Autophagic dysfunction contributes to α-synuclein accumulation in dopaminergic neurons induced by paraquat

Abstract

Objective: To investigate the effects of Paraquat on autophagy level in SH-SY5Y cell and the mechanism of abnormal aggregation of α-synuclein. Methods: Human neuroblastoma cell (SH-SY5Y cell) was used as model of dopaminergic neurons in vitro. The cells were treated with different concentrations of PQ (0, 18.75, 37.5, 75, 150, 300, 600 μmol/L) for 24 hours, and induced by 150 μmol/L PQ for 0, 12, 24, 36, 48, 60, 72, 96 hours to detect the relative survival rate of cells and determine dose/time-effect relationship. The cells were treated with concentration of 0, 75, 150, 300, 600 μmol/L PQ for 24 hours, and induced by 150 μmol/L PQ for different hours to detect intracellular LDH activity. The expression levels of autophagy-related proteins(LC3I, LC3II, Beclin1 , Vps34 and p62) and α-synuclein were detected by Western blot. The gene expression level of α-synuclein was assayed by Real-time quantitative PCR. The expression level of α-synuclein was also evaluated by immunofluorescence. The cells were pretreated with 100 nmol/L autophagy inducer rapamycin (RAPA) for 6 hours. The expression levels of autophagy-related proteins and α-synuclein were detected by Western blot. Results: CCK8 assay showed PQ induced cell survival rate decrease in a time and dose dependent manner; Compared with control group, the activity of LDH in the cell supernatant increased significantly after PQ exposure (P<0.05) ; Western blot analysis showed the ratio of autophagy-related protein LC3II/LC3I, Beclin1 and Vps34 protein expression were significantly lower after PQ treatment while the expression of p62 protein was higher (P<0.05) ; The protein and gene expression of α-synuclein were increased significantly after PQ treatment (P <0.05) ; Immunofluorescence showed the fluorescence intensity of α- synuclein in cells was significantly enhanced (P <0.05) . Compared with PQ group, the expression levels of autophagy-related proteins LC3II/LC3I and Beclin1 were significantly increased whlie α-synuclein protein level was decreased after RAPA induction (P<0.05) . Similarly, the IF result showed the fluorescence signal of α- synuclein significantly decreased after RAPA induction (P<0.05) . Conclusion: Paraquat induced autophagy dysfunction in SH-SY5Y cells, which leads to an abnormal aggregation of α-synuclein.