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

项目摘要/摘要 正在进行的阿片类药物流行是我们这个时代最重要的公共卫生危机之一，吸毒过量 自1999年以来，死亡人数翻了两番，超过了近年来的持枪杀人案。给出了个人 和阿片类药物依赖的社会负担，有必要描述 可以有针对性地治疗和干预的依赖。项目2中提出的工作结合了 多组学资源，以检验关于阿片类药物功能脑结构的特定假说 成瘾(OA)。在现有理论的指导下，涉及前额叶皮质(PFC)、伏隔核(NAC)、 和杏仁核在物质滥用的循环中，我们建议在以下位置生成和合并现有数据 使用阿片类药物过量收集的死后人体组织在每个脑区的多个组学水平 病例和配对对照。我们使用的测量方法是全基因组的，在每个 三个脑区和每个生物域：单核苷酸多态(SNP)、表观基因组(DNA 甲基化和组蛋白乙酰化)和转录本(通过RNA-seq)在我们的联合样本(N=641)中 后续单细胞验证/复制。 我们将分三个目标实现这些目标。在目标1中，在现有的合并样本中，我们将填充 找出目前在特定大脑/区域或生物域中未评估的大脑区域，并执行 分析以检测特定区域和一般(跨脑区域)甲基化、组蛋白乙酰化和 病例组和对照组的基因表达差异。此外，我们将进行针对以下目标的分析 跨越这些生物领域的整合。在目标2中，我们将对目标1的发现进行单细胞追踪 要准确定位PFC、NAC和杏仁核中导致目标1中确定的差异的所有细胞类型，请使用 RNA-seq在荧光激活细胞分选(FACS)分离细胞和RNA-FISH中的验证。在AIM 3、我们将在需要的地方对样本进行基因分型，并进行遗传关联图谱-组蛋白修饰， DNA甲基化和基因表达QTL定位-以确定导致 在AIM 1中观察到的病例和对照之间的调控差异。这些QTL方法将允许我们 确定遗传基础上的调控差异，与作为结果而获得的身体上的差异 长期使用阿片类药物、暴露或其他环境因素。 我们的总体目标是创建一个框架，在这个框架中，我们可以在大脑中映射出 基因组集(基因、途径、特征)并生成可在项目1和 Synergy Core通过mapOA，并在项目3中通过选定的基因，目标是在 利用基因工程、病毒载体注射和药理学方法在dlPFC和NAC中进行活体实验 老鼠。通过这种协同作用，我们将区分诱因和随之而来的暴露 并朝着机械性理解的方向发展，从而推动新的OA治疗方法。

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.