Mitochondrial depolarization, mitophagy, and mitochondrial DAMPs in ALD

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Alcohol-associated liver disease (ALD) is the leading cause of liver-related mortality. How ethanol (EtOH) damages the liver remains poorly understood, and therapies are lacking or unproven. A better understanding of the mechanisms by which the liver handles, processes, and responds to EtOH is needed to develop strategies to avoid and treat the dangerous hepatic ramifications of alcohol, particularly fibrosis and cirrhosis. EtOH consumption produces a swift increase in alcohol metabolism (SIAM) that is associated with a commensurate increase of mitochondrial respiration. Our work reveals reversible hepatic mitochondrial depolarization (mtDepo), increased mitophagic burden, and release of mitochondrial DNA (mtDNA) in mice after EtOH treatment, that precedes hepatic steatohepatitis and fibrosis. Here, we build on these findings to characterize the signals, pathways, and mechanisms of 1) onset and recovery from EtOH-induced mtDepo, 2) mtDepo- induced mitophagy, and 3) stellate cell activation and fibrosis downstream of mtDepo. Overall, we hypothesize that mtDepo is an adaptive response stimulating more rapid mitochondrial NADH oxidation to supply NAD+ required for EtOH metabolism, but chronically, mtDepo becomes maladaptive, causing disordered mitophagy and release of mitochondrial damage-associated molecular patterns (mtDAMPs) like mtDNA, leading ultimately to hepatic end stage liver disease. In Specific Aim 1, we will characterize the mechanisms of onset and recovery of EtOH-induced mtDepo, specifically the role of opening and closing of different proton leak pathways in inducing mtDepo and of mitochondrial biogenesis to restore functional mitochondria after EtOH is metabolically eliminated. In Specific Aim 2, we will characterize the manner of mitophagy after EtOH treatment. Specifically, we will determine if mitophagy initiated by EtOH-induced mtDepo involves the classical PINK1/Parkin ubiquitination pathway, is triggered by mitochondrial swelling leading to inner membrane herniation through a ruptured outer membrane with release of mtDNA as observed in preliminary work, or both. In Specific Aim 3, we will determine pathways by which mtDAMPs (like mtDNA) (possibly in synergism with acetaldehyde generated from hepatic EtOH metabolism) elicit a profibrogenic response in stellate cells with particular attention of the role of mtDNA-sensing toll-like receptor-9 (TLR9) whose deficiency decreases alcohol-induced liver injury. Together, these aims will increase our understanding of mechanisms underlying both the onset and recovery from mtDepo and how mtDepo-induced dysregulated mitophagy leads to mtDAMP release causing stellate cell activation and ultimately hepatic fibrosis. The findings of this project will allow development of new mechanism-based therapeutics to treat and prevent alcoholic liver disease.