α-Synuclein Up-regulation and Aggregation during MPP+-induced Apoptosis in Neuroblastoma Cells

1-Methyl-4-phenylpyridinium (MPP+) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP+-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of α-synuclein (α-syn) in MPP+-induced oxidative stress and apoptosis. Results show that MPP+ inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H2O2 generation, TfR expression and α-syn expression/aggregation. Pretreatment with cell-permeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, α-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing α-syn exacerbated MPP+ toxicity, whereas antisense α-syn treatment totally abrogated MPP+-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of α-syn in MPP+-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP+-induced iron signaling is responsible for intracellular oxidant generation, α-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed. 1-Methyl-4-phenylpyridinium (MPP+) is a neurotoxin that causes Parkinson's disease in experimental animals and humans. Despite the fact that intracellular iron was shown to be crucial for MPP+-induced apoptotic cell death, the molecular mechanisms for the iron requirement remain unclear. We investigated the role of transferrin receptor (TfR) and iron in modulating the expression of α-synuclein (α-syn) in MPP+-induced oxidative stress and apoptosis. Results show that MPP+ inhibits mitochondrial complex-1 and aconitase activities leading to enhanced H2O2 generation, TfR expression and α-syn expression/aggregation. Pretreatment with cell-permeable iron chelators, TfR antibody (that inhibits TfR-mediated iron uptake), or transfection with glutathione peroxidase (GPx1) enzyme inhibits intracellular oxidant generation, α-syn expression/aggregation, and apoptotic signaling as measured by caspase-3 activation. Cells overexpressing α-syn exacerbated MPP+ toxicity, whereas antisense α-syn treatment totally abrogated MPP+-induced apoptosis in neuroblastoma cells without affecting oxidant generation. The increased cytotoxic effects of α-syn in MPP+-treated cells were attributed to inhibition of mitogen-activated protein kinase and proteasomal function. We conclude that MPP+-induced iron signaling is responsible for intracellular oxidant generation, α-syn expression, proteasomal dysfunction, and apoptosis. Relevance to Parkinson's disease is discussed. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; carboxy-H2DCFDA, carboxy-2′,7′-dichlorodihydrofluorescein diacetate; DCF, dichlorofluorescein; FeTBAP, Fe(III) tetrakis (4-benzoic acid) porphyrin; l-NAME, l-nitroarginine-N-methyl ester; DPBS, Dulbecco's phosphate buffered saline; HBED, N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid; TfR, transferrin receptor; α-syn, α-synuclein; MAP, mitogen-activated protein; FBS, fetal bovine serum; RT, reverse transcriptase; GPx1, glutathione peroxidase; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein. is one of the major human neurodegenerative disorders that is characterized by a progressive loss of dopaminergic neurons and the formation of Lewy body aggregates (1Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Crossref PubMed Scopus (1264) Google Scholar, 2Hornykiewicz O. Neurology. 1998; 51: S2-S9Crossref PubMed Google Scholar). Increasing evidence suggests that α-synuclein (α-syn), parkin, ubiquitin, and iron constitute a major fraction of Lewy body aggregates (3Kaur D. Andersen J.K. Ageing Cell. 2002; 1: 17-21Crossref PubMed Scopus (47) Google Scholar). Defects in mitochondrial complex-1 activity and dopamine deficiency were detected in patients suffering from PD (4Parker Jr., W.D. Boyson S.J. Parks J K. Ann. Neurol. 