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, Hoˆpital 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 forms of ␣-synuclein (␣Sp22) and Pael-R (10). Both ␣Sp22 and midbrain dopamine neurons and the presence of cytoplasmic Pael-R have been found to accumulate in AR-JP brains (11). The inclusions called Lewy bodies. Mutations in several genes including absence of detectable parkin in the brain of AR-JP patients with ␣-synuclein and parkin have been linked to familial PD. The loss of exon 3 or 4 deletions in the parkin gene (12) and the recessive parkin’s E3-ligase activity leads to dopaminergic neuronal degen- mode of inheritance indicate that a loss of function of parkin is eration in early-onset autosomal recessive juvenile parkinsonism, likely to be responsible for AR-JP. Conversely, several reports suggesting a key role of parkin for dopamine neuron survival. To have described neuroprotective effects of parkin in vitro against evaluate the potential neuroprotective role of parkin in the patho- endoplasmic reticular stress (13, 14), ␣-synuclein or Pael-R genesis of PD, we tested whether overexpression of wild-type rat overexpression (15–17), proteasomal inhibition (15), excitotox- parkin could protect against the toxicity of mutated human A30P icity (18), and polyglutamine toxicity (19). Similarly, parkin ␣-synuclein in a rat lentiviral model of PD. Animals overexpressing prevents dopaminergic cell loss in both ␣-synuclein and Pael-R parkin showed significant reductions in ␣-synuclein-induced neu- transgenic flies (20). These findings support an essential role of ropathology, including preservation of tyrosine hydroxylase- parkin in the survival of dopaminergic neurons. In contrast to positive cell bodies in the substantia nigra and sparing of tyrosine sporadic and dominant familial PD, Lewy bodies are generally hydroxylase-positive nerve terminals in the striatum. The parkin- absent in parkin mutation carriers (12, 21–23), suggesting that mediated neuroprotection was associated with an increase in parkin may also be involved in the genesis of Lewy bodies. hyperphosphorylated ␣-synuclein inclusions, suggesting a key role Despite previous evidence that parkin might be neuroprotective, for parkin in the genesis of Lewy bodies. These results indicate that this property has never been tested in a mammalian model of PD. parkin gene therapy may represent a promising candidate treat- Thus, we have evaluated the lentiviral delivery of parkin in an ment for PD. ␣-synuclein rat model of PD (24). In contrast to ␣-synuclein transgenic mouse models, expression of human ␣-synuclein with gene therapy ͉ lentivirus ͉ neurodegenerative disease ͉ Lewy lentiviral or adeno-associated viral vectors induces a progressive body ͉ neuroprotection degeneration of dopamine neurons in the substantia nigra (24–28). In the present study, we report that lentiviral-mediated arkinson’s disease (PD) is one of the most common neuro- expression of parkin in the substantia nigra protects dopamine Ϸ neurons against A30P ␣-synuclein-induced neurotoxicity. Using Pdegenerative disorders, affecting 2% of the population ␣ over the age of 60. Loss of dopaminergic neurons in the a specific Ab for Ser-129-phosphorylated -synuclein, Pser129, we also show that overexpression of parkin promotes the for- substantia nigra pars compacta and subsequent striatal dopa- ␣ mine depletion causes motor impairments including akinesia, mation of inclusions containing phosphorylated -synuclein reminiscent of Lewy bodies in PD brain (29). Thus, parkin may resting tremor, muscle rigidity, and gait and postural deficits. play a central role in mitigating the pathogenesis of PD by The neuropathological hallmark of PD is the appearance of promoting the survival of dopaminergic neurons through the proteinaceous intracellular deposits identified as Lewy bodies detoxification of misfolded proteins in both soluble and aggre- and Lewy neurites. Although the mechanism leading to the gated forms. selective degeneration of nigral dopamine neurons in sporadic PD remains unknown, clues about the pathogenesis of familial Methods forms of PD are emerging because of the discovery of various Lentiviral Vector Production. The cDNAs encoding nuclear- gene mutations. Two