α-突触核蛋白通过抑制Wnt/β-catenin信号通路参与帕金森病发病机制
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摘要
目的探讨帕金森病动物模型和细胞模型中α-突触核蛋白与Wnt/β-catenin信号通路的相关关系及其导致神经元损伤的可能机制。方法在动物水平和细胞水平上应用western blotting方法、流式细胞术、免疫荧光法等方法研究α-synuclein与Wnt/β-catenin信号通路关键信号分子GSK-3β的表达关系及其对细胞活性的影响。结果α-syn转基因小鼠脑组织中α-synuclein、p-GSK-3β蛋白表达量均明显增加,与对照组和MPTP组相比具有统计学差异(P <0. 05);α-syn过表达组SH-SY5Y细胞中α-synuclein、p-GSK-3β表达量较对照组及MPP+损伤组明显升高,结果具有统计学差异(P <0. 05);阿立哌唑预处理组α-synuclein较对照组及MPP+组明显增加,p-GSK-3β与对照组及MPP+组差异不具有统计学差异;阿立哌唑预处理组细胞活性明显高于α-syn过表达组,细胞凋亡率较α-syn过表达组明显下降,差异具有统计学意义(P <0. 05);免疫荧光法示α-synuclein与p-GSK-3β存在共定位关系。结论α-synuclein可能通过抑制Wnt/β-catenin信号通路导致神经元损伤,从而参与帕金森病的发病机制。
Objective To investigate the effect of α-synuclein( α-syn) on Wnt/β-catenin signaling pathway in Parkinson's disease animal models and cell models and the possible mechanism of neuronal injury. Methods Western blotting,flow cytometry,immunofluorescence and other methods were used to detect the expression of α-synuclein and the key signal molecule GSK-3β in Wnt/β-catenin signaling pathway and its effect on cell viability at the animal and cell levels.Results The expressions of α-synuclein and p-GSK-3β in the brain tissue of α-syn transgenic mice increased significantly,compared with the control group and MPTP group( P < 0. 05). The expression levels of α-synuclein and p-GSK-3β in SH-SY5 Y cells of α-syn overexpression group were significantly higher than those in the control group and MPP + injury group,and the results were statistically significant( P < 0. 05). The α-synuclein in the aripiprazole pretreatment group was significantly increased compared with the control group and the MPP + group. There was no significant difference in pGSK-3β between the control group and the MPP + group. The cell viability of aripiprazole pretreatment group was significantly higher than that of α-syn overexpression group,and the apoptosis rate was significantly lower than that of α-syn overexpression group,the difference was statistically significant( P < 0. 05). Immunofluorescence showed a colocalization relationship between α-synuclein and p-GSK-3β. Conclusion α-synuclein may participate in the pathogenesis of Parkinson's disease by inhibiting the Wnt/β-catenin signaling pathway leading to neuronal damage.
Objective To investigate the effect of α-synuclein( α-syn) on Wnt/β-catenin signaling pathway in Parkinson's disease animal models and cell models and the possible mechanism of neuronal injury. Methods Western blotting,flow cytometry,immunofluorescence and other methods were used to detect the expression of α-synuclein and the key signal molecule GSK-3β in Wnt/β-catenin signaling pathway and its effect on cell viability at the animal and cell levels.Results The expressions of α-synuclein and p-GSK-3β in the brain tissue of α-syn transgenic mice increased significantly,compared with the control group and MPTP group( P < 0. 05). The expression levels of α-synuclein and p-GSK-3β in SH-SY5 Y cells of α-syn overexpression group were significantly higher than those in the control group and MPP + injury group,and the results were statistically significant( P < 0. 05). The α-synuclein in the aripiprazole pretreatment group was significantly increased compared with the control group and the MPP + group. There was no significant difference in pGSK-3β between the control group and the MPP + group. The cell viability of aripiprazole pretreatment group was significantly higher than that of α-syn overexpression group,and the apoptosis rate was significantly lower than that of α-syn overexpression group,the difference was statistically significant( P < 0. 05). Immunofluorescence showed a colocalization relationship between α-synuclein and p-GSK-3β. Conclusion α-synuclein may participate in the pathogenesis of Parkinson's disease by inhibiting the Wnt/β-catenin signaling pathway leading to neuronal damage.
引文
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[9]Vargas JY,Ahumada J,Arrázola MS,et al. WASP-1,a canonical Wnt signaling potentiator,rescues hippocampal synaptic impairments induced by Abeta oligomers[J]. Exp Neurol,2015,264:14-25.
[10]Zhang L,Bahety P,Ee PLR. Wnt co-receptor LRP5/6 overexpression confers protection against hydrogen peroxide-induced neurotoxicity and reduces tau phosphorylation in SH-SY5Y cells[J]. Neurochem Int,2015,87(8):13-21.
[11]Sancho RM,Law BMH,Harvey K. Mutations in the LRRK2 RocCOR tandem domain link Parkinson’s disease to Wnt signalling pathways[J]. Hum Mol Genet,2009,18(20):3955-3968.
[12]Berwick DC,Harvey K. LRRK2 functions as a Wnt signaling scaffold,bridging cytosolic proteins and membrane-localized LRP6[J].Hum Mol Genet,2012,21(22):4966-4979.
