The Role Of MTOR Signaling Pathway In Apoptosis Induced By Manganese In Dopaminergic Neurons

Objective:To investigate the role of mTOR signaling pathway in apoptosis induced by manganese in dopaminergic neurons and to provide theoretical and experimental basis to prevention and also treatment of manganese-induced neurotoxicity.Methods:In this research,N27 rat dopaminergic neuron cell lines?N27 cells?,lentivirus-mediated silencing mTOR gene N27 cells?shRNA-mTOR?and its blank control cells?shRNA-GFP?were chosen.Different concentrations of MnCl₂?0,200,400,800?M?were administrated to these cells for 24 h.We observed cell morphology after manganese exposure under inverted microscope;tested cell viability using CCK8 kit;detected cell apoptotic rate with AnnexinV Alexa Fluor 647/PI apoptotic detection kit;and detected the expression of apoptotic related proteins such as Cleaved Caspase-3,mTOR and its downstream effector proteins such as S6K1,4E-BP1,Akt and SGK1 were detected by Western blot.Results:We can see from CCK8 result that the effect of manganese on the activity of N27 cells was time-and dose-dependent.The activity of N27 cells decreased with the increase of manganese concentrations and exposure time?P<0.05?.2.The morphology of N27 cells were observed under inverted microscope.We found the number of cells with morphological abnormalities and dead cells increased with the accumulation of manganese concentration.3.AnnexinV Alexa.Fluor 647/PI and Western blot results turned out that the apoptotic rate and Cleaved Caspase-3expression increased with the increase of manganese exposure.4.Manganese treatment induced the change of protein expression in mTOR signaling pathway of N27 cells.The expression of p-mTOR and its downstream p-S6K1,p-4E-BP1,p-Akt,p-SGK1 increased?P<0.05?;5.After silencing mTOR gene,the total protein expression of mTOR decreased.The expression of p-mTOR was almost wholly inhibited,and the phosphorylation of downstream proteins S6K1,4E-BP1,Akt and SGK1 was inhibited.6.Silencing mTOR gene can resist manganese-induced cell damage,shrinkage,roundness,and decrease of floating cells and cell debris,indicating that the pathological morphology changes of cells were increased comparing with negative controls,the apoptotic rate of shRNA-mTOR cells detected by Annexin V Alexa Fluor647/PI double staining flow cytometry was lower?P<0.05?;Western blot analysis showed that the expression of Cleaved-Caspase-3 was significantly lower than that in control?P<0.05?;whether mTOR was knocked out or not,p-mTOR and p-mTOR in the mTOR signaling pathway of middle and high dose groups were higher than that of control cells?P<0.05?.The downstream contents of p-S6K1,p-4E-BP1,p-Akt and p-SGK1 were all increased?P<0.05?.Conclusion:Manganese induces apoptosis by activating mTOR signaling pathway in dopaminergic neuron cells.