β-Synuclein Displays an Antiapoptotic p53-dependent Phenotype and Protects Neurons from 6-Hydroxydopamine-induced Caspase 3 Activation
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m306083200
ISSN1083-351X
AutoresCristine Alvès da Costa, Eliezer Masliah, Frédéric Checler,
Tópico(s)Parkinson's Disease Mechanisms and Treatments
ResumoWe have established stable transfectants expressing β-synuclein in TSM1 neurons. We show that in basal and staurosporine-induced conditions the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)-positive β-synuclein-expressing neurons was drastically lower than in mock-transfected TSM1 cells. This was accompanied by a lower DNA fragmentation as evidenced by the reduction of propidium iodide incorporation measured by fluorescence-activated cell sorter analysis. β-Synuclein strongly reduces staurosporine-induced caspase 3 activity and immunoreactivity. We establish that β-synuclein triggers a drastic reduction of p53 expression and transcriptional activity. This was accompanied by increased Mdm2 immunoreactivity while p38 expression appeared enhanced, indicating that β-synuclein-induced p53 down-regulation likely occurs at a post-transcriptional level. We showed previously that α-synuclein displays an antiapoptotic function that was abolished by the dopaminergic derived toxin 6-hydroxydopamine (6OHDA). Interestingly, β-synuclein retains its ability to protect TSM1 neurons even after 6OHDA treatment. Furthermore, β-synuclein restores the antiapoptotic function of α-synuclein in 6OHDA-treated neurons. Altogether, our data document for the first time that β-synuclein protects neurons from staurosporine and 6OHDA-stimulated caspase activation in a p53-dependent manner. Our observation that β-synuclein contributes to restoration of the α-synuclein antiapoptotic function abolished by 6OHDA may have direct implications for Parkinson's disease pathology. In this context, the cross-talk between these two parent proteins is discussed. We have established stable transfectants expressing β-synuclein in TSM1 neurons. We show that in basal and staurosporine-induced conditions the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)-positive β-synuclein-expressing neurons was drastically lower than in mock-transfected TSM1 cells. This was accompanied by a lower DNA fragmentation as evidenced by the reduction of propidium iodide incorporation measured by fluorescence-activated cell sorter analysis. β-Synuclein strongly reduces staurosporine-induced caspase 3 activity and immunoreactivity. We establish that β-synuclein triggers a drastic reduction of p53 expression and transcriptional activity. This was accompanied by increased Mdm2 immunoreactivity while p38 expression appeared enhanced, indicating that β-synuclein-induced p53 down-regulation likely occurs at a post-transcriptional level. We showed previously that α-synuclein displays an antiapoptotic function that was abolished by the dopaminergic derived toxin 6-hydroxydopamine (6OHDA). Interestingly, β-synuclein retains its ability to protect TSM1 neurons even after 6OHDA treatment. Furthermore, β-synuclein restores the antiapoptotic function of α-synuclein in 6OHDA-treated neurons. Altogether, our data document for the first time that β-synuclein protects neurons from staurosporine and 6OHDA-stimulated caspase activation in a p53-dependent manner. Our observation that β-synuclein contributes to restoration of the α-synuclein antiapoptotic function abolished by 6OHDA may have direct implications for Parkinson's disease pathology. In this context, the cross-talk between these two parent proteins is discussed. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; LB, Lewy body; 6OHDA, 6-hydroxydopamine; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. is one of the most common and devastating diseases in the elderly (1Fratiglioni L. Launer L.J. Andersen K. Breteler M.M. Copeland J.R.M. Dartigues J.F. Lobo A. Martinez-Lage J. Soininen H. Hofman A. Neurology. 2000; 54: 10-15PubMed Google Scholar, 2Giampaoli S. Aging Clin. Exp. Res. 2000; 12: 93-105Crossref Google Scholar). This pathology is characterized by intracellular aggregates, called Lewy bodies (LB) (3Goedert M. Spillantini M.G. Davies S.W. Curr. Opin. Neurobiol. 