ii

Dedication

This thesis work is dedicated to my husband, Bruce, who has been a constant
source of support and encouragement during the challenges of graduate
school and life. I am truly thankful for having you in my life. This work is also
dedicated to my parents, William and Karen Ryan, who have always loved me
unconditionally and whose good examples have taught me to work hard for
the things that I aspire to achieve.
 iii

Curriculum Vitae

Deborah Anne Ryan was born in Buffalo, NY on July 15, 1981. She

graduated from Williamsville East High School in 1999. She attended

Vanderbilt University from 1999 to 2003, and graduated magna cum laude

with a Bachelor of Arts degree in Neuroscience. Deborah entered graduate

school at the University of Rochester School of Medicine and Dentistry in the

fall of 2003. She worked in the lab of Dr. Howard J. Federoff from 2005 until

his departure from the University in 2007, at which time she continued her

studies on immunotherapeutic approaches to Alzheimers disease in the

laboratory of Dr. William J. Bowers. She was awarded the Interdepartmental

Neuroscience Training Grant from 2004 to 2006 and received the Ruth L.

Kirschstein National Research Service Award from 2008 to 2009. Deborah

earned a Masters of Science degree from the Interdepartmental Graduate

Program in Neuroscience at the University of Rochester in May 2007.

 iv

Publications

Ryan DA, Mastrangelo MA, Narrow WC, Sullivan MA, Federoff HJ, Bowers
WJ (2010) A-directed single-chain antibody delivery via a serotype-1 AAV
vector improves learning behavior and pathology in Alzheimers disease mice.
Molecular Therapy (in submission).

Desai MK, Mastrangelo MA, Ryan DA, Sudol KL, Narrow WC, Bowers WJ
(2010) The oligodendrocyte lineage represents an amyloid-beta sensitive cell
population during early Alzheimers disease. The American Journal of
Pathology (in submission).

Ryan DA, Federoff HJ, Bowers WJ (2009) An improved method for

generating consistent soluble amyloid-beta oligomer preparations for in vitro
neurotoxicity studies. Journal of Neuroscience Methods (in submission).

Ryan DA, Federoff HJ (2009) Immune Responses to Herpes Viral Vectors.

Human Gene Therapy 20(5):434-41.

Chiu YG, Bowers WJ, Lim ST, Ryan DA, Federoff HJ (2009) Effects of HSV
amplicon transduction on murine dendritic cells. Human Gene Therapy
20(5):442-52.

Ryan DA and Federoff HJ (2007) Translational considerations for CNS gene

therapy. Expert Opinion on Biological Therapy 7(3):305-318.

Weimer JM, Benedict JW, Elshatory YM, Short DW, Ramirez-Montealegre D,

Ryan DA, Alexander NA, Federoff HJ, Cooper JD, Pearce DA (2007)
Alterations in striatal dopamine catabolism precede loss of substantia nigra
neurons in a mouse model of juvenile neuronal ceroid lipofuscinosis. Brain
Research 1162:98-112.
 v

Meeting Abstracts

Ryan DA, Sullivan MA, Federoff HJ, Bowers WJ (2008) In vitro and in vivo
characterization of amyloid beta-specific single chain fragment variable
antibodies. Society for Neuroscience 542.6 O2 (poster).

Ryan DA, Sullivan MA, Federoff HJ, Bowers WJ (2007) Isolation and
characterization of oligomeric amyloid beta-specific single chain fragment
variable antibodies. Society for Neuroscience 886.3 L19 (poster).
 vi

Acknowledgments

I am very fortunate to have performed my graduate work at a university

as collaborative as the University of Rochester; therefore, there are many

people to thank for their part in my success. I would first like to thank my

advisor, Dr. William J. Bowers, for giving me a home in his lab and support

over the years. I am grateful for his guidance and the opportunities he has

afforded me. He is incredibly organized and a great problem solver, both of

these qualities were immensely helpful in moving my project forward. Under

his mentorship I have learned the particulars of grant writing, which is an

invaluable tool to have as my career moves forward. Bill is also exceptionally

generous and would frequently take his staff and students on outings to let us

know our work is appreciated. I will remember my time in the lab and these

outings very fondly.

I would also like to thank my thesis committee members, Dr. Stephen

Dewhurst, Dr. John Olschowka, and Dr. John Frelinger, for their contributions

to this work. Over the years, each has given me superb scientific guidance,

many insightful suggestions and demonstrated a sincere interest in my work.

I am fortunate to have such a group of intelligent scientists, who are also

student advocates, on my committee. A former thesis committee member,

Dr. Mark Sullivan, was instrumental to the initiation of this project. Dr.

Sullivan generated the human scFv phage display library used in these

experiments and instructed me on phage selection and scFv production

 vii

techniques. He deserves many thanks for his continued interest and

guidance throughout my work.

