Project Summary There is strong association between cannabis use and depression and suicide, but the underlying cellular links are poorly understood. This presents a challenge for understanding how drug use can lead to additional psychiatric disorders. It is believed that cannabis use alters the development of key brain regions which in turn makes the brain more susceptible to additional stressors that could lead to major depressive disorder (MDD). These findings are particularly concerning given the popularity of cannabis legalization. One of the key unsolved problems is how to link drug use with specific molecular changes in the brain that can lead to depression. In this proposal, we take the first steps to bridging this gap. First, we will use innovative single cell type postmortem genomics to identify the molecular (transcriptomic and chromatin) changes occurring in the cannabis use disorder (CUD) brain. We will focus our studies on two discreet brain regions: the dorsolateral prefrontal cortex and the ventral striatum in 120 donors with CUD, MDD, and neurotypical controls. We will use reverse genetic approaches to identify genomic alterations in the CUD brain that harbor GWAS risk signals for MDD. We hypothesize that these alterations lead to widespread epigenomic changes that make the brain vulnerable to stress and developing MDD. We will prioritize a credible set of genomic regions and genes and validate their function in a human iPSC brain organoid model using massively parallel reporting assays (MPRA). We will also test the effect THC (the active compound in cannabis) has on these organoids by comparing THC-exposed organoids from donors with a history of CUD (but not MDD) with our MDD postmortem brain genomics to identify convergent molecular mechanism changes. These studies will uncover genes driving MDD pathogenesis and establish if cannabis use shares and drives these mechanisms. We expect this project will open new lines of exploration in the comorbidity of substance use disorders and major depression and contribute broadly to understanding the relationship between gene regulation and functional roles in cannabis use, which may identify new therapeutic targets.