Caspase-3-derived C-terminal Product of Synphilin-1 Displays Antiapoptotic Function via Modulation of the p53-dependent Cell Death Pathway*

Parkinson disease is the second most frequent neurodegenerative disorder after Alzheimer disease. A subset of genetic forms of Parkinson disease has been attributed to α-synuclein, a synaptic protein with remarkable chaperone properties. Synphilin-1 is a cytoplasmic protein that has been identified as a partner of α-synuclein (Engelender, S., Kaminsky, Z., Guo, X., Sharp, A. H., Amaravi, R. K., Kleiderlein, J. J., Margolis, R. L., Troncoso, J. C., Lanahan, A. A., Worley, P. F., Dawson, V. L., Dawson, T. M., and Ross, C. A. (1999) Nat. Gen. 22, 110–114), but its function remains totally unknown. We show here for the first time that synphilin-1 displays an antiapoptotic function in the control of cell death. We have established transient and stable transfectants overexpressing wild-type synphilin-1 in human embryonic kidney 293 cells, telecephalon-specific murine 1 neurons, and SH-SY5Y neuroblastoma cells, and we show that both cell systems display lower responsiveness to staurosporine and 6-hydroxydopamine. Thus, synphilin-1 reduces procaspase-3 hydrolysis and thereby caspase-3 activity and decreases poly(ADP-ribose) polymerase cleavage, two main indicators of apoptotic cell death. Furthermore, we establish that synphilin-1 drastically reduces p53 transcriptional activity and expression and lowers p53 promoter transactivation and mRNA levels. Interestingly, we demonstrate that synphilin-1 catabolism is enhanced by staurosporine and blocked by caspase-3 inhibitors. Accordingly, we show by transcription/translation assay that recombinant caspase-3 and, to a lesser extent, caspase-6 but not caspase-7 hydrolyze synphilin-1. Furthermore, we demonstrate that mutated synphilin-1, in which a consensus caspase-3 target sequence has been disrupted, resists proteolysis by cellular and recombinant caspases and displays drastically reduced antiapoptotic phenotype. We further show that the caspase-3-derived C-terminal fragment of synphilin-1 was probably responsible for the antiapoptotic phenotype elicited by the parent wild-type protein. Altogether, our study is the first demonstration that synphilin-1 harbors a protective function that is controlled by the C-terminal fragment generated by its proteolysis by caspase-3.

Parkinson disease (PD) 3 is characterized by the presence of intracytoplasmic inclusions, named Lewy bodies (LB), and by a massive loss of dopaminergic neurons in the substantia nigra (2). Most PD cases are of sporadic origin, but about 15% are associated with genetic causes. From the various loci associated with PD, named PARKs, six proteins have been identified so far. They are linked to either autosomal dominant (␣-synuclein, UCHL1, and LRRK2/dardarin) or autosomal recessive (parkin, DJ-1, and PINK-1) transmission (3)(4)(5), the latter forms being more severe and characterized by an early onset. The above proteins are linked to the three major dysfunctions observed in PD, which are oxidative stress, mitochondrial failure, and proteasomal dysfunction (6). Interestingly, these dysfunctions are associated with exacerbated cell death in PD (7)(8)(9).
Among the proteins responsible for the familial forms of PD, ␣-synuclein has received particular attention, not only because it was the first gene product implicated in familial forms of the disease but also because it is the major fibrillar protein of the LB (10,11). Even if its function is far from being completely elucidated, ␣-synuclein seems to play a major role in cell death processes. Thus, we have shown that wild-type ␣-synuclein triggered an antiapoptotic response in TSM1 neurons and that this phenotype could be abolished by familial PD mutations and 6-hydroxydopamine (6OH-DOPA) (12,13), a natural endogenous dopaminergic toxin (14) that is frequently used to induce PD in vivo (15)(16)(17).