I would like to recognize the members of the Bowers lab who have all

contributed to the progress I have made. Over the years, Mike Mastrangelo

has given his time to teach me several techniques and aid in experiments.

Lou Lotta and Mike both deserve huge thanks for dealing with animal

husbandry. I would like to acknowledge Wade Narrow for his rAAV

packaging expertise and wish him luck as he has already taken over the

oligomer-specific scFv project. Also, Clark and Marilyn contributed to my

work by performing the thankless task of ordering much-needed reagents and

dealing with administrative staff always with a cheerful demeanor. I would

like to thank the former graduate students, Dr. Michelle Janelsins for sharing

her enthusiasm for science with me, Dr. Suresh DeSilva for being an

incredible resource for molecular biology and cloning advice, and Dr. Keigan

Park for his help with techniques and always being excited to talk about

scientific problems or data. Maya Desai, my bay-mate, has always been

there to talk if ever something is bothering me about my experiments or in life.

I would like to express my appreciation for the optimistic attitude that Sara

Montgomery brings to lab; her insightful questions usually provoke interesting

scientific discussions. The Bowers lab members are a wonderful group of

people who I have enjoyed working with and learning from.

 viii

I would like to thank the former Federoff lab members and associates

who have contributed to my development as a scientist, including Dr. Howard

Federoff, Dr. David Rempe, Dr. Kathy Maguire-Zeiss, Dr. Tim Mhyre, Dr.

Christina Liliehook, Dr. Jason Hamilton, Dr. Charles Wuertzer, Dr. Carolyn

Tyler, and Doug Short. Each person has had a significant impact on my

graduate career that I will not forget. I would like to give special thanks to

Rita Giuliano, who always made time to instruct and help with my cell culture

problems, without her expertise I would not have accomplished as much.

I would like to acknowledge the support from the National Institute of

Health for funding my National Research Service Award, Targeting Amyloid

Beta Oligomers in the 3xTg-AD Mouse Model with scFv Antibodies

(F31NS059283). I would like to thank investigators from outside the

University for their generous gifts of key reagents: Dr. Charles Glabe at UC

Irvine for A11 and I11 antibodies and Dr. William Klein at Northwestern

University for the NU-4 antibody. I would like to recognize Dr. Semyon

Papernov at the University of Rochester Laboratory for Laser Energetics, who

instructed me on how to use the atomic force microscope.

The atmosphere at the University of Rochester, especially within the

Neuroscience community, is incredibly collaborative. I would like to extend

my appreciation to the people who have fostered this wonderful learning

environment. I would like to express thanks to Ania Dworzanski for

completing my thesis paperwork and doing her job extremely well as to make
 ix

the life of neuroscience graduate students easier. I would like to recognize

the CNDD administrative staff, especially a former member, Anne Dickinson,

who performed her job so competently it appeared effortless.

Finally, I would like to thank my friends and family for their continued

support and encouragement. The individuals I have met in graduate school

that I consider friends are too numerous to name. There are a few, however,

that cannot go unmentioned, I would specifically like to recognize Carolyn

Tyler, Keigan Park, Grace Vangeison, Laurie Robak, Jason Hamilton, Clark

Burris, Jill Weimer, Pete & Seasson Vitiello, and William Mowery. These

friends have been there for me when the challenges of graduate school

seemed too great to overcome. Although many have moved away from

Rochester, I will never forget the experiences weve shared and hope to stay

in touch.

I would like to express the deepest gratitude to my family. Mom, Dad,

Julie, Jeffrey, Erin and Greg you have all provided support, encouragement

and interest in my thesis work. Thanks for listening to my problems and

providing perspective. I would not be who am I today without you all. Finally,

I would like to thank my husband, Bruce. You have been continually

supportive of my graduate education. Thank you for the little things youve

done like brining me dinner when I worked late nights. Thank you for those

weekends when you sat at the computer in the mouse-scented behavioral

suite to cue-up each mouses file so that my work would go a little quicker.
 x

You have been patient with me when Im frustrated, you celebrate with me

when even the littlest things go right, and you are there whenever I need you

to just listen.
 xi

Abstract

Alzheimers disease (AD) is a progressive dementing disorder

characterized by age-related amyloid-beta (A) deposition, neurofibrillary

tangles, as well as synapse and neuronal loss. It is the leading cause of

dementia in the United States with an estimated 5.3 million Americans

currently afflicted. As the population ages, AD becomes an increasingly

important public health concern. Implementing an effective treatment for AD

would alleviate the emotional and economic burden, but currently approved

interventions primarily target symptoms and not disease pathogenesis. It is

widely recognized that A is a principal pathogenic mediator of AD, and may

represent a viable therapeutic target.