1989; 26: 719-723Crossref PubMed Scopus (973) Google Scholar, 5Fahan S. Przedborski S. Rowland L.P. Merritt's Neurology. Lippincott, Williams & Wilkins, New York2000: 679-695Google Scholar). Mitochondrial complex-1 inhibitors such as 1-methyl-4-phenylpyridinium (MPP+), a metabolic product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone have been shown to induce symptoms similar to those of PD in experimental animals and humans (6Schmidt N. Ferger B. J. Neural. 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MPP+ is transported into dopaminergic neurons by the dopamine transporter and is concentrated into mitochondria, where it causes ATP depletion by inhibiting complex-1 activity, leading to alterations in the mitochondrial membrane potential and finally to cell death (11Bates T.E. Heales S.J. Davies S.E. Boakye P. Clark J.B. J. Neurochem. 1994; 63: 640-648Crossref PubMed Scopus (103) Google Scholar). Recent studies show that MPTP and/or MPP+ and rotenone induce α-syn expression and its aggregation (12Lee H.J. Shin S.Y. Choi C. Lee Y.H. Lee S.J. J. Biol. Chem. 2002; 277: 5411-5417Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 13Vila M. Vukosavic S. Jackson-Lewis V. Neystat M. Jakowec M. Przedborski S. J. Neurochem. 2000; 74: 721-729Crossref PubMed Scopus (347) Google Scholar). α-syn aggregation is considered to be one of the pathological hallmarks of PD (14Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6469-6473Crossref PubMed Scopus (2462) Google Scholar, 15Baba M. Nakajo S. Tu P.H. Tomita T. Nakaya K. Lee V.M. Trojanowski J.Q. Iwatsubo T. Am. J. Pathol. 1998; 152: 879-884PubMed Google Scholar). However, the role of α-syn in cellular toxicity is not well understood. In some cells, α-syn overexpression was found to be protective whereas in others α-syn overexpression elicited increased toxicity (16Seo J.H. Rah J.C. Choi S.H. Shin J.K. Min K. Kim H.S. Park C.H. Kim S. Kim E.M. Lee S.H. Lee S. Suh S.W. Suh Y.H. FASEB J. 2002; 16: 1826-1828Crossref PubMed Google Scholar, 17Zhou W. Schaack J. Zawada W.M. Freed C.R. Brain Res. 2002; 926: 42-50Crossref PubMed Scopus (125) Google Scholar), but the general consensus is that α-syn overexpression is ultimately toxic to dopaminergic neurons (18Xu J. Kao S.Y. Lee F.J. Song W. Jin L.W. Yankner B.A. Nat. Med. 2002; 8: 600-606Crossref PubMed Scopus (649) Google Scholar). However, the mutant forms of α-syn are more toxic compared with the wild type α-syn (19Tabrizi S.J. Orth M. Wilkinson J.M. Taanman J.W. Warner T.T. Cooper J.M. Schapira A.H. Hum. Mol. Genet. 2000; 9: 2683-2689Crossref PubMed Google Scholar, 20Ostrerova-Golts N. Petrucelli L. Hardy J. Lee J.M. Farer M. Wolozin B. J. Neurosci. 2000; 20: 6048-6054Crossref PubMed Google Scholar). Additional studies elucidating the molecular signaling events that lead to the up-regulation and aggregation of α-syn in cells are, therefore, warranted. Reports indicate that MPP+ promotes the aggregation of α-syn in neuroblastoma cells (21Kakimura J. Kitamura Y. Takata K. Kohno Y. Nomura Y. Taniguchi T. Eur. J. Pharmacol. 2001; 417: 59-67Crossref PubMed Scopus (78) Google Scholar). Iron and H2O2 exacerbated α-syn aggregation in vitro (22Hashimoto M. Hsu L.J. Xia Y. Takeda A. Sisk A. Sundsmo M. Masliah E. Neuroreport. 1999; 10: 717-721Crossref PubMed Scopus (386) Google Scholar). Previously, we showed that MPP+ up-regulates transferrin receptor (TfR) expression and iron uptake, resulting in oxidative stress and apoptosis (23Kalivendi S.V. Kotamraju S. Cunningham S. Shang T. Hillard C.J. Kalyanaraman B. Biochem. J. 2003; 371: 151-164Crossref PubMed Scopus (112) Google Scholar). In the present study, we sought to determine whether MPP+-induced transferrin iron plays a critical role in the up-regulation/aggregation of α-syn leading to enhanced apoptosis in human neuroblastoma cells. Materials—Carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) was purchased from Molecular Probes, Inc. (Eugene, OR). Fe(III) tetrakis (4-benzoic acid) porphyrin (FeTBAP) was synthesized according to the published procedure (24Day B.J. Shawen S. Liochev S.I. Crapo J.D. J. Pharmacol. Exp. Ther. 1995; 275: 1227-1232PubMed Google