization (McKee et al., 1989) associated with the reexpression of a
number of developmentally regulated proteins involved in morphoregulation indicating that molecules,
overexpressed in AD, might play a role in structural remodeling of the adult brain (Arendt, 2001b).
Based on this evidence, we have suggested previously that the process of continuous synaptic reorgani-
zation becomes defective in AD (Arendt, 1993). In this pathogenetic process, a subset of neurons retaining a
high degree of plasticity and that are presumably in a labile state of differentiation, are forced into a
condition of dedifferentiation that is characterized by an expression of developmental regulated genes,
posttranslational modifications, and an accumulation of gene products to an extent that goes beyond those
observed during regeneration. This replay of developmentally regulated mechanisms might be the end stage
of disturbed structural brain selforganization and a slowly progressing morphodysregulation. This
process of dedifferentiation involves molecular events that, in dividing cell populations, would lead to
cellular transformation and is thus, not compatible with the state of a neuron being irreversibly blocked
from reentry into the cell cycle. It might, therefore, lead to neuronal death. From this hypothesis, it can be
predicted that these molecular events that are involved in neoplastic transformation might also play a key
role in the pathomechanism of AD (Heintz, 1993). These mechanisms are notably a dysfunction of
mitogenic signal transduction and cellcycle control.

3.2.4 CellCycle Activation in Alzheimers Disease and Other Neurodegenerative Disorders

Proteins that normally function to control the cellcycle progression in actively dividing cells may play roles
in the death of terminally differentiated postmitotic neurons (Farinelli and Greene, 1996; Park et al., 1996).
Thus, a dysregulation of Cdks and their regulating partners, such as cyclins and Cdk inhibitors, indicating
an activation of the cell cycle in postmitotic neurons has been observed in AD (Smith et al., 1995; Arendt
et al., 1996, 1998b, c, 2000; McShea et al., 1997; Vincent et al., 1997; Nagy et al., 1997a, 1988; JordanSciutto
et al., 1999; Arendt, 2000; Dranovsky et al., 2001), cerebral ischemia, and during trophic factor deprivation
(Gill and Windebank, 1998; Tomasevic et al., 1998; van Lookeren Campagne and Gill, 1998; Timsit
et al., 1999; Osuga et al., 2000; Sakurai et al., 2000; Katchanov et al., 2001) (> Figures 8-12 and > 8-13,
> Table 8-4). Correspondingly, pharmacological inhibitors of cell cycle or ectopic expression of Cdkis can

protect neurons against death (Farinelli and Greene, 1996; Kranenburg et al., 1996; Park et al., 1996;
Giovanni et al., 1999; Padmanabhan et al., 1999; Osuga et al., 2000; Katchanov et al., 2001; Knockaert et al.,
2002) (> Figure 8-14).

3.2.4.1 CyclinDependent Kinases Several cyclindependent kinases critical for progression through the cell
cycle (Koh et al., 1995) such as Cdk1 (Cdc2), Cdk4, and Cdk5 are deregulated in AD (Ledesma et al., 1992;
Vincent et al., 1997; Busser et al., 1998; Nagy et al., 1998; Patrick et al., 1999; Arendt et al., 2000). The Cdk1
(Cdc2) kinase, moreover, is able to phosphorylate t protein at sites known to be phosphorylated in AD, and
thus potentially contributes to the generation of PHFt (Ledesma et al., 1992; Kobayashi et al., 1993). APP,
furthermore, is phosphorylated both in vitro and in intact cells by a Cdk1 (Cdc2)like kinase in a cellcycle
dependent manner, which is associated with altered production of potentially amyloidogenic fragments
containing the entire b/A4 domain (Suzuki et al., 1994). Ab in turn might potentially act as a proliferating
signal, driving cultured rat primary neurons into the cell cycle (Copani et al., 1999), although in vivo, this
has not been replicated (Gartner et al., 2003).

3.2.4.2 Cyclins The level of cyclin D1, a critical regulator of the transition from the G0 to G1 phase of the
cell cycle that acts through activation of Cdk4, is increased in neurons prone to neurodegeneration in AD
(Busser et al., 1998; Arendt et al., 2000). Cyclins other than D1 such as cyclins E and A involved in
regulation of G1/S transition as well as cyclin B regulating G2/M transition (Nagy et al., 1997a; Vincent
et al., 1997; Smith et al., 1999; Arendt et al., 2000) are also elevated.
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 191

. Figure 8-12
The cell division cycle and its major regulatory elements. Main switches for the activation and progression
of the cell cycle are elevated in neurons prone to neurofibrillary degeneration early in the course of AD (shown
in dark gray)

INK4a
3.2.4.3 Cdk Inhibitors p16 , a prominent representative of the INK4 family, involved in pathways for
control of cell growth and proliferation (Kamb et al., 1994), is also increased in AD as are other members of
the INK4 family of the cyclindependent kinase inhibitors interacting with Cdk4/6 such as p15INK4b,
p18INK4c, and p19INK4d. However, alterations of p21Cip1 and p27Kip1 are less constant (Arendt et al., 1996,
1998b, c).
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8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

. Figure 8-13
Major regulators of the activation and orderly progression through the cell cycle are expressed in vulnerable
neurons in AD (compare also > Figure 7-11). They are already highly abundant in pyramidal neurons prior to
PHF formation. In more advanced stages of the disease, they are associated with PHFs (Brodmann area 22).
Modified after Arendt et al. (2000), Neurobiol Aging 21, 783796

3.2.4.4 Retinoblastoma Protein In AD, increased immunoreactivity for hyperphosphorylated pRb and for
E2F has been described (JordanSciutto et al., 2002; Hoozemans et al., 2004). The upregulation of E2F1 as
it has been detected in pyramidal neurons of Downs patients exhibiting the neuropathological features of
AD at the same time as Ab deposition occurs might thus, be of potential relevance to the process of
neurodegeneration (Motonaga et al., 2001).

3.2.4.5 The Cell Cycle Integrates Intercellular and Intracellular Signaling and Links Development, Oncogenesis,
and Neurodegeneration The high capacity of structural neuronal plasticity in the adult brain might
predispose neurons to tangle formation in AD (Arendt et al., 1995a, 1998a; Arendt 2001a). This high
potential of neuroplasticity associated with the necessity of synaptic turnover and reorganization might
require properties inherent to both growth cones and synaptic connections (Pfenninger et al., 1991). Highly
plastic neurons might thus retain immature features and might not be fully differentiated or truly
postmitotic, i.e., arrested in G0, an assumption supported by recent findings on the expression of cyclin B
and E in hippocampal neurons of healthy elderly (Nagy et al., 1997a; Smith et al., 1999).
It is, therefore, suggested that the reexpression of developmentally regulated genes, the induction of
posttranslational modifications and the accumulation of gene products to an extent, which goes beyond
that observed during regeneration, and the aborted attempt of differentiated neurons to activate the cell
cycle, apparently is a critical event in the pathomechanism of AD (Arendt, 1993, 2001a; Arendt et al., 1998a,
1998c, 2000; Busser et al., 1998; McShea et al., 1997; Masliah et al., 1993c; Nagy et al., 1997a, b, 1998; Smith
et al., 1999; Smith and Lippa, 1995; Vincent et al., 1996, 1997, 1998). This condition is associated with a
re-expression of developmentally regulated genes, the induction of posttranslational modifications and
accumulation of gene products to an extent which goes beyond that observed during regeneration and
might, thus, be due to a loss of differentiation control that normally is involved in the regulation of
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 193

. Table 8-4
Evidence for cell-cycle activation in neurodegenerative disorders
Alzheimers disease
increased neuronal expression of cell-cycle regulators and proliferation Arendt et al. (1996, 1998d, e,
associated proteins 2000)
Cdk1 Blalock et al. (2004)
Cdk4 Busser et al. (1998)
Cdk7 Ding et al. (2000)
Dranovsky et al. (2001)
cyclin A Harris et al. (2000)
cyclin B Husseman et al. (2000)
cyclin C Illenberger et al. (1998)
cyclin D1 Jenkins et al. (1998)
cyclin E Jordan-Sciutto et al. (1999, 2002)
cyclin G1 Kondratick and Vandre (1996)
Ledesma et al. (1992)
Cdk5/p35, p25 Masliah et al. (1993)
McShea et al. (1997)
polo-like kinase Nagy et al. (1997a, b, 2002)
Ogawa et al. (2003)
CDC25A Pei et al. (2002)
CDC25B Pruess and Mandelkow (1998)
Ranganathan et al. (2001)
neuronal CIP-1-associated regulator of cyclin B (CARB) Smith and Lippa (1995)
Smith et al. (1999)
p15INK4b Stieler et al. (2001)
p16INK4a Suzuki et al. (1994)
p18INK4c Tsujioka et al. (1999)
p19INK4d Ueberham et al. (2003)
p21Cip1 Urcelay et al. (2001)
p27Kip1 Vincent et al. (1996, 1997, 1998,
2001)
p105 Yang et al. (2001)
Ki-67 Zhu et al. (2000, 2004)
PCNA
increased phosphorylation of Rb
mitotic phosphoepitopes are absent in immature neurons of the human brain
but reappear in potentially vulnerable neurons prior to neurofibrillary
degeneration
PHFs are associated with mitotic specific phosphoepitopes
regulation of the phosphorylation and metabolism of APP is cell-cycle
dependent
partial tetraploidy of neurons
abnormal mitotic regulation in peripheral cells such as lymphoblasts,
peripheral lymphocytes, or skin fibroblasts
Mild Cognitive Impairment (MCI)
increased neuronal expression of cell-cycle regulators/proliferation associated Yang et al. (2003)
proteins
cyclin D
cyclin B
PCNA
194
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

. Table 8-4 (continued)

Vascular dementia
increased neuronal expression of cell-cycle regulators
cyclin B Smith et al. (1999)
Stroke
increased neuronal expression of cell-cycle regulators Love (2003)
Cdk4/
Cdk2
cyclin D1
Parkinsons disease
increased neuronal expression of cell-cycle regulators/proliferation associated Brion and Couck (1995)
proteins
Cdk5 Jordan-Sciutto et al. (2003)
ppRb Smith and Lippa (1995)
Ki-67
Lewy body disease
increased neuronal expression of cell-cycle regulators/proliferation associated Brion and Couck (1995)
proteins
Cdk5 Smith and Lippa (1995)
Ki-67
Picks disease
increased neuronal expression of cell-cycle regulators/proliferation associated Nagy et al. (1997-Acta 94)
proteins
Cdk1 Husseman et al. (2000)
cyclin B Nagy et al. (1997-Acta 93)
Ki-67 Smith and Lippa (1995)
Frontotemporal dementia with Parkinsonism (FTDP-17)
increased neuronal expression of cell-cycle regulators Husseman et al. (2000)
Cdk1
cyclin B
Downs syndrome
increased neuronal expression of cell-cycle regulators/proliferation associated Hermon et al. (2001)
proteins
Cdk1 Husseman et al. (2000)
Ki-67 Nagy et al. (1997a, b)
cyclin B Petersen et al. (2000)
higher incidence of leukemia in Downs patients Schneider and Epstein (1972)
higher incidence of colo-rectal cancer in siblings of Downs patients Smith and Lippa (1995)
altered mitotic index in fibroblasts from Downs patients Vincent et al. (1997)
maternal meiosis II errors in Downs patients are associated with presenilin-1
polymorphism
Temporal lobe epilepsy
increased neuronal expression of cell-cycle regulators Nagy and Esiri (1998)
cyclin B
Progressive supranuclear palsy
increased neuronal expression of cell-cycle regulators/proliferation associated Husseman et al. (2000)
proteins
Cdk1/cyclin B Borghi et al. (2002)
Cdk5 Smith and Lippa (1995)
Ki-67
 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders
8 195

. Table 8-4 (continued)

Corticobasal degeneration
increased neuronal expression of cell-cycle regulators Husseman et al. (2000)
Cdk1
cyclin B
Huntingtons disease
increased neuronal expression of cell-cycle regulators Husseman et al. (2000)
Cdk1
cyclin B
Amyotrophic lateral sclerosis
increased neuronal expression of cell-cycle regulators Ranganathan and Bowser (2003)
cyclin D Husseman et al. (2000)
cyclin B1 Nakamura et al. (1997)
Cdk5 Bajaj et al. (1998)
phosphorylation of Rb Patzke and Tsai (2002)
Ranganathan et al. (2001)
NiemannPicks disease type C (NPC)
increased neuronal expression of cell-cycle regulators/proliferation associated Husseman et al. (2000)
proteins
Cdk1 Bu et al. (2002 a, b)
cyclin B
Cdk5/p25
presence of mitotic phosphoepitopes: TG-3, MPM-2, H-5

. Figure 8-14
Neuroprotection by ectopic expression of the cyclindependent kinase inhibitor p16INK4a. Microexplants
prepared from rat embryonic day 17 were challenged with 10nM OA (24h), a strong inducer of apoptosis.
Explants transfected with p16INK4a prior to OA treatment showed a much reduced rate of apoptosis (TUNEL,
dUTPrhodamine)
196
8 Neuronal cellcycle regulation during development and agerelated neurodegenerative disorders

neuronal plasticity. A direct link between cellcycle reactivation and cell death is supported by observations
on the neuroprotective action of overexpression of the cyclindependent kinase inhibitor p16INK4a, which
locks neurons in a differentiated stage and prevents cellcycle reentry (Arendt, 2003).
Thus, it might be a labile fixation of plastic neurons in G0, which allows for ongoing morphoregu-
latory processes after development is completed. The delicate balance, however, between G0arrest and
G1entry might be prone to a variety of potential disturbances during the lifetime of an individual.
Morphodysregulation in AD, accompanied by aberrancies in intracellular mitogenic signaling, might
thus be a slowly progressing dysfunction, which eventually overrides this differentiation control and results
in dedifferentiation, a condition in conflict with the otherwise mature background of the nervous
system. Cell cycle and differentiation control might thus provide the link between structural brain self
organization and neurodegeneration (Arendt, 1993; Heintz, 1993) both of which in the human brain have
reached a phylogenetic level unique in nature.

4 Conclusions

Taken together, in multicellular organisms, the cell number is regulated spatially by extracellular signals
through cell interactions controlling proliferation and survival in local neighborhood. Instructions from
neighboring cells can induce cell proliferation, differentiation, or death. These stimuli include cellcell and
cellECM adhesion, growth factors, cytokines, neuropeptides, and mechanical factors. Signals from G
proteincoupled receptors, tyrosine kinase receptors, and integrins cooperate to integrate information from
multiple stimuli, which regulate cellcycle progression. To allow for a regulation of these processes, a tight
link is necessary between cell attachment mechanisms and control of proliferation and differentiation, i.e.,
the cellcycle machinery.
Contrary to many other cells that make up a multicellular organism, neurons use the molecular
circuitry developed to sense their relationship with other cells to reorganize their connectivity according
to the requirements for information processing within a cellular network. To form connections and to
reshape them continuously, cell division needs to be shut down. The molecular machinery behind it is now
used to regulate synaptic plasticity. This switch from proliferation control to plasticity control, which
occurs during the process of differentiation of a neuron, implies the permanent risk of erroneously
converting signals derived from plastic synaptic changes into positional cues that will activate the cell
cycle (Arendt, 2003). This replay of developmental mechanisms, i.e., phylogenetic and ontogenetic regres-
sion in the adult nervous system, contrary to development, ultimately results in cell death. Maintaining
neurons in a differentiated but still highly plastic phenotype will thus be the challenge to prevent
neurodegeneration.

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9 mRNA Modulations in
Stress and Aging
E. Meshorer . H. Soreq

1 The Mammalian Stress ResponseNeurochemical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216

1.1 Defining Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
1.2 The HPA Axis: Neuroendocrine Response to Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
1.3 Glucocorticoid Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
1.4 Other StressRelated Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
1.5 Stress and the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

2 PremRNA Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

2.1 The Splicing Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
2.2 SR Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

3 Splicing and Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

3.1 Constitutive Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
3.2 Alternative Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
3.3 StressInduced Changes in SRRelated Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

4 StressAssociated Dendritic Translocation of mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

5 Defects in premRNA Splicing as Causes of and Predisposition to Diseases . . . . . . . . . . . . . . . . . . . 225

6 Splicing and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

6.1 PremRNA Splicing Modulations in Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
6.2 PremRNA Splicing Modulations in AgingRelated Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

7 Candidate Gene Study: ACHE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

7.1 The Molecular Biology of AChE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
7.2 AChE in Multitissue Stress Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

8 AChEs 30 Alternative Splicing under Stress: LongTerm Implications . . . . . . . . . . . . . . . . . . . . . . . . . . 233

9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

# 2008 Springer ScienceBusiness Media, LLC.

216
9 mRNA modulations in stress and aging

Abstract: Ample information suggests multileveled relationships between mRNA modulations and long
term stress responses, especially with regard to alternative splicing. The nature of these relationships is
predictably complex and multileveled. Here, we describe the contribution of both hormonal and stress
induced changes to alternative splicing, and address the molecular pathways that lead to the longlasting
stressassociated modulation in alternative splicing as well as the cellular changes that follow. We cover the
general areas of stress responses (both to psychological and chemical insults) and their longterm con-
sequences, the mechanism of splicing and alternative splicing, and the phenomenon of neuritic transloca-
tion of messenger RNA (mRNA). To present a more detailed example, we describe the alternative splicing
patterns of acetylcholinesterase (AChE) premRNA, which is neuronally expressed, stressresponsive, and
yields dendritically translocated products, and therefore served as an adequate case study for this purpose.
The ACHE gene and its variant AChE protein products are thus presented as both subjects to and putative
mediators of longterm consequences of stress and aging. We attempt to illustrate, citing from the current
literature, how these diverse areas are nonetheless intertwined.

List of Abbreviations: ACh, Acetylcholine; AChE, Acetylcholinesterase; ACTH, Adrenocorticotropic

hormone; AD, Alzheimers disease; ALS, Amyotrophic lateral sclerosis; AP-1, Activator protein 1; ASAP,
Apoptosis- and splicing-associated protein; ATF-3, Activating transcription factor 3; ATM, Ataxia-telangi-
ectasia; BBB, Blood brain barrier; BDNF, Brain derived neurotrophic factor; CaMK, Calcium-calmodulin
dependent protein kinase; CBG, Corticosteroid-binding globulin; CNS, Central nervous system; ColQ,
Collagen Q; CRF, Corticotrophin-releasing factor; CSF, Cerebrospinal fluid; DFP, Diisopropylfluoropho-
sphonate; ESE, Exonic splicing enhancer; ESS, Exonic splicing suppressor; FTDP-17, Frontotemporal
dementia with parkinsonism linked to chromosome 17; GCs, Glucocorticoids; GPI, Glycosylphosphatidyl
inositol; GR, Glucocorticoid receptor; HnRNP, Heterogeneous nuclear ribonucleoprotein; HPA, Hypotha-
lamic-pituitary-adrenal; HRE, Hormone response element; HSP, Heat shock protein; ISE, Intronic splicing
enhancer; ISS, intronic splicing suppressor; LTP, Long-term potentiation; MAP, Microtubule-associated
protein; NCAM, Neural cell adhesion molecule; NFB, Nuclear transcription factor B; NO, Nitric oxide;
PET, Positron emission tomography; PFC, Prefrontal cortex; PRiMA, Proline-rich membrane anchor; PS1,
Presenilin 1; PTB, Polypyrimidine tract binding protein; PTSD, Post-traumatic stress disorder; PVN,
Paraventricular nucleus; RRM, RNA recognition motif; Py, Polypyrimidine; SMaRT, Spliceosome-mediated
RNA trans-splicing; SPECT, Single photon emission computed tomography; SnRNA, Small nuclear RNA;
SnRNP, Small nuclear ribunucleoprotein particle; U2AF, U2 auxiliary factor; UTR, Untranslated region

1 The Mammalian Stress ResponseNeurochemical Aspects

1.1 Defining Stress

Adopted from the field of physics, in which stress is referred to as a physical pressure, biological stress, by
nature, is a subjective idiom. It was hence neglected for a long time, even years after the fields pioneers
Walter Cannon and Hans Selye advanced stress to the laboratory bench, coining famous terms such as
fight or flight (Cannon, 1939), homeostasis (Cannon, 1939), or general adaptation syndrome (GAS)
(Selye, 1978), referring to the collection of stress symptoms in an individual. Stress in biology is regarded
today as the physiological and/or behavioral responses to a challenge (stressor) that involve adaptive,
hormonal, or behavioral changes, which inevitably link to neurochemical processes. The myriad types of
stresses that may lead to such responses are largely dictated by ones own capacity to perceive and interpret
the stressful signal(s). This results in hormonal and molecular responses influencingmildly or strongly,
acutely or chronically, and sometimes indefinitelythe individuals responses. Early in life, Hans Selye
(1936) regarded physical insults as the main culprit causing the diverse effects of stress, but his later reports
highlight the power of psychological stress (Selye, 1978). Nowadays, it is widely accepted that different
psychological challenges are among the most powerful stressors. For example, anticipation of punishment
activates a much stronger stress response than the punishment itself (McEwen, 2002).
 mRNA modulations in stress and aging
9 217

1.2 The HPA Axis: Neuroendocrine Response to Stress

Stressful experiences include, but are not limited to, physical and psychological traumatic events (grief, car
accidents, war trauma, etc.), physical and psychological abuse, prolonged fatigue, and life threatening
events (Gershuny and Thayer, 1999). In essence, therefore, life itself is a stressful experience, and the process
of aging resembles the longterm consequences of stressful events. In principle, stress responses are vital for
surviving the immediate challenge; a fleeing animal or person turn on the immediate stress response
(Sapolsky et al., 2000). This includes the release of catecholamines (epinephrine and norepinephrine) from
the sympathetic nervous system, and the stimulation of the hypothalamicpituitaryadrenal (HPA) axis
(> Figure 9-1). HPA activation results in hypothalamic secretion of corticotrophinreleasing factor (CRF)

. Figure 9-1
The HPA axis. The regions involved in the activation of the HPA axis are shown. The area enlarged is the
hypothalamic region. The hypothalamus is shaded gray in the inset. See text for details. PVN, paraventricular
nucleus; CRF, corticotrophinreleasing factor, ACTH, adrenocorticotropic hormone; PFC, prefrontal cortex; GC,
glucocorticoids

and vasopressin (AVP) from the cell bodies of the parvicellular hypophysiotropic neurons of the hypothalamic
paraventricular nucleus (PVN) (Sapolsky et al., 2000). The axons of these neurons terminate at the hypotha-
lamic median eminence, where CRF secreted from these neuronal processes diffuses into the hypophyseal
portal circulation, and through the binding of CRF to its specific receptor, CRFR1, it triggers the anterior
pituitary gland to release adrenocorticotropic hormone (ACTH) from specialized cells called corticotropes
into the general circulation (Sapolsky et al., 2000). ACTH then reaches the kidneys adrenal cortex, where it
binds specific cell surface receptors on the adrenal cells in the zona fasciculata, and through cAMPdependent
pathways stimulates the synthesis and secretion of glucocorticoids (GCs) (cortisol in humans and corticoste-
rone in rodents) and mineralocorticoids (aldosterone) into the general circulation (Sapolsky et al., 2000).
GCs, acting back on the hypothalamic PVN, activate a negative feedback loop, which inhibits CRF and hence
ACTH production, consequently decreasing the synthesis and secretion of GCs from the adrenal gland.
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9 mRNA modulations in stress and aging

1.3 Glucocorticoid Actions

About 95% of the GCs in the circulation are bound to proteins, mainly corticosteroidbinding globulin
(CBG), and also albumin, to a lesser extent. Free GCs, and only free GCs, can readily enter cells and bind
their specific intracellular corticosteroid receptors (Beato et al., 1996). Bound GCs can also be functional,
for example, in cases where the carrier protein interacts with cell surface receptors, or following enzymatic
cleavage of CBG by serine proteases in inflamed tissues. Two distinct subtypes of intracellular corticosteroid
receptors have been identified: the highaffinity mineralocorticoid receptor (MR, type I) and the low
affinity glucocorticoid receptor (GR, type II). Although MR binds both GCs and mineralocorticoids
with high and almost equal affinity (Kd
0.52 nM), GR displays weaker binding and is selective for
GCs (Kd
1030 nM) (Sapolsky et al., 2000).
Following the binding of GCs to GRs, the GRs dimerize and the hormonereceptor complex subse-
quently translocates into the nucleus, where it recognizes and binds consensus DNA sequences known as
hormone response elements (HRE) (Chandler et al., 1983; Beato et al., 1996). This binding usually activates
transcription, but may also interfere with the activity of other transcription factors, such as activator
protein 1 (AP1) (Heck et al., 1994) or may have dual functions of activation or repression, depending on
the promoter context, such as with nuclear transcription factor kB (NFkB) (Hofmann and Schmitz, 2002).
The GCsresponding genes include cytokines (Arimura et al., 1994; Licinio and Wong, 1999), transcription
factors (Persico et al., 1993; Jolly and Morimoto, 1999), synaptic proteins (Persico et al., 1995), brain
receptors (Duman et al., 1999), and neurotrophins (Altar, 1999), to name a few.
In addition to acting as transcription factors, GCs exert multiple effects on the organism. In general, the
actions of GCs can be divided into permissive or proactive, and suppressive or protective (Buckingham,
2000). The permissive roles maintain the basal activity of the HPA axis, setting its threshold for stress
responsiveness, and normalizing the bodys response to stress by priming defense mechanisms. The
protective roles include antiinflammatory actions, redirection of metabolism to meet energy needs during
the stress response, promotion of specific memory processes (Furey et al., 2000), and suppression of
nonessential functions such as digestion, growth, and reproduction (Sapolsky et al., 2000).

1.4 Other StressRelated Hormones

As evident from GCs actions, the stress response avoids wasting expensive energy on reproduction,
digestion, and growth, while concentrating on stress ridding. Prolonged stress responses may, therefore,
have harmful effects on the general maintenance of many different properties. However, GCs do not act
alone. In order to obtain a regulated response, additional hormones, acting directly on specific target
tissues, also participate in the mammalian stress response. Such hormones include prolactin (Kjaer et al.,
1991), which suppresses the reproductive system; endorphins and enkephalines (Amir et al., 1980), which
blunt pain receptors and promote alertness; and vasopressin (Robertson, 1976), which acts to accelerate
heartbeat and to elevate blood pressure. Other hormones are suppressed. These include growth hormone
(Brown et al., 1978), estrogen (Ballinger, 1990), testosterone (Jeong et al., 1999), progesterone (Magiakou
et al., 1997), and insulin (KeltikangasJarvinen et al., 1998).
The picture that emerges is that of a complex response, which affects most, if not all, of the mammalian
systems. The complexity can be specifically illustrated by the example of the immune response. Following
stress, GCs induce granulocytosis (Stefanski, 2000), yet quench inflammatory responses in a longlasting
manner. It was demonstrated, for example, that psychological stress renders individuals more susceptible to
the common cold (Cohen et al., 1991). These immunosuppressive properties of GCs have made them
desirable antiinflammatory agents in wide clinical use. However, whereas chronic stress clearly suppresses
immune responses, it was shown that some stress conditions such as shortduration stressors can have
stimulating actions on immune functions (Dhabhar and McEwen, 1999). Different stressors can lead to
differential activation of certain proinflammatory cytokines (e.g., IL1, IL6, and TNFa) while suppressing
others (usually IL2, IL3, and IL5). An additional level of complexity is added by the ability of the immune
system to act back on the HPA axis: for instance, it was demonstrated that proinflammatory cytokines such
 mRNA modulations in stress and aging
9 219

as IL1 and IL6, the same ones that are suppressed by GCs or conversely abundantly produced in response
to immunological stimuli (microbes, lipopolysaccharides), can activate the HPA axis and exert additional
effects on the central nervous system (Pruett, 2003).

1.5 Stress and the Brain

The stressbrain connection was only made in 1968 with the discovery of GRs in the rat hippocampus
(McEwen et al., 1968). A plethora of studies followed, establishing the hippocampus as a major stress
responding area having the highest concentration of GRs in the brain. As is the case with the immune
system, stress can exert multiple, sometimes contrasting or even opposite effects on the central nervous
system. For example, the release of epinephrine during stress is known to modulate memory consolidation,
enhancing memory for many different training experiences (McGaugh, 2000). On the other hand, it was
shown that stress can impair neurogenesis in the hippocampus (Gould et al., 1998), lead to hippocampal
atrophy in primates (Uno et al., 1989), and significantly impair the hippocampusrelated declarative
memory function (Lupien et al., 1998). Hippocampal longterm potentiation (LTP) impairments, reflecting
compromised memory, were also observed following various stress paradigms, especially chronic stress
(Diamond and Rose, 1994; McEwen, 1999a, b). Stressinduced hippocampal plasticity is a key factor in
repeated and chronic stress responses (McEwen, 1999a, b). Chronic restraint stress was shown to alter
synaptic terminal structures in the hippocampus, mainly in the CA3 region (Magarinos et al., 1997), and
cause atrophy of apical dendrites in this region (Magarinos et al., 1996). Chronic stress also leads to
impaired feedback mechanisms, which are associated with a decreased expression of hippocampal corticoid
receptor genes. In addition, or in parallel, longterm stress accelerates the accumulation of a number of
biological markers of aging, increases the excitability of CA1 pyramidal neurons through a calcium
dependent mechanism, and causes loss of hippocampal pyramidal neurons. Also, the persistence of excita-
tory amino acid release after the termination of a stressful experience may render the aging hippocampus
more vulnerable (McEwen, 1999a, b).
In humans, atrophy of the hippocampus was reported as a result of both elevated GCs (Lupien et al., 1998)
and severe traumatic stress (Bremner, 1999). A schematic diagram of the hippocampus is shown in
> Figure 9-2. Related syndromes include Cushings disease (Starkman et al., 1992), major depression

(Sheline et al., 1996), schizophrenia (Bogerts et al., 1993), agingrelated syndromes (Convit et al., 1995),
Alzheimers disease (de Leon et al., 1993), and finally posttraumatic stress disorder (PTSD) (Bremner et al., 1995).
It is tempting to speculate that GCs and the processes induced under their excess accumulation are
responsible for the hippocampal atrophy associated with the syndromes listed here. Indeed, longlasting

. Figure 9-2
The hippocampus and prefrontal cortex (PFC). Two of the major stressresponding areas in the human brain are
illustrated. The hippocampus is shown as a dark crescent and the PFC is marked with a circle.
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9 mRNA modulations in stress and aging

elevated glucocorticoid levels were shown to associate with diverse psychological syndromes such as
anxiety, depression, and related mood disorders (HoehnSaric et al., 1991; Yehuda et al., 1993; Calvo and
Volosin 2001; Kalinichev et al., 2002). However, considerable controversy still exists regarding the specific
contributions of the HPA axis to PTSD. Thus, PTSD patients may have normal to low levels of circulating
GCs, while presenting features reminiscent of those of normal individuals with excess GCs (reviewed in
(Yehuda, 2002). The contribution of the initial burst of GCs during trauma or stress leading to the PTSD
phenotype, hence, remained enigmatic.
An additional brain region central to the stress response is the prefrontal cortex (PFC, > Figure 9-2).
Similar to the hippocampus, the PFC is rich in GRs and participates in the feedback regulation loop of the
HPA (Lupien and Lepage, 2001). The PFC is known to serve cognitive and emotional functioning, both of
which are disrupted by stress (Moghaddam, 2002), and chronic stress was shown to induce impairments of
spatial working memory, a type of memory most associated with the PFC (Mizoguchi et al., 2000). Using
in vivo brain imaging methods, such as positron emission tomography (PET) or single photon emission
computed tomography (SPECT), the PFC was identified as a major area affected in a variety of depressive
and mental states (Ebert and Ebmeier, 1996). Indeed, PFC malfunctioning was shown in patients with
PTSD (Shin et al., 2001). PFC efferents and most of the afferents to the PFC, especially those that arrive
from the thalamus and the hippocampus, are glutamatergic. Glutamatemediated neurotransmission is
hence a major player in regulating PFC stress responses (Bagley and Moghaddam, 1997). Nevertheless, the
PFC is by no means restricted to glutamatergic neurotransmission. Levels of other neuromodulators were
found to be modified following stress in the PFC, attesting to the complex functionality of the PFC during
stress. These include serotonin (Kawahara et al., 1993), dopamine (Hamamura and Fibiger, 1993), mono-
amines (Flugge et al., 1997), excitatory amino acids (Moghaddam, 1993), norepinephrine (Nakane et al.,
1994), GABAA (Gruen et al., 1995), and acetylcholine (Mark et al., 1996). The latter poses an interesting
and important link between the glutamatergic and the cholinergic systems in the brain. Cholinergic
glutamatergic interactions have been associated with higher brain functions such as LTP, memory, and
behavior (Aigner, 1995), all of which are affected by stress.

2 PremRNA Splicing

In eukaryotic cells, especially in mammalian cells, the newly transcribed premRNA is subjected to a series
of processing events before leaving the nucleus, reaching its subcellular location, and translating into
protein. These include 50 capping, splicing, cleavage, and 30 polyadenylation (Rio, 1992). The precise
regulation of these events is critical for ensuring the accuracy and the diversity of gene expression.
Particularly interesting is the splicing machinery, which recognizes and removes introns to produce the
mature mRNA (Hastings and Krainer, 2001). In up to 60% of the genes, the splicing machinery further
generates different transcripts from the same transcription unit by selecting or excluding different exons,
a process known as alternative splicing. The different transcripts created must then be spatially and
temporally regulated. Alternative splicing is the key process responsible for the large proteomic diversity,
estimated to produce hundreds of thousands of proteins (Hodges et al., 2002), from merely 20,000 to
30,000 genes by alternative splicing of the majority of the premRNAs (Lander et al., 2001). This estimation
is based on the comparison of expressed sequence tags (ESTs) with genomic data. Therefore, the real
number is likely to be higher, as numerous lowabundance mRNAs are not taken into account, and because
ESTs are biased toward the mRNA 30 ends (Lander et al., 2001). One extreme example includes the
Drosophila Down syndrome cell adhesion molecule (Dscam), encoding an axon guidance receptor shown
to possess more than 38,000 alternatively spliced transcripts (Schmucker et al., 2000).

2.1 The Splicing Machinery

The splicing reaction involves two transesterification steps. In the first reaction, the 50 end of the intron
binds to the branchpointa conserved adenine upstream from the 30 splice siteto form an intron lariat
 mRNA modulations in stress and aging
9 221

intermediate. In the second reaction, the two exons are ligated and the lariat is released for nuclear
degradation. In general, for an intron to be recognized, it must include, in addition to the conserved
branchpoint sequence, conserved 50 and 30 splice sites (usually GU and AG, respectively), and a polypyr-
imidine (Py) tract, located close to the 30 end of the intron. These elements recruit the required molecules
carrying out the splicing reaction.
The splicing machinery is mainly governed by small nuclear ribonucleoprotein particles (snRNPs),
which recognize and bind conserved elements within the premRNA. SnRNPs are RNAprotein complexes
made out of five small nuclear RNAs (snRNAs) (U1, U2, U4, U5, and U6) and associated proteins, which
form the core spliceosome (Hastings and Krainer, 2001). As shown in > Figure 9-3, the first steps of

. Figure 9-3
Spliceosome assembly. Early complex (E) involves binding of U1 snRNP to the 50 SS (GU) and the two subunits
(65 and 35) of U2AF to the Py tract and 30 SS (AG), respectively. Recognition of the branch point (A) by U2 snRNP
creates the A complex. Further binding of the U4/U6U5 trisnRNP breaches the association of U1, which is
released with U4, to form the B complex. The 50 splice site then juxtaposes near the branch point to form the C
complex, and the lariatshaped intron is removed

spliceosome assembly involve 50 splice site recognition by U1 and binding of U2 auxiliary factor (U2AF) to
the Py tract and 30 splice site. These early interactions form the E complex. Further base pairing of U2 with
the branchpoint forms the A complex. Subsequent steps include association of the U4/U6U5 trisnRNP
with the spliceosome. Before the first catalytic step, the U4U6 interaction is disrupted and an invariant
motif in U6 replaces U1 snRNA at the 50 splice site (forming the B complex). A specific base pairing between
U2 and U6 snRNAs then juxtaposes the branchpoint and the 50 splice site. U5 snRNP holds onto the 50 exon
intermediate, which has been cut free, and the spliceosome is reconfigured to bring the 30 splice site into the
catalytic core and align the exons for the second catalytic step (C complex).
The spliceosome involves, apart from the above, some additional 150 spliceosomeassociated proteins
(Zhou et al., 2002). NonsnRNPs relevant for splicing catalysis include heterogeneous nuclear ribonucleo-
proteins (hnRNPs), additional RNAbinding proteins, and most important, a family of SR proteins (Zahler
et al., 1992), which promote splicing activity (Zahler et al., 1993) and affect alternative splicing patterns
(Caceres et al., 1994).