missense mutations in ␣-synuclein (A53T localized yellow fluorescent protein (YFP) (BD Biosciences and A30P) were the first to be identified, and these are respon- Clontech), A30P human ␣-synuclein, and rat parkin were cloned sible for early-onset autosomal dominant PD (1, 2). The subse- into the SIN-W-PGK lentiviral transfer vector, and the viral quent findings that ␣-synuclein is a major component of Lewy particles (lenti-YFP, lenti-A30P, and lenti-parkin) were pro- bodies in sporadic PD (3, 4) and that ␣-synuclein locus triplica- duced as described in refs. 24 and 30. The viral suspensions tion causes autosomal dominant PD (5), suggest that accumu- lenti-A30P͞lenti-YFP and lenti-A30P͞lenti-parkin were pre- 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). This paper was submitted directly (Track II) to the PNAS office. Mutations in the parkin gene are associated with autosomal Abbreviations: AR-JP, autosomal recessive juvenile parkinsonism; PD, Parkinson’s disease; recessive juvenile parkinsonism (AR-JP), a disease character- TH, tyrosine hydroxylase; TH-IR, TH-immunoreactive; YFP, yellow fluorescent protein. ized by juvenile onset of typical parkinsonian symptoms and †Present address: Waisman Center, University of Wisconsin, Madison, WI 53705. pathology (8). Parkin is an E3 ubiquitin ligase, and parkin ¶To whom correspondence should be addressed at: Institute of Neuroscience, SG-AAB 132, mutations found in AR-JP patients lead to partial or complete Swiss Federal institute of Technology Lausanne, Ecole Polytechnique Fe´de´ rale de Lau- loss of this activity (9). Several substrates of parkin have been sanne, CH-1015 Lausanne, Switzerland. E-mail: patrick.aebischer@epfl.ch. identified such as CDCrel-1, synphilin-1, and o-glycosylated © 2004 by The National Academy of Sciences of the USA

17510–17515 ͉ PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405313101 Downloaded by guest on September 30, 2021 pared by mixing viruses at 1:1 ratios. Viral particle content was normalized to 360,000 ng of p24 per ml for each lentiviral suspension.

Stereotaxic Injection. Lentiviral vectors were stereotaxically in- jected in the right substantia nigra of adult female Wistar rats (Iffa-Credo, Charles River Laboratories) weighing Ϸ200 g. Viral suspensions (2.5 ␮l volumes) were injected with a 10-␮l Ham- ilton syringe at a speed of 0.2 ␮l͞min 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 86͞609͞EEC for the care and use of laboratory animals.

Immunohistochemistry. Six weeks after the injection of lentiviral vector suspensions, animals were killed with a sodium pentobarbital overdose and transcardially perfused with saline and 4% parafor- maldehyde. Brains were removed and postfixed in 4% paraformal- Fig. 1. Lentiviral-mediated expression of human A30P ␣-synuclein (green) dehyde for Ϸ24 h, cryoprotected in 25% sucrose͞0.1 M phosphate and parkin (red) in the rat substantia nigra. (A) A large proportion of double- infected neurons overexpresses both A30P ␣-synuclein and parkin. Quantifi- buffer for 48 h, and processed as described in ref. 24. Ϯ The following primary Abs were used as described previously: cation of double-immunostained cells revealed 72 3% coinfected cells. (B) Confocal images of triple labeling for TH (red), parkin (green), and A30P a tyrosine hydroxylase (TH) sheep Ab (1:500) (Pel-Freez Bio- ␣ ␣ human -synuclein (blue) in the rat substantia nigra injected with lenti-A30P logicals), an -synuclein polyclonal rabbit Ab (24), the LB509 and lenti-parkin. Higher magnification (Lower) reveals the presence of nu- human ␣-synuclein-specific monoclonal Ab (1:500) (Zymed), merous TH neurons still expressing A30P human ␣-synuclein and parkin at 6 the Pser129 Ab specifically recognizing phospho-Ser-129 of weeks after lentiviral injection. (Scale bars: A, 100 ␮m; B, 250 ␮m.) ␣-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– injected in the substantia nigra of rats. Control animals received biotin complex method as described in ref. 24. For fluorescent either lenti-parkin alone or a 2-fold higher dose of lenti-YFP multiple labeling, the secondary Abs conjugated to Cy2, Cy3, and (lenti-YFP2X) (to match the total amount of viral particles Cy5 were purchased from Jackson ImmunoResearch. TOPRO-3 relative