Derick S. Han, Ph.D. - US grants

DESCRIPTION (provided by applicant): It is my long term goal to become an independent investigator and study the pathogenesis of liver disease including alcoholic liver disease. Obtaining a K01 grant would be an important step for me to become an independent researcher since it will provide me an opportunity and time to overcome many weaknesses (lack of preliminary data and publications in alcohol research, previous lack of focus on a clinically important model system, inexperience in cell signaling, cell imaging, and molecular biology techniques) in my background to become competitive for an R01 in alcohol research in the near future. By providing my salary and research funding, the K01 grant will free me from working on other PIs'projects and allow me to really focus on alcohol, as well as become more experienced in cell signaling, cell imaging, and molecular biology. The area of science I have chosen to pursue in the upcoming period is at the interface of redox biology, signal transduction and hepatotoxicity using alcohol as a model of liver injury. TNF-induced damage to hepatocytes remains a central point in alcohol-induced liver damage. Recent evidence from our laboratory suggests that redox alterations through GSH modulation or oxidant treatment sensitize hepatocytes to TNF-induced apoptosis by inhibiting NF-?B transcriptional activity. This suggests a link between TNF secretion and redox alterations in mediating damage to liver. The hypothesis to be tested is that redox alterations caused by alcohol sensitize hepatocytes to TNF-induced apoptosis through inhibition of NF-?B dependent signaling pathways. The hypothesis will be tested with the following specific aims: 1) Determine how alcohol-induced alterations of cellular redox status modulates NF-?B signaling and TNF-induced apoptosis in cultured primary hepatocytes. The question of whether alcohol-induced redox changes inhibit NF-?B dependent gene expression in the presence of TNF in cultured primary hepatocytes will be explored. 2) Determine the effect of chronic alcohol consumption on hepatic redox status, NF-?B redox status, NF-?B signaling pathways, N binding to DNA, and NF-?B dependent gene expression in liver. The relationship between NF-?B signaling and redox status will be explored following chronic alcohol treatment in vivo. A focus of this application is to utilize imaging techniques (e.g., confocal microscopy, flow cytometry) to study redox status and NF-?B signaling simultaneous in the same cell. These methods will allow me to assess possible heterogeneity in redox status and NF-?B signaling that alcohol may induce to the hepatocyte population. The outcome of these studies will provide new insights into the mechanism by which alcohol causes liver damage. Public Health Relevance: The outcome of these studies will provide new insights into the mechanism by which alcohol causes liver damage. The findings of this application may shed light on new therapeutic strategies involving thiol antioxidants for prevention or treatment of alcohol related diseases.

ABSTRACT The overarching goal of this proposal is to elucidate the role that mitochondrial remodeling in the liver plays in alcoholic liver disease (ALD). Our recent exciting findings suggest that chronic alcohol feeding causes dynamic mitochondrial remodeling in the liver that enhances mitochondrial bioenergetic activity as an adaptation to alcohol. We observed two major types of mitochondrial remodeling following alcohol feeding: 1) increased biogenesis of key mitochondrial constituents (e.g. expression of respiratory complex proteins, pyridine nucleotide levels) and, 2) alterations in liver mitochondrial morphology through changes in mitochondrial fusion-fission rates. Our findings add new insights to the established dogma that ALD primarily involves mitochondrial dysfunction. While this paradigm of mitochondrial dysfunction in ALD has been widely accepted for decades, it represents an incomplete picture of mitochondrial dynamics in the liver. Chronic alcohol feeding causes some mitochondrial dysfunction, but it also induces a great deal of mitochondrial remodeling in the liver as an adaptation to the stress induced by alcohol intake. In this proposal, we will examine the significance of mitochondrial remodeling in ALD by modulating two distinct pathways in the liver. Peroxisome proliferator-activated receptor gamma coactivator-1? (PGC-1?) is the master regulator of mitochondrial biogenesis, and our preliminary data suggests that it plays a role in alcohol- induced mitochondrial biogenesis, particularly of respiratory complexes in the liver. Consequently, we will silence PGC-1? using antisense (ASO) to determine if mitochondrial biogenesis plays a beneficial (i.e. adaptation) or deleterious role in the liver with chronic alcohol feeding. During ALD, like in many pathologies, mitochondrial fusion-fission alters to increase mitochondrial heterogeneity, which produces subpopulations of mitochondria that may have different properties (respiration, reactive oxygen species generation, JNK binding). We will modulate mitochondrial fusion-fission rates using ASO (e.g. Mfn-2 - produces fragmented mitochondria; Opa-1 - produces larger mitochondria) to alter mitochondrial heterogeneity to determine its significance in the pathogenesis of ALD. The proposal has two specific aims: 1) Determine the significance of mitochondrial biogenesis in the pathogenesis of ALD, and 2) Determine the extent and significance of mitochondrial heterogeneity that occurs in the liver with alcohol feeding. Overall, by modulating two different aspects of mitochondrial remodeling (biogenesis and fusion-fission), our proposal should provide new insights on the role mitochondrial remodeling plays in ALD. Although our proposal focuses on alcohol, we believe the findings of this study will have broader implications in liver pathophysiology, since many liver diseases, such as non-alcoholic fatty liver disease, are also associated with mitochondrial remodeling.

