Study On The Mechanisms Of Wnt And Related Pathway In Parkinson's Disease

PartⅠThe change of expression of GSK-3βin certain brain regions of1-methyl-4-phenvl-1,2,3,6-tetrahvdropvridine Parkinson disease model micesObjective To investigate the change of expression of Glycogen Synthase Kinase-3β(GSK-3β) in certain brain regions on 1-methyl-4-phenvl-1,2,3,6-tetrahvdropvridine (MPTP)lesioned Parkinson disease model mice.Methods①Establishing MPTP lesion of PD model mice: At the beginning of theexperiments, the mice (5 per group) received an intraperitoneal(i.p.) injection ofMPTP-HC1 in saline at 24h intervals for continuous 5 days. The control group was injectedwith saline only. The mice were sacrificed at 1, 2, 4, 7, 14 and 21 days after last MPTPinjection.②Western blot: Western blot was carried out using total protein isolated fromventral mesencephalon, striatum and cortex, which were removed rapidly on ice fromdecapitated mice. Phosphorylated GSK-3β(p-GSK-3β) and GSK-3βwere examined.③Immunohistochemistry: Immunohistochemistry experiments were carried out using tissuesection prepared from ventral mesencephalon, striatum and cortex. The expression ofGSK-3βwas detected.④Immunofluorescence: immunofluorescence experiments werecarried out using tissue section prepared from ventral mesencephalon coexpression ofGSK-3βand Hochest33258 were examined. Results TH-ir was decreased significantly in midbrain after MPTP treatment. The modelwas successively established. We observed the expression of GSK3βof MPTP model.Western blot showed that the levels of total GSK3βin the midbrain was increased by day4,7,14,21 after last injection of MPTP(P＜0.05 vs the control groups respectively). And thelevels of phosphorylated GSK-3βin the midbrain was also increased from day 0 to 21(P＜0.05 vs the control groups respectively). The ratio of p-GSK-3βand total GSK-3βisdecreased by day 0 to 14. So non-phosphorylated GSK-3βwas increased during the time(P＜0.05 vs the control groups respectively). Mean while, immunohistchemistry also detectedthat total GSK-3βwas observed and expressed intensively in ventral mesencephalon.Immunofluorecence found that coexpression of GSK-3βand hochest33258 was increased.Conclusion: GSK3βis highly expressed in the ventral midbrain of MPTP model. Theexpression and the activity of GSK3βare changed in the model suggesting that GSK3βmaybe play important roles in the process of MPTP intoxication in mice. PartⅡThe change of expression ofβ-catenin in certain brain regions of1-methyl-4-phenvl-1,2,3,6-tetrahvdropvridine Parkinson disease model micesObjective To investigate the change of expression ofβ-catenin in certain brain regions on1-methyl-4-phenvl-1,2,3,6-tetrahvdropvridine (MPTP) lesioned Parkinson disease modelmice.Methods①Establishing MPTP lesion of PD model mice: At the beginning of theexperiments, the mice (5 per group) received an intraperitoneal (i.p.) injection ofMPTP-HC1 in saline at 24h intervals for continuous 5 days. The control group was injectedwith saline only. The mice were sacrificed at 1,2,4,7,14 and 21 days after last MPTPinjection.②Western blot: Western blot was carried out using total protein isolated fromventral mesencephalon, striatum and cortex, which were removed rapidly on ice fromdecapitated mice. Phosphorylatedβ-catenin (p-β-catenin) andβ-catenin were examined.③Immunohistochemistry: Immunohistochemistry experiments were carried out using tissuesection prepared from ventral mesencephalon, striatum and cortex.④Immunofluorencence:immunofluorescence experiments were carried out using tissue section prepared fromventral mesencephalon coexpression ofβ-catenin and hochest33258 were examined.Results We observed the expression ofβ-catenin of MPTP model. Western blot showed thatthe levels of totalβ-catenin in the midbrain was increased after last injection of MPTP. Andthe levels of phosphorylatedβ-catenin in the midbrain was not changed significantly. Meanwhile, immunohistchemistry also detected that totalβ-catenin was expressed intensivelyand transferred to nuclear in ventral mesencephalon. Mean while, expression ofβ-cateninwas not changed significantly in striatum and cortex. Immunofluorencence found that therewas no coexpression ofβ-catenin and Hochest33258. Conclusion:β-catenin is highly expressed in the ventral mesencephalon of MPTP model.