Our goal was to develop an immunotherapeutic-based approach,

which would specifically facilitate the clearance and/or neutralization of A

within the parenchymal space. Efficacy of the therapy to mitigate

downstream pathological cascades, and lead to improved cognitive function

was assessed in the triple transgenic mouse model (3xTg-AD). These mice

develop age-related amyloid deposition, neurofibrillary tangle pathology,

synaptic dysfunction and cognitive deficits reminiscent of human AD.

Using a human single-chain variable fragment (scFv) antibody phage

display library, a novel scFv antibody specific to A was isolated via selection

on synthetic A oligomer preparations. The specificity and therapeutic activity

 xii

of A-scFv was characterized in vitro, and its open reading frame

subsequently cloned into a recombinant adeno-associated virus (rAAV)

vector. At 3 months of age, prior to the onset of observable pathology, 3xTg-

AD mice were intrahippocampally infused with serotype-1 rAAV vectors

encoding A-scFv or a non-relevant control scFv using convection-enhanced

delivery. Mice infused with rAAV1-A-scFv harbored lower levels of insoluble

A and hyperphosphorylated tau, and exhibited improved cognitive function

as measured by the Morris Water Maze spatial memory task. These results

underscore the potential of gene-based passive vaccination for AD, and

provide further rationale for the development of A-targeting strategies for this

debilitating disease.
 xiii

Table of Contents

Curriculum Vitae iii

Publications iv

Acknowledgments vi

Abstract xi

Table of Contents xiii

List of Tables xvi

List of Figures xvii

List of Abbreviations xix

Chapter 1:
Introduction

1.1 Alzheimers Disease 2

1.2 Current treatments 3

1.3 Familial versus sporadic AD 4

1.4 Amyloid precursor protein processing 5

1.5 Alzheimers disease mouse models 7

1.6 The dual faces of amyloid-beta: a regulator of physiological

function or a pathogenic mediator 10

1.7 Abnormal hyperphosphorylation of tau 13

1.8 Therapeutics targeting A 14

1.9 Immunotherapeutic approaches to AD 16

1.10 Thesis project aims 19

 xiv

Chapter 2:
Generation of Consistent Soluble Amyloid-beta Oligomer Preparations

2.1 Introduction 28

2.2 Synthesis of amyloid-beta oligomer preparations 29

2.3 Structural characterization of AOs 30

2.4 Neurotoxic action of AO preparations 36

2.5 Discussion 37

2.6 Experimental Methods 42

Chapter 3:
Isolation and in vitro Characterization of A
-specific
Single-chain Variable Fragment Antibodies

3.1 Introduction 59

3.2 Isolation of A-specific scFv antibodies 62

3.3 In vitro characterization of an A-specific scFv antibody 65

3.4 A-scFv primary sequence 66

3.5 A-scFv binds A and prevents toxicity in vitro 67

3.6 Discussion 68

3.7 Experimental Methods 73

Chapter 4:
Passive Immunotherapeutic Approach to Alzheimers Disease

4.1 Introduction 94
 xv

4.2 In vivo eGFP delivery to the brains of 3xTg-AD mice via

serotype-1 rAAV vector 97

4.3 In vivo A-scFv delivery to 3xTg-AD mice via a serotype-1

rAAV vector 98

4.4 Impact of rAAV1 A-scFv on learning and memory 99

4.5 Pathological assessment of A-scFv in the 3xTg-AD mouse 101

4.6 Discussion 103

4.7 Experimental Methods 109

Chapter 5:
Discussion and Future Directions

5.1 Different forms of A may have distinct pathological mechanisms 139

5.2 Anti-A antibodies employed in passive immunization require

stringent characterization 143

5.3 AAV immune/inflammatory responses 148

5.4 Conclusion 153

Appendix

Continuation of In Vitro Characterization of Oligomer-specific Single-

chain Variable Fragment Antibodies

A Candidate oligomer-specific scFv antibodies are retained within

cells 160

B Candidate oligomer-specific scFv antibodies prevent secretion

of A 162

References 164
 xvi

List of Tables

Table 3-1 Overview of scFv expression and A immunoreactivity 83

Table 5-1 An overview of select studies utilizing A-specific

full-length antibodies for passive immunization 154
 xvii

List of Figures

Figure 1-1 Regulated processing of the amyloid precursor protein 20

Figure 1-2 Triple transgenic AD mouse pathological timeline 22

Figure 1-3 Fibrillogenesis of A 24

Figure 2-1 Flow chart of A oligomer preparation method 48

Figure 2-2 Structural characteristics of AO preparations 50

Figure 2-3 AO preparations analyzed by atomic force microscopy 52

Figure 2-4 Thioflavine T fluorescence assay of AO preparations 54

Figure 2-5 AO preparations are neurotoxic 56

Figure 3-1 Human scFv phage display library vector system and
selection schematic 79