to dual-virus treatments). As an initial step, we evaluated (Molecular Probes) was used as a nucleus marker. Sections were the substantia nigra transduction efficiencies of lentiviral vector then analyzed by confocal microscopy (TCS SP2 AOBS, Leica, mixes encoding A30P ␣-synuclein and parkin. Double staining Heidelberg). specific for human ␣-synuclein and parkin (Fig. 1A) showed that The FD NeuroSilver kit (FD Neuro-Technologies, Baltimore) the majority of transduced cells overexpressed both A30P was used according to the manufacturer’s protocol to detect ␣-synuclein and parkin in the injected side. No staining for degenerating neurons (23). endogenous ␣-synuclein and parkin was observed in the nonin- jected side (data not shown). Both proteins were localized in the Quantification of TH-Positive Neurons and Phosphorylated Inclusions. cell bodies and axons of transduced neurons. The percentage of The percentage of TH-immunoreactive (TH-IR) neurons rela- double-transduced cells was determined by confocal microscopy tive to the contralateral side was determined by fluorescence on four coronal sections of four animals and was revealed to be microscopy in a blind manner as described in refs. 24 and 30. To 72 Ϯ 3%. Almost all transduced cells have a typical neuronal determine the density of TH-IR terminals, striatal fibers were morphology confirming the strong tropism of lentiviral vectors stained for TH with the ABC kit (Vector Laboratories), and the for neuronal cells (31). Thus, coinjection of two lentiviral vectors corresponding tissue optical densities were evaluated with NIH 1.4 represented a feasible strategy to investigate the relationship software as described in ref. 24. To determine the numbers of between these two proteins. neurons containing phosphorylated ␣-synuclein inclusions, five sections throughout the substantia nigra were stained with the Expression of Parkin Protects Dopamine Neurons. Lentiviral- Pser129 Ab with the avidin–biotin complex method, and adja- mediated expression of either wild-type or mutated human cent sections were stained for ␣-synuclein. ␣-synuclein was described previously to induce a selective do- Statistical analysis was performed by one-way ANOVA fol- paminergic cell death in the rat (24). Animals expressing A30P lowed by a Scheffe´’sprobable least-squares difference post hoc ␣-synuclein showed a 33% loss of TH-IR neurons in the sub- test (STATISTICA 5.1, StatSoft). The significance level was set at stantia nigra, and almost all dopaminergic neurons expressing P Ͻ 0.05. human ␣-synuclein died within 6 weeks of postviral injection (24). Strikingly, confocal microscopy analysis of triple labeling Results for TH, human ␣-synuclein, and parkin revealed that animals Lentiviral-Mediated Expression of Parkin and Human A30P injected with lenti-A30P͞lenti-parkin retained numerous dopa- ␣-Synuclein. To evaluate the neuroprotective effect of parkin on minergic neurons still expressing A30P ␣-synuclein and overex- ␣-synuclein-induced nigrostriatal neurodegeneration, a 1:1 mix- pressing rat parkin at 6 weeks after lentiviral injection (Fig. 1B). tures of lentiviral vector suspensions encoding A30P human Higher magnification of the substantia nigra pars compacta

␣-synuclein and wild-type rat parkin (lenti-A30P͞lenti-parkin) indicates that both viruses homogeneously diffused in the sub- NEUROSCIENCE or A30P ␣-synuclein and YFP (lenti-A30P͞lenti-YFP) were stantia nigra region. Animals injected with lenti-YFP2X or

Lo Bianco et al. PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 ͉ 17511 Downloaded by guest on September 30, 2021 Fig. 2. Overexpression of parkin reduces A30P ␣-synuclein-induced dopa- 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 (YFP2ϫ), rat parkin (Parkin), and A30P human ␣-synuclein (A30P). (B) Histo- Fig. 3. Parkin prevents the ␣-synuclein-induced dopaminergic fiber loss in grams represent the loss of TH-IR nigral neurons at 6 weeks relative to the the striatum of rats. (A) Striatal sections stained for the TH marker from rats contralateral side in rats unilaterally injected with the different lentiviral that received intranigral injection of rats injected with different solutions of constructs. Values refer to means Ϯ SEM; n ϭ 5 animals for YFP2ϫ or parkin; lentiviral vectors encoding for YFP (YFP2ϫ), rat parkin (Parkin), and A30P n ϭ 10 for A30P͞YFP; n ϭ 18 for A30P͞parkin; *, P Ͻ 0.05, compared with human ␣-synuclein (A30P). (B) Histograms represent the loss of TH-IR fibers lenti-YFP-injected animals; §, P Ͻ 0.005, compared with A30P͞YFP-expressing neurons at 6 weeks relative to the contralateral side in rats unilaterally animals. (Scale bar: 350 ␮m.) injected with the different lentiviral suspensions. Animals injected with lenti- A30P͞lenti-YFP showed a considerable reduction of dopaminergic innerva- tion on the ipsilateral side of the striatum. Values refer to means Ϯ SEM; n ϭ lenti-parkin showed no dopaminergic nigrostriatal lesion (Fig. 5 animals for YFP2ϫ or parkin; n ϭ 12 for A30P͞YFP; n ϭ 13 for A30P͞parkin; 2A). In contrast, animals expressing A30P͞YFP revealed a *, P Ͻ 0.05, compared with lenti-YFP2ϫ-injected animals; §, P Ͻ 0.005, com- significant loss of nigral TH-IR cells. Interestingly, overexpres- pared with A30P͞YFP-expressing animals. sion of parkin rescued TH-IR neurons from the A30P ␣-synuclein neurotoxicity. Quantification of the percent of nigral appearance of silver-positive degenerating neurons. No silver TH-IR neuron loss compared to the contralateral noninjected staining was detected on the noninjected side of animals ex- side showed that parkin significantly decreases the TH-IR cell pressing A30P͞YFP or in animals injected with lenti-YFP or loss from 31% (A30P͞YFP) to 9% (A30P͞parkin) (Fig. 2B). ␣ lenti-parkin. These results show that parkin prevents Parkin expression also prevented -synuclein-induced loss of ␣-synuclein-induced neurodegeneration in the substantia nigra striatal TH-IR fibers (Fig. 3A). Quantification of TH-positive of rats expressing PD-linked mutated human ␣-synuclein. nerve terminal densities showed that parkin significantly de- ͞ creased the loss of dopaminergic terminals from 16% (A30P Parkin Increases the Number of Phosphorylated ␣-Synuclein Inclu- ͞ YFP) to 4% (A30P parkin) (Fig. 3B). No reduction in TH sions. Lewy bodies are present in almost all forms of PD with the staining was observed in the striatum of animals injected with exception of AR-JP patients with parkin mutations, suggesting lenti-YFP2X or lenti-parkin. A similar protective effect of an essential role of the E3 ligase parkin in the formation of Lewy parkin was observed with striatal sections stained for the vesic- bodies (12, 21–23). Because of the difficulty of discriminating ular monoamine transporter type 2 (data not shown). between pathological inclusions and accumulation of ␣-synuclein in subcellular regions associated with general Parkin Prevents A30P-Induced Neurodegeneration. Neurons under- ␣-synuclein staining, inclusions were detected with the Ab going degeneration become argyrophilic and are therefore spe- Pser129 specific for the phosphorylated form of ␣-synuclein at cifically detected with silver staining (32). Lentiviral-mediated position 129 (29). This form of ␣-synuclein selectively and expression of mutated human ␣-synuclein was shown to induce abundantly accumulates in Lewy bodies of synucleinopathy a strong neuritic and cellular pathology detected with silver lesions (29), whereas only a small fraction of ␣-synuclein is staining (24). Animals expressing A30P͞YFP showed the pres- phosphorylated in normal human and rat brains (29). A very low ence of silver-positive dark structures in both neurites and cell level of phosphorylated rat ␣-synuclein was observed in the bodies (Fig. 4). A similar ␣-synuclein expression was observed in substantia nigra of noninjected rats (Fig. 5A) and those over- the brains of animals expressing A30P͞YFP or A30P͞parkin. expressing YFP (data not shown). In contrast, lentiviral- Coexpression of parkin with A30P ␣-synuclein prevented the mediated expression of A30P ␣-synuclein led to the formation of

17512 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405313101 Lo Bianco et al. Downloaded by guest on September 30, 2021 Fig. 4. Parkin prevents A30P ␣-synuclein-induced neurodegeneration. Brain sections from animals expressing A30P͞YFP (A–C) or A30P͞parkin (D–F) were 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 A30P͞YFP (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.)