Scholar). N,N′-Bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED) was obtained as a gift from Dr. Cherakuri Muralikrishna (National Institutes of Health). MPP+ iodide, l-nitroarginine-N-methyl ester (l-NAME) and other chemicals for buffer preparations were purchased from Sigma. LipofectAMINE reagent was obtained from Invitrogen. Adenovirus containing full-length GPx1 (Ad.CMV-GPx1) was a generous gift from Dr. Larry Oberley (University of Iowa). Ad-CMV-GFP was obtained from the adenoviral core facility, Medical College of Wisconsin (Milwaukee, WI). MAP kinase inhibitor U0126 was obtained from Calbiochem. Monoclonal antibody, 42/6, against human TfR was a gift from Dr. Ian Towbridge (Salk Institute, San Diego, CA). Culturing of Human Neuroblastoma Cells—Human neuroblastoma cells (SH-SY5Y) obtained from the American Type Cell Collection were transferred to 75-cm2 filter vent flasks (Costar, Cambridge, MA), grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS), l-glutamine (4 mmol/liter), penicillin (100 units/ml), and streptomycin (100 μg/ml) and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. For cells were in and grown to a of the of medium was with Dulbecco's modified Eagle's medium containing The were to of the in this Cell were in neuroblastoma cells in Cells were for with the or U0126 and inhibitors by the of MPP+ to a of a cell were and cells were with Dulbecco's and by The cell were with and used for For the neuroblastoma cells were with MPP+, and cells were by at In some neuroblastoma cells were with H2O2 for activity was in neuroblastoma cell using the cells were in of buffer in the and a to The was at °C for at and of the was used for the activity according to the The in the at was considered as of caspase-3 cells were with and with buffer containing and in The activity of aconitase was measured in containing at 37 for of at was used J. Biol. Chem. Full Text PDF PubMed Google Scholar). activity of mitochondrial was measured as Oberley Biochem. Biophys. 1999; PubMed Scopus (125) Google Scholar). were and in buffer phosphate of the was to of phosphate buffer containing (100 bovine serum and in a at 37 The in the of at was used as of complex-1 these the activity was by of with GPx1 in neuroblastoma cells was in a medium for at a of of S. S. T. Oberley L. 2001; PubMed Scopus Google Scholar), by the of of medium containing and was for The medium was with a medium containing FBS, and the cells were with transfection with as with GPx1 activity was measured as S. S. T. Oberley L. 2001; PubMed Scopus Google Scholar). of α-syn and TfR the neuroblastoma cells were with and in of buffer a of the the were and The cells were by the a The was at for and the was used for were by the and was used for the were a 10% and were with containing and 5% bovine serum to the protein were incubated with against human α-syn or a TfR antibody μg/ml) in containing and bovine serum for at and incubated with for at α-syn and TfR were detected using the was obtained using the the of the medium was and of reagent was to cells in and was using the of was used for the using a with in the in a of of the was used to for α-syn, TfR, and by a using the by the Inc. of and was from neuroblastoma cells using a reagent according to the α-syn was using using the and reverse containing and in type and the product was with and and into that was with the in to the for overexpressing α-syn The full-length product was into to the that antisense for The were by the of the of and α-syn in Neuroblastoma Neuroblastoma cells in were with of containing the and antisense α-syn using the LipofectAMINE reagent as by the cells were with in of Dulbecco's modified Eagle's medium and for by the of of Dulbecco's modified Eagle's medium 10% FBS, and and cells were incubated for with a cells were to for and used for the these the transfection was found to be using the of of intracellular oxidant was the of to the fluorescent