Alcohol-induced Golgiphagy is triggered by the downregulation of Golgi GTPase RAB3D.

ABSTRACT

The development of alcohol-associated liver disease (ALD) is associated with disorganized Golgi apparatus and accelerated phagophore formation. While Golgi membranes may contribute to phagophores, association between Golgi alterations and macroautophagy/autophagy remains unclear. GOLGA4/p230 (golgin A4), a dimeric Golgi matrix protein, participates in phagophore formation, but the underlying mechanism is elusive. Our prior research identified ethanol (EtOH)-induced Golgi scattering, disrupting intra-Golgi trafficking and depleting RAB3D GTPase from the trans-Golgi. Employing various techniques, we analyzed diverse cellular and animal models representing chronic and chronic/binge alcohol consumption. In trans-Golgi of non-treated hepatocytes, we found a triple complex formed between RAB3D, GOLGA4, and MYH10/NMIIB (myosin, heavy polypeptide 10, non-muscle). However, EtOH-induced RAB3D downregulation led to MYH10 segregation from the Golgi, accompanied by Golgi fragmentation and tethering of the MYH10 isoform, MYH9/NMIIA, to dispersed Golgi membranes. EtOH-activated autophagic flux is evident through increased WIPI2 recruitment to the Golgi, phagophore formation, enhanced LC3B lipidation, and reduced SQSTM1/p62. Although GOLGA4 dimerization and intra-Golgi localization are unaffected, loss of RAB3D leads to an extension of the cytoplasmic N terminal domain of GOLGA4, forming GOLGA4-positive phagophores. Autophagy inhibition by hydroxychloroquine (HCQ) prevents alcohol-mediated Golgi disorganization, restores distribution of ASGR (asialoglycoprotein receptor), and mitigates COL (collagen) deposition and steatosis. In contrast to short-term exposure to HCQ, extended co-treatment with both EtOH and HCQ results in the depletion of LC3B protein via proteasomal degradation. Thus, (a) RAB3D deficiency and GOLGA4 conformational changes are pivotal in MYH9-driven, EtOH-mediated Golgiphagy, and (b) HCQ treatment holds promise as a therapeutic approach for alcohol-induced liver injury.Abbreviation ACTB actin, beta; ALD alcohol-associated liver disease; ASGR asialoglycoprotein receptor; AV autophagic vacuoles; EM electron microscopy; ER endoplasmic reticulum; EtOH ethanol; HCQ hydroxychloroquine; IP immunoprecipitation; KD knockdown; KO knockout; MYH10/NMIIB myosin, heavy polypeptide 10, non-muscle; MYH9/NMIIA myosin, heavy polypeptide 9, non-muscle; PLA proximity ligation assay; ORO Oil Red O staining; PM plasma membrane; TGN trans-Golgi network; SIM structured illumination super-resolution microscopy.