Pink1, Parkin, DJ-1 and mitochondrial dysfunction in Parkinsons

disease
Mark W Dodson
1,2
and Ming Guo
1,2,3
Mutations in PARKIN, PTEN-induced kinase 1 (PINK1) and
DJ-1 are found in autosomal recessive forms and some
sporadic cases of Parkinsons disease. Recent work on these
genes underscores the central importance of mitochondrial
dysfunction and oxidative stress in Parkinsons disease. In
particular, pink1 and parkin loss-of-function mutants in
Drosophila show similar phenotypes, and pink1 acts upstream
of parkin in a common genetic pathway to regulate
mitochondrial function. DJ-1 has a role in oxidative stress
protection, but a direct role of DJ-1 in mitochondrial function
has not been fully established. Importantly, defects in
mitochondrial function have also been identied in patients
who carry both PINK1 and PARKIN mutations, and in those
who have sporadic Parkinsons disease. Future studies of the
biochemical interactions between Pink1 and Parkin, and
identication of other components in this pathway, are likely to
provide insight into Parkinsons disease pathogenesis, and
might identify new therapeutic targets.
Addresses
1
Department of Neurology and Brain Research Institute, David Geffen
School of Medicine, University of California, Los Angeles, CA 90095,
USA
2
Molecular Biology Institute, University of California, Los Angeles,
CA 90095, USA
3
Department of Pharmacology, David Geffen School of Medicine,
University of California, Los Angeles, CA 90095, USA
Corresponding author: Guo, Ming (mingy@ucla.edu)
Current Opinion in Neurobiology 2007, 17:331337
This review comes from a themed issue on
Signalling mechanisms
Edited by Stuart Cull-Candy and Ruediger Klein
Available online 11th May 2007
0959-4388/$ see front matter
Published by Elsevier Ltd.
DOI 10.1016/j.conb.2007.04.010
Introduction
Parkinsons disease pathology is characterized most pro-
minently by loss of dopaminergic neurons in the sub-
stantia nigra and formation of intraneuronal protein
aggregates called Lewy bodies [1]. Over the past decade,
mutations that mediate familial forms of Parkinsons
disease have been denitively identied in ve genes:
those that encode a-Synuclein (SNCA, also known as
PARK1) and Leucine-rich repeat kinase 2 (LRRK2, also
known as PARK8), which mediate autosomal dominant
forms of Parkinsons disease, and those that encode
Parkin (PARKIN, also known as PARK2), DJ-1 (also
known as PARK7) and PTEN-induced kinase 1 (PINK1,
also known as PARK6), which mediate autosomal reces-
sive forms [1]. Although familial forms of Parkinsons
disease account for only 510% of all cases, mutations in
several of these genes have also been identied in spora-
dic Parkinsons disease [1], suggesting that studies of
these genes might provide insight into both familial
and sporadic disease.
Mitochondrial dysfunction and oxidative stress were
originally implicated in Parkinsons disease pathogenesis
because exposure to environmental toxins that inhibit
mitochondrial respiration and promote production of
reactive oxygen species (ROS) cause loss of dopaminergic
neurons in humans and animal models [2]. Recent
demonstrations that pink1, parkin and DJ-1 have crucial
roles in mitochondrial function and resistance to oxidative
stress reinforce the central importance of these themes in
Parkinsons disease pathogenesis and have begun to
enable understanding of these processes at the mecha-
nistic level. This reviewhighlights work fromthe past two
years on the roles of parkin, pink1 and DJ-1 in mitochon-
drial function.
Parkin: an E3 ubiquitin ligase essential for
mitochondrial function
The Parkin protein bears two RING-nger motifs and has
E3 ubiquitin ligase activity in vitro [3]. It is not yet known
whether the ligase activity of Parkin is required for its
protective role with respect to Parkinsons disease patho-
genesis. Nonetheless, because protein aggregation is a
pathological feature of Parkinsons disease, a major hy-
pothesis is that mutations in parkin result in the aberrant
accumulation of toxic substrates via dysfunction of the
ubiquitinproteasome system [4]. Several putative Parkin
substrates have been identied in vitro [4]. However, only
two of these, the aminoacyl-tRNA synthetase cofactor
p38 [5