1998; 8: 619-632Crossref PubMed Scopus (228) Google Scholar, 4Trojanowski J.Q. Goedert M. Iwatsubo T. Lee V.M.-Y. Cell Death Differ. 1998; 5: 832-837Crossref PubMed Scopus (248) Google Scholar) that are thought to be responsible for final dementia occurring not only in PD but also in Lewy body diseases. The main component of LB was identified as α-synuclein (5Spillantini M.G. Schmidt M.L. Lee V.-Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6267) Google Scholar), a 140-amino acid-long synaptic protein (6Uéda K. Fukushima H. Masliah E. Xia Y. Iwai A. Yoshimoto M. Otero D.A.C. Kondo J. Ihara Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11282-11286Crossref PubMed Scopus (1239) Google Scholar) that accumulates within these neuropathological hallmarks (7Wakabayashi K. Matsumoto K. Takayama K. Yoshimoto M. Takahashi H. Neurosci. Lett. 1997; 239: 45-48Crossref PubMed Scopus (287) Google Scholar, 8Takeda A. Mallory M. Sundsmo M. Honer W. Hansen L. Masliah E. Am. J. Pathol. 1998; 152: 367-372PubMed Google Scholar, 9Hashimoto M. Masliah E. Brain Pathol. 1999; 9: 707-720Crossref PubMed Scopus (221) Google Scholar). The central role of α-synuclein in PD pathology has been emphasized by the observation that familial forms of PD were due to two mutations borne by α-synuclein (10Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnston W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar, 11Krüger R. Kuhn W. Müller T. Woitalla D. Graeber M. Kösel S. Przuntek H. Epplen J.T. Schöls L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3344) Google Scholar). Interestingly, these mutations trigger alterations of the biophysical properties of α-synuclein, leading to an exacerbation of misfolding and aggregation (12Giasson B.I. Uryu K. Trojanowski J.Q. Lee V.M.-Y. J. Biol. Chem. 1999; 274: 7619-7622Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Therefore, as with many other neurodegenerative diseases such as Alzheimer's and prion diseases, among others (13Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar, 14Ferrigno P. Silver P.A. Neuron. 2000; 26: 9-12Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 15Wolozin B. Behl C. Arch. Neurol. 2000; 57: 793-796Crossref PubMed Scopus (71) Google Scholar), PD can be documented as a disease associated with protein misfolding. The fact that such aggregates of proteins were recently shown to exhibit an intrinsic toxic potential (16Bucciantini M. Giannoni E. Chiti F. Baroni F. Formigli L. Zurdo J. Taddei N. Ramponi G. Dobson C.M. Stefani M. Nature. 2002; 416: 507-511Crossref PubMed Scopus (2164) Google Scholar) could lead to a reunifying theory linking misfolding and neurodegeneration. Interestingly, we have shown that α-synuclein displays an antiapoptotic phenotype that was abolished by PD-related mutations (17Alves da Costa C. Ancolio K. Checler F. J. Biol. Chem. 2000; 275: 24065-24069Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This antiapoptotic function was also prevented when neuronal cells were exposed to 6-hydroxydopamine (6OHDA), a toxin derived from dopamine (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). This was associated with an increased expression of α-synuclein, and we postulated that the protein exerted a physiological antiapoptotic phenotype that was abolished in conditions of drastic overexpression and aggregation (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). This hypothesis was in line with the observation that overexpression of α-synuclein and its aggregation could lead to a PD-like phenotype in transgenic mice (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar) and Drosophila (20Feany M.B. Bender W.W. Nature. 2000; 404: 394-398Crossref PubMed Scopus (1725) Google Scholar). β-Synuclein is another member of the synuclein family (21Nakajo S. Tsukada K. Omata K. Nakamura Y. Nakaya K. Eur. J. Biochem. 