The hunt for putative physiological binding partners of a protein often gives insight into its function. This strategy has led to the identification of a novel ␣-synuclein cellular partner named synphilin-1, the function of which remains completely unknown (1). Synphilin-1 is a cytoplasmic protein of 919 amino acids that has been identified by a yeast two-hybrid approach (1). The putative relevance of this protein to PD pathology is emphasized by several studies. Thus, synphilin-1 interacts with two proteins linked to familial PD (e.g. ␣-synuclein and parkin) (1,18) and is expressed in 80 -90% of the LB detected in PD brain samples (19). In vitro studies have shown that the co-expression of ␣-synuclein and synphilin-1 favor the formation of cytoplasmic inclusions that resemble LB in vivo (1,20,21). Synphilin-1 is located within a region of the chromosome 5q23.1-23.3 that is characterized by evocative lod scores for PD in distinct whole genome scans (22)(23)(24). Indeed, * This work was supported by the Centre National de la Recherche Scientifique by European Union Contract LSHM-CT-2003-503330 (Apopis) and by the Fondation pour la Recherche Mé dicale. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed. mutation analysis of the synphilin-1 gene in familial and sporadic German PD patients allowed the identification of the R621C mutation in two sporadic PD patients, suggesting a putative role of synphilin-1 as a genetic susceptibility factor for the disease (25). Due to the implication of synphilin-1 in PD and to the modulation of cell death by ␣-synuclein and parkin, two privileged binding partners of synphilin-1, we investigated the role of synphilin-1 in cell death control. We show that synphilin-1 lowers HEK293 cells, TSM1 neurons, and SH-SY5Y neuroblastoma responsiveness to staurosporine and 6OH-DOPA by decreasing caspase-3 activity and poly(ADP-ribose) polymerase and by down-regulating the p53-dependent proapoptotic pathway. In addition, in silico examination of the synphilin-1 sequence revealed a consensus site for a caspase-3 cleavage. Accordingly, we demonstrate the cleavage of synphilin-1 by cellular and purified caspase-3 and the abolishment of its antiapoptotic function by site-directed mutagenesis of the caspase-3 site in its sequence. Finally, we demonstrate that the C-terminal fragment of synphilin-1 generated by caspase-3 is indeed responsible for the antiapoptotic phenotype of synphilin-1.
The cDNA encoding the V5-tagged caspase-3-derived C-terminal fragment of synphilin-1 was engineered by introducing an ATG codon in position 454 after the putative consensus cleavage site of caspase-3 (oligonucleotide 5Ј-TA-CCC-AAG-CTT-ATG-CAG-GAT-GGC-3Ј). An additional HindIII restriction site was also added, adjacent to the ATG codon, for further subcloning of the construction in pcDNA3.1/V5/His-TOPO.
Cell Systems and Transfections-TSM1 neurons (26), HEK293 human cells, and SH-SY5Y neuroblastoma were cultured as previously described (27,28). Stable transfectants expressing empty vector (mock) and wild-type and mutated synphilin-1 in HEK293 cells were obtained after transfection with 2 g of each cDNA (all in pcDNA3) by means of calcium phosphate precipitation. TSM1 neurons expressing empty vector (mock) and wild-type synphilin-1 were obtained after the transfection with 2 g of each cDNA by means of Superfect reactive according to the manufacturer's conditions. Positive clones were screened for their synphilin-1-like immunoreactivity as described below. Transient transfections were carried out by means of 2 g of cDNA by calcium phosphate precipitation (HEK293 cells) or Lipofectamine (4 l; TSM1 and SH-SY5Y).
Wild-type and Mutated Synphilin-1 Degradation-Wild-type and D454A-synphilin-1-overexpressing HEK293 and TSM1 cells were preincubated for 16  Then cells were lysed and analyzed for synphilin-1-like immunoreactivity by Western blot using anti-V5 antibodies as described below.
Western Blot Analysis-For the detection of wild-type and mutated synphilin-1, equal amounts of protein (50 g) were separated on 8% gels and Western blotted with the anti-V5 mouse monoclonal antibodies (Invitrogen). For the detection of procaspase-3, human and mouse PARP, and ␤-tubulin immunoreactivities, equal amounts of protein (25 g) were separated on 8 or 12% gels and Western blotted with antihuman procaspase-3 antibodies (Interchim) and anti-human (Euromedex) and anti-mouse (BD Biosciences) PARP antibodies. Anti-␤-tubulin and anti-actin monoclonal antibodies were from Sigma. Immunological complexes were revealed as previously described (29).
p53 Expression, Activity, and Promoter Transactivation-The activity of p53 was analyzed after transient transfection of the PG13-luciferase (PG13) cDNA designed and kindly provided by Dr. B. Vogelstein (Baltimore, MD) (30). The transcriptional activation of the human p53 promoter (hpp53) was measured after transfection of the cDNA coding for the human p53 promoter sequence in frame with luciferase (provided by Dr. M. Oren, Rehovot, Israël). All activities were measured after co-transfection of 0.5-1 g of the above cDNAs and 0.25-0.5 g of ␤-galactosidase cDNA, in order to normalize transfection efficiencies. p53 immunoreactivity was analyzed by Western blot using an anti-p53 mouse monoclonal antibody (1:10,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in nuclear extracts prepared as previously described for cytochrome c translocation experiments (13).
