Translational examination of alcohol-associated epigenetic signatures: from primates to rodents

酒精相关表观遗传特征的转化检查：从灵长类动物到啮齿类动物

• 批准号: 10056068
• 负责人: Verginia Carmella Cuzon Carlson
• 金额: $ 23.52万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10056068
• 关键词: Age; Alcohol abuse; Alcohol consumption; Alcoholic beverage heavy drinker; Alcohols; American; Anatomy; Anterior; Antibodies; Astrocytes; Behavioral; Biological Assay; Brain region; Brodmann' s area; Cause of Death; Cells; Consumption; Corpus striatum structure; Cytosine; DNA Methylation; DNA methylation profiling; Data; Data Set; Development; Dorsal; Dose; Electrophysiology (science); Elements; Engineering; Epigenetic Process; Equilibrium; Ethanol; Extracellular Matrix; FDA approved; Female; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Targeting; Genes; Genomic Segment; Glutamates; Goals; Heavy Drinking; Individual; Lead; Link; Macaca; Macaca mulatta; Maps; Methylation; Modeling; Modification; Molecular; Monkeys; Mus; Neurons; Nucleic Acid Regulatory Sequences; Nucleus Accumbens; Pathway Analysis; Pharmaceutical Preparations; Pharmacology; Play; Primates; Process; Regulation; Regulator Genes; Relapse; Rodent; Role; Sampling; Self Administration; Signal Transduction; Slice; Specificity; Synapses; Technology; Testing; Tissues; Validation; Viral; Viral Vector; Work; alcohol effect; alcohol response; alcohol use disorder; annotation system; base; behavioral genomics; bisulfite sequencing; brain tissue; cell type; chronic alcohol ingestion; cingulate cortex; design; drinking behavior; effective therapy; epigenome; epigenomics; genetic manipulation; genome-wide; male; methylome; mouse genome; neural circuit; neuroadaptation; neurotransmission; nonhuman primate; novel; postsynaptic; preference; synaptic function; tool; transcriptome sequencing; transcriptomics; translational approach; transmission process

PROJECT ABSTRACT Although approximately 54% of Americans over the age of 18 consume alcohol, only 6.5% meet the criteria for an alcohol use disorder (AUD). To date, there are three FDA approved medications to treat AUD, each having varying efficacy on an individual basis. Understanding the molecular mechanisms underlying circuit-specific changes that lead to AUD is essential to developing novel, targeted, and effective treatments for AUD. In advancement of this goal, we have begun to identify genome-wide DNA methylation (DNAm) signals within the nucleus accumbens core (NAcC) that distinguish low and heavy ethanol drinking monkeys. A subset of these differential DNAm (D-DNAm) signals were associated with the expression of genes that play a role in modulating neurotransmission. In particular, we found D-DNAm signals in genes that are functionally associated with different compartments of the tetrapartite synapse that includes pre- and postsynaptic elements, astroglial processes, and the extracellular matrix. Glutamatergic inputs into the NAcC arise from brain regions outside of the NAcC, including Brodmann area 32 (A32, in primates; prelimbic cortex (PL) in rodents). By investigating A32 inputs into NAcC, we will begin to elucidate circuit-specific DNAm signals that distinguish low from heavy/very heavy ethanol drinkers. We will use to advantage the highly relevant and well- characterized nonhuman primate (NHP) alcohol drinking model in which rhesus macaques imbibe alcohol daily for over 12 months and self-select into low and heavy drinkers. Genome-wide DNAm sequencing (GW-DNAm) will be used to identify alcohol-dose associated differentially methylated cytosines (DMCs) and regions (DMRs) in the A32 and NAcC. Using a combination of a neuron specific antibody (NeuN) with a fluorescent tag and fluorescence-activated cell sorting, we will begin to elucidate cell specificity (i.e. neuron-specific) in the DNAm signals of the NHP A32. In parallel, we will examine the DNAm state of these same NHP A32 targets using amplicon bisulfite sequencing in PL in high-ethanol preference mice that have consumed alcohol for 3 months and self-select into low and heavy drinkers. These studies will yield gene/regulatory regions as targets that are highly correlated with ethanol dose, based on differences in consumption, conserved across species and play a role in the tetrapartite synapse. Using engineered viral vectors that alter the expression, function or methylation level of gene targets, we will perform a mouse functional assay that will test their role in ethanol drinking (self-administration of ethanol for 3 months) and in the PL-NAcC circuit (tested using ex vivo slice electrophysiology). The most efficacious targets from the mouse functional assay will be tested in ex vivo slices obtained from chronic ethanol drinking NHPs for their ability to alter A32 and NAcC circuitry. In total, this work will identify alcohol dose-dependent DNAm modifications that are specific to the A32/PL-NAcC circuit, and provide functional support for the design of promising new AUD treatments.

项目摘要 虽然大约54%的18岁以上的美国人饮酒，但只有6.5%的人符合标准， 酒精使用障碍（AUD）迄今为止，有三种FDA批准的药物用于治疗AUD，每种药物具有 在个体基础上改变功效。了解特定电路的分子机制 导致AUD的变化对于开发针对AUD的新型、靶向和有效治疗至关重要。在 为了推进这一目标，我们已经开始识别基因组范围内的DNA甲基化（DNAm）信号。 神经核核心（NAcC），区分低和高酒精饮用猴。其中一部分 差异DNAm（D-DNAm）信号与基因的表达相关，这些基因在 调节神经传递特别是，我们发现D-DNAm信号存在于功能上 与包括突触前和突触后的四分突触的不同隔室相关 成分、星形胶质细胞过程和细胞外基质。进入NAcC的谷氨酸能输入来自 NAcC外的脑区，包括灵长类动物的布罗德曼32区（A32）;灵长类动物的前边缘皮层（PL）。 啮齿动物）。通过研究A32对NAcC的输入，我们将开始阐明电路特异性DNAm信号， 区分低酒精饮用者和重度/重度酒精饮用者。我们将利用高度相关和良好的- 特征非人灵长类动物（NHP）饮酒模型，其中恒河猴每天饮酒 12个月以上，并自我选择成低和重度饮酒者。全基因组DNAm测序（GW-DNAm） 将用于识别酒精剂量相关的差异甲基化胞嘧啶（DMC）和区域（DMR） 在A32和NAcC中。使用神经元特异性抗体（NeuN）与荧光标签的组合， 荧光激活细胞分选，我们将开始阐明DNA中的细胞特异性（即神经元特异性）。 NHP A32的信号。同时，我们将使用以下方法检查这些相同NHP A32靶标的DNAm状态： 在已经饮用酒精3个月的高乙醇偏好小鼠中PL中的扩增子亚硫酸氢盐测序 并自我选择成低度和重度饮酒者。这些研究将产生基因/调控区作为靶点， 与乙醇剂量高度相关，基于消费差异，在物种和游戏中保持不变 在四分突触中的作用。使用工程化的病毒载体， 基因靶点的甲基化水平，我们将进行小鼠功能测定，以测试它们在乙醇中的作用。 饮用（自我给予乙醇3个月）和在PL-NAcC回路中（使用离体切片测试 电生理学）。将在离体切片中测试来自小鼠功能测定的最有效靶标 从慢性乙醇饮用NHP获得，因为它们能够改变A32和NAcC回路。总的来说，这项工作 将鉴定对A32/PL-NAcC回路特异性的酒精剂量依赖性DNAm修饰， 为设计有前景的新AUD治疗方法提供功能支持。