] and far upstream element-binding protein 1 [6]

have been found to accumulate in parkin null mice, and
their roles in Parkinsons disease pathogenesis remain
unclear. Interestingly, recent studies suggest that the
ubiquitin ligase activity of Parkin can have proteasome-
independent functions, offering a potential explanation
for the lack of accumulation of some putative substrates in
parkin null mice. In addition to lysine48 (K48)-linked
polyubiquitination, which generally targets substrates for
proteasomal degradation, Parkin is able to catalyze both
monoubiquitination [7,8] and K63-linked polyubiquitina-
tion [9,10]. These modications can inuence cellular
processes such as signal transduction, transcriptional
www.sciencedirect.com Current Opinion in Neurobiology 2007, 17:331337
regulation, and protein and membrane trafcking,
without promoting substrate degradation [11]. For
example, it was recently reported that ubiquitination of
the endocytic protein Eps15 by Parkin modulates the
kinetics of endocytosis of epidermal growth factor re-
ceptor (EGFR) and, downstream of EGFR, signaling by
phosphoinositide 3-kinase and Akt [12

].
Important clues to the cellular processes that rely on
parkin function have come from studies of parkin loss-
of-function mutants in Drosophila. parkin mutant ies
exhibit dramatic mitochondrial defects swollen mito-
chondria that have severely fragmented cristae in
several energy-intensive tissues, including the male
germline and adult ight muscle [13,14]. The ight
muscles ultimately die, and their death shows features
of apoptosis [14]. parkin mutant ies also display a small
but signicant degeneration of a subset of dopaminergic
neurons [15]. Although severe defects in mitochondrial
morphology are not observed in parkin knockout mice
[16], these animals display reduced mitochondrial respir-
atory activity, which is associated with signs of oxidative
tissue damage in the brain [16]. Interestingly, defects of
the mitochondrial respiratory chain have also been
detected in peripheral tissues taken from human Parkin-
sons disease patients who have parkin mutations [17].
Together, these observations suggest that parkin has a
crucial and evolutionarily conserved role in mitochondrial
function.
pink1 and parkin function in a common
genetic pathway to regulate mitochondrial
function
Recent studies of pink1 and its interaction with parkin
strengthen the idea that parkin regulates mitochondrial
function. We and others reported that pink1 loss-of-
function mutants in Drosophila are viable, but exhibit
increased stress sensitivity and mitochondrial morphologi-
cal defects in testes and muscle [18

,19

,20

]. pink1
mutants also show reduced ATP levels and mitochondrial
DNA (mtDNA) content [18

,19

,20

]. As in parkin
mutant ies, mitochondria in pink1 mutant ight muscle
are swollen with fragmented cristae, and these cells
ultimately undergo apoptotic death [18

,19

,20

].
Muscle degeneration and reduced ATP and mtDNA
content can be rescued by overexpression of buffy, a y
Bcl-2 homolog [19

], but it is unclear if buffy overexpres-

sion exerts protective effects through direct inhibition of
apoptosis or through other effects on mitochondrial physi-
ology. Mitochondria within dopaminergic neurons inpink1
mutants also display aberrant morphology [19

], and a
small but statistically signicant loss of a subset of these
neurons occurs with age [19

,20

,21]. The striking

similarity between the phenotypes of pink1 and parkin
mutant ies led us and others to search for genetic
interactions between the two genes. parkin overexpression
in ies suppresses all pink1 mutant phenotypes tested
[18

,19

,20

] andpink1 overexpressiondoes not compen-

sate for loss of parkin function [18

,19

]. Furthermore,
double mutants in which both pink1 and parkin are absent
have phenotypes identical to, rather than stronger than,
either single mutant [18

