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• W2013085111 abstract "Recent works suggest that α-synuclein could play a central role in Parkinson's disease (PD). Thus, two mutations were reported to be associated with rare autosomal dominant forms of the disease. We examined whether α-synuclein could modulate the caspase-mediated response and vulnerability of murine neurons in response to various apoptotic stimuli. We established TSM1 neuronal cell lines overexpressing wild-type (wt) α-synuclein or the PD-related Ala-53 → Thr mutant α-synuclein. Under basal conditions, acetyl-Asp-Glu-Val-Asp-aldehyde-sensitive caspase activity appears significantly lower in wt α-synuclein-expressing cells than in neurons expressing the mutant. Interestingly, wt α-synuclein drastically reduces the caspase activation of TSM1 neurons upon three distinct apoptotic stimuli including staurosporine, etoposide, and ceramide C2 when compared with mock-transfected cells. This inhibitory control of the caspase response triggered by apoptotic agents was abolished by the PD-related pathogenic mutation. Comparison of wild-type and mutated α-synuclein-expressing cells also indicates that the former exhibits much less vulnerability in response to staurosporine and etoposide as measured by the sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid assay. Altogether, our study indicates that wild-type α-synuclein exerts an antiapoptotic effect in neurons that appears to be abolished by the Parkinson's disease-related mutation, thereby suggesting a possible mechanism underlying both sporadic and familial forms of this neurodegenerative disease. Recent works suggest that α-synuclein could play a central role in Parkinson's disease (PD). Thus, two mutations were reported to be associated with rare autosomal dominant forms of the disease. We examined whether α-synuclein could modulate the caspase-mediated response and vulnerability of murine neurons in response to various apoptotic stimuli. We established TSM1 neuronal cell lines overexpressing wild-type (wt) α-synuclein or the PD-related Ala-53 → Thr mutant α-synuclein. Under basal conditions, acetyl-Asp-Glu-Val-Asp-aldehyde-sensitive caspase activity appears significantly lower in wt α-synuclein-expressing cells than in neurons expressing the mutant. Interestingly, wt α-synuclein drastically reduces the caspase activation of TSM1 neurons upon three distinct apoptotic stimuli including staurosporine, etoposide, and ceramide C2 when compared with mock-transfected cells. This inhibitory control of the caspase response triggered by apoptotic agents was abolished by the PD-related pathogenic mutation. Comparison of wild-type and mutated α-synuclein-expressing cells also indicates that the former exhibits much less vulnerability in response to staurosporine and etoposide as measured by the sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid assay. Altogether, our study indicates that wild-type α-synuclein exerts an antiapoptotic effect in neurons that appears to be abolished by the Parkinson's disease-related mutation, thereby suggesting a possible mechanism underlying both sporadic and familial forms of this neurodegenerative disease. N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine acetyl-Asp-Glu-Val-Asp-aldehyde 7-amino-4-methylcoumarin sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid β-amyloid precursor protein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Parkinson's disease neuropathology is mainly characterized by proteinaceous deposits called Lewy bodies (1Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1256) Google Scholar). The main component of these brain lesions is α-synuclein (2Spillantini 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, 3Arima K. Uéda K. Sunohara N. Hirai S. Izumiyama Y. Tonozuka-Uehara H. Kawai M. Brain Res. 1998; 808: 93-100Crossref PubMed Scopus (218) Google Scholar), a 140-amino acid peptide first identified as the precursor of the “non-amyloidogenic component” (4Uéda 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) of the senile plaques invading