GSK-3β-SIRT2 Pathway Regulates 6-OHDA Induced Neuronal Death In Parkinson’s Disease

Parkinson’s disease(PD)is the second most common neurodegenerative disease.The loss of dopaminergic neurons within the substantia nigra pars compacta(SNpc)is the pathological hallmark of PD.PD affects more than 1%people over the age of 65in the world.PD seriously affects the quality of life,family and social burden of patients.So far PD treatment is still based on drugs,but these drugs have aimed only at improving or alleviating symptoms,but cannot slow,prevent or reverse the duration of PD.Therefore,it is very important to find drugs that can slow,prevent and reverse the course of PD.Emerging evidences confirmed that the phosphorylated level of the ninth Serine residue in GSK-3β(p-GSK-3β Ser9)was markedly reduced and GSK-3βwas activatedin PD.The activated GSK-3p increased the alpha-Synuclein expressionand aggregation formation of inclusion bodies,therefore down-regulation of antiapoptotic factor Bcl-2 and up-regulation of proapoptotic factor GSK-3βexpression,cell death.SIRT2 is a NAD+ dependent histone deacetylase,mainly located in the cytoplasm.Inhibition of SIRT2 reduced the number of alpha-synuclein and regulates oligomer size,reduce the toxicity of alpha-synuclein in PD model rat,improve motor behavior,prevent the level of dopamine(DA)in striatum and the level oftyrosine hydroxylasepositive(TH+)neurons in substantia nigrato decrease,enhance glutathione(GSH)and malondialdehyde(MDA)level,reduce Caspase-3activity to fulfill neuroptotective effect.Whether the activity of SIRT2 was changed and how to regulated the activity has not been reported.So we firstly study whether the activity of SIRT2 is regulated by GSK-3i and how to GSK-3p kinase regulate SIRT2 activity in SH-SY5Ycells after 6-OHDA treatment.Firstly,the SH-SY5Y cells model of 6-OHDA was well established.And 100μM 6-OHDAsignificantly lead the SH-SY5Y cell death in 4h.Then we detected the level of p-GSK-3β Ser9 in SH-SY5Ycells and primary cortical neurons after 6-OHDA treatment by Western Blotting.The Western Blot analyzed that the level of p-GSK-3βSer9 was reducedsignificantly and the GSK-3β was activatedin SH-SY5Ycells and primary cortical neurons.The level of total SIRT2 was not changed,but the nuclear SIRT2 level was increased.The activity ofSIRT2 was increased significiently in SH-SY5Y cells after 6-OHDA treatment,and SB216763 could reverse the change.These results showed that the activity of GSK-3β was increased and the level of SIRT2was not changed in PD cell model,but the activity of SIRT2 was increased significiently.We then demonstrated that SIRT2could be phosphorylated byGSK-3β kinase in vitro.We constructed the GST-SIRT2 plasmid which SIRT2 with GST(pGEX-4T-3)tag.AfterGST-SIRT2fusion expression,the GST-SIRT2 protein was combined to GST-Beads to obtain the purified SIRT2 protein.Then we used GSK-3βkinase to phosphorylate SIRT2in vitro and autoradiography assay indicated that the GSK-3βcould phosphorylate SIRT2,specific locus of SIRT2 Ser327,331,335 which was phosphorylated by GSK-3β were identificated by mass spectrometry.Then we used GST-SIRT2 plasmid as template to construct point mutation plasmid,GST-SIRT2 S327A,GST-SIRT2 S331A,GST-SIRT2S335A,respectively.AfterGST-SIRT2 S327A,GST-SIRT2 S331A,GST-SIRT2 S335A fusion expression,the GST-SIRT2S327A,GST-SIRT2 S331A,GST-SIRT2 S335Aprotein was respectively combined to GST-Beads to obtain the purified SIRT2 S327A,SIRT2 S331A,SIRT2 S335A protein.Then we used GSK-3βkinase to phosphorylate SIRT2 S327A,SIRT2 S331A,SIRT2 S335A protein in vitro and autoradiography assay indicated that the phosphorylation level of allthe SIRT2 point mutation protein were significantly lower by GSK-3βkinase.These results confirmed that SIRT2 could be phosphorylated byGSK-3β,and the relationship between GSK-3βand SIRT2 was preliminarilyestablished.The result of co-immunoprecipitationin(Co-IP)suggested that GSK-3β and SIRT2 can bind mutully in SH-SY5Y cells.In the PD cell model,the level of SIRT2 phosphorylation was determined by Western Blotting and showed that the level of SIRT2 phosphorylation was significiently increased after 6-OHDA treatment in SH-SY5Y cells after immunoprecipitation.SB216763,a special inhibitor of GSK-3βreversed the level of phosphorylation of SIRT2 after 6-OHDAtreatment in SH-SY5Y cells.In PD cell model,theactivity of SIRT2 was increased markely after 6-OHDA treatment,and SB216763 can reverse the change.In PD cell model,GSHwas significantly decreased,MDAwas significantly increased after 6-OHDA treatment;SB216763 and SIRT2 sh-RNA which knockdown endogenous SIRT2expression in SH-SY5Y cells can increase the level of GSH and reduce the level of MDA.We demonstrated:1)In PD cell model,the activity of GSK-3β was increased significantly.2)In PD cell model,the total level of SIRT2 was not changed after 6-OHDA treatment,but the nuclear SIRT2 was increased.The level of phosphorylationof SIRT2 was increased after 6-OHDA treatment and activity of SIRT2 in the nucleus was increased.3)In 6-OHDA cell model,GSK-3βphosphorylated SIRT2 and promote its activity in the nucleus.4)In 6-OHDA cell model,the activity of SIRT2 was increased and increased the level of intracellular oxidative stress and therefore caused neurotoxicity in SH-SY5Y cells.