The Herbicide Paraquat Induces Dopaminergic Nigral Apoptosis through Sustained Activation of the JNK Pathway

Abstract

Environmental exposure to the oxidant-producing herbicide paraquat has been implicated as a risk factor in Parkinson's disease. Although intraperitoneal paraquat injections in mice cause a selective loss of dopaminergic neurons in the substantia nigra pars compacta, the exact mechanism involved is still poorly understood. Our data show that paraquat induces the sequential phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun and the activation of caspase-3 and sequential neuronal death both in vitro and in vivo. These effects are diminished by the specific JNK inhibitor SP600125 and the antioxidant manganese(III) tetrakis (4-benzoic acid) porphyrin in vitro. Furthermore, JNK pathway inhibitor CEP-11004 effectively blocks paraquat-induced dopaminergic neuronal death in vivo. These results suggest that the JNK signaling cascade is a direct activator of the paraquat-mediated nigral dopaminergic neuronal apoptotic machinery and provides a molecular linkage between oxidative stress and neuronal apoptosis. Environmental exposure to the oxidant-producing herbicide paraquat has been implicated as a risk factor in Parkinson's disease. Although intraperitoneal paraquat injections in mice cause a selective loss of dopaminergic neurons in the substantia nigra pars compacta, the exact mechanism involved is still poorly understood. Our data show that paraquat induces the sequential phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun and the activation of caspase-3 and sequential neuronal death both in vitro and in vivo. These effects are diminished by the specific JNK inhibitor SP600125 and the antioxidant manganese(III) tetrakis (4-benzoic acid) porphyrin in vitro. Furthermore, JNK pathway inhibitor CEP-11004 effectively blocks paraquat-induced dopaminergic neuronal death in vivo. These results suggest that the JNK signaling cascade is a direct activator of the paraquat-mediated nigral dopaminergic neuronal apoptotic machinery and provides a molecular linkage between oxidative stress and neuronal apoptosis. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; MKK, MAP kinase kinases; MKKK, MAP kinase kinase kinases; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MnTBAP, manganese(III) tetrakis (4-benzoic acid) porphyrin; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Z, benzyloxycarbonyl; MPP+, 1-methyl-4-phenylpyridium ion; fmk, fluoromethyl ketone; AFC, 7-amino-4-trifluoromethyl coumarin; CMK, chloromethyketone; PVDF, polyvinylidene difluoride. is a progressive neurodegenerative disorder that is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the midbrain. It affects over one million people in North America (1Forno L.S. J. Neuropathol. Exp. Neurol. 1996; 55: 259-272Google Scholar, 2Lang A.E. Lozano A.M. N. Engl. J. Med. 1998; 339: 1044-1053Google Scholar). Epidemiological evidence has suggested that environmental agents in combination with genetic susceptibility may be responsible for the associated neurodegeneration in PD (3Tanner C.M. Ottman R. Goldman S.M. Ellenberg J. Chan P. Mayeux R. Langston J.W. J. Am. Med. Assoc. 1999; 281: 341-346Google Scholar). Exposure to agricultural chemicals via living in a rural environment, drinking well water, or occupational exposure has been postulated to be a potential environmental risk factor for the disease (4Hertzman C. Wiens M. Bowering D. Snow B. Calne D. Am. J. Ind. Med. 1990; 17: 349-355Google Scholar, 5Jimenez-Jimenez F.J. Mateo D. Gimenez-Roldan S. Movement Disorders. 1992; 7: 149-152Google Scholar, 6Semchuk K.M. Love E.J. Lee R.G. Neurology. 1992; 42: 1328-1335Google Scholar, 7Hubble J.P. Cao T. Hassanein R.E. Neuberger J.S. Koller W.C. Neurology. 1993; 43: 1693-1697Google Scholar, 8Liou H.H. Tsai M.C. Chen C.J. Jeng J.S. Chang Y.C. Chen S.Y. Chen R.C. Neurology. 