round hyperphosphorylated Pser129-positive inclusions (Fig. mutant ␣-synuclein-induced dopaminergic cell loss in vivo and 5B) and occasional phosphorylated neurites (Fig. 5C). Triple promotes the formation of hyperphosphorylated ␣-synuclein labeling with a nuclear marker, Pser129, and human ␣-synuclein- inclusions. Consistent with these results, inactivation of the specific Abs showed that the intracytoplasmic phosphorylated gene encoding the E6-AP ubiquitin ligase leads to an increase inclusions are strongly immunopositive for human ␣-synuclein in neurotoxicity and a decrease in the number of nuclear (Fig. 5D). To explore the effect of parkin in the formation of inclusions in a transgenic model of spinocerebellar ataxia type ␣-synuclein inclusions, the number of cells containing hyper- 1 (34). Recently, the E3-ligase CHIP (carboxyl terminus of the phosphorylated inclusions was quantified in animals overex- Hsc70-interacting protein) was reported to attenuate tau- pressing A30P͞YFP and A30P͞parkin. Animals coexpressing induced cell death and also to facilitate hyperphosphorylated parkin with A30P ␣-synuclein showed a 45% increase in the tau aggregation (35, 36). Parkin, CHIP and E6-AP may number of neurons containing hyperphosphorylated inclusions similarly enhance cell survival by eliminating soluble toxic (Fig. 5E). A 41% increase in cells containing hyperphosphory- proteins in favor of insoluble aggregates. Additionally, in vitro lated inclusions was observed when only cells positive for mammalian cell and transgenic fly studies also attributed a ␣-synuclein were analyzed (data no shown). To determine protective role to parkin in dopamine neuron survival (15, 20). whether other posttranslational modifications of synuclein ag- Overexpression of parkin in the ␣-synuclein transgenic fly gregates might also correlate with the neuroprotection afforded induces, on the contrary, a decrease in the number of non- by parkin, we also examined the brains of A30P͞YFP- and phosphorylated ␣-synuclein inclusions. In the present study, A30P͞parkin-overexpressing animals for the presence of ubi- however, we analyzed the effect of parkin in the formation of quitinated inclusions. These experiments showed no ubiquiti- more mature posttranslationally modified ␣-synuclein- nated inclusions in the brains of A30P-expressing animals either containing inclusions. The presence of phosphorylated with or without parkin overexpression (data not shown). The ␣-synuclein was recently recognized as a pathological hallmark finding that not all ␣-synuclein inclusions are immunoreactive of ␣-synucleinopathy lesions (29, 37, 38). After 6 weeks of for ubiquitin in patients with PD indicates that ubiquitin is not ␣-synuclein expression, not all ␣-synuclein-positive cells de- a prerequisite for the ␣-synuclein pathology (4). veloped hyperphosphorylated inclusions. Because the forma- tion of hyperphosphorylated ␣-synuclein inclusions is a pro- Discussion gressive time- and dose-dependant process (37, 39), the Abnormal accumulation of ␣-synuclein is considered to be a majority of inclusions are likely nonphosphorylated in the key pathological event in the process leading to selective short time period of 6 weeks. However, detecting these Pser129 dopaminergic degeneration in ␣-synuclein-linked and sporadic inclusions has the advantage over classic ␣-synuclein staining PD, but the neurotoxic role of inclusions in PD is highly of better differentiating true inclusions from nonaggregate