2.2 SR Proteins
SR proteins are characterized by a modular structure with a conserved Cterminal arginine/serinerich
domain (RS domain) that interacts with components of the basic splicing machinery, and one or two
RNArecognition motifs (RRMs) (Tacke and Manley, 1999). The RRMs recognize loosely conserved RNA
222
9 mRNA modulations in stress and aging

sequences, identified as either exonic or intronic splicing enhancers (ESE/ISE) or suppressors (ESS/ISS)
(Cartegni et al., 2002). When SR proteins bind to their corresponding ESEs, they recruit the early splicing
component, i.e., U1 snRNP and U2AF to their correct sites (> Figure 9-4). In alternative splicing, ESEs are

. Figure 9-4
ESESR protein interactions. A premRNA containing both introns (white) and exons (dark) is shown. Exons
contain exonic splicing enhancers (ESE) recognized and bound by the RNA recognition motif (RRM) of SR
proteins (ovals). SR proteins recruit U1 snRNP to the 50 splice site and U2AF to the Py tract and the 30 splice site.
Subsequently, U2AF recruits U2 snRNP to the branchpoint (A). SR proteins can also bind intronic splicing
enhancers (ISE) and mediate proteinprotein interactions through their RS domain

usually found in the alternatively spliced exon, and function by recruiting splicing factors to a suboptimal 30
splice site, thus stimulating splicing of an upstream intron or inclusion of an alternative exon (Cartegni
et al., 2002). It was demonstrated, in vitro, that SR proteins alone can influence splice site selection and
hence regulate alternative splicing (Ge and Manley, 1990; Krainer et al., 1990). In vivo, it is likely that the
finetuned balance between SR proteins, hnRNPs, splice sites, and enhancer/silencer elements can be
modulated to achieve a change in the exon usage of premRNA (Hastings and Krainer, 2001). The levels
of SR proteins and hnRNPs vary among tissues (Hanamura et al., 1998) and can be further modulated by
releasing proteins from intranuclear storage compartments, such as the speckle domains at the nuclear
internal boundaries through protein phosphorylation (Mintz and Spector, 2000) and by tissuespecific
factors, especially in the nervous system (Dredge et al., 2001).
Using computational algorithms together with in vivo and in vitro validation, several functional ESEs
were identified (Schaal and Maniatis, 1999; Liu et al., 2000; Cartegni et al., 2002). These were found to be
68 bases long, purinerich sequences that are usually degenerate. In addition to serving in alternative
splicing, these elements may have crucial roles in identifying correct exons for constitutive splicing, as the
other relevant conserved sequences in exons (i.e., splice sites, Py tract, and branchpoint) are weak. If this is
indeed the case, it would explain the somewhat paradoxically high occurrence of ESEs in exons rather than
introns, and would support the model, which suggests that the recognition unit of the spliceosome is the
exon itself rather than the intron (Robberson et al., 1990).

3 Splicing and Stress

3.1 Constitutive Splicing

The initial observations that cellular stress leads to splicing modifications were made about two decades ago
with the discovery that a brief severe heatshock impairs the splicing process, leading to the accumulation of
unspliced RNA products. This inhibition of splicing could be partly avoided in cells that were previously
exposed to a mild heat shock, an adaptation process that involved the cellular activation of heatshock
proteins (HSPs) (Yost and Lindquist, 1986). This first set of experiments suggested a controllable associa-
tion of heatshockrelated proteins with components of the splicing machinery. Interestingly, whereas the
 mRNA modulations in stress and aging
9 223

splicing of HSP27 was impeded during heat shock (Bond, 1988), the splicing of the small HSP21 remained
unaffected but the length of its poly(A) tail, which usually confers longer halflife of mammalian mRNAs
(Soreq et al., 1974; Jacobson and Peltz, 1996), was increased (Osteryoung et al., 1993), demonstrating a
finely regulated mechanism and suggesting stressinduced stabilization of HSP21 mRNA. Later, two of the
HSPs participating in the reactivation of splicing following heat shock were identified as HSP70 and
HSP104 (Vogel et al., 1995).
Additional factors contributing to stressinduced splicing alterations include the family of hnRNPM
proteins and the splicingrelated protein 2H9, shown to participate in basal splicing processes as well as in
the early stages of stressinduced splicing arrest (Gattoni et al., 1996; Mahe et al., 1997). Other reports
showed disruption of the U4/U6U5 trisnRNPs complex in HeLa cells under heat stress (Bond, 1988;
Shukla et al., 1990), as well as inactivation of a splicing factor associated with U4/U6U5 (Utans et al., 1992).
Indeed, a heatshockinduced shift from speckled to diffusive distribution of snRNP antigens was reported
in the nucleus of mouse fibroblasts, whereas the splicing factor SC35 retained its normal distribution
(Spector et al., 1991). Remarkably, spatial association between splicing factors and transcriptionally active
genes in nuclear sites of transcription was shown to be intronindependent, as evident by the association of
both an intronrich, HSP90 alpha and the intronless HSP70 protein with nuclear speckles upon exposure
of cells to heat shock (Jolly et al., 1999). These findings indicate an intrinsic, finely tuned targeting capacity
for splicing proteins to potential splice sites during cellular stress.

3.2 Alternative Splicing

In addition to splicing arrest, various stressful stimuli were shown to involve regulated, specific modula-
tions in alternative splicing. Splicing arrest may result in the accumulation of introncontaining transcripts,
which under strict evolutionary pressure can be beneficial for the cell. Differential availability of splicing
related proteins could further result in a regulatedlike fashion of alternative splicing, because the fine
balance between the splicing factors, more than the precise concentration of any specific protein, is the key
regulatory element (Hastings and Krainer, 2001). This phenomenon may be viewed as a beneficial adapta-
tion strategy that modifies a finely tuned regulation process; alternatively, or in addition, these changes
might reflect adverse or neutral consequences of the uncontrolled processing of premRNA.
In recent years, a growing body of evidence links diverse stress responses of cells and tissues to
alternative splicing modulations. Although the cellular signaling pathways affecting alternative splicing
under modified stimuli are poorly understood, recent efforts by several investigators highlight interesting
possibilities. In one study, activation of the MKK3/6p38 cascade was shown to modify the subcellular
distribution of hnRNP A1, resulting in modified alternative splicing of target premRNAs (van der Houven
van Oordt et al., 2000). This demonstrated that the stressinduced biochemical cascades can influence the
patterning of alternative splicing through modifying the phosphorylation state of upstream molecules,
thereby affecting their cellular compartmentalization. In another example, the stressactivated ERK MAP
kinase pathway was shown to regulate the phosphorylation state, and subsequent cytoplasmic localization
of hnRNP K, an RNAbinding protein that silences mRNA translation (Habelhah et al., 2001). The same
ERK/MAPkinase pathway was also shown to be involved, upon activation of T cell lymphocytes, in the
alternative splicing of the cell membrane adhesion glycoprotein CD44 by retaining its exon v5 sequence in
the mature mRNA (WegRemers et al., 2001), demonstrating how kinase cascades can regulate different
posttranscriptional events simultaneously by activating a single stressinduced pathway.
Stressinduced changes in alternative splicing have been demonstrated for the transcripts of numerous
genes. The GR serves as a most relevant example in our case. Perinatal manipulations and postnatal
handling were both shown to selectively elevate GR mRNA containing a hippocampusspecific exon,
which facilitates adaptation to modified stimuli (McCormick et al., 2000). GCs also regulate the splicing
pattern of the murine Slo gene encoding a brain potassium channel, which depends on neuronal depolari-
zation (Xie and McCobb, 1998). Hypoxia was shown to induce the alternative splicing of the presenilin
2 (PS2) gene, generating an isoform also found extensively in the brain of Alzheimers disease patients
(Sato et al., 1999). A detailed study of Tra2b1 and additional SR proteins under ischemic conditions in the
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9 mRNA modulations in stress and aging

brain revealed that some of them translocate from the nucleus to the cytosol after an ischemic event, which
could explain the observed changes in splice site selection (Daoud et al., 2002). Activating transcription
factor 3 (ATF3) was shown to possess a specific stressassociated splice variant, ATF3DeltaZip2, modulating
the activity of the normal ATF3 protein during stress responses (Hashimoto et al., 2002). Splice site
selection can hence serve both as a physiologic adaptation to a change in the external conditions and as a
contribution to pathophysiologic events.
Intriguingly, some of the evoked changes in alternative splicing produce transcripts that have antago-
nistic cellular actions. For example, the heatshock factor 4 transcript can generate either an activator or a
repressor of downstream heatshock genes by alternative splicing (Tanabe et al., 1999). Also, alternative
splicing of the apoptotic gene, bclx, substitutes a large protein product, BclxL, which inhibits cell death,
for a smaller one, BclxS, which antagonizes this property under certain conditions (Boise et al., 1993). The
gene encoding neural cell adhesion molecule (NCAM) was shown to undergo alternative splicing during
nitric oxide (NO)induced apoptosis and during neuronal differentiation, producing a smaller protein
product than the normal one, through the action of cJun/AP1 (Feng et al., 2002). This smaller species,
NCAM140, protects cells against NOinduced apoptosis, illustrating the significance of alternative splicing
regulation during stress responses. Most important, a direct link between splicing and apoptosis was
recently shown with the isolation of a protein complex termed the apoptosis and splicingassociated
protein (ASAP). ASAP was shown to disassemble during apoptosis, releasing the splicing activator RNPS1,
which could possibly participate in the splicing and/or alternative splicing of target genes that are activated
during apoptosis (Schwerk et al., 2003), strengthening the stress/apoptosis and splicing relationship.
An examination of the cases studied so far (e.g., GR, Slo, PS2, ATF3, HSF4, Bclx, NCAM, and AChE,
see later) supports the notion that alternative splicing serves as an adaptation strategy. Thus, alternative
splicing in these cases yields specific splice variants, which facilitate beneficial stress responses, at least in the
short range. For example, the alternative products of GR, Slo, ATF3, HSF4, Bclx, and NCAM actively
participate in adapting to the novel conditions, either by generating an antagonizer to the action of its
normally spliced counterpart (e.g., ATF3, HSF4, Bclx) or by generating a protective protein (e.g., GR,
NCAM). In the case of the potassium ion channel, Slo, a calciumcalmodulin dependent protein kinase
(CaMK) IV responsive RNA element (CaRRE) was identified. To demonstrate its function, this element was
experimentally transferred from the 30 splice site upstream of STREX, the alternatively spliced product of
Slo, to a heterologous gene. The otherwise constitutive acceptor sequence became sensitive to CaMK IV
(Xie and Black, 2001). This sets an intriguing example for the role of the premRNA sequence itself in
stress responses, which in this particular case, introduces longterm adaptive changes to the neuronal
electrophysiological properties.

3.3 StressInduced Changes in SRRelated Proteins

As stated earlier, several proteins emerge as particularly relevant to stress responses. These include, but are
not limited to, the splicing activator RNPS1, the splicingassociated complex ASAP, and the SRrelated
protein Tra2b1. Another potentially involved member of the SR protein family worth mentioning is SC35.
SC35 possesses an RRM and an RS domain, and was shown to affect both splice site selection and
alternative splicing (Wang et al., 2001). In addition, the SC35 premRNA itself undergoes alternative
splicing (Sureau et al., 1997) and the SC35 protein further promotes splicing events that destabilize its
own mRNA, exercising a selfcontrol mechanism that limits its durability (Sureau et al., 2001). This, in turn,
suggests that to maintain its longlasting high levels, SC35 mRNA should be continuously overproduced in
the poststress brain.
ASF/SF2, the first SR protein to be identified (Ge and Manley, 1990; Krainer et al., 1990), was also
shown to affect both alternative splicing and choice of splice sites (Mayeda and Krainer, 1992; Sun et al.,
1993; Wang et al., 1998). Interestingly, the 32 kD subunit of the splicing factor ASF/SF2, termed SF2p32
inhibits ASF/SF2 activity through inhibition of ASF/SF2 phosphorylation even in cells that maintain
unchanged levels of the ASF/SF2 protein (PetersenMahrt et al., 1999). As ASF/SF2 antagonizes the splicing
activity of SC35 (Gallego et al., 1997), SF2p32 increases should potentially facilitate increases in the ratio
 mRNA modulations in stress and aging
9 225

between the alternative RNA targets of ASF/SF2 and SC35. Both proteins should hence be tested in the
poststress brain.

4 StressAssociated Dendritic Translocation of mRNAs

Identified more than 15 years ago (Davis et al., 1987), dendritic transport of mRNAs is known today to be a
feature of over 400 different transcripts, comprising about 5% of the total expressed neuronal mRNAs
(Eberwine et al., 2001). Dendritic targeting of mRNAs and local protein synthesis were both shown to be
associated with hippocampal synaptic plasticity (Kang and Schuman, 1996), as well as with memory storage
(Mayford et al., 1996), predicting involvement with aging and stress.
As expected from a process regulating synaptic function and plasticity, targeting of mRNAs to dendrites
was shown to be stimulusinduced (Tongiorgi et al., 1997; Steward et al., 1998). Yet more specifically,
potassium depolarization was demonstrated to induce calciumdependent transport of the mRNAs for
brain derived neurotrophic factor (BDNF) and the tropomyosinrelated kinase B (TrkB) BDNF receptor
into dendrites. This translocation did not require newly synthesized RNA, yet was followed by local protein
synthesis (Tongiorgi et al., 1997). In another study, the mRNA for the immediateearly gene activity
regulated cytoskeletonassociated protein (Arc) was shown to translocate selectively to activated dendritic
segments in the dentate gyrus. In this case as well, mRNA translocation was followed by new synthesis of the
Arc protein (Steward et al., 1998). Activitydependent mRNA targeting may thus provide a mechanism for
synaptic modulation, which requires local protein translation of specific transcripts. The recent generation
of mice lacking dendritic transport ability and local protein synthesis of calcium and calmodulin
dependent protein kinase II alpha (CaMKIIa) provided a direct proof that local translation is required
for LTP and memory consolidation (Miller et al., 2002).
The translocation of mRNAs into neuronal processes is generally considered to be associated with cis
acting elements, primarily within the 30 untranslated region (UTR) of the mRNA (Steward and Schuman,
2001). However, the length of the sequence required for dendritic targeting varies considerably. For
example, a 21 nucleotide long sequence in the 30 UTR of the myelin basic protein (MBP) mRNA was
shown to be sufficient for dendritic translocation (Ainger et al., 1997), whereas in another study,
640 nucleotides from the 30 UTR of the microtubuleassociated protein 2 (MAP2) were shown to be essential
for mRNA targeting (Blichenberg et al., 1999). For aCaMKII, 94 nucleotides from the 30 UTR were shown
to mediate its dendritic localization in cultured hippocampal neurons (Mori et al., 2000), whereas in a
different study, a 1,230 nucleotide region of the 30 UTR was shown to be responsible (Blichenberg et al.,
2001). These apparent discrepancies could be settled if the secondary structure, rather than primary
sequence, was responsible for targeting. Indeed, careful analyses demonstrated that dendritically localized
mRNAs do not share a common consensus sequence (Tiedge et al., 1999). It is therefore likely that
RNA folding into secondary structures might act as the driving mechanism (Ainger et al., 1997; Blichenberg
et al., 1999).

5 Defects in premRNA Splicing as Causes of and Predisposition to Diseases

Alternative splicing of a growing number of gene products can be found in numerous types of cancerous
cells, although not abundant in the corresponding normal cells (HerreraGayol and Jothy, 1999; Caballero
et al., 2001; Perry et al., 2002), probably reflecting a general modification of the processing machinery
during tumorogenesis (Scorilas et al., 2001; Stoilov et al., 2002). In humans, SC35 expression is altered
under other diseases as well, e.g., HIV infection (Maldarelli et al., 1998) and also during pregnancy (Nie
et al., 2000). ASF/SF2 too is spatiotemporally regulated in the uterine myometrium during pregnancy
(Pollard et al., 2000), again, probably insinuating a more general phenomenon for the regulation of
splicingrelated genes during various stresses or under adaptation to altered conditions. A striking example
of a splicingrelated protein, which directly affects the severity of disease state, is illustrated by the putative
splicing factor SCNM1 (sodium channel modifier 1). A single point mutation changes a donor splice site in
226
9 mRNA modulations in stress and aging

the sodium channel gene Scn8a leading to chronic movement disorder. In one mouse strain, C57BL/6J, an
additional modifier nonsense mutation in SCNM1 exacerbates the Scn8a disorder into a lethal neurological
disease. The effect of the modifier mutation is a reduction of the abundance of correctly spliced sodium
channel transcripts below the threshold that is essential for survival (Buchner et al., 2003). This example
demonstrates how genetic variation in a splicing factor influences disease susceptibility.
A large number of mutations lead to aberrant splicing (Stoilov et al., 2002). These can directly disrupt
or create acceptor or donor splice sites, or alternatively, disrupt exonic or intronic splicing enhancers or
suppressors. Intriguingly, in a few cases, diseaseassociated alternative or aberrant splicing was reported, but
the culprit gene was found to be devoid of any splicingassociated mutations. These are particularly relevant
examples for this discussion, because a splicing regulatory protein is likely to be at fault, causing the
abnormal phenotype. We present several examples to illustrate each of these cases.
Familial hypercholesterolemia is an autosomal dominant genetic lipoprotein disorder caused by defects
within the lowdensity lipoprotein receptor gene. Some of its many mutations were shown to disrupt
acceptor splice sites: an A to G substitution in the penultimate nucleotide of intron 16 (Lombardi et al.,
1993) and a G to C transposition at the last nucleotide of intron 7 (Yu et al., 1999). In both cases, a cryptic
splice site was activated, leading to the formation of a mutated receptor protein. Another example of a splice
site mutation that leads directly to a disease phenotype is molybdenum cofactor deficiency (MoCoD), an
inherited autosomal recessive disease that leads to early childhood death. Two bicistronic genes, MOCS1
and MOCS2, are responsible for the generation of the molybdoenzymes, sulfite oxidase, xanthine dehy-
drogenase, and aldehydeoxidase. An MOCS1 splice site mutation leads to the deficiency of all these
molybdenum cofactorrelated enzymes (Reiss et al., 1999).
An increasing number of alterations, both in normal and in alternative splicing, are being linked to
defects in enhancer/silencer sequences (Philips and Cooper, 2000; Cartegni et al., 2002). A number of these
mutations are present in an exon, but do not change the protein sequence. Because they cause aberrant
splice site selection, they can cause a disease, although they do not modify mRNA translation. Examples of
these mutations include a T to C (L284L) mutation in tau exon 10 that disrupts an exonic splicing enhancer,
causing frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17) (DSouza et al.,
1999), a C to G (R28R) mutation in porphobilinogen deaminase that results in exon 3 skipping, and that
causes one of the many forms of porphyria (Llewellyn et al., 1996) and an A to G mutation in pyruvate
dehydrogenase resulting in Leighs encephalomyelopathy (De Meirleir et al., 1994).
Mutations that alter the splicing process can occur outside of both splice sites and enhancer/silencer
elements. An example is the mutation described in a patient with ataxiatelangiectasia (ATM), which
represents a deletion in an intronsplicing processing element crucial for accurate intron removal (Pagani
et al., 2002). The deletion of four nucleotides in this element abolishes a binding site for U1 snRNP and
leads to activation of a cryptic exon, thus producing an abnormal mRNA transcript.
Changes in splicing factors can also be phenotypic. The appearance of splicingassociated diseases is
often correlated with impaired plasticity and longevity of the affected cells. For example, mutations in the
human homologs of the splicing factors PRP31 or PRPC8 may cause retinitis pigmentosa, a progressive loss
of rods and cones, which causes loss of over 90% of vision during childhood (McKie et al., 2001; Vithana
et al., 2001).
An example of a disease with unexplained aberrant splicing includes sporadic amyotrophic lateral
sclerosis (ALS), which was shown to be associated with a change in the splicing patterns of the mRNAs for
the glutamate transporter EAAT2 (Lin et al., 1998), as well as nitric oxide synthase (NOS) (Catania et al.,
2001). This indicates a basic defect in the premRNA processing machinery, which gives rise to altered
isoforms associated with the disease.
To a certain extent, premRNA processing thus appears to have the potential to adapt the information
stored in the genome to the physiologic requirements of circumstance, place, and time. The failure of such
adaptation is frequently traced to defects in processing. These defects can manifest themselves directly in a
disease or may remain silent until some internal or environmental stimulus (e.g., stress) or time (e.g., old
age) allows the mutation to become apparent. Furthermore, the sequential nature of premRNA processing
raises the interesting possibility that pleiotropic diseases with a variable phenotype may be caused by
specific compositions of transacting factors.
 mRNA modulations in stress and aging
9 227

6 Splicing and Aging

6.1 PremRNA Splicing Modulations in Senescence

Splicing alterations are particularly important during prolonged altered conditions associated with chronic
cellular stress states, which include aging (Meshorer and Soreq, 2002). From the viewpoint of a molecular
biologist, aging reflects gradual deterioration of the molecular components, checkpoints, and/or events, the
concerted functioning of which is vital for cell viability and proliferation. The complexity of alternative
splicing makes this process particularly vulnerable to senescence, leading to both transient changes and
chronic agingrelated diseases. Available examples of modified alternative splicing events during aging
predict a generally modified state of premRNA processing machinery in senescent cells, leading to altered
expression levels of premRNA processing factors and their downstream target mRNAs and corresponding
proteins during aging.
With the emergence of microarray technologies, changes in gene expression profiles during aging have
recently become the focus of extensive research. However, relatively little is yet known about changes in
alternative splicing, a key premRNA processing mechanism during cellular and organismal senescence.
The gradual aging process is often ascribed to passive accumulation of lateonset mutations due to lack
of natural selection beyond the age of reproduction. Alternatively, aging is viewed as the outcome of active
selection of traits that are beneficial at an early stage of life but have deleterious effects later on (reviewed in
Partridge and Gems, 2002). The idea that aging is a genetically programmed mechanism was also subject to
some debate. Planned or sporadic, passive or active, aging also involves changes in the pathway for gene
expression that are not necessarily associated with mutations. This is exemplified in that most agingrelated
diseases, representing the beststudied cases of aging, involve aberration in the alternative splicing of
premRNA.
Agingrelated modifications in the alternative splicing of specific gene products were sought for by
several researchers, primarily as means to search for the molecular origin(s) of agingrelated deterioration
in the processes they were studying. Examples include neural adhesion processes, dopaminergic neuro-
transmission, insulin responses, and gastric cytoprotection (> Table 9-1). Together, the picture that

. Table 9-1
Agingassociated changes in alternative splicing
Gene Organism Tissue Affected process Reference
APP, APLPs rat Brain amyloid plaque (Sandbrink et al., 1997)
Dopamine D2 rat Neostriatal dopaminergic (Merchant et al., 1993)
receptor subregions neurotransmission
NCAM rat Heart neural adhesion (Andersson et al., 1993)
Insulin receptor rat Liver, muscle, heart insulin responses (Vidal et al., 1995)
MGF rat Skeletal muscle insulin responses (Owino et al., 2001)
COX1 rat Stomach gastric cytoprotection (Vogiagis et al., 2000)
NF1 Human Blood rRas signaling, (Wimmer et al., 2000)
neural architecture

emerges from these studies is of a general control switch that causes multiple aginginduced impairments
in alternative splicing. For example, such impairments could all reflect one or a few changes in regulatory
upstream factor(s). Support for this concept can be found in more recent microarray screens for changes in
gene expression during aging. Significant agerelated tissuespecific changes were found in the expression
levels of several RNA processing genes in aged, compared to young, gastrocnemius muscle, neocortex, and
cerebellum of C57BL/6 mice (Lee et al., 1999). Changes spanned SR proteins, hnRNPs, as well as 30 end
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9 mRNA modulations in stress and aging

. Table 9-2
Agingassociated alterations in the expression of murine premRNA processing genes
Fold Affected premRNA Reference to
Gene changea Tissue processing step reported function
SF3A2 3.4 Cerebellum U2 snRNP binding (Ruby et al., 1993)
(SAP62,
PRP11)
U2AF65 3.2 Gastrocnemius muscle Splice site recognition, splicing (Wang et al., 1995)
Sox17 2.4 gastrocnemius muscle Transcription, Splicing (Ohe et al., 2002)
PRP22 (HRH1) 2.5 neocortex Spliceosome disassembly, (Company et al.,
mRNA release 1991)
PTB 2.3 cerebellum Alternative splicing (Wagner and
GarciaBlanco 2001)
PolyA RNA 2.1 gastrocnemius muscle 30 end processing, mRNA
export protein export
hnRNP H3 2.0 cerebellum Splicing, heatshock splicing (Mahe et al., 1997)
(hnRNP 2H9) arrest
CStF 0.7 neocortex Transcript cleavage
DDX18 0.7 neocortex DEADbox protein, specific
function unknown
PRP16 0.6 cerebellum Aberrant lariat discard (Burgess and
Guthrie 1993)
PABI 0.4 cerebellum mRNA stability
PRP22 0.4 cerebellum Spliceosome disassembly, (Company et al.,
mRNA release 1991)
a
From Lee et al. (1999)

processing factors (> Table 9-2). Because both SR proteins and hnRNPs influence splice site selection,
misdirected regulation of their expression levels can lead to alternative or aberrant splicing of many
downstream target sequences.
One of the first essential splicing factors to be identified was the 65 kD subunit of the U2 auxiliary factor
(U2AF65). U2AF65 binding to the conserved Py tract at the 30 end of introns is required for the subsequent
binding of U2 snRNA to the 50 splice site. Another subunit protein of this complex, U2AF35, binds the 30
splice site. U2AF65 mRNA levels were found to increase by over threefold in the gastrocnemius muscle of
aged mice (> Table 9-2). As U2AF65 contains both the RNA recognition motif and the RS domain of
SRproteins, it can regulate splicing in a concentrationdependent manner. Uncontrolled regulation of the
expression level of U2AF65 may therefore lead to aberrant regulation of splicing activity and alternative
splicing of numerous target genes (Wang et al., 1995) (> Figure 9-5).
The Py tractbinding protein (PTB, hnRNP I) also binds intronic Py tracts. PTB was shown to possess a
direct role in premRNA alternative splicing (Wagner and GarciaBlanco, 2001), and its expression levels
were reported to increase over twofold in the cerebellum of aged mice, which can potentially harm finely
regulated alternative splicing of many premRNAs (> Figure 9-5).
Other factors that may have a direct or indirect role in influencing splicing during aging are PRP22,
PRP16, SF3A2, DDX18, and hnRNP H3 (Table 1-2). The expression level of the splicing factor PRP22, for
example, is 2.5fold increased in the neocortex of aged C57BL/6 mice, whereas the same factor displays
2.7fold decrease in the cerebellum of these mice. Although the function of several of these factors is still
largely obscure, this expanded list points to a central involvement of splicing changes in the aging process
(> Figure 9-5).
Another checkpoint, which may affect agingrelated changes in premRNA splicing, lies upstream, at
the earlier phases controlling the initiation and efficacy of transcription. This involves the expression levels
 mRNA modulations in stress and aging
9 229

. Figure 9-5
Misdirected regulation of premRNA processing during aging. Factors that were reported to change during
aging are depicted in the figure, along with the different steps of premRNA processing, in which they are
involved in normal and aged cells. a: The naked premRNA transcript. Exons are dark and introns are marked
white. The 50 (GU) and the 30 (AG) splice sites, the branchpoint (A), and the polypyrimidine tract (Py) are shown.
The 50 end of the premRNA is capped shortly after initiation of transcription, b: Initial snRNP complex
formation. U2AF65 and PTB were shown to be significantly overproduced in the gastrocnemius muscle and
cerebellum, respectively, of aged C57BL/6 mice, thus, potentially affecting splicing, c: Spliceosome assembly
and attraction of SR proteins. The splicingrelated factor/RNA helicase PRP16 was shown to be twofold
decreased in the cerebellum, d: Intron release and exon joining. Altered expression level of the different
factors shown earlier may lead to the alternatively spliced transcript shown, e: 30 end processing. The cleavage
and polyadenylation complex, which consists of CPSF, which recognizes and binds the poly(A) site (AAUAAA),
CStF, CFI, CFII, and PAP, which catalyze the first, slow, polyadenylation step are shown. PABII then binds the
short, newly formed, poly(A) tail and rapidly adds additional 200250 adenines. CStF and PAP were both about
1.5fold decreased in the neocortex of C57BL/6 aged mice, potentially harming the 30 end processing pathway
and the splicing pathway. Abbreviations: Py, polypyrimidine tract; snRNP, small nuclear ribonucleoprotein
particle; U2AF, U2 auxiliary factor; PTB, polypyrimidine tractbinding protein; SRproteins, serine, argininerich
proteins; CPSF, cleavage and polyadenylation specificity factor; CStF, cleavage stimulation factor; CF, cleavage
factor; PAP, poly(A) polymerase; PAB, poly(A)binding protein.
230
9 mRNA modulations in stress and aging

of a large number of transcription factors, also shown to be modified in aged tissues. As transcription
and splicing are tightly coupled processes, such changes are likely to affect the splicing machinery as well.
The transcription factor SOX17, for example, with a direct role in premRNA splicing (Ohe et al., 2002),
demonstrated 2.4fold overexpression in the aged gastrocnemius muscle. Altered expression of tran-
scriptionassociated genes during aging thus adds further support to the concept of premRNA splicing
as an agingaffected process. Indeed, agingrelated diseases often reflect abnormal upstream factors,
impaired regulation, or aberrant finetuning of mRNA processing in general and premRNA splicing in
particular.

6.2 PremRNA Splicing Modulations in AgingRelated Diseases

Agerelated, lateonset diseases are especially vulnerable to splicing variations, of several different origins.
These include, for example, mutations affecting the correct splicing of a particular gene. Unlike diseases of
early onset, the phenotype associated with these mutations displays a delayed onset. Agerelated macular
degeneration (AMD) serves as an example of an agingrelated disease that is associated with a lateonset
splicing mutation. AMD is the most common cause of acquired visual impairments in the elderly. The
occurrence of AMD is often associated with mutations within the Stargardt disease gene (STGD1 or ABCR),
coding for a photoreceptorspecific member of the ATPbinding cassette (ABC) superfamily of transporter
proteins. One of these mutations, a G for A substitution at position 5196, was found to be a donor splice site
mutation (Allikmets et al., 1997). The late onset of the disease phenotype, in this case, may be explained by
agerelated modifications in the functioning efficacy and/or the composition of premRNA processing
factors.
Alzheimers disease (AD) is the most common neurodegenerative disorder of aging, characterized by
progressive memory loss and cognitive deterioration. AD involves premature death of cholinergic neurons,
associated with the formation of amyloid plaques the appearance of which in the brain of patients is
facilitated by mutations in several different genes. Misdirected splicing regulation of relevant gene products
due to specific mutations and aberrant processing of such products with no known mutations were both
demonstrated for AD. Presenilins provide examples for both cases: mutations within the fourth intron
of presenilin 1 (PS1) were shown to impair PS1 splicing and cause early onset AD; moreover, an exon
5lacking splice variant of apparently normal PS2, which accumulates following hypoxia in cultured
neuroblastoma cells, was found to be prevalent in the brains of AD patients, indicating that this unique
splice variant is inducible, rather than inherent in the genome (Sato et al., 1999).
Tauopathies are a family of lateonset neurodegenerative diseases associated with mutations within the
microtubuleassociated protein (MAP) tau gene (Lee et al., 2001). Progressive accumulation of filamentous
tau inclusions causes neural degeneration in specific brain regions of patients with tauopathies. The late
onset of this phenotype suggests that an agerelated molecular change is responsible. Tau is alternatively
spliced in the adult human brain (Lee et al., 2001) and mice overexpressing the human shortest tau
variant display agedependent CNS deterioration reminiscent of human tauopathies (Ishihara et al.,
1999). Thus, impairments in premRNA processing and/or splicing might be the cause. As shown earlier,
FTDP17 serves as an example of a tauopathy associated with changes in alternative splicing. In this case, as
well, the mutated genotype leads to an abnormal phenotype of a delayed onset, hinting at a change in a yet
unidentified agerelated factor involved in premRNA processing.

7 Candidate Gene Study: ACHE

Transcriptional and posttranscriptional poststress responses induce a cascade of changes in the levels and
properties of certain brain proteins that may play major roles in the subsequent events. This particularly
relates to proteins involved in relevant neurotransmission pathways. For example, acetylcholine (ACh)
levels were shown to be transiently elevated in the mammalian brain during stress responses (Imperato
et al., 1991). Similar increases in ACh were also reported following exposure to cholinesterase inhibitors
 mRNA modulations in stress and aging
9 231

(Dazzi et al., 1995). Indeed, psychological stress elicits strikingly similar neuropsychological effects to those
observed after acute or chronic exposure to anticholinesterases (Rosenstock et al., 1991). In addition,
acute stress and pharmacological inhibition of acetylcholinesterase (AChE), the ACh hydrolyzing enzyme
(> Figure 9-6), suppress the production of the AChsynthesizing enzyme choline acetyltransferase (ChAT)

. Figure 9-6
Acetylcholine hydrolysis by AChE. AChE cleaves acetylcholine to acetate and choline at a rate of 300,000
molecules per minute, making it one of the most efficient enzymes in nature. Its active gorge includes a serine
residue responsible for the enzymes catalytic activity (Soreq and Seidman, 2001)

(Kaufer et al., 1998; Anguelova et al., 2000), while inducing rapid increases in AChE mRNA and protein
levels in response to forced swim stress in the mouse brain (Kaufer et al., 1998). This suggests a bimodal
mechanism for suppressing the cholinergic excitation, which follows stress insults.