the cortical and subcortical areas of both sporadic and familial Alzheimer's disease-affected brains. Of the most interest was the recent demonstration that rare cases of Parkinson's disease were of genetic origin and that two mutations identified on α-synuclein were likely responsible for these autosomal forms of the disease (5Polymeropoulos 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 lorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar, 6Krüger 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). It has been suggested that part of the disease etiology is derived from the accelerated aggregation process triggered by the two mutations (7Narhi 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). El Agnaf et al. (8El-Agnaf O.M.A. Jakes R. Curran M.D. Middleton D. Ingenito R. Bianchi E. Pessi A. Neill D. Wallace A. FEBS Lett. 1998; 440: 71-75Crossref PubMed Scopus (333) Google Scholar) showed that β-sheets-related aggregates of wild-type and mutant α-synucleins could trigger apoptotic cell death in human neuroblastoma cells. More recently, Kholodilov et al. (9Kholodinov N.G. Oo T.F. Burke R.E. Neurosci. Lett. 1999; 275: 105-108Crossref PubMed Scopus (37) Google Scholar) demonstrated that α-synuclein expression was decreased in the rat substantia nigra after induction of apoptosis by intrastriatal injection of 6-hydroxydopamine. Although these two studies established a possible link between α-synucleins and apoptosis, nothing is really known concerning the genuine function of α-synuclein. We have taken advantage of the design of a clonal cell line from neocortical origin (TSM1 cells (10Chun J. Jaenisch R. Mol. Cell. Neurosci. 1996; 7: 304-321Crossref PubMed Scopus (61) Google Scholar)) to examine the possible influence of α-synuclein in the control of the neuronal apoptotic response and to establish a putative modulation of such a function by the Parkinson's disease pathogenic mutation. We set up TSM1 neurons stably overexpressing wild-type α-synuclein or its Parkinson's disease-associated Ala-53 → Thr mutant to examine their caspase response to various apoptotic stimuli. We show here that wild-type α-synuclein displays antiapoptotic properties that are abolished by the Parkinson's disease-related mutation. A cDNA library was prepared from total RNA derived from human adult cortex. A polymerase chain reaction product was obtained by means of the following primers, 5′-CGCAAGCTTAGGAATTCATTAGCCATGGATGTATTCAT-3′ containing the HindIII restriction site and 5′-TTTCTCGAGTATTTCTTAGGCTTCAGGTTCGTAGTC-3′ containing the XhoI site. The polymerase chain reaction fragment was cut with HindIII and XhoI and then subcloned in pcDNA3, and the identity of α-synuclein was confirmed by entire sequencing analysis (11Ancolio K. Alves da Costa C. Uéda K. Checler F. Neurosci. Lett. 2000; 285: 79-82Crossref PubMed Scopus (104) Google Scholar). The Ala-53 → Thr mutation was introduced according to the uracylated single strand strategy as described previously (12Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar) and confirmed by sequencing. TSM1 neuronal cells were cultured as described (13Ancolio K. Dumanchin C. Barelli H. Warter J.M. Brice A. Campion D. Frébourg T. Checler F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4119-4124Crossref PubMed Scopus (169) Google Scholar). TSM1 cells were stably transfected with superfect agent (Qiagen) containing 2 μg of pcDNA3 vector either empty or encoding wild-type or Ala-53 → Thr α-synucleins. Transfectants were screened by Tris-Tricine1 gel analysis and Western blotting (see below). Positive clones overexpress a 18–19-kDa immunoreactive protein in agreement with a previous study (4Uéda 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). TSM1 cells were scraped and lysed in RIPA 1× buffer (10 mm Tris, pH 7.5, containing 150 mm NaCl, 5 mm EDTA, 0.1% SDS, 0.5% deoxycholate, and 1% Nonidet P-40), and then proteins were analyzed by a 16.5% Tris-Tricine gel electrophoresis and Western blotted as described (13Ancolio K. Dumanchin C. Barelli H. Warter J.M. Brice A. Campion D. Frébourg T. Checler