Lentiviral vector delivery of parkin prevents
dopaminergic degeneration in an ?-synuclein rat
model of Parkinson’s disease
Christophe Lo Bianco*, Bernard L. Schneider*†, Matthias Bauer*, Ali Sajadi*, Alexis Brice‡, Takeshi Iwatsubo§,
and Patrick Aebischer*¶
*Institute of Neuroscience, Swiss Federal Institute of Technology Lausanne, Ecole Polytechnique Fe ´de ´rale de Lausanne, CH-1015 Lausanne, Switzerland;
‡Institut National de la Sante ´ et de la Recherche Me ´dicale U289, Ho ˆpital de la Salpe ´trie `re, 75651 Paris, France; and§Department of Neuropathology and
Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan
Edited by Fred H. Gage, The Salk Institute for Biological Studies, San Diego, CA, and approved October 29, 2004 (received for review July 22, 2004)
Parkinson’s disease (PD) is characterized by a progressive loss of
midbrain dopamine neurons and the presence of cytoplasmic
?-synuclein and parkin have been linked to familial PD. The loss of
parkin’s E3-ligase activity leads to dopaminergic neuronal degen-
eration in early-onset autosomal recessive juvenile parkinsonism,
suggesting a key role of parkin for dopamine neuron survival. To
evaluate the potential neuroprotective role of parkin in the patho-
genesis of PD, we tested whether overexpression of wild-type rat
parkin could protect against the toxicity of mutated human A30P
?-synuclein in a rat lentiviral model of PD. Animals overexpressing
parkin showed significant reductions in ?-synuclein-induced neu-
ropathology, including preservation of tyrosine hydroxylase-
positive cell bodies in the substantia nigra and sparing of tyrosine
hydroxylase-positive nerve terminals in the striatum. The parkin-
mediated neuroprotection was associated with an increase in
hyperphosphorylated ?-synuclein inclusions, suggesting a key role
for parkin in the genesis of Lewy bodies. These results indicate that
parkin gene therapy may represent a promising candidate treat-
ment for PD.
gene therapy ? lentivirus ? neurodegenerative disease ? Lewy
body ? neuroprotection
over the age of 60. Loss of dopaminergic neurons in the
substantia nigra pars compacta and subsequent striatal dopa-
mine depletion causes motor impairments including akinesia,
resting tremor, muscle rigidity, and gait and postural deficits.
The neuropathological hallmark of PD is the appearance of
proteinaceous intracellular deposits identified as Lewy bodies
and Lewy neurites. Although the mechanism leading to the
selective degeneration of nigral dopamine neurons in sporadic
PD remains unknown, clues about the pathogenesis of familial
forms of PD are emerging because of the discovery of various
gene mutations. Two missense mutations in ?-synuclein (A53T
and A30P) were the first to be identified, and these are respon-
sible for early-onset autosomal dominant PD (1, 2). The subse-
quent findings that ?-synuclein is a major component of Lewy
bodies in sporadic PD (3, 4) and that ?-synuclein locus triplica-
tion causes autosomal dominant PD (5), suggest that accumu-
lation of wild-type ?-synuclein is sufficient to cause PD. Other
PD-linked mutations in genes encoding for parkin, UCH-L1,
DJ-1, and PINK1 have also been identified (6, 7).
Mutations in the parkin gene are associated with autosomal
recessive juvenile parkinsonism (AR-JP), a disease character-
ized by juvenile onset of typical parkinsonian symptoms and
pathology (8). Parkin is an E3 ubiquitin ligase, and parkin
mutations found in AR-JP patients lead to partial or complete
loss of this activity (9). Several substrates of parkin have been
identified such as CDCrel-1, synphilin-1, and o-glycosylated
arkinson’s disease (PD) is one of the most common neuro-
degenerative disorders, affecting ?2% of the population
forms of ?-synuclein (?Sp22) and Pael-R (10). Both ?Sp22 and
absence of detectable parkin in the brain of AR-JP patients with
exon 3 or 4 deletions in the parkin gene (12) and the recessive
mode of inheritance indicate that a loss of function of parkin is
likely to be responsible for AR-JP. Conversely, several reports
have described neuroprotective effects of parkin in vitro against
endoplasmic reticular stress (13, 14), ?-synuclein or Pael-R
overexpression (15–17), proteasomal inhibition (15), excitotox-
icity (18), and polyglutamine toxicity (19). Similarly, parkin
prevents dopaminergic cell loss in both ?-synuclein and Pael-R
transgenic flies (20). These findings support an essential role of
parkin in the survival of dopaminergic neurons. In contrast to
sporadic and dominant familial PD, Lewy bodies are generally
absent in parkin mutation carriers (12, 21–23), suggesting that
parkin may also be involved in the genesis of Lewy bodies.