1993; 217: 1057-1063Crossref PubMed Scopus (151) Google Scholar, 22Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (908) Google Scholar) that lacks the non-amyloidogenic component (NAC) domain that appears to be responsible for the aggregating properties of α-synuclein (23Jensen P.H. Sorensen E.S. Petersen T.E. Gliemann J. Rasmussen L.K. Biochem. J. 1995; 310: 91-94Crossref PubMed Scopus (90) Google Scholar). β-Synuclein is therefore considered to be a non-amyloidogenic homolog of α-synuclein. Interestingly, it was demonstrated that the ratio of β-synuclein to α-synuclein appeared altered in LB disease (24Rockenstein E. Hansen L.A. Mallory M. Trojanowski J.Q. Galasko D. Masliah E. Brain Res. 2001; 914: 48-56Crossref PubMed Scopus (144) Google Scholar). Because β-synuclein can not seed α-synuclein aggregation (25Biere A.L. Wood S.J. Wypych J. Steavenson S. Jiang Y. Anafi D. Jacobsen F.W. Jarosinski M.A. Wu G.M. Louis J.-C. Martin F. Nahri L.O. Citron M. J. Biol. Chem. 2000; 275: 34574-34579Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), it was postulated that β-synuclein could act as a physiological inhibitor of α-synuclein aggregation (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Indeed, it was reported that β-synuclein-derived peptides behave as anti-aggregating agents (26Windisch M. Hutter-Paier B. Rockenstein E. Hashimoto M. Mallory M. Masliah E. J. Mol. Neurosci. 2002; 19: 63-69Crossref PubMed Scopus (40) Google Scholar). That this β-synuclein property could lead to a therapeutic strategy was validated by the observation that β-synuclein inhibited α-synuclein aggregation in transgenic models of PD pathology (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Of the greatest importance was the demonstration that this was accompanied by an amelioration of motor deficits and neurodegenerative alterations (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Nothing is known concerning the cellular function of β-synuclein and, more particularly, whether, as is the case for α-synuclein, β-synuclein could control neuronal cell death. We establish here that β-synuclein is antiapoptotic in TSM1 neurons and that this function is linked to a drastic down-regulation of p53 expression and activity. We show that β-synuclein still protects neurons from 6OHDA insult. Finally, we demonstrate that β-synuclein contributes to the α-synuclein function and, more particularly, restores its antiapoptotic phenotype in 6OHDA-treated cells, an experimental condition that normally abolishes α-synuclein antiapoptotic function. Cell Systems and Transfections—TSM1 neurons (27Chun J. Jaenisch R. Mol. Cell. Neurosci. 1996; 7: 304-321Crossref PubMed Scopus (61) Google Scholar) were cultured as described previously (17Alves da Costa C. Ancolio K. Checler F. J. Biol. Chem. 2000; 275: 24065-24069Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Stable transfectants expressing wild-type β-synuclein were obtained after transfection with 2 μg of β-synuclein cDNA (in pcDNA3) by means of DCA30 (Eurogentec) according to the manufacturer's recommendations. Positive transfectants were screened for their β-synuclein-like immunoreactivity as described below. Transient transfections were also performed in TSM1 neurons with DCA30 containing either 2 μg of cDNA encoding α-synuclein and β-synuclein, alone (28Ancolio K. Alves da Costa C. Uéda K. Checler F. Neurosci. Lett. 2000; 285: 79-82Crossref PubMed Scopus (104) Google Scholar) or in combination. Cells were used 48 h after their transfection. Western Blot Analyses—For the detection of α- and β-synucleins, equal amounts of protein (50 μg) were separated on Tris-tricine gels and Western blotted with the anti-α- and β-synuclein rabbit polyclonal antibodies (Affiniti Research Products). Active caspase 3, p53, Mdm2 phospho-p38, and β-tubulin immunoreactivities were analyzed by Western blot performed by means of anti-active caspase 3 (rabbit polyclonal; R&D Systems), anti-p53 (mouse monoclonal; Santa Cruz Biotechnology), anti-Mdm2 (mouse monoclonal; provided by Dr. R. Fahraeus), anti-phospho-p38 (rabbit polyclonal; Promega), and anti-β-tubulin (Sigma). Immunological complexes were revealed as described previously (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 29Paitel E. Fahraeus R. Checler F. J. Biol. Chem. 2003; 278: 10061-10066Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Caspase 3 Activity Measurements—Stable or transient transfectants were preincubated without or with staurosporine (1 μm) or 6OHDA (0.2 mm) for 2 or 7 h, respectively, and then caspase-3-like activity was fluorometrically measured as detailed extensively (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Caspase 3-like activity is considered as the Ac-DEVD-al sensitive Ac-DEVD-7AMC-hydrolyzing activity. p53 Transcriptional Activity—The PG13-luciferase gene reporter construct (kindly provided by Dr. B. Vogelstein) has been described previously (30El-Deiry W. Kern S. Pietenpol J. Kinzler K. Vogelstein B. Nat. Genet. 