1997; 48: 1583-1588Google Scholar, 9Gorell J.M. Johnson C.C. Rybicki B.A. Peterson E.L. Richardson R.J. Neurology. 1998; 50: 1346-1350Google Scholar, 10Stephenson J. J. Am. Med. Assoc. 2000; 283: 3055-3056Google Scholar). The widely used herbicide 1,1′-dimethyl-4,4′-bipyridium (paraquat) has been suggested as a prime risk factor for PD based both on reports of parkinsonism correlated with exposure to the agent (4Hertzman C. Wiens M. Bowering D. Snow B. Calne D. Am. J. Ind. Med. 1990; 17: 349-355Google Scholar, 8Liou H.H. Tsai M.C. Chen C.J. Jeng J.S. Chang Y.C. Chen S.Y. Chen R.C. Neurology. 1997; 48: 1583-1588Google Scholar) and on the similarity of the chemical structure of paraquat to the active metabolite of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridium ion (MPP+). Although recent studies have shown that paraquat, either alone or in combination with the dithiocarbamate fungicide manganese ethylenebisdithiocarbamate, results in preferential degeneration of the nigrostriatal system (11Thiruchelvam M. Richfield E.K. Baggs R.B. Tank A.W. CorySlechta D.A. J. Neurosci. 2000; 20: 9207-9214Google Scholar, 12McCormack A.L. Thiruchelvam M. Manning-Bog A.B. Thiffault C. Langston J.W. Cory-Slechta D.A. Di Monte D.A. Neurobiol. Dis. 2002; 10: 119-127Google Scholar), the molecular mechanisms by which paraquat induces dopaminergic neuronal cell death are not yet fully understood. Understanding the mechanism that mediates paraquat-selective degeneration of nigral dopaminergic neurons may provide insights into more effective therapeutic approaches for the disease. A variety of extracellular stimuli elicit cellular activities such as survival, proliferation, differentiation, and apoptosis through the activation of a family of mitogen-activated protein kinases (MAPKs) consisting of the extracellular signal-regulated kinases (ERKs), the p38 MAPK, and the c-Jun N-terminal kinases (JNKs) (13Davis R.J. Cell. 2000; 103: 239-252Google Scholar, 14Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Google Scholar). The JNKs, also known as stress-activated protein kinases (SAPKs), serve as phosphorylation substrates for MAP kinase kinases (MKKs) such as MKK4/7, which are activated in turn by phosphorylation via MAP kinase kinase kinases (MKKKs). JNK is activated in response to many different stress factors including heat shock, inflammatory cytokines, protein synthesis inhibitors, growth factor withdrawal, chemotherapeutic drugs, and ultraviolet irradiation (15Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar, 16Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell Biol. 2002; 3: 663-672Google Scholar). However, the role of JNK signaling pathway in cell death is highly controversial. JNK activation has been implicated in both cell survival and cell death in response to stress depending on the cell type or the stimulus (13Davis R.J. Cell. 2000; 103: 239-252Google Scholar, 15Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar). Several studies have suggested that the JNK signal transduction pathway may be involved in PD and that blocking the activity of the JNK protein kinases may be effective in preventing disease-related neuropathology (for a review, see Ref. 17Peng J. Andersen J.K. IUBMB Life. 2003; 55: 267-271Google Scholar). For example, the activation of the JNK signaling pathway converging on the phosphorylation of c-Jun has been associated with the induction of apoptosis in MPTP-treated mice (18Saporito M.S. Thomas B.A. Scott R.W. J. Neurochem. 2000; 75: 1200-1208Google Scholar, 19Xia X.G. Harding T. Weller M. Bieneman A. Uney J.B. Schulz J.