7.1 The Molecular Biology of AChE

The single AChE gene in vertebrates generates at least three different premRNAs with distinct 30 regions
due to alternative splicing (Soreq and Seidman, 2001). These include the synaptic, AChES (also known
as tailed AChET), the erythrocytic AChEE (also known as hydrophobic, AChEH), and the
readthrough, AChER (> Figure 9-7). As mentioned earlier, the latter is the stressinduced form, rarely
found in nave adult tissues. Conversely, AChES mRNA is generally ubiquitously expressed and is subject
to transcriptional and posttranscriptional developmentrelated regulation, whereas AChEE mRNA is
primarily expressed in red blood cell progenitors. The three different 30 transcripts give rise to three protein

. Figure 9-7
30 alternative splicing of AChE. The distal enhancer glucocorticoid response element (GRE), the human AChE
gene, and the three 30 splicing products (S, E, R) are shown. Linkage of exons 2, 3, and 4 is common to all
variants (a). The R transcript includes at its 30 terminus pseudointron 4 (bottom) and exon 5; The E transcript is
generated by splicing out intron 4, and option b yields the S transcript by connecting exon 4 to 6 (top).
232
9 mRNA modulations in stress and aging

isoforms, which differ at their Ctermini, dictating different cell adherence and noncatalytic properties for
each of the three. The Cterminus of human AChES consists of a 40 residue peptide, which includes a
cysteine 4 residues upstream from the Cterminus. This cysteine enables the formation of dimers and
tetramers, which are able to further bind a cholinesterasespecific collagen tail (ColQ) unique to AChE in
neuromuscular junctions (Massoulie, 2002) or a prolinerich membrane anchor (PRiMA) in the central
nervous system (Perrier et al., 2002). The former generates asymmetric AChE forms through interactions
between the ColQ tails of two or three different tetrameric units, and the two structural subunits anchor
AChES to synapses and neuromuscular junctions (Massoulie, 2002). AChEE forms dimers exclusively,
through a cysteine residue at its 43 amino acid Cterminus (position 8 of 43). This peptide is subsequently
cleaved after amino acid 14 (557 from the Nterminus) to enable linkage of the remaining Cterminus
through a glycosylphosphatidyl inositol (GPI) to the erythrocytic membrane. AChER has a shorter,
26 amino acids long, Cterminal peptide, which lacks a cysteine residue and hence remains monomeric
and soluble (Soreq and Seidman, 2001).
The core sequence of human and rodent AChE, common to all variants, consists of 543 amino acids
encoded by exons E2, E3, and E4. Exon 2 codes for the three amino acids responsible for the catalytic
activity of AChE inside the active gorge. This catalytic triad consists (in humans) of Ser203, Glu334, and
His447. Thus, AChES and AChER share the same capacities to hydrolyze acetylcholine (Soreq and
Seidman, 2001); however, the catalytic function of AChER in vivo should be extrasynaptic because it
does not attach to the synaptic membrane. This variant is hence more likely to tune the modulatory roles
of acetylcholine, presumed to reside outside synapses (Legay et al., 1995).
Several lines of evidence suggest that AChES and AChER have distinct roles in the normal brain,
as well as in the poststress processes (Seidman et al., 1995; Karpel et al., 1996; Shohami et al., 2000;
Sternfeld et al., 2000; Birikh et al., 2003; Brenner et al., 2003). Transgenic mice overexpressing one of the
corresponding human AChE isoforms (Beeri et al., 1995; Sternfeld et al., 1998) display distinct character-
istics. Mice overexpressing AChES show accelerated stressrelated pathology (Sternfeld et al., 2000)
including loss of dendritic arborizations and spines, leading to progressive deterioration (Beeri et al.,
1997), neuromotor malfunctions (Andres et al., 1997), hypersensitivity to anticholinesterases (Shapira
et al., 2000), and to closedhead injury (Shohami et al., 2000), and vulnerability to a switched dayandnight
cycle (Cohen et al., 2002). Intriguingly, transgenic mice expressing human AChES, which display changes
in the balance of their cholinergic neurotransmission (Erb et al., 2001) possess high levels of HSP70
(Sternfeld et al., 2000) associated with neuronal splicing abnormalities, reflected in high levels of their
murine AChER mRNA variant (Cohen et al., 2002). Further research will be required to explore the
possibility of an involvement of cholinergic signaling pathways in the maintenance of neuronal mRNA
splicing. AChERexpressing mice display normal neuromuscular function and their brains are relatively
protected from the stressassociated hallmarks of pathology, which predict agedependent neurodeteriora-
tion (Sternfeld et al., 2000). They further display intensified LTP, parallel to the stressinduced LTP
enhancement that associates with AChER mRNA accumulation in the hippocampus of nontransgenic
FVB/N mice (Nijholt et al., 2003). Although it is not yet clear whether this stressinduced alternative
splicing of AChE occurs as a cause or an effect, this phenotype may predict physiological relevance for the
exclusive overexpression of AChER following stress in the consolidation of fearful memories. Apart from
this apparent protective function, AChER may mediate at least some of the adverse cellular changes
associated with delayed stress responses. Intriguingly, AChER mRNA was found in apical dendrites of
neurons from all cortical layers following exposure to cholinesterase inhibitors (Kaufer et al., 1998),
suggesting possible local regulation and translation of AChE in the synapse (Steward and Schuman,
2001) also in noncholinergic neurons.

7.2 AChE in Multitissue Stress Responses

The apparent link between stress and AChE was investigated in a variety of other tissues and systems. AChE
mRNA overexpression was observed in the mouse intestinal epithelium and the intestinal gland 2 h
following intraperitoneal (i.p.) injection of the antiAChE diisopropylfluorophosphonate (DFP) (Shapira
 mRNA modulations in stress and aging
9 233

et al., 2000). In addition, repeated forced swim stress induced AChE mRNA overproduction in spermatozoa
during early spermatogenesis and elevated AChE protein levels at later stages of sperm formation (Mor
et al., 2001). In mice subjected to forced swim stress, serum AChE was subject to Cterminal proteolysis
whereas human CD34 hematopoietic progenitor cells presented cortisolinduced AChE mRNA synthesis
(Grisaru et al., 2001). Also in humans, increased levels of AChE were reported in the cerebrospinal fluid
(CSF) of hospitalized patients, but not in agematched controls. These increases were associated with blood
brain barrier (BBB) disruption, increased CSF albumin levels, and elevated serum cortisol values (Tomkins
et al., 2001). Interestingly, all the abovementioned instances of AChE overexpression involve elevations of a
single isoform of this enzyme, AChER, produced by 30 alternative splicing of the AChE premRNA.
The stressinduced changes in the alternative splicing of AChE premRNA and the overproduction of
AChER were shown to be beneficial under acute stress responses, when AChER tunes down the stress
induced hyperarousal (Kaufer and Soreq, 1999). However, prolonged stress responses likely present a
different case. Numerous physiological functions are adversely suppressed under prolonged stress
responses, with critical consequences for the affected organism sometimes. Likewise, transgenic animal
studies (Cohen et al., 2002; Birikh et al., 2003), animal disease models (Shohami et al., 2000; Brenner et al.,
2003), and human clinical trials with an antisense agent destroying AChER mRNA (Argov et al., 2005)
suggest that prolonged overproduction of AChER may take its toll.

8 AChEs 30 Alternative Splicing under Stress: LongTerm Implications

The major product of AChEs 30 alternative splicing, AChES, was found to be abundantly expressed in
numerous tissues and developmental states. As the main variant responsible for terminating cholinergic
transmission, AChES forms tetramers through its unique Cterminus, allowing increased density of the
anchored enzyme in the synaptic cleft (25003000 moleculesmm2, (Anglister et al., 1994). This involves
interaction with structural proteins, such as ColQ (Krejci et al., 1997) or PRiMA (Perrier et al., 2002).
AChER expression, on the other hand, was found to increase during brain development, under poststress
situations (Soreq and Seidman, 2001; Massoulie, 2002), and in glial tumors (Perry et al., 2002). The
overexpression of AChER following stress could possibly be beneficial for shortterm responses, ridding
of excess ACh secreted now at higher rates. However, the longterm excess of this protein is likely to be
causally involved in the longterm hypersensitization associated with prolonged stress responses (Meshorer
et al., 2002). AChE is also modified in AD, shifting its protein product from tetramers to monomers (Talesa,
2001), and AD patients treated with anticholinesterases display increased levels of AChER in their CSF
(DarrehShori et al., 2004).
Studies in our laboratory demonstrated rapid, yet longlasting stressinduced shift from neuronal
AChES to AChER mRNA transcripts, which was associated with the accumulation of their protein
products in neurites, diverting the composition of neuritic AChE from the Cterminally adhered synaptic
membrane isoform to AChER, the variant equipped with a hydrophilic nonadhered Cterminus (Meshorer
et al., 2002). This shift was common to different neurons and stressors, suggesting that stimulusinduced
changes in alternative splicing and dendritic mRNA translocationboth considered major contributors to
neuronal plasticityare intimately interrelated for AChE. In vivo, such changes were associated with long
term cholinergic hypersensitivity in both nonstressed controls and transgenics overproducing human
AChES and murine AChER (Meshorer et al., 2002), indicating that they may contribute toward the
longknown neuronal hypersensitivity that follows stressful experiences (Antelman et al., 1980) for which
there was no mechanistic explanation.
Several steps along the pathway for gene expression may contribute toward the neuritic AChER
overexpression under stressful insults. First, elevated glucocorticoid levels activate transcription through
GRE interactions (Sapolsky et al., 2000). That corticosterone induces AChE overproduction in cultured
PC12 cells and primary cerebellar neurons suggests their participation in the initiation of neuronal AChER
mRNA overexpression.
Second, stressinduced alternative splicing [e.g., through Nova, Buckanovich et al. (1993)] might
regulate the production of AChER by binding to the Nova short consensus intronic motif, UCAUY (Jensen
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9 mRNA modulations in stress and aging

et al., 2000), whereas the brainenriched polypyrimidine tractbinding protein (brPTB) can antagonize this
Novagenerated alternative splicing (Polydorides et al., 2000). AChE premRNA possesses both the UCAUY
motif and a possible polypyrimidine tract in conserved 30 intronic locations of both the human (positions
7,2427,246 and 7,2207,245 respectively in GeneBank accession number AF002993) (> Figure 9-8) and
mouse ACHE genes (positions 13,24213,246 and 13,21013,244, AF312033), suggesting their possible
relevance to this process.

. Figure 9-8
Novarelated sequences on AChE premRNA. Two Nova recognition sites (UCAUY) and a possible polypyrimi-
dine (Py) tract, which can potentially be recognized by the brainenriched polypyrimidine tractbinding protein
(brPTB), which antagonizes Novainduced alternative splicing, are shown. Note that among the variants of
AChE, these elements are absent in AChES mRNA

Third, stressinduced AChER mRNA accumulation is accompanied by neuritic translocation and most
likely, local AChER synthesis. Translocation into neuronal processes presumably depends on cisacting
elements, primarily within the 30 untranslated region (Wallace et al., 1998). Dendritic targeting sequences,
however, do not share common consensus motifs (Tiedge et al., 1999) and the 30 UTR of AChER mRNA
includes none of the known dendritic targeting sequences. Rather, RNA secondary structures were
proposed to direct targeting (Ainger et al., 1997; Blichenberg et al., 1999). The differential regulation of
AChER mRNA targeting into dendrites may thus be associated with its unique 30 sequence and secondary
structure. Alternatively, or additionally, AChER mRNA translocation may reflect the stressinduced
accumulation of many more nascent AChER mRNA transcripts. This is compatible with the intense
nuclear labeling of AChER mRNA, reflecting a higher premRNA:mRNA ratio for this transcript.
Fourth, neuritic stabilization of AChER mRNA may be involved. The normally short halflife charac-
teristic of AChER mRNA (Chan et al., 1998) is reminiscent of that of cytokines, where an AUUUA motif
or a longer Urich element in the 30 UTR is responsible for mRNA destabilization through the binding
of transacting proteins [reviewed by (Guhaniyogi and Brewer, 2001)]. A candidate Urich element
(UUUAUUUUUUUUUU) is present in the 30 UTR of mouse AChER mRNA (positions 13,02013,007
in GeneBank accession number AF312033) but not in AChES mRNA; however, its importance to the
stability of these transcripts remains to be tested.
Neuritic AChER mRNA is further resistant to antisense oligonucleotidecatalyzed degradation
(Meshorer et al., 2002). This further supports the notion of its sitedependent stabilization, and is probably
due to the low nuclease levels (especially RNase H, which targets DNAmRNA hybrids) in neurites. The
subcellular distribution of RNase H in neurons is not yet known, but tagged overexpressed chimeric RNase
H displayed in the protozoa Crithidia fasciculata a gradient pattern, from high levels in the nucleus to low
levels in the cytoplasm (Engel et al., 2001), which may extrapolate to low RNase H levels in neurites.
Cholinergicglutamatergic interactions were demonstrated in several brain regions (Giovannini et al.,
1994) to affect CNS functions such as LTP, memory, and behavior (Aigner, 1995), functions that show
hyperexcitation following stress. Electrophysiological recordings in the hippocampal CA1 region (Meshorer
et al., 2002) demonstrated weekslong hypersensitivity of prestressed mice to anticholinesterases, attribut-
ing a role for these changes in the cholinergic impact over poststress sensitization (Bell et al., 1994).
 mRNA modulations in stress and aging
9 235

Stressinduced neuronal hypersensitivity was shown to occur through intensification of cholinergic

glutamatergic interactions (Meshorer et al., 2002), and intensified LTP was found in AChER overexpres-
sing brain (Nijholt et al., 2004). This sheds new light on the wellknown phenomenon of sensitization
following stress (Orsini et al., 2001). The glutamatergic nature of the hypersensitized response may be
relevant to the additional functions attributed to neuritic AChER, especially noncatalytic capacities to
compete with and mediate cellcell and cellmatrix interactions, based on AChEs electrotactin properties
(Botti et al., 1998). Neuroligin 1, for example, is a postsynaptic cell adhesion molecule of excitatory synapses
(Song et al., 1999), which includes an extracellular catalytically inactive AChEhomologous domain. When
overexpressed, AChER shares the neuritogenic properties of neuroligin 1 (Grifman et al., 1998), possibly
through interaction with its partner protein neurexin b2 (Nguyen and Sudhof, 1997). However, the
distinct Ca2binding domains in AChE and neuroligin (Tsigelny et al., 2000) predict differences in their
adherence properties, suggesting that excess AChER may modify glutamatergic functions by impairing
neurexinneuroligin interactions.
The experiments given here suggest that stressinduced increases in ACh and neuronal sensitization are
tightly linked with AChE splice variations, implying a novel mechanism for the stressinduced neuronal
hypersensitivity (Meshorer et al., 2002). This involves neuritic replacement of the membranebound AChE
S protein, an indispensable enzyme continuously needed to maintain cholinergic neurotransmission, with
its Cterminally hydrophilic and relatively unstable counterpart, AChER. At the short term, the increased
diameter allowing local AChER production and release would dampen more inhibitor molecules than in
the nonstressed brain, leading to an initial phase of apparent protection (Kaufer et al., 1998). However,
AChER overproducing neurites may fail to promote local AChES synthesis under repeated stimulus due
to limited translational capacity and the shifted AChER:AChES mRNA ratio. Neurites of a previously
stressed or exposed organism would therefore produce Cterminally hydrophilic AChER monomers, rather
than possess Cterminally membraneadhered AChE multimers, impairing the capacity of dendritic
synapses to respond to repeated cholinergic stimuli.
AChER properties are likely to be, at least partially, speciesspecific. Nevertheless, when expressed in
transgenic mice, human AChER acts as a crossspecies protective agent, preventing the accumulation of
pathological stress hallmarks (Sternfeld et al., 2000). In contrast, transgenics expressing human AChES
respond by overexpressing endogenous murine AChER and display progressive dendrite and spine loss
(Beeri et al., 1997), compensatory increases in hippocampal ACh release (Erb et al., 2001), and anticholin-
esterase hypersensitivity (Shapira et al., 2000). Their antisensesuppressible vulnerability to closedhead
injury (Shohami et al., 2000) and working memory failure (Cohen et al., 2002) further support the notion
that AChER is causally involved with the failure to address cholinergic stimuli. Altogether, this may
indicate that the stimulusinduced choice between the splicing decisions, which lead to production of
these two AChE isoforms, is at fault.

9 Conclusions

The realm of premRNA splicing modulations in aging, disease, and stress is now beginning to emerge.
Although accumulating evidence creates a clear association between splicing aberrations and disease states,
the biochemical, cellular, and molecular pathways are far from being understood. Harnessing the use of
highthroughput techniques, such as splicingspecific microarrays (Clark et al., 2002) and fiberoptic arrays
(Yeakley et al., 2002) over the coming years promises to bring about novel and exciting examples to add to
this growing list, and to shed new light on the intracellular pathways that generate alternatively spliced
products during cellular crisis. Studies aimed at the production of abnormal transcripts will pave the way
for novel RNAtargeted therapies that are already being tested in various disease models. These include
antisense oligonucleotides, ribozymes, RNAi, or spliceosomemediated RNA transsplicing (SMaRT)
(Stoilov et al., 2002). The use of such technologies will hopefully enable delicate manipulations of gene
expression in disease and aging, suppressing the alternatively spliced culprits, while maintaining accurate
levels of the normal proteins.
236
9 mRNA modulations in stress and aging
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10 Molecular Mechanisms of
Dendritic Spine Plasticity in
Development and Aging
M. R. Kreutz . I. Konig . M. Mikhaylova . C. Spilker . W. Zuschratter

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

2 The Structure of Dendritic Spine Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

3 The Postsynaptic Density of the Spinous Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

4 Synaptogenesis and the Formation of Dendritic Spine Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

5 Molecular Dynamics of the Plastic Spine or What are Spines Good for . . . . . . . . . . . . . . . . . . . . . . . . . 251

6 Molecular Mechanisms of Spine Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

7 Structural Alterations of Spine Number and Formation in Brain Disease States . . . . . . . . . . . . . . . 254

8 The Dendritic Spine in the Aging Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

# 2008 Springer ScienceBusiness Media, LLC.

246 10 Molecular mechanisms of dendritic spine plasticity in development and aging

Abstract: The postsynaptic density (PSD) of spinous excitatory synapses is characterized by an electron
dense filamentous meshwork of cytoskeletal proteins that serve three major functions: (1) the organization
of glutamate receptors, (2) the clustering of synaptic adhesion molecules, and (3) the coupling of synaptic
membrane proteins to intracellular signaling cascades. Consequently, in recent years a plethora of protein
protein interactions of PSD scaffolding proteins has been mapped and their possible function for the
formation and integrity of the synapse delineated. It is assumed that structural alterations in the PSD
precede morphological alterations of the spine. Detailed studies of such processes are of particular
significance because they will probably lead to a new conceptual framework in our appreciation of how
changes in the protein composition and proteinprotein interactions in the PSD can regulate spine
morphogenesis. This should provide novel answers to the longstanding questionwhat molecular
mechanisms underlie synaptic plasticity and in turn learning and memory processes?
List of Abbreviations: AKAP79, Akinase anchor protein 79; CaMKII, calcium/calmodulindependent
protein kinase II; CNS, central nervous system; GAP, GTPaseactivating protein; GEF, guanine nucleotide
exchange factors; LTD, longterm depression; LTP, longterm potentiation; NCAM, neural cell adhesion
molecule; PAK, p21activated kinase; PSD, postsynaptic density; SER, smooth endoplasmic reticulum;
SynCAM, synaptic cell adhesion molecule

1 Introduction

Synapses are sites of a specialized cellcell contact between neuronal cells and represent the major structure
involved in chemical neurotransmission in the nervous system. In higher mammals, the majority of brain
excitatory synapses using the neurotransmitter glutamate is found on the principal neuron of the cortex
and hippocampus, the pyramidal cell. Pyramidal cells are characterized by a complex dendritic cytoarch-
itecture harboring approximately 104105 synaptic contact sites with other neurons. It is estimated that only
1% of all synaptic contacts of cortical pyramids is concerned with the wiring to subcortical areas
(Creutzfeldt, 1995), implying that the predominant synapse of the mammalian telencephalon is concerned
with input from a closely related neuron in terms of cell lineage, morphology, and functional character-
istics. This fact is mainly emphasized because our knowledge about synaptic plasticity is to a large degree
based on this type of synaptic input.
Within the central nervous system (CNS), glutamatergic synapses are the major means by which excitatory
signals are conveyed from one neuron to another. As such, glutamatergic synapses are crucially important for
CNS function. Furthermore, it is widely believed that glutamatergic synapses are important loci for modifying
the functional properties of CNS networks, possibly providing the basis for phenomena collectively referred to
as learning and memory. Given their importance, it is not surprising that enormous efforts are being made to
understand the formation, structure, function, and regulation of glutamatergic synapses. To date, significant
progress has been made in our understanding of their ultrastructure, molecular composition, and physiological
properties, as well as the principles of how these synapses are initially assembled.
The term synaptic plasticity covers many different aspects of usedependent synaptic modifications.
Synaptic plasticity is therefore used in this chapter in a broader sense covering aspects of synaptic signal
transmission as well as structural alterations in the molecular makeup of the synapse related to synaptic
signaling events. The scope of this chapter is therefore (1) to introduce to current concepts on the molecular
mechanisms in the formation of spine synapses, (2) to survey current concepts on activitydependent
remodeling of the synaptic cytoskeleton, and (3) to summarize the present knowledge about changes in
spine synapses in brain disease states and during aging. Given that the topic is rather broad, extensive
reference to other recent reviews that cover certain aspects in more detail is given.

2 The Structure of Dendritic Spine Synapses

Spines are dendritic membrane protrusions that build synaptic contacts to the presynaptic membrane
compartment. The spine density of a typical pyramidal neuron varies between 4 and 10 spines/10 mm
dendritic length. As outlined above, gross calculations suggest that an average principal neuron harbors up
 Molecular mechanisms of dendritic spine plasticity in development and aging 10 247

to 10,000 synaptic contacts. > Figure 10-1 depicts a reconstruction of such a pyramidal neuron with its
spine protrusions in two dimensions to give an impression of the morphological complexity that is imposed
by the existence of spinous synaptic contacts.

. Figure 10-1
A Lucifer yellowfilled neuron from the cortex of a gerbil (Meriones unguiculatus) shows numerous dendritic spines
along its dendric arborization. Note the variable shape and size of the dendritic spines in the inset. Imaging of
the neuron and dendrite was done by a confocal laser scanning microscope, with which approximately 80 focal
planes were taken from the specimen, and subsequently volume rendered by a 3D imaging program

Spines have different shapes and their threedimensional structure varies substantially. Mature spines
are typically 0.52 mm in length but can also reach up to 56 mm. Accordingly, the spine volume ranges from
less than 0.01 mm3 to 0.8 mm3. Large spines contain mitochondria and also smooth endoplasmic reticulum
(SER), an internal store of Ca2 (> Figure 10-2). Many attempts have been made in the past to assign spines
into discrete groups. At present, spine synapses are classified commonly into one of the following: filopodia
like protrusions, thin, stubby, mushroomshaped, or cupshaped spines (Hering and Sheng, 2001) (see
> Figure 10-3). This classification scheme, however, should be treated with some caution. Spines can

change their morphology rather rapidly (see also below) and in many cases it is unclear whether certain
spine types exist only as a developmental transition state. Moreover, not all spines can be clearly assigned to
one group, and it is also unclear whether this classification reflects differences in spine function. Accord-
ingly, recent measurements of spine dimension do support the existence of distinct spine categories
(Wallace and Bear, 2004). Despite these limitations, the obvious question that arises is how the different
shape and the molecular dynamics of dendritic spines are brought about.

3 The Postsynaptic Density of the Spinous Synapse

An important role for the threedimensional organization of the spine synapse is played by an electron
dense structure beneath the postsynaptic membrane termed postsynaptic density (PSD) (> Figure 10-3).
The PSD of the spinous synapse consists of a specialized cytoskeleton, which is crucially involved in the
248 10 Molecular mechanisms of dendritic spine plasticity in development and aging

. Figure 10-2
Dendritic spine and shaft synapse from human hippocampus. Left: A dendritic spine (S) forms an asymmetric
contact with a presynaptic terminal (Pre), while another synaptic bouton contacts the dendritic shaft (D). Note,
both synaptic terminals are filled with numerous clear synaptic vesicles. In addition, the spine synapse contains
some mitochondria (Mi). Scale bar: 0.5 mm. Right: A dendritic shaft synapse, which contains a huge number of
clear synaptic vesicles and mitochondria (Mi), forms multiple contacts with a dendrite (D). Arrowheads indicate
postsynaptic contact zones. Scale bar: 0.5 mm

. Figure 10-3
Different morphologies of dendritic spines depending on type of neurons, developmental stage, and activity
status. From left to right: filopodium, thin, stubby, mushroomshaped, and cupshaped spines

organization of the neurotransmitter receptive apparatus and the adhesion of the postsynapse to presynap-
tic terminals (Sheng, 2001) (see > Figure 10-4). Furthermore, this structure contains specialized and
elaborate molecular scaffolds that link synaptic neurotransmission to various signaling cascades and the
cytoskeleton (McGee and Bredt, 2003; Kennedy et al., 2005). This is of particular importance since the spine
head is highly enriched in actin filaments (Matus et al., 1982), which mediate spine shape changes and
motility (Fischer et al., 1998). Fluoresancerecovery after photobleaching (FRAP) experiments of EGFP
actin in dendritic spines indicate that 85% of the actin in dendritic spines is dynamic, with an average cycle
 Molecular mechanisms of dendritic spine plasticity in development and aging 10 249

. Figure 10-4
Molecular structure of the postsynaptic density (PSD) of excitatory synapses. Since excitatory synapses contain
thousands of proteins that form a complex, highly ordered functional meshwork, only a subset of molecules
and protein interactions are shown, focusing on the PSD. Among the key components of the PSD are on one
hand proteins of the MAGUK familylike SAP90/PSD95, Chapsyn110/PSD93, SAP97, and SAP102, which form
complexes with NMDA and AMPAtype glutamate receptors, kainate receptors, cell adhesion proteins like
neuroligin, or signaling proteins like AKAP79 (Akinase anchor protein 79) and kalirin7. AKAP79 is able to
target protein kinase A (not shown) to specific substrates in the PSD. Kalirin7 is a GDP/GTP exchange factor
(GEF) for the small GTPase Rac1, which belongs to the Rho subfamily of small GTPases. Additional signaling
proteins illustrated here are calcium/calmodulindependent protein kinase II (CaMKII), which can interact with
NMDA receptors, and caldendrin, which was shown to bind to Ltype calcium channels. On the other hand,
proteins of the ProSAP/Shank family are important scaffolding molecules that connect neurotransmitter
receptors in the postsynaptic membrane via direct or indirect protein interactions with the actin cytoskeleton
and are therefore believed to play a crucial role in the functional organization of the PSD during synaptogen-
esis, synaptic plasticity, and also in spine morphology. Among the proteins that interact directly with ProSAPs/
Shanks are, for example, Homer proteins, which provide a connection to metabotropic glutamate receptors
(mGluR1a) in the postsynaptic membrane and to inositol1,4,5trisphosphate receptors (InsP3R) in the mem-
brane of the SER. Cortactin and afodrin both connect ProSAPs/Shanks directly to the actin cytoskeleton. GKAP/
SAPAPs provide the link between MAGUK proteins and ProSAPs/Shanks. Recently, it was shown that ProSAPiP,
a novel ProSAPinteracting protein, binds to SPAR (spineassociated Rap GTPaseactivating protein). This
interaction provides an additional link for ProSAPs/Shanks to the actin cytoskeleton via SPAR and also to
MAGUKs, since SPAR was shown to be directly associated with PSD95 or PSD93. Additionally, it is assumed
that SPAR can bind to cellsurface proteins like ephrins or neuroligins
250 10 Molecular mechanisms of dendritic spine plasticity in development and aging

time of 44 s (Star et al. 2002). The actin cytoskeleton is linked to the PSD via a number of protein
interactions including the actinbinding proteins, cortactin and afodrin, and it is thought that this
interface will be responsible for the molecular dynamics of the spine (Oertner and Matus, 2005). At present
it is not yet entirely clear which regulatory events within the PSD control these events (but see also below).
Molecular and ultrastructural analysis of the PSD has lead to the identification of several hundred PSD
constituents (see Li et al., 2004) and to the notion that the PSD is a multilayered complex, composed of
membrane molecules (such as glutamate receptors), primary scaffolding molecules (such as SAP90/PSD95),
and higherorder scaffolding molecules (such as ProSAPs/Shanks) that bind subcomplexes and cytoskeletal
components into even more elaborate structures (> Figure 10-4). These scaffolding molecules harbor a
variety of domains for proteinprotein interactions, including ankyrin repeats, a GUK domain, a SH3
domain, a PDZ domain, a SAM domain, and a prolinerich region (> Figure 10-4). Accordingly, a large
number of proteinprotein interactions have been ascribed to these domains supporting the view that their
multidomain structure underlies their supposed function as scaffolders of the PSD (see > Figure 10-4 for
more details). Part of this function is to integrate signaling molecules like kinases or GTPases into the
complex network of the synaptic cytoskeleton. Such signaling components will eventually connect synaptic
activation to its downstream effectors and can also feedback to the structural organization of the PSD and
the morphology of the spine.

4 Synaptogenesis and the Formation of Dendritic Spine Synapses

Much progress has been made in recent years in understanding the mechanisms that underlie synaptogen-
esis of excitatory brain synapses (for reviews see Garner et al., 2002; Ziv and Garner, 2004). With the
identification of the protein components of the synapse, their functional characterization with molecular
and genetic approaches, and with recent advances in optical imaging of living neurons it has become
increasingly clear that synapse assembly is a multistep process. In most cases, the development of dendritic
spines occurs concurrently with the growth of the presynaptic terminals, suggesting that cellcell interac-
tions and extrinsic cues likely induce the formation of dendritic spines. Thus, spines and filopodia actively
contact presynaptic afferents (Ziv and Smith, 1996) and can subsequently induce the formation of the
releasecompetent presynaptic membrane, but also the opposite, i.e., ingrowing axons initiating
the emergence of dendritic protrusions, which has been also described (Jontes et al., 2000). Moreover, it
was also suggested that dendritic filopodia participate in the formation of shaft synapses by recruitment of
the presynaptic contact to the dendrite (Sorra and Harris, 2000). Unexpectedly, it was shown that
synaptogenesis in brain as well as in culture can proceed in the complete absence of synaptic transmission
(Verhage et al., 2000; Varoqueaux et al., 2002). This implies that synaptic activity is not required for the
establishment of synaptic contacts. In any case, however, the question remains how the initial contact site
between the axon and the dendrite is specified.
The molecular mechanisms of synapse formation in the brain are not yet completely understood. For
the formation of the presynaptic membrane, synaptic precursor vesicles have been described in the axon
that are 80 nm in diameter and characterized by a dense core appearance. They contain the large presynaptic
multidomain proteins Piccolo and Bassoon, which participate in the formation of the cytoskeletal matrix at
the active zone of mature synapses (Ziv and Garner, 2004). Additionally, other core components of the
active zone are present on these transport vesicles indicating that larger parts of the presynapse will already
arrive as one complex at the initial site of synaptic contact. Interestingly, no corresponding precursor
transport vesicles have been found for the postsynapse (Bresler et al., 2004). Whether a particular molecule
determines the precise site on a dendrite where an axon will form a synapse remains also a largely
unresolved issue. An important role, however, seems to be played by the cell recognition molecules
neurexins and neuroligins (Dean et al., 2003). The most convincing evidence for an instructive role of
the postsynaptic compartment comes from the work of Scheiffele and coworkers (2000), who showed that
expression of the postsynaptic cell adhesion molecule neuroligin in nonneuronal cells is sufficient to trigger
the development of presynaptic structures in contacting axons. An induction of synapse formation after
 Molecular mechanisms of dendritic spine plasticity in development and aging 10 251

overexpression of neuroligin by activation of its binding partner neurexin was also found in cultured
hippocampal neurons (Dean et al., 2003).
Many other cell recognition molecules accumulate at synapses during development, which indirectly
suggests that they also might be involved in synaptogenesis or synapse stabilization through their adhesive
properties. These molecules include Ncadherin, which is initially associated with all types of synapses in
cell culture but then becomes restricted to excitatory synapses (Benson and Tanaka, 1998), and members of
the immunoglobulin superfamily, such as synaptic cell adhesion molecule (SynCAM) (Biederer et al.,
2002), nectins (Mizoguchi et al., 2002), and neural cell adhesion molecule (NCAM) (Sytnyk et al., 2002).
Like neuroligin, overexpression of SynCAM in nonneuronal cells induces formation of synapses on the
transfected cells by axons of cocultured neurons (Biederer et al., 2002), whereas inhibition of nectinbased
adhesion results in a decrease of synapse size and a concomitant increase in synapse number (Mizoguchi
et al., 2002). Finally, the ephrinB and EphB tyrosine kinase receptor system has also been implicated in the
development of excitatory synapses through phosphorylation of the cellsurfaceexposed heparin sulphate
proteoglycan syndecan (Ethell et al., 2001) or interaction with the NMDA receptor (Dalva et al., 2000).
These findings suggest that EphB receptors participate not only during the initial formation of synapses but
also in their maturation.

5 Molecular Dynamics of the Plastic Spine or What are Spines Good for

Computer modeling as well as experimental evidence supports the idea that the dendritic spine serves as a
biochemical microcompartment. Ca2 transients can be restricted to single spines, thus isolating the effect
of activation of specific synapses (Yuste et al., 2000; Sabatini et al., 2002). Under physiological conditions,
Ca2 diffusion across the spine neck is negligible, and the spine head functions as a separate compartment
on long timescales, allowing localized Ca2 buildup during trains of synaptic stimuli (Sabatini et al., 2002).
Along these lines it has been proposed by many authors that morphology of synapses and dendritic spines
affects postsynaptic integration of signals, for example, allowing neurons to compensate for the attenuation
of signals from synapses distant from the soma (reviewed by Hausser, 2001). It was also reported that thick
spines (mushroom type) whose head is fully developed remain mostly stable (reviewed by Hering and
Sheng, 2001). Therefore, it was proposed that memory in the cerebral neuronal circuit is possibly stored in
the spine morphology (Oertner and Matus, 2005) and that the spine separation is a specialization for
regulating anatomical plasticity.
A stunning observation made with the invention of novel in vivo imaging techniques in the last decade
is, however, that the structure of dendritic spines is plastic, in the sense that spines can take various shapes
both during neuronal activity and at rest. Spine movements based on alterations in the cytoskeleton have
been postulated on theoretical grounds first by Crick (1982). This hypothesis was based on the idea that
spine structurestabilityfunction relationships are well suited for a molecular account of usedependent
modifications in the brain largely referred to as learning and memory. The first observation that spines are
very mobile structures that seem to constantly change their shape and size at least in culture was made by
the pioneering work of Andrew Matus and coworkers (Fischer et al., 1998, 2000). Furthermore, de novo
spine formation was shown to occur after induction of hippocampal longterm potentiation (LTP) (Engert
and Bonhoeffer, 1999; Toni et al., 1999; but see also Fiala et al., 2002a for contradictory results).
More recent work showed that synapse formation and elimination occur throughout life, although the
magnitude of such changes at distinct developmental stages seems to be quite different in vivo. Interestingly,
thin spines appear and disappear over a few days, while most thick spines persist for months (Grutzendler
et al., 2002; Holtmaat et al., 2005; Zuo et al., 2005). Utilizing transcranial twophoton microscopy, it was
shown that during postnatal week two to four spine retractions exceed additions, resulting in a net loss of
spines (Grutzendler et al., 2002). The fraction of persistent spines grows gradually during development and
into adulthood providing evidence that synaptic circuits continue to stabilize even in the adult brain, long
after the closure of known critical periods (Grutzendler et al., 2002; Holtmaat et al., 2005). Moreover,
filopodialike dendritic protrusions, extending and retracting over hours, are abundant in young animals
252 10 Molecular mechanisms of dendritic spine plasticity in development and aging

but virtually absent in the adults (Trachtenberg et al., 2002; Holtmaat et al., 2005; Zuo et al., 2005)
(> Figure 10-5). In summary, the published data suggest that spines, initially plastic during development,
become remarkably stable in the adult with the majority of them lasting throughout life, and providing a
potential structural basis for longterm information storage. This, however, does not seem to preclude
synapse formation in the adult cortex in the context of experiencedependent plasticity. Thus, experience
dependent plasticity of cortical receptive fields was reported to be accompanied by increased synapse
turnover (Trachtenberg et al., 2002).