1992; 1: 45-49Crossref PubMed Scopus (1752) Google Scholar). One microgram of PG13-luciferase cDNA was co-transfected with 0.5 μg of a β-galactosidase transfection vector (to normalize transfection efficiencies) in mock- or β-synuclein-expressing TSM1 neurons. Forty-eight hours after transfection, luciferase and β-galactosidase activities were measured as described (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). TUNEL Analysis—This procedure has been extensively described (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Briefly, cells were fixed for 20 min in 4% paraformaldehyde (in phosphate-buffered saline), rinsed in phosphate-buffered saline, left overnight in 70% ethanol, and then processed for the dUTP nick end labeling TUNEL technique as recommended (Roche Applied Science kit). Staining was assessed with a peroxidase-conjugated antibody, and DNA label is revealed with 3,3′-diaminobenzidine as black spots. All cells are stained with erythrosine B to quantify the percentage of total neurons undergoing cell death. Stably transfected TSM1 neurons overexpress a 19-kDa protein, a molecular mass corresponding to that expected for β-synuclein (Fig. 1A). The responsiveness of these cells to staurosporine, an apoptotic effector, has been examined. TSM1 cells display about 20% of TUNEL-positive neurons in basal conditions (Fig. 1B). This number increases drastically upon staurosporine treatment (Fig. 1, A and B). Interestingly, β-synuclein expression significantly reduces the number of TUNEL-positive cells under both basal and staurosporine-stimulated conditions (Fig. 1, B and C). This histological feature was reinforced by the observation that the propidium iodide incorporation measured upon staurosporine stimulation by flow cytometry revealed a lower DNA fragmentation in β-synuclein-expressing cells (data not shown). The β-synuclein-mediated antiapoptotic phenotype was associated with a drastic reduction of staurosporine-induced caspase 3 activation (Fig. 2A). This was accompanied by a lower active caspase 3 immunoreactivity (Fig. 2B). It should be noted that identical data were obtained with various independent β-synuclein-expressing clones (not shown).Fig. 2β-Synuclein decreases caspase 3-like activity. A, caspase 3-like activity was fluorometrically recorded in mock (black bars) and β-synuclein-expressing cells (beta-SYN, white bars) in the absence (Basal) or presence of staurosporine (STS; 1 μm, 2h). Bars represent the Ac-DEVD-al sensitive caspase 3-like activity and are the means ± S.E. of eight determinations carried out in duplicate. Statistical analysis indicates that p < 0.001 by t test analysis for both basal and STS conditions when comparing mock- versus β-synuclein- stably transfected cells. B, active caspase-3-like immunoreactivity (Casp-3 act.) was analyzed in basal (–) and stimulated (+) conditions (STS; 1 μm, 2h) in the indicated cell lines as described under "Experimental Procedures." Protein charge was monitored by β-tubulin analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) p53-like immunoreactivity is lower in β-synuclein-expressing neurons than in mock-transfected cells (Fig. 3, A and B). Interestingly, reduced p53 expression is associated with a statistically significant reduction of p53 transcriptional activity (Fig. 4). As p53 expression can be controlled at post-transcriptional levels by several proteins, we examined the influence of β-synuclein on the expression of Mdm2 and phosphorylated p38 (Fig. 5). Thus Mdm2 controls p53 expression mainly by regulating its ubiquitination/degradation rates (31Yin Y. Luciani M.G. Fahraeus R. Nat. Cell Biol. 2002; 4: 462-467Crossref PubMed Scopus (241) Google Scholar). Conversely, phosphorylated p38 activates p53 by phosphorylation (32Zhu Y. Mao X.O. Sun Y. Xia Z. Greenberg D.A. J. Biol. Chem. 2002; 277: 22909-22914Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In line with our observed decreased p53 expression, we found higher Mdm2-like immunoreactivity and reduced p38 expression in β-synuclein-expressing cells in both basal and staurosporine-stimulated conditions (Fig. 5). This suggests a possible control of p53 by β-synuclein at a post-transcriptional level. These data, however, do not totally rule out the possibility that β-synuclein could also control p53 expression at a transcriptional level.Fig. 4β-Synuclein overexpression lowers p53 transcriptional activity in TSM1 neurons. Mock- and β-synuclein-expressing TSM1 transfectants neurons were transiently co-transfected with β-galactosidase and PG13-luciferase cDNAs. Forty-eight hours after transfection, luciferase and galactosidase activities were monitored as described under "Experimental Procedures." Bars represent the means ± S.E. of 12 determinations carried out in duplicate of four independent transfections. Statistical analysis by t test indicates that p < 0.0001 when comparing mock versus β-synuclein (beta-syn) stably transfected cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5β-Synuclein expression increases Mdm2 immunoreactivity and lowers p38 expression. Mdm2 and active p38-like immunoreactivities in β-synuclein-expressing neurons were monitored as described under "Experimental Procedures." The gel corresponds to one of three representative densitometric analysis of the Mdm2 and p38-active (p38act) immunoreactivities. Protein charge is indicated by β-tubulin (Tub.) analysis. Ct, control; STS, staurosporine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As we showed previously, 6OHDA, a dopaminergic toxin, triggers drastic caspase 3 activation (Fig. 6). β-synuclein fully prevents 6OHDA-induced caspase activation, with the activity remaining close to the control value (Fig. 6). Furthermore, β-synuclein fully prevents the 6OHDA-induced increase in caspase 3 immunoreactivity observed in mock-transfected cells (Fig. 7C). Therefore, β-synuclein retains its antiapoptotic potential with distinct caspase activators, namely staurosporine and 6OHDA. This is not the case for α-synuclein. Thus, although α-synuclein displays a clear protective effect toward staurosporine in neurons (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), our data on caspase activity (Fig. 7A) and immunoreactivity (Fig. 7C) indicate that 6OHDA abolishes the α-synuclein-induced antiapoptotic phenotype in agreement with our previous study (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). However, of the most interest is our observation that β-synuclein restores the α-synuclein-like antiapoptotic phenotype when both proteins are co-expressed (Fig. 7A). Thus, caspase activity (Fig. 7B) and immunoreactivity (Fig. 7C) returned the values observed with β-synuclein alone. It should be noted that in both basal and 6OHDA-treated neuronal cells the two proteins do not trigger additive protection (Fig. 7B), suggesting that they were likely using the same molecular pathways and that their expression could be redundant, at least for this effect.Fig. 7β-Synuclein restores α-synuclein anti-apoptotic function. TSM1 neurons were transiently transfected with empty vector (pcDNA3) or vector containing the cDNAs of α-synuclein (alpha-syn), β-synuclein (beta-syn), or α-synuclein and β-synuclein (alpha/beta-syn) as described under "Experimental Procedures." After 48 h of transfection they were treated for 8 h in the absence or the presence of 0.2 mm 6OHDA and recovered for subsequent analysis of synuclein immunoreactivities (A) and caspase-3 activity (B), as described under "Experimental Procedures." Statistical analysis of the means represented by the bars (four independent experiments carried in duplicate) show p < 0.05 by t test analysis (*). Note that the means concerning cells transfected either with pcDNA3 or α-synuclein when treated with 6OHDA are not significantly different (NS). Caspase 3 immunoreactivity and caspase 3 activity (Caspase-3 act.) in the transiently transfected cells of a representative experiment after treatment with 0.2 mm 6OHDA are shown (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The synucleins constitute a family of small proteins, including α-, β-, and γ-synucleins (for review, see Ref. 33Alves da Costa C. Curr. Mol. Med. 2003; 3: 17-24Crossref PubMed Scopus (19) Google Scholar). Recently, α-synuclein draw particular attention because of the demonstration that this presynaptic protein (34Iwai A. Masliah E. Yoshimoto M. Ge N. Flanagan L. Rohan de Silva H.A. Kittel A. Saitoh T. Neuron. 