Chem. Res. PubMed Scopus Google Scholar). treatment of cells with MPP+, the were and cells were with and in of cell medium without The was to a of and cells were incubated for The cells were with and in of was at of and The from of were using the and of was measured as K. A. Biol. Med. 2002; PubMed Scopus Google Scholar). cells were with buffer and with buffer containing of were with buffer to a of The was in a of activity was measured by the of the fluorescent and at and MPP+ α-syn and in Neuroblastoma of MPP+ to neuroblastoma cells a in α-syn protein in protein expression was detected in MPP+-treated cells that to the the molecular to the and forms of α-syn were and the as by the in α-syn MPP+ The of α-syn to as as MPP+ treatment that this observed is not to in as detected using the indicate that α-syn expression is at and in to MPP+ with increased α-syn expression and forms of α-syn increased in MPP+-treated cells MPP+ TfR and of aconitase in MPP+-treated neuroblastoma cells was measured as of oxidative was a with in the aconitase activity a treatment in cells with MPP+ the in the expression of TfR was observed TfR increased in MPP+-treated cells and to The obtained these not show in TfR in the activity was detected in neuroblastoma cells with in caspase-3 activity was a of in MPP+-treated cells that MPP+ causes oxidative stress as by the in aconitase activity by TfR expression and finally to apoptosis. oxidative in apoptosis in neuroblastoma cells. of Iron and MPP+-induced α-syn and we that MPP+ intracellular H2O2 and iron in neuroblastoma cells (23Kalivendi S.V. Kotamraju S. Cunningham S. Shang T. Hillard C.J. Kalyanaraman B. Biochem. J. 2003; 371: 151-164Crossref PubMed Scopus (112) Google Scholar). we that H2O2 and iron be responsible for MPP+-induced overexpression and aggregation of In MPP+ treatment in the aconitase activity in cells a treatment with MPP+ Pretreatment of neuroblastoma cells with that is a of or a cell-permeable iron HBED, the aconitase activity in neuroblastoma cells with MPP+ of cells with GPx1 that intracellular H2O2 MPP+-induced of aconitase of cells with l-NAME, a inhibitor of or oxidative of aconitase in MPP+-treated cells that MPP+-induced of aconitase was by intracellular and more the of α-syn aggregation and the of intracellular we the of a TfR antibody to the extracellular of TfR and inhibits of iron into Pretreatment of cells with TfR antibody α-syn expression and aggregation B and as well as the of α-syn that iron transported TfR is responsible for α-syn expression and The MPP+-induced as measured by the of was by FeTBAP, TfR antibody, and GPx1 overexpression these a role for transferrin iron and H2O2 in MPP+-induced α-syn and apoptosis. we sought to determine whether treatment α-syn and apoptosis in neuroblastoma cells. of α-syn that the protein were increased in cells to H2O2 for and The in α-syn the with increased expression of α-syn, the caspase-3 activity was enhanced in cells with H2O2 of α-syn MPP+-induced and determine whether α-syn is responsible for apoptosis or whether α-syn up-regulation by MPP+ is a we and neuroblastoma cells. this neuroblastoma cells were with of containing the and antisense α-syn using the LipofectAMINE reagent as The transfection was using the shown in α-syn protein in the increased in neuroblastoma cells with and the in the antisense cells. We measured caspase-3 in these cells treatment with MPP+ for shown in the caspase-3 by MPP+ was in antisense cells. In the of MPP+ to cells enhanced the caspase-3 activity compared with cells indicate that α-syn is for apoptosis. we measured the oxidative stress in and cells. and that the aconitase and mitochondrial complex-1 activities were by in cells with MPP+ for However, caspase-3 the aconitase and mitochondrial complex-1 activities in to of α-syn in neuroblastoma cells. MPP+ treatment of and cells not aconitase or complex-1 activities and Previously, we that MPP+-induced