Project Summary Alcohol is the most commonly used and misused substance amongst adolescents and young adults. Alcohol consumption during early life can produce persistent neurobiological consequences given that adolescence/young adulthood is a critical period characterized by significant and ongoing brain maturation. Investigating the neuropathophysiology salient to alcohol use (AU) is challenging given that brain tissue from living humans is not readily accessible for longitudinal measurement. We propose to circumnavigate this challenge by investigating the role of extracellular vesicle cargo in AU neurobiology of young people. ECVs are membrane-bound sacs that transport biologically active materials throughout the body.8 They are released by all tissues as a normal part of physiology, including brain neurons, and deviations in blood plasma ECV characteristics have been associated with psychiatric, substance use, and cognitive disorders. In particular, comparative ECV cargo analyses are of interest because ECV cargo reflects tissue of origin and may be used to infer functional roles for ECVs in target cells, pathophysiology, and treatment responsiveness. MicroRNAs (miRNAs) comprise a majority of blood plasma ECV cargo36, making them the best initial target for basic science ECV research. MiRNAs are attractive candidates for understanding the genetic architecture of AU in their own right and have been associated with AU in animal and human studies. Importantly, ECVs from brain cell lineages can be accessed in peripheral blood plasma, providing an opportunity to index brain biological processes that may be salient to AU neurobiology. We propose to leverage existing biospecimens to measure ECV- miRNAs and investigate AU pathophysiology in a sample of adolescents and young adults during a period of high AU. Specifically, this proposal will leverage data collected from an existing cohort (Parent R01 MH101518; Ntotal=860 twins; ages 15-22; 57% female) to improve our understanding of ECV-miRNA cargo, particularly those deriving from neurons, to add to the current understanding of the neurobiology of early life AU. We will select N=313 participants (ages 15-22; 52% female) from the parent R01, which included a broad and rich assessment of alcohol use, concurrent substance use, psychiatric history and symptoms, and environmental exposures (e.g., parenting, life stressors/adversity). Participants in the parent R01 also provided blood from which plasma was separated. This R21 proposal will address critical basic science questions about the nature of circulating miRNAs in young people and their relationship to AU early in life.