1995; 14: 467-475Abstract Full Text PDF PubMed Scopus (1134) Google Scholar) coexisted with the amyloid β-peptide as the main non-amyloidogenic component of the senile plaques in Alzheimer's disease (6Uéda K. Fukushima H. Masliah E. Xia Y. Iwai A. Yoshimoto M. Otero D.A.C. Kondo J. Ihara Y. Saitoh T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11282-11286Crossref PubMed Scopus (1239) Google Scholar, 35Masliah E. Iwai A. Mallory M. Ueda K. Saitoh T. Am. J. Pathol. 1996; 148: 201-210PubMed Google Scholar, 36Iwai A. Biochim. Biophys. Acta. 2000; 1502: 95-109Crossref PubMed Scopus (70) Google Scholar). More puzzling was the observation that α-synuclein was also the main component of Lewy bodies (5Spillantini M.G. Schmidt M.L. Lee V.-Y. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (6267) Google Scholar) (37Baba M. Nakajo S. Tu P.-H. Tomita T. Nakaya K. Lee V.M.-Y. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar), the intracellular inclusions that accumulate in Parkinson's disease and other dementia-related diseases (38Goedert M. Nat. Rev. Neurosci. 2001; 2: 492-501Crossref PubMed Scopus (1112) Google Scholar). That this protein played a central role in PD was suggested by the observation that a subset of familial PD was indeed due to autosomal dominant mutations borne by α-synuclein (10Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnston W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar, 11Krüger R. Kuhn W. Müller T. Woitalla D. Graeber M. Kösel S. Przuntek H. Epplen J.T. Schöls L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3344) Google Scholar). Indeed, these mutations abolish various physiological functions elicited by the protein (for review, see Ref. 33Alves da Costa C. Curr. Mol. Med. 2003; 3: 17-24Crossref PubMed Scopus (19) Google Scholar), and this was thought to be due to an exacerbation of its aggregation properties (39Iwai A. Yoshimoto M. Masliah E. Saitoh T. Biochemistry. 1995; 34: 10139-10145Crossref PubMed Scopus (194) Google Scholar, 40Han H. Weinreb P.H. Lansbury J.P.T. Chem. Biol. 1995; 2: 163-169Abstract Full Text PDF PubMed Scopus (287) Google Scholar) triggered by the mutation (41Narhi L. Wood S.J. Steavenson S. Jiang Y. Wu G.M. Anafi D. Kaufman S.A. Martin F. Sitney K. Denis P. Louis J.-C. Wypych J. Biere A.L. Citron M. J. Biol. Chem. 1999; 274: 9843-9846Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar). Interestingly, this aggregated profile was also observed in transgenic mice overexpressing α-synuclein, leading to motor activity impairment mimicking some of that observed in PD pathology (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). We recently showed that, under physiological conditions, α-synuclein reduces neuronal responsiveness to various apoptotic stimuli, leading to a decreased caspase 3 activation (17Alves da Costa C. Ancolio K. Checler F. J. Biol. Chem. 2000; 275: 24065-24069Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This phenotype appeared to be p53-dependent (18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Here again, it was of the greatest interest that this anti-apoptotic phenotype was abolished by a pathogenic mutation and 6OH-DOPA (17Alves da Costa C. Ancolio K. Checler F. J. Biol. Chem. 2000; 275: 24065-24069Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 18Alves da Costa C. Paitel E. Vincent B. Checler F. J. Biol. Chem. 2002; 277: 50980-50984Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), two distinct "effectors" triggering PD or used for mimicking this pathology (42Beal M.F. Nat. Rev. Neurosci. 