Real Time Quantitative PCR-Total RNA from cells was extracted at the indicated times using the RNeasy kit following the instructions from the manufacturer (Qiagen). After treatment with DNase I, 2 g of total RNA were reverse transcribed using oligo(DT) priming and avian myeloblastosis virus reverse transcriptase (Promega). Real time PCR was performed in an ABI PRISM 5700 Sequence Detector System (Applied Biosystems) using the SYBR Green detection protocol as outlined by the manufacturer. Gene-specific primers were designed using the Primer Express software (Applied Biosystems). Relative expression level of target genes was normalized for RNA concentrations with two different housekeeping genes (human glyceraldehyde-3-phosphate dehydrogenase, mouse ␥-actin) according to the cell specificity.

In Vitro Transcription/Translation of Wild-type and Mutated Synphilin-1 and Cleavage by Caspase-3, -6, and -7 in a Cell-free System-
Wild-type and D454A synphilin-1 were transcribed and translated using the Promega TNT coupled reticulocyte lysate system in the presence of [ 35 S]methionine (ICN) as extensively described (31). Briefly, 2.5 l of reticulocyte lysates were incubated in 50 l of 25 mM HEPES, pH 7.5, 0.1% CHAPS, 5.0 mM dithiothreitol with 25 ng of recombinant caspase-3, -6, and -7 (Sigma) for 8 h at 37°C. In some experiments, the effect of the caspase inhibitor benzyloxycarbonyl-VAD (10 M) was examined. Proteins were then electrophoresed on 11% polyacrylamide gels and autoradiographed using Amersham Biosciences hyperfilms.
Statistical Analysis-Statistical analysis was performed with PRISM software (Graphpad Software, San Diego, CA), by using the Newman-Keuls multiple comparison tests for one-way analysis of variance and Student's t test.

Wild-type Synphilin-1 (WT-synphilin-1) but Not D454A-synphilin-1 Undergoes Cellular Proteolysis by Ac-DEVD-CHO-sensitive Caspaselike Activity in HEK293 Human Cells and Is Cleaved by Purified
Caspase-3 in Vitro-We have established stable transfectants overexpressing WT-synphilin-1 and mutated D454A-synphilin-1 in human embryonic kidney (HEK293) cells. The design of mutated D454A-synphilin-1 is based on an in silico study that identified a consensus cleavage site for caspase-3 ( 451 DEVD 454 ) on the WT-synphilin-1 sequence. Fig. 1A shows several of the wild-type and mutated stable transfectants obtained that overexpress a 120-kDa protein, a molecular mass corresponding to that expected for the V5-tagged synphilin-1 (1). Clones 10 and 11 (Fig. 1B), which display similar levels of wild-type and mutated synphilin-1 protein expression, were selected for the follow-up of our study. Fig. 2 illustrates the susceptibility of WT-synphilin-1 to various protease inhibitors. Pepstatin (acidic protease inhibitor), ALLN (calpain inhibitor), and o-phenanthroline (metalloprotease inhibitor) were unable to affect WT-synphilin-1 expression (Fig. 2, A and B). Ac-DEVD-CHO (caspase-3, -6, and -7 inhibitor) significantly increased WT-synphilin-1 immunoreactivity (Fig. 2, A and B) in a time-dependent manner (Fig. 2C), suggesting a processing of this protein by caspases. It is interesting to note that E64 (cysteine/serine protease inhibitor) also slightly but significantly potentiated WT-synphilin-1 expression (Fig. 2, A and B), in agreement with the fact that caspases activities belong to the class of cysteine proteases (32,33). Interestingly, D454A-synphilin-1 remained completely insensitive to both E64 and caspase inhibitor (Fig. 2, A and C). These data first confirm that the D454A mutation renders synphilin-1 resistant to proteolysis in HEK293 cells and indicates that caspase-like activities mainly contributed to synphilin-1 catabolism in HEK293 cells.