F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4119-4124Crossref PubMed Scopus (169) Google Scholar). Nitrocellulose sheets were heated in boiling phosphate buffer and then capped with 5% skim milk in phosphate-buffered saline. Membranes were then rinsed and incubated with a 1/5000 dilution of anti-human α-synuclein (SA3400 from Affiniti). Membranes were then incubated with protein A coupled to peroxidase (2 μg/ml), and then immunological complexes were revealed by ECL (Amersham Pharmacia Biotech) as described previously (13Ancolio K. Dumanchin C. Barelli H. Warter J.M. Brice A. Campion D. Frébourg T. Checler F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4119-4124Crossref PubMed Scopus (169) Google Scholar). TSM1 cells were cultured in 12-well plates for various times at 37 °C in the absence or in the presence of various concentrations of etoposide, ceramide C2, or staurosporine. In some cases, cells were incubated with 100 μm Ac-DEVD-al for 24 h. Cells were then rinsed, gently scraped, pelleted by centrifugation, and then resuspended in 40 μl of lysis buffer (25 mm Hepes, pH 7.5, containing 5 mm MgCl2, and dithiothreitol, 2 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/ml pepstatin A, and leupeptin). Cell lysates were submitted to two freezing/thawing cycles and then centrifuged (16,000 ×g for 5 min). Caspase activity of supernatants (10 μl, about 50 μg of proteins) was measured in 96-well plates according to manufacturer's recommendations (Promega). Briefly, reaction mixtures containing 48 μl of water, 32 μl of assay buffer (312 mm Hepes, pH 7.5, 31.25% sucrose, 0.31% Chaps), 10 μl of 100 mm dithiothreitol, and 2 μl of Me2SO were incubated for various times with 2 μl of 2.5 mm Ac-DEVD-AMC (caspase substrate). In some assays, proteins were preincubated for 30 min at 37 °C in the absence or in the presence of 2 μl of Ac-DEVD-al (2.5 mm). Fluorimetry was recorded at 360 and 460 nm for excitation and emission wavelengths, respectively. Caspase-specific activity was calculated from the linear part of fluorimetry recording and expressed in arbitrary units/h/mg or proteins (established by the Bio-Rad procedure). One arbitrary unit corresponds to 4 nmol of AMC released. TSM1 neurons were grown in a 6.5% CO2 atmosphere in 96-well microtiter plates in a 100-μl culture medium (see above) and treated with 100 μmetoposide or 1 μm staurosporine for 24 h at 37 °C. XTT-metabolizing activity was determined mainly according to the manufacturer's recommendations (Roche Molecular Biochemicals). Briefly, after cell treatment, 50 μl of XTT labeling mixture was added to each well and further incubated at 37 °C. Absorbances were recorded after successive 10-min intervals (for a total time of 60 min) and measured at 452 nm on a microtiter plate reader (lab system). Statistically analyses were performed with PRISM software (Graphpad Software, San Diego, CA) using the Newman Keuls multiple comparison test for one-way analysis of variance. Ac-DEVD-AMC and Ac-DEVD-al were purchased from Neosystem. Anti-human α-synuclein (SA3400) was from Affiniti. XTT kit was from Roche Molecular Biochemicals. Etoposide, staurosporine, protease inhibitors, and protein A-peroxidase were from Sigma. Ceramide C2 was from Biomol. ECL was from Amersham Pharmacia Biotech. Mock-transfected TSM1 neurons were examined for the modulation of their caspase activity in response to various apoptotic stimuli. In basal conditions, TSM1 neurons display an Ac-DEVD-7AMC hydrolyzing activity that is virtually fully abolished by prior treatment with the caspase inhibitor, Ac-DEVD-al (Fig. 1,N.St). Ac-DEVD-al-sensitive activity was drastically enhanced after treatment of mock-transfected TSM1 neurons with staurosporine, etoposide, and ceramide-C2 (Fig. 1), three classical pro-apoptotic effectors. We set up stably transfected TSM1 neurons overexpressing wild-type and Ala-53 → Thr α-synucleins. As expected from the use of antibody specificities against the human species, mock-transfected TSM1 neurons do not display any α-synuclein-like immunoreactivity (Fig.2). We obtained several positive