2001; 2: 325-332Crossref PubMed Scopus (513) Google Scholar). Unlike, α-synuclein, nothing was known about the putative function of β-synuclein. It was, however, suggested that this protein could act as a regulator of the α-synuclein aggregation process and thereby control putative sliding toward a PD-like pathogenesis. Thus, β-synuclein shares some common structural properties with α-synuclein but lacks the non-amyloidogenic component domain thought to make α-synuclein prone to aggregation (23Jensen P.H. Sorensen E.S. Petersen T.E. Gliemann J. Rasmussen L.K. Biochem. J. 1995; 310: 91-94Crossref PubMed Scopus (90) Google Scholar). Therefore, β-synuclein is considered to be a non-amyloidogenic member of the α-synuclein family (43El-Agnaf O. Irvine G.B. J. Struct. Biol. 2000; 130: 300-309Crossref PubMed Scopus (87) Google Scholar). β-Synuclein not only can resist aggregation but can also interfere with the aggregating process of α-synuclein. Thus, it was reported that β-synuclein-derived peptides could display anti-aggregating properties (26Windisch M. Hutter-Paier B. Rockenstein E. Hashimoto M. Mallory M. Masliah E. J. Mol. Neurosci. 2002; 19: 63-69Crossref PubMed Scopus (40) Google Scholar). That these in vitro properties could have implications for an anti-Parkinsonian therapeutic strategy was suggested by the ability of β-synuclein to lower the density of Lewy bodies (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar) and prevent the behavioral PD-like phenotypic alterations triggered by the overexpression of α-synuclein in doubly transgenic mice (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar). Although the macroscopic and behavioral examination of the above transgenic mice indicate a cross-talk between α- and β-synuclein, the molecular mechanisms by which β-synuclein could control α-synuclein function remained poorly understood. As we demonstrated that α-synuclein could display a physiological anti-apoptotic phenotype abolished in the pathology, we first questioned whether β-synuclein could be involved in the control of cell death. Second, we examined whether this protein could influence the α-synuclein-mediated anti-apoptotic function and behave as a neuroprotective effector for its parent protein. We clearly establish, for the first time, that β-synuclein drastically reduces the neuronal responsiveness to staurosporine. β-Synuclein lowers the number of TUNEL-positive cells and DNA fragmentation and diminishes both caspase 3 activity and immunoreactivity. It was very interesting to note that the down-regulation of staurosporine-induced caspase activation was accompanied by a reduction of p53 expression and transcriptional activity. We demonstrate that the expression of Mdm2, a protein responsible for p53 ubiquitination and, thereby, degradation by the proteasome (31Yin Y. Luciani M.G. Fahraeus R. Nat. Cell Biol. 2002; 4: 462-467Crossref PubMed Scopus (241) Google Scholar), was enhanced. This indicated that β-synuclein likely controls p53 levels at a post-transcriptional level. In agreement with this hypothesis, phosphorylated p38 immunoreactivity, a clue to p53 activation (32Zhu Y. Mao X.O. Sun Y. Xia Z. Greenberg D.A. J. Biol. Chem. 2002; 277: 22909-22914Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), was lowered in β-synuclein-expressing neurons. That α-and β-synuclein act as physiological anti-apoptotic proteins via the identical lowering of p53-dependent caspase 3 activation is likely not anecdotal and suggests that β-synuclein could eventually counterbalance α-synuclein deficiency, likely explaining at a molecular level the selectivity of dopaminergic cell death taking place in PD (for review, see Ref. 44Barzilai A. Melamed E. Trends Mol. Med. 2003; 9: 126-132Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This could be the case particularly when the α-synuclein phenotype is abolished by 6OHDA, one of the dopaminergic, toxic, derived products (45Curtius H.C. Wolhensberger M. Steinmann B. Redweik S. J. Chromatogr. 1974; 99: 529-540Crossref PubMed Scopus (122) Google Scholar, 46Irwin I. Langston J.W. Ellenberg J.H Koller W.C. Langston J.W. Etiology of Parkinson's Disease. Marcel Dekker, New York1995: 153-201Google Scholar, 47Jellinger K. Linert L. Kienzl E. Herlinger E. Youdim M.B.H. J. Neural. Transm. 