. Figure 10-5
Time lapse 3D imaging of a GFPtransfected hippocampal neuron grown in vitro for 10 days. The 3D image
sequence shows ejecting and retracting filopodia as well as highly motile spines along a dendrite during an
observation period of approximately 2 h
 Molecular mechanisms of dendritic spine plasticity in development and aging 10 253

6 Molecular Mechanisms of Spine Plasticity

To this end, it is largely unknown how synaptic signaling is transferred to the underlying alterations in the
spinous actin cytoskeleton and how altered spine morphology may feedback onto the efficacy of neuro-
transmission. It is assumed that structural alterations in the PSD precede the morphological alterations and
some important mechanisms have been investigated. To this end, it was shown that activitydependent
remodeling of the PSD is accompanied by altered protein turnover, which is remarkably rapid and robust
(Ehlers, 2003). In turn, this remodeling occurs with corresponding increases or decreases in ubiquitin
conjugation of synaptic proteins and requires proteasomemediated degradation (Colledge et al., 2003;
Ehlers, 2003). Functionally, these modifications alter synaptic signaling to major downstream effectors (Pak
et al., 2001, Pak and Sheng, 2003). In addition, a longlasting modification of synaptic strength requires
protein synthesis (Kang and Schuman, 1996; Steward and Worley, 2002), suggesting that newly synthesized
proteins contribute to the modification of synaptic efficacy. The detection of polyribosomes beneath
synapses at the base of dendritic spines (Steward and Levy, 1982) indicates that protein synthesis may
occur locally in dendrites under the control of the synapse itself. This concept implies that mRNAs are
present in dendrites, and that dendritically synthesized proteins can mediate activitydependent structural
and functional modifications of the postsynaptic apparatus. Therefore, active protein synthesis at or in
spines has been proposed to function as a synaptic tag for transcriptiondependent memory formation
(Frey and Morris, 1997). A variety of experimental evidence has underscored the involvement of local
protein synthesis in paradigms of synaptic plasticity. Ostroff and coworkers (2002) could show that
polyribosomes redistribute from dendritic shafts to synapses during LTP in rat hippocampal slices. The
importance of dendritic protein translation for synaptic plasticity has been demonstrated for calcium/
calmodulindependent protein kinase IIa (CaMKIIa) (Miller et al., 2002).
Another important mechanism of synaptic plasticity is through movement of neurotransmitter recep-
tors and regulatory proteins to and from the synapse (Malinow and Malenka, 2002; Kasai et al., 2003).
Several activitytriggered biochemical signaling mechanisms control these movements and their outcome
can therefore be quite diverse (Sheng and Kim, 2002; Kennedy et al., 2005). Activation of glutamate
receptors can cause an increase or decrease of spine size depending upon the activation scheme. These
differing responses may be due to activation of diverse downstream molecules. For example, spine collapse
requires calcineurin activation (Halpain et al., 1998), whereas an increase in size, which is likely due to an
increased recruitment of AMPA receptors to the postsynaptic membrane, requires CaMKII (Jourdain et al.,
2003; Matsuzaki et al., 2004). Interestingly, this pattern is reminiscent of longterm depression (LTD) and
LTP regulation and their morphological correlates in the regulation of spine size (Matsuzaki et al., 2004;
Zhou et al., 2004). LTD has been shown to be accompanied by shrinkage of spine size while induction of
NMDA receptordependent LTP had the opposite effect (Matsuzaki et al., 2004; Zhou et al., 2004). The
preferential sites of LTP induction are small spines, which express a small number of AMPA receptors. In
accord, it was hypothesized that they might correspond to the postsynaptic structures of socalled silent
synapses (Matsuzaki et al., 2004). During LTPinduction, NMDA receptor activation might also engage
molecules that directly affect actin redistribution. For instance, profilin, an actinbinding protein, moves to
the spine head following NMDA receptor activation and suppresses actin dynamics (Ackermann and
Matus, 2003), which leads to spine stabilization. Another actinbinding protein, cortactin, shows a similar
movement to the dendrite after NMDA activation (Hering and Sheng, 2003). A knockdown of cortactin,
however, leads to a decrease in spine density, whereas overexpression results in an elongation of spines
(Hering and Sheng, 2003). In addition to its binding site for actin, cortactin also binds to ProSAPs/Shanks,
thereby providing a physical link between the NMDA receptor and the actin cytoskeleton that could
potentially control spine morphology (Naisbitt et al., 1999). Accordingly, the protein levels of ProSAPs/
Shanks at the synapse control spine shape (Naisbitt et al., 1999).
Other components of the PSD, like SAP90/PSD95, also indirectly link glutamate receptors to the actin
cytoskeleton. This is brought about by its interaction with the small GTPase Ras, through the GTPase
activating protein (GAP) SynGAP (Chen et al., 1998), and Rap, through another GAP, SPAR (Pak et al.,
2001). Other small GTPases, specifically those of the Rho family, play a role in the regulation of spine
254 10 Molecular mechanisms of dendritic spine plasticity in development and aging

motility and morphogenesis (Tashiro et al., 2000). They are activated by guanine nucleotide exchange
factors (GEFs). For one of these GEFs, kalirin7, it was shown that its overexpression can increase spine
density (Penzes et al., 2001), whereas reduced kalirin7 levels are accompanied by shortened dendrites and
significantly lower numbers of dendritic spines (Ma et al., 2003). The putative downstream effectors of
small GTPases that control spine morphology include the p21activated kinase (PAK). Phosphorylated PAK
colocalizes with SAP90/PSD95 in dendritic spines, and transgenic mice expressing a dominant negative
form of PAK show a decreased spine density, morphological spine abnormalities, as well as enhanced LTP
and reduced LTD (Hayashi et al., 2004). Moreover, recent studies show that the interaction of kalirin7 with
Eph tyrosine kinase receptor has important consequences for spine morphology and incidence. These
effects are most likely mediated by the downstream activation of PAK and small GTPases. Transsynaptic
binding of the ligand ephrinB to the EphB receptor leads to a recruitment of kalirin7 to the activated
spine. Kalirin7 subsequently activates Rac1 via its GEF activity, which then results in enhanced PAK activity
and actin remodeling (Penzes et al., 2003), leading to an increased spine density and size. In summary, these
studies are especially important because they will probably lead to a new conceptual framework in our
understanding of how changes in the protein composition of the PSD can regulate spine morphogenesis.
Further work will provide novel answers for the old questionwhich molecular mechanisms underlie
synaptic plasticity and thereby indirectly learning and memory processes?

7 Structural Alterations of Spine Number and Formation in Brain Disease

States

First accounts of altered spine morphology in brain diseases date back to the 1980s. Given the delicate balance
between the supply and breakdown of PSD molecules, it is likely that seemingly minor genetically or environ-
mentally related perturbations of postsynaptic maintenance mechanisms may eventually lead to synaptic
dysfunction, disintegration, and ultimately elimination, resulting in pathological synaptic loss and degenerative
conditions. Indeed, PSD and dendritic spine pathologies have been associated with a rapidly increasing number
of CNS diseases, ranging from neurodegenerative disease states to epilepsy and schizophrenia (> Figure 10-6).
For example, the loss of synaptic contacts is an early hallmark of both Alzheimer and Huntington disease
(Selkoe, 2002; Smith et al., 2005). The major hereditary mental retardation syndromes, fragile X and Down,
are accompanied by changes in spine morphology, in particular a decrease in mature dendritic spines with well
formed PSDs and an increase in filopodialike protrusions that lack PSDs (Kaufmann and Moser, 2000;
Irwin et al., 2000). The development of epileptic seizures is associated with rather drastic changes in the protein
composition of the PSD (Wyneken et al., 2001, 2003) and the loss of dendritic spines (Mizrahi et al., 2004),
whereas chronic administration of the psychostimulants cocaine and amphetamine produce increased
spine density in the nucleus accumbens (Robinson and Kolb, 1997, 1999). In schizophrenia, one of the
most prevalent psychiatric diseases, dysplastic events at the dendritic spine level not only are a common
observation but also are thought to be causally related to the onset of the disease (Costa et al., 2001;
Blanpied and Ehlers, 2004). More recently, downregulation of the expression of the synaptic scaffolding
protein SAP90/PSD95 has been causally linked to synaptic dysfunction in cocaineinduced drug addic-
tion (Yao et al., 2004). This list could be extended further, although a question arises whether such
alterations are really the cause or merely a consequence of a particular disease (Fiala et al., 2002b).

8 The Dendritic Spine in the Aging Brain

At present, our knowledge about alterations in dendritic spines of the aged brain is still sparse. Most reports
are concerned with the morphology and number of spines in the aged brain. But very little is known about
the underlying mechanisms of such alterations. Aging is often accompanied by cognitive impairments like
memory and spatiallearning deficits in senescent humans, nonhuman primates, and rodents. In contrast to
initial studies of neuronal densities, more recent studies based on stereological methods and counting of
 Molecular mechanisms of dendritic spine plasticity in development and aging 10 255

. Figure 10-6
Spine pathologies. (b) A reduction of the density of dendritic spines as compared to (a) the spines occur in cases of
deafferentation, most mental retardation syndromes, severe alcohol abuse, epilepsy, Alzheimers disease, and
others. (c) An increase in dendritic spine density was shown for some types of deafferentation syndromes, the
fragile X syndrome, sudden infant death syndrome, and the abuse of psychostimulants. (d) Sensory deprivation
leads to a reduction of spine size. The same spine phenotype is described for Downs syndrome and schizophrenia.
Morphological changes that result in a (e) distortion of spine shape are also typical for most mental retardation
symptoms, epilepsy, severe alcohol abuse, poisoning with neurotoxicants, and spongiform encephalitis

total neuron number of brain material of several species have established that only little, if any, loss of
excitatory and inhibitory neurons occurs in different brain regions in normal aging and that this loss cannot
account for the observed agerelated functional deficits in multiple neurotransmitter systems. Studies have
demonstrated subregional specificities in sensitivity to the effects of advancing age, and it has become
increasingly clear that the mechanisms underlying agerelated changes are of a more subtle nature and
sometimes compensatory rather than detrimental to cognitive functioning (Morrison and Hof, 1997).
Disruption and agerelated structural alterations of the myelin sheaths of neocortical axons have been
described and correlated with age and degree of impairment in monkeys (Peters et al, 2000). In both
rodents and humans, changes have been reported in dendritic arbor, spines, and synapse morphology
(Morrison and Hof, 1997). Spine number and density seem to decrease with age (Duan et al., 2003), and
dendritic length seems to decline in cortical neurons (Jacobs et al., 1997). Rather than changes in spine
number, reduction in size of perforated postsynaptic densities, whose formation is postulated to be a
structural correlate of enhanced efficacy of synaptic transmission, has been reported for hippocampal
axospinous synapses (Nicholson et al., 2004). Studies in the monkey revealed decreased synaptic excitation
and increased synaptic inhibition of layer 2 and 3 pyramidal cells in the prefrontal cortex (Luebke et al.,
2004). Thus, alterations first appear on the level of synaptic connectivity and further manifest themselves on
the subcellular and molecular level.
BertoniFreddari and coworkers (2003, 2004, 2005) recently described ageinduced changes in cellular
energy metabolism. They observed an increase in the number of enlarged synaptic mitochondria with
generally decreased metabolic competence in cerebellar cortex of adult rats, as well as an agerelated decline
256 10 Molecular mechanisms of dendritic spine plasticity in development and aging

in metabolic competence of small and mediumsized synaptic mitochondria. Age also affects Ca2
homeostasis and changes in regulation of intracellular Ca2 concentrations underlie some agerelated
deficits in plasticity and cognition (reviewed in Foster, 1999; Foster and Kumar, 2002; Rosenzweig and
Barnes, 2003; Toescu and Verkhratsky, 2004). A number of mechanisms for handling Ca2 are thought to be
modified during aging, including intracellular buffering, extrusion, and influx of Ca2. Consequently,
hippocampal synaptic function is altered as seen by a shift in synaptic plasticity thresholds with LTP
requiring a higher stimulation threshold for induction and more induction sessions to saturate
LTP mechanisms in aged animals, and LTD showing a reduction in threshold, such that robust LTD is
observed for lower frequency stimulation patterns in aged compared with young animals. Thus, aged
animals acquire new information only slowly and with more repetition and forget rapidly. In this context, it
is hypothesized that a postsynaptic increase in Ca2 influx through Ltype VDCCs and reduced Ca2 influx
through NMDARs with age not only modifies the threshold for induction of Ca2dependent synaptic
plasticity in favor of LTD induction but also increases the duration and amplitude of afterhypolarization
following cell discharge activity and with that alters information processing. The effects seem to be
mediated by agerelated changes in the Ca2signaling pathways, including the activity of protein phos-
phatases and kinases involved in the expression of synaptic plasticity. The Ca2 and CaMdependent
phosphatase calcineurin and protein phosphatase 1, which is regulated by calcineurin, are enzymes critical
for LTD and according to Foster and coworkers (2001), activity of both increases with advanced age in an
Lchannel related manner. They showed that the phosphorylation state of calcineurin substrates (e.g., of
CREB) was reduced and that increased calcineurin activity correlated with memory function. This finding
is challenged by a more recent study by Agbas and coworkers (2005) who showed that calcineurin activity
decreases in the aging brain due to increased aggregation of the catalytic subunit of calcineurin caused by
oxidation.
Other components of the synaptic transmission machinery have also been analyzed for agerelated
changes. Whereas several groups examined the expression of membrane channel subtypes and receptor
subunits in cortical and hippocampal neurons of different species and documented regional and subtype/
subunit and sometimes species specific alterations (Tanaka and Ando, 2001; Hof et al., 2002; Bai et al., 2004;
Iwamoto et al., 2004), levels of presynaptic proteins like synaptotagmin1, synaptophysin, and syntaxin in
rat cerebral cortex were not significantly reduced with age (Iwamoto et al., 2004). Interestingly, in animal
models some of the described agerelated changes and effects on cognitive function can be reversed either
directly or in a complementary way by the administration of trophic factors that otherwise decline with age,
like insulinlike growth factor1 and estrogen (Sonntag et al., 2000; Adams and Morrison, 2003; Adams
et al., 2004; Shi et al., 2005).

Acknowledgments

The work of M.R.K. is supported by the BMBF, DAAD, DFG, the Land SachsenAnhalt, and the Schram
Foundation.

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11 Alzheimers Disease
BACE Proteases
S. Roner . S. F. Lichtenthaler

1 Introduction to Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

2 The Discovery of BACE Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

3 BACE Gene Structure and Alternative Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

4 Transcription and Translation of BACE1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

5 Posttranslational Modifications, Intracellular Transport, and Binding Partners of BACE1 . . . 271

5.1 Posttranslational Modifications and Intracellular Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
5.2 BACE1 Interacting Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272

6 BACE1 Expression in Aging and AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

6.1 Localized BACE1 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
6.2 Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
6.3 APP Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
6.4 BACE1 Expression in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
6.5 Astrocytic BACE1 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

7 Biological Function and Substrate Spectrum of BACE1 and BACE2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 276

7.1 Substrates of BACE Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
7.2 BACE1 Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

# 2008 Springer ScienceBusiness Media, LLC.

262
11 Alzheimers disease BACE proteases

Abstract: The aberrant proteolytical processing of the amyloid precursor protein (APP) gives rise to
bamyloid peptides, which accumulate as perivascular or parenchymal deposits in brains of Alzheimers
disease (AD) patients. The generation of bamyloid peptides is initiated by APP cleavage by an aspartyl
protease named betasite APPcleaving enzyme 1 (BACE1), followed by gsecretase cleavage. The discovery
of BACE1 some years ago and subsequent studies on the regulation of its expression and enzymatic activity
allowed for a better understanding of APP processing, bamyloid generation and the pathogenesis of AD in
general. Some of the unique features of BACE1, such as regulatory elements in the 50 region of BACE1
mRNA and factors that contribute to its cell typespecific activation helped to explain epidemiological
observations and opened new rational therapeutic opportunities. However, although BACE1 has been
shown to be the only protease in brain with significant bsecretase activity, there are a number of alternative
substrates in addition to APP, indicating that a careful estimation of possible side effects of BACE1 inhibi-
tors is required. Nevertheless, experimental data from transgenic mouse models of AD, BACE1 knockout
mice and pharmacological studies corroborate the potential usefulness of drugs that interfere with BACE1
expression and/or enzymatic activity for the treatment of AD.
List of Abbreviations: Ab, bamyloid peptide; AD, Alzheimers disease; ADAM, a disintegrin and metal-
loprotease; APLP, amyloid precursorlike protein; APP, amyloid precursor protein; BACE, betasite APP
cleaving enzyme; CCS, copper chaperone for superoxide dismutase1; ChAT, choline acetyltransferase; DS,
Downs syndrome; ER, endoplasmatic reticulum; FAD, familial forms of Alzheimers disease; GFAP, glial
fibrillary acidic protein; GGA, Golgilocalized gearcontaining ARF binding; HEK, human embryonic
kidney; MDCK, MadinDarby canine kidney; NSAIDs, nonsteroidal antiinflammatory drugs; PLSCR 1,
phospholipid scramblase 1; PPARg, peroxisome proliferatoractivated receptorg; PPRE, PPARg responsive
element; pro, prodomain; RTN3, reticulon 3; RTN4b, reticulon 4b; TGN, transGolgi network; SP,
signal peptide; TACE, tumor necrosis factor aconverting enzyme; TMD, transmembrane domain;
50 UTR, 50 untranslated region; uORF, upstream open reading frame

1 Introduction to Alzheimers Disease

Alzheimers disease (AD) is the most prevalent neurodegenerative disease, affecting about 20 million people
worldwide [for an overview see Selkoe and Schenk (2003)]. It is characterized by progressive loss of
memory, declining cognitive function and, ultimately, leads to decreasing physical functions and death.
The neuropathological hallmarks of AD are the senile plaques and the neurofibrillary tangles, which are
protein aggregates deposited in the brain. The neurofibrillary tangles represent intraneuronal bundles of
paired helical filaments, which mainly consist of the microtubuleassociated protein tau in an abnormally
phosphorylated form [(for a review see Iqbal et al. (2005)]. The extracellular amyloid plaques mainly
consist of the 42 residue long amyloid bpeptide (Ab, bA4) (Glenner and Wong, 1984; Masters et al., 1985),
which is proteolytically derived from the much larger amyloid precursor protein (APP, bAPP) (Kang et al.,
1987). The generation and subsequent aggregation of Ab seem to be at the origin of the disease and are
believed to trigger a complex pathological cascade that causes neuronal dysfunction, the appearance of
the neurofibrillary tangles, inflammatory processes, neuronal loss, cognitive impairment, and finally the
onset of the disease. This pathological cascade, which is now widely accepted, is also referred to as the
amyloid hypothesis of AD [for overview see Hardy and Selkoe (2002), Citron (2004)].
Although drugs are currently available that may ameliorate latestage symptoms like the cognitive deficits
for a short time, no drugs are on the market that specifically target the cellular mechanisms of the disease,
namely the proteolytic generation of the Ab peptide from APP. APP is a type I membrane protein with unclear
biological function. Suggested functions for APP include a role in cell adhesion and copper homeostasis [for a
review see De Strooper and Annaert (2000)], cellular motility (Sabo et al., 2001) and being a cell surface
receptor (Cao and Sudhof, 2001) or a receptor for the motor protein kinesin (Kamal et al., 2001).
The proteolytic processing of APP involves protease activities that are referred to as a, b, and
gsecretase because their molecular identity was initially not known. The three proteases process APP in
two different pathways (> Figure 11-1a), one of which is called antiamyloidogenic because it prevents Ab
generation. The other one is termed amyloidogenic as it leads to the generation of the Ab peptide. Both
 Alzheimers disease BACE proteases
11 263

. Figure 11-1
Proteolytic processing pathways of APP. (a) Amyloidogenic pathway: Cleavage of APP by the protease
bsecretase (BACE1) occurs at the Nterminus of the Abdomain and yields the secreted sAPPb and a Cterminal
fragment of APP of 99 amino acids (C99). C99 is further cleaved within its transmembrane domain by
gsecretase, leading to the secretion of the Ab peptide and the generation of the APP intracellular domain
(AICD). The consecutive cleavage of APP by b and gsecretase constitutes the amyloidogenic pathway as it
generates Ab. Antiamyloidogenic pathway: Cleavage of APP by asecretase or potentially BACE2 within the Ab
domain yields the neurotrophic and neuroprotective sAPPa as well as the Cterminal fragment C83. The
asecretase is a member of the ADAM family of metalloproteases. Similar to the processing of C99, C83 is
further processed by gsecretase within its transmembrane domain, leading to the generation of AICD and to
the secretion of the p3 peptide that is not deposited in amyloid plaques. (b) Amino acid sequence of APP within
and around the Ab peptide domain. Indicated are the major cleavage sites of the different protease activities
and the double mutation directly preceding the bsecretase cleavage site. This mutation is responsible for a
familial form of AD found in a Swedish family. Amino acids are shown in the oneletter code and numbered
according to the Ab sequence

pathways and their cleavage products (including Ab) are part of the normal proteolytic processing of APP
[for an overview see Selkoe and Schenk (2003)], which occurs in most cells. In the amyloidogenic pathway,
APP is first cleaved by the bsecretase at the Nterminus of the Ab domain (> Figure 11-1a). This cleavage
generates the soluble sAPPb and a Cterminal fragment, which undergoes a second cleavage by a protease
called gsecretase. The bsecretase was recently identified as the membranebound aspartyl protease BACE1
(also named memapsin2, Asp2). gSecretase also belongs to the aspartyl family of proteases but is an
unusual protease activity in that it cleaves APP within its hydrophobic transmembrane domain. gSecretase
is a heterotetrameric protein complex consisting of presenilin, nicastrin, PEN2, and APH1 [for a review
see Haass (2004)]. gSecretase cleavage occurs mainly after residue 40 and to a lower extent after residue
42 of the Ab sequence, thereby generating the 40 and 42 amino acid long Ab40 and Ab42 peptides (> Figure
11-1b). Due to its more hydrophobic nature and its higher tendency to aggregate, it is the Ab42 peptide but
not the Ab40 peptide, which is assumed to be the key player in the pathogenesis of AD. In fact, the amyloid
plaques mainly consist of Ab42. The physiological relevance of b and gsecretase in Ab generation is clearly
established. Mice deficient in the bsecretase or the gsecretase activity do not generate Ab, making both
proteases suitable drug targets for AD [reviewed in Haass (2004)].
264
11 Alzheimers disease BACE proteases

In contrast, the antiamyloidogenic pathway starts with APP cleavage by asecretase, which cuts
within the Ab domain (> Figure 11-1a, b). Thus, asecretase cleavage precludes Ab generation. Following
acleavage the Cterminal APP fragment undergoes intramembrane cleavage by the gsecretase complex.
This leads to the generation of the p3 peptide (Haass et al., 1993) (> Figure 11-1a), which is assumed to be
benign as it is not found in the AD amyloid plaques. aSecretase is a member of the ADAMfamily
of proteases (A disintegrin and metalloprotease) [for a review see Allinson et al. (2003)], and is either
ADAM10 (Lammich et al., 1999), ADAM17/tumor necrosis factor aconverting enzyme (TACE) (Buxbaum
et al., 1998) or even ADAM9 (Koike et al., 1999). At present, it is unclear, whether only one of them or all
three together constitute the physiologically relevant asecretase. An asecretaselike proteolytic cleavage at
residues 19 and 20 of Ab (> Figure 11-1b) is catalyzed by the protease BACE2 (Farzan et al., 2000; Yan et al.,
2001), which is a close homolog of the bsecretase BACE1. Because BACE2 cleaves within the Ab sequence,
it does not contribute to Ab formation, which is consistent with the finding, that a BACE1 knockout
alone in mice is sufficient to completely suppress Ab generation (Cai et al., 2001; Luo et al., 2001; Roberds
et al., 2001).
The essential role of the secretases in the generation or prevention of the Ab peptide is underlined by
genetic evidence. Mutations in the APP gene linked to familial forms of Alzheimers disease (FAD) are
found at or very close to the secretase cleavage sites and generally increase Ab generation. The Swedish
double mutation, which was found in a Swedish FAD family, is located directly Nterminally to the
bsecretase cleavage site (> Figure 11-1b) and allows a much more efficient bsecretase cleavage of APP
and turnover to Ab. Mutations close to the gsecretase site do not increase the total amount of Ab being
generated but increase the amount of the longer Ab42 species relative to the Ab40 peptide. Additionally,
FAD mutations were found close to the asecretase cleavage site, and appear to reduce asecretase cleavage,
resulting in an increased bsecretase cleavage of APP and thus in more Ab generation.
The following chapters highlight various aspects of BACE1 and BACE2 protease biology, starting with a
description of the discovery of both proteases.

2 The Discovery of BACE Proteases

Early on in the research of the APP secretases, cell biological and pharmacological studies revealed that
bsecretase is an enzyme different from a and gsecretase. It became clear that the bsecretase cleavage
requires an acidic environment and is localized both in endosomes and in the secretory pathway (Koo and
Squazzo, 1994; Haass et al., 1995). Moreover, it only cleaved membranebound APP, suggesting that
bsecretase itself is a membrane protein. Additionally, bsecretase activity was found in most tissues
analyzed and showed a particularly strong activity in brain. Moreover, it cleaved Swedish mutant APP
much more efficiently than wildtype APP (Citron et al., 1992, 1995). Over the years, different candidate
bsecretases were suggested, but none fulfilled all the criteria for bsecretase.
All three APP secretases were identified within a short time. After the identification of ADAM17 and
ADAM10 as asecretases (Buxbaum et al., 1998; Lammich et al., 1999), the presenilins were shown in early
1999 to be the catalytic subunit of gsecretase (Wolfe et al., 1999). At the end of the same year, bsecretase
was the last secretase to be identified. Vassar and colleagues were the first ones to report the identification of
the bsecretase enzyme, which they named bsite APP cleaving enzyme (BACE1, also called memapsin2 or
Asp2) (Vassar et al., 1999). Four subsequent reports by other groups identified the same enzyme (Hussain
et al., 1999; Sinha et al., 1999; Yan et al., 1999; Lin et al., 2000). The fact that all groups used different
experimental approaches to identify BACE1 made a strong case for BACE1 being bsecretase.
Vassar and colleagues used an expression cloning approach to identify bsecretase from a cDNA library
derived from human embryonic kidney 293 cells (HEK293) (Vassar et al., 1999). As a reporter cell line they
used HEK293 cells stably expressing Swedish mutant APP and transiently transfected them with pools of
cDNAs. Pools that increased Ab secretion into the conditioned medium were further subdivided by the sib
selection approach until they finally identified the individual cDNA that increased Ab generation. This
cDNA turned out to be BACE1.
 Alzheimers disease BACE proteases
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Another group used an affinitypurification strategy and identified bsecretase from human brain using
a peptidebased transitionstate analog of the APP sequence (Sinha et al., 1999). This compound inhibits
bsecretase activity and binds tightly to the protein. After several enrichment steps the enzyme was
Nterminally sequenced. The obtained amino acid sequence was then used to clone the cDNA of bsecretase.
Three additional groups hypothesized that bsecretase is an aspartyl protease and applied a genomics
approach searching EST databases to identify BACE1 (Hussain et al., 1999; Yan et al., 1999; Lin et al., 2000).
BACE1 is a type I membrane protein with 501 amino acids (> Figure 11-2). Its gene is encoded on
chromosome 11. The Nterminal signal peptide is followed by a prodomain, a catalytic domain comprising
the two catalytic aspartic acid residues [amino acids 9396 (DSGT) and amino acids 289292 (DTGS)]
characteristic for aspartyl proteases, a transmembrane domain, and a short cytoplasmic tail. Several
experiments show that BACE1 fulfills all criteria for being bsecretase (Hussain et al., 1999; Sinha et al.,
1999; Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000). Mutation of the catalytic active site aspartic acid

. Figure 11-2
Domain structures of BACE1 and BACE2. N and Ctermini (N, C) of both proteins are indicated as well as the
transmembrane domain (TMD) and the cleavage sites of the signal peptide (SP) and the prodomain (pro). Also
shown are the two catalytic aspartic acid residues (D) and the Cterminal sequences of both proteins

residues abolishes its activity, providing evidence that BACE1 is indeed an aspartyl protease. Moreover,
BACE1 acts as a protease in vitro assays and cleaves APP at the correct peptide bond. The catalytic residues
are in the luminal domain of BACE1, and thus have the correct topology for cleaving within the luminal
domain of APP. Overexpression of BACE1 increases bsecretase cleavage of APP and Ab generation.
Conversely, BACE1 knockout mice do not generate Ab anymore (Cai et al., 2001; Luo et al., 2001; Roberds
et al., 2001). BACE1 is ubiquitously expressed, with higher expression levels observed in brain and
particularly in neurons, which is in agreement with the increased bsecretase activity found in this cell
type. Additionally, BACE1 has an acidic pHoptimum of 4.5, and localizes to the Golgi, TGN, and the
endosomes, where bsecretase activity is found. Most important, BACE1 cleaves the Swedish mutant APP
much more efficiently than wildtype APP (Lin et al., 2000; GruningerLeitch et al., 2002). Taken together,
BACE1 fulfills the criteria for being bsecretase.
Shortly after the discovery of BACE1, a homolog was identified by EST database searches and named
BACE2 (also referred to as memapsin1, Asp1, or DRAP) (Saunders et al., 1999; Yan et al., 1999; Acquati
et al., 2000; Lin et al., 2000; Solans et al., 2000). The BACE2 protein contains 518 amino acids, and its
sequence shares 46% identity and 62% similarity with the corresponding sequence of BACE1. Both
proteases are evolutionarily highly conserved. The sequence identity between the human and the mouse
protein is 95% for BACE1 and 89% for BACE2. BACE1 and BACE2 belong to the pepsin family of aspartyl
proteases, and are the only membranebound family members. BACE2 shares the same domain structure as
BACE1 (> Figure 11-2).

3 BACE Gene Structure and Alternative Splicing

Initial important information on the regulation of BACE1/BACE2 expression and APP processing might be
obtained from the BACEs gene structure and alternative splicing events. Because BACE1 is the predominant
bsecretase in brain, and BACE2 is expressed peripherally under basal conditions, most studies performed
so far focused on BACE1 only.
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11 Alzheimers disease BACE proteases

BACE1 mRNA encoding the fulllength, 501 aa BACE1 protein is expressed at high levels in brain and in
pancreas, but significant concentrations of enzymatically active BACE1 proteins are found exclusively
in brain (Hussain et al., 1999; Sinha et al., 1999). BACE1 mRNAs are synthesized as nine exons and eight
introns from a 30.6 kb region of the human chromosome 11q23.211q23.3 (Saunders et al., 1999) and the
presence of multiple mRNAs that terminate at different sites has been attributed to variable polyadenylation
(Sambamurti et al., 2004). Additionally, fulllength BACE1 mRNA may undergo alternative splicing to give
rise to three truncated BACE1 mRNAs with the deletion of 132, 75, and 207 nucleotides, respectively
(Tanahashi and Tabira, 2001; see also > Figure 11-3). Removal of 132 bp of exon 3 results in the expression

. Figure 11-3
Alternative splicing of BACE1 mRNA. Schematic representation of BACE1 coding exons and alternatively spliced
transcripts. Alternative splicing leads to the removal of 132 (44), 75 (25) or 207 (69) nucleotides (amino acids)
and results in the expression of BACE1457; BACE1476, and BACE1432 isoforms. In the transcript BACE1457
twothirds of exon 3 are spliced, resulting in the removal of two glycosylation sites (arrows) of the
corresponding protein. In the transcript BACE1476, 75 nucleotides of exon 4 are spliced and in the BACE1
432 mRNA both mentioned nucleotide sequences of exon 3 and exon 4 are removed. Asterisks, activesite
motifs; SP, signal peptide sequence; pro, prodomain; TMD, transmembrane domain

of a 457 aa BACE1 protein lacking two glycosylation sites between the two active site motifs (> Figure 11-3).
This BACE1457 mRNA was detected at high levels not only in pancreas (Bodendorf et al., 2001) but also at
much lower concentrations in brain (Tanahashi and Tabira, 2001). The resultant BACE1457 protein is
retained in a proenzymatic and endoglycosidase Hsensitive state in the endoplasmatic reticulum (ER;
Bodendorf et al., 2001; Tanahashi and Tabira, 2001). Similar observations were made for the BACE1476
isoform lacking 75 nucleotides of exon 4 (Tanahasi and Tabira, 2001). Because the BACE1501 isoform is
resistant to endoglycosidase H treatment (Capell et al., 2000; Huse et al., 2000), different posttranslational
modifications of the distinct BACE1 isoforms are discussed (see also > Section 5). Most importantly, the
deletion of 25 or more aa between the two activesite motifs of BACE1 impairs APP processing at the
bsecretase site. This might partly explain the discrepancy between high levels of BACE1 mRNA but low
BACE1 enzymatic activity with regard to APP cleavage in pancreas. However, despite the existence of
truncated BACE1 mRNAs in pancreas, the fulllength BACE1 isoform has been shown by RNAse protection
assays to be the major pancreatic BACE1 transcript (Ehehalt et al., 2002). On the other hand, the presence of
BACE1 isoforms with reduced or lacking bsecretase activity might be interpreted as a first hint for the
subsistence of alternative BACE1 substrates, which are processed by tissuespecific BACE1 isoforms in
distinct organs.
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What is the function of BACE2? Although it cleaves APP more efficiently within the bamyloid
sequence than at the bsecretase site (see also > Figure 11-1), it might play a role in specific forms of AD.
When comparing by database analysis the chromosomal localization of the BACE2 gene in different species
it came to our attention that BACE2 (but not BACE1) and APP map to the same chromosome in mice, rats,
and humans (> Figure 11-4). The localization of the APP gene on human chromosome 21 (Kang et al.,

. Figure 11-4
Chromosomal localization of BACE1, BACE2, and APP in mouse, rat, and human. BACE2, but not BACE1, and its
substrate APP map to the same chromosome in all three species

1987; Tanzi et al., 1987) and the finding that virtually all patients more than the age of 40 suffering Downs
syndrome (DS; trisomy 21) invariantly develop the clinical and pathological characteristics of AD, support
the hypothesis that increased APP expression plays a significant role in the progression of the disease [for
review see Kola and Hertzog (1997)]. In addition to APP, its secretase BACE2 is localized on human
chromosome 21 and maps to the Down critical region in 21q22.3 (Acquati et al., 2000; Solans et al., 2000)
and, therefore, the increased expression of both, substrate and its processing secretase might increase the
generation of Ab peptides and contribute to the pathogenesis of AD. Indeed, the increased secretion of
BACE2 from fibroblasts of patients suffering from DS has been reported (Barbiero et al., 2003). BACE2
overexpression was also observed in the DS fetal brains and in human neural embryonic DS stem cells in
which conditioned media BACE2 was secreted (Barbieri et al., 2003). Moreover, changes in the expression
of BACE2 were observed immunohistochemically in the frontal cortex of DS patients. The immunoreac-
tivity for BACE2 was particularly detected in neurofibrillary tanglebearing neurons from the elderly DS
brains with ADtype neuropathology, but was not detected in those of DS brains without ADtype
neuropathology or in those of control brains of any age (Motonaga et al., 2002). This suggests the possibility
that the elevated expression of BACE2 is involved in the ADtype neuropathology of DS. However, recent
studies using transfection and knockdown strategies revealed that overexpression of BACE2 reduces
amyloidogenic APP processing and increases the generation of nonamyloidogenic APP fragments whereas
elimination of BACE2 produced opposite effects (Sun et al., 2005, 2006a). It has to be established in
transgenic mice how BACE2 functions in brain in vivo. One way to address that question is to overexpress
BACE2 neuronally on the background of hAPPtg/BACE1
/
mice and to quantify Ab generation and Ab
plaque formation. An alternative mechanism possibly contributing to AD pathology in DS patients was
revealed recently. DS brains displayed an higher ratio of mature to immature forms of BACE1 in the Golgi,
resulting in increased bsecretase activity (Sun et al., 2006b). This indicates that overproduction of Ab in DS
might be caused by abnormal BACE1 protein trafficking and maturation.
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11 Alzheimers disease BACE proteases

The transcriptional regulation of BACE2 appears to be quite different from that of BACE1. The BACE2
gene is controlled by a TATAless promoter and although SP1 can regulate BACE1 and BACE2 genes there is
very little similarity between the two promoters as revealed by comparative sequence analysis and tran-
scription factor prediction (Sun et al., 2005). Moreover, these authors identified a neuronspecific repressive
element between
652 bp to
200 bp of the BACE2 promoter. Although it is not known yet under which
conditions the neuronal BACE2 repression can be overruled, such information might be important with
regard to diseaserelated APP processing (see earlier) and a potential upregulation of neuronal BACE2
expression as an AD treatment strategy.