Despite previous evidence that parkin might be neuroprotective,
Thus, we have evaluated the lentiviral delivery of parkin in an
?-synuclein rat model of PD (24). In contrast to ?-synuclein
transgenic mouse models, expression of human ?-synuclein with
lentiviral or adeno-associated viral vectors induces a progressive
degeneration of dopamine neurons in the substantia nigra
(24–28). In the present study, we report that lentiviral-mediated
expression of parkin in the substantia nigra protects dopamine
neurons against A30P ?-synuclein-induced neurotoxicity. Using
a specific Ab for Ser-129-phosphorylated ?-synuclein, Pser129,
we also show that overexpression of parkin promotes the for-
mation of inclusions containing phosphorylated ?-synuclein
reminiscent of Lewy bodies in PD brain (29). Thus, parkin may
play a central role in mitigating the pathogenesis of PD by
promoting the survival of dopaminergic neurons through the
detoxification of misfolded proteins in both soluble and aggre-
Lentiviral Vector Production. The cDNAs encoding nuclear-
localized yellow fluorescent protein (YFP) (BD Biosciences
Clontech), A30P human ?-synuclein, and rat parkin were cloned
into the SIN-W-PGK lentiviral transfer vector, and the viral
particles (lenti-YFP, lenti-A30P, and lenti-parkin) were pro-
duced as described in refs. 24 and 30. The viral suspensions
lenti-A30P?lenti-YFP and lenti-A30P?lenti-parkin were pre-
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: AR-JP, autosomal recessive juvenile parkinsonism; PD, Parkinson’s disease;
TH, tyrosine hydroxylase; TH-IR, TH-immunoreactive; YFP, yellow fluorescent protein.
†Present address: Waisman Center, University of Wisconsin, Madison, WI 53705.
¶To whom correspondence should be addressed at: Institute of Neuroscience, SG-AAB 132,
Swiss Federal institute of Technology Lausanne, Ecole Polytechnique Fe ´de ´rale de Lau-
sanne, CH-1015 Lausanne, Switzerland. E-mail: firstname.lastname@example.org.
© 2004 by The National Academy of Sciences of the USA
December 14, 2004 ?
vol. 101 ?
pared by mixing viruses at 1:1 ratios. Viral particle content was
normalized to 360,000 ng of p24 per ml for each lentiviral
Stereotaxic Injection. Lentiviral vectors were stereotaxically in-
jected in the right substantia nigra of adult female Wistar rats
(Iffa-Credo, Charles River Laboratories) weighing ?200 g. Viral
suspensions (2.5 ?l volumes) were injected with a 10-?l Ham-
ilton syringe at a speed of 0.2 ?l?min with an automatic injector
(Stoelting), and the needle was left in place for an additional 10
min before being withdrawn. Stereotaxic injections were deliv-
ered to two sites within the substantia nigra with the following
coordinates in millimeters: anterior, lateral, and ventral, 4.8, 2,
and 7.7 and 5.5, 1.7, and 7.7 for the first site and second site,
respectively. The anterior and lateral coordinates were calcu-
lated from the Bregma, and the ventral coordinates were cal-
culated from the skull surface. The experiments were carried out
in accordance with European Community Council Directive
86?609?EEC for the care and use of laboratory animals.
Immunohistochemistry. Six weeks after the injection of lentiviral
overdose and transcardially perfused with saline and 4% parafor-
maldehyde. Brains were removed and postfixed in 4% paraformal-
dehyde for ?24 h, cryoprotected in 25% sucrose?0.1 M phosphate
buffer for 48 h, and processed as described in ref. 24.
The following primary Abs were used as described previously:
a tyrosine hydroxylase (TH) sheep Ab (1:500) (Pel-Freez Bio-
logicals), an ?-synuclein polyclonal rabbit Ab (24), the LB509
human ?-synuclein-specific monoclonal Ab (1:500) (Zymed),
the Pser129 Ab specifically recognizing phospho-Ser-129 of
?-synuclein (29) (1:100), and a rabbit Ab to the C terminus of
parkin (1:500) (Cell Signaling Technology, Beverly, MA). For
light microscopy, sections were stained by the classic avidin–
biotin complex method as described in ref. 24. For fluorescent
Cy5 were purchased from Jackson ImmunoResearch. TOPRO-3
(Molecular Probes) was used as a nucleus marker. Sections were
then analyzed by confocal microscopy (TCS SP2 AOBS, Leica,
The FD NeuroSilver kit (FD Neuro-Technologies, Baltimore)
was used according to the manufacturer’s protocol to detect
degenerating neurons (23).
