Manganese Induced Mitophagy Of Dopaminergic Neuron Through BNIP3 Mediated Oxidative Stress

Objective To explored oxidative stress and especially BNIP3 mediated MnCl₂-induced mitophagy in dopaminergic SH-SY5Y human neuroblastoma cells.Methods Logarithmic phase of Human neuroblastoma SH-SY5Y cells were used for subsequent mechanism experiments.The SH-SY5Y cells were treated with different concentrations of MnCl₂ for different time and cell viability was detected by CCK-8 kit.After treated with different concentrations of MnCl₂ for 24h,the change of mitochondrial membrane potential in SH-SY5Y cells were measured by Flow cytometry.The expression of mitophagic receptor protein BNIP3,mitochondrial outer membrane marker protein TOMM20 and the conversion of autophagic marker protein LC3-I to LC3-II were detected by Western blot after treated with MnCl₂ for 24h.Transmission electron microscopy was used to observed the change of mitochondrial morphology and the formation of mitophagic body in SH-SY5Y cells after treated with400?mol/LMnCl₂ for 24h.Autophagic inhibitor 3-MA was used to reverse MnCl₂ induced mitophagy.Confocal fluorescence was used to measured whether mitochondria can be degraded by lysosomes efficiently.The generation of ROS in SH-SY5Y cells was detected by microplate reader and the antioxidant NAC was used to reverse MnCl₂ could increase ROS generation.After pretreatment with antioxidants NAC,the expression of mitophagic receptor protein BNIP3,mitochondrial outer membrane marker protein TOMM20 and the conversion of autophagic marker protein LC3-I to LC3-II were detected by Western blot.The application of transmission electron microscopy and the autophagic inhibitor 3-MA were used to further detect ROS was involved in MnCl₂-induced mitophagy in SH-SY5Y cells.The co-immunoprecipitation was used to verify the mitophagic receptor protein BNIP3 and autophagic marker protein LC3 could bind to each other in SH-SY5Y cells,and the BNIP3 shRNA was used to reduced BNIP3 expression in SH-SY5Y cells to observe the effects of BNIP3 lower expression on the change of mitochondrial morphology and the formation of mitophagic body,ROS generation,the conversion of autophagic marker protein LC3I to LC3II and the expression of mitochondrial outer membrane marker protein TOMM20 in MnCl₂ treated SH-SY5Y cells.Results MnCl₂ reduced the viability of SH-SY5Y cells in time and concentrations-dependent manner,and decreased the mitochondrial membrane potential in concentrations-dependent manner.MnCl₂ increased the expression of mitophagic receptor protein BNIP3 and the conversion of autophagic marker protein LC3-I to LC3-II,moreover,it decreased the expression of mitochondrial outer membrane marker protein TOMM20 in a concentrations-dependent manner.Mitochondrial swelling,dissolution,aggregation,as well as denaturation and mitochondrial autophagosomes formation increased after treated with400?mol/L MnCl₂ for 24 hours in SH-SY5Y cells.Besides,MnCl₂ increased the co-localization of mitochondria and lysosome.Furthermore,co-treated with400?mol/L MnCl₂,autophagic inhibitor 3-MA can reduced the formation of mitochondrialautophagosomesinSH-SY5Y cellsand reducedthe co-localization of mitochondria and lysosome.Interestingly,it could also reduced the expression of mitophagic receptor protein BNIP3 and the conversion of autophagic marker protein LC3-I to LC3-II.Moreover,it also increased the expression of mitochondrial outer membrane marker protein TOMM20.The ROS generation in SH-SY5Y cells increased followed by the MnCl₂concentrations increased.Pretreated with antioxidant NAC could reduced the generation of ROS in SH-SY5Y cells which cultured with 400?mol/L MnCl₂,while NAC could also reduced the expression of mitophagic receptor protein BNIP3 and the conversion of autophagic marker protein LC3-I to LC3-II.Moreover,it could also increased the expression of the mitochondrial outer membrane marker protein TOMM20.In addition,pretreated with NAC also reduced 400?mol/L MnCl₂ induced the formation of mitochondrial autophagosomes in SH-SY5Y cells,whereas the autophagic inhibitor 3-MA did not inhibit ROS generation.Co-immunoprecipitation results showed that the mitophagic receptor protein BNIP3 and autophagic marker protein LC3 in SH-SY5Y cells can bind to each other effectively.Knockouted BNIP3 could reduced the conversion of autophagic marker protein LC3-I to LC3-II and increased the expression of mitochondrial outer membrane marker protein TOMM20.Meanwhile,the decreased of BNIP3 expression can also reduced mitochondrial autophagosome formation after treated with 400?mol/L MnCl₂for 24h in SH-SY5Y cells.After treated with MnCl₂ for 24h,the generation of ROS and mitochondrial membrane potential losses were decreased in BNIP3lower expression SH-SY5Y cells.Conclusion As a mitophagic receptor protein,BNIP3 mediated MnCl₂induced mitophagy in dopaminergic SH-SY5Y cells by controled ROS release.This function of BNIP3 may be a potential mechanism of manganese induced neurotoxicity.