Activation Of Akt By Insulin Prevents Apoptosis In Transfected And Untransfected PC12 Cells

Parkinson's disease(PD) is a common neurodegenerative disease in the aged and its exact pathogenesis is unknow.Some factors are associated with disease,including aging,oxidative stress and mitochondrial dysfunction.Insulin that acts as a neurotrophic factor in central nervous system(CNS) protects neurons from oxidative damage.Dysregulation of insulin signaling in the brain may lead to PD.We have previously explored insulin signaling pathway,and documented that insulin prevents from MPP⁺-induced apoptosis by activating insulin receptor and phosphoinositide 3-kinase(PI 3-K).We now extend the work to study elements of insulin signaling pathway,including PI 3-K cascades in PC12 cells,and to investigate the mechanism responsible for the protection.Partâ… Insulin Prevents MPP⁺-Induced Apoptosis in PC12 CellsAim:To investigate the neuroprotection in MPP⁺-induced PC12 cells.Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was performed to evaluate the change of PC12 cells viability when cells were exposed to MPP+ and the insulin.The apoptosis induced by MPP⁺ was determined by HOECHST 33258 staining method and flow cytometric analysis.The phosphorylation of protein kinase B(Akt),glycogen synthase kinase-3β(GSK3β) and tau after insulin treatment was measured by microtiter immunocytochemical ELISA assay(MICE assay).Results:The results of MTT assay suggested that insulin pre-treatment protected PC12 cells against serum deprivation(P＜0.001).Insulin showed a protection effect against treatment with 100μM MPP⁺ in PC12 cells(P=0.04).PI 3-K inhibitor LY294002 reversed the protective effect of insulin.HOECHST 33258 staining method and flow cytometric analysis confirmed apoptosis by MPP+ and anti-apoptosis by insulin.Expression of phosphorylated Akt(pS473) was increased treated with 100nM insulin(P=0.008).Accordingly,expression of phosphorylated GSK3β(pS9) was increased(P=0.005).Insulin decreased expression of phosphorylated tau at Ser199,not at Thr231.Conclusion:insulin could protect PC12 cells from the cytotoxity of MPP⁺ by activating Akt and GSK3β.Partâ…¡Construction of PLenti6/V5-α-synuclein Expressing Plasmid and Expression of Wild and Mutantα-synuclein by Lentivirus in PC12 CellsAim:To construct the pLenti6/V5-α-synuclein expressing plasmid,produce the pseudolentivirus in 293FT cells and transfect wild and mutantα-synuclein by lentivirus in PC12 cells.Methods:Total RNA was extracted from human brain. The sequence encoding human wild-typeα-synuclein was amplificated by RT-PCR. The SNCA-WT pDrive plasmid was constructed by transformed into Top 10 cells. The resulting pLenti6/v5-SNCA-WT,pLenti6/v5-SNCA-A30P and pLenti6/v5-SNCA-A53T vectors were set up by the digestion of Pstâ… and Xhoâ… double restriction enzyme and ligation of T4 ligase.The plasmids were transformed into Top10 cells.Several clones were picked for enzymatic digestion and only the clones with correct result of enzymatic digestion were chosen for sequencing. Co-transfecting the 293FT with the pLenti6/V5-SNCA-WT,pLenti6/V5-SNCA-A30P, or pLenti6/V5-SNCA-A53T and the packaging mix by Lipofectamine 2000,the purified plasmid DNA from the positive clone was used to produce the lentivirus. Viral supernatant was harvested.Lentivirus-engineered SYN infect PC 12 cells.Stably transfected PC12 cell lines were established by selecting for blasticidin-resistant cells. Results:The pLenti6/V5-SNCA-WT,pLenti6/V5-SNCA-A30P,and pLenti6/V5-SNCA-A53T were constructed and confirmed by sequencing.The lentivirus expressing GDNF in-frame with the V5 epitope tag was produced. Conclusion:Lentivirus-engineeredα-synuclein can be constructed successfully and wild- and mutant-type virus stock was produced.Partâ…¢Survival-Promoting Effects of insulin in Wild and Mutantα-synuclein Transfected PC12 CellsAim:To evaluate protective effect of insulin on the wild and mutantα-synuclein transfected PC12 cells,and clarify the role of insulin mediated cell survival.Methods: Neurotoxic effects of MPP⁺ or 6-OHDA were compared on PC12 cells to the effects on a wild typeα-synuclein and 2α-synuclein mutants by MTT assay.Cell viability was measured after insulin treatment.The apoptosis was determined by HOECHST 33258 staining method and flow cytometric analysis.Phosphorylation of Akt,GSK3βand tau after insulin treatment was assessed by MICE assay.Immunofluorescence analysis of Phosphorylation of tau was detected after treating with different drugs. Results:Wild- and mutant-type PC12 cells expressed normal cell proliferation in complete medium.Cell viability of various cell lines was decreased treated with MPP⁺ or 6-OHDA for 24h.A30P and A53T mutation impaired cell viability more significantly compared with untransfected cells and wild-type cells.The increase of cell survival was shown after insulin treatment in all type cells(P＜0.05).LY294002 reversed the protective effects of insulin.Expression of phosphorylated Akt(pS473) was increased treated with 100nM insulin in all type cells.Insulin did not influenced the phosphorylation of GSK3β(pS9) and tau(pS199 and pT231).The incubation of cells with MPP⁺,MG132,and NH₄Cl significantly increased thioflavin-S positive aggregation in cells.Conclusion:Transfectedα-synuclein did not alter cell survival. Transfected cells,especially mutant-type cells,impaired cell survival with MPP⁺ treatment.Increased expression of phosphorylated Akt activated by insulin prevented from MPP⁺-induced apoptosis.Phosphorylation of GSK3βwas not influenced by insulin in mutant cells,and it may be the cause that these ceils did not express enough resistance to neurotoxin.1.Insulin protects transfected and untransfected PC12 cells from MPP⁺-induced apoptosis.2.The protection of insulin is associated with activated Akt and GSK3βcascades.3.PC12 cells expressed mutantα-synuclein are susceptible to neurotoxin.