Quantification of TH-Positive Neurons and Phosphorylated Inclusions.
The percentage of TH-immunoreactive (TH-IR) neurons rela-
tive to the contralateral side was determined by fluorescence
microscopy in a blind manner as described in refs. 24 and 30. To
determine the density of TH-IR terminals, striatal fibers were
stained for TH with the ABC kit (Vector Laboratories), and the
correspondingtissueopticaldensitieswereevaluatedwith NIH 1.4
software as described in ref. 24. To determine the numbers of
neurons containing phosphorylated ?-synuclein inclusions, five
sections throughout the substantia nigra were stained with the
Pser129 Ab with the avidin–biotin complex method, and adja-
cent sections were stained for ?-synuclein.
Statistical analysis was performed by one-way ANOVA fol-
lowed by a Scheffe ´’s probable least-squares difference post hoc
test (STATISTICA 5.1, StatSoft). The significance level was set at
P ? 0.05.
?-Synuclein. To evaluate the neuroprotective effect of parkin on
?-synuclein-induced nigrostriatal neurodegeneration, a 1:1 mix-
tures of lentiviral vector suspensions encoding A30P human
?-synuclein and wild-type rat parkin (lenti-A30P?lenti-parkin)
or A30P ?-synuclein and YFP (lenti-A30P?lenti-YFP) were
Expressionof Parkinand Human A30P
injected in the substantia nigra of rats. Control animals received
either lenti-parkin alone or a 2-fold higher dose of lenti-YFP
(lenti-YFP2X) (to match the total amount of viral particles
the substantia nigra transduction efficiencies of lentiviral vector
mixes encoding A30P ?-synuclein and parkin. Double staining
specific for human ?-synuclein and parkin (Fig. 1A) showed that
the majority of transduced cells overexpressed both A30P
?-synuclein and parkin in the injected side. No staining for
endogenous ?-synuclein and parkin was observed in the nonin-
jected side (data not shown). Both proteins were localized in the
cell bodies and axons of transduced neurons. The percentage of
double-transduced cells was determined by confocal microscopy
on four coronal sections of four animals and was revealed to be
72 ? 3%. Almost all transduced cells have a typical neuronal
morphology confirming the strong tropism of lentiviral vectors
for neuronal cells (31). Thus, coinjection of two lentiviral vectors
represented a feasible strategy to investigate the relationship
between these two proteins.
Expression of Parkin Protects Dopamine Neurons. Lentiviral-
mediated expression of either wild-type or mutated human
?-synuclein was described previously to induce a selective do-
paminergic cell death in the rat (24). Animals expressing A30P
?-synuclein showed a 33% loss of TH-IR neurons in the sub-
stantia nigra, and almost all dopaminergic neurons expressing
human ?-synuclein died within 6 weeks of postviral injection
(24). Strikingly, confocal microscopy analysis of triple labeling
for TH, human ?-synuclein, and parkin revealed that animals
injected with lenti-A30P?lenti-parkin retained numerous dopa-
minergic neurons still expressing A30P ?-synuclein and overex-
pressing rat parkin at 6 weeks after lentiviral injection (Fig. 1B).
Higher magnification of the substantia nigra pars compacta
indicates that both viruses homogeneously diffused in the sub-
stantia nigra region. Animals injected with lenti-YFP2X or
and parkin (red) in the rat substantia nigra. (A) A large proportion of double-
infected neurons overexpresses both A30P ?-synuclein and parkin. Quantifi-
cation of double-immunostained cells revealed 72 ? 3% coinfected cells. (B)
Confocal images of triple labeling for TH (red), parkin (green), and A30P
human ?-synuclein (blue) in the rat substantia nigra injected with lenti-A30P
and lenti-parkin. Higher magnification (Lower) reveals the presence of nu-
merous TH neurons still expressing A30P human ?-synuclein and parkin at 6
weeks after lentiviral injection. (Scale bars: A, 100 ?m; B, 250 ?m.)
Lentiviral-mediated expression of human A30P ?-synuclein (green)
Lo Bianco et al.
December 14, 2004 ?
vol. 101 ?
no. 50 ?
lenti-parkin showed no dopaminergic nigrostriatal lesion (Fig.
