Gene Regulation in the Opioid Dependent Human Brain (Project 2)

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PROJECT SUMMARY/ABSTRACT The ongoing opioid epidemic is one of the most important public health crises of our time with overdose fatalities quadrupling since 1999 and surpassing those of gun homicides in recent years. Given the individual and societal burden of opioid dependence, it is necessary to characterize the biological pathways to dependence that can be targeted for treatment and intervention. The work proposed in Project 2 combines multi-omics resources to examine specific hypotheses regarding the functional brain architecture of opioid addiction (OA). Guided by existing theory implicating the prefrontal cortex (PFC), nucleus accumbens (NAc), and amygdala in the cycle of substance abuse, we propose to generate and combine existing data at multiple omics levels in each brain region using postmortem human tissue collected from opioid overdose cases and matched controls. The measurement approaches we use are genome-wide, in each of the three brain regions and in each biological domain: single nucleotide polymorphism (SNP), epigenome (DNA methylation and histone acetylation), and transcript (via RNA-seq) in our combined sample (N=641) with follow-up single-cell validation/replication. We will accomplish these goals across three aims. In Aim 1, in the existing combined sample, we will fill out the brain regions not currently assessed in specific brain/regions or biological domains and perform analyses to detect region-specific and general (across-brain regions) methylation, histone acetylation, and gene expression differences between cases and controls. In addition, we will perform analyses aimed at integrating across those biological domains. In Aim 2, we will perform single-cell follow-up of Aim 1 findings to pinpoint all cell types within the PFC, NAc, and amygdala that drive the differences identified in Aim 1, with validation by RNA-seq in fluorescence activated cell sorting (FACS) separated cells and RNA-FISH. In Aim 3, we will genotype samples where needed and perform genetic association mapping—histone modification, DNA methylation, and gene expression QTL mapping—to identify genetic variants that account for the regulatory differences observed between cases and controls in Aim 1. These QTL approaches will allow us to identify regulatory differences with a genetic basis, versus those somatically acquired as a consequence of chronic opiate use, exposure, or other environmental factors. Our overall goal is to create a framework in which we can map, in brain, the OA functional relevance of genomic sets (genes, pathways, features) and generate results that can be leveraged in Project 1 and the Synergy Core through mapOA, and in Project 3 through selected genes, targeted for functional validation in vivo in the dlPFC and NAc using genetic engineering, viral vector injection, and pharmacology approaches in mice. Through this synergy we will distinguish between predisposing and exposure consequent dysregulation and move toward mechanistic understanding that can drive new treatments of OA.