4 Transcription and Translation of BACE1

Observations on altered BACE1 protein concentrations in brains of sporadic AD patients (see later) indicate
transcriptional and/or translational regulation of BACE1 expression in brain. The BACE1 promoter is
highly conserved between rat, mouse, and human (> Figure 11-5) and contains a number of putative
transcription factor binding sites (LangeDohna et al., 2003). This would suggest that regulatory mechan-
isms of BACE1 expression are common between these species and that mice and rats represent valuable
animal models to reveal such mechanisms and to test therapeutic strategies aimed at reducing BACE1
expression in AD. Interestingly, the BACE1 promoter contains several putative transcription factor binding
sites that are known to be involved in the expression of other ADrelated proteins. For example, NFkB sites
are present in the promoters of APP (Grilli et al., 1995), choline acetyltransferase (ChAT; ToliverKinsky
et al., 2000) and presenilins [for review see Glasgow and PerezPolo (2000)]. Likewise, SP1 sites have been
reported for the APP (Lahiri and Robakis, 1991; Pollwein, 1993; Lukiw et al., 1994; Hoffman and Chernak,
1995), ChAT (Hahn et al., 1992; Inoue et al., 1993), presenilin1 (Mitsuda et al., 1997), and presenilin
2 (Pennypacker et al., 1998) promoters. Based on results derived from luciferase reporter assays it was
suggested that an upstream NFkB site might act as a repressor of BACE1 transcription (LangeDohna et al.,
2003). A detailed analysis using BACE1 promoter constructs carrying mutations of the NFkB site revealed
a unique cell typespecific regulation of BACE1 promoter activity. In neuronlike cells, such as retinoic acid
differentiated SHSY5Y cells or nerve growth factordifferentiated PC12 cells, the NFkB site indeed acts
as repressor of the BACE1 promoter whereas in C6 glioma cells it acts as activator (Bourne et al., 2005). This
has important therapeutic implications and might explain in part the cell typespecific upregulation BACE1
in reactive astrocytes of APP transgenic mice with amyloid plaque formation (Roner et al., 2001),
in chronic lesion paradigms (HartlageRubsamen et al., 2003) and in brains of AD patients (Hartlage
Rubsamen et al., 2003; Leuba et al., 2005). These results are also consistent with observations demonstrating
that nonsteroidal antiinflammatory drugs (NSAIDs) decreased the BACE1 mRNA levels, protein expres-
sion, and also BACE1 enzymatic activity (Sastre et al., 2003). Pharmacological studies suggested that this
effect is mediated through activation of the peroxisome proliferatoractivated receptorg (PPARg; Sastre
et al., 2003). In line with that, the absence of PPARg potentiates BACE1 mRNA levels by increasing BACE1
promoter activity. Conversely, overexpression of PPARg as well as NSAIDs and pioglitazone, a PPARg
activator, reduced BACE1 promoter activity (Sastre et al., 2006). These effects appear to be mediated by a
PPARg responsive element (PPRE) within the BACE1 promoter. Mutagenesis of this PPRE element
abolished the binding of PPARg to the PPRE and increased BACE1 gene promoter activity (Sastre et al.,
2006). Furthermore, proinflammatory cytokines decreased the transcription of the PPARg gene, an effect
that was suppressed by NSAIDs. Interestingly, brain extracts from AD patients showed decreased PPARg
expression and reduced binding to PPRE in the BACE1 promoter. From these results, it was concluded that
the protective mechanism of NSAIDs in AD is exerted through activation of PPARg and decreased BACE1
gene transcription (Sastre et al., 2006). This conclusion is supported by data from a transgenic mouse
model of AD, in which treatment with NSAIDs or pioglitazone suppressed proinflammatory markers,
reduced BACE1 mRNA and protein levels and lowered Ab plaque load (Heneka et al., 2005). Transcrip-
tional regulation of BACE1 expression occurs also via a functional SP1 element 850 nucleotides upstream
of the transcription start site (Christensen et al., 2004). In HEK293T cells and in PC12 cells this SP1
site appears to act as an activator of BACE1 expression as shown by reduced BACE1 expression after
 Alzheimers disease BACE proteases
11 269

. Figure 11-5
Rat, mouse, and human BACE1 promoter sequences. Comparison of the rat BACE1 promoter sequence with that
of its mouse and human homologs. There is 92% similarity between rat and mouse and 81% similarity between
rat and human within the region from bp
1 to
600. The similarity between rat and mouse by about 86%
extends between bp
600 and
1000, but the similarity between the rat and the human sequence is only 39%
in this fragment. At a greater distance than bp
1000, this similarity is reduced significantly (after LangeDohna
et al., 2003; modified)
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11 Alzheimers disease BACE proteases

targeted disruption of the SP1 gene and by the potentiated BACE1 expression after overexpression of SP1
(Christensen et al., 2004).
In addition to a possible regulation of BACE1 expression by transcriptional mechanisms, there is ample
evidence for posttranscriptional modulation of BACE1 enzymatic activity. For example, BACE1 protein
levels are significantly upregulated up to 2.7fold in the brains of sporadic AD patients compared with
nonAD controls (Fukumoto et al., 2002; Holsinger et al., 2002; Yang et al., 2003), whereas mRNA levels were
unchanged (Yasojima et al., 2001; Holsinger et al., 2002; Preece et al., 2003). This indicates that posttran-
scriptional mechanisms, such as translational control or altered BACE1 protein degradation, regulate
BACE1 protein levels. In fact, tissue culture experiments have provided evidence for both mechanisms in
controlling BACE1 protein levels. An altered BACE1 degradation has been described by Kovacs and
coworkers (Puglielli et al., 2003). They reported that the lipid second messenger ceramide, which is elevated
in the brain of AD patients, increases the halflife of BACE1 and thereby increases Ab generation (Puglielli
et al., 2003). Translational control of BACE1 expression has been observed independently by different
groups (De Pietri Tonelli et al., 2004; Lammich et al., 2004; Rogers et al., 2004). The studies were based on
the observation that the 50 untranslated region (50 UTR) of the BACE1 mRNA contains unusual sequence
features that are often found in mRNAs showing tight translational control. In most vertebrate mRNAs, the
50 UTR is 10200 nucleotides long, is unstructured, not very GCrich and does not contain upstream open
reading frames (uORFs). In contrast, less than 10% of vertebrate genes, including many regulatory proteins
like protooncogenes, have 50 UTRs that are longer than 200 nucleotides and are often GCrich (7090%).
This indicates a high degree of secondary structure, which may impede the efficient scanning of the
ribosome (Kozak, 1987; Willis, 1999). Additionally, these long 50 UTRs may contain uORFs [for a review
see Clemens and Bommer (1999)] that can inhibit the translation of the main ORF. The 50 UTR of BACE1,
which is highly conserved in the human, rat, and murine sequence, belongs to the class of the long 50 UTRs
similar to many regulatory genes. It is 446 nucleotides long, has a GCcontent of 77%, and contains 3 uORFs
(> Figure 11-6). Importantly, all three different BACE1 mRNA species detected by Northern Blot analysis

. Figure 11-6
The mRNA domain structure of BACE1. The BACE1 mRNA consists of the 50 untranslated region (50 UTR),
followed by the coding region (open reading frame with the start codon AUG) and the 30 UTR. The 50 UTR has
the indicated high GCcontent and contains three upstream open reading frames (uORFs), represented as boxes

contain the 50 UTR (Lammich et al., 2004). The role of the 50 UTR in a possible translational control of
BACE1 mRNA was tested in different cell lines as well as in vitro translation experiments (De Pietri Tonelli
et al., 2004; Lammich et al., 2004; Rogers et al., 2004). The use of BACE1 constructs lacking part or all the
50 UTR revealed that the presence of the 50 UTR decreased BACE1 translation by up to 90%. Moreover, when
fused to luciferase, the 50 UTR of BACE1 strongly suppressed luciferase translation and activity, revealing
that the translation repressing activity must be encoded within the 50 UTR. The uORFs within the 50 UTR
were not the major reason for the inhibition of translation. Instead, the experiments suggest that due to its
high GCcontent the 50 UTR forms a constitutive translation barrier that prevents the ribosomes from
efficiently translating the BACE1 mRNA. Additionally, Zacchetti and colleagues showed that the 50 UTR
dependent translational repression may be alleviated in activated astrocytes (De Pietri Tonelli et al., 2004),
leading to an increased expression of BACE1 protein. In fact, activated astrocytes expressing elevated levels
of BACE1 are found around amyloid plaques in an animal model of AD and in the AD brain (Roner et al.,
 Alzheimers disease BACE proteases
11 271

2001; HartlageRubsamen et al., 2003) (see also > Section 6). Thus, pathophysiological conditions leading
to the activation of astrocytes may increase BACE1 expression and thereby Ab generation and could
exacerbate AD pathogenesis.

5 Posttranslational Modifications, Intracellular Transport, and Binding

Partners of BACE1

A variety of cell biological studies have provided important insights into the posttranslational modifications
of BACE1 and the control of its intracellular transport. Like in the studies addressing transcriptional and
translational control of BACE1 expression (> Section 4), an elucidation of BACE1 modifications or
transport may allow to find new therapeutic approaches for AD. They may be used to lower BACE1 activity
or shift the APP cleavage away from the Ab generating bsecretase toward an increased asecretase cleavage
that prevents Ab generation.

5.1 Posttranslational Modifications and Intracellular Transport

On cotranslational integration of BACE1 into the ER the signal peptide is cleaved after amino acid 21
(> Figure 11-2). The immature form of BACE1 (proBACE1) in the ER is Nglycosylated and has an
apparent molecular weight of 60 kDa. On rapid transport through the Golgi, BACE1 undergoes matura-
tion. Complex sugars are added (Charlwood et al., 2001), which become sulfated (Benjannet et al., 2001),
and BACE1 becomes endoglycosidase H resistant (Capell et al., 2000). Furin or a furinlike prohormone
convertase removes the prodomain by cleaving after residue 45 (> Figure 11-2) (Bennett et al., 2000; Capell
et al., 2000; Benjannet et al., 2001). The maturation of BACE2 follows a similar pattern as BACE1. However,
its prodomain cleavage is autocatalytic (Hussain et al., 2001). The prodomain of many proteases, such as
ADAM10 and ADAM17, inhibit the proteolytic activity until they are removed. In this regard, the prodo-
main of BACE1 is unusual in that it does not strictly inhibit BACE1 activity (Shi et al., 2001). In fact,
overexpressed proBACE1 can cleave APP in the ER (Huse et al., 2002). The role of the prodomain of BACE1
seems to be the facilitation of the correct folding of BACE1 (Shi et al., 2001). Oglycosylation has not been
observed for BACE1 (Haniu et al., 2000). BACE1 contains four Nglycosylation sites (> Figure 11-3), which
all carry sugar moieties (Haniu et al., 2000; Charlwood et al., 2001). The Nglycosylation affects BACE1
activity, since a BACE1 mutant engineered to contain only two out of the four sites shows reduced
BACE1 activity (Charlwood et al., 2001). It remains unclear, though, whether this is a direct effect on BACE1
activity or a more indirect effect due to alterations in folding, stability, or solubility of the enzyme. BACE1
contains six cysteine residues in its ectodomain, which form disulfide bridges in a pattern that is conserved
in its homolog BACE2, but that is not found in the other members of the pepsin family of aspartyl proteases
(Haniu et al., 2000). At the boundary between transmembrane and cytoplasmic domain BACE1 has three
cysteine residues, which are palmitoylated (Benjannet et al., 2001). This modification reduces the ectodo-
main shedding of BACE1, but does not seem to have a major effect on BACE1 activity, as revealed by the
analysis of BACE1 mutants lacking these cysteine residues (Benjannet et al., 2001).
Mature BACE1 with an apparent molecular weight of 70 kDa is a relatively stable enzyme with a half
life of > 9 h in HEK293 cells (Haniu et al., 2000; Huse et al., 2000). Another form of posttranslational
modification of BACE1 is ectodomain shedding. On overexpression of BACE1, secreted BACE1 lacking its
transmembrane and cytoplasmic domain has been detected in the conditioned medium of HEK293 cells
(Benjannet et al., 2001). The secretion could be reduced by inhibitors of metalloproteases, suggesting that
members of the ADAMfamily or matrix metalloproteases might mediate the shedding of BACE1 (Hussain
et al., 2003). Whether this shedding also occurs under conditions not involving BACE1 overexpression,
remains to be analyzed. In contrast to BACE1 shedding, fulllength BACE1 has been detected in the
cerebrospinal fluid of AD patients and controls, with the BACE1 levels being higher in the AD patients
(Holsinger et al., 2004). How fulllength BACE1 including its transmembrane and cytoplasmic domain can
be released into the CSF remains unknown. An additional proteolytic cleavage on a surface exposed helix
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11 Alzheimers disease BACE proteases

within the ectodomain of BACE1 has been described between residues Leu228 and Ala299. This leads to two
BACE1 fragments, which remain bound to each other and which retain BACE1 activity. This cleavage was
mainly observed in liver, pancreas, and muscle, but not in brain (Huse et al., 2003).
Like APP, mature BACE1 cycles between the TGN, the plasma membrane, and the endocytic pathway,
particularly early and late endosomes (Huse et al., 2000; Walter et al., 2001). In fact, APP and BACE
colocalize at the plasma membrane and in endosomes (Kinoshita et al., 2003). BACE2 shows a similar
cellular distribution as BACE1, but a larger proportion of BACE2 is found at the plasma membrane
(Hussain et al., 2000; Yan et al., 2001). Close to its Cterminus BACE1 contains an evolutionarily conserved
dileucine amino acid motif (> Figure 11-2), which conforms to endocytic trafficking signals found in other
proteins, such as mannose6 phosphate receptor (Johnson and Kornfeld, 1992). Deletion of this motif alters
BACE1 trafficking, such that more BACE1 is localized at the cell surface and less is found in endosomes
(Huse et al., 2000).
Phosphorylation of BACE1 by casein kinase 1 at Ser498 seems to be another way of regulating BACE1
sorting in the cell. Replacing Ser498 by aspartic acid (Ser498Asp), which mimics phosphorylated BACE1 is
predominantly found in the TGN and in late endosomes, whereas an alanine at position 498 (Ser498Ala)
mimics nonphosphorylated BACE1 and is mainly localized in early endosomes (Walter et al., 2001;
Pastorino et al., 2002). Despite the differences in cellular sorting, both BACE1 mutants have similar effects
on APP processing when transfected into HEK293 cells, revealing that the phosphorylation of BACE1 does
not have a major effect on its catalytic activity (Pastorino et al., 2002).
In cultured cells, overexpressed APP and BACE1 have been detected partly within and partly outside
cholesterolrich membrane domains, socalled detergentresistant membrane microdomains or rafts. The
question whether BACE1 cleavage of APP occurs within or outside the cholesterolrich domains is of
particular interest, since cholesterol lowering drugs have been shown to reduce Ab generation. In contrast
to initial studies suggesting that APP cleavage by BACE1 occurs within the detergentresistant membranes
(Riddell et al., 2001; Tun et al., 2002; Cordy et al., 2003; Ehehalt et al., 2003), a recent report suggests that
this cleavage event takes place outside the detergentresistant membranes, at least under endogenous
expression levels (AbadRodriguez et al., 2004). Further studies are needed to fully understand the role of
cholesterol and cholesterolrich membrane domains in BACE1 activity and AD pathogenesis.

5.2 BACE1 Interacting Proteins

An increasing number of proteins have been reported to interact with BACE1. The two gsecretase complex
subunits nicastrin and presenilin coimmunoprecipitate with BACE1 when overexpressed (Hattori et al.,
2002; Hebert et al., 2003). Whether the interaction is also seen at endogenous expression levels, remains to
be analyzed.
Another binding partner of BACE1, the phospholipid scramblase 1 (PLSCR 1), was identified in a yeast
two hybrid screen using the short, 24 amino acid long cytoplasmic tail of BACE1 as the bait (Kametaka
et al., 2003). The type II membrane protein PLSCR 1 colocalizes with BACE1 in the Golgi and in endosomes
and was shown to coimmunoprecipitate with BACE1 at endogenous expression levels. The interaction
between both proteins requires the Cterminal dileucine trafficking motif of BACE1, suggesting that
PLSCR may be involved in the intracellular distribution of BACE1.
Other proteins binding to the dileucine trafficking motif of BACE1 are members of the GGA (Golgi
localized gearcontaining ARF binding) family, namely GGA1 and GGA2. The interaction was shown
in pulldown experiments and using peptides corresponding to the sequence of the BACE1 Cterminus (He
et al., 2002). The interaction was confirmed in cultured cells using a fluorescence resonance energy transfer
assay (von Arnim et al., 2004). Because GGAs are intracellular trafficking proteins that also interact with
mannose6phosphate receptors, the authors suggested that the interaction with GGAs may influence
BACE1 intracellular transport. Phosphorylation of the serine residue directly preceding the dileucine
motif seems to enhance the interaction between BACE1 and GGAs (He et al., 2003; Shiba et al., 2004).
Experiments knockingdown GGA expression resulted in accumulation of BACE1 in early endosomes,
 Alzheimers disease BACE proteases
11 273

suggesting that the GGABACE1 interaction is required for the recycling of endocytosed BACE1 to the cell
surface (He et al., 2005).
Another binding partner of BACE1 that was identified in a yeast two hybrid screen is the brain specific
type II membrane protein BRI3, which colocalizes with BACE1 in HEK293 cells but also in brain (Wickham
et al., 2005). Moreover, BRI3 coimmunoprecipitates with endogenous BACE1 in HEK293 cells. The
maximal interaction between both proteins requires the cytoplasmic domain of BACE1 to be palmitoylated.
The functional consequences of this interaction and the function of BRI3 itself are as yet unknown.
Given the link of copper to the pathogenesis of AD [for an overview see Multhaup et al. (2002),
Cuajungco et al. (2005)], the identification of the copper chaperone for superoxide dismutase1 (CCS) is of
particular interest. CCS binds to the cytoplasmic tail of BACE in rat brain extracts, and both proteins are
cotransported through axons. Surprisingly, the cysteine residues in the cytoplasmic domain of BACE1 bind
a single Cu(I) atom (Angeletti et al., 2005). Whether the binding of copper or CCS to BACE1 has an effect
on APP processing remains to be studied.
Proteins that alter BACE1 activity by binding to BACE1 are reticulon 3 (RTN3) and its homolog RTN4b
(NoGo) and heparan sulfate. Heparan sulfate was found to be a natural extracellular ligand for BACE1
(Scholefield et al., 2003). This interaction inhibits BACE1 cleavage of APP but not the asecretase cleavage
of APP. The underlying molecular mechanism is not yet known. Heparan sulfate and BACE1 colocalize in
the Golgi and at the plasma membrane and coimmunoprecipitate at endogenous expression levels in HEK
293 cells.
RTN3 binds BACE1 in neurons, whereas RTN4b binds BACE1 in oligodendrocytes. The interaction of
BACE1 with RTN3 was not only found in cells overexpressing both proteins but also at endogenous expression
levels in brain. Most important, increasing the expression of RTN3 inhibited the secretion of Ab, whereas
lowering the expression of RTN3 by RNA interference increased the secretion of Ab, suggesting that reticulon
proteins are negative modulators of BACE1 by blocking access of BACE1 to APP (He et al., 2004).
Yet another binding partner of BACE1 is BACE1 itself (Schmechel et al., 2003; Westmeyer et al., 2004).
The homodimerization of BACE1 was described to occur already in the ER and to increase BACE1
proteolytic activity (Westmeyer et al., 2004). The dimerization takes place between the ectodomains and
requires BACE1 to be membraneanchored. Homodimerization of BACE1 was observed both in cultured
cells overexpressing BACE1 and in brain tissue.
Taken together as shown in > Table 111, several BACE1 binding proteins have been identified. Some of
them seem to influence BACE1 intracellular trafficking (PLSCR 1, GGAs), whereas other ones affect BACE1

. Table 11-1
Posttranslational modifications and binding partners of BACE1
Posttranslational modifications of BACE1 Binding partners of BACE1
Removal of signal peptide in the ER gsecretase (presenilin and nicastrin)
Nglycosylation at four sites Phospholipid scramblase 1 (PLSCR 1)
Removal of prodomain GGA (Golgilocalized gearcontaining ARF binding)
Three disulfide bridges BRI3
Ectodomain shedding Copper chaperone for superoxide dismutase 1 (CCS)
Palmitoylation Heparan sulfate
Phosphorylation Reticulon 3 and 4b (NoGo)
Sulfated at mature Nsugars BACE1 (homodimer formation)
Proteolytic cleavage within its ectodomain in liver,
pancreas and muscle

activity (heparan sulfate, RTNs, BACE1 homodimerization). Yet another group consists of proteins with
poorly understood function with respect to their binding to BACE1 (gsecretase subunits nicastrin and
presenilin, CCS, BRI3).
274
11 Alzheimers disease BACE proteases

6 BACE1 Expression in Aging and AD

6.1 Localized BACE1 Expression

It is widely accepted that neurons are the primary source of Ab peptides in brain. Therefore, it was believed
for a long time that the bsecretase crucial for Ab generation is expressed by neurons. After BACE1 was
identified in 1999 as the long searched for bsecretase, its cell typespecific expression and localization in
brain was investigated. In mouse brain, BACE1 is primarily expressed by neurons as shown by in situ
hybridization (Vassar et al., 1999; Bigl et al., 2000; Marcinkiewicz and Seidah, 2000) and by immunohis-
tochemistry (Roner et al., 2001). As expected, BACE1 expression is robust in neocortical and hippocampal
brain regions characterized by high expression levels of its major substrate APP and by the suscept-
ibility to Ab plaque formation. However, BACE1 mRNA (Bigl et al., 2000; Marcinkiewicz and Seidah, 2000)
and protein (Roner et al., 2001) are also detected at high quantities in brain areas that are almost devoid of
APP and which barely develop any Ab plaques, namely thalamus and striatum. This might be a first hint
for the existence of alternative BACE1 substrates in different brain regions.

6.2 Aging
During aging BACE1 mRNA (Bigl et al., 2000; Irizarry et al., 2001; Apelt et al., 2004) and protein levels
(Roner et al., 2001; Apelt et al., 2004; Fukumoto et al., 2004) are stable in brains of mice and humans.
BACE1 enzymatic activity, in contrast, is increased with aging in mouse, monkey, and human brain
(Fukumoto et al., 2004). In the light of aging being the most stringent risk factor for developing AD and
the concentrations of Ab peptides in brain strongly correlating with age, this is an important observation
and indirect evidence for the contribution of posttranslational mechanisms in the regulation of BACE1
activity and the pathogenesis of AD (see also > Section 5).

6.3 APP Transgenic Mice

In the recent decade several strains of APP transgenic mice were developed and widely used to study the
consequences of APP overexpression and/or Ab plaque formation on the generation of other histologi-
cal and biochemical AD markers. The Tg2576 mouse strain is characterized by a five to sevenfold
overexpression of hAPP carrying the Swedish double mutation and by an agedependent formation of
Ab plaques (Hsiao et al., 1996). Despite the APP overexpression, the BACE1 mRNA levels (Bigl et al.,
2000; Irizarry et al., 2001; Apelt et al., 2004), BACE1 protein levels (Roner et al., 2001; Gau et al., 2002;
Fukumoto et al., 2004), and BACE1 enzymatic activities (Fukumoto et al., 2004) were not different between
wildtype and Tg2576 mice. This indicates that BACE1 is not the ratelimiting enzyme for Ab peptide
generation. As a consequence, a partial reduction of BACE1 enzymatic activity in the AD brain is unlikely to
result in a significant depletion of Ab peptides.

6.4 BACE1 Expression in AD

In the AD brain, increases in both BACE1 protein concentrations and enzymatic activities have been re-
ported (Fukumoto et al., 2002, 2004; Holsinger et al., 2002; Sun et al., 2002; Yang et al., 2003). Interestingly,
the soma of BACE1 expressing neurons did not directly associate with Ab plaques in hippocampal and
neocortical areas characterized by robust Ab deposition (Sun et al., 2002), a finding which is consistent with
observations from brains of APP transgenic mice (Roner et al., 2001). The increased bsecretase activity in
AD brain is accompanied by a reduction of asecretase activity (Tyler et al., 2002). Interestingly, when
plotting cortical a or bsecretase activities against ChAT activity in cortex of AD patients a strong positive
(to asecretase) or negative (to bsecretase) correlation can be established. Of course, this observation leaves
open the possibility that a primary cholinergic deficit induces alterations in secretase activities. On the other
 Alzheimers disease BACE proteases
11 275

hand, it is well established that a quantitative shift APP processing toward the generation of Ab peptides
interferes with cholinergic neurotransmission. For example, solubilized Ab peptides strongly inhibit the
potassiumstimulated release of acetylcholine from hippocampal slices (Kar et al., 1996) and decreases
ChAT activity but not acetylcholinesterase activity in the cholinergic SN56 cell line (Pedersen et al., 1996).
Ab peptides also decrease the intracellular acetylcholine concentration (Hoshi et al., 1997) and impair
M1associated signaling (Kelly et al., 1996) in primary septal or cortical cultures. All these effects of Ab on
markers of cholinergic neurotransmission were observed at pM to nM concentrations and without obvious
evidence of neurotoxicity. The effects of Ab on reduction of pyruvate dehydroxygenase activity (Hoshi et al.,
1997), the key enzyme for the generation of acetylcholine used for neurotransmitter synthesis and citrate
cycle, might explain both, cholinergic hypoactivity and metabolic dysfunction of cholinergic neurons after
exposure to Ab at low concentrations.

6.5 Astrocytic BACE1 Expression

In addition to quantitative changes of BACE1 activity in AD, there are also remarkable alterations with
regard to the cell typespecific expression of BACE1. As stated earlier and consistent with the notion that
neurons are the primary source of Ab peptides in brain, BACE1 is exclusively expressed by neurons under
normal conditions. However, reactive astrocytes in proximity to Ab plaques also display BACE1 immuno-
reactivity indicating that astrocytes in their activated state might contribute to Ab plaque formation
(HartlageRubsamen et al., 2003; Leuba et al., 2005). This astrocytic BACE1 expression is not only limited
to the brains of AD patients, but was also observed in brains of APP transgenic Tg2576 mice with Ab plaque
pathology. This was not found in transgenic mice before the onset of Ab plaque formation (> Figure 11-7)
(Roner et al., 2001). Furthermore, in a number of chronic but not acute lesion models, reactive astrocytes
express BACE1 (HartlageRubsamen et al., 2003) indicating that augmented BACE1 expression by chroni-
cally activated astrocytes may contribute to a localized increase in amyloidogenic APP fragments or plaque
formation. This scenario is supported by studies using different experimental paradigms [for review see
Roner et al. (2005)]. For example, a single intrahippocampal LPSinjection fails to increase the Ab load in
APP/presenilin1 transgenic mice (DiCarlo et al., 2001), but the chronic intracerebroventricular infusion of

. Figure 11-7
Astrocytic BACE1 expression in Tg2576 mice and AD. Confocal images of double immunofluorescence labeling
for BACE1 and GFAP in the parietal cortex of 17monthold Tg2576 mice and in Area 22 of the AD cortex. BACE1
immunoreactivity is shown by the green fluorescence; GFAPimmunoreactivity is encoded by red fluorescence.
In the overlay channel, colocalization of BACE1 and GFAP is indicated by yellow/orange color. Reactive astro-
cytes expressing BACE1 are indicated by arrowheads; the asterisks mark the position of Ab plaques (after
HartlageRubsamen et al., 2003; modified)
276
11 Alzheimers disease BACE proteases

LPS accelerates Ab deposition in APPV717F transgenic mice (Qiao et al., 2001). However, at the present
stage it is not clear to what extent reactive astrocytes contribute to the production of Ab peptides and to Ab
plaque formation. First, although activity measurements indicate that reactive astrocytes do express
enzymatically active BACE1 (Roner et al., 2005), it still has to be shown that the fulllength, nontruncated
BACE1 isoform is expressed. Second, to contribute to Ab plaque formation, the substrate APP has to be
available to astrocytic BACE1. It is known that primary astrocytes themselves do express APP and generate
significant amounts of Ab peptides (Gray and Patel, 1993; Amara et al., 1999; Beck et al., 2000; Blasko et al.,
2000; Docagne et al., 2004). Additionally, APP is also expressed by reactive astrocytes in experimental
models of chronic gliosis [see, e.g., Martins et al. (2001)] and this induced astrocytic APP expression results
in the increased generation of Ab peptides and BACE1 cleavagederived A4CT fragments (Bates et al., 2002;
Lesne et al., 2003). Alternatively, astrocytic BACE1 might be secreted and cleave membranous APP at the
neuronal surface. Such a shedding of BACE1 has been reported recently (Benjannet et al., 2001; Hussain
et al., 2003).

7 Biological Function and Substrate Spectrum of BACE1 and BACE2

7.1 Substrates of BACE Proteases

What is the biological function of BACE1? Knowing the answer to this question will allow not only a better
evaluation of the therapeutic potential but also of the liabilities of BACE1 inhibition. In case that BACE1
cleaves a number of different substrates, the inhibition of BACE1 may cause side effects by interfering with
the function of these proteins.
Do BACE1 and BACE2 only cleave APP or do they contribute to the proteolytic cleavage of a larger
number of membrane proteins, similar to a and gsecretase? The asecretases ADAM10 and ADAM17
mediate the proteolytic conversion of a variety of membrane proteins to their soluble counterparts [for a
review see Hooper et al. (1997), Blobel (2002)]. This process is referred to as ectodomain shedding and
has been described for growth factors, cytokines and their receptors, cell adhesion proteins, and proteins of
unknown biological function, such as APP. Likewise, gsecretase mediates the intramembrane proteolysis of
an increasing number of type I membrane proteins [reviewed in Selkoe and Kopan (2003)]. In contrast
to a and gsecretase, very few substrates have been identified for BACE1 and even less for BACE2. Both
proteases were initially identified as APP cleaving enzymes. Although additional substrates were not imme-
diately identified, many scientists assumed that more substrates for BACE1 and possibly BACE2 should
exist. This assumption was based on the following theoretical considerations. In the polarized Madin
Darby canine kidney cell line (MDCK), BACE1 is predominantly found on the apical side (Capell et al.,
2002), whereas APP is mainly transported to the basolateral side (Haass et al., 1994; Lo et al., 1994;
De Strooper et al., 1995). Given this differential localization of APP and BACE1, it seemed reasonable to
assume that APP is not the main substrate for BACE1 and that BACE1 may have one or several other
substrates, which should also be found on the basolateral side. Additionally, in vitro experiments addressing
the substrate specificity of BACE1 revealed that wildtype APP is a much poorer substrate for BACE1 than
APP carrying the socalled Swedish double mutation, which is found in a large Swedish pedigree affected by
an inherited form of Alzheimers disease. Carriers of this genetic mutation have the two amino acids lysine
and methionine (KM) preceding the BACE1 cleavage site replaced by asparagine and leucine (NL)
(> Figure 11-1b), allowing a much more efficient cleavage by BACE1 in vitro experiments using short
peptides (Lin et al., 2000; GruningerLeitch et al., 2002).
The prediction that BACE1 should have additional substrates in addition to APP turned out to be true.
A number of additional substrates have been identified over the past few years, namely the APPhomologs
APLP1 and APLP2 (Li and Sudhof, 2004; Pastorino et al., 2004), the sialyltransferase ST6Gal I (Kitazume
et al., 2001, 2003, 2005), the Pselectin glycoprotein ligand1 (PSGL1; Lichtenthaler et al., 2003), beta
subunits of voltagegated sodium channels (VGSCb) (Wong et al., 2005), and the LDL receptorrelated
protein (von Arnim et al., 2005). Both PSGL1 and ST6Gal I function in the immune system, where BACE1
is expressed. The type I membrane protein PSGL1 is involved in the inflammatory response, and
 Alzheimers disease BACE proteases
11 277

contributes to the rolling of leukocytes along the vessel wall before they transmigrate through the
endothelium. The type II membrane protein ST6Gal I is involved in B cell expansion and is required for
the correct glycosylation of CD22 ligands. VGSCs are an abundant type of ion channels that are responsible
for the initiation and propagation of action potentials in neurons. ST6Gal I, PSGL1, and VGSCb meet
three basic criteria for BACE1 substrates: (a) their cleavage occurs under conditions of endogenous BACE1
expression, (b) exogenous expression of BACE1 strongly increases their cleavage and, importantly, (c) their
cleavage is reduced or abolished in BACE1
/
cells or animals (Lichtenthaler et al., 2003; Kitazume et al.,
2005; Wong et al., 2005), which establishes the physiological relevance of their cleavage. Interestingly, both
ST6Gal I and PSGL1 are cleaved Cterminally of a leucine residue, which is in good agreement with the in
vitro substrate specificity of BACE1 and with the fact that BACE1 cleavage of the Swedish mutant APP also
occurs after a leucine residue. Thus, additional substrates for BACE1 to be identified may also be cleaved
preferentially after a leucine residue. The exact cleavage sites of BACE1 in APLP1 and APLP2 remain to be
determined.
BACE1 is not only able to cleave type I membrane proteins (APP, PSGL1, APLP1, and APLP2) but also
a type II membrane protein (ST6Gal I), suggesting that, similar to the ADAM proteases, BACE1 may
contribute to the ectodomain shedding of a larger number of membrane proteins of type I or type II
membrane orientation. However, BACE1 is likely to have a more restricted set of substrate proteins, because
it is not involved in the shedding of two other ADAM protease substrates, the TNFa receptor 2 and the cell
adhesion protein Lselectin (Lichtenthaler et al., 2003). The proteolytic processing of APP and PSGL1
reveals additional insights into cleavage preferences of BACE1. APP and PSGL1 are both cleaved by BACE1,
but also by an ADAM metalloprotease. For both proteins most of the cleavage seems to occur by the ADAM
proteases, whereas BACE1 cleavage constitutes only a smaller fraction of the total cleavage. An additional
similarity is the localization of the BACE1 cleavage site in the membraneproximal domain. In contrast to
APP, it is the BACE1 cleavage site in PSGL1 that is located more closely to the membrane than the putative
ADAM protease cleavage site (Lichtenthaler et al., 2003). This is in opposite order compared with the
proteolytic processing of APP. However, this finding agrees well with the known properties of BACE1 and
ADAM proteases. The ADAM protease cleavage of APP occurs without a strict sequence specificity but at a
fixed distance from the membrane (Sisodia, 1992), whereas the BACE1 cleavage seems to be more sequence
specific but not strongly dependent on the distance of the cleavage site from the membrane (Citron et al., 1995).
Taken together, it is clear now, that BACE1 does not exclusively cleave APP, but has a wider substrate
spectrum. Much less is known about BACE2 substrates. It is not known whether the three BACE1 substrates
ST6Gal I, APLP1, and APLP2 can be cleaved by BACE2, but BACE2 clearly cleaves two other BACE1
substrates, namely APP and PSGL1. This raises the possibility, that BACE1 and BACE2 may have similar
or identical substrates. However, both proteases do not necessarily cleave their substrates at identical peptide
bonds. For example, BACE1 cleaves APP at the Nterminus of Ab, whereas BACE2 cleavage occurs within the
Ab domain and thus functions as an alternative asecretase, precluding Ab formation. However, BACE2
cleavage of APP may not reduce Ab levels in the brain, given the low expression level of BACE2 in this organ.
At present, the specific function of BACE1 in the cleavage of its substrates remains unclear, but different
possibilities are plausible. Given that several BACE1 substrates are also substrates for ADAM metallopro-
teases, it is possible, that both proteases cleave the substrates for different purposes. BACE1 seems to be
constitutively active, whereas ADAM proteases are tightly regulated in their activity by different cellular
signal transduction pathways (Allinson et al., 2003). Therefore, BACE1 cleavage, which partly occurs in
the secretory pathway, could provide a lowlevel constitutive cleavage and secretion of membrane
proteins. In contrast, under conditions where a high level of secretion is needed (e.g., of a growth factor
or cytokine) the activity of ADAM proteases might be upregulated. Another function of BACE1 could be
the initiation of substrate protein degradation. In this scenario, BACE1 would cleave off most of the
ectodomain of its substrate and leave the remaining membranebound stub for additional degradation by
the gsecretase omplex. This idea is in agreement with the finding that BACE1 cleavage occurs after APP
endocytosis in the endosomes and that the resulting secreted ectodomain of APP (sAPPb) does not have
any known biological functions. In contrast, sAPPa has neuroprotective and neurotrophic properties
(Furukawa et al., 1996; Meziane et al., 1998) and is generated by ADAM proteases at or close to the
plasma membrane.
278
11 Alzheimers disease BACE proteases

7.2 BACE1 Deficient Mice

For many proteins, the use of knockout mice has strongly contributed to elucidating the proteins function.
This approach was very successful in understanding the in vivo function and the main in vivo substrates of
gsecretase and of the two asecretases ADAM10 and ADAM17. Deficiencies in ADAM10 (Hartmann et al.,
2002), ADAM17 (TACE) (Peschon et al., 1998) or in subunits of gsecretase (De Strooper et al., 1998)
resulted in severe phenotypic changes and embryonic or perinatal lethality in mice, revealing an essential
function of these proteases during development. A more detailed analysis showed that the phenotype of
these mice strongly correlated with the lack of cleavage of the major protease substrate during development.
Thus, gsecretase and ADAM10deficient mice show a phenotype characteristic for reduced or abolished
proteolytic processing of Notch, whereas ADAM17 knockout phenotype was caused by the lack of TGFa
cleavage and the resulting impaired EGFR signaling.
In contrast to mice deficient in a or gsecretase activity, BACE1 and BACE2 deficient mice show a very
mild phenotype (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001). BACE1
/
mice are viable and fertile,
ruling out an essential function for BACE1 during embryonic development. BACE1
/
mice do not
generate Ab, clearly implicating BACE1 as the physiological bsecretase. Despite the lack of overt neuro-
logical or physiological defects, even in older mice (Luo et al., 2003), BACE1deficient mice seem to have
more subtle defects. Harrison and colleagues recently reported that BACE1
/
mice are more timid and
anxious than control mice and show a less exploratory behavior correlating with increased 5HT turnover
in the hippocampus (Harrison et al., 2003). Whether BACE1 has a direct role in neurotransmitter
metabolism remains to be analyzed. Another study found that BACE1 deficiency rescued memory deficits
and cholinergic dysfunctions observed in an animal model of AD, the APP transgenic Tg2576 mouse
(Ohno et al., 2004). The same authors also found differences in spatial working memory between wildtype
and BACE1
/
mice. Clearly, more studies are needed to fully understand the BACE1 knockout phenotype,
in particular with regard to memory, cognition, and behavior. An additional field, where BACE1
/
mice
may have deficiencies, is the immune system, given that the two BACE1 substrates PSGL1 and ST6Gal I
are proteins with immune function. Because these mice have not yet been immunologically challenged
extensively, an immunological phenotype may not have been detected. Interestingly, BACE1 is only found
in vertebrates but not in C. elegans and D. melanogaster, suggesting that BACE1 may have special functions
in cellular processes developed later in evolution such as the elaborate immune system or complex brain
functions of vertebrates.