clones expressing various levels of an 18–19-kDa immunoreactive protein in agreement with the expected molecular weight of α-synuclein (4Uéda 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). We have selected two clones expressing virtually identical amounts of wild-type (clone T1) and mutated (clone K1) α-synucleins (Fig. 2) for further analysis. T1 clone exhibits a drastically lower basal Ac-DEVD-al-sensitive caspase activity than mock-transfected cells, suggesting that wild-type α-synuclein exerts an inhibitory control on basal caspase activity (Fig. 3). Interestingly, another clone (T6) displaying lower wild-type α-synuclein-like immunoreactivity than T1 clone also displays a reduced basal caspase activity, although to a lower extent (not shown). Very strikingly, this inhibitory tonus was not observed with the K1 clone, the basal caspase activity of which resembles that measured in mock-transfected cells (Fig. 3). Fig. 4 indicates that the treatment of T1 and K1 clones with the Ac-DEVD-al did not not modify the α-synuclein-like immunoreactivities, indicating that the distinct basal apoptotic caspase-mediated response of K1 and T1 clones could not be accounted for a distinct susceptibility of wild-type and mutated α-synucleins to caspase proteolysis. This agrees well with previous studies showing that α-synucleins are long-lived proteins in various cell types including PC12 and HEK293 cells (14Okochi M. Walter J. Koyama A. Nakajo S. Baba M. Iwatsubo T. Meijer L. Kahle P.J. Haass C. J. Biol. Chem. 2000; 275: 390-397Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar) and that both wild-type and Ala-53 → Thr α-synucleins resist proteolysis by the proteasome in TSM1 neurons (11Ancolio K. Alves da Costa C. Uéda K. Checler F. Neurosci. Lett. 2000; 285: 79-82Crossref PubMed Scopus (104) Google Scholar). We further examined the caspase activation of TSM1 transfectants upon stimulation by various apoptotic stimuli. TableI indicates that T1 clone responsiveness to staurosporine, etoposide, and ceramide C2 was 22–35% of those observed with mock-transfected TSM1 neurons. Here again, the K1 clone displays a caspase response close to that observed with the mock-transfected cells. Of most importance was the fact that the Ac-DEVD-7AMC hydrolyzing activities were virtually abolished by Ac-DEVD-al whatever the stimulus examined (Table I). It should be noted here that apoptotic stimuli do not modify the immunoreactivity of wild-type and α-synucleins in TSM1-transfected cells (not shown), excluding the possibility that the distinct responses could be due to a modulation of α-synuclein expression by apoptotic agents.Table IEffect of various apoptotic stimuli on the Ac-DEVD-al-sensitive caspase activity of TSM1 transfectantsEffectorMockK1T1DEVDΔDEVD%DEVD%−+−+−+Basal1669 ± 65336 ± 9.613331868 ± 85396 ± 33110680 ± 93260 ± 4631STS10069 ± 223508 ± 54956110399 ± 664395 ± 581042751 ± 300409 ± 6125ETO11965 ± 1010476 ± 491148911400 ± 1045618 ± 52994206 ± 227333 ± 5035CER9925 ± 1546269 ± 1896566359 ± 551329 ± 20622439 ± 236326 ± 1522Mock-transfected TSM1 neurons (Mock) and K1 or T1 clones were cultured without (−) or with (+) Ac-DEVD-al in the absence (basal) or in the presence of stauroporine (STS, 1 μm, 2 h), etoposide (ETO, 50 μm, 24 h) or ceramide C2 (CER, 100 μm, 24 h). After incubations, caspase activity was assayed as detailed under “Experimental Procedures.” Δ corresponds to the Ac-DEVD-al-sensitive Ac-DEVD-AMC-hydrolyzing activity. % are the Δ obtained with T1 and K1 cells expressed as the percent of Δ obtained with mock-transfected TSM1 neurons. Values are the mean ± S.E. of duplicate determinations of 8–12 independent experiments. Open table in a new tab Mock-transfected TSM1 neurons (Mock) and K1 or T1 clones were cultured without (−) or with (+) Ac-DEVD-al in the absence (basal) or in the presence of stauroporine (STS, 1 μm, 2 h), etoposide (ETO, 50 μm, 24 h) or ceramide C2 (CER, 100 μm, 24 h). After incubations, caspase activity was assayed as detailed under “Experimental Procedures.” Δ corresponds to the Ac-DEVD-al-sensitive Ac-DEVD-AMC-hydrolyzing