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10433-10438Google Scholar). These and related findings prompted us to assess the changes in c-Jun and the JNK pathway activity in dopaminergic neurons during paraquat-induced cell death, a recently established model of the disease (12McCormack A.L. Thiruchelvam M. Manning-Bog A.B. Thiffault C. Langston J.W. Cory-Slechta D.A. Di Monte D.A. Neurobiol. Dis. 2002; 10: 119-127Google Scholar). We report here evidence that suggests a role for the JNK pathway in paraquat-mediated apoptosis of midbrain dopaminergic neurons. Reagents—Paraquat, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), protease inhibitor mixture, and monoclonal anti-β-actin antibody were purchased from Sigma. Polyvinylidene difluoride (PVDF) membrane and SDS-PAGE gels were obtained from Bio-Rad. Rabbit polyclonal anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-JNK, anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-p38 MAPK (Thr180/Tyr182), anti-phospho-c-Jun (Ser63), anti-c-Jun, and anti-cleaved caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Rabbit and sheep anti-tyrosine hydroxylase polyclonal antibodies were obtained from Chemicon (Temecula, CA). Media and sera were from Invitrogen. The ERK inhibitor PD98059, the p38 inhibitor SB202190, and the JNK inhibitor SP600125 were from Calbiochem. Benzyloxycarbonyl-DEVD-fluoromethylketone (Z-DEVD-fmk) was purchased from BD Biosciences. Rabbit anti-4-hydroxynonenal antibody was from Alpha Diagnostic International (San Antonio, TX). Manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) was purchased from OXIS International Inc. (Portland, Oregon). CEP-11004 was a gift from Cephalon (West Chester, PA). Cell Culture—N27 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin. Cell viability was determined by MTT incorporation (20Peng J. Wu Z. Wu Y. Hsu M. Stevenson F.F. Boonplueang R. Roffler-Tarlov S.K. Andersen J.K. J. Biol. Chem. 2002; 277: 44285-44291Google Scholar). To evaluate the effect of signaling pathways on cell death, the inhibitors listed above were added 1 h prior to paraquat. Caspase-3 Activity Assay—Caspase-3 activity was examined using a commercially available kit according to the manufacturer's directions (Bio-Rad) (20Peng J. Wu Z. Wu Y. Hsu M. Stevenson F.F. Boonplueang R. Roffler-Tarlov S.K. Andersen J.K. J. Biol. Chem. 2002; 277: 44285-44291Google Scholar). Briefly, 50 μg of protein from each sample was incubated with the synthetic substrate cabobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (Z-DEVD-AFC) for 2 h at 37 °C. Fluorescence was measured using a microplate spectrofluorometer (excitation at 400 nm, emission at 530 nm). Serial dilutions of AFC were used as standards. A negative control in which caspase-3 inhibitor (Ac-DEVD-CMK) was added and a positive control containing apopain were used to test the efficacy of the assay. Paraquat Administration—Eight-week-old male C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were intraperitoneally injected with either saline or 7 mg/kg paraquat dichloride hydrate (dissolved in saline) at 2-day intervals for a total of 10 doses. Animals were killed at day 7 or 8 after the last administration. The JNK pathway inhibitor CEP-11004 (1 mg/kg) was administered to mice by subcutaneous injection 1 day prior to paraquat administration at 2-day intervals for a total of 10 doses and for 7 days following the last paraquat injection on alternative days. Experimental protocols were in accordance with the National Institutes of Health Guidelines for Use of Live Animals and were approved by the Animal Care and Use Committee at the Buck Institute. Primary Mesencephalic Cultures—Primary mesencephalic cell cultures were prepared from embryonic gestation day 14 mouse embryos. Ventral mesencephalic tissue was mechanically dissociated by mild trituration in ice-cold calcium and magnesium-free Hanks' balanced saline solution (Invitrogen) and incubated with 0.05% trypsin/EDTA at 37 °C for 15 min. Dissociated cells were transferred to neurobasal medium (Invitrogen) containing 2% B27 supplement, 2 mm glutamate, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded onto poly-d-lysine-coated 24-well culture plates at a density of 7 × 105 cells/well. Cultures were maintained at 37 °C in a humidified atmosphere containing 95% air and 