,19

]. Together, these data

provide compelling in vivo evidence that pink1 and parkin
act in a linear pathway that affects mitochondrial function,
with parkin downstream of pink1.
Several lines of evidence suggest that these observations
on pink1 and parkin function are relevant to humans. First,
expression of human PINK1 [18

,20

] or PARKIN (LJ
Pallanck, personal communication) in Drosophila sup-
presses phenotypes caused by loss-of-function of pink1
or parkin, respectively, suggesting that the human and y
proteins are functionally conserved. Second, in addition
to being expressed in the brain, human PINK1 and
PARKIN are highly expressed in testes and muscle
[22,23], the tissues in which mitochondrial defects are
most prominently seen in pink1 and parkin mutant ies.
Third, pathological changes and defects in mitochondrial
respiration have been detected in peripheral tissues from
patients with PINK1 [24] or PARKIN [17] mutations, as
well as those with sporadic Parkinsons disease [2527],
suggesting that such defects accompany loss of dopamin-
ergic neurons as part of a more global pathology in
Parkinsons disease. Finally, Parkinsons disease patients
who harbor mutations in PINK1 or PARKIN are clinically
indistinguishable [28], a nding consistent with the hy-
pothesis that these genes function in a common genetic
pathway.
What is the biochemical relationship
between Pink1 and Parkin?
An important unanswered question is whether Pink1 and
Parkin directly bind to each other, and whether Parkin is a
substrate for Pink1 kinase activity. We have found that
Pink1 and Parkin can physically interact in cultured
Drosophila cells (R Feldman and M Guo, unpublished
data), although the functional consequences of this inter-
action remain unclear. No substrates for Pink1 kinase
activity have been identied to date but the ubiquitin
ligase activity of Parkin can be modulated by phosphoryl-
ation [29]. Thus, Pink1 might regulate Parkin activity
post-translationally, either by direct phosphorylation or
by activation of intermediate signaling proteins
(Figure 1). Alternatively, it has been reported that knock-
down of pink1 by RNA interference (RNAi) in Drosophila
results in reduced abundance of the Parkin protein [20

],
suggesting that Pink1 functions directly (perhaps through
modulation of Parkin autoubiquitination and degra-
dation) or indirectly (through actions on other proteins)
to maintain Parkin levels. The aforementioned genetic
data are also consistent with a model in which Pink1 and
Parkin act in series on shared targets. For example, Pink1-
dependent phosphorylation of a substrate might facilitate
the interaction of this protein with Parkin (Figure 1).
332 Signalling mechanisms
Current Opinion in Neurobiology 2007, 17:331337 www.sciencedirect.com
Further genetic and biochemical characterization of the
Pink1Parkin interaction is required to clarify the mech-
anisms by which this pathway functions.
Where do Pink1 and Parkin function?
Several lines of evidence suggest that Pink1 functions
within mitochondria. Pink1 bears an N-terminal mito-
chondrial targeting sequence (MTS) and colocalizes pre-
dominantly with mitochondrial markers in cultured
mammalian cells [19

,30] and in vivo [18

]. In human
and rat brain, endogenous Pink1 fractionates with mar-
kers of the outer and inner mitochondrial membranes but
not with those of the matrix or intermembrane space [31].
Immunoelectron microscopy studies show Pink1 to be
closely associated with the inner mitochondrial mem-
brane [32]. Removal of the outer membrane exposes
Pink1 to protease digestion [32], suggesting that at
least a portion of the protein is exposed to the intermem-
brane space. Pink1 contains a predicted transmembrane
domain after its N-terminal MTS, suggesting that it
might be an integral membrane protein [32], although
this hypothesis has yet to be tested directly. Interestingly,
an N-terminally truncated form of the protein can be
detected in the cytosolic fraction when pink1 is over-
expressed in cultured mammalian cells [33], raising the
possibility that mature Pink1 is released from the mito-
chondrion under certain conditions [33].
Unlike Pink1, Parkin does not contain a MTS and loca-
lizes predominantly to the cytosol and endoplasmic reti-
culum [34,35]. However, part of the cellular Parkin pool
associates with the cytoplasmic surface of the outer mito-
chondrial membrane, as determined by limited protease
digestion of mitochondria-enriched fractions from cul-
tured mammalian cells [35]. Furthermore, although Par-
kin had not previously been identied within
mitochondria, one recent study using immunoelectron
microscopy has detected Parkin in association with mito-
chondrial cristae in a proliferating neuronal cell line
[36