2A). In contrast, animals expressing A30P?YFP revealed a
significant loss of nigral TH-IR cells. Interestingly, overexpres-
sion of parkin rescued TH-IR neurons from the A30P
TH-IR neuron loss compared to the contralateral noninjected
side showed that parkin significantly decreases the TH-IR cell
loss from 31% (A30P?YFP) to 9% (A30P?parkin) (Fig. 2B).
Parkin expression also prevented ?-synuclein-induced loss of
striatal TH-IR fibers (Fig. 3A). Quantification of TH-positive
nerve terminal densities showed that parkin significantly de-
creased the loss of dopaminergic terminals from 16% (A30P?
YFP) to 4% (A30P?parkin) (Fig. 3B). No reduction in TH
staining was observed in the striatum of animals injected with
lenti-YFP2X or lenti-parkin. A similar protective effect of
parkin was observed with striatal sections stained for the vesic-
ular monoamine transporter type 2 (data not shown).
Parkin Prevents A30P-Induced Neurodegeneration. Neurons under-
going degeneration become argyrophilic and are therefore spe-
cifically detected with silver staining (32). Lentiviral-mediated
expression of mutated human ?-synuclein was shown to induce
a strong neuritic and cellular pathology detected with silver
staining (24). Animals expressing A30P?YFP showed the pres-
ence of silver-positive dark structures in both neurites and cell
bodies (Fig. 4). A similar ?-synuclein expression was observed in
the brains of animals expressing A30P?YFP or A30P?parkin.
Coexpression of parkin with A30P ?-synuclein prevented the
appearance of silver-positive degenerating neurons. No silver
staining was detected on the noninjected side of animals ex-
pressing A30P?YFP or in animals injected with lenti-YFP or
lenti-parkin. These results show that parkin prevents
?-synuclein-induced neurodegeneration in the substantia nigra
of rats expressing PD-linked mutated human ?-synuclein.
Parkin Increases the Number of Phosphorylated ?-Synuclein Inclu-
sions. Lewy bodies are present in almost all forms of PD with the
exception of AR-JP patients with parkin mutations, suggesting
an essential role of the E3 ligase parkin in the formation of Lewy
bodies (12, 21–23). Because of the difficulty of discriminating
between pathological inclusions and accumulation of
?-synuclein in subcellular regions associated with general
?-synuclein staining, inclusions were detected with the Ab
Pser129 specific for the phosphorylated form of ?-synuclein at
position 129 (29). This form of ?-synuclein selectively and
abundantly accumulates in Lewy bodies of synucleinopathy
lesions (29), whereas only a small fraction of ?-synuclein is
phosphorylated in normal human and rat brains (29). A very low
level of phosphorylated rat ?-synuclein was observed in the
substantia nigra of noninjected rats (Fig. 5A) and those over-
expressing YFP (data not shown). In contrast, lentiviral-
mediated expression of A30P ?-synuclein led to the formation of
mine nigral neuron loss. (A) TH expression at 6 weeks in the substantia nigra
of rats injected with different mixes of lentiviral vectors encoding for YFP
(YFP2?), rat parkin (Parkin), and A30P human ?-synuclein (A30P). (B) Histo-
grams represent the loss of TH-IR nigral neurons at 6 weeks relative to the
contralateral side in rats unilaterally injected with the different lentiviral
constructs. Values refer to means ? SEM; n ? 5 animals for YFP2? or parkin;
n ? 10 for A30P?YFP; n ? 18 for A30P?parkin;*, P ? 0.05, compared with
lenti-YFP-injected animals; §, P ? 0.005, compared with A30P?YFP-expressing
animals. (Scale bar: 350 ?m.)
Overexpression of parkin reduces A30P ?-synuclein-induced dopa-
the striatum of rats. (A) Striatal sections stained for the TH marker from rats
that received intranigral injection of rats injected with different solutions of
lentiviral vectors encoding for YFP (YFP2?), rat parkin (Parkin), and A30P
human ?-synuclein (A30P). (B) Histograms represent the loss of TH-IR fibers
neurons at 6 weeks relative to the contralateral side in rats unilaterally
injected with the different lentiviral suspensions. Animals injected with lenti-
A30P?lenti-YFP showed a considerable reduction of dopaminergic innerva-
tion on the ipsilateral side of the striatum. Values refer to means ? SEM; n ?
