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 尽管约有18岁以上的美国人中约有54％的美国人饮酒，但只有6.5％的美国人符合标准 酒精使用障碍（AUD）。迄今为止，有三种FDA批准的药物可以治疗AUD，每个药物都有 单独的疗效变化。了解电路特异性的分子机制 导致AUD的变化对于开发新颖，有针对性和有效治疗AUD至关重要。在 该目标的进步，我们已经开始确定全基因组DNA甲基化（DNAM）信号 伏隔核（NACC）区分低和重型乙醇饮用猴子。这些子集 差异DNAM（DNAM）信号与在中发挥作用的基因的表达相关 调节神经传递。特别是，我们在功能上发现了基因中的d-dnam信号 与包括突触前和突触后的四边形突触的不同隔室有关 元素，星形胶质过程和细胞外基质。 NACC的谷氨酸能输入来自 NACC以外的大脑区域，包括Brodmann区域32（A32，灵长类动物； 啮齿动物）。通过调查A32输入到NACC中，我们将开始阐明电路特异性的DNAM信号 区分低/非常沉重的乙醇饮用者。我们将利用高度相关和良好的优势 表征非人类灵长类动物（NHP）酒精饮酒模型，其中猕猴每天都在 超过12个月，自选为低饮酒者。全基因组DNAM测序（GW-DNAM） 将用于鉴定酒精剂量相关的差异甲基化细胞（DMC）和区域（DMR） 在A32和NACC中。使用具有荧光标签的神经元特异性抗体（NEUN）和 荧光激活的细胞分选，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，我们将使用这些相同NHP A32目标的DNAM状态 在高乙醇偏好小鼠中饮酒3个月的高乙醇偏好小鼠中的PL中的Bisulfite测序 并自我选择为低饮酒者。这些研究将产生基因/调节区域作为目标 基于消费量的差异，与乙醇剂量高度相关 在四方突触中的作用。使用更改表达式，功能或 基因靶标的甲基化水平，我们将执行小鼠功能测定法，该测定将测试其在乙醇中的作用 饮酒（乙醇的自我给药3个月）和在PL-NACC电路中（使用离体切片进行测试 电生理学）。小鼠功能测定最有效的靶标将在离体切片中测试 从慢性乙醇饮用NHP的能力从慢性乙醇中获得的能力改变了A32和NACC电路。总共这项工作 将确定与A32/PL-NACC电路相关的酒精剂量依赖性DNAM修饰，并且 为有希望的新AUD治疗设计提供功能支持。