β-catenin was transferred to nuclear. It suggests thatβ-catenin may be play important rolesin the process of MPTP intoxication in mice. PartⅢThe role of Akt/GSK-3βsignaling pathway in the protective effect ofIGF-1 on MPP+ induced neurotoxicity in PC12 cellsObjective To investigate the role of Akt/GSK-3βand Wnt/β-catenin signaling pathway inthe protective effect of IGF-1 on MPP~+ induced neurotoxicity in PC12 cells.Method: PC12 cells impaired by MPP~+ were used as the cell model of Parkinson's disease.The cultured cells were divide into such groups: control group received the administrationof F12 medium; LY294002 group, LiCl group and MPP~+ group were identical to the controlgroup except that LY294002(10μmol/L), LiCl(20mmol/L) or MPP~+(250μmol/L) wasadministrated, respectively; MPP~++IGF-1 group was treated with MPP~+(250μmol/L) andIGF-1 (100nmol/L);MPP~++LiCl group received LiCl(20mmol/L) 1h before treatment withMPP~+(250μmol/L);MPP~++IGF-1+LY294002 group was identical to the MPP~++IGF-1 groupexcept that LY294002 was given 1h before MPP~+ and IGF-1. After incubation for 24h,methyl thiazolyl tetrazolium (MTT) was used to assay the viability of the PC12 cells andthe AO/EB was used to analyze the apoptosis ration of PC12 cells. After incubation for15min, 30min, 1h, 2h, 4h, 6h, 8h, western blot was used to detect the expression level ofAkt, p-Akt, GSK-3β, p-GSK-3β, p-β-catenin andβ-catenin.Results①Enhanced levels of p-AKT were detected IGF-1 was added to the culture andthere was a significant increase in the phosphorylation of Akt in MPP~+-treated cells byIGF-1.LY294002 thoroughly abolished IGF-1-induced phosphorylation of Akt.②The effectof rescuing the cells from death induced by IGF-1 was lost with addition of LY294002, aspecific inhibitor of PI3K.③Phosphorylation of GSK-3βwas enhanced followingIGF-1-treated and there was a significant increase in the phosphorylation of GSk-3βinMPP~+-treated cells by IGF-1. LY294002 pretreatment abolished phosphorylation ofGSK-3βby EPO.④Inhibition of GSK-3βby LiCl promoted viability and reduced apoptosis in PC12 cells subjected to MPP~+.⑤The expression ofβ-catenin and p-β-cateninwas not changed after treatment of MPP~+ and IGF-1+MPP~+.Conclusion These finding indicate that IGF-1 protects against apoptosis in PC12 cellsexposed to MPP~+, through the AKT/GSK-3βsignaling pathway in this model system. PartⅣThe role of ERK signaling pathway in the protective effect of IGF-1 onMPP~+ induced neurotoxicity in PC12 cellsObjective To investigate the role of ERK signaling pathway in the protective effect ofIGF-1 on MPP~+ induced neurotoxicity in PC12 cells.Method PC12 cells impaired by MPP~+ were used as the cell model of Parkinson's disease.The cultured cells were divide into such groups: control group received the administrationof F12 medium; PD98059 group and MPP+ group were identical to the control groupexcept that PD98059(50μmol/L), or MPP+(250μmol/L) was administrated instead of F12medium, respectively; MPP~++IGF-1 group was treated with MPP~+(250μmol/L) andIGF-1 (100nmol/L); MPP~++IGF-1+PD98059 group was identical to the MPP~++IGF-1 groupexcept that PD98059 was given 1h before MPP~+ and EPO. After incubation for 24h, MTTwas used to assay the viability of the PC12 cells and the AO/EB stain were used to analyzethe apoptosis ratio of PC12 cells. After incubation for 15min,30min, 1h,2h,4h,6h,8h,western blot was used to detect the expression level of ERK, p-ERK.Results①Transienly enhanced levels of p-ERK were detected when MPP~+ were added tothe culture. But p-ERK were not changed when MPP~+ and IGF-1 were treated. PD98059thoroughly abolished IGF-1-induced phosphorylation of ERK.②PD98059, a specificinhibitor of ERK, does not alter the survival effect of IGF-1 in this model system.Conclusion These findings indicate that IGF-1 protects against apoptosis in PC12 cellsexposed to MPP~+ transiently increased ERK1/2 activity in PC12 cells, the ERK signalingpathway is not involved in this model system.