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neprilysin in Alzheimer disease and normal brain. Brain
Res 919: 115-121.
12 Protein Misfolding, a Common
Mechanism in the Pathogenesis of
Neurodegenerative Diseases
L. Vergara . K. Abid . C. Soto

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

2 Role of Protein Misfolding and Aggregation in Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

3 Clinical, Neuropathological, and Molecular Features of Neurodegenerative Diseases . . . . . . . . . 288

3.1 Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
3.2 Parkinson Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
3.3 Huntington Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
3.4 Amyotrophic Lateral Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
3.5 Prion Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

4 Animal Models of Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

5 Structural and Mechanistic Basis of Protein Misfolding and Aggregation . . . . . . . . . . . . . . . . . . . . . 294

6 Mechanism of Brain Degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

7 Targets for Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

8 Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

# 2008 Springer ScienceBusiness Media, LLC.

286 12 Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

Abstract: Neurodegenerative diseases are some of the most debilitating disorders, affecting abstract
thinking, skilled movements, emotional feelings, cognition, and memory. Recent and compelling evi-
dence indicates that the misfolding, aggregation, and accumulation of proteins in the brain may be the
cause of these diseases. The aim of this chapter is to review the literature around the molecular mechanism
and role of misfolded protein aggregates in neurodegeneration and the possibilities for therapeutic
intervention.

List of Abbreviations: AD, Alzheimers disease; ALS, amyotrophic lateral sclerosis; APP, amyloid-
precursor protein; BACE, b-amyloid cleaving enzyme 1; BSE, bovine spongiform encephalopathy; CJD,
Creutzfeldt-Jakob disease; CWD, chronic wasting disease; fCJD, Familial CJD; GSS, Gerstmann-Straussler-
Scheinker; HD, Huntington disease; iCJD, Iatrogenic CJD; ND, neurodegenerative diseases; NFT,
Neurofibrillary tangles; PD, Parkinson disease; RNAi, RNA interference; SCA, spinocerebellar ataxia;
sCJD, Sporadic CJD; SOD1, superoxide dismutase 1; TSE, transmissible spongiform encephalopathies;
vCJD, Variant CJD

1 Introduction

As a consequence of the longer life expectancy brought by the advances in medical care, the last decade has
observed an increase in the incidence of a number of neurodegenerative diseases (ND) including Alzhei-
mers disease (AD), Parkinson disease (PD), Huntington disease (HD) (and related polyglutamine dis-
orders including several forms of spinocerebellar ataxia or SCA), transmissible spongiform
encephalopathies (TSEs, which include several human and animal diseases), and amyotrophic lateral
sclerosis (ALS). This diverse group of diseases can affect abstract thinking, skilled movements, emotional
feelings, cognition, memory, and other abilities (Martin, 1999). Despite their differences in clinical
symptoms and disease progression, these disorders do share some common features: most of them (except
HD and SCA) have both sporadic and inherited origins, all of them appear later in life (usually after the
fourth or fifth decade), and their pathology is characterized by neuronal loss and synaptic abnormalities
(Martin, 1999).
Recent, but compelling evidences suggest that protein misfolding and aggregation is the most likely
cause of ND (Soto, 2003). The physiology of cells, tissues, and organisms depends on the correct activity of
a network of thousands of proteins. The function of a protein depends on its three-dimensional structure,
which is determined by its amino acid sequence. In ND, some normal proteins become improperly folded
and begin a process of polymerization to form large protein aggregates, commonly referred to as amyloid-
like deposits (Soto, 2003). Amyloid was the name originally given to extracellular protein deposits found in
AD and systemic amyloid disorders, but it has been recently used to refer also to some of the intracellular
aggregates. Amyloid is a generic term that refers to aggregates organized in a cross-b structure, which
contain specific morphological and tinctorial characteristics, such as binding to congo red and thioflavin S,
higher resistance to proteolytic degradation, and a fibrillar appearance under electron microscopy (straight,
unbranched, 10-nm-wide fibrils) (> Figure 12-1) (Glenner, 1980; Sipe, 1992).
Misfolding and aggregation of different proteins is responsible for distinct diseases, and no
structural homology or similar amino acid sequences have been demonstrated among the proteins
implicated in different ND. Protein misfolding and aggregation not only occurs in the brain, and indeed
several systemic disorders have also this molecular mechanism of pathogenesis, including diabetes type II,
serpin deficiency disorders, hemolytic anemia, amyloid polyneuropathy, cystic fibrosis, dialysis-related
amyloidosis, secondary or reactive amyloidosis, and more than 15 other diseases (Carrell and Gooptu,
1998; Soto, 2001).
In this chapter we will review the characteristics and molecular mechanisms implicated in the
pathogenesis of ND. We will also outline some of the strategies under development for the treatment of
these yet incurable disorders.
Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases 12 287

. Figure 12-1
Morphological, tinctorial, and structural characteristic of amyloid. (a) Tissue staining with antibodies specific for
the protein forming the aggregates. (b) Electron microscopy shows typical straight, unbranched fibrils, which
are 510 nm in diameter and 100 nm in length. (c) Structure of the typical amyloid-binding dyes Congo red and
Thioflavine T. (d) Cong red staining visualized under polarized light shows green/yellow birefringency. X-ray
fiber diffraction studies show a characteristic pattern known as cross-b, represented by signals at 4.7A and 9.7A.
(e) A diagrammatic picture for the cross-b structure showing the periodicity observed in X-ray diffraction
studies. (f) Computer modeling of the putative tridimensional structure of a prototype amyloid fibril viewed
from the side and from the top

2 Role of Protein Misfolding and Aggregation in Disease

Neuropathologic and genetic studies, along with the development of transgenic animal models, have
provided strong evidence for the involvement of protein misfolding in ND. Nearly 100 years ago, the
German neuropathologist Alois Alzheimer described for the first time the typical aggregates observed in the
288 12 Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

brain parenchyma of demented people (Alzheimer et al., 1995). These aggregates have now been
well characterized as formed by protein fibrils called amyloid plaques. The end point of protein misfolding
is usually aberrant protein aggregation and accumulation as amyloid-like deposits in the brain (Glenner,
1980; Sipe, 1992; Soto, 2003; Dimakopoulos and Dimakopoulos, 2005; Ross and Poirier, 2005). The
involvement of amyloid deposition in the pathogenesis of ND has been suggested by the colocalization
of protein aggregates with sites of tissue degeneration and its correlation with the symptoms of the
disease. Furthermore, protein deposits have become so frequent in affected individuals that their presence
is used for definitive diagnosis (Westermark, 1995; Gillmore et al., 1997). On the other hand, absolute
proof is still awaiting confirmation that aggregation itself is the culprit of the disease and not only an
inseparable epiphenomenon (Carrell and Gooptu, 1998; Tran and Miller, 1999; Goldberg and Lansbury,
2000; Caughey and Lansbury, 2003). Indeed, today the most accepted view is that the process of misfolding
and early stages of oligomerization, rather than the mature compacted aggregates deposited in the brain, are
the real culprits in neurodegeneration (Glabe and Kayed, 2006; Lansbury and Lashuel, 2006; Haass
and Selkoe, 2007).
Genetic studies have provided strong support for the role of protein misfolding in ND (Hardy and
Gwinn-Hardy, 1998; Selkoe et al., 2002). In addition to sporadic and infectious origin, ND can also be
caused by inherited defects. Epidemiological analysis has shown the presence of several loci in the human
genome responsible for diverse ND. Importantly, in most of the inherited cases, the mutated gene encodes
the protein that undergoes misfolding and aggregation. Thus it is believed that specific mutations in critical
amino acids may destabilize the normal protein folding, increasing its propensity to misfold and aggregate.
Mutations in the respective fibrillar proteins have been associated with familial forms of all ND, and also
with many other forms of systemic and brain disorders associated with protein misfolding and aggregation,
including amyloid polyneuropathy, cardiac amyloidosis, visceral amyloidosis, cerebral hemorrhage with
amyloidosis of the Dutch and Icelandic type, cerebral amyloidosis of the British and Danish type,
thromboembolic disease, angioedema, emphysema, sickle cell anemia, and diabetes type 2 (Jacobson and
Buxbaum, 1991; Kelly, 1996; Buxbaum and Tagoe, 2000). The familial forms usually have an earlier onset
and greater severity than sporadic cases.
As described later in this chapter, another strong evidence for the role of protein misfolding and
aggregation in ND has been the successful production of animal models by solely introducing the mutated
human gene encoding for the protein undergoing misfolding. In most of cases, this is enough to produce a
disease in the animals with similar pathological, clinical, and biochemical features as the human disease.

3 Clinical, Neuropathological, and Molecular Features of

Neurodegenerative Diseases

3.1 Alzheimers Disease

AD is the most prevalent ND, known to affect at least 5 million people in the United States alone, with more
than 300,000 new cases each year. About 10% of people over the age of 65 are affected by AD, and the
proportion increases dramatically with age. The patients suffer from progressive memory impairments,
disordered cognitive function, and decline in language function among other signs of dementia (Small and
Cappai, 2006).
The neuropathological features of AD include extensive neuronal loss and synaptic alterations mostly in
areas implicated in memory and cognition, and massive deposition of amyloid plaques and neurofibrillary
tangles (NFT) (Selkoe, 2004a; Small and Cappai, 2006). Amyloid plaques are composed of deposits of the
amyloid-b peptide (Ab). Ab is derived from a larger precursor protein, termed amyloid precursor protein
(APP), by sequential proteolytic cleavages (Selkoe, 1998). Ab deposits, in the form of insoluble fibrils, are
mostly found in the brain parenchyma and on the walls of cerebral blood vessels (Selkoe, 2001). The familial
forms of AD, although representing less than 5% of the patients affected, have provided strong evidence that
Ab is involved in the disorder and have been used to develop in vitro and experimental animal models of
AD. Several gene mutations have been identified to be involved in early-onset AD, including those encoding
Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases 12 289

for APP and the presenilins (PS1 and PS2) (Selkoe, 2001, 2004b). The presence of the e4 allele of
apolipoprotein E (ApoE) has been shown to be a major predisposition factor for the late-onset form of
AD (Rebeck et al., 1993; Gearing et al., 1996; Lane and Farlow, 2005). APP is a transmembrane cell surface
glycoprotein that can be found in several forms arising both from alternative splicing and posttranslational
modifications (Walter et al., 1997; Selkoe, 2004b). The APP695 isoform is the most abundant in neurons;
other forms are expressed in other cell types of the brain, as well as in other tissues. Although the function
of APP is unclear, its soluble form has been described as an autocrine (Saitoh et al., 1989) and a
neuroprotective (Mattson et al., 1993) factor. The presence of an ectodomain containing a KPI (serine
protease inhibitor domain of Kunitz type) motif has raised the possibility that APP may function as
a protease inhibitor. Indeed, APP has been shown to inhibit the serine protease factor XIa in the clotting
cascade (Smith et al., 1990). Other suggested functions include participation in cellcell and cellsubstrate
adhesions (Schubert et al., 1989; Qiu et al., 1995), and more recent findings have shown that APP may be
involved in the brain regulation of neural progenitor cell proliferation (Caille et al., 2004; Conti and
Cattaneo, 2005) as well as in axonal outgrowth after injury in a fly model (Leyssen et al., 2005). No evidence
has been obtained to support the hypothesis that a loss of cellular function of APP may explain AD
pathogenesis. Indeed, knockout mice with the APP gene are viable and fertile; however, they exhibit a
decreased locomotor activity and forelimb grip strength, both events occurring later in adult life (Zheng
et al., 1995).
APP can be cleaved by three different complexes of proteases; a, b, and g secretases. The Ab fragment
comprises the first 1214 residues of the transmembrane domain plus 28 extracellular residues (Sisodia,
1992). Cleavage by a-secretase gives rise to a large soluble fragment (sAPPa) and a smaller peptide of 83 aa
(c83), which is retained in the membrane. The c83 peptide can be further cleaved by g-secretase to generate
a small peptide (p3) that encompasses two-thirds of Ab, but is not involved in amyloid formation (Chyung
et al., 2005; Agiostratidou et al., 2006). Alternative cleavage of APP is carried out by b-secretase, also called
b-amyloid cleaving enzyme 1 (BACE), an aspartyl protease. BACE cleavage gives rise to the sAPPb (which is
soluble and secreted from the cell membrane) and the 99 c-terminal amino acids retained in the membrane.
The c99 fragment is heterogeneously cleaved by g-secretase to produce Ab 40 and 42 peptides, ending at the
amino acid 40 and 42, respectively (Selkoe, 2004a). Ab42 is the most prevalent form found in amyloid
plaques. It is more hydrophobic than Ab40 and thus more prone to aggregation, despite the fact that it is
normally less abundant than its counterpart in healthy individuals (Selkoe, 1998, 2004a). Even though Ab is
embedded in the membrane at the time cleavage occurs, it is subsequently released to the extracellular space
were it is able to initiate the formation of fibrils resulting in the genesis of amyloid plaques. Mutations
observed in APP-, PS1-, and PS2-encoding genes result in an increased production of total Ab peptides,
selective increase of the Ab42 variant, or in Ab forms more prone to misfold (Soto, 1999; Selkoe, 2001).
Presenilin 1 missense mutations have been shown to be responsible of causing the earliest and most
aggressive forms of AD. The genes of PS1 and PS2 were shown to encode for the two presenilins that
form the catalytic core of the g-secretase complex (Wolfe et al., 1999). PS1 and PS2 are highly homologous
and functionally redundant (Wolfe et al., 1999; Haass and Steiner, 2002; Brunkan and Goate, 2005).
Mutations within the PS genes might modify the structure of the presenilin, affecting the precision of the
g-secretase activity and usually resulting in an increase in production of the Ab42 peptide (Selkoe, 2001).
Even though more than 100 mutations that cause AD have been found for the PS1 gene alone, genetically
based AD represents a minority compared with sporadic cases.
NFTs, the other specific characteristic lesion found in the brain of AD patients, are long bundles of
fibers that form large vesicles within the cytoplasm. These fibers, structurally organized in pairs of
approximately 10 nm in length, are composed of the microtubule-associated Tau proteins, whose main
function is microtubule stabilization, but it has also been reported to be involved in membrane interaction
or acting as an anchor for enzymes (Sontag et al., 1999). The majority of the Tau proteins found in NFTs
are highly insoluble and hyperphosphorylated (Grundke-Iqbal et al., 1986; Kosik et al., 1986). NFTs are
biochemically similar to amyloid plaques, although these two well-known lesions of AD have been observed
independent of each other in affected patients. Unlike amyloid plaques, NFT formation has not been found
to be because of known mutations within the genes associated with familial AD (Armstrong, 2006).
Inherited mutations in the protein Tau lead to fronto-temporal dementia with parkinsonism (Goedert
290 12 Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

and Spillantini, 2000). In this disease, mutated Tau proteins accumulate in the cytoplasm as NFT without
extracellular amyloid plaques. Even though this disease is far less common than AD, it leads to similar
clinical manifestations (Hutton, 2001).

3.2 Parkinson Disease

PD is less prominent than AD; in the elderly it is estimated to affect around 1% of individuals over
65 years old (Savitt et al., 2006). PD was first characterized by James Parkinson in 1817, but more than a
century passed before the neuropathological features of PD were revealed. PD is a slowly progressive
motor disorder, characterized by tremor at rest, rigidity, slowness, absence of voluntary movements, and
freezing, as well as depression and dementia in older patients (Savitt et al., 2006). The disease is character-
ized by the progressive degeneration of dopaminergic cells and the presence of intracellular proteinaceous
aggregates known as Lewy bodies (Forno, 1996). These two events occur in a region of the brain termed
substantia nigra. Biochemical and neuropathological studies have identified a-synuclein as being the
major constituent of the intracellular aggregates (Spillantini et al., 1997). a-Synuclein is a highly conserved
140 amino acids protein found in the central nervous system, particularly abundant in the presynaptic
terminals in close association with synaptic vesicles (Clayton and George, 1998). The physiological role of
a-synuclein is still unclear, but it may regulate synaptic vesicle function, or be involved in synaptic plasticity
(Clayton and George, 1998). a-Synuclein has also been identified in several other neurologic disorders
known as a-synucleinopathies (Spillantini et al., 1997; Spillantini and Goedert, 1998) and in the
filamentous glial and neuronal inclusions of multiple-system atrophy (Goedert et al., 1998; Trojanowski
and Lee, 2003).
Genetic determinants have also been identified in PD, with familial cases representing less than 5% of
the affected individuals. Three different genes have been identified to contain mutations correlated with the
disease. In addition to a-synuclein, they encode parkin and the carboxy-terminal hydrolase L1 (UCHL1)
(Bertoli-Avella et al., 2004; Gandhi and Wood, 2005). Several additional loci have been shown to be
involved as risk factors for PD. Interestingly, as it is the case for AD, certain apolipoprotein E polymorph-
isms have been shown to represent a risk factor for sporadic PD (Huang et al., 2004, 2006). Parkin is a large
protein of 456 amino acids, mainly expressed in the brain and muscles, and functions as an E3 ubiquitin
ligase, a component of the ubiquitinproteasome system that plays a role in targeting misfolded proteins
to the proteasome for degradation (Sherman and Goldberg, 2001). UCHL1 is a thiol protease known to
hydrolyze ubiquitin chains back to the ubiquitin monomer (Wilkinson, 2000).

3.3 Huntington Disease

HD is an autosomal dominant inherited disorder characterized by uncontrolled movements, loss of
intellectual faculties, and emotional disturbance (Vonsattel and DiFiglia, 1998). In the United States
alone, about 30,000 people have HD; estimates of its prevalence are about 1 in every 10,000 persons. The
rate of disease progression and the age at onset vary from person to person. Adult-onset HD, with its
disabling, uncontrolled movements, most often begins in middle age. Some individuals develop symptoms
of HD when they are very youngbefore age 20. The terms early-onset or juvenile HD are often used
to describe this form of the disease (Gonzalez-Alegre and Afifi, 2006). Symptoms of juvenile HD include
subtle changes in handwriting and slight problems with movement, such as slowness, rigidity, tremor, and
myoclonus. Several of these symptoms are similar to those seen in PD, and they differ from the chorea seen
in individuals who develop the disease as adults. People with juvenile HD may also have seizures and mental
disabilities. The earlier the onset, the faster the disease seems to progress, and death often follows within
10 years.
The neuropathologic abnormalities include massive neuronal death mostly in the basal ganglia, which
is a brain region that participates in coordinating movement (Vonsattel and DiFiglia, 1998). Within the
Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases 12 291

basal ganglia, HD especially targets neurons of the striatum, particularly those in the caudate nuclei and the
pallidum. In severe cases, the brain cortex is also largely affected. Nuclear protein aggregates composed of
the huntingtin protein are a typical feature of HD (DiFiglia et al., 1997).
The large majority of HD cases are inherited in an autosomal dominant way, due to a triplet
repeat expansion of CAG (coding for the amino acid glutamine) in exon 1 of the gene encoding for
the protein huntingtin (Wanker, 2000). The HD gene comprises a region coding for a stretch of poly-
glutamine residues close to the N terminus of the protein. In the case of the triplet expansion observed in
patients, the longer glutamine portion promotes protein aggregation in the brain (Wanker, 2000). These
deposits are known to trigger oxidative and endoplasmic stress along with proteosomal and mitochondrial
dysfunction (Meriin et al., 2002; Nishitoh et al., 2002). Individuals with 35 or fewer triplet repeats
do not develop HD, in contrast, those with more than 40 repeats always develop the disease. Several
biological activities have been attributed to huntingtin, including cytoskeletal anchoring, axonal transport,
endocytosis, and intracellular trafficking (Cattaneo et al., 2001; Smith et al., 2005). Evidence also points to a
role in brain development and neuroprotection. Ablation of huntingtin in transgenic mice results in
embryonic death, and conditional deletion results in neurodegeneration, suggesting that huntingtin has a
protective role (Reiner et al., 2003). Furthermore, huntingtin has been shown to function as a caspase
substrate that is actively cleaved during apoptosis in cultured cells (Cattaneo et al., 2001). In vitro
experiments have revealed the ability of huntingtin to protect CNS cells from a variety of apoptotic stimuli,
including serum withdrawal, death receptors, and pro-apoptotic proteins activation (Cattaneo et al., 2001;
Reiner et al., 2003).

3.4 Amyotrophic Lateral Sclerosis

ALS, also called Lou Gehrigs disease, is a rapidly progressive, fatal ND that attacks motor neurons in the
brain and spinal cord. ALS is one of the most common neuromuscular diseases, which most commonly
affects people between 40 and 60 years of age, but younger and older people also can develop the disease
(Pasinelli and Brown, 2006). It is estimated that around 20,000 people in the United States have ALS, and
around 5,000 new cases are diagnosed every year in this country alone. The disease appears mostly in a
sporadic form, but around 510% of the cases are inherited. About 20% of all familial cases result from
mutations in the gene encoding for the enzyme known as Cu/Zn superoxide dismutase 1 (SOD1) (Pasinelli
and Brown, 2006). Recently, several other genes have been linked to familial ALS, including the vascular
endothelial growth factor, Angiogenin, and the vesicle-associated membrane protein B (Lambrechts et al.,
2006). The other genes responsible for familial ALS remain to be identified.
ALS is characterized by progressive degeneration of both the upper and lower motor neurons. The
neuronal degeneration produces a decreased muscle function, leading to the gradual weakening of the
muscles, which finally waste away and twitch (Price et al., 1996; Pasinelli and Brown, 2006). Individuals
with ALS lose their ability to start and control voluntary movements, and the ability to move their arms,
legs, and body. When muscles in the diaphragm and chest wall fail, individuals lose the ability to breathe
without external support. Patients in the later stages of the disease may become totally paralyzed, yet, for the
vast majority of people, their sensorial and cognitive abilities remain unaffected. Most people with ALS die
from respiratory failure, usually within 35 years from the onset of symptoms.
Cytoplasmic protein aggregates in cell bodies and axons of motor neurons are observed in both
sporadic and familial ALS cases (Bruijn et al., 1997). However, in ALS the role of protein misfolding and
aggregation has been studied much lesser than that in other ND, and the term aggregate has been used to
refer to a variety of different morphological structures. The major component of ALS aggregates appears to
be SOD1, but they also contain substantial quantities of ubiquitin and other proteins. ALS pathology has
been observed in mice bearing the human mutated SOD1 gene (Gurney et al., 1994). These mice develop
motor neuron dysfunction and typical pathological alterations, including the presence of hyaline inclusion
bodies in degenerating axons, muscle atrophy, astrocytic damage, and extensive loss of large myelinated
axons of motor neurons (Gurney et al., 1994).
292 12 Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

3.5 Prion Diseases

Prion diseases, also called TSE, are a group of fatal neurodegenerative disorders that affect humans and
other mammals (Collinge, 2001). A unique feature of these diseases is that they can have three different
origins: sporadic, inherited, and infectious. The clinical, epidemiological, and neuropathological features
can be very different in each of the diseases, but they are classified together because the key molecular event
appears to be the same, that is, the misfolding of the prion protein (Prusiner, 1998; Collinge, 2001).
CreutzfeldtJakob disease (CJD) is the most common form of TSE in humans. CJD is a rare disease with an
estimated incidence rate of about one new case per million people each year. There are four different forms
of CJD: (1) Sporadic CJD (sCJD) refers to those cases in which there is no known infectious source and no
evidence of the disease in the prior or subsequent generations of the patients family. This is the most
common subtype corresponding to about 85% of cases. Patients are usually between 50 and 75 years old
and typical clinical features include a rapidly progressive dementia and myoclonus followed by loss of the
ability to move or speak (Johnson and Gibbs, 1998). The disease is 100% fatal and in most of the cases death
occurs within a year since the appearance of first clinical symptoms (Brown et al., 1986; Johnson and Gibbs,
1998; Weber, 2000; Collinge, 2001). (2) Familial CJD (fCJD) is an inherited disease that represents
approximately 1015% of the total CJD cases. In most of these kindreds, point mutations, deletions, or
insertions are found in the coding sequence of the prion protein gene (Prusiner and Scott, 1997; Kovacs
et al., 2002). More than 30 mutations in this gene have been described that are associated with phenotypes
mimicking typical CJD or that induce distinctive progressive diseases with spongiform changes in the
nervous system (Prusiner and Scott, 1997; Kovacs et al., 2002). In general, fCJD has an earlier age of onset
and a more prolonged course than sporadic disease. (3) Iatrogenic CJD (iCJD) represents less than 5% of
the cases and results from transmission of the causative agent via medical or surgical interventions using
accidentally contaminated materials (Brown et al., 2000; Will, 2003). Iatrogenic transmission of CJD has
occurred in cases involving corneal transplants, implantation of electrodes in the brain, dura mater grafts,
contaminated surgical instruments, and treatment with human growth hormone derived from cadaveric
pituitaries (Brown et al., 2000; Will, 2003). (4) Variant CJD (vCJD) is a new disease, which was first
described in March 1996 (Will et al., 1996). In contrast to typical cases of sCJD, this variant form affects
young patients (average age 27 years old) and has a relatively long duration of illness (median 14 months vs.
4.5 months in traditional sCJD). vCJD patients usually experience psychiatric symptoms early in the illness,
which most commonly take the form of depression or less often a schizophrenia-like psychosis (Will et al.,
2000). Compelling evidence suggest that vCJD has an infectious origin and is acquired by consumption or
exposition to cattle meat contaminated by the cow form of TSE, known as bovine spongiform encephalop-
athy (BSE) (Bruce et al., 1997; Smith et al., 2004). Insufficient information is available at present to make
any well-founded prediction about the future number of vCJD cases (Hilton, 2000; Balter, 2001; Smith
et al., 2004). In addition to CJD, three other clinically or pathologically similar prion diseases have been
recognized in humans, including kuru, GerstmannStrausslerScheinker (GSS) disease, and fatal insomnia
(Collinge, 2001).
TSEs are known to affect various animal species including sheep, goats, mink, deer, elk cows, cats and
exotic felines, and ungulates (Collinge, 2001). The most common animal TSE is scrapie, a disorder of sheep
and goats that was first recognized in 1730 and became an endemic problem in several countries (Detwiler,
1992). However, the most worrisome zoonotic TSEs from the point of view of public health are BSE, the
cattle disease that is known to have infected humans, and chronic wasting disease (CWD), a disease of wild-
range and captive cervids that has spread uncontrollably in North America. The typical neuropathological
alterations in TSEs are vacuolation of the neuropil in the gray matter, prominent neuronal loss, exuberant
reactive astrogliosis, and a variable degree of cerebral accumulation of prion protein aggregates (Wells,
1993; Budka et al., 1995; MacDonald et al., 1996). The most specific of these abnormalities is the
vacuolation, giving the brain the appearance of a sponge and hence the name of spongiform encephalo-
pathies. A characteristic feature of TSE neuropathology is the large degree of variation in the distribution
and magnitude of the abnormalities (Budka et al., 1995; Jeffrey et al., 1995). These variations have been
attributed to several factors including the kinetics of prion propagation, cell tropism, differential toxicity,
host neuro-anatomy, and genetic makeup.
Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases 12 293

The nature of the transmissible agent is perhaps the most extensively studied aspect of TSE research
(Soto and Castilla, 2004). Compelling evidence suggest that the infectious agent is composed mainly (or
exclusively) of a misfolded protein that can propagate in the absence of nucleic acid. The proteinaceous
nature of the agent led to its denomination as prion, and extensive research has enabled to identify that a
b-sheet-rich misfolded form of the prion protein (termed PrPSc) is the main component (Prusiner, 1998).
Infection with PrPSc produces the autocatalytic conversion of the normal form of the prion protein
(termed PrPC) into more of PrPSc in a process that results in the formation of a toxic structure responsible
for brain damage and disease. The fact that administration of highly purified PrPSc (>99%) is sufficient to
induce the disease in experimental animals provides a strong support not only for the prion hypothesis, but
also for the key role of protein misfolding in neurodegeneration.

4 Animal Models of Neurodegenerative Diseases

Several animal models have provided tremendous amount of information in understanding the mechan-
isms underlying ND (Bilen and Bonini, 2005; Marsh and Thompson, 2006). Drosophila has been used for
modeling most syndromes, because the availability of powerful genetic tools allows testing many possible
genetic modifiers. In addition, the architecture of the fly nervous system presents several similarities with
the mammalian CNS, like compartmentalized brain functions such as olfaction, vision, or memory. Fly
models are engineered by cloning the genes responsible for the diseases observed in humans into transpo-
sable p-element vectors under the control of a yeast transcription system and injected into fly embryos
to obtain transgenic animals (Bilen and Bonini, 2005). Neuronal degeneration is typically measured
by observation of the structure of the photoreceptor neurons of the eyes and motor function. Like in
humans, transgenic drosophila models for human genes involved in ND develop progressive neuropathol-
ogy (Bilen and Bonini, 2005; Marsh and Thompson, 2006). For example, upon expressing polyQ-
tract extended protein or mutant alpha synuclein, tau, or Ab the number of intact photoreceptors
rapidly decreased after the day of eclosion (Jackson et al., 1998). Aggregates of misfolded proteins are
also found in transgenic flies, expressing the mutant human proteins, supporting the fact that fly is a
suitable model for neurodegeneration (Bilen and Bonini, 2005; Marsh and Thompson, 2006). However,
major limitations exist, the most significant ones including differences in morphology and functionality of
the brain, differences in metabolism, a lack of or reduced inflammatory response, or the absence of the
bloodbrain barrier.
Another animal model used to study ND is the worm Caenorhabditis elegans (Link, 2006; Brignull et al.,
2007). This organism has an invariant number of 959 cells, of which, remarkably around one-third (302)
are neuronal. Microinjection of mutated proteins results in several models of human disease (Kraemer
et al., 2003; Miyasaka et al., 2005; Wu et al., 2005). Transgenic C. elegans expressing Ab presents deposits of
the peptide in muscle cells with formation of vacuolation and motor impairment (Link, 1995). The
Huntingtin gene fused with expanded triplet repeats has been shown to aggregate in the cytoplasm of the
worm and to induce progressive cell death. Interestingly, the toxicity of polyQ-expanded protein was shown
to occur at a threshold of approximately 40 glutamine residues, similar to the human disease (Morley et al.,
2002). RNA interference (RNAi) technology has recently demonstrated that up to 186 genes might be
involved in the response to protein misfolding events in transgenic worms expressing yellow fluorescent
protein fused with an expanded polyQ tract (Nollen et al., 2004).
Considering the complexity of neurological diseases, it is no surprise that transgenic mice have been the
most popular model to study the contribution of protein misfolding in the pathogenesis of these diseases
(Price et al., 2000). Several pathological, biochemical, and clinical features of diverse ND have been
reproduced in transgenic mice models expressing mutant forms of the human genes encoding the
corresponding fibrillar protein (Tom Van Dooren et al., 5 A.D.; Araki et al., 1994; Janson et al., 1996; Price
et al., 1998; Weissmann et al., 1998; Emilien et al., 2000; Gurney, 2000; Price et al., 2000; Grieb, 2004).
Transgenic mice overexpressing the human APP containing diverse mutations responsible for AD progres-
sively develop many of the pathological hallmarks observed in humans, including cerebral amyloid
deposits, neuritic dystrophy, astrogliosis, and cognitive and behavioral alterations (Duff, 1998; Price
294 12 Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

et al., 1998; Emilien et al., 2000). Similarly, transgenic mice overexpressing the human-mutated SOD1 gene
involved in ALS pathology have shown to develop signs corresponding to the disease found in ALS patients
(Price et al., 1997, 1998). Some of these mice developed motor neuron dysfunction and typical pathological
alterations, including the presence of hyaline inclusion bodies in degenerating axons, muscle atrophy,
astrocyte damage, and extensive loss of large myelinated axons of motor neuronal cells (Grieb, 2004).
Transgenic mice expressing the wild-type form of the human a-synuclein gene develop several of the
clinicopathological features of PD (Hashimoto et al., 2003). Among them are Lewy bodies accumulated in
neurons of the neocortex, hippocampus, and substantia nigra, loss of dopaminergic terminals in the basal
ganglia, and the associated motor impairments (Klement et al., 1998; Masliah et al., 2000). The transgenic
mice model of HD expressing exon 1 of the human huntingtin and carrying 115156 CAG repeat
expansions develop pronounced neuronal intranuclear inclusions (Davies et al., 1997). These typical
cerebral lesions were found to contain huntingtin and ubiquitin protein aggregates that appeared prior
to the development of the neurological phenotype and were strikingly similar to those observed in HD
patients. Furthermore, these mice develop a progressive neurological dysfunction with a movement
disorder and weight loss similar to HD (Sathasivam et al., 1999). One of the first animal models mimicking
a human misfolding protein disorder was obtained when the mutated gene PrnP, coding for human prion
protein (PrP), was overexpressed in a transgenic mice (Hsiao et al., 1990). Spontaneous neurologic disease
with spongiform degeneration was observed in these mice. Moreover, these abnormalities were found to be
transmissible to some transgenic mice by inoculation of the sick brain homogenate.
Thus, the critical role for protein misfolding and aggregation as the culprit of neurodegeneration is
strongly supported by transgenic animal models in which expression of the human proteins involved in
abnormal folding resulted in typical clinical and pathological features. However, time course studies of the
appearance of neurological signs of the disorder in some of the transgenic models have shown that visible
protein aggregates appear after clinical symptoms and significant tissue damage (Janson et al., 1996;
Moechars et al., 1999; Van Leuven, 2000). These observations suggest the presence of a misfolded soluble
intermediate that does not form large detectable aggregates and is not accumulated in the brain, which
could be the real culprit for the pathogenesis (Tran and Miller, 1999; Goldberg and Lansbury, 2000;
Caughey and Lansbury, 2003). This hypothesis implies that the formation of the characteristic aggregates
could be considered as a protective mechanism working by sequestering the possible toxic misfolded
proteins (Caughey and Lansbury, 2003).