5% carbon dioxide. After 4 days, one-half of the medium was replaced with fresh medium. Cells were grown an additional 2 days and then treated with 40 μm paraquat for 18 or 24 h. Immunocytochemistry—Cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min at room temperature and incubated with blocking buffer (2% horse serum, 1% bovine serum albumin, and 0.1% Triton X-100 in phosphate-buffered saline, pH 7.5). Primary antibodies were added at 4 °C overnight and included the following: sheep anti-tyrosine hydroxylase (TH) polyclonal antibody (1:200), rabbit polyclonal anti-JNK (1:100), rabbit polyclonal anti-phospho-c-Jun (1:100), and rabbit polyclonal anti-cleaved caspase-3 (1:100). The secondary antibodies were rhodamine-conjugated rat-absorbed donkey and was used to and were using a Cell in Primary Mesencephalic total of neurons in mesencephalic cultures was in well in at after 24 h of paraquat The of neurons as with that of cells was used to evaluate paraquat and activated neurons neurons were the well were with cultures from tissue or cell cultures were on in prepared buffer containing pH 50 mm mm 1% 1 mm and a of protease The was by at × for 30 min at 4 °C. The protein of the was measured using a commercially available protein kit of protein from each sample were on a SDS-PAGE and transferred to were with 5% in buffer mm pH mm and and incubated with antibodies JNK, overnight at 4 °C. was using secondary antibody and an kit The protein gels were with to of with saline and 4% paraformaldehyde in phosphate-buffered saline, were in the and in Serial were on a For were incubated with blocking solution (2% horse serum, 1% bovine serum albumin, and 0.1% Triton X-100 in phosphate-buffered saline, pH and then with antibodies at 4 °C overnight by secondary antibodies in blocking solution at room temperature for 2 h. The antibodies used were sheep polyclonal antibody (1:200), rabbit polyclonal anti-JNK (1:100), rabbit polyclonal anti-phospho-c-Jun (1:100), and rabbit polyclonal anti-cleaved caspase-3 (1:100). The secondary antibodies were rhodamine-conjugated rat-absorbed donkey and were with and were using a For were through the midbrain. neurons were by the tissue with a rabbit polyclonal antibody and horse and following the by the of the kit in combination with neuronal were by using a system consisting of a with a neurons were in the of the of the midbrain was at and the was by using the of at a the of neurons was at After from the were the total of neurons in the was by the of neurons the with the of the of the and the of the Neurosci. 1999; Scholar). data are as for the of of the data was using of and of were as Paraquat Cell in is a dopaminergic cell by of midbrain neurons from mesencephalic cultures S. J. C. A. Cell Biol. Scholar). The cell and the tyrosine hydroxylase and the cell has been used for the effects of in dopaminergic neurons and is a model system to the role that paraquat-mediated oxidative stress may have on JNK activation as to Parkinson's disease. of cells with of paraquat for 24 h cell shown in paraquat cell viability in a Cell death was by an in of the caspase-3 protein in cells 18 h after paraquat a in as with the control protein suggests that paraquat-induced cell death is at in Paraquat JNK ERK and p38 in is known to J.S. Health 55: Scholar). The specific signal transduction pathways involved in cellular to with cell For example, cells with a activation of both p38 and JNK Mol. Cell. 2003; Scholar), ERK and p38 are activated in S. B. R. J. Biol. Chem. 2003; Scholar). To specific MAPK signal transduction pathways are involved in cellular to paraquat, measured changes in the of of JNK, and antibodies JNK, and ERK, that exposure to paraquat for h in a progressive in for antibody total JNK paraquat-induced in paraquat not the of either p38 or JNK activation has been suggested to be involved in the induction of apoptosis by different (13Davis R.J. Cell. 2000; 103: 239-252Google