5 animals for YFP2? or parkin; n ? 12 for A30P?YFP; n ? 13 for A30P?parkin;
*, P ? 0.05, compared with lenti-YFP2?-injected animals; §, P ? 0.005, com-
pared with A30P?YFP-expressing animals.
Parkin prevents the ?-synuclein-induced dopaminergic fiber loss in
www.pnas.org?cgi?doi?10.1073?pnas.0405313101Lo Bianco et al.
round hyperphosphorylated Pser129-positive inclusions (Fig.
5B) and occasional phosphorylated neurites (Fig. 5C). Triple
labeling with a nuclear marker, Pser129, and human ?-synuclein-
specific Abs showed that the intracytoplasmic phosphorylated
inclusions are strongly immunopositive for human ?-synuclein
(Fig. 5D). To explore the effect of parkin in the formation of
?-synuclein inclusions, the number of cells containing hyper-
phosphorylated inclusions was quantified in animals overex-
pressing A30P?YFP and A30P?parkin. Animals coexpressing
parkin with A30P ?-synuclein showed a 45% increase in the
number of neurons containing hyperphosphorylated inclusions
(Fig. 5E). A 41% increase in cells containing hyperphosphory-
lated inclusions was observed when only cells positive for
?-synuclein were analyzed (data no shown). To determine
whether other posttranslational modifications of synuclein ag-
gregates might also correlate with the neuroprotection afforded
by parkin, we also examined the brains of A30P?YFP- and
A30P?parkin-overexpressing animals for the presence of ubi-
quitinated inclusions. These experiments showed no ubiquiti-
nated inclusions in the brains of A30P-expressing animals either
with or without parkin overexpression (data not shown). The
finding that not all ?-synuclein inclusions are immunoreactive
for ubiquitin in patients with PD indicates that ubiquitin is not
a prerequisite for the ?-synuclein pathology (4).
Abnormal accumulation of ?-synuclein is considered to be a
key pathological event in the process leading to selective
dopaminergic degeneration in ?-synuclein-linked and sporadic
PD, but the neurotoxic role of inclusions in PD is highly
debated (33). The major findings of this report are that gene
therapy delivery of parkin efficiently prevents PD-linked
mutant ?-synuclein-induced dopaminergic cell loss in vivo and
promotes the formation of hyperphosphorylated ?-synuclein
inclusions. Consistent with these results, inactivation of the
gene encoding the E6-AP ubiquitin ligase leads to an increase
in neurotoxicity and a decrease in the number of nuclear
inclusions in a transgenic model of spinocerebellar ataxia type
1 (34). Recently, the E3-ligase CHIP (carboxyl terminus of the
Hsc70-interacting protein) was reported to attenuate tau-
induced cell death and also to facilitate hyperphosphorylated
tau aggregation (35, 36). Parkin, CHIP and E6-AP may
similarly enhance cell survival by eliminating soluble toxic
proteins in favor of insoluble aggregates. Additionally, in vitro
mammalian cell and transgenic fly studies also attributed a
protective role to parkin in dopamine neuron survival (15, 20).
Overexpression of parkin in the ?-synuclein transgenic fly
induces, on the contrary, a decrease in the number of non-
phosphorylated ?-synuclein inclusions. In the present study,
however, we analyzed the effect of parkin in the formation of
more mature posttranslationally modified ?-synuclein-
containing inclusions. The presence of phosphorylated
?-synuclein was recently recognized as a pathological hallmark
of ?-synucleinopathy lesions (29, 37, 38). After 6 weeks of
?-synuclein expression, not all ?-synuclein-positive cells de-
veloped hyperphosphorylated inclusions. Because the forma-
tion of hyperphosphorylated ?-synuclein inclusions is a pro-
gressive time- and dose-dependant process (37, 39), the
majority of inclusions are likely nonphosphorylated in the
short time period of 6 weeks. However, detecting these Pser129
inclusions has the advantage over classic ?-synuclein staining
of better differentiating true inclusions from nonaggregate
subcellular accumulations of the protein (37). We observed
that the neuroprotective effect of parkin is associated with a
stained for ?-synuclein. A similar expression of A30P human ?-synuclein was observed in both groups. No ?-synuclein staining was observed for the noninjected
side (NI), lenti-YFP-injected, or lenti-parkin-injected (data not shown) animals. Silver staining (B, C, and E–I) was performed on adjacent nigral sections to detect
degenerating neurons. Higher magnification shows the presence of silver-positive dark structures in both cell body and axon of nigral neurons from animals
expressing A30P?YFP (C). Coexpression of parkin with A30P ?-synuclein prevents the appearance of silver-positive degenerating neurons (F). Noninjected side
(NI) (G) and animals expressing YFP (H) or parkin (I) did not show any specific silver staining. (Scale bars: A, B, D, E, and G–I, 140 ?m; C and F, 40 ?m.)