5 Structural and Mechanistic Basis of Protein Misfolding and Aggregation

No sequence or structural homology has been found among the native proteins implicated in ND. However,
there is accumulating evidence that the aggregates formed by the different misfolded proteins have a similar
molecular structure (Soto, 2003; Glabe and Kayed, 2006; Sawaya et al., 2007). The process of misfolding and
aggregation of most of the proteins involved in ND has been modeled in vitro. Large secondary structural
differences between the monomeric native protein and the aggregated material have been observed using
low-resolution structural studies (Makin and Serpell, 2005). The native protein is generally folded into a a-
helix structure while the misfolded protein is rich in b-sheet conformation. High-resolution studies have
been difficult due to the insoluble and noncrystalline nature of the aggregated proteins. However, recent
studies using X-ray fiber diffraction and solid-state nuclear magnetic resonance have confirmed the b-sheet
structure of the protein aggregates implicated in ND (Makin and Serpell, 2005; Nelson and Eisenberg, 2006;
Tycko, 2006).
Structural studies performed on misfolded proteins reveal that amyloid-like fibrils are composed of
several protofilaments that consist of hydrogen-bonded b-sheets with the b-strands running perpendicular
to the long fiber axis, a structure known as a cross-b conformation (Makin and Serpell, 2005; Makin et al.,
2005). These structural analyses demonstrate that a large conformational rearrangement of the polypeptide
chain occurs during misfolding and aggregation. However, it is presently unclear whether the misfolding
triggers protein aggregation or whether it is the protein oligomerization that induces the conformational
changes. This issue is of the highest interest, especially for the design of effective therapeutic strategies.
Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases 12 295

Although the detailed mechanism for the formation of fibrillar amyloid-like aggregates is not entirely
clear, the initiating event is protein misfolding, which results in the formation of aggregation-prone
structures that can eventually grow by an autocatalytic mechanism (Soto, 2003). Kinetic studies have
suggested that the critical event is the formation of protein oligomers that act as seeds to further propagate
protein misfolding (Soto et al., 2006). This is the basis for the currently accepted nucleation-dependent
polymerization model of amyloid formation (Gajdusek, 1994; Harper and Lansbury, 1997; Soto et al.,
2006). Diverse proteins have been shown to follow this crystallization-like process, including Ab,
huntingtin, and a-synuclein among others. According to this model, aggregation starts after the protein
concentration exceeds a point known as the critical concentration (Harper and Lansbury, 1997). Unfavor-
able interactions between monomers determine a slow phase (termed lag phase) in which oligomers are
formed, providing an ordered nucleus to catalyze further growth of the polymers. The addition of
preformed nuclei (seeds) serves as templates for the reaction and, as a result, the initial, slow phase of
primary nucleation is eliminated (Harper and Lansbury, 1997; Soto et al., 2006).
In vitro studies have been useful for understanding the structural requirements for protein misfolding
and the fragments of the protein mostly implicated in the conformational changes. Using mutated peptides
or shorter Ab fragments it has been shown that the internal hydrophobic region between amino acids 17
and 21 is essential in the early steps of Ab misfolding and aggregation (Soto, 1999). This finding
demonstrates that Ab assembly is partially driven by hydrophobic interactions (Hilbich et al., 1992; Soto
et al., 1995; Wood et al., 1995; Lazo et al., 2005), which is consistent with the higher ability of Ab peptides to
aggregate with two or three extra hydrophobic amino acids at the carboxy terminus (Jarrett et al., 1993).
Indeed, hydrophobic forces appear to be crucial in the aggregation process of most of the misfolded
proteins. In PrPc misfolding, the hydrophobic fragment 106126 of PrPc has been mapped to represent the
most relevant sequence for protein oligomerization (Tagliavini et al., 1993; Ziegler et al., 2006). A similar
observation has been reported concerning the fibrillogenesis of a-synuclein. Although less is known about
this process, evidence indicates that the amino-terminal fragment 187 might be crucial (Serpell et al., 1995,
2000). However, the aggregation process involved in huntingtin and other polyglutamine-containing
proteins seems to be different. In HD and other polyglutamine diseases, both disease and protein aggrega-
tion are associated with an inherited expansion of CAG (the codon for glutamine) repeats. In the case of
HD, aggregation of huntingtin in vitro depends on the length of the polyglutamine repeat (Scherzinger
et al., 1997; Burke et al., 2003), and the aggregation process is driven by the glutamine residue, which
contains an amide group that provides a polar side chain and the potential to form a hydrogen bond with
water in a model termed polar zipper (Perutz et al., 1994; Chan et al., 2005). In this model, b-sheets are
formed and stabilized by the collective strength of cooperative hydrogen bonding involving the amide
groups of the glutamine residues. Therefore, protein aggregation can arise from two different (and, in some
respects, opposite) driving forces: hydrophobic interactions and polar hydrogen bonding among side-chain
groups.
In addition to mature fibrils, several other structures have been described as part of the protein
misfolding and aggregation process, including soluble oligomers, pores, annular structures, spherical
micelles, and protofibrils (Caughey and Lansbury, 2003; Glabe and Kayed, 2006; Haass and Selkoe,
2007). Interestingly, these diverse structures have been identified in the amyloidogenesis process of various
disease-associated proteins, suggesting common misfolding pathways (Caughey and Lansbury, 2003; Glabe
and Kayed, 2006; Haass and Selkoe, 2007). However, currently, the biological relevance of these inter-
mediates is not clear, and it is even unknown whether or not they exist in the diseased brain. Furthermore,
although it is likely that these metastable species assemble in a stepwise process, the relative implication of
each of them is difficult to assess because they are too unstable to study (Glabe and Kayed, 2006; Teplow
et al., 2006). Recently, a set of antibodies that recognize specifically different type of aggregated species, such
as oligomers, annular assemblies, protofibrils, and fibrils, have been produced (Glabe and Kayed, 2006;
Kayed and Glabe, 2006). Strikingly, these antibodies are not sequence specific, but conformation specific
and thus they can recognize intermediate species formed by different proteins (Glabe and Kayed, 2006;
Kayed and Glabe, 2006). In summary, the biophysical studies of the intermediates in the amyloid formation
process indicate that diverse species with progressive degree of aggregation are present simultaneously and
in a dynamic equilibrium between each other (Caughey and Lansbury, 2003; Glabe and Kayed, 2006;
296 12 Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

Teplow et al., 2006). This makes evaluation of the relative contribution of different protein structures to
neurodegeneration very difficult.

6 Mechanism of Brain Degeneration

Besides of protein deposits, the typical pattern of brain damage in ND includes selective neuronal death,
synaptic alterations, and astrogliosis (Martin, 1999). The different clinical picture associated with distinct
ND is most likely determined by the diverse brain regions most affected in each case. Although it was widely
thought that neuronal apoptosis was the most important problem in neurodegeneration, recent evidence
from different diseases suggest that extensive neuronal death may not be the initial cause of the disease
(Haass and Selkoe, 2007). Indeed, clinical symptoms can be observed before significant neuronal loss and a
better temporal and topographic correlation is found with synaptic dysfunction (Haass and Selkoe, 2007).
Although protein misfolding and aggregation is undoubtedly associated with neurodegeneration and
disease, the mechanism by which this process leads to brain damage is unknown. The most widely accepted
theory of brain degeneration in ND proposes that misfolding and aggregation results in the acquisition of a
neurotoxic function by the misfolded protein (Soto, 2003). Although the possibility that neuronal damage
may be produced by the loss of activity of the protein upon the conformational changes has been extensively
studied, most of the evidence argues against this mechanism (Soto, 2003). The mechanism by which
misfolded aggregates produce synaptic dysfunction and neuronal death is unknown. It is also unknown
which of the different polymeric structures formed in the process of amyloidogenesis is the triggering factor
of brain damage (Lansbury and Lashuel, 2006; Haass and Selkoe, 2007) (> Figure 12-1). For many years it
was thought that big amyloid-like protein deposits were the species responsible for brain damage. However,
the hypothesis that large aggregates accumulated in the brain are toxic has been challenged by histopatho-
logical, biochemical, and cell biology studies (Lansbury and Lashuel, 2006; Haass and Selkoe, 2007). The
current view is that the process of misfolding and early stages of oligomerization, rather than the mature
compacted aggregates deposited in the brain, are the real culprits in neurodegeneration (Glabe and Kayed,
2006; Lansbury and Lashuel, 2006; Haass and Selkoe, 2007). This hypothesis is supported by results showing
that purified oligomeric species and protofibrils are toxic to cultured neurons, inhibit hippocampal long-
term potentiation, impair synaptic functions, and disrupt cognition and learned behavior in rats (for list of
references, see Glabe and Kayed (2006), Lansbury and Lashuel (2006), and Haass and Selkoe (2007)).
Several mechanisms have been proposed for the neurotoxic activity of misfolded oligomers, and it is
likely that different pathways operate, depending on whether the proteins accumulate intra- or extracellu-
larly (Soto, 2003). Extracellular oligomers might activate a signal transduction pathway leading to apoptosis
by interacting with specific cellular receptors. Intracellular polymers might damage cells by, for example,
recruiting factors essential for cell viability. Another well-supported mechanism by which oligomeric and
annular pore-like structures may damage cells is by membrane disruption and depolarization, resulting in
alterations of ion homeostasis and disregulation of cellular signal transduction (Glabe and Kayed, 2006).
Finally, misfolded proteins could induce cellular oxidative stress by producing free radical species, resulting
in protein and lipid oxidation, elevation of intracellular calcium, and mitochondrial disfunction (Behl et al.,
1994; Hsu et al., 2000).

7 Targets for Therapy

In spite of the extensive knowledge collected in recent years toward understanding the pathogenesis of ND,
there are no effective therapies directed to prevent or reverse the disease progression. If protein misfolding
and aggregation play a central role in the pathogenesis of ND, then the strategy to follow should be the
prevention and/or the correction of the misfolding process. At least three different strategies have been
proposed to intervene against protein misfolding and aggregation (Soto, 2003): (1) stabilization of the
native protein conformation; (2) inhibition and reversion of protein conformational changes; and (3)
increase the clearance of the misfolded protein.
Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases 12 297

Stabilization of the native folding of the normal protein has been attempted in several protein
misfolding disorders. In the case of AD, nicotine and cotinine have been shown to inhibit Ab amyloid
formation by specifically binding to the peptide and stabilizing its alpha helix structure (Salomon et al.,
1996; Ono et al., 2002). In prion-infected neuroblastoma cells, treatment with chemical chaperones
(reagents known to stabilize native conformation of proteins) results in prevention of PrP misfolding
(Tatzelt et al., 1996; Bennion et al., 2004). Protein engineering has been proposed as an approach to create
sequence-modified proteins with higher stability, lower tendency to misfold, and ability to trans-suppress
the aggregation of wild-type proteins (Villegas et al., 2000). Although the idea is attractive, its therapeutic
application would require sophisticated gene therapy technologies.
Several small molecules, peptide fragments, specific antibodies, and proteins capable of interacting with
b-sheet aggregates have been shown to specifically interact and inhibit protein aggregation (Aguzzi et al.,
2001; LeVine, 2002; Sacchettini and Kelly, 2002; Bose et al., 2005; Soto and Estrada, 2005). Several small
chemical molecules have been reported to have this activity, including Congo red, 40 -iodo-40 -deoxydoxor-
ubicin, curcumin, rosmarinic acid, rifampicine, polyphenol, tetracycline, minocyclin, and ferulic acid
among others (Forloni et al., 2001, 2002; Cardoso et al., 2003; Ono et al., 2005; Familian et al., 2006).
The majority of the active compounds produce their effect by competitively blocking proteinprotein
interactions. These compounds can be classified in two classes depending on their ability to either interact
with the complexes in between monomers or to bind at the edge of b-sheet oligomers, preventing their
growth. One of the major problems with this approach is to determine the mechanism of action of
these competitive inhibitors. Depending on their activity, it is likely that some inhibitors may lead to
accumulation of toxic intermediates and thus may aggravate the disease. Another class of inhibitors
compounds act by destabilization of the pathological b-sheet conformation of misfolded proteins. One
example of this strategy are b-sheet breaker peptides that have been shown to inhibit and reverse protein
misfolding in vitro and in vivo (Soto and Estrada, 2005). b-Sheet breakers are short synthetic peptides that
can specifically interact with the protein fragment that is undergoing misfolding but has been designed to
be unable to get incorporated into the b-sheet structure. b-Sheet breaker peptides have been designed
to block the conformational changes and aggregation of both Ab and PrP in vitro and in various animal
models (Soto et al., 1998, 2000; Permanne et al., 2002).
Another strategy is to increase the clearance of misfolded and aggregated proteins. This alternative
represents an interesting approach based on findings showing that accumulation of protein aggregates
is dependent on a balance between deposition and clearance. In a conditional transgenic model of
HD, preventing the production of mutant fragments caused nuclear inclusions to disappear, indicating
that cells can metabolize the aggregated material (Yamamoto et al., 2000). Perhaps the most promising
strategy to increase the clearance of misfolded proteins is the immunization approach first described for
AD (Schenk et al., 1999). Aggregates of synthetic Ab protein were used as antigens to induce the immune
system to produce antibodies to clear them. Immunization can reduce amyloid load, cerebral damage,
and behavioral impairments in transgenic animal models of AD (Schenk et al., 1999; Janus et al., 2000;
Morgan et al., 2000; Boche et al., 2005; Gandy and Heppner, 2005; Goni and Sigurdsson, 2005; McGeer
et al., 2005; McGavern, 2006). A similar approach has been used for the treatment of TSE (Sigurdsson et al.,
2002; Cashman and Caughey, 2004; Boche et al., 2005; Goni and Sigurdsson, 2005; Magri et al., 2005).
However, a clinical trial to evaluate the efficacy of the immunization strategy in humans affected by AD was
stopped because of several cases of meningoencephalitis (Schenk, 2002). Little is known about the reason
for this side effect, but future research should bring both more knowledge about this problem and also
should result in new strategies to minimize brain inflammation after vaccination. Immunization with
fragments of Ab that preferentially stimulate the B cells (and not the T cells) or passive immunization
with antibodies against the misfolded proteins might be safer strategies (Schenk, 2002). Enhancement of
the clearance of amyloid-like deposits has been also attempted by removing some accessory constituents
found in plaques. The underlying idea is that other factors that tightly bind to the aggregates might
increase their insolubility and resistance to proteolytic degradation. Strategies for removing specifically the
amyloid-P component, proteoglycans, and metal ions have shown the best results in models of AD
and systemic amyloidosis (Kisilevsky et al., 1995; Cherny et al., 2001; Pepys et al., 2002; Lee et al., 2004;
Maynard et al., 2005).
298 12 Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

8 Conclusions and Perspectives

Over the last 10 years considerable progress has been made in our understanding of the molecular
mechanisms responsible for ND. The observation that several syndromes are histologically characterized
by accumulation in the brain of normal proteins has led to the hypothesis that most ND are caused by a very
similar cascade of events. Misfolding of distinct proteins appears to be the initiating step that eventually
leads to neuronal dysfunction and clinical symptoms specific for each disease. Formation of amyloid
structures in the brain is the visible consequence of abnormalities in protein folding, but recent data
suggest that the plaques themselves may not be the triggering factor in neurodegeneration. The similarities
observed in the different ND provide hope for a common therapeutic strategy to treat these devastating
illnesses. The development of a therapy based on the misfolding concept may also enable a better
understanding of the mechanisms that eventually lead to amyloid formation.
The concept that protein misfolding leads to severe illness is relatively new; thus, one important
research for the future is to find out whether other diseases are associated with similar alterations. For
example, a report demonstrated that atherosclerosis might indeed be a consequence of ApoB misfolding
induced by high cholesterol level (Ursini et al., 2002). This raises the possibility that other common diseases
might directly or indirectly be a consequence of the misfolding of proteins. During many years it was
thought that only certain proteins could undergo misfolding and eventually form amyloid-like structures,
but this concept has been reconsidered since recent studies have shown that many, if not all, proteins can
form amyloid-like structures under appropriate conditions (Fandrich et al., 2001; Stefani and Dobson,
2003). Even though most of the data were obtained for proteins incubated in nonphysiological pH or salt
concentrations, it cannot be excluded that in certain cellular compartments, these conditions could be
reached transitorily, leading to a seeding event that would trigger the aggregation process. One important
open question is whether protein misfolding always leads to disease or rather a similar phenomenon may
play a normal biological function. The recent demonstration that several proteins can misfold and aggregate
into amyloid-like structures (Lindquist, 1997; Wickner et al., 1999; Iconomidou et al., 2000; Wosten and de
Vocht, 2000; Chapman et al., 2002; Kenney et al., 2002; Berson et al., 2003; Bieler et al., 2005), changing the
biological activity of the protein suggest that the acquisition of alternative protein folding is indeed not only
a disease-associated process. The possibility that many proteins may adopt multiple conformations to exert
different functions might revolutionize our understanding of biology.

Acknowledgments

This project was supported in part by NIH grant R01 AG028821 and an award from CART Foundation.

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13 AntiAging Strategies
J. A. Joseph . J. R. PerezPolo

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

2 Muscarinic Receptors and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

3 Muscarinic Receptors and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

4 Consequences of Aging on Cholinergic Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

5 The Effects of Fruit and Vegetable Supplementation on Behavioral and Neuronal

Deficits in Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

# 2008 Springer ScienceBusiness Media, LLC.

306 13 Antiaging strategies

Abstract: There are age-associated motor and cognitive deficits, even in the absence of neurodegenerative
disease, that result from alterations in the striatal dopamine or cholinergic systems, respectively. In both
instances, oxidative stress and inflammation are contributing factors to the observed behavioral impair-
ments. Muscarinic acetycholine receptors (mAChR) play important roles in neuronal and vascular func-
tion. There is a loss of sensitivity in classic receptor systems that include the dopaminergic and muscarinic
systems. It appears that a major factor that may be important in the loss of sensitivity in mAChR with
aging may be increased sensitivity to oxidative stress that is at least partially determined by changes in
membranes. Also possibly contributing to the loss of cholinergic integrity and ultimately behavioral
function in aging are perturbations in ChAT function. There are fewer cholinergic neurons in the aged
basal forebrain that express lower levels of cholinergic activity. For example, decreased ACh synthesis in
cholinergic neurons of the aged rat basal forebrain may result in part from altered binding of NF-kB to a
cognate DNA-binding repressor sites within the ChAT promoter. Given that NF-kB DNA-binding activity is
elevated in the aged rat basal forebrain and hippocampus, the result is age-associated decreases in ChAT
expression that have consequences on cognitive function in the aged. Given the role of oxidative stress in
aging, Anti-oxidant based fruit and vegetable supplementation could forestall the consequences of oxidative
stress on behavioral and neuronal deficits associated with aging.

List of Abbreviations: BC, black currant; BY, boysenberry; CB, cranberry; CHL, cholesterol; DA,
dopamine; DP, dried plums; ERK, extracellular signalregulated kinase; GR, grape; MPT, mitochondrial
pore transition; ORAC, oxygenradical absorbance capacity assay; PKC, protein kinase Ca; RIP, receptor
interacting protein; SAM, Sadenosylmethionine; SB, strawberry; TNFR, tumor necrosis factor receptor;
TRAF2, TNFRassociated factor 2; VaD, vascular disease

1 Introduction

There is a plethora of research indicating the occurrence of numerous behavioral deficits during aging, even
in the absence of neurodegenerative disease. These decrements can be expressed, ultimately, as alterations in
both motor (Joseph et al., 1983; Kluger et al., 1997) and cognitive behaviors (Bartus, 1990). The alterations
in motor function may include decreases in balance, muscle strength, and coordination (Joseph et al.,
1983), while memory deficits are seen on cognitive tasks that require the use of spatial learning and memory
(Ingram et al., 1994; ShukittHale et al., 1998). Indeed, these characterizations have been supported by a
great deal of research both in animals (Bartus, 1990; Ingram et al., 1994; ShukittHale et al., 1998) and
humans (West, 1996; Muir, 1997). Agerelated deficits in motor performance are thought to be the result of
alterations in the striatal dopamine (DA) system as the striatum shows marked neurodegenerative changes
with age (Joseph, 1992) or in the cerebellum which also shows agerelated alterations (Bickford et al., 1992;
Bickford, 1993). Memory alterations appear to occur primarily in secondary memory systems and are
reflected in the storage of newly acquired information (Bartus et al., 1982; Joseph, 1992). It is thought that
the hippocampus mediates allocentric spatial navigation (i.e., place learning), and that the prefrontal cortex
is critical for acquiring the rules that govern performance in particular tasks (i.e., procedural knowledge),
while the dorsomedial striatum mediates egocentric spatial orientation (i.e., response and cue learning)
(McDonald and White, 1994; Zyzak et al., 1995; Devan et al., 1996). It appears that oxidative stress (OS)
(ShukittHale, 1999) and inflammation (HaussWegrzyniak et al., 1999, 2000) are contributing factors to
the behavioral decrements seen in aging.
There are numerous factors involved in mediating these behavioral deficits in aging that include
decrements in calcium buffering (Landfield and Eldridge, 1994), as well as declines in the sensitivity of
several receptor systems including the (1) adrenergic (Gould and Bickford, 1997) and (2) dopaminergic
(Joseph et al., 1978; Levine and Cepeda, 1998) systems. However, one of the most notable changes are those
involving the muscarinic cholinergic system. There is a long association between cognitive behavior and
cholinergic functioning in the literature (Wortwein et al., 1998), and there are many reports of significant
declines in cholinergic functioning in aging. While we realize that there is an involvement of the nicotinic
 Antiaging strategies 13 307

cholinergic system in learning and memory that mediates cognition, those changes that have been observed
in the muscarinic system in aging and the possible mechanisms involved in these changes have not been
well defined.

2 Muscarinic Receptors and Aging

Research indicates that muscarinic acetylcholine receptors (mAChRs) are intimately involved in various
aspects of both neuronal (e.g., amyloid precursor protein (APP) processing) (Rossner et al., 1998) and
vascular functioning (Elhusseiny et al., 1999). The finding concerned with mAChR/vascular interaction is
important, since it also appears that vascular disease and Alzheimers disease (AD) can occur in concert in
aging and that vascular disease may exacerbate cognitive dysfunction in AD (Morris et al., 2000; Deschamps
et al., 2001). In fact, data suggest that there may be complex interactions between vascular dementia (VaD)
and AD (Morris, 2000; Deschamps et al., 2001).
There is a long history of research showing a loss of sensitivity in classic receptor systems including the
dopaminergic and muscarinic (see Joseph et al., 1992). Numerous studies have shown that the PIlinked
muscarinic receptors (mAChRs) show deficits in signal transduction, which contributes to their loss in
sensitivity (Joseph and Roth, 1991). Joseph et al. (1991) have shown that mAChR agonist enhancement of
IP3 formations is reduced in striatal tissue obtained from senescent Wistar rats. Additional studies have
suggested that the site of the deficit in agonist responsiveness in mAChR in aging may be at the mAChR/G
protein interface (Joseph et al., 1988), which may involve deficits in mAChR/Gprotein coupling/uncou-
pling (Yamagami et al., 1992). This is an important consideration since research also indicates that
decrements in mAChR/Gprotein coupling/uncoupling, as assessed via carbacholstimulated GTPase activ-
ity, may also be greater in (Cutler et al., 1994) than that seen in aging. Additionally, there also appear to be
reductions in anterior, cortical PI levels, as well as a 70% loss of IP3 binding in the temporal cortex and
hippocampus (Young et al., 1988) and a decrease in the number of mAChRs that are coupled to G proteins
in the highaffinity states (Flynn et al., 1991).
The mechanisms involved in these deficits have yet to be determined, but such factors as decreases
in membrane fluidity may also be involved. Findings showed that when striatal tissue was incubated with
Sadenosylmethionine (SAM), a potent membranefluidizing agent, it increased the sensitivity of the
mAChR to agonist stimulation in striatal slices, while exposure of the slices to cholesterol (CHL), which
increases membrane viscosity, decreased this sensitivity (Joseph et al., 1991). There are decreases in
membrane fluidity in aging that involves the translocation of CHL to the outer leaflet of the membrane
where it is more readily oxidized. It also appears that sphingomyelin metabolites such as sphingosine1
phosphate may increase sensitivity to OS in aging. Recent studies have also suggested an involvement of
lipid rafts with OS sensitivity (Shen et al., 2004). The findings of Shen and coworkers (2004) indicate that
receptorinteracting protein (RIP) and tumor necrosis factor receptor (TNFR)associated factor
2 (TRAF2), which are two primary molecules involved TNF signaling, may be necessary for ROSinduced
cell death of TNFR1. The colocalization of RIP with a membrane lipid raft marker (GM1) suggested that
these rafts might participate in the formation of a complex between RIP and TRAF2 during OS. The study
suggests that this complex may be part of a novel signaling pathway that mediates oxidantinduced cell
death. Thus, it appears that a major factor that there may be important in the loss of sensitivity in mAChR
with aging may be increased sensitivity to OS, which is at least partially determined by changes in
membranes.
Another component of the cholinergic signaling system that is affected in AD pathophysiology is
regulation of the deposition of bamyloid (Ab) peptides due to increased APP processing, resulting in
increased levels of Ab. For example, AChE inhibitor treatments increase the secretion of nonamyloidogenic
processing products and decreases Ab formation. (Mori et al., 1995; Pakaski et al., 2001). There is also
in vivo evidence that APP processing underlies cholinergic regulation in the rat brain (Rossner et al., 1998).
When M1MAChRs are selectively stimulated in tissue slices, the nonamyloidogenic processing of
APP increases, suggesting that activation of the M2MAChR subtype results in a suppression of the
308 13 Antiaging strategies

nonamyloidogenic processing pathway (Farber et al., 1995). Furthermore, BACE1 expression is regulated
in different ways through MAChR. Since increased M1MAChR increases BACE1 expression but increased
M2MAChR decreases BACE1 expression (Zuchner et al., 2004), cholinergic function is important for APP
processing and AD pathophysiology. This is also consistent with the observation that there is demonstrable
downregulation of M2MAChR in those brain regions of AD patients which contain senile plaques (Boulay
et al., 1996; Lai et al., 2001; Apelt et al., 2003).

3 Muscarinic Receptors and Oxidative Stress

Several experiments conducted over the last several years have indicated that there are considerable parallels
between the loss of sensitivity seen in aging in muscarinic receptors and those seen following heavyparticle
irradiation. Thus, studies have shown that, just as was seen in aging, striatal tissue obtained from young rats
exposed to various doses of 56Fe irradiation (0.11 Gy) showed reductions in oxotremorine enhancement of
Kevoked dopamine release (KEDAR) (Joseph et al., 1992), as well as decreases in carbacholstimulated
IP3 activity (Joseph et al., 1993a) and carbacholstimulated GTPase activity, an indicator of receptor/
Gprotein coupling/uncoupling. Further studies showed that the deficit appeared to be at the muscarinic
receptor/Gprotein interface (Joseph et al., 1993a). As early as 1991, Stark postulated that certain free
radicals were able to gain access into the interior of cellular membranes and interact with the lipid matrix or
with membranebound proteins, altering their integrity. Importantly, in this regard, subsequent research
indicated that as was seen previously in aging, increasing membrane fluidity via exposure of striatal tissue
obtained from the irradiated animals to SAM reversed the irradiatedinduced deficits in KERDA ( Joseph
et al., 1999b).
Even more important are recent findings that suggest that there may be differences among MAChR
subtypes in response to OS in aging. Clearly, if this is the case, it might provide some explanation for
findings such as those showing that there is differential aging among various brain regions. Areas such as
the hippocampus (Nyakas et al., 1997; Kaufmann et al., 2001), cerebellum (Hartmann et al., 1996;
Kaufmann et al., 2001), and striatum (Joseph et al., 1996; Kaasinen et al., 2000) show profound alterations
in aging in such factors as morphology, electrophysiology, and receptor sensitivity. Interestingly, studies
that have examined regional differences in mAChR subtypes have shown that the mAChRs that show
increased vulnerability to OS appear to be localized in areas that show profound alterations in aging; these
include the dentate, striatum, and forebrain (e.g., see Levey, 1996 for review). Thus, we assessed differential
vulnerability to OS among the various mAChR subtypes by transfecting one of five MAChR subtypes
(M1M5) in COS7 cells. The results showed that when the cells were exposed to DA (Joseph et al., 2002) or
Ab (Joseph and Fisher, 2003), differences in OS sensitivity, expressed as a function of Ca2 buffering as assessed
by examining the ability of the cell to extrude or sequester Ca2 following oxotremorineinduced depolari-
zation, were observed. The COS7 cells transfected with M1, M2, or M4AChRs showed greater sensitivity to
DA or Abinduced disruptions in calcium buffering than those transfected with M3 or M5MAChRs. An
additional study ( Joseph et al., 2004) has suggested that the locus of the differential sensitivity to OS among
the various receptor subtypes may involve the i3 loop, and studies are being carried out to determine
possible differences among the i3 loops of these mAChR subtypes that could contribute to these differences.
Importantly, the findings with respect to COS7 cells suggest that, as mentioned above, brain areas
showing increased effects of aging contain high concentrations of M1, M2, or M4 receptors (Flynn and
Mash, 1993; Levey, 1994; Hersch et al., 1999). Additionally, it should be noted that the loss in calcium
buffering could lead to further decrements in cell functioning and OS, since the intracellular compartmen-
talization of high amounts of calcium (Brini, 2003) and mitochondrial pore transition (MPT) (Green and
Kroemer, 2004) can mediate both cell necrosis and apoptosis, with MPT possibly releasing cytochrome
c. Thus, in the case of aging, OS appears to impinge on systems that are already compromised in their
ability to regulate Ca2 flux. Additionally, it also appears that the membrane alterations and decrements
in calcium buffering may reduce the effectiveness of endogenous antioxidants in the aged organism
(Joseph et al., 1998a, 2001).
 Antiaging strategies 13 309

4 Consequences of Aging on Cholinergic Function

Also possibly contributing to the loss of cholinergic integrity and ultimately behavioral function in aging
are perturbations in choline acetyltransferase (ChAT) function. There are fewer cholinergic neurons in the
aged basal forebrain, and they express lower levels of ChAT and ACh uptake than younger rats. These
differences are consistent with ageassociated increases in proapoptotic markers and decreases in ChAT
activity (Drachman and Leavitt, 1974; Davies and Maloney, 1976; Drachman and Sahakian, 1980; Bartus
et al., 1982; Whitehouse et al., 1982; Davies, 1985; Fischer et al., 1987; Williams et al., 1993; De Lacalle et al.,
1996; Clemens et al., 1997). For example, cholinergic deafferentations carried out by transection of the
fimbria fornix or selective killing of basal forebrain cholinergic neurons by immunolesion fail to increase
neurotrophin protein levels in aged rat brains (Whitehouse et al., 1982; Fischer et al., 1987; Wortwein et al.,
1998). Taken together, it would appear that there is an agingassociated impairment of stress response
mechanisms. Determining the different contributions of cholinergic cell losses versus changes in the
cholinergic phenotype in the aged brain remains a disputed area.
For example, it could be that the activities of transcription factors that regulate gene expression
responsible for cholinergic function and apoptotic responses to OS and trauma are altered in the aged
brain (Tong et al., 2002). Although the activation of the transcription factor NFkB has been determined in
the aged rat and AD brains, the assessments have been limited to binding to oligonucleotide preparations
displaying the IgGkB promoter consensus sequence and have not differentiated among the specific
sequences present in individual promoters.
Choline acetyltransferase is rate limiting for acetylcholine synthesis (Nachmansohn and Machado,
1943). ChAT is expressed in basal forebrain cholinergic neurons, important in memory and cognition,
whose deterioration is associated with cognitive deficits. Decreases in ChAT and acetylcholine release are
pathological markers in AD and agingassociated dementias (Armstrong et al., 1983; Davies, 1985; Williams
et al., 1993). The cDNAs and regulatory regions for mouse, rat (Ishii et al., 1990), human (Toussaint et al.,
1992), and porcine (Berrard et al., 1987) ChATs have been cloned. They are highly homologous, and while
the 50 flanking sequences have greater interspecies variation, they share common regulatory elements, such
as a silencer element restricting expression to cholinergic neurons (Lonnerberg et al., 1996). ChAT
promoters also have a general enhancer and an NGFresponsive enhancer (Bejanin et al., 1992; Pu et al.,
1993). The mouse ChAT promoter 50 flanking sequence is alternatively spliced to yield seven different
mRNAs (R14, N12, and the most predominant M form) that differ in their 50 noncoding regions
(Misawa et al., 1992). Misawa and coworkers (1992) cloned a 4,014 base pair (bp) region of the promoter,
including the 1,470 nucleotides reported by Pu and coworkers (1993). Here, promoter positions are
numerically referenced as described by Misawa and coworkers (1993). The 50 flanking region lacks a
consensus TATA box, but contains TATAlike elements and multiple transcription initiation start sites.
There is a strong enhancer element that is not modulated by NGF (3021 to 2706) and an NGFinducible
enhancer (3416 to 3021). Region 3516 to 3148 confers celltype specificity, acting as a repressor
element in noncholinergic cells (Pu et al., 1993). Although there are AP1 (2934 to 2928 and 2858 to
2852), serum response element (3159 to 3145), cAMP response element (3083 to 3076), NGFIA
(3190 to 3182), and NFkB (3174 to 3165) consensus sites within the NGFresponsive element, it is not
known if the cognate transcription factors regulate ChAT. Transcription of Rtype mRNAs is controlled by
regulatory elements upstream of the R promoter, and transcription of N and Mtype mRNAs by elements
surrounding the N and M promoters. The most common form of ChAT mRNA, which is present in the
brain and relevant to this project, is the Mform. Misawa and coworkers (1992) cloned a 4014bp flanking
region, which includes the N and Mtype exons and the 1470 nucleotides reported by Pu and coworkers
(1993); numbering of mouse ChAT promoter nucleotide positions will be as in Misawa et al. (1992).
The transcription factor NFkB belongs to a family of homo and heterodimeric proteins that are
related by a conserved
300 residue NH2 terminal Rel/homology domain and includes p50, p65 (or RelA),
p52 (or p49), cRel, and RelB proteins (Bours et al., 1993; Nolan et al., 1993; Ruben et al., 1991; Schmid
et al., 1991; Bours et al., 1993). Heterodimerization of NFkB proteins produces species with different
intrinsic DNAbinding specificities (p52, p50) and transactivation properties (p65, cRel), an important
310 13 Antiaging strategies

functional distinction (Baeuerle, 1991; Siebenlist et al., 1994; Chen et al., 1998). The most commonly
described active subunit combination (Flohe et al., 1997) is the p50/p65 heterodimer, present in the
cytoplasm, bound to an inhibitory IkB subunit. There are several inhibitory IkB proteins (IkBa, IkBb,
IkBe, IkBg, and Bcl3). Upon stimulation, IkBa is phosphorylated, ubiquitinated, and degraded (Ghosh
and Baltimore, 1990; Liu et al., 1993), exposing nuclear localization signals on NFkB proteins, which allow
translocation to the nucleus for DNA binding. NFkB is stimulated by OS or receptor ligands via increased
IkBa degradation and NFkB nuclear translocation, but also via an independent pathway involving Bcl3
(Gozal et al., 1998; Zhang et al., 1998; Qiu et al., 2001). Bcl3, of the IkB family, functions in the nucleus by
drawing NFkB p50 or p52 homodimers away from NFkBbinding sites in promoters (Bours et al., 1993;
Franzoso et al., 1993; Fujita et al., 1993; Nolan et al., 1993; Bundy and McKeithan, 1997). Thus, Bcl3 can
inhibit p50 or p52 homodimer binding to promoter sites (Franzoso et al., 1993; Nolan et al., 1993;
Siebenlist et al., 1994; Zhang et al., 1998), allowing cRel/p50 or p65/p50 binding and activation (Heissmeyer
et al., 1999; Qiu et al., 2001).
We and others have shown that NFkB DNAbinding activity is elevated in the aged rat basal forebrain
(ToliverKinsky et al., 1997; 2000). Our hypothesis is that ageassociated alterations in these two regulatory
factors contribute to the ageassociated decreases in ChAT expression, which has consequences on cognitive
function in the aged. We have shown that a 20bp ChAT (NFkB) oligonucleotide probe containing the
ChAT (NFkB) site binds to NFkB p49, somewhat less to p65, but not at all to p50, cRel, or RelB. This
observation supports the idea that different consensus sequences are regulated by different subunits in a
genespecific and tissuespecific manner. When PC12 cells are transfected with two ChAT promoter
reporter constructs containing the wildtype ChAT promoter and a mutant construct where the NFkB
binding site is replaced with a Bgl2 restriction site, there was a threefold increase in promoter activity in
the cultures transfected with the mutant reporter plasmid as compared with those transfected with the
wildtype plasmid, consistent with the assignment of a repressor function to the (ChAT) NFkB site.
Furthermore, the NFkB proteins p49, p65, and p50 were overexpressed in NGFtreated PC12 cells, and
ChAT promoter and enzymatic activities were measured. The overexpression of p49 and of p49 and p65
significantly decreased total ChAT activity as compared with controls. The assignment of a repressor role to
NFkB on the ChAT promoter agrees with reports of increased NFkB binding and decreased ChAT in aged
and AD brains (Coyle et al., 1983; Vile et al., 1995; Helenius et al., 1996; Taglialatela et al., 1996; Terai et al.,
1996; Boissiere et al., 1997a, b; Kitamura et al., 1997). We measured NFkB activity by EMSA and Western
blots on nuclear fractions from brains of 3 and 30monthold male F344  BN hybrid rats. p49 appeared to
be higher in the basal forebrain nuclear extracts prepared from 30monthold rats.
Thus, NFkB DNAbinding activity increases in the aged basal forebrain where it may contribute to
decreased ChAT expression. Interestingly, this increase supports the hypothesis that changes in NFkB are
gradual and proportional to the aging process.