activity. % are the Δ obtained with T1 and K1 cells expressed as the percent of Δ obtained with mock-transfected TSM1 neurons. Values are the mean ± S.E. of duplicate determinations of 8–12 independent experiments. All apoptotic effectors activate the Ac-DEVD-al-sensitive caspase activity in a dose-dependent manner (Fig.5). At all concentrations examined, the T1 clone displays a caspase response drastically lower than those exhibited by mock-transfected cells or K1 clone (Fig. 5). Time course analysis of caspase activation upon apoptotic effectors further confirms the much lower Ac-DEVD-al-sensitive activity detectable at any time of the kinetics in cells expressing wild-type α-synucleins when compared with other transfectants (Fig.6).Figure 6Time course analysis of various apoptotic stimuli on the caspase activity of TSM1 transfectants.Mock-transfected TSM1 neurons (triangles), K1 (squares), or T1 (circles) clones were cultured without or with Ac-DEVD-al in the absence or in the presence of staurosporine (STS, 1 μm), etoposide (ETO, 50 μm), or ceramide C2(CER, 100 μm) for the indicated times. After incubations, caspase activity was assayed as detailed under “Experimental Procedures.” Each point corresponds to the Ac-DEVD-al-sensitive Ac-DEVD-AMC hydrolyzing activity and is the mean of duplicate determinations of 3 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The measurement of Ac-DEVD-al-sensitive caspase activity is a specific cell response that can be likely ascribed to programmed cell death. In order to examine a more global response, we studied the etoposide and staurosporine-induced vulnerability of mock-transfected TSM1 neurons and compared it with those of T1 and K1 clones. Both effectors trigger an ∼50% decrease in cell viability of mock-transfected neurons (Fig.7). Wild-type α-synuclein clearly enhances neurons viability in response to staurosporine (71.2%,n = 14, p < 0.001 compared with mock) and etoposide (75.8%, n = 8, p < 0.001, see Fig. 7, A and B). Interestingly, K1 clone viability is highly affected by both agents and appears even more susceptible than mock-transfected cells (32% staurosporine,n = 14, p < 0.01 and 23% etoposide,n = 8, p < 0.001, see Fig. 7,A and B). A dense network of histological and biochemical evidence indicates that programmed cell death could contribute to Parkinson's disease neuropathology. Thus, Mochizuki et al. (15Mochizuki H. Goto K. Mori H. Mizuno Y. J. Neurol. Sci. 1996; 137: 120-123Abstract Full Text PDF PubMed Scopus (432) Google Scholar) reported on the presence of nick end-labeled apoptotic stigmata in the midbrains of late and early onset affected patients. This was confirmed by a morphological study showing typical degenerating neurons in the nigro-striatal area (16Ziv I. Barzilai A. Offen D. Nardi N. Melamed E. J. Neural Transm. Suppl. 1997; 49: 69-76PubMed Google Scholar, 17Tompkins M.M. Basgall E. Zamrini E. Hill W. Am. J. Pathol. 1997; 150: 119-131PubMed Google Scholar). Several animal models used to study Parkinson's disease pathology led to the in situ detection of apoptotic nuclei (18Tatton N. Kish S. Neuroscience. 1997; 77: 1037-1048Crossref PubMed Scopus (409) Google Scholar) as it can also be evidenced in several cell models including human neuroblastoma (19Fall C. Bennett J. J. Neurosci. Res. 1999; 55: 620-628Crossref PubMed Scopus (115) Google Scholar), PC12 (20Offen D. Ziv I. Sternion H. Melamed E. Hochman A. Exp. Neurol. 1996; 141: 32-39Crossref PubMed Scopus (225) Google Scholar, 21Ruberg M. France-Lanord V. Brugg B. Lambeng N. Michel P. Anglade P. Hunot S. Damier P. Faucheux B. Hirsch E. Agid Y. Rev. Neurol. (Paris). 1997; 153: 499-508PubMed Google Scholar), or primary cultures of mesencephalic neurons (21Ruberg M. France-Lanord V. Brugg B. Lambeng N. Michel P. Anglade P. Hunot S. Damier P. Faucheux B. Hirsch E. Agid Y. Rev. Neurol. (Paris). 