Scholar, 15Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar), examined paraquat was to JNK in cells through phosphorylation of specific substrate c-Jun shown in the of as the of paraquat exposure to 18 h. phosphorylation not from the of c-Jun as protein were to of JNK Cell in the between paraquat induction of MAPK signaling pathways and paraquat-induced cell death, used a of kinase inhibitors to the ERK, and JNK We that a kinase were for the effects of paraquat on cell death, activity shown in with the JNK inhibitor SP600125 paraquat of caspase-3 the ERK inhibitor and the p38 inhibitor were the phosphorylation of p38 and the phosphorylation of ERK in cells the manganese(III) tetrakis (4-benzoic acid) porphyrin and a specific caspase-3 inhibitor also paraquat-mediated caspase-3 MnTBAP, and also cells from paraquat-induced death, the inhibitors were in The above results suggest that both and JNK activation are for paraquat-induced cell death of cells in vitro. Paraquat JNK Activity in Primary Mesencephalic is a cell that may have survival mechanisms that are in dopaminergic neurons. to the role of the JNK signaling pathway in paraquat-induced cell death on a cellular in the effects of paraquat in mesencephalic cultures via with antibodies specific for both tyrosine hydroxylase and and activated with control was of and activated caspase-3 in neurons mesencephalic cultures treated with 40 μm paraquat for 18 of and activated caspase-3 was in neurons To the between paraquat induction of the JNK signaling pathway and paraquat-induced neuronal death, treated neurons with or the JNK inhibitor SP600125 1 h prior to paraquat that both and SP600125 the of and and activated caspase-3 with neurons after 18 h of paraquat Cells were for at 24 h after paraquat and cells were shown in with the JNK inhibitor SP600125 neurons from paraquat-induced death, the ERK inhibitor and the p38 inhibitor were and also paraquat-mediated death These results suggest that via paraquat results in the activation of the JNK signaling pathway and apoptotic neuronal death of dopaminergic midbrain neurons. Paraquat in JNK in in JNK is activated in neurons after paraquat administration in using as a dopaminergic midbrain from mouse and were and in nigral dopaminergic neurons However, in from mice treated with paraquat, neurons a that more by 7 days after the last paraquat administration. the after paraquat the of activated caspase-3 in the of a of dopaminergic neurons using an antibody caspase-3 results were with to the in of JNK c-Jun and caspase-3 activation via Paraquat the of the neurodegenerative effects of paraquat administration on dopaminergic neurons in also midbrain with shown (12McCormack A.L. Thiruchelvam M. Manning-Bog A.B. Thiffault C. Langston J.W. Cory-Slechta D.A. Di Monte D.A. Neurobiol. Dis. 2002; 10: 119-127Google Scholar), a of neurons in the at day 8 after the last injection in neurons was in the as with that in using the of nigral neurons in mice at day 8 after the last administration. shown in paraquat administration results in a in nigral neurons in mice as with administration of CEP-11004 prior to paraquat and the of neurons were not different in the substantia nigra of mice injected with paraquat CEP-11004 as with the role of the JNK signal pathway in paraquat-mediated nigral dopaminergic neuronal The of is the role for in to the selective dopaminergic neuronal death by paraquat. Our data that neurons paraquat-induced apoptosis activated the activator factor activation was by mechanisms through the activation of the JNK of c-Jun activity in dopaminergic neurons caspase-3 activation and neuronal These to have in dopaminergic neuronal survival and death from and environmental The mechanism of paraquat is via oxidative Paraquat by both with and via with molecular J.S. Health 55: Scholar, Scholar, Mol. 1993; 43: Scholar). studies that an role in paraquat M. 1996; Scholar). Several including MPP+, and in and related and activation of the JNK which apoptosis J. Neurochem. 