1995; 46: 297-314Google Scholar). In this context, we demonstrate that unlike α-synuclein, interestingly, β-synuclein fully retains its anti-apoptotic function in 6OHDA-treated neurons. Therefore, one can envision that β-synuclein could complement α-synuclein deficiency at least in the first stages of PD neuropathology. Furthermore, α-synuclein remains anti-apoptotic in the presence of β-synuclein in 6OHDA-treated neurons. Therefore, one could consider that β-synuclein could also act as a neuroprotective factor via the preservation of α-synuclein antiapoptotic properties. In this context, it is noteworthy that the ratio of β-synuclein over α-synuclein is altered in Lewy bodies (24Rockenstein E. Hansen L.A. Mallory M. Trojanowski J.Q. Galasko D. Masliah E. Brain Res. 2001; 914: 48-56Crossref PubMed Scopus (144) Google Scholar). As α- and β-synucleins have been shown to physically interact (19Hashimoto M. Rockenstein E. Mante M. Mallory M. Masliah E. Neuron. 2001; 32: 213-223Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar), it is tempting to hypothesize that lowered levels of β-synuclein in PD could result first in an alteration of intrinsic β-synuclein-mediated control of cell death and, second, in a defective α-synuclein anti-apoptotic phenotype normally controlled by β-synuclein. The above data have clear implications for PD and strengthen the track of searching for β-synuclein derivatives able to protect α-synuclein from aggregation, thereby preserving its beneficial antiapoptotic properties. This strategy could perhaps be extrapolated to other neurodegenerative diseases. Thus, we previously established that presenilin 2, its mutated counterpart (48Alves da Costa C. Paitel E. Mattson M.P. Amson R. Telerman A. Ancolio K. Checler F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4043-4048Crossref PubMed Scopus (114) Google Scholar), and its C-terminal fragment (49Alves da Costa C. Mattson M.P. Ancolio K. Checler F. J. Biol. Chem. 2003; 278: 12064-12069Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) trigger a pro-apoptotic phenotype that could, to some extent, account for some of the neuropathological stigmata observed in Alzheimer's disease (50Cotman C.W. Anderson A.J. Mol. Neurobiol. 1995; 10: 19-45Crossref PubMed Scopus (360) Google Scholar, 51Cotman C.W. Neurobiol. Aging. 1998; 19: S29-S32Crossref PubMed Scopus (136) Google Scholar). Amazingly, in prion-related diseases we also recently showed that overexpression of PrPc led to a drastic Mdm2-regulated, p53-dependent caspase 3 activation (29Paitel E. Fahraeus R. Checler F. J. Biol. Chem. 2003; 278: 10061-10066Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Therefore, p53 can be seen as a key factor acting as a common denominator involved in various neurodegenerative diseases. Targeting p53 can be seen as a therapeutic strategy. Thus, p53 inhibitors were recently shown to protect dopaminergic neurons and improve motor deficits observed in an experimental animal model of Parkinsonism (52Duan W. Zhu X. Ladenheim B. Yu Q.-S. Guo Z. Oyler J. Cutler R.G. Cadet J.-L. Greig N.H. Mattson M.P. Ann. Neurol. 2002; 52: 597-606Crossref PubMed Scopus (192) Google Scholar). Furthermore, p53 deletion protects mice from methamphetamine-induced toxicity of dopaminergic terminals and cell bodies (53Hirata H. Cadet J.L. J. Neurochem. 1997; 69: 780-790Crossref PubMed Scopus (107) Google Scholar). However, the means of specific cell-permeant inhibitors of p53 such as pifithrin-α (54Komarov P.G. Komarova E.A. Kondratov R.V. Christov-Tselkov K. Coon J.S. Chernov M.V. Gudkov A.V. Science. 1999; 285: 1733-1737Crossref PubMed Scopus (1122) Google Scholar) should be considered cautiously with respect to putative side effects due to widespread and nonspecific accessibility to neuronal cells. Alternatively, a strategy aimed at delivering β-synuclein to a specifically targeted and disease-affected neuronal population could be envisioned as a less dramatic mean to down-regulate p53 in PD but also in other p53-related pathologies such as Alzheimer's and prion diseases. We thank Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD) for providing us with the PG13-luciferase construct. We also thank Dr. R. Fahraeus (Dundee, UK) for providing anti-Mdm2 antibodies.
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