1997; 153: 499-508PubMed Google Scholar). Several biochemical clues of a link between Parkinson's disease and actors of the apoptotic pathways have also been reported. Bcl2 expression is modulated in Parkinson's disease-affected brains (22Mogi M. Harada M. Kondo T. Mizuno Y. Narabayashi H. Riederer P. Nagatsu T. Neurosci. Lett. 1996; 215: 137-139Crossref PubMed Scopus (103) Google Scholar,23Marshall K. Daniel S. Cairns N. Jenner P. Halliwell B. Biochem. Biophys. Res. Commun. 1997; 240: 84-87Crossref PubMed Scopus (76) Google Scholar). Prostate apoptosis response-4 levels increase in neurons of the dopaminergic pathway after exposure of mice or monkeys to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (24Duan W. Zhang Z. Gash D. Mattson M.P. Ann. Neurol. 1999; 46: 587-597Crossref PubMed Scopus (99) Google Scholar), a neurotoxin classically used to elicit dopaminergic neurons cell death. Furthermore, oxidative stress seems to contribute to the Parkinson's disease pathogenesis (25Jenner P. Olanow C. Neurology. 1996; 47: 161-170Crossref PubMed Google Scholar), as corroborated by subsequent works demonstrating that a mitochondrial impairment (26Merad-Boudia M. Nicole A. Santiard-Baron D. Saille C. Ceballos-Picot I. Biochem. Pharmacol. 1998; 56: 645-655Crossref PubMed Scopus (204) Google Scholar) and ceramide-dependent apoptosis (27France-Lanord V. Brugg B. Michel P. Agid Y. Ruberg M. J. Neurochem. 1997; 69: 1612-1621Crossref PubMed Scopus (157) Google Scholar) occurred in the neuronal cell line NS20Y as well as in PC12 cells. We previously used TSM1 neurons to establish a protein-kinase A-regulated α-secretase cleavage of β-amyloid precursor protein (β-APP) (28Marambaud P. Chevallier N. Ancolio K. Checler F. Mol. Med. 1998; 4: 715-723Crossref PubMed Google Scholar, 29Marambaud P. Ancolio K. Alves da Costa C. Checler F. Br. J. Pharmacol. 1999; 126: 1186-1190Crossref PubMed Scopus (23) Google Scholar). This cell model also allowed us to confirm the unusual phenotypic alteration triggered by a novel mutation of β-APP associated with familial form of Alzheimer's disease in agreement with that seen in HEK293 cells (13Ancolio K. Dumanchin C. Barelli H. Warter J.M. Brice A. Campion D. Frébourg T. Checler F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4119-4124Crossref PubMed Scopus (169) Google Scholar). Altogether, these data indicate that TSM1 neurons represent a most suitable cell system from central origin to examine the putative function of protein candidates involved in neurodegenerative disease. This cell model was used to demonstrate that α-synuclein could negatively control the Ac-DEVD-al-sensitive caspase activation of TSM1 neurons in response to various stimuli, the pharmacological spectrum of which strongly suggests an apoptotic rather than necrotic mechanism. Of most interest is the observation that this negative control of caspase activity is fully abolished by the Ala-53 → Thr mutation of α-synuclein responsible for autosomic dominant forms of the disease (5Polymeropoulos 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 lorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6734) Google Scholar). The cellular mechanism underlying the antiapoptotic function of α-synuclein still remains to be elucidated. However, one could postulate on the involvement of the chaperoning property of α-synuclein. Thus, α-synuclein has been shown to bind tau proteins (30Jensen H., P. Hager H. Nielsen M.S. Hojrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25485Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) and to display the ability to interact with brain vesicles, a property that is abolished by Parkinson's disease mutations (30Jensen H., P. Hager H. Nielsen M.S. Hojrup P. Gliemann J. Jakes R. J. Biol. Chem. 1999; 274: 25481-25485Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Most interesting was the recent report (31Ostrerova N. Petrucelli L. Farrer M. Mehta N. Choi P. Hardy J. Wolozin B. J. Neurosci. 1999; 19: 5782-5791Crossref PubMed Google Scholar) indicating that α-synuclein exhibits a 40% homology with members of the 14-3-3 chaperone protein family. 