Lead(Pb) Induced ATM-dependent Mitophagy Via PINK1/Parkin Pathway

Lead（Pb）,a widely distributed heavy metal in environment,can result in serious risks to human health.Pb can be absorbed into human body via gastrointestinal tract,respiratory tract and skin.Pb has toxic effects on diverse organs through lipid peroxiadation,apoptosis,autophagy and so on,and is able to cause severe impairment to mitochondria which can further result in the imbalance of cell homeostasis.Mitophagy,an important way to maintain mitochondria quality control,is responsible for the elimination of dysfunctional mitochondria which mainly mediated by PTEN-induced putative kinase 1（PINK1）/Parkin pathway.Whether Pb could induce PINK1/Parkin-mediated mitophagy is currently unclear.In addition,ataxia-telangiectasia mutated（ATM）,an important regulator in maintenance of cell homeostasis,is recently reported to be linked with PINK1/Parkin pathway and participate in the regulation of mitophagy,however,its exact role remains enigmatic.In the present research,human embryonic kidney cells HEK293,human skin fibroblasts GM00637 and male Kunming mice were employed to investigate the role of lead acetate（Pb（Ac）₂）in PINK1/Parkin-mediated mitophagy and to further detect how ATM regulates PINK1/Parkin pathway by the use of RNAi and transient transfection with YFP-PINK1and YFP-Parkin.The results were as follows:1.Exposure of 1 mM Pb（Ac）₂ within 24 h remarkably induced the overproduction of reactive oxygen species（ROS）（HEK293 cells,p<0.01;GM00637 cells,p<0.05）,decreased the mitochondrial membrane potential（p<0.01）and induced autophagy（HEK293 cells,p<0.01;GM00637 cells,p<0.05）in HEK293 and GM00637 cells without significant decline of cell viability or dramatic cell death.2.1 mM Pb（Ac）₂ treatment for 12 h and 24 h induced the formation of mitophagosome and mitolysosome,the accumulation of full-length PINK1（24 h,p<0.01）and mitochondrial Parkin,indicating the activation of PINK1/Parkin pathway.In HEK293 cells,the level of mitochondrial inner membrane protein COX IV and mitochondrial matrix protein HSP60 was decreased significantly under Pb（Ac）₂ exposure（COX IV:12 h,p<0.05,24 h,p<0.01;HSP60:24 h,p<0.01）.In addition,the COX IV level and HSP60 level were also dramatically declined in Pb（Ac）₂-exposed GM00637 cells（COX IV:12 h,p<0.01,24 h,p<0.01;HSP60:12 h,p<0.05,24 h,p<0.01）.These altogether suggested that Pb（Ac）₂ could trigger PINK1/Parkin-mediated mitophagy.3.Though the mice body weight and viscera index of kidney,liver and brain were not remarkably influenced when mice were exposed to Pb（Ac）₂（0,2.4,4.8,9.6 mg/kg）intraperitoneally for 7 d,4.8 mg/kg and 9.6 mg/kg exposure led to the elevation of PINK1 level（p<0.01）and the decrease of HSP60（4.8 mg/kg:p<0.05;9.6 mg/kg:p<0.01）in kidney cortex.Besides,2.4,4.8 and 9.6 mg/kg treatment promoted the translocation of Parkin to mitochondria and significantly declined the expression of COX IV（p<0.01）in kidney cortex as well.Additionally,in liver,9.6 mg/kg treatment increased the accumulation of PINK1（p<0.05）and mitochondrial Parkin and decreased the level of COX IV（p<0.01）,while 4.8 mg/kg and 9.6 mg/kg exposure resulted in the dramatic decline of HSP60 level（4.8 mg/kg:p<0.05;9.6 mg/kg:p<0.01）.However,no significant change of the expression level of mitophagy-related proteins was found in brain.Collectively,these results demonstrated that mitophagy took place in kidney cortex and liver of Pb（Ac）₂-exposed mice.4.Pb（Ac）₂ activated ATM which is indicated by the elevation of total p-ATM S1981 in HEK293 and GM00637 cells as well as in mice kidney cortex and liver,and the cytoplasmic p-ATM S1981 could be observed clearly in both cells.5.With elevated level of conventional autophagy which was shown as the increase of LC3-II/LC3-I ratio（p<0.05）and the decrease of p62 level（p<0.01）,knockdown of ATM by RNAi in HEK293 cells enhanced the accumulation of PINK1（p<0.01）and mitochondrial Parkin at basal level and Pb（Ac）₂ treatment could not change the expression of PINK1,Parkin and COX IV significantly further,indicating that Pb（Ac）₂-induced mitophagy was blocked.6.Pb（Ac）₂ promoted p-ATM S1981 co-localizing with PINK1 and Parkin in cytosol of HEK293 and GM00637 cells and facilitated ATM interacting with PINK1 at endogenouse level in HEK293 cells.7.Pb（Ac）₂ enhanced the phosphorylation of PINK1 and Parkin,while under the circumstance of ATM knockdown,the phosphorylation level of PINK1 and Parkin were already very high and could not be changed significantly by Pb（Ac）₂ treatment further,suggesting that ATM influenced the phosphorylation of PINK1 and Parkin indirectly.Altogether,the current research indicates that Pb can induce PINK1/Parkin-mediated mitophagy and activate ATM simultaneously.Pb-activated ATM colocalizes with PINK1 and Parkin and can further interact with PINK1 at endougenous level.Additionally,ATM facilitates PINK1/Parkin-mediated mitophagy and can indirectly regulate the phosphorylation of PINK1 and Parkin.These findings reveal a novel mechanism for Pb toxicity and suggest the regulatory importance of ATM in PINK1/Parkin-mediated mitophagy.