Staurosporine and 6OH-DOPA have been shown to increase caspase-3 activity in various cell systems (13). We therefore examined whether treatment of WT-synphilin-1-expressing cells with these two proapoptotic effectors could enhance WT-synphilin-1 degradation. Indeed, Fig. 2D shows that staurosporine and 6OH-DOPA both decreased the expression of WT-synphilin-1, the levels of which appeared drastically increased upon Ac-DEVD-CHO treatment of the cells (Fig. 2D), in agreement with the above data suggesting an implication of caspases in the processing of synphilin-1. In order to identify the caspases involved in the cleavage of synphilin-1, we examined its susceptibility to proteolysis by recombinant caspase-3, -6, and -7 in vitro. Fig. 3A shows that WT-synphilin-1 is cleaved by recombinant caspases-3 and, to a much lesser extent, by caspase-6, whereas caspase-7 appeared unable to cleave WT-synphilin-1 (Fig. 3A). Ac-DEVD-CHO fully prevented caspase-3 and caspase-6-mediated hydrolysis of WT-synphilin-1 (Fig. 3A). Interestingly, D454A-synphilin-1 fully resisted proteolysis by recombinant caspase-3 (Fig. 3B). It should be noted that WT-synphilin-1 resisted proteolysis by recombinant and cellular overexpressed caspase-8 (not shown), in agreement with the fact that the site cleaved in synphilin-1 (DEVD2Q) and mutated in D454A-synphilin-1 is canonical for caspase-3 but not caspase-8.
WT-synphilin-1 but Not D454A-synphilin-1 Reduces Staurosporine-and 6OH-DOPA-induced Caspase-3 Activation in HEK293 Cells and Lowers the p53-dependent Proapoptotic Pathway-The implication of caspases in the processing of synphilin-1 led us to investigate whether WT-synphilin-1 could control cell death and whether the caspase site mutation could influence such a phenotype. We analyzed the responsiveness of transiently or stably transfected WT-synphilin-1 and D454A-synphilin-1-expressing HEK293 cells to staurosporine (STS) and 6OH-DOPA and, more particularly, the levels of caspase-3. Staurosporine was used as a broad and nonspecific proapoptotic inducer, whereas 6OH-DOPA is a natural dopaminergic toxin that triggers neurodegenerescence that mimics that observed in PD pathology (2). First, we confirmed that STS (Fig. 4, A, C, and G) and FIGURE 1. Immunological analysis of WT-synphilin-1-and mutated D454A-synphilin-1expressing HEK293 cells. HEK293 cells were stably transfected with empty pcDNA3 vector (Mock), wild-type-synphilin-1 (WTSynp), or D454A-synphilin-1 (D454ASynp) cDNA as described under "Experimental Procedures." Synphilin-1-like immunoreactivities of wild-type (clones WT) and mutated synphilin-1 (clones D454A) were analyzed by electrophoresis on a 8% Tris-glycine gel, Western Blot, and incubation with anti-V5 primary antibodies as described under "Experimental Procedures" (A). Actin immunoreactivity was monitored as a control of protein charge (see "Experimental Procedures"). In B, the bars correspond to the densitometric analyses of the various clones normalized for actin expression.

The C-terminal Product of Synphilin-1 Is Antiapoptotic
6OH-DOPA (Fig. 4, B, D, and H) stimulate caspase-3 activity in a time-and dose-dependent manner. Interestingly, WT-synphilin-1 expression drastically reduced caspase-3 activity, whereas the D454A mutation drastically reverted this inhibitory control of caspase-3 activity (Fig. 4, A-D, G, and H). Accordingly, STS-and 6OH-DOPA-induced synphilin-1 catabolites were only observed in cells expressing the wild-type protein (Fig. 4I). Overall, these data indicate that caspase-resistant D454A-synphilin-1 was unable to modulate cell death in HEK293 cells and, therefore, that the antiapoptotic response elicited by synphilin-1 was controlled by its proteolysis by caspase-3.
In order to further confirm the influence of WT-synphilin-1 on caspase-3 modulation, we analyzed the immunoreactivities of the inactive procaspase-3 in control and STS-or 6OH-DOPA-stimulated conditions. Procaspase-3 is the inactive precursor of caspase-3 that is catalytically activated by caspase-8 and caspase-9 during apoptosis. Thus, a reduction of its immunoreactivity reflects an activation of cell death processes. As expected, STS (Fig. 4E) and 6OH-DOPA (Fig. 4F) treat-ment of mock-transfected cells drastically lowers procaspase-3 expression (Fig. 4, E and F). It should be noted that the extent of proteolytic maturation of procaspase-3 by 6OH-DOPA was more important than the one triggered by STS, in agreement with caspase-3 activity measurements (see Fig. 4, A-D, G, and H). WT-synphilin-1 elicited a reduction of procaspase-3 cleavage in stimulated conditions (Fig. 4, E and F), whereas D454A-synphilin-1-expressing cells still displayed procaspase-3 reduction (not shown). It should be noted that WT-synphilin-1 reverted procaspase-3 immunoreactivity to nearly control levels (Fig. 4, E and F). This suggests that the bulk of WT-synphilin-1-induced effects observed on "caspase-like" activities as well as its protection by the Ac-DEVD-CHO indeed reflects a functional link between WT-synphilin-1 and genuine caspase-3 rather than another caspase-like activity.
