Pharmacological manipulations of autophagy modulate paraquat-induced cytotoxicity in PC12 cells.

Environmental exposure to paraquat has been reported to be associated with Parkinson's disease (PD). In experimental animal models paraquat reproduces features of PD, however, the exact mechanism of PD-induced neurotoxicity has not been fully established. This study was designed to investigate paraquat-mediated interference with mitochondrial function and autophagy, and determine the impact of the modulation of autophagy flux on paraquat-induced cell toxicity. Rat adrenal pheochromocytoma PC12 cells were treated with paraquat for 24 h to establish a cellular mode of PD induced neurotoxicity. Pre-incubation of PC12 cells with an antioxidant N-acetyl-L-cysteine (NAC) or autophagy modulators rapamycin and chloroquine was conducted to determine the effect of modulation of oxidative status and autophagy flux on paraquat-elicited cytotoxicity. Mitochondrial functions and dynamics were analyzed by measuring oxygen consumption in a high-resolution oxygraph and imaging with a fluorescent mitochondrial dye (MitoTracker). Reactive oxygen species was determined by flow cytometry using fluorescent probe DCFH-DA. Autophagic flux was determined by Western blot analysis of autophagy marker LC3-II as well as p62 expression. It was found that treatment of cells with paraquat caused a concentration-dependent loss of cell viability that was accompanied by a decrease in cell respiration and reduction of polarized mitochondria, which was prevented by pretreatment of cells with NAC. Analysis of autophagy showed that NAC inhibited basic autophagy flux of PC12 cells, as evidenced by a decrease in LC3-II level and an increase in p62 expression. However, this modulation of autophagy by NAC may not be implicated into its cellular protective mechanism over paraquat cytotoxicity as inhibition of autophagy by chloroquine significantly enhanced paraquat induced cytotoxicity. Furthermore, the autophagy inducer rapamycin dramatically decreased paraquat induced cellular toxicity in PC12 cells. The present study demonstrates that basal autophagy plays a protective role in paraquat-induced cell toxicity. Antioxidant NAC confers protective role in paraquat toxicity mainly through maintaining mitochondrial dynamics and function, other than a modulation of autophagy flux.