Figure 3-2 Isolation of A-specific scFv antibodies from a human scFv

phage display library 81

Figure 3-3 Characterization of an A-specific scFv antibody 85

Figure 3-4 A-scFv amino acid sequence 88

Figure 3-5 A-scFv antibody reduces detection of secreted A and

mitigates A-mediated toxicity in vitro 90

Figure 4-1 rAAV1-GFP distribution and long-term expression in vivo 117

Figure 4-2 Experimental design and in vivo A-scFv expression from

rAAV1 constructs 119

Figure 4-3 Schematic of Morris water maze 122

Figure 4-4 3xTg-AD mice receiving rAAV1 A-scFv learn a new target
location sooner than those receiving rAAV1-Phe-scFv
during training on the Morris water maze 124
 xviii

Figure 4-5 3xTg-AD mice receiving rAAV1 A-scFv use spatial

search strategies more frequently than mice receiving
rAAV1-Phe-scFv during training on the Morris water maze 127

Figure 4-6 3xTg-AD mice receiving rAAV1-A-scFv and

rAAV1-Phe-scFv exhibit no differences in performance
on MWM probe trials to test short and long-term memory 129

Figure 4-7 rAAV1 vector-mediated expression of A-scFv antibody

decreases levels of insoluble A in the hippocampus of
3xTg-AD mice 131

Figure 4-8 rAAV1 vector-mediated expression of the A-scFv

antibody increases number and activation of microglia 134

Figure 4-9 rAAV1 vector-mediated expression of the A-scFv antibody

decreases levels of A and hyperphosphorylated tau in the
hippocampus of 3xTg-AD mice 136

Figure 5-1 Potential therapeutic mechanisms by which A-scFv prevents

pathology in vivo 156

Appendix A Candidate oligomer-specific scFv antibodies are retained

within cells 160

Appendix B Candidate oligomer-specific scFv antibodies prevent

secretion of A 162
 xix

List of Abbreviations

3xTg-AD Triple transgenic AD

6xHIS hexahistidine tag

A amyoid-beta

AM amyloid-beta monomer

AOs amyloid-beta oligomers

AD Alzheimer's disease

ADAM10 a disintegrin and metalloprotease 10

ADAM17 a disintegrin and metalloprotease 17

ADDLs A-derived diffusible ligands

AFM atomic force microscopy

AICD APP intracellular domain

ANOVA analysis of variance

APOE apolipoprotein E

APP amyloid precursor protein

APPswe APP Swedish mutation

syn synuclein

BACE -site APP cleaving enzyme

BBB blood-brain barrier

BHK baby hamster kidney

 xx

CA1 cornu Ammonis 1

cdk5 cyclin-dependent kinase 5

CDR complementarity determining region

CED convection-enhanced delivery

CFC contextual fear conditioning

CM culture media

CMV cytomegalovirus

CNS central nervous system

CSF cerebrospinal fluid

CTF C-terminal fragment

DAB 3,3-diaminobenzidine

DG dentate gyrus

DIV day in vitro

DMEM Dulbeccos modified eagle medium

E. coli Escherichia coli

eGFP enhanced green fluorescent protein

ELISA enzyme linked immunosorbent assay

ER endoplasmic reticulum

FAD familial AD

FBS fetal bovine serum

Fc fragment crystallizable
 xxi

FTDP-17 frontotemporal dementia with parkinsonism linked to

chromosome 17

FWR framework region

GFAP glial fibrillary acidic protein

GSK-3 glycogen-synthase kinase-3

HBSS Hank's balanced salt solution

HEK human embryonic kidney

HFIP Hexafluoroisopropanol

HRP horseradish peroxidase

i.c.v intracerebroventricular

i.p. intraperitoneal

IAPP islet amyloid polypeptide

IHC immunohistochemical

ITRs inverted terminal repeats

kb kilobases

KD dissociation constant

kDa kilodalton

LOAD late-onset AD

LRP low-density lipoprotein receptor related protein 1

LTP long-term potentiation

MAP microtubule associated protein

Monsub monomer pre-incubation

 xxii

MRI magnetic resonance imaging

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)
-2-(4-sulfophenyl)-2H-tetrazolium

MWM Morris water maze

NEP neprilysin

NFDM non-fat dried milk

NGS normal goat serum

NMDA N-methyl-D-aspartate

NSAID non-steroidal anti-inflammatory drug

PBS phosphate buffered saline

Phe phenobarbital

PI3-kinase phosphatidylinositol-3-kinase

PKA cAMP-dependent protein kinase

PMS phenazine methosulfate

PSEN presenilin

PVDF polyvinylidene difluoride

rAAV recombinant adeno-associated virus

scFv single-chain variable fragment

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

sLRP soluble LRP

TACE tumor necrosis factor -converting enzyme

 xxiii

TBS tris buffered saline

TBST tris buffered saline with Tween-20

ThioT thioflavine T

VH variable heavy chain

VL variable light chain