Project summary Chronic alcohol drinking has been linked to the development and progression of alcoholic liver disease (ALD) by means of damaging cellular DNA, which results in cell death of hepatocytes, contributing to alcoholic steatohepatitis, liver fibrosis, and potentially liver cancers. However, it remains unclear how alcohol promotes DNA damage responses in ALD development and understanding the underlying mechanisms may yield insight into therapeutic approaches toward treating ALD. In the pilot studies, it was demonstrated that aspartate beta- hydroxylase (ASPH) is down-regulated in the rodent livers derived from the ethanol-fed group. In addition, DNA damage markers, including γ-H2aX (a phosphorylated form of H2aX) and GADD45α, were found elevated when compared to the control-diet fed group. The liver is predominantly composed of hepatocytes whose function is critical in the maintenance of homeostasis thereof. Thus, we investigated whether there is a correlation between ASPH and DNA damage in human hepatocytes. We ectopically expressed ASPH and challenged hepatocytes with DNA damage inducers. Intriguingly, the expression of ASPH almost eliminated γ-H2aX expression; however, knockdown of ASPH substantially promoted DNA damage. These observations suggest a protective role of ASPH in the DNA damage response. Based on these findings, we hypothesize that chronic alcohol consumption leads to ASPH down-regulation in the liver contributing to DNA damage responses and hepatocyte cell death in chronic liver disease progression. Our long-term goal is to understand how ASPH is involved in ethanol-induced hepatocyte DNA damage and the consequent impacts on ALD progression. To test our hypothesis and achieve this goal, two specific aims are proposed. In aim 1, we will investigate how the expression of ASPH alters ALD progression in vivo. We will determine if targeting hepatic ASPH using an AAV strategy will exacerbate DNA damage and cell death in the hepatocytes of ethanol-fed mice. Furthermore, we investigate if overexpression of ASPH protects hepatocytes in an ALD model. In aim 2, we will decipher the mechanism by which ASPH protects hepatocytes from DNA damage induced cell death. We will determine how ASPH is involved in ethanol-mediated DNA damage by using CRISPR/Cas9 to knock out ASPH or ectopically expressing ASPH via a lentiviral system. To delineate the mechanisms involved in this regulation, we will determine how the enzymatic functions of ASPH are associated with the ASPH-mediated DNA damage responses and cell death. Using the proteomic approach, we will also illustrate innovative ASPH downstream targets upon ethanol treatment. The results will significantly advance our understanding of the molecular mechanisms in hepatocyte cell death and pathophysiological mechanisms underlying ALD progression. We also anticipate that it will broaden our knowledge of ASPH expression and its relationship to hepatocyte function in general.

Project Narrative: Liver cirrhosis is one of the leading causes of death worldwide, and alcoholic liver disease (ALD) accounts for half of cirrhosis-associated death. Alcohol-induced DNA damage and the consequent hepatocyte cell death have been shown to participate in ALD progression. Clarifying the involvement of ALD-induced aspartate beta- hydroxylase down-regulation in exacerbating hepatocyte DNA damage and cell death may identify the potential therapeutic approach for ALD patients.