Parkin prevents A30P ?-synuclein-induced neurodegeneration. Brain sections from animals expressing A30P?YFP (A–C) or A30P?parkin (D–F) were
Lo Bianco et al.
December 14, 2004 ?
vol. 101 ?
no. 50 ?
significant increase in the number of hyperphosphorylated
?-synuclein inclusions. We therefore hypothesize that parkin
helps dopamine neurons survive by promoting the sequestration
of toxic prefibrillar oligomers in mature hyperphosphorylated
inclusions. Interestingly, cytosolic dopamine has been shown to
interact with ?-synuclein to form adducts that stabilize the forma-
tion of toxic protofibrils, suggesting a potential mechanism for the
selective degeneration of dopaminergic neurons (40). One toxic
vesicles (41, 42).
Other findings also indicate that toxicity and aggregation are
two distinct phenomena in ?-synuclein-induced pathology. A
recent study has reported behavioral impairments linked to
neuronal dysfunction without aggregate formation in transgenic
mice expressing A53T human ?-synuclein (43). Additionally,
toxicity induced by overexpression of human ?-synuclein in
primary midbrain cells is not associated with the presence of
visible protein aggregates (15).
Brains of AR-JP patients with parkin mutations generally
show dopaminergic neurodegeneration without Lewy bodies,
the neuronal proteinaceous cytoplasmic inclusions that are
typically found in PD. This finding suggests the requirement of
parkin in the genesis of Lewy bodies. Our observation that
coexpression of parkin with A30P human ?-synuclein increases
the number of neurons containing hyperphosphorylated in-
clusions is consistent with this hypothesis. Parkin may also act
indirectly in the formation of inclusions by blocking the
cellular death pathway triggered by A30P human ?-synuclein
and consequently compelling the resistant cells to accumulate
?-synuclein in their cytoplasm and eventually form inclusions.
Interestingly, parkin was shown to increase the formation of
ubiquitinated inclusions when synphilin-1 and ?-synuclein
were coexpressed in vitro (44). Furthermore, the protective
effect of parkin against ?-synuclein-induced toxicity in cul-
tured cells was also described to be associated with the
appearance of higher-molecular-weight species of ?-synuclein,
suggesting that parkin promotes the aggregation of ?-synuclein
(16). We also recently investigated the ability of lentiviral
vectors encoding glial cell line-derived neurotrophic factor to
prevent nigral dopaminergic degeneration associated with the
lentiviral-mediated expression of the A30P mutant human
?-synuclein (45). Contrary to parkin, expression of this neu-
rotrophic factor does not prevent ?-synuclein-induced toxicity.
This difference in neuroprotection may reflect a particular
relationship between the two PD-linked proteins, ?-synuclein
and parkin, and their implication in a common cellular path-
way. Dissecting the molecular mechanism of parkin’s protec-
tion against ?-synuclein toxicity should provide important
clues about the unique vulnerability of dopamine neurons in
PD. These results also indicate that gene therapy delivery of
parkin or pharmacological agents increasing parkin expression
may constitute a potential therapeutic strategy for PD. Inter-
estingly, brain delivery of human ?-synuclein with viral vectors
has recently been scaled up to non-human primates, opening
the potential to evaluate the neuroprotective property of
parkin in a genetic primate model of PD (27, 46). The demonstra-
tion that parkin is able to overcome the considerable difficulty of
improving the pathological phenotypes observed in genetic animal
models of disease further strengthens the case for parkin-based
strategies as promising treatments for patients with PD.
We thank Nicole De ´glon, William Pralong, and Ruth-Luthi Carter for
helpful comments; Philippe Colin, Christel Sadeghi, Anne Maillard, and
Maria Rey for excellent technical help; and Dr. Michel Goedert for the
A30P human ?-synuclein cDNA. This work was supported by the Swiss
National Science Foundation and the Michael J. Fox Foundation.
1. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra,
A., Pike, B., Root, H., Rubenstein, J., Boyer, R., et al. (1997) Science 276,
2. Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek,
H., Epplen, J. T., Schols, L. & Riess, O. (1998) Nat. Genet. 18, 106–108.
3. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R. &
Goedert, M. (1997) Nature 388, 839–840.
4. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. & Goedert, M.