PARP is an enzyme implicated in the reparation of DNA that is proteolytically inactivated by caspase-3 during apoptosis. Thus, an augmentation of its 89-kDa cleavage product or a lowering of the precursor versus product ratio reflects an increase of caspase-3 activity and subsequent caspase-3-dependent apoptotic process. As expected, STS or 6OH-DOPA treatment of mock-transfected cells drastically augments the recovery of PARP product with concomitant virtual abolishment of PARP precursor immunoreactivity (Fig. 4, E and F). In both STS-and 6OH-DOPA-stimulated conditions, WT-synphilin-1 expression enhances PARP precursor immunoreactivity (Fig. 4, E and F), thereby leading to an augmentation of precursor versus product ratio. Altogether, our data demonstrate by both enzymatic and immunological approaches that WT-synphilin-1 triggers and antiapoptotic response by controlling caspase-3 activity and that this phenotype is fully reverted by site-directed mutagenesis of the synphilin-1 caspase-3 cleavage consensus site.
In order to further delineate the cellular intermediates involved in the WT-synphilin-1 antiapoptotic phenotype, we examined the influence of WT-synphilin-1 on the p53-dependent pathway. Fig. 5 shows that WT-synphilin-1-expressing HEK293 cells display drastically reduced p53 transcriptional activity (Fig. 5A) and nuclear expression (Fig. 5B). Furthermore, WT-synphilin-1 lowers the transactivation of the p53 promoter (Fig. 5C), in very good agreement with the reduced p53 mRNA levels established by real time PCR (Fig. 5D). Of most interest is  our observation that the down-regulation of the p53 pathway was not observed in cells expressing mutated D454A-synphilin-1 (Fig. 5, A-D).

The Caspase-3-derived C-terminal Fragment of Synphilin-1 Lowers HEK293 and TSM1 Responsiveness to STS-and 6OH-DOPA-induced
Caspase-3 Activation-The fact that WT-synphilin-1 undergoes caspase-3-mediated proteolysis together with the observation that the mutation that renders WT-synphilin-1 resistant to this cleavage also abolished its antiapoptotic phenotype strongly suggested that the C-terminal fragment of WT-synphilin-1 (synphilin-1-CTF) generated by caspase-3 could indeed be responsible for the WT-synphilin-1-associated protective phenotype. In order to directly examine this possibility, we have designed the V5-tagged synphilin-1-CTF (Fig. 8), and we have assessed its influence after transient transfection in HEK293 cells and TSM1 neurons. Synphilin-1-CTF lowers the STS-induced (Fig. 8, A and C) and  6OH-DOPA-induced (Fig. 8, B and D) caspase-3 activation in both HEK293 cells (Fig. 8, A and B) and TSM1 neurons (Fig. 8, C and D).

DISCUSSION
PD-affected brains exhibit selective loss of substantia nigra pars compacta neurons and are invaded at late stages by cytoplasmic inclusions called Lewy bodies (LB) (38 -40). Dopaminergic neuron cell death is apparently linked to exacerbated oxidative stress and p53-dependent apoptosis (41)(42)(43)(44) that could be the consequence of the accumulation and aggregation of misfolded proteins. Thus, it has been demonstrated that aggregated proteins display inherent toxicity (45) and harbor the ability to inhibit the proteasome (46). In this context, when the cellular capacity of refolding, recovery, and degradation are saturated, misfolded proteins accumulate, aggregate (47), and ultimately kill the cells.