2001; Scholar). with data show that MnTBAP, a of paraquat-induced JNK activation and apoptosis. including and have also been shown to apoptotic death via an oxidative JNK signaling pathway Y. R. J. Biol. Chem. 1998; Scholar). paraquat the following of JNK activation and phosphorylation of These effects and of caspase-3 which in turn to the apoptotic The that of activated ERK by and p38 by not apoptosis suggests that ERK and p38 are not involved in the paraquat-induced apoptotic pathway of dopaminergic neurons. the of JNK activation by the JNK inhibitor SP600125 A. S. Y. A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: Scholar) activation of caspase-3 and that JNK activation of The results in the signaling by which the activation of JNK after paraquat to apoptosis in dopaminergic neurons. JNK is involved in many cellular such as proliferation, differentiation, and apoptosis (13Davis R.J. Cell. 2000; 103: 239-252Google Scholar, 15Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Google Scholar). It is that the of activation may be in the signaling JNK activation in the dopaminergic neuronal apoptosis by (18Saporito M.S. Thomas B.A. Scott R.W. J. Neurochem. 2000; 75: 1200-1208Google Scholar, 19Xia X.G. Harding T. Weller M. Bieneman A. Uney J.B. Schulz J.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10433-10438Google Scholar, J. Neurochem. 2001; Scholar, Y. R. J. Biol. Chem. 1998; Scholar). oxidative stress via of in cells in vitro was to JNK J.P. D. A.M. C.M. J. Neurochem. 1999; Scholar). We have shown that phosphorylation of JNK, by phosphorylation of is an in the apoptotic signaling cascade by paraquat. The activation of JNK results in c-Jun that protein kinase an role in c-Jun c-Jun is implicated in many neuronal cell death T. P. M. Neurosci. 1997; 20: Scholar, J. A. J. B. 2000; Scholar). of c-Jun has been shown to be for dopaminergic neuronal apoptosis by X.G. Harding T. Weller M. Bieneman A. Uney J.B. Schulz J.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10433-10438Google Scholar). of c-Jun activity by antibody and genetic blocks neuronal apoptosis by growth factor J. C. J. C.M. D. M. Scholar, M. S. C. Johnson R. J. Cell Biol. 2002; Scholar). neurons a c-Jun that the N-terminal phosphorylation and are to apoptosis by the A. M. Nat. 1999; Scholar). the effects of a selective caspase-3 inhibitor as well as the JNK inhibitor SP600125 in preventing paraquat-mediated cell death and and activator of or are of the cell death recent studies have shown that activated JNK is involved in or into the with factor 1 and the activation of C. P. J. A. D. R.J. Science. 2000; Scholar, D. T. C. N. N. S. J. Biol. Chem. 2003; Scholar). in and the results in the of additional cellular to the changes associated with including and Nat. Rev. 2002; Scholar). of the that the JNK signaling cascade is a direct activator of the paraquat-mediated dopaminergic neuronal death, that paraquat-induced death is by CEP-11004 of kinases in the JNK pathway has that CEP-11004 by kinases C. M. B.A. Thomas B.A. M.S. D. Med. Chem. 2002; Scholar). CEP-11004 has been to apoptosis in neuronal apoptosis including growth factor and C. M. B.A. Thomas B.A. M.S. D. Med. Chem. 2002; Scholar). Furthermore, CEP-11004 has been shown to be an effective in the model of PD in C. M. B.A. Thomas B.A. M.S. D. Med. Chem. 2002; Scholar, P. Wu A. S. M. S. Proc. Natl. Acad. Sci. U. S. A. 2003; Scholar). data the that the activation of JNK is a of the mechanism of of dopaminergic neurons to paraquat-mediated apoptosis. both paraquat-induced JNK activation and that paraquat-induced oxidative stress is Understanding the mechanisms to paraquat-induced neuronal death may to in the of neurodegenerative such as the JNK inhibitor an of is in as a for Our the of the pathway in yet model of the the that inhibitor may be an effective for the disease. We are to for on