Oxidative Stress Regulates Mitophagy Through ALS-linked TBK1 And OPTN

Aim:To explore how ALS-related proteins OPTN and TBK1 regulate mitophagy under normal conditions and oxidative stress,Methods:(1)In this project,we mainly used HEK 293 cell as model in the early stage.We constructed the mitophagy models expressing wild-type OPTN and pathological mutants of OPTN by drug intervention;(2)We developed new techniques,such as multi-channel live cell imaging to examine the functions and differences of wild-type OPTN and pathological mutants in mitophagy;(3)Under stress-induced condition,such as treatment of cells with the oxidative damage inducer H2O2,the translocation of OPTN in cells was checked by subcellular fractionation and immunofluorescence;(4)using the mitophagy reporter tool mt-mKeima and western blotting,we detected the effect of nuclear translocation of wild-type and mutant OPTN on mitophagy under oxidative stress;(5)Immunoprecipitation and immunofluorescence were applied to identify the binding of OPTN-TBK1.Under oxidative stress,We explored the relationship between TBK1-OPTN and mitochondrial dissociation and nuclear transport during this process by cell biology and biochemical experiments.(6)Simulating the physiological environment by primary culture of neurons,we examined the relationship between nuclear transport of OPTN and oxidative stress,and studied how OPTN-TBK1 influence mitophagy and the molecular mechanism of ALS during this process.Results:(1)When mitochondria are damaged,OPTN significantly promotes autophagosomes to recognize ubiquitinated mitochondria,and the mitophagy efficiency is significantly improved.(2)Compared with wild-type OPTN,OPTN-E50K pathological mutant enhances the recognition of damaged mitochondria by autophagosomes,but impairs the autophagic clearance of damaged mitochondria.(3)E478G mutant in UBAN domain abolishs autophagosomal recruitment and therefore impairs the autophagic clearance of damaged mitochondria.(4)WT-OPTN and mutant OPTN can accumulate into nuclear after H2O2-treatment.(5)During oxidative stress,TBK1 regulates the dissociation of OPTN from mitochondria and carries OPTN together into the nucleus,ultimately blocking parkin-mediated mitophagy.Conclution:During oxidative stress,TBK1 regulates the dissociation of OPTN from mitochondria and carries OPTN together into the nucleus.The nuclear translocation of OPTN disrupts the Parkin-OPTN mitophagic signal axis and ultimately blocks the clearance of damaged mitochondria.