DESCRIPTION (provided by applicant): The central nervous system is susceptible to many environmental insults and like many organs can be affected by alcohol. Alcohol impacts the brain in a variety of ways including short-term cognitive changes, development of dependence, memory deficits, neuronal loss and initiation of neuroinflammation. An emerging mechanism being studied in the field of central nervous system (CNS) inflammation, extracellular vesicle communication, has not yet been investigated in alcohol-related neuroinflammation and offers the potential for therapeutic intervention. Key components of alcohol-induced neuroinflammation, the cytokines IL-1β and HMGB1, are thought to be released from cells via extracellular vesicles. This study will explore the hypothesis that alcohol alters the release of extracellular vesicles within the CNS and that these vesicles contain content critical to the inflammatory process. Our Preliminary Data reveals that EVs are released by CNS cell types and can be taken up by unstimulated cells. First, we examined the effect of alcohol exposure on microglia and astrocytes in vitro and found that exosomes were stimulated for release at either 50 or 100mM alcohol. These findings were confirmed with western blot against exosome marker CD63 in the supernatant. Next, we used the membrane dye PKH26 to label membranes of microglia which were then stimulated to release EVs by alcohol. Those EVs were transferred to untreated/unlabeled cells and the dye was seen to incorporate in recipient cells, suggesting that those EVs were taken up by the untreated cells. Specific Aim 1 will investigate the effect of alcohol on extracellular vesicle release from primary mouse CNS cells (neurons, microglia or astrocytes) in single cell-type cultures in vitro. Nanoparticle tracking analysis will be used to measure released vesicles size, which will allow for quantification of the two types of released vesicles: exosomes (<150nm diameter) or microvesicles (150nm-1μm). Proinflammatory cytokines IL-1β and HMGB1 will then be measured in vesicles secreted from CNS cell types after alcohol exposure. These experiments will provide important knowledge regarding alcohol's impact on vesicle release as well as vesicle content. As extracellular vesicles are believed to transmit intercellular signals, Specific Aim 2 will explore the effect of transferring alcohol-induced vesicles onto naïve cells. First, extracellular vesicle uptake by primary CNS cell types will be measured. Next brain slices maintained in culture will be exposed to vesicles derived from alcohol-exposed cells and activation of inflammatory pathways will be examined. Finally, IL-1β or HMGB1 will be individually knocked down or overexpressed in CNS cell types and alcohol-induced vesicles will be transferred onto brain slices. These experiments will test the effect that alcohol-induced extracellular vesicles have on other cells, as well as the contribution made by cargo cytokines. Specific Aim 3 will elucidate the impact that alcohol-induced vesicles have on the brain in vivo. First, we will investigate the concentrations of EVs required for intracranial injection and uptake in the brain by using fluorescently-labeled vesicles. Next, vesicles will be stimulated in vitro from primary mouse CNS cells exposed to alcohol. After isolating those vesicles, they will be injected into the brains of naïve mice. Brain tissue will b examined for increases in immune cell activation and upregulation of inflammatory signals. This experiment will provide important information regarding the impact of extracellular vesicles on inflammation in vivo. The first year of this fellowship will be dedicated to quantifying and qualifying the vesicles released by CNS cells after alcohol exposure. Specific Aim 2 will be investigated in years two and three of the fellowship, while Specific Aim 3 will be completed in year three. The final two years of the fellowship will be dedicated to completing the clinical rotations for my MD training as well as any necessary follow up experiments needed for publishing this proposed work.

PUBLIC HEALTH RELEVANCE This proposal seeks to explore mechanisms of alcohol-induced brain inflammation. This project will explore how alcohol may influence small vesicles released by brain cells, the messenger molecules that they carry and their impact on surrounding tissue. Knowledge about how these pathways may be therapeutically targeted to inhibit changes that occur in the brain following alcohol consumption and will provide new information about how the immune system functions in the brain.