Histone Deacetylase HDAC6 Regulates Mitophagy

AIMS: Mitophagy is a strategy that eukaryotes evolve to selectively remove damaged or unwanted mitochondria in response to the bioenergetic crisis and oxidative stress.Mitochondria are encapsulated by double-membrane vesicles to form mitophagosomes during mitophagy,and then transported to lysosomes for degradation.Mitophagy consists of four sequential steps: initiation,phagophore nucleation,mitophagosome formation,and fusion of mitophagosome with lysosome.There are two types of mitophagy pathways: PINK1/Parkin/ubiquitin-dependent and receptor-dependent.Aberrant mitophagy contributes to the pathogenesis of several human diseases,including neurodegenerative diseases,cancer,and metabolic diseases.Post-translational modifications of mitophagy machinery ensure spatiotemporal regulation of mitophagy flux,while the precise regulation of post-translational modifications remains elusive.HDAC6 is a unique class II HDAC as it predominately localizes in the cytoplasm and contains two homologous catalytic domains.Upon mitochondria depolarization,HDAC6 translocates onto mitochondria and helps clean mitochondrial protein.However,the regulatory mechanism of HDAC6 in mitophagy and the involvement of its deacetylase activity remains unknown.METHODS: HDAC6-knockout HEK293 cells were constructed utilizing the CRISPR-Cas9 strategy for functional study.Immunofluorescence of GFP-LC3 puncta,western blot of LC3B-II level,flow cytometry,and electron microscopy were combined to monitor mitophagosome formation.The target pathway was defined by comparing autophagosome formation in various mitophagy and autophagy models.HDAC6 inhibitors were utilized to study the importance of deacetylase activity for neuronal mitochondria quality control,ubiquitin phosphorylation,and PINK1 stability.Immunoprecipitation was performed to elucidate the interacting pattern between HDAC6 and PINK1 or ubiquitin.Combining in vitro chemical acetylation,deacetylation assay,and mass spectrum,the substrates of HDAC6 were analyzed.The acetylmimic mutant was constructed and introduced into cells to evaluate the impact of substrate acetylation on mitophagy.RESULTS: Firstly,we aim to determine the mitophagic stage in which HDAC6 is involved.HDAC6 deficiency hinders the clearance of stressed mitochondria induced by uncoupling agent CCCP in HDAC6-knockout HEK293 cells and cultured mouse dopaminergic neurons.This is due to lowered mitophagosome production revealed by a reduction of GFP-LC3 puncta and electron microscopy.However,mitophagosome remains normal in hypoxia-induced mitophagy and rapamycin-activated autophagy in HDAC6-knockout cells.The second part focuses on the mitophagy pathways regulated by HDAC6.Ubiquitination of mitochondrial proteins and translocation of the ULK1 complex are impeded in HDAC6-knockout cells,indicating an abnormality in the ubiquitin-dependent mitophagy pathway.The current ubiquitin-dependent mitophagy model is as: a drop of mitochondrial membrane potential causes accumulation of full-length PINK1 at mitochondrial outer membrane;PINK1 phosphorylates ubiquitin and Parkin,boosting E3-ligase activity of Parkin,which then decorates mitochondria with ubiquitin coat;mitochondrial ubiquitination recruits autophagic receptors,autophagic initiation machinery ULK1 and LC3-coated phagophore to form mitophagosome.In this study,we further notice a declination of phosphor-ubiquitin in HDAC6 KO cells.Ubiquitin phosphorylation correlates with HDAC6 deacetylase activity as deacetylase inhibitors phenocopy HDAC6 KO condition.Mechanistically,we find that HDAC6 gathers around damaged mitochondria.HDAC6 interacts with PINK1 through its C-terminal BUZ domain.PINK1 protein level,phosphor-ubiquitin,and phosphor-Parkin decrease in HDAC6 KO cells and HDAC6 inhibitor treatment,indicating that HDAC6 guarantees the surge in ubiquitin phosphorylation by stabilizing PINK1.Meanwhile,the binding affinity between HDAC6 and ubiquitin increases under CCCP treatment,and the HDAC6 inhibitor enhances ubiquitin acetylation,suggesting an additional regulatory mechanism between HDAC6 and ubiquitin.By combining in vitro chemical acetylation,deacetylase assay,and LC-MS/MS,we confirm that HDAC6 catalyzes ubiquitin deacetylation at lysine 29(K29).By introducing acetyl-mimic ubiquitin K29 Q into cells,we find that the K29 Q mutant has a lower affinity with PINK1 and blocks the phosphorylation at S65,indicating that HDAC6 could deacetylate ubiquitin to facilitate ubiquitin phosphorylation.CONCLUSIONS: Our study demonstrates that HDAC6 involves in the initiation of ubiquitin-dependent mitophagy.HDAC6 regulates phosphorylation of ubiquitin,mitochondrial ubiquitination and autophagic machinery recruitment.Mechanistically,HDAC6 stabilizes PINK1 via its deacetylase activity.HDAC6 is also capable of catalyzing ubiquitin deacetylation to boost phosphorylation.Our work provides a theoretical basis for developing new treatments for mitophagy-related diseases.