]. Thus, it is likely that both Pink1 and Parkin are

present within or at the surface of the mitochondrion in at
least some contexts. Given that Pink1 localizes predomi-
nantly within mitochondria and is required for mitochon-
drial function, the most parsimonious hypothesis is that
Pink1, Parkin, DJ-1 and mitochondrial dysfunction in Parkinsons disease Dodson and Guo 333
Figure 1
Mechanisms by which Pink1 and Parkin might interact. Pink1 associates with the inner mitochondrial membrane [31,32] and is exposed to the
intermembrane space [32]. Parkin localizes predominantly to the cytosol [34,35] but also associates with the mitochondrial outer membrane
[35] and has been detected within the mitochondrion in association with mitochondrial cristae under certain conditions [36

] (depicted in the top

panel only, for simplicity). Pink1 might modulate the activity or stability of Parkin by directly binding to and phosphorylating Parkin, either within
the mitochondrion or in the cytosol, as Pink1 might be released from mitochondria under certain conditions [33] (top). Pink1 might phosphorylate
an intermediate signaling protein that has a downstream effect on the activity or stability of Parkin (middle). Alternatively, Pink1 and Parkin might
act sequentially on a shared target. For example, Pink1-mediated phosphorylation of a substrate might facilitate the interaction of this substrate
with Parkin, possibly enabling its ubiquitination by Parkin (bottom).
www.sciencedirect.com Current Opinion in Neurobiology 2007, 17:331337
Pink1 functions directly at the mitochondrion, perhaps
through direct phosphorylation of Parkin or of other
signaling molecules that regulate the activity or abun-
dance of Parkin. Alternatively, because Pink1 might be
released from the mitochondrion under certain con-
ditions, it remains possible that Pink1 has extra-
mitochondrial functions, perhaps involving interaction
with cytosolic Parkin, that regulate mitochondrial func-
tion (Figure 1). By the same logic, the partial mitochon-
drial localization of Parkin might reect a direct function
on mitochondria, but it might also be that actions carried
out by Parkin in other cellular compartments have a
downstream effect on mitochondrial function.
Which aspects of mitochondrial function rely
on Pink1 and Parkin?
Mitochondria have crucial roles in multiple cellular pro-
cesses, including ATP production, regulation of cell
death, Ca
2+
homeostasis and cellular signaling [37]. pink1
mutant Drosophila exhibit decreased ATP content
[18

,19

,20

], and deciencies in mitochondrial respir-

atory chain activity have been detected in parkin knock-
out mice [16] and in human Parkinsons disease patients
who have mutations in PARKIN [17] or PINK1 [24].
Furthermore, parkin loss-of-function sensitizes cultured
mammalian cells [38] and Caenorhabditis elegans [39] to
inhibition of mitochondrial complex I, whereas parkin
overexpression increases complex I activity [40]. It is
unclear whether these defects in respiration and energy
production reect direct or indirect roles of pink1 and
parkin in these processes. For example, y pink1 mutants
display additional mitochondrial defects, such as loss of
cristae [18

,19

,20

] and mtDNA [19

], which could
account for the reduced ATP production. In the Droso-
phila ight muscle, pink1 [18

,19

] and parkin [14] are

required for proper mitochondrial maintenance, because
mitochondria initially appear normal during pupal de-
velopment but progressively deteriorate as the y ages.
Given this, it will be interesting to determine whether
biochemical defects in mitochondrial function precede
mitochondrial morphological defects, or vice versa. One
possible mechanism by which pink1 and parkin could
inuence mitochondrial physiology is suggested by the
intriguing observation that Parkin can associate with
mitochondrial transcription factor A (TFAM) and posi-
tively regulate the transcription of mtDNA-encoded
respiratory chain complexes [36

