PROJECT SUMMARY/ABSTRACT Neuronal dysfunction underlies the general pathophysiological mechanisms related to heavy alcohol use and development of alcohol use disorder (AUD). Homeostatic glia-neuron communications underlie the development and functional maintenance of the central nervous system (CNS). Alcohol-induced neuropathology has been demonstrated to involve dysregulation of glia-neuron interactions, but the exact molecular and cellular mechanisms remain elusive. Evidence suggests involvement of extracellular vesicles, including exosomes, in CNS communication and the pathogenesis of CNS diseases. Recent studies point to the exosome as a potential mediator of astrocyte-neuron interactions. It has been shown that exosomes released from astrocytes are internalized by neurons and also that neuronal activity-dependent exosome secretion from astrocytes may modulate the efficacy of synaptic transmission and the preservation of axonal health. These suggest an important contribution of astrocyte-neuron communication by exosomes to neuronal integrity. Moreover, the utility of brain- derived plasma exosomes as a biomarker for neuropathology has been demonstrated. Thus, it has been shown that genetic information associated with brain tumor or brain injury is detectable in plasma/serum microvesicles of patients, suggesting that brain-derived exosomes may serve as cargo for diagnostic biomarkers. Astrocytic exosomes may also play significant roles in the pathophysiology of AUD, but it is unknown if heavy alcohol exposure affects the biogenesis of astrocytic exosomes, their trafficking and distribution, and further biological functions. This R21 exploratory grant application is designed to address our hypothesis that chronic alcohol affects the biogenesis and distribution of astrocyte-derived exosomes and their molecular components thereby affecting astrocyte-neuron communication. We propose two Specific Aims to achieve our immediate goals: (1) develop an in vivo model of astrocyte-derived exosome tracking with which to profile the astrocyte-derived exosomal RNAs, and (2) determine the effect of chronic intermittent ethanol (CIE) vapor exposure on the biogenesis, distribution, and RNA signatures of astrocyte-derived exosomes. By achieving these aims, we will be able to determine the potential roles of astrocyte exosomes in transducing the effects of chronic alcohol exposure onto neuronal cells. We will also gain insight into the potential utility of brain-origin exosomes and their contents as biomarkers of alcohol dependence-related abnormalities. This reporter model may then be used to study the mechanisms of astrocyte involvement in various CNS homeostatic functions and numerous other pathological conditions.