debated (33). The major findings of this report are that gene subcellular accumulations of the protein (37). We observed NEUROSCIENCE therapy delivery of parkin efficiently prevents PD-linked that the neuroprotective effect of parkin is associated with a

Lo Bianco et al. PNAS ͉ December 14, 2004 ͉ vol. 101 ͉ no. 50 ͉ 17513 Downloaded by guest on September 30, 2021 activity of these protofibrils appears to be the disruption of synaptic 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 Fig. 5. Parkin increases the number of phosphorylated ␣-synuclein inclu- ␣ ␣ -synuclein (45). Contrary to parkin, expression of this neu- sions. Sections from the substantia nigra of rats expressing A30P -synuclein rotrophic factor does not prevent ␣-synuclein-induced toxicity. and parkin were immunostained with the Pser129 Ab specific for phospho- Ser-129 of ␣-synuclein that selectively and extensively accumulates in human This difference in neuroprotection may reflect a particular Lewy bodies. (A) A very weak Pser129 staining was observed in the noninjected relationship between the two PD-linked proteins, ␣-synuclein side corresponding to the physiological level of phosphorylated rat and parkin, and their implication in a common cellular path- ␣-synuclein. (B) The substantia nigra of animals overexpressing either A30P͞ way. Dissecting the molecular mechanism of parkin’s protec- YFP or A30P͞parkin revealed the presence of numerous hyperphosphorylated tion against ␣-synuclein toxicity should provide important inclusions reminiscent of Lewy bodies. (C) Pser129-positive neurites were clues about the unique vulnerability of dopamine neurons in occasionally observed in animals overexpressing A30P͞parkin. (D) Triple stain- ␣ PD. These results also indicate that gene therapy delivery of ing with Pser129 Ab (green), LB509 human -synuclein specific Ab (red), and parkin or pharmacological agents increasing parkin expression TOPRO-3 nuclear marker (blue) shows that phosphorylated inclusions abun- dantly contain human ␣-synuclein. (E) The number of neurons containing may constitute a potential therapeutic strategy for PD. Inter- Pser129-positive inclusions were quantified in the substantia nigra of rats estingly, brain delivery of human ␣-synuclein with viral vectors expressing A30P͞YFP or A30P͞parkin. Values refer to means Ϯ SEM; n ϭ 6 has recently been scaled up to non-human primates, opening animals per group; *, P Ͻ 0.05; **, P Ͻ 0.005. (Scale bars: A and B, 150 ␮m; C, the potential to evaluate the neuroprotective property of 40 ␮m; D,10␮m.) 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 significant increase in the number of hyperphosphorylated ␣ models of disease further strengthens the case for parkin-based -synuclein inclusions. We therefore hypothesize that parkin strategies as promising treatments for patients with PD. helps dopamine neurons survive by promoting the sequestration of toxic prefibrillar oligomers in mature hyperphosphorylated We thank Nicole De´glon,William Pralong, and Ruth-Luthi Carter for inclusions. Interestingly, cytosolic dopamine has been shown to helpful comments; Philippe Colin, Christel Sadeghi, Anne Maillard, and interact with ␣-synuclein to form adducts that stabilize the forma- Maria Rey for excellent technical help; and Dr. Michel Goedert for the tion of toxic protofibrils, suggesting a potential mechanism for the A30P human ␣-synuclein cDNA. This work was supported by the Swiss selective degeneration of dopaminergic neurons (40). One toxic National Science Foundation and the Michael J. Fox Foundation.

1. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, 5. Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., et al. (1997) Science 276, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., et al. (2003) Science 2045–2047. 302, 841. 2. Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, 6. Dawson, T. M. & Dawson, V. L. (2003) Science 302, 819–822. H., Epplen, J. T., Schols, L. & Riess, O. (1998) Nat. Genet. 18, 106–108. 7. Valente, E. M., Abou-Sleiman, P. M., Caputo, V., Muqit, M. M., Harvey, K., 3. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R. & Gispert, S., Ali, Z., Del Turco, D., Bentivoglio, A. R., Healy, D. G., et al. (2004) Goedert, M. (1997) Nature 388, 839–840. Science 304, 1158–1160. 4. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. & Goedert, M. 8. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, (1998) Proc. Natl. Acad. Sci. USA 95, 6469–6473. S., Yokochi, M., Mizuno, Y. & Shimizu, N. (1998) Nature 392, 605–608.

17514 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405313101 Lo Bianco et al. Downloaded by guest on September 30, 2021 9. Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S., Minoshima, S., 28. Lauwers, E., Debyser, Z., Van Dorpe, J., De Strooper, B., Nuttin, B. & Shimizu, N., Iwai, K., Chiba, T., Tanaka, K. & Suzuki, T. (2000) Nat. Genet. 25, Baekelandt, V. (2003) Brain Pathol. 13, 364–372. 302–305. 29. Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., 10. Cookson, M. R. (2003) Neuron 37, 7–10. Goldberg, M. S., Shen, J., Takio, K. & Iwatsubo, T. (2002) Nat. Cell Biol. 4, 11. Cookson, M. R. (2003) Neuromol. Med. 3, 1–13. 160–164. 12. Shimura, H., Hattori, N., Kubo, S., Yoshikawa, M., Kitada, T., Matsumine, H., 30. Deglon, N., Tseng, J. L., Bensadoun, J. C., Zurn, A. D., Arsenijevic, Y., Pereira Asakawa, S., Minoshima, S., Yamamura, Y., Shimizu, N. & Mizuno, Y. (1999) de Almeida, L., Zufferey, R., Trono, D. & Aebischer, P. (2000) Hum. Gene Ann. Neurol. 45, 668–672. Ther. 11, 179–190. 13. Imai, Y., Soda, M. & Takahashi, R. (2000) J. Biol. Chem. 275, 35661–35664. 31. Blomer, U., Naldini, L., Kafri, T., Trono, D., Verma, I. M. & Gage, F. H. (1997) 14. Darios, F., Corti, O., Lucking, C. B., Hampe, C., Muriel, M. P., Abbas, N., Gu, J. Virol. 71, 6641–6649. W. J., Hirsch, E. C., Rooney, T., Ruberg, M. & Brice, A. (2003) Hum. Mol. 32. Beltramino, C. A., de Olmos, J. S., Gallyas, F., Heimer, L. & Zaborszky, L. Genet. 12, 517–526. (1993) NIDA Res. Monogr. 136, 101–126; discussion 126–132. 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 33. Goldberg, M. S. & Lansbury, P. T., Jr. (2000) Nat. Cell Biol. 2, E115–E119. 36, 1007–1019. 34. Cummings, C. J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y., Ciechanover, 16. Oluwatosin-Chigbu, Y., Robbins, A., Scott, C. W., Arriza, J. L., Reid, J. D. & A., Orr, H. T., Beaudet, A. L. & Zoghbi, H. Y. (1999) Neuron 24, 879–892. Zysk, J. R. (2003) Biochem. Biophys. Res. Commun. 309, 679–684. 