(1998) Proc. Natl. Acad. Sci. USA 95, 6469–6473.
5. Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus,
J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., et al. (2003) Science
6. Dawson, T. M. & Dawson, V. L. (2003) Science 302, 819–822.
7. Valente, E. M., Abou-Sleiman, P. M., Caputo, V., Muqit, M. M., Harvey, K.,
Gispert, S., Ali, Z., Del Turco, D., Bentivoglio, A. R., Healy, D. G., et al. (2004)
Science 304, 1158–1160.
8. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima,
S., Yokochi, M., Mizuno, Y. & Shimizu, N. (1998) Nature 392, 605–608.
sions. Sections from the substantia nigra of rats expressing A30P ?-synuclein
and parkin were immunostained with the Pser129 Ab specific for phospho-
Ser-129 of ?-synuclein that selectively and extensively accumulates in human
side corresponding to the physiological level of phosphorylated rat
?-synuclein. (B) The substantia nigra of animals overexpressing either A30P?
inclusions reminiscent of Lewy bodies. (C) Pser129-positive neurites were
ing with Pser129 Ab (green), LB509 human ?-synuclein specific Ab (red), and
TOPRO-3 nuclear marker (blue) shows that phosphorylated inclusions abun-
dantly contain human ?-synuclein. (E) The number of neurons containing
Pser129-positive inclusions were quantified in the substantia nigra of rats
expressing A30P?YFP or A30P?parkin. Values refer to means ? SEM; n ? 6
animals per group;*, P ? 0.05;**, P ? 0.005. (Scale bars: A and B, 150 ?m; C,
40 ?m; D, 10 ?m.)
Parkin increases the number of phosphorylated ?-synuclein inclu-
www.pnas.org?cgi?doi?10.1073?pnas.0405313101 Lo Bianco et al.
9. Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S., Minoshima, S.,
Shimizu, N., Iwai, K., Chiba, T., Tanaka, K. & Suzuki, T. (2000) Nat. Genet. 25,
10. Cookson, M. R. (2003) Neuron 37, 7–10.
11. Cookson, M. R. (2003) Neuromol. Med. 3, 1–13.
12. Shimura, H., Hattori, N., Kubo, S., Yoshikawa, M., Kitada, T., Matsumine, H.,
Asakawa, S., Minoshima, S., Yamamura, Y., Shimizu, N. & Mizuno, Y. (1999)
Ann. Neurol. 45, 668–672.
13. Imai, Y., Soda, M. & Takahashi, R. (2000) J. Biol. Chem. 275, 35661–35664.
14. Darios, F., Corti, O., Lucking, C. B., Hampe, C., Muriel, M. P., Abbas, N., Gu,
W. J., Hirsch, E. C., Rooney, T., Ruberg, M. & Brice, A. (2003) Hum. Mol.
Genet. 12, 517–526.
15. Petrucelli, L., O’Farrell, C., Lockhart, P. J., Baptista, M., Kehoe, K., Vink, L.,
Choi, P., Wolozin, B., Farrer, M., Hardy, J. & Cookson, M. R. (2002) Neuron
16. Oluwatosin-Chigbu, Y., Robbins, A., Scott, C. W., Arriza, J. L., Reid, J. D. &
Zysk, J. R. (2003) Biochem. Biophys. Res. Commun. 309, 679–684.
17. Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y. & Takahashi, R. (2001)
Cell 105, 891–902.
18. Staropoli, J. F., McDermott, C., Martinat, C., Schulman, B., Demireva, E. &
Abeliovich, A. (2003) Neuron 37, 735–749.
19. Tsai, Y. C., Fishman, P. S., Thakor, N. V. & Oyler, G. A. (2003) J. Biol. Chem.
20. Yang, Y., Nishimura, I., Imai, Y., Takahashi, R. & Lu, B. (2003) Neuron 37,
21. Takahashi, H., Ohama, E., Suzuki, S., Horikawa, Y., Ishikawa, A., Morita, T.,
Tsuji, S. & Ikuta, F. (1994) Neurology 44, 437–441.
22. Hayashi, S., Wakabayashi, K., Ishikawa, A., Nagai, H., Saito, M., Maruyama,
M., Takahashi, T., Ozawa, T., Tsuji, S. & Takahashi, H. (2000) Movement
Disorders 15, 884–888.
23. van de Warrenburg, B. P., Lammens, M., Lucking, C. B., Denefle, P.,
Wesseling, P., Booij, J., Praamstra, P., Quinn, N., Brice, A. & Horstink, M. W.
