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 尽管大约54%的18岁以上的美国人饮酒，但只有6.5%的人符合 酒精使用障碍(AUD)。到目前为止，FDA批准的治疗AUD的药物有三种，每种药物都有 在个体基础上不同的功效。了解电路特定的潜在分子机制 导致AUD的变化对于开发新的、有针对性的和有效的AUD治疗方法至关重要。在……里面 为了推进这一目标，我们已经开始在基因组范围内识别DNA甲基化(DNaM)信号 伏隔核核心(NACC)，用于区分低酒精饮酒和高酒精饮酒猴子。其中的一个子集 差异dNaM(D-dNaM)信号与起作用的基因的表达有关 调节神经传递。特别是，我们在具有功能的基因中发现了D-dNaM信号 与包括突触前和突触后的四部分突触的不同隔室相关的 元素、星形胶质细胞突起和细胞外基质。谷氨酸能传入NACC的来源是 NACC以外的脑区，包括Brodmann区32(灵长类为A32；大脑皮层(PL) 啮齿动物)。通过研究A32对NACC的输入，我们将开始阐明特定于电路的dNaM信号 区分低度酒精饮用者和重度酒精饮用者。我们将利用高度相关和良好的- 描述了非人灵长类(NHP)饮酒模型，猕猴每天在该模型中饮酒 超过12个月，自己挑选出酗酒和酗酒的人。全基因组dNaM测序(GW-dNaM) 将用于识别与酒精剂量相关的差异甲基化胞嘧啶(DMC)和区域(DMR) 在A32和NACC上。使用神经元特异性抗体(Neun)与荧光标记的组合，以及 荧光激活的细胞分类，我们将开始阐明dNaM中的细胞特异性(即神经元特异性) NHP A32的信号。同时，我们将使用以下命令检查这些相同NHP A32目标的dNaM状态 饮酒3个月的高酒精偏好小鼠PL亚硫酸氢盐扩增序列测定 自己挑选出酗酒和酗酒的人。这些研究将产生作为靶点的基因/调控区域 与酒精剂量高度相关，基于消费差异，在不同物种和游戏中保存 在四部分突触中扮演一个角色。使用工程病毒载体改变表达、功能或 基因靶标的甲基化水平，我们将进行小鼠功能测试，以测试它们在乙醇中的作用 饮酒(自我注射酒精3个月)和PL-NACC回路(使用体外切片进行测试 电生理学)。小鼠功能测试中最有效的靶点将在体外切片中进行测试 从长期饮用乙醇的NHP中获得，因为它们有能力改变A32和NACC电路。总的来说，这项工作 将确定针对A32/PL-NACC电路的依赖酒精剂量的dNaM修饰，以及 为设计有前景的AUD新疗法提供功能支持。