35. Shimura, H., Schwartz, D., Gygi, S. P. & Kosik, K. S. (2004) J. Biol. Chem. 279, 17. Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y. & Takahashi, R. (2001) 4869–4876. Cell 105, 891–902. 36. Petrucelli, L., Dickson, D., Kehoe, K., Taylor, J., Snyder, H., Grover, A., De 18. Staropoli, J. F., McDermott, C., Martinat, C., Schulman, B., Demireva, E. & Lucia, M., McGowan, E., Lewis, J., Prihar, G., et al. (2004) Hum. Mol. Genet. Abeliovich, A. (2003) Neuron 37, 735–749. 13, 703–714. 19. Tsai, Y. C., Fishman, P. S., Thakor, N. V. & Oyler, G. A. (2003) J. Biol. Chem. 37. Neumann, M., Kahle, P. J., Giasson, B. I., Ozmen, L., Borroni, E., Spooren, W., 278, 22044–22055. Muller, V., Odoy, S., Fujiwara, H., Hasegawa, M., et al. (2002) J. Clin. Invest. 20. Yang, Y., Nishimura, I., Imai, Y., Takahashi, R. & Lu, B. (2003) Neuron 37, 110, 1429–1439. 911–924. 38. Iwatsubo, T. (2003) J. Neurol. 250, Suppl. 3, 11–14. 21. Takahashi, H., Ohama, E., Suzuki, S., Horikawa, Y., Ishikawa, A., Morita, T., 39. Takahashi, M., Kanuka, H., Fujiwara, H., Koyama, A., Hasegawa, M., Miura, Tsuji, S. & Ikuta, F. (1994) Neurology 44, 437–441. M. & Iwatsubo, T. (2003) Neurosci. Lett. 336, 155–158. 22. Hayashi, S., Wakabayashi, K., Ishikawa, A., Nagai, H., Saito, M., Maruyama, 40. Conway, K. A., Rochet, J. C., Bieganski, R. M. & Lansbury, P. T., Jr. (2001) M., Takahashi, T., Ozawa, T., Tsuji, S. & Takahashi, H. (2000) Movement Science 294, 1346–1349. Disorders 15, 884–888. 41. Volles, M. J., Lee, S. J., Rochet, J. C., Shtilerman, M. D., Ding, T. T., Kessler, 23. van de Warrenburg, B. P., Lammens, M., Lucking, C. B., Denefle, P., J. C. & Lansbury, P. T., Jr. (2001) Biochemistry 40, 7812–7819. Wesseling, P., Booij, J., Praamstra, P., Quinn, N., Brice, A. & Horstink, M. W. 42. Volles, M. J. & Lansbury, P. T., Jr. (2002) Biochemistry 41, 4595–4602. (2001) Neurology 56, 555–557. 24. Lo Bianco, C., Ridet, J. L., Schneider, B. L., Deglon, N. & Aebischer, P. (2002) 43. Gispert, S., Del Turco, D., Garrett, L., Chen, A., Bernard, D. J., Hamm- Proc. Natl. Acad. Sci. USA 99, 10813–10818. Clement, J., Korf, H. W., Deller, T., Braak, H., Auburger, G. & Nussbaum, 25. Klein, R. L., King, M. A., Hamby, M. E. & Meyer, E. M. (2002) Hum. Gene R. L. (2003) Mol. Cell. Neurosci. 24, 419–429. Ther. 13, 605–612. 44. Chung, K. K., Zhang, Y., Lim, K. L., Tanaka, Y., Huang, H., Gao, J., Ross, 26. Kirik, D., Rosenblad, C., Burger, C., Lundberg, C., Johansen, T. E., Muzyczka, C. A., Dawson, V. L. & Dawson, T. M. (2001) Nat. Med. 7, 1144–1150. N., Mandel, R. J. & Bjorklund, A. (2002) J. Neurosci. 22, 2780–2791. 45. Lo Bianco, C., De´glon,N., Pralong, W. & Aebischer, P. (2004) Neurobiol. Dis., 27. Kirik, D., Annett, L. E., Burger, C., Muzyczka, N., Mandel, R. J. & Bjorklund, in press. A. (2003) Proc. Natl. Acad. Sci. USA 100, 2884–2889. 46. Kirik, D. & Bjorklund, A. (2003) Trends Neurosci. 26, 386–392. NEUROSCIENCE

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