]. Thus, it is possible
that parkin and pink1 have a direct role in the mainten-
ance of proper mitochondrial respiratory activity. Given
that pharmacological inhibition of complex I recapitulates
key features of Parkinsons disease [2], it is conceivable
that defects in mitochondrial respiration caused by
mutations in PARKIN or PINK1 could contribute to
Parkinsons disease pathogenesis by a similar mechanism.
In addition, mitochondria undergo dynamic fusion and
ssion events, which are linked to the maintenance of
proper mitochondrial function and the regulation of cell
death [41]. Therefore, it is not unreasonable to speculate
that parkin and pink1 also contribute to the regulation of
mitochondrial dynamics.
DJ-1 functions in resistance to oxidative
stress
Unlike pink1 and parkin, there is little evidence that DJ-1
has a direct role in mitochondrial function. However,
studies in both cell culture and animal models have
demonstrated that DJ-1 deciency increases sensitivity
to cell death induced by oxidative stress, whereas over-
expression is protective [42]. Interestingly, these effects
seem to be specic to oxidative stress because DJ-1
deciencysensitizes ies to H
2
O
2
andthe ROS-generating
compound paraquat, but not to various non-oxidative
stresses [43]. Cysteine residues in both human [44,45]
and Drosophila [46

] DJ-1 are modied to cysteine-sulnic

and cysteine-sulfonic acids under oxidative conditions;
this modication is required for the protective function
of DJ-1 [45,46

]. These data suggest that, throughcysteine

modication, DJ-1 senses the redox state of the cell and
under oxidative conditions is activated to exert protective
effects. DJ-1 has been suggested to function through
various mechanisms, including as a transcriptional coacti-
vator, a protease or a molecular chaperone [42], but exactly
how DJ-1 exerts its protective effects remains unclear.
Endogenous DJ-1 can be found in the mitochondrial
matrix and intermembrane space in mouse brain, although
its localization is predominantly cytosolic [47]. This partial
mitochondrial localization might reect a role for DJ-1 in
mitochondrial function, but mitochondrial morphological
defects due to DJ-1 loss-of-function have not been
reported in any model organism. Interestingly, in vitro
studies have reported physical interactions of DJ-1 with
Pink1 [48] and, under oxidative conditions, with Parkin
[49]; however, it remains unclear whether these associ-
ations occur invivo. Overexpressionof DJ-1does not rescue
muscle phenotypes [20

] or male sterility (M Guo, unpub-

lished data) that is due to lack of pink1, or muscle degener-
ation that is caused by lack of parkin (LJ Pallanck, personal
communication), suggesting that DJ-1 might function in a
distinct pathway from that of pink1 and parkin. Interest-
ingly, the mild neuronal death caused by loss of pink1
function [21] or parkin function [15] in Drosophila can be
suppressed by overexpression of genes that encode anti-
oxidant proteins, suggesting that oxidative stress is also a
component of pathology due to pink1 and parkin loss-of-
function. Because defective mitochondrial respiration can
increase ROS production, a reasonable hypothesis is that
DJ-1 functions to detect and/or defend against oxidative
stress associated with mitochondrial respiration.
A convergence between mitochondrial
dysfunction and protein aggregation?
Two major processes, mitochondrial dysfunction and
protein aggregation, have been implicated in Parkinsons
334 Signalling mechanisms
Current Opinion in Neurobiology 2007, 17:331337 www.sciencedirect.com
disease pathogenesis. A major question that remains to be
answered is whether these two mechanisms intersect.
Recent work suggests a previously unappreciated inter-
action between a-Synuclein and mitochondrial dysfunc-
tion. a-Synuclein is a brillar aggregation-prone protein
that is a main component of Lewy bodies and is believed
to contribute to Parkinsons disease by toxic gain-of-
function effects. Expression of mutant human SNCA in
mice causes mitochondrial degeneration associated with
mtDNA damage and reduced activity of the respiratory
chain complex [50

