Downregulation of Rab3D: Critical Role in Golgi Disorganization and the Pathogenesis of Alcoholic Liver Disease

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The goal of this VA Merit is to examine how ethanol exposure results in impaired function of the Golgi apparatus. The Golgi apparatus (also called the Golgi body or Golgi complex) packages proteins into membrane bound vesicles inside the cell before the vesicles are sent to their destination. As such, this organelle resides at the intersection of the secretory, lysosomal, and endocytic pathways; it is known to be of particular importance in processing proteins for secretion. Previous work from our laboratory has identified multiple defects in endocytosis, protein trafficking, and secretion, after alcohol administration, but we have not until now, examined a role for altered Golgi function in these processes. Because the incidence of alcoholic liver disease (ALD) is greater in the Veteran population and more than half of all medical admissions in VA Medical Centers across the country are linked to alcohol abuse, we are focusing efforts towards the identification of potential targets to intervene during the progressive injury which occurs after chronic alcohol administration, and perhaps the Golgi will prove to be such a target. Of central importance to our study is the role of a small GTPase, Rab3D, which is involved in exocytosis, secretion and vesicle trafficking. We have shown that Rab3D protein content was significantly decreased after alcohol administration, and recently we have obtained exciting new preliminary data that ethanol-impaired Rab3D function plays an important role in Golgi disorganization and fragmentation. The studies proposed in this application will extend our ongoing investigation of how ethanol alters hepatocyte biology, specifically in protein processing, to an examination of its role in transport through the Golgi. We provide a concept as to how alcohol-induced remodeling of Golgi morphology is a significant impairment of post-Golgi trafficking, and this leads to utilization of trans-Golgi membranes for the formation of autophagosomes. These recent and novel findings provide an excellent foundation for this proposal and support our central hypothesis that EtOH-induced down-regulation of Rab3D disrupts the assembly and function of Golgi apparatus leading to impaired protein trafficking and metabolism, contributing to liver injury. To examine this hypothesis, we have proposed three specific aims; in Aim 1 we will determine how the function of Rab3D regulates the integrity of the Golgi, in Aim 2 we will examine how autophagy is linked to EtOH-induced Golgi disorganization, and in Aim 3 we will explore recent preliminary data examining recovery of compact Golgi and reconstitution of trans-Golgi membranes. Altogether, successful completion of these aims will characterize the effect of EtOH on Golgi disorganization, and establish a role for altered Rab3D during this process. We will be able to correlate mechanisms of alcohol-mediated liver cell trafficking impairments with impaired Golgi function and provide key information that could lead to therapeutic strategies aimed at reducing or eliminating liver injury. Dr. Casey is the principal investigator, and she is joined by three outstanding co-investigators (Drs. Petrosyan, Thomes and Rasineni). The investigators have complementary strengths which are essential for the success of the project. All four are experienced with alcoholic liver injury, and Dr. Casey has specific expertise in protein trafficking and endocytosis, while Dr. Rasineni is currently examining Rab3D function in another organ, the pancreas. Dr. Petrosyan is an expert in Golgi morphology, and Dr. Thomes brings years of experience in autophagy for the project. Together with this expertise and our exciting and innovative approach, we anticipate that we will be successful in these studies and will be able to contribute to improved healthcare for Veteran patients by the identification of mechanisms involved in the alcoholic liver injury.