(2001) Neurology 56, 555–557.
24. Lo Bianco, C., Ridet, J. L., Schneider, B. L., Deglon, N. & Aebischer, P. (2002)
Proc. Natl. Acad. Sci. USA 99, 10813–10818.
25. Klein, R. L., King, M. A., Hamby, M. E. & Meyer, E. M. (2002) Hum. Gene
Ther. 13, 605–612.
26. Kirik, D., Rosenblad, C., Burger, C., Lundberg, C., Johansen, T. E., Muzyczka,
N., Mandel, R. J. & Bjorklund, A. (2002) J. Neurosci. 22, 2780–2791.
27. Kirik, D., Annett, L. E., Burger, C., Muzyczka, N., Mandel, R. J. & Bjorklund,
A. (2003) Proc. Natl. Acad. Sci. USA 100, 2884–2889.
28. Lauwers, E., Debyser, Z., Van Dorpe, J., De Strooper, B., Nuttin, B. &
Baekelandt, V. (2003) Brain Pathol. 13, 364–372.
29. Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E.,
Goldberg, M. S., Shen, J., Takio, K. & Iwatsubo, T. (2002) Nat. Cell Biol. 4,
30. Deglon, N., Tseng, J. L., Bensadoun, J. C., Zurn, A. D., Arsenijevic, Y., Pereira
de Almeida, L., Zufferey, R., Trono, D. & Aebischer, P. (2000) Hum. Gene
Ther. 11, 179–190.
31. Blomer, U., Naldini, L., Kafri, T., Trono, D., Verma, I. M. & Gage, F. H. (1997)
J. Virol. 71, 6641–6649.
32. Beltramino, C. A., de Olmos, J. S., Gallyas, F., Heimer, L. & Zaborszky, L.
(1993) NIDA Res. Monogr. 136, 101–126; discussion 126–132.
33. Goldberg, M. S. & Lansbury, P. T., Jr. (2000) Nat. Cell Biol. 2, E115–E119.
34. Cummings, C. J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y., Ciechanover,
A., Orr, H. T., Beaudet, A. L. & Zoghbi, H. Y. (1999) Neuron 24, 879–892.
35. Shimura, H., Schwartz, D., Gygi, S. P. & Kosik, K. S. (2004) J. Biol. Chem. 279,
36. Petrucelli, L., Dickson, D., Kehoe, K., Taylor, J., Snyder, H., Grover, A., De
Lucia, M., McGowan, E., Lewis, J., Prihar, G., et al. (2004) Hum. Mol. Genet.
37. Neumann, M., Kahle, P. J., Giasson, B. I., Ozmen, L., Borroni, E., Spooren, W.,
Muller, V., Odoy, S., Fujiwara, H., Hasegawa, M., et al. (2002) J. Clin. Invest.
38. Iwatsubo, T. (2003) J. Neurol. 250, Suppl. 3, 11–14.
39. Takahashi, M., Kanuka, H., Fujiwara, H., Koyama, A., Hasegawa, M., Miura,
M. & Iwatsubo, T. (2003) Neurosci. Lett. 336, 155–158.
40. Conway, K. A., Rochet, J. C., Bieganski, R. M. & Lansbury, P. T., Jr. (2001)
Science 294, 1346–1349.
41. Volles, M. J., Lee, S. J., Rochet, J. C., Shtilerman, M. D., Ding, T. T., Kessler,
J. C. & Lansbury, P. T., Jr. (2001) Biochemistry 40, 7812–7819.
42. Volles, M. J. & Lansbury, P. T., Jr. (2002) Biochemistry 41, 4595–4602.
43. Gispert, S., Del Turco, D., Garrett, L., Chen, A., Bernard, D. J., Hamm-
Clement, J., Korf, H. W., Deller, T., Braak, H., Auburger, G. & Nussbaum,
R. L. (2003) Mol. Cell. Neurosci. 24, 419–429.
44. Chung, K. K., Zhang, Y., Lim, K. L., Tanaka, Y., Huang, H., Gao, J., Ross,
C. A., Dawson, V. L. & Dawson, T. M. (2001) Nat. Med. 7, 1144–1150.
45. Lo Bianco, C., De ´glon, N., Pralong, W. & Aebischer, P. (2004) Neurobiol. Dis.,
46. Kirik, D. & Bjorklund, A. (2003) Trends Neurosci. 26, 386–392.
Lo Bianco et al.
December 14, 2004 ?
vol. 101 ?
no. 50 ?