] (see also the Update section of this

review). Mutant SNCA expression also increases sensi-
tivity to inhibitors of mitochondrial complex I, whereas
loss-of-function of a-Synuclein promotes resistance to
these compounds [51]. Intriguingly, pharmacological
inhibition of complex I of the mitochondrial respiratory
chain in rodents causes aggregation of a-Synuclein
[52,53]. Together, these data suggest a possible conver-
gence between mitochondrial function and protein aggre-
gation. One may envisage a positive feedback model in
which mitochondrial dysfunction promotes a-Synuclein
aggregation, which promotes further mitochondrial dys-
function. In such a model, either stimulus mitochon-
drial dysfunction or a-Synuclein aggregation could
initiate the cycle but the end result would be similar.
Conclusions
Recent work has underscored the central importance of
mitochondrial dysfunction in Parkinsons disease. In
particular, studies from Drosophila have demonstrated
that pink1 and parkin act in a linear genetic pathway
required for the maintenance of mitochondrial function.
Meanwhile, cumulative data suggest that DJ-1 functions
in protection from oxidative stress but does not directly
affect mitochondrial morphology. Whereas a large body of
evidence suggests that Pink1 is present within the mito-
chondrion, Parkin localizes predominantly to the cytosol,
although recent studies have demonstrated mitochondrial
localization for a portion of the cellular Parkin pool. Thus,
it remains unclear how Pink1 and Parkin act together to
regulate mitochondrial function. It is crucial that future
studies address the mechanisms through which Pink1 and
Parkin interact with each other to affect mitochondrial
physiology. Towards this end, genetic approaches in
Drosophila have tremendous potential to identify further
components of the pink1parkin pathway as enhancers or
suppressors of the robust phenotypes associated with
mutations in these genes. Future studies on the pink1
parkin pathway will provide further insight into Parkin-
sons disease pathogenesis, and have the potential to
identify new therapeutic targets.
Update
Recently, Stichel et al. have identied defects in mito-
chondrial morphology in dopaminergic neurons in parkin
knockout mice expressing mutant human SNCA [54

].
These mice display degenerative mitochondrial changes,
such as dilated cristae, which are absent in young mice
but frequent in 1214 month old mice. The same types of
age-dependent mitochondrial structural defects were also
seen in mice either mutant for parkin or expressing
mutant SNCA more frequently than in wild type controls,
but the number of damaged mitochondria only reached
statistical signicance in mice with both genetic modi-
cations. These results suggest that the effects of parkin
loss-of-function and SNCA expression on mitochondrial
morphology are additive. These data also extend the
ndings of Martin et al. [50

] by demonstrating that
mutant human SNCA expression in mice causes mito-
chondrial degeneration in dopaminergic neurons in the
substantia nigra.
Acknowledgements
We apologize to those whose work has not been cited because of space
constraints. We thank Marie-Francoise Chesselet, Bruce Hay, Leo
Pallanck, Ira Clark and Guo laboratory members for helpful comments on
the manuscript. This work is supported by an NIH/NINDS NRSA
predoctoral fellowship to MWD and by NIH KO8 and RO1 grants and an
Alfred Sloan Foundation Fellowship to MG.
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336 Signalling mechanisms
Current Opinion in Neurobiology 2007, 17:331337 www.sciencedirect.com
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Stichel CC, Zhu XR, Bader V, Linnartz B, Schmidt S, Lubbert H:

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The authors demonstrate that mitochondria from parkin knockout mice
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age-dependent defects in mitochondrial morphology within dopaminer-
gic neurons. These defects were also seen in mice either mutant for parkin
or expressing SNCA more frequently than in wild type mice, but the
number of damaged mitochondria only reached statistical signicance in
mice with both genetic modications.
Pink1, Parkin, DJ-1 and mitochondrial dysfunction in Parkinsons disease Dodson and Guo 337
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