14-3-3 proteins interact with BAD, a pro-apoptotic oncogene that remains inactive when sequestered in the cytosol (32Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2257) Google Scholar). To explain the antiapoptotic function of α-synuclein by its chaperone activity, one could therefore envision that, under physiological conditions, wild-type α-synuclein interacts with cellular intermediates of the apoptotic pathways. In the pathology, α-synuclein accumulates and aggregates as has been documented by the high concentration of the protein in Lewy bodies invading Parkinson's disease brains. Under these pathological conditions, the wild-type α-synuclein-mediated inhibitory tonus on caspase activity could be abolished, thereby contributing to increased cell death. This hypothesis is in agreement with the observation that aggregated α-synuclein triggers cell death in human neuroblastoma cells (8El-Agnaf O.M.A. Jakes R. Curran M.D. Middleton D. Ingenito R. Bianchi E. Pessi A. Neill D. Wallace A. FEBS Lett. 1998; 440: 71-75Crossref PubMed Scopus (333) Google Scholar). In this context, mutated α-synucleins could accelerate the pathogenesis because of the absence of neuroprotection to apoptotic stimuli (our study), its higher susceptibility to aggregation (7Narhi 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), and its ability to trigger apoptotic cell death when aggregated (8El-Agnaf O.M.A. Jakes R. Curran M.D. Middleton D. Ingenito R. Bianchi E. Pessi A. Neill D. Wallace A. FEBS Lett. 1998; 440: 71-75Crossref PubMed Scopus (333) Google Scholar). It should be noted in support of this hypothesis that at a cellular level, α-synuclein is almost exclusively found to be associated with normal neurons but not with those exhibiting an apoptotic phenotype (9Kholodinov N.G. Oo T.F. Burke R.E. Neurosci. Lett. 1999; 275: 105-108Crossref PubMed Scopus (37) Google Scholar). Some authors (33Kholodilov N.G. Neystat M. Oo T.F. Lo S.E. Larsen K.E. Sulzer D. Burke R.E. J. Neurochem. 1999; 73: 2586-2599Crossref PubMed Scopus (94) Google Scholar) demonstrated that in the target injury model, α-synuclein expression was up-regulated, suggesting that this could correspond to a compensatory response of neurons designed to promote their survival, in agreement with a physiological antiapoptotic function. It is interesting to emphasize the parallels between Parkinson's disease and Alzheimer's disease pathology. Thus, familial Alzheimer's disease cases are mostly due to mutations located on two proteins, namely the β-amyloid precursor protein and presenilin 1 (for reviews see Refs. 34Van Broeckhoven C. Nat. Genet. 1995; 11: 230-232Crossref PubMed Scopus (207) Google Scholar and 35Hutton M. Hardy J. Hum. Mol. Genet. 1997; 6: 1639-1646Crossref PubMed Scopus (166) Google Scholar). It has been demonstrated that wild-type presenilin 1 displays antiapoptotic function that is abolished by presenilin 1 bearing familial Alzheimer's disease mutations (for reviews see Refs. 36Haass C. Neurons. 1997; 18: 687-690Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar and 37Checler F. IUBMB Life. 1999; 48: 33-39Crossref PubMed Google Scholar). Identical observations indicate that β-APP confer resistance to p53-induced cell death, but the familial Alzheimer's disease-associated V717I β-APP did not (38Xu X. Yang D. Wyss-Coray T. Yan J. Gan L. Mucke L. Proc. Natl. Acad. Sci. U. S. A. 1999; 22: 7547-7552Crossref Scopus (79) Google Scholar). Our work opens a possible track to slow down or stop the progression of the neurodegeneration taking place in Parkinson's disease. Thus, one can envision the design of peptides or chemically designed agents displaying α-synuclein anti-aggregating properties. According to our hypothesis, such effectors may prevent α-synuclein deposits and should maintain it as a physiological antiapoptotic modulator. We thank Drs. Patrick Auberger for critical reading of the manuscript. We sincerely thank Allelix Biopharmaceutical Inc. (Missisauga, Canada) for TSM1 cell line." @default.
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• W2013085111 title "Wild-type but Not Parkinson's Disease-related Ala-53 → Thr Mutant α-Synuclein Protects Neuronal Cells from Apoptotic Stimuli" @default.
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