PROJECT SUMMARY The long-term goal of this proposal is to determine how chronic alcohol intake modulates the liver-to-brain axis to induce and/or promote Alzheimer's disease (AD) pathology. Studies have largely focused on the direct action of alcohol on the brain and studies looking outside the brain to understand how alcohol modulates AD pathology are lacking. Our exciting preliminary data have identified two potential alcohol-induced changes to the liver that could induce and/or promote AD pathology in the brain. First, we have discovered that chronic alcohol feeding reduces hepatic low-density lipoprotein receptor-1 (LRP1), a receptor essential in removing peripheral amyloid-beta (A?). Since peripheral A? can be transported across the blood-brain barrier (BBB) by receptor for advanced glycation end products (RAGE) and become deposited in the brain, it is conceivable that altered LRP1- mediated hepatic A? clearance can significantly affect brain A? load. Second, our work shows that peripheral tumor necrosis factor-? (TNF-?) secreted by the liver and other organs during alcohol-induced injury can greatly impact the BBB and AD pathology. In AD transgenic mice, peripheral TNF-? blockage by the TNFR-Fc fusion protein (etanercept) reduces AD pathology, and our exciting preliminary data shows enhanced A?(1-42) migration across the brain endothelium due to TNF-?-mediated increase in BBB-permeability in vitro. This proposal will explore these novel findings to provide an integrated examination of how alcohol intake may alter the liver-to-brain axis to induce and/or promote AD pathology. The proposal has two specific aims: 1) Delineate the effect of alcohol on A? clearance by the liver and examine its impact on peripheral-to-central A? homeostasis. Our working hypothesis is that alcohol intake alters hepatic peripheral A? clearance through LRP1 downregulation to increase peripheral-to-central A? load. 2) Characterize the effect of alcoholic-liver-injury- induced peripheral inflammation on neurovascular- and neuronal-degeneration, and its impact on AD pathology. Our working hypothesis is that alcoholic-liver-injury-induced peripheral inflammation causes BBB dysfunction, increases AD hallmark pathology (A? and tau-tangles), and modulates neuroinflammation, thereby inducing and/or potentiating AD pathology. The proposal will combine specialists in the areas of liver/alcohol research with those in AD/BBB research to provide a comprehensive exploration of the liver-to-brain axis utilizing state- of-the-art in vivo and in vitro techniques and models, which will increase synergy and likelihood of success. The resulting new knowledge will enable the identification of new therapeutic-targets and provide mechanistic insight into alcohol-dependent AD, and will delineate the importance of the liver-to-brain axis in AD pathology, an unexplored concept in the emerging field of alcohol-dependent AD.