of to is intracellular H2O2 and iron (23Kalivendi S.V. Kotamraju S. Cunningham S. Shang T. Hillard C.J. Kalyanaraman B. Biochem. J. 2003; 371: 151-164Crossref PubMed Scopus (112) Google Scholar). MPP+-induced was by GPx1 cell-permeable iron chelators, and TfR antibody, that iron and intracellular H2O2 were responsible for increased intracellular was in antisense neuroblastoma cells and MPP+-induced was not by α-syn overexpression or transfection with antisense α-syn these we conclude that MPP+-induced oxidative stress is of α-syn expression and not α-syn overexpression in cells induce apoptotic signaling by mechanisms other intracellular oxidative of MAP and in that α-syn can the activities of MAP and A. M. N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. C. Lee J. A. Wolozin B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). that MPP+-induced α-syn role by inhibiting as been A. M. N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), and that the antisense cells against MPP+-induced the MAP kinase with this we found that treatment of cells with a kinase abrogated the by the antisense α-syn MPP+-induced apoptosis Results showed that activity with the was in MPP+-treated cells and in cells the antisense α-syn treatment the activity in of indicate that α-syn can with kinase and/or proteasomal leading to enhanced apoptosis in neuroblastoma However, it is not whether the are In this study, we showed that MPP+, inhibitor of mitochondrial transferrin oxidative up-regulation of α-syn, protein and apoptosis in neuroblastoma cells. Pretreatment with antibody or cell-permeable iron or GPx1 overexpression transferrin iron uptake, oxidative α-syn expression, and apoptosis in cells with MPP+-induced apoptosis was in neuroblastoma cells α-syn and in cells. The toxic effects of α-syn be to the inhibition of MAP kinase and proteasomal these a critical role for α-syn in MPP+-induced apoptosis MPP+-induced and Iron α-syn aggregation is to the of neurodegenerative M. E. L. Masliah E. Med. 2003; PubMed Scopus Google Scholar). been that MPP+, of the neurotoxin MPTP, promotes α-syn expression in neuroblastoma cells (12Lee H.J. Shin S.Y. Choi C. Lee Y.H. Lee S.J. J. Biol. Chem. 2002; 277: 5411-5417Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 13Vila M. Vukosavic S. Jackson-Lewis V. Neystat M. Jakowec M. Przedborski S. J. Neurochem. 2000; 74: 721-729Crossref PubMed Scopus (347) Google Scholar). with the we found that MPP+ α-syn expression and aggregation in a and In the observed in α-syn expression is not to its as Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), but at the α-syn increased with in to MPP+ treatment The of α-syn in the brain is a pathological of Parkinson's disease Y.H. Pharmacol. 2002; PubMed Scopus Google Scholar). Iron have been to be in the of PD patients D. M. L. P. G. J. Neurochem. 2001; PubMed Scopus Google Scholar). Iron and H2O2 exacerbated α-syn aggregation in vitro (22Hashimoto M. Hsu L.J. Xia Y. Takeda A. Sisk A. Sundsmo M. Masliah E. Neuroreport. 1999; 10: 717-721Crossref PubMed Scopus (386) Google Scholar). We have shown that MPP+ H2O2 in neuroblastoma cells a the inhibition of a mitochondrial complex-1 (23Kalivendi S.V. Kotamraju S. Cunningham S. Shang T. Hillard C.J. Kalyanaraman B. Biochem. J. 2003; 371: 151-164Crossref PubMed Scopus (112) Google Scholar). the from mitochondrial to in generation. The mitochondrial and of mitochondrial complex-1 and aconitase is well J. Biol. Chem. Full Text PDF PubMed Google Scholar). this the is to the by and the formation of or in The role of iron signaling is in neurodegenerative Y. Neurol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, D. G. P. O. Res. 2002; PubMed Scopus Google Scholar). Mitochondrial that and H2O2 formation of cellular iron (23Kalivendi S.V. Kotamraju S. Cunningham S. Shang T. Hillard C.J. Kalyanaraman B. Biochem. J. 2003; 371: 151-164Crossref PubMed Scopus (112) Google Scholar, B. J. S. S. E. Kotamraju S. Mol. Cell. Biochem. 