/// PROJECT SUMMARY \\\ Alcohol use disorder (AUD) is a medical condition characterized by an impaired ability to stop or control alcohol use despite adverse social, occupational, or health consequences. AUD is common with a 12-month and lifetime prevalence in the US of 13.9% and 29.1%, respectively. Gene expression studies provide promising opportunities to better understand AUD etiology. As cells differ in their function, gene expression will typically also differ across cell-types. When studying bulk tissue, failure to account for cell diversity has a detrimental impact on the ability to detect disease associations. For example, case-control differences will be “diluted” if they affect only one cell-type, may cancel out if the differences are of opposite signs across cell-types, and may be undetectable if the differences involve low abundance cells. Furthermore, identifying the specific cell- types from which the association signals originate is key to formulating refined hypotheses of AUD etiology, designing proper follow-up experiments and, eventually, developing novel clinical interventions. Our overall goal is to perform a rigorous study in post-mortem brain samples to detect differentially expressed genes in AUD at a fine-grained cellular level, and identify the genetic elements that regulate these differences. To achieve this goal, we propose a strategy that has become available with the recent evolution of single nucleus RNA sequencing (snRNA-seq) technology that can characterize the expression levels of thousands of individual nuclei with a single reaction. Our pilot data shows that our lab is able to produce snRNA-seq data of the highest quality and illustrates the promise of snRNA-seq to detect biologically relevant findings. Using careful selection procedures, we obtained a unique collection of relatively severe cases that were diagnostically homogenous while having minimal comorbidities, and that were matched to the controls on key variables. We will focus on three brain regions that are heavily implicated in AUD and capture different aspects of the disease. Study design features were guided by a series of power analyses that are grounded in empirical observations from our pilot study. To ensure robust results, findings will be replicated using snRNA- seq data from independent individuals and validated with a different technology. Finally, we propose a series of follow-up analyses aimed at identifying potential regulators of replicating findings, (functionally) characterizing existing robust AUD GWAS associations, and detecting blood biomarkers of AUD disease processes in brain. Successful completion of this project will yield (i) unprecedented insights into AUD disease mechanisms, (ii) possible blood biomarkers of genes differentially expressed in brain, and (iii) lay the foundation for functional follow-up studies and, eventually, novel highly specific clinical interventions for improving AUD treatment.