5 The Effects of Fruit and Vegetable Supplementation on Behavioral

and Neuronal Deficits in Aging

We have attempted to outline the possible role of OS in mediating decrements in cholinergic functioning
in aging. It is evident that methods to reduce stress may forestall or even reduce these deficits in the
aged organism. While there are numerous studies suggesting that various antioxidant supplements (see
Casadesus et al., 2002 for review) may be effective in this regard, our research suggests that the combina-
tions of antioxidant/antiinflammatory polyphenolics found in fruits and vegetables may show efficacy in
aging. All plants, including fruit or vegetable bearing plants, synthesize a vast array of chemical compounds
that are not necessarily involved in the plants metabolism. These secondary compounds instead serve a
variety of functions that serve to enhance the plants survivability. These compounds may be responsible for
the putative multitude of beneficial effects of fruits and vegetables on healthrelated issues, two of the most
important of which may be their antioxidant and antiinflammatory properties.
The anthocyanins are among the plant polyphenols that have potent antioxidant and antiinflammatory
activities. These are natural pigments responsible for the orange, red, and blue colors of fruits, flowers,
 Antiaging strategies 13 311

vegetables, and other storage tissues in plants (Wang et al., 1999; Seeram et al., 2001a, b). Anthocyanins
have been reported to affect many of the parameters discussed above by inhibiting lipid peroxidation and
the activity of cycloxygenase1 and 2 (COX) enzymes (Seeram et al., 2001b, 2003).
The chemistry of the anthocyanins can be reduced to six major anthocyanidins: delphinidin, cyanidin,
pelargonidin, petunidin, peonidin, and malvidin. Among berry fruits, blueberries contain high levels of a
wide variety of anthocyanins including glycosides of four of the six major anthocyanidins: malvidin,
petunidin, peonidin, and cyanidin (Kalt et al., 1999). Therefore, blueberries may have very potent effects
on the fires of aging.
Anthocyanins are a subset of a larger class of polyphenols known as flavonoids. Over 4000 flavonoids
have been identified in plants. They are also abundant in seeds, fruits, and plantderived oils such as olive
oils, as well as tea and red wine. Thus, they are part of the human diet, and plants and spices containing
them have been used for many years in Eastern medicine. As might be expected from the above discussion
of the anthocyanins, flavonoids have been reported to inhibit lipid peroxidation in several biological
systems including mitochondria and microsomes (Bindoli et al., 1977; Cavallini et al., 1978) as well as
erythrocytes (Sorata et al., 1984; MaridonneauParini et al., 1986) and liver (Kimura et al., 1984). They
appear to be potent inhibitors of both NADPH and CCl4induced lipid peroxidation (Afanasev et al.,
1989). It appears that the ironchelating ability of the flavonoids may be very important in mediating their
potent inhibitory effects on 5LOX (Hoult et al., 1994), while CO inhibition appears to involve other
mechanisms.
The antioxidant effects of flavonoids may be derived in part from their ability to upregulate antioxidant
enzymes (e.g., glutathione) or enzymes related to glutathione synthesis. One mechanism that may be opera-
tional in these beneficial effects may be the direct enhancement of transcription factors that enhance antioxi-
dant enzymes or their signaling cascades. It is known, for example, that the enzymes for glutathione (reviewed
in Zipper and Mulcahy, 2000; Schroeter et al., 2002) or heme oxygenase (Chen and Maines, 2000) synthesis
exhibit extracellular signalregulated kinase 1/2 (ERK1/2) dependency in the regulation of their expression,
while Cu/ZnSOD is regulated by ELK1 (Chang et al., 1999), and MnSOD expression contains binding sites for
Sp1, AP1, and CREB (Das et al., 1995; Chang et al., 1999), which are ERK1/2 (Sgambato et al., 1998; Chang
and Karin, 2001) regulated. Finally, it also appears that flavonoids regulating ERK1/2 may influence iNOS
activity. Thus, there is a great deal of evidence to suggest that a possible link exists between the antioxidant
activity of flavonoids and their putative MAPkinases altering activity.
Since MAPKs are involved in numerous biological activities, the findings that flavonoids may influence
such signaling suggests that their potential benefits may involve properties other than those involving
antioxidant or antiinflammatory effects. As examples, delphinidin inhibits endothelial cell proliferation and
cellcycle progression by ERK1/2 activation (Martin et al., 2003), while grape seed proanthocyanidin can
reduce ischemia/reperfusioninduced activation of JNK1 and cJun and reduce cardiomyocyte apoptosis
(Sato et al., 2001). Additional research indicates that phytochemicals can regulate MAP kinase and other
signaling pathways at the level of transcription (Frigo et al., 2002).
These findings, coupled with a plethora of studies showing the involvement of ERK in diverse forms
of memory, such as contextual fear conditioning (English and Sweatt, 1996), longterm potentiation
(English and Sweatt, 1997), striataldependent learning and memory (Mazzucchelli and Brambilla, 2000),
hippocampaldependent spatial memory (Selcher et al., 1999), and inhibitory avoidance (Schafe et al.,
1999), suggest that interventions that influence MAPK signaling may have beneficial effects on cognition.
Given the findings reviewed above showing alterations in signaling as a function of age, the putative
signaling modifying properties of flavonoids may prove to be invaluable in altering the neuronal
and behavioral effects of aging. Thus, we believed that given multiple properties of fruits and vegetables,
they might show considerable efficacy in reducing the deleterious effects of aging on neuronal function
and behavior.
In the mAChRtransfected COS7 cells, research showed that M1MAChR transfected cells that had
been pretreated with various high antioxidants (boysenberry (BY); cranberry (CB); black currant (BC);
strawberry (SB); dried plums (DP); and grape (GR)) and exposed to Ab2535 or DA. Calcium buffering was
examined following oxotremorineinduced depolarization in M1MAChRtransfected COS7 cells, and on
cell viability following DA (4 h) exposure. The results suggested that, while there were differential levels of
312 13 Antiaging strategies

protection among the various fruit extracts, all of them decreased to varying degrees, the decrements in
calcium buffering induced by Ab or DA (Joseph et al., 2004).
Extending these findings, additional research showed that feeding high antioxidants (via the oxygen
radical absorbance capacity assay (ORAC)) (Cao et al., 1997; Wang et al., 1996; Prior et al., 1998) could both
prevent the onset of agerelated deficits in several indices (e.g., cognitive behavior, Morris water maze
performance) (Joseph et al., 1998) or reverse them (Joseph et al., 1999).
Interestingly, examinations of various brain regions from the supplemented animals showed only small
levels of antioxidant activity, which was insufficient to account for the observed significant beneficial effects
of blueberry (BB) supplementation on motor and cognitive function. Findings from this (Joseph et al.,
1999) and a subsequent study (Youdim et al., 2000) suggested that there are beneficial properties, in
addition to those involving antioxidants, of BBs on both motor and cognitive behavior; these may involve
alterations in neuronal signaling and communication.
This was observed recently in a study (Joseph et al., 2003) carried out in APP/PS1 transgenic mice,
which serve as a model for AD since these mutations promote the production of Ab, and subsequently
Alzheimerlike plaques in several brain regions, which are accompanied in middle age by cognitive
deficits. A group of these mice was given BB supplementation beginning at four months of age (as in
Joseph et al., 1999) and continued until they were 12 months of age, when their performance was tested in a
Ymaze. The results indicated that mice supplemented with BB exhibited Ymaze performance that was
similar to those seen in nontransgenic mice and significantly greater than that seen in the nonsupplemented
transgenic animals. Interestingly, there was a dichotomy between the plaque burden and behavior in the
BBsupplemented transgenic mice. No differences between the supplemented and nonsupplemented APP/
PS1 mice in the number of plaques were observed, even though behavioral declines were prevented in the
BBsupplemented animals.
One possible reason that the behavior did not reflect the morphology may be that there was enhanced
signaling present in the BBsupplemented transgenic mice, which acted to prevent or circumvent any
putative deleterious effects of the amyloid plaques on behavior. The evidence for this possibility is provided
by data showing that the BBsupplemented APP/PS1 mice exhibited greater levels of hippocampal ERK as
well as striatal and hippocampal protein kinase Ca (PKC) than that seen in the transgenic mice maintained
on the control diet. As pointed out above, ERK and PKC have been shown to be important in mediating
cognitive function, especially conversion of shortterm to longterm memory (Micheau and Riedel, 1999).
Enhancement was also seen in the BBsupplemented group in the sensitivity of muscarinic receptors
(i.e., increasing striatal, carbacholstimulated GTPase activity), which has been found to be associated
with learning and memory in numerous studies.
These findings, combined with additional preliminary research showing that BB supplementation in
addition to altering ERK activity may also increase hippocampal neurogenesis (Casadesus et al., 2004),
suggests that at least part of the efficacy of the BB supplementation may be to enhance neuronal function in
areas of the brain affected by aging or disease. This would allow more effective intra and interarea
communication and ultimately facilitate both cognitive and motor function.

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Index
Acetylcholine (Ach), 220, 230232
AChE, 230235
ACTH, 217
AD. See Alzheimers disease
Advanced glycosylation end products
(AGEs), 125
Aging, 217, 219, 225, 227230
age-related increases in iron
levels, 73
age-related loss of myelin, 71
ferritin, 73
iron content, 73
reduction of fast conducting CNS
axons, 71
Aging brain and dendritic spine,
254256
Aging processes
causes of, 104
free-radical theory, 105
genetic theories on evolution
of, 133
mitochondrial theory of aging,
108110
ALS. See Amyotrophic lateral sclerosis
Alzheimer disease, 78, 82, 83, 85, 105,
219, 223, 230, 286
aging, 274
amyloid, 149
amyloid beta peptides (Ab)
deposit, 71
Ab toxicity in, 73
amyloid precursor protein, 262
cell
cycle, 181, 190196
death, 189, 190, 196
damaging free radicals in
pathogenesis of myelin, 73, 74
effects of developmental-
dependent oligodendrocyte
myelination in, 74
genetic factors, 148, 149
Hsps proteins in, 121
mitotic catastrophe in, 150
# 2008 Springer ScienceBusiness Media, LLC.
myelin breakdown in, 71, 73 hybridization, in situ, 274
neurodegeneration, 181, 182, 184, immunohistochemistry, 274
186, 190 interaction, protein-protein,
neuropathological and clinical 272, 273
features of, 288290 isoforms, 266
pathogenesis of, 125 localization, subcellular, 274, 276
secretases, 264 mice
tau phosphorylation, 150, 151 knock-out, 265, 278
AMD, 230 transgenic, 274276
Amyloid beta-peptide, 125, 288 modification, posttranslational
b-amyloid cleaving enzyme 1, 289 glycosylation, 271
b-amyloid peptide phosphorylation, 272
amyloid precursor protein, 307 mRNA, alternative splicing,
free radicals, 308 265, 266
Amyloid precursor protein, 288 promoter, 268, 269
Amyotrophic lateral sclerosis, 226, regulation
286, 291 transcriptional, 268
due to abnormal proteins, translational, 268
107, 108 structure, genomic, 265
Anticholinesterases, 231, 233, 234 transport, intracellular,
Antioxidant proteins 271, 272
cellular expression and activity of, BACE. See b-amyloid cleaving enzyme 1
104, 110 BBB. See Blood brain barrier
HO, 113 bdnf gene, transgenic mouse, 12, 13
Antisense, 233235 BDNF. See Brain-derived neurotrophic
Apolipoprotein E (ApoE), 289 factor
Apoptosis Betaine, 83
anoikis, 178 homocysteine
Bcl-2, 178180 methyltransferase, 80
caspases, 178 Bilirubin
p53, 179 as antioxidant, 121, 122
transcription factors, 161, end products of heme
180, 181 degradation, 113
APP. See Amyloid precursor protein nonconjugated,
ASF/SF2, 224, 225 supraphysiological levels
AVP, 217 of, 121
Ab. See Amyloid beta-peptide Biliverdin, antinitrosative
BACE characteristics of, 122
expression Biomarker, of DNA damage, 106
cell type-specific, 268, Blood brain barrier, 233
274, 275 Brain
mRNA, 266, 268, 270, 274 GSH system in, 120
protein, 268 heme oxygenase in, 117, 120
320 Index
Brain aging
DNA oxidative damage
genetic instability, 107
methylating agents, 106
production of ROS, 106
protein oxidative damage
neurodegenerative diseases
due to, 107, 108
proteolysis, 107
Brain damage mechanism, in
ND, 296
Brain-derived neurotrophic factor
(BDNF)
cerebellar granule neuron
migration, role in, 10
motor neuron death by, 5
mutant mouse
cortical layer thickness, 8
motor neuron survival, 5
neuronal migration in neocortex,
7, 8
Brain disease, spine number and
formation in, 254
Brain stress tolerance
heat-shock pathway of
Hsp27, Ubiquitin, and
Hsp47, 116
Hsp60 and Hsp32, 117
Hsp70, stress proteins,
115, 116
in mammalian cells, 115
Branchpoint, 220222, 229
Caenorhabditis elegans, 293
Calcium/calmodulin-dependent
protein kinase II-a, 253
Caloric restriction
rate of aging and cellular
pathology, 122
signaling pathway, 123
CaMKII-a. See Calcium/calmodulin-
dependent protein kinase II-a
Cannon, W., 216
Carbon monoxide
antiinflammatory properties,
113115
neurogasobiology of, 110, 111
Cardiolipin, 109
Cardiovascular disease, 78, 81
Caspase-1, 91
Caspr2, 7173
Cell cycle
in Alzheimer disease, 149152
check points, 159, 167, 168,
179, 181
control elements, 149151
cyclin(s), 150, 160163, 165167,
169, 172175, 181, 190
cyclin dependent kinases, 150,
160162, 175, 190
inhibitors of cyclin dependent
kinases, 165, 174, 175, 180, 181,
190, 191
mitotic catastrophe, 150
restriction point, 160, 167, 168
retinoblastoma protein, 161, 166,
167, 192
tau phosphorylation, 150, 151
Cell differentiation
de-differentiation, 175, 190, 196
development, 166, 172175
mechanisms, molecular, 167, 168,
173, 186
Cellular apoptosis, 90
Central nervous system (CNS), 90
development, TrkB and TrkC
role in, 5
neuronal survival, neurotrophins
role in, 3
neurons in, 11
NOS and its isoforms in, 111
Central nervous system disorders
associated with ROS/RNS-
mediated damage, 123
AD, 125126
ALS, 124
DS, 132
FA, 130132
HD, 132
ischemia/reperfusion, 133
MS, 128130
PD, 126, 127
Cerebellar granule neurons, migration
of, 9, 10
Cerebellar purkinje cells, dendritic
differentiation in, 12
Cerebellum, layered structure
formation of, 9
Cerebral cortex, layered architecture
formation of
during development and
adulthood, 8
postmitotic neuron migration, 7
Cerebrovascular disease, 78, 82
Chaperone-buffered silent
mutations, 105
Cholinergic system
acetylcholine, 309
aging, 309, 310
alzheimers disease, 309, 310 DFP, 232
choline acetyltransferase,
309, 310
cognitive deficits, 309
Chronic wasting disease, 292
CJD. See Creutzfeldt-Jakob disease
CNS disorders. See Central nervous
system disorders
CO. See Carbon monoxide
Cortical neuronal migration, Trk
signaling pathways in, 9
Corticosterone, 217, 233
Cortisol, 217, 233
COX enzyme, 95
COX-2. See Cyclooxygenase-2
Creutzfeldt-Jakob disease, 292
CRF, 217
Cu/Zn-superoxide dismutase
(Cu/Zn-SOD)
antioxidative genes,
overexpression of, 105, 106
cytosolic form, mutation in, 124
in DS patients, 132
CWD. See Chronic wasting disease
Cyclooxygenase-2, 93
g-cystathionase, 7981
Cystathionine, 7881, 84
Cystathionine b-synthase, 7981, 84
Cystathioninuria, 81
Cysteic acid, 7880
Cysteine, 7981, 83, 84
Cysteine oxidase, 79, 80
Cysteinesulfinic acid, 7880
Cysteinesulfinic acid decarboxylase,
79, 80
Cytochrome oxidase, mitochondrial,
activity and aging process, 106
Cytokine-induced nitrosative stress, 116
Cytokine responses and cell survival,
91, 92
Decoy treatments, 96
Dendritic arborization, 11
Dendritic development, neurotrophin
role in
in BDNF mouse strain, 12, 13
neurotrophin receptors and
downstream signaling molecules,
13, 14
non-pyramidal interneuron
dendrites, 11, 12
pyramidal neurons, 11
Dendritic spine
and aging brain, 254256
synapses
formation of, 250, 251
structure of, 246, 247
Dendritic transport, 225
DNA damage
enzmes to repair, 107
due to oxidative stress, 106
DNA polymerases, 107
Dopamine, 220, 227

Downs syndrome, 132
Drosophila, in neurodegenerative
disease modeling, 293
DS. See Downs syndrome
EAE. See Experimental allergic
encephalomyelitis
Electron traffic jam upstream, 106
Embryogenesis, TrkB expression
during, 8
Endothelial NOS (eNOS), 95
Epinephrine, 217, 219
ESEs, 222
ESTs, 220
Experimental allergic
encephalomyelitis, HO-1 expression
in, 121
FA. See Friedreichs ataxia
Familial CJD (fCJD), 292
Fight or flight, 216
Fluoresance-recovery after
photobleaching, 248
Folic acid, 8082, 85
Forebrain development
Emx1-positive
neuroepithelium, 67
fate mapping studies, 66
genomic screened homeobox2
(Gsh2), 62, 67
lateral ganglionic eminence
(LGE), 62, 66, 67
medial ganglionic eminence
(MGE), 62, 6668
Nkx 2.1, 67, 68
Nkx2.1-Cre transgenic mouse
line, 67
FRAP. See Fluoresance-recovery after
photobleaching
Free-radical hypothesis of aging
causative factors, 105
oxidative damage, DNA and
proteins, 106, 107
oxidized dysfunctional proteins,
accumulation of, 108
proteasomal dysfunction, 107
Friedreichs ataxia, 130, 131
Fruits and vegetables
anthocyanins, 310, 311
flavonoids, 311
polyphenols, 310, 311
FTDP-17, 226, 230
GABAergic interneurons
in medial ganglionic eminence
(MGE), 7
tangential migration of, NT-4 and
BDNF role in, 9
GAP. See GTPase activating protein
GCs. See Glucocorticoids
GEFs. See Guanine nucleotide exchange
factors
Gene expression regulation and NGF,
2233
General adaptation syndrome, 216
Genome-based theory, 104
Gerstmann-Straussler-Scheinker, 292
GFP-transfected hippocampal neuron,
3D imaging of, 252
Gliosis
Glucocorticoids, 217, 218, 220, 233
Glutamate, 220, 226
Glutathione, 80, 84
cellular detoxification, 119
reduced and oxidized, 120
GR, 218, 223, 224
GSH. See Glutathione
GSS. See Gerstmann-Straussler-
Scheinker
GTPase activating protein, 253
Guanine-nucleotide exchange factor(s),
13, 254
HD. See Huntington disease
Heme oxygenase (HO) pathway
defensive mechanism, 120
in antidegenerative mechanism,
AD, 105
HNE. See 4-hydroxy-trans-nonenal
hnRNPs, 221, 222, 227, 228
HO system
colocalization of, 118
endogenous, 121
expression, regulation of
HO-1, 119
HO-2, 118
intracellular modulators for,
119, 120
induction of, 118
inhibition of, 121
isoforms of, 117
NADPH cytochrome c P450
reductase activity, degradation of
heme, 117
upregulation in nigral
neurons, 121
Homeostasis, 90, 216
Homocysteic acid, 78, 83
Homocysteine
age, 82
aging, 82
alzheimers disease, 78, 82, 83, 85
amyloid beta, 83
amyotrophic lateral sclerosis, 83
apoptosis, 82, 83
atherosclerosis, 81, 85
cardiovascular disease, 81
cerebrovascular disease, 78, 82
Index 321
cognitive function, 81, 82
dementias, 82
depression, 82
early development, 80, 82
fetal death, 82
hip fractures, 82
ischemic stroke, 81, 82
neural tube defects, 78, 81, 82
NMDA receptor, 83, 84
programmed cell death, 82, 83
protein bound, 84
thromboembolic episodes, 81
vascular dementia, 82
venous thrombosis, 82
Homocysteine methyltransferase,
79, 80
Homocystinuria, 78, 81, 82, 84
HPA, 217220
Hsp 27, 116
HSP70, stress proteins family
components of, 115
pathological condition
induction, 116
Huntington disease, 132, 286,
290, 291
Hydrophobic interactions, in protein
aggregation, 295
4-hydroxy-trans-nonenal, 108
Hypersensitivity, 232235
Hypotaurine, 7880
Hypoxia, 82
Hypoxia ischemia (HI), 90
Iatrogenic CJD (iCJD), 292
IL-1 RI. See IL-1 type-I receptor
IL-1 type-I receptor, 91
Imaging, 220
Inducible nitric oxide synthase (NOS),
93, 95
Inflammatory gene expression, 95, 96
Inflammatory responses, 91
Inherited genetic defects, 78
iNOS. See Inducible nitric oxide
synthase; Inducible NOS
Intron, 220223, 226, 229231
Ischemic brain damage, 133
Juxtaparanode junction, cytoskeletal
proteins
connexin 29, 71
Kv1.1, 71, 73
Kv1.2, 71
Kv1.6, 71
Kv2, 71
tranasient axonal glycoprotein-1
(Tag1), 71
Lipid peroxidation, in
neurodegenerative disorders, 108
Lipopolysaccharides (LPS), 94
322 Index
Longevity assurance processes
genes, 134
maintenance and repair process,
brain cells, 110
Long-term potentiation, 219, 220, 225,
232, 234, 235, 251
Lou Gehrigs disease. See Amyotrophic
lateral sclerosis
L-1 retrotransposon sequence,
retrotransposons, 118
LTP. See Long-term potentiation
Luteinizing hormone
in Alzheimer disease, 152
estrogen, 151, 152
gender differences in, 151, 152
HPG axis, 152
leuprolide acetate, 152
receptors, 152
Manganese superoxide dismutase, 93
MCAO. See Middle cerebral arterial
occlusion
Memory, 218220, 225, 230, 234, 235
Mesencephalic trigeminal nucleus, CNS
neuronal survival in, 5
Methionine, 7883
Methionine adenosyltransferase, 79, 81
Mg2+-ATPase, 83
Middle cerebral arterial occlusion, 92
Misfolded protein clearance, increase
of, 297
Mitochondrial dysfunction, role in
aging and neurodegenerative
diseases, 104, 105
Mitochondrial theory of aging
free radicals generation, 108
mtDNA mutations, 109
Mixed disulfide, 84
MnSOD. See Manganese superoxide
dismutase
Monoamines, 220
Motor neuron survival, BDNF, NT-3
and NT-4 role in, 5
mRNA, 220235
MS. See Multiple sclerosis
mtDNA
age-related disease due to, 109
mutations, 109
MTHFR 677C!T, 81, 82
MTN. See Mesencephalic trigeminal
nucleus
Multiple sclerosis
clinical symptoms of, 128
CNS stress response, 121
pathogenesis of, 129
Myelination
adenosine, 70
g-ratio, 70
inner mesaxons, 72
leukemia inhibitory factor, 70
neuregulin (NRG)-ErbB
receptor, 70
outer mesaxons, 72
salutatory conduction, 71
Schmidt-Lanterman incisures, 72
Schwann cells, 69, 70, 72
septate junction, 73
Myelin composition
cholesterol, 69, 71, 73
2,3-cyclic nucleotide-3-
phosphohydrolase (CNP), 63
glycosphingolipids, 69
myelin basic protein (MBP),
62, 63
preteolipid proteins
(PLP/DM20), 67, 69
NADPH cytochrome c P450 reductase
degradation of heme, 117
Na+, K+-ATPase, 83
Native protein folding, stabilization
of, 297
NCAM. See Neural cell adhesion
molecule
ND. See Neurodegenerative diseases
Nerve growth factor, 22
central cholinergic neuron
survival, role in, 4
family of trophic factors, 3
mechanism, in gene expression,
2233
responsive transcripts, 33
Nervous system development
cerebellum, 9
dendritic development,
neurotrophin role in
in BDNF mouse strain, 12, 13
neurotrophin receptors and
downstream signaling
molecules, 13, 14
non-pyramidal interneuron
dendrites, 11, 12
pyramidal neurons, 11
neurons, 2
neurotrophin receptors,
expression of, 3
Neural cell adhesion molecule, 251
Neural tube defects, 78, 81, 82
Neurodegeneration
Alzheimer disease, 148, 149
Amyloid-b, 148
genetic factors
amyloid b protein precursor,
148, 149
apolipoprotein E, 149
presenilins, 149
mitochondria, 151
tau phosphorylation, 150
Neurodegenerative diseases, 230
animal models for, 293, 294
brain damage mechanism, 296
causes of, 104
clinical, neuropathological, and
molecular features of, 288293
development of therapy for, 298
pathogenesis of, 110
protein misfolding and, 287288
therapeutic strategies for, 296, 297
types and causes of, 286
Neurofibrillary tangles, 288
Neuronal heterotopias, 8
Neuronal migration
modulation in
cerebellum, 9, 10
cerebral cortex, 79
radial, postmitotic neurons, 8
Neuronal NOS, 95
Neurotransmission, 220, 227, 230,
232, 235
Neurotrophic hypothesis, 2
Neurotrophin-3 and Neurotrophin-4.
See NT-3 and NT-4
Neurotrophins
binding selectivity to Trk
receptors and p75, 2, 3
competition among neurons
for, 2
effects on
neuronal dendritic
development, 12, 13
non-pyramidal interneuron
dendrites, 11, 12
pyramidal neurons, 11
and neuronal activity,
relationship between, 14
role in CNS neuronal
migration, 711
survival, 35
signaling pathways, 9
NFT. See Neurofibrillary tangles
NGF. See Nerve growth factor
Nitric oxide
neurogasobiology of, 110, 111
as neurotransmitter, 111
protective effect of, 112
redox activities elicited by, 112
Nitric oxide synthase, 95
and its isoforms, in CNS, 111
Nitrosative stresses
cytokine-induced, 116
gene expression regulation,
112, 113
protein damage, 108

N5, N10-methylenetetrahydrofolic
Acid, 80
N5-methyltetrahydrofolate, 80, 81
nNOS. See Neuronal NOS
NO. See Nitric oxide
Nodes of Ranvier
Cytoskeleton proteins
act-binding protein spectrin
b-IV (bIV), 70
ankyrin G, 70, 73
gliomedin, 70
neurofascin-186 (Nf186),
70, 73
neuronal-adhesion molecules
(Nrcam), 70
Kcnq2, 70, 73
Kv3.1, 70, 73
Nav1.2, 70, 73
Nav1.6, 70, 73
synantocytes, 70
Norepinephrine, 217, 220
NOS. See Nitric oxide synthase
NT-3 and NT-4, neuronal migration,
radial, 7, 8
NT-3 mutant mice
mesencephalic trigeminal nucleus
neurons loss in, 6
motor neuron survival in, 5
NT-4 mutant mice, motor neuron
survival in, 5
Nutritional antioxidants, therapeutic
potential of, 123
8-OH-dG. See Oxidized nucleotide
8-hydroxy-deoxyguanosine
Oligodendrocyte development
basic fibroblast growth factor
(FGF), 62
2,3-cyclic nucleotide-3-
phosphohydrolase (CNP), 63
developing brain homeobox gene
(Dbx1), 65
galactocerebroside glycolipid
(GC), 62, 63
ganglioside (GD3), 63
ganglioside (O4, O1), 63
glial restricted progenitor, 66, 67
glutothione S-Transferases pi
(GSTpi), 63
glycerol 3-phosphate
dehydrogenase (GPDH), 63
glycerolphosphate dehydrogenase
(GPDH), 63
Msx3, 65, 66
myelin basic protein (MBP),
62, 63
myelin oligodendrocyte
glycoprotein (MOG), 63
neurogenin2, 64
neuron restricted progenitor,
62, 66
NG2, 6769
Nkx 2.2, 65
Nkx6 transcription factors, 64
Olig1, 64, 67
Olig2, 6469
paired box 7 (Pax7), 65
platlet-derived growth factor
(PDGF), 62, 65, 67, 68
platlet-derived growth factor
receptor-alpha (PDGFRa), 62, 65
polysialic acid-NCAM,
ganglioside (A2B5), 63, 67
proteolipid protein (PLP), 62,
63, 69
sonic hedgehog (SHH), 62, 64
stage specific embryonic
antigen-1 (SSEA1), 63
transferrin (Tf), 63
Oxidative molecular damage
to DNA and proteins, 106, 107
endogenous, 122, 123
gene activation by, 110
gene expression regulation by,
112, 113
in mtDNA, 107
in senescence, 104, 105
Oxidative stress, 82, 83
cholinergic function, 309, 310
muscarinic receptors, 308
nicotinic cholinergic system,
306, 307
Oxidized nucleotide 8-hydroxy-
deoxyguanosine, 106
p21-activated kinase, 254
p21ras, 169, 180, 185, 186, 188, 189
p75 mutant mouse
hippocampal pyramidal
neurons, dendritic complexity
of, 13
septal region volume and
cholinergic neuron numbers, 4
p75 receptor, 2
putative role in basal forebrain
cholinergic neurons, 4
PAK. See p21-activated kinase
Paranodal junction, cytoskeletal
proteins
ankyrin B, 71
contactin, 7173
contactin-associated protein
(CASPR), 71
neurofascin-155 (Nf155), 71, 73
protein 4.1B, 71
a/bII spectrin, 71
Index 323
Parkinsons disease, 286, 290
due to abnormal proteins,
107, 108
glutathione peroxidase and
catalase activities, 127
pathogenesis of, 126
PC12 cells gene regulation, SAGE in,
3452
PD. See Parkinson disease
PET. See Positron emission tomography
PFC. See Prefrontal cortex
Phosphothiorated oligonucleotides, 96
PI3K pathway, 9
Plasticity
mechanisms, molecular, 182
neurodegeneration, 182, 186
neuronal vulnerability, 184
Polar hydrogen bonding, in protein
aggregation, 295
Polyamines, 79, 80
Positron emission tomography, 220
Postsynaptic density, 246, 247
Prefrontal cortex, 217, 219, 220
Presenilins, 289
Prion diseases, 292, 293
Prion protein, 293, 294
Pro-neurotrophins, mature proteins
release, 2
Protein conformational changes,
inhibition and reversion of, 297
Protein misfolding and aggregation, in
ND, 286288
animal models in, 293, 294
and brain damage mechanism,
296
development of therapy for, 298
studies on, 294296
therapeutic strategies for, 296, 297
PrP. See Prion protein
PS. See Presenilins
PSD. See Postsynaptic density
PTSD, 219, 220
Putrescine, 79
PVN, 217
Radial glia-independent radial
migration, 7
Real-time polymerase chain reaction, 92
Receptor tyrosine kinases, Trk family of,
neurotrophin actions, 2
Remethylation pathway, 81
Reperfusion, 133
Respiratory complex activities, 105
Ribonuclease protection assay, 92
RNA interference, 293
RNAi. See RNA interference
RNase H, 234
RPA. See Ribonuclease protection assay
324 Index

RRMs, 221 snRNPs, 221, 223 Tiam1, guanine-nucleotide exchange

RS domain, 221, 222, 224, 228
RT-PCR. See Real-time polymerase
chain reaction
S-adenosylhomocysteine, 79, 80
S-adenosylhomocysteine hydrolase,
79, 80
S-adenosylmethionine, 79
S-adenosylmethionine
decarboxylase, 79
SAGE. See Serial analysis of gene
expression
SC35, 223225
SCA. See Spinocerebellar ataxia
sCJD. See Sporadic CJD
Selye, H., 217
Serial analysis of gene expression, 22
in PC12 cells gene regulation,
3452
Serine Hydroxymethyltransferase,
79, 80
Serotonin, 220
Signaling mechanisms, regulatory
proteins, 110
Signal transduction
extracellular matrix, 170
integrins, 161, 170, 172
mitogen activated protein
kinase, 159
mitogenic signaling, 190
neurotrophins, 186
S-nitrosation, 112
snRNAs, 221
SOD1. See Superoxide dismutase 1
Spermidine, 79
Spermine, 79
Spine plastic
molecular dynamics of, 251, 252
molecular mechanisms of,
253, 254
Spinocerebellar ataxia, 286
Spinous synapse, postsynaptic density
of, 247250
Splicing, 220235
Sporadic CJD, 292
SR proteins, 221223, 227229
Stress, 216220, 222227, 230235
Striatal cholinergic neurons, 4
Sulfur containing amino acids, 7785
Superoxide dismutase 1, 291
SVZ development
GFAP-positive type B
Astrocytes, 68
subventricular zone (SVZ), 62, 67
Synapse formation and elimination, 251
Synaptic cell adhesion molecule, 251
Synaptogenesis and dendritic spine
synapses, 250, 251
SynCAM. See Synaptic cell adhesion
molecule
a
Synuclein protein, 290
Tauopathies, 230
Taurine, 7880
Testes, HO-2 in, 117
Tetrahydrofolic acid, 79, 80
factor
activation, 13
as TrkB effector, 14
TNF-a/b. See Tumor necrosis
factor-a/b
Transcription factors, NF-kB,
9295, 309
Transmissible spongiform
encephalopathies, 286
Transsulfuration, 80
TrkB mutant mice, brain-derived
neurotrophic factor (BDNF), motor
neuron death by, 5
TrkB/TrkC mutant mice
granule cell migration delay
in, 10
neuronal apoptosis in, 6
spinal cord motor neurons, 5
Trk neurotrophin receptors, 13
Trk receptor fusion proteins and
neurotrophin receptor, 12
Trk signaling pathways, 9, 13
TSEs. See Transmissible spongiform
encephalopathies
Tumor necrosis factor-a/b, 91
Ubiquitin, 116
Variant CJD (vCJD), 292
Vitagenes, 110
redox regulation of gene
expression, 135
role in life span prolongation,
133, 134