2002; Scopus Google Scholar). of the cellular iron is used to and in R. K. A. W. C. G. Biol. Chem. 1999; PubMed Scopus Google Scholar). of mitochondrial aconitase and is a for iron of mitochondrial iron is responsible for the cellular by the expression of TfR and iron with this was a in the aconitase activity and in TfR expression as as MPP+ treatment in neuroblastoma cells and However, we not in TfR similar treatment be to its J. 2002; PubMed Scopus Google Scholar). is that mechanisms are for TfR protein expression in to as J. 2002; PubMed Scopus Google Scholar). However, these events apoptosis as measured by caspase-3 that intracellular overexpression of GPx1 and TfR antibody (that to the extracellular of TfR and inhibits iron into MPP+-induced α-syn at and and apoptotic signaling that MPP+-induced H2O2 is responsible for α-syn we neuroblastoma cells with H2O2 and found the of α-syn and protein to apoptosis showed that intracellular iron and TfR overexpression oxidative stress and apoptosis Y. Kotamraju S. C.R. S.V. A. J. Kalyanaraman B. Res. 2003; PubMed Scopus Google Scholar). with this iron by overexpression of the protein or of iron to oxidative in dopaminergic cells and D. S. J. J.Q. R. V. R. L. D. L. R.A. Andersen J.K. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). is a protein containing with and oxidative α-syn and formation Lee V.M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). these that MPP+-induced α-syn aggregation in neuroblastoma cells is by from iron and we not MPP+-induced oxidative stress and iron a role in up-regulation of α-syn but it is that oxidative are or the and of α-syn be by similar to that for the protein J.T. J.D. L. J. T. T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). α-syn MPP+-induced but are with to the potential toxicity of α-syn overexpression in cells, α-syn overexpression in dopaminergic neurons to increased (18Xu J. Kao S.Y. Lee F.J. Song W. Jin L.W. Yankner B.A. Nat. Med. 2002; 8: 600-606Crossref PubMed Scopus (649) Google Scholar). α-syn were found to be to MPTP toxicity W. N. M. R. R. K. Y. M. Jackson-Lewis V. S. D. Przedborski S. R. R. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar). shown in this study, α-syn overexpression to apoptosis in neuroblastoma we not a in mitochondrial complex-1 and aconitase activities in cells and The of MPP+ to cells enhanced apoptosis as compared with MPP+ treatment MPP+ α-syn expression to the of we the of antisense α-syn treatment MPP+-induced oxidative stress and apoptosis. the antisense α-syn treatment abrogated MPP+-induced oxidant in these cells, as measured by the of aconitase and complex-1 activities and enhanced intracellular α-syn its effects in neuroblastoma cells to oxidative stress and not of MAP and in show that and forms of α-syn the proteasomal at by with the and K. C. Lee J. A. Wolozin B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google and that the proteasomal inhibition exacerbated MPP+-induced toxicity G. A. M. B. J. Neurochem. 2003; PubMed Scopus Google Scholar). present indicate that the activity of the was by MPP+ treatment and α-syn In cells, these effects were abrogated that the proteasomal a critical role in apoptosis. that α-syn cell death by inhibiting MAP kinase activity A. M. N. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). that α-syn overexpression as well as MPP+ treatment induce apoptosis by inhibiting kinase cells with a inhibitor of abrogated the protective effects by antisense α-syn transfection inhibitors were shown to signaling Y. J. Biochem. 2002; PubMed Scopus Google Scholar), it is not whether these as in this study, are or In as in the present that MPP+ α-syn expression at and intracellular H2O2 formation and intracellular that H2O2 or intracellular iron MPP+-induced α-syn expression and apoptosis. α-syn promotes apoptosis to oxidative proteasomal and/or MAP kinase