Study On Molecular Mechanism Of Neuronal Apoptosis Mediated By Rotenone-induced Autophagy Inhibition

The present study,using cellular and molecular biology methods and techniques including cell culture、DAPI and MDC staining、RNA interference,fluorescent protein labeling,Western blotting,etc.,and employing PC 12 cells an primary murine neurons as experimental objects,synthetically studied the effect of rotenone on the level of autophagy in neuronal cells,and deeply discussed the relation of rotenone-induced apoptosis to autophagy inhibition in neuronal cells.The detailed results were summarized as follows:1 Rotenone induces autophagy inhibition in neuronal cellsPC 12 cells and primary murine neurons,or PC 12 cells and primary murine neurons infected with Ad-GFP-LC3,were treated with 0-1 μM rotenone for 24 h,or with 1 μM rotenone for 0-24 h.The change of cellular GFP-LC3 puncta was detected,and the expression of Atg5,p62 and LC3 proteins was evaluated using Western blotting.The results showed that rotenone induced a decreased number of GFP-LC3 puncta,as well as LC3-Ⅱ reduction and p62 increase in the cells in a concentration-and d time-ependent manner.These results indicate that rotenone induces autophagy inhibition in neuronal cells.2 Rotenone induces autophagy inhibition contributing to neuronal apoptosisPC 12 cells and primary murine neurons,or PC 12 cells infected with shRNA LC3-Ⅰ/Ⅱ,shRNA Atg5,shRNA GFP,Ad-GFP-LC3,respectively,were treated with 0-1 μM rotenone for 24 h,or pretreated with/without rapamycin(0.2 μg/ml)and then exposed to 1 μM rotenone for 24 h.The changes of autophagosomes were evaluated by fluorescent imaging using MDC staining and GFP-LC3 labeling,cell apoptosis was assayed by DAPI staining,and the expression of Atg5,p62,LC3,cleaved-caspase-3 and cleaved-PARP proteins was evaluated using Western blotting.The results showed that rapamycin reversed rotenone-induced decrease of MDC fluorescence intensity and increase of apoptosis in neuronal cells;downregulation of LC3-Ⅰ/Ⅱ or Atg5 potentiated rotenone-induced decrease of MDC fluorescence intensity or GFP-LC3 puncta,as well as increase of apoptosis in neuronal cells.The data suggest that rotenone induces autophagy inhibition contributing to neuronal.