[13]Jantas D,Greda A,Golda S,et al. Neuroprotective effects of metabotropic glutamate receptor group II and III activators against MPP(+)-induced cell death in human neuroblastoma SH-SY5Y cells:the impact of cell differentiation state[J]. Neuropharmacology,2014,83(8):36-53.
[14]Pariyar R,Lamichhane R,Jung HJ,et al. Chronic administration of aripiprazole activates GSK3beta-dependent signalling pathways,and up-regulates GABAA receptor expression and CREB1 activity in rats[J]. Sci Rep,2016,6(7):30040.
[15]Rawal N,Corti O,Sacchetti P,et al. Parkin protects dopaminergic neurons from excessive Wnt/beta-catenin signaling[J]. Biochem Biophys Res Commun,2009,388(3):473-478.
[16]Lou HY,Montoya SE,Alerte TNM,et al. Serine 129 phosphorylation reduces the ability of alpha-synuclein to regulate tyrosine hydroxylase and protein phosphatase 2A in vitro and in vivo[J]. J Biol Chem,2010,285(23):17648-17661.
[17]Farrell KF,Krishnamachari S,Villanueva E,et al. Non-motor parkinsonian pathology in aging A53T alpha-synuclein mice is associated with progressive synucleinopathy and altered enzymatic function[J]. J Neurochem,2014,128(4):536-546.
[2]Libro R,Bramanti P,Mazzon E. The role of the Wnt canonical signaling in neurodegenerative diseases[J]. Life Sci,2016,158(8):78-88.
[3]Nusse R,Clevers H. Wnt/beta-catenin signaling,disease,and emerging therapeutic modalities[J]. Cell,2017,169(6):985-999.
[4]Maschio DA,Oliveira RB,Santos MR,et al. Activation of the Wnt/beta-catenin pathway in pancreatic beta cells during the compensatory islet hyperplasia in prediabetic mice[J]. Biochem Biophys Res Commun,2016,478(4):1534-1540.
[5]L’Episcopo F,Tirolo C,Caniglia S,et al. Targeting Wnt signaling at the neuroimmune interface for dopaminergic neuroprotection/repair in Parkinson’s disease[J]. J Mol Cell Biol,2014,6(1):13-26.
[6]Yuan YH,Sun JD,Zhao M,et al. Overexpression of alpha-synuclein down-regulates BDNF expression[J]. Cell Mol Neurobiol,2010,30(6):939-946.
[7]Dehay B,Decressac M,Bourdenx M,et al. Targeting alpha-synuclein:Therapeutic options[J]. Mov Disord,2016,31(6):882-888.
[8]Boonen RA,Van Tijn P,Zivkovic D. Wnt signaling in Alzheimer’s disease:up or down,that is the question[J]. Ageing Res Rev,2009,8(2):71-82.
[9]Vargas JY,Ahumada J,Arrázola MS,et al. WASP-1,a canonical Wnt signaling potentiator,rescues hippocampal synaptic impairments induced by Abeta oligomers[J]. Exp Neurol,2015,264:14-25.
[10]Zhang L,Bahety P,Ee PLR. Wnt co-receptor LRP5/6 overexpression confers protection against hydrogen peroxide-induced neurotoxicity and reduces tau phosphorylation in SH-SY5Y cells[J]. Neurochem Int,2015,87(8):13-21.
[11]Sancho RM,Law BMH,Harvey K. Mutations in the LRRK2 RocCOR tandem domain link Parkinson’s disease to Wnt signalling pathways[J]. Hum Mol Genet,2009,18(20):3955-3968.
[12]Berwick DC,Harvey K. LRRK2 functions as a Wnt signaling scaffold,bridging cytosolic proteins and membrane-localized LRP6[J].Hum Mol Genet,2012,21(22):4966-4979.
[13]Jantas D,Greda A,Golda S,et al. Neuroprotective effects of metabotropic glutamate receptor group II and III activators against MPP(+)-induced cell death in human neuroblastoma SH-SY5Y cells:the impact of cell differentiation state[J]. Neuropharmacology,2014,83(8):36-53.
[14]Pariyar R,Lamichhane R,Jung HJ,et al. Chronic administration of aripiprazole activates GSK3beta-dependent signalling pathways,and up-regulates GABAA receptor expression and CREB1 activity in rats[J]. Sci Rep,2016,6(7):30040.
[15]Rawal N,Corti O,Sacchetti P,et al. Parkin protects dopaminergic neurons from excessive Wnt/beta-catenin signaling[J]. Biochem Biophys Res Commun,2009,388(3):473-478.
[16]Lou HY,Montoya SE,Alerte TNM,et al. Serine 129 phosphorylation reduces the ability of alpha-synuclein to regulate tyrosine hydroxylase and protein phosphatase 2A in vitro and in vivo[J]. J Biol Chem,2010,285(23):17648-17661.
[17]Farrell KF,Krishnamachari S,Villanueva E,et al. Non-motor parkinsonian pathology in aging A53T alpha-synuclein mice is associated with progressive synucleinopathy and altered enzymatic function[J]. J Neurochem,2014,128(4):536-546.