LB reflect such an accumulation process in PD. These structures are mainly composed of ubiquitin, a number of elements of the proteosomal machinery and aggregated proteins among which ␣-synuclein is the main component (48). ␣-Synuclein, one of the key proteins implicated in familial PD (10,49,50), has a high propensity to aggregate in vitro and in vivo, and several studies showed that ␣-synuclein aggregation can be exacerbated by pathogenic mutations and by different factors, including the dopaminergic derivative prooxidant toxin 6OH-DOPA (for reviews, see Refs. [51][52][53]. Interestingly, ␣-synuclein aggregation impairs its function. Thus, the A53T familial-associated PD mutation and 6OH-DOPA both trigger ␣-synuclein aggregation and abolish its antiapoptotic function (13). ␣-Synuclein displays remarkable chaperone properties (53), and recently, synphilin-1 has been characterized as one of its binding partners (1,19). Interestingly, synphilin-1 accumulates in LB (19), and the co-overexpression of ␣-synuclein and synphilin-1 favors the formation of eosinophil cytoplasmic inclusions that resemble LB (1,21). Therefore, the possible implication of synphilin-1 in the formation of the LB and its possible functional link with ␣-synuclein led us to study the role of synphilin-1 in cell death.
We have established that wild-type synphilin-1 has a protective phenotype in human HEK293 cells, TSM1 neurons, and SH-SY5Y neuroblastoma cells. Thus, synphilin-1 reduces STS-and 6OH-DOPA-induced caspase-3 activation and PARP cleavage. In agreement with its protective function, WT-synphilin-1 also drastically down-regulated the proapoptotic p53 pathway. Interestingly, synphilin-1 function appears regulated by its proteolysis. Thus, we show that cellular synphilin-1 degradation is enhanced by the proapoptotic effectors STS and 6OH-DOPA and reduced by caspase-3 inhibitor. In agreement, we found that synphilin-1 is cleaved preferentially by caspase-3 in vitro. Interestingly, D454A-synphilin-1, a mutant in which a consensus cleavage site for caspase-3 had been abolished, fully resisted proteolysis by recombinant caspase-3.
What is the molecular influence of caspase-3 cleavage on synphilin-1 function? At least two theoretical hypotheses could stand. First, synphilin-1 holoprotein itself would be responsible for the protective phenotype, and caspase-3 cleavage could be seen as an inactivating process. Second, synphilin-1-associated antiapoptotic phenotype would be associated with one of its caspase-3-derived proteolytic products. Our data strongly argue in favor of the latter view. Thus, synphilin-1-induced antiapoptotic phenotype is drastically reduced when synphilin-1 is rendered resistant to caspase-3 proteolysis by mutagenesis of a caspase-3 cleavage site consensus sequence. This observation strongly suggested a role of caspase-3 in the generation of a synphilin-1-derived product with  antiapoptotic properties. Indeed, we have shown that the caspase-3derived C-terminal fragment of synphilin-1 lowered staurosporine-and 6OH-DOPA-induced caspase-3 activation. In this context, one could envision that cellular stress or environmental factors trigger caspase-3 activation and associated cell death but also provide a means to downregulate apoptosis by concomitantly increasing the production of caspase-3-derived synphilin-1 proteolytic fragment. It should be noted that this type of regulation has already been documented for other proteins. Thus, presenilins (54,55) and ␤-amyloid precursor protein (56 -60) undergo caspase-derived cleavages, generating proteolytic fragments controlling cell death. More related to PD, parkin, another binding partner of synphilin-1 (18,61) displaying an antiapoptotic phenotype (62,63), is also cleaved by caspases, but unlike for synphilin-1, this endoproteolysis leads to a loss of function of this protein (64).
It is worth noting that although both ␣-synuclein and synphilin-1 protect human cells and neurons from STS stimulation (12,13), only synphilin-1 keeps its protective function in the presence of 6OH-DOPA (this work). This phenotype is reminiscent of the one associated with ␤-synuclein, the homologue of ␣-synuclein. Thus, both synphilin-1 and ␤-synuclein remain protective toward 6OH-DOPA (55) and lower the p53 pathway. Furthermore, ␤-synuclein restores the protective activity of ␣-synuclein, even in the presence of 6OH-DOPA (55). Whether synphilin-1 restores the antiapoptotic potential of ␣-synuclein in the presence of the dopaminergic derivative remains to be established. However, it should be noted that ␣-synuclein and synphilin-1 co-localize in LB at the late stages of the pathology and that aggresomes formed by ␣-synuclein and synphilin-1 are cytoprotective (65). These observations together with the present demonstration of a protective function of synphilin-1 argue in favor of a caspase-3-regulated protective role of synphilin-1 and for a functional cross-talk between ␣-synuclein and synphilin-1 within LB.