2/2 NADIA U24 Epigenetic/Molecular Core

 描述（由申请人提供）：酒精是青少年最广泛使用的成瘾药物之一，持续使用和滥用可导致精神疾病的发展，包括成年后的酗酒。与成年人相比，青少年对酒精表现出不同的敏感性。遗传和环境风险因素在酒精中毒的发展中发挥作用。对人类和动物酒精中毒模型的遗传研究已经确定了可能在酒精中毒病理生理学中至关重要的基因。表观遗传机制，参与基因表达的调节，最近出现的神经精神疾病的研究领域，使我们能够更好地了解人类疾病的分子机制，包括精神和酒精使用障碍。表观遗传过程，如组蛋白乙酰化和DNA甲基化机制，已被证明在神经成熟中发挥作用，有助于大脑发育过程中基因表达的稳定性。NADIA（青少年成年饮酒神经生物学）研究已经确定了关键大脑区域中的几个基因和分子通路，这些基因和分子通路被青少年间歇性乙醇（AIE）暴露改变，并参与神经炎症，突触可塑性和神经发生。然而，表观遗传学机制在成人AIE诱导的病理病因学仍在探索。表观遗传/分子核心的主要目标是为NADIA的每个研究组成部分的基因靶点子集的实验分析提供资源，以了解在研究中AIE诱导的分子和行为变化中起作用的表观遗传机制。我们假设，由于AIE的表观遗传过程的扰动可能会导致关键的大脑回路（前额叶皮层，杏仁核，海马，下丘脑，隔，腹侧被盖区，和核丘脑）的转录的动态变化，负责持续的行为和神经化学表型在成年期。以下具体目的将检验这一假设：1）使用实时定量PCR（qPCR）检查NADIA每个研究组分的选定靶基因mRNA水平的AIE诱导变化。2)使用染色质免疫沉淀（ChIP）和qPCR检查AIE诱导的与每个项目的靶基因启动子相关的组蛋白修饰（组蛋白H3-K9乙酰化/甲基化）。3)使用基于甲基DNA免疫沉淀（MeDIP）或甲基-CpG结合结构域（MBD）的测定（MethylMiner（tm））和qPCR检查靶基因启动子的DNA甲基化状态。Core还将使用5-羟甲基胞嘧啶免疫沉淀（hMeDIP）试验检查靶基因启动子的5-羟甲基胞嘧啶水平。该核心将能够检查NADIA中各个脑区中AIE介导的基因表达变化的表观遗传机制。这项工作将确定表观基因组内的共同分子靶点，这可能导致开发新的治疗策略，用于治疗青少年酗酒导致的成人精神病理学。

 DESCRIPTION (provided by applicant): Alcohol is one of the most widely used addictive drugs in adolescence and continued use and abuse can lead to the development of psychiatric disorders including alcoholism in adulthood. Adolescents show differential sensitivity to alcohol as compared to adults. Both genetic and environmental risk factors play roles in the development of alcoholism. Genetic studies in human and animal models of alcoholism have identified genes that may be critical in the pathophysiology of alcoholism. Epigenetic mechanisms, involved in the regulation of gene expression, have recently emerged as a promising area of research into neuropsychiatric illnesses and enabled us to better understand the molecular mechanisms of human diseases, including psychiatric and alcohol use disorders. Epigenetic processes, such as histone acetylation and DNA methylation mechanisms, have been shown to play a role in neuromaturation by contributing to the stability of gene expression during brain development. NADIA (Neurobiology of Adolescent Drinking in Adulthood) studies have identified several genes and molecular pathways in key brain regions that are altered by adolescent intermittent ethanol (AIE) exposure and that are involved in neuroinflammation, synaptic plasticity and neurogenesis. However, the epigenetic mechanisms operative in the etiology of AIE-induced pathology in adulthood are still underexplored. The major objective of the Epigenetic/Molecular core is to provide the resources for experimental analyses of a subset of gene targets to each Research Component of NADIA, in order to understand epigenetic mechanisms that are operative in AIE- induced molecular and behavioral changes under investigation. We hypothesize that perturbations of epigenetic processes due to AIE may lead to dynamic changes in transcription in key brain circuitries (prefrontal cortex, amygdala, hippocampus, hypothalamus, septum, ventral tegmental area, and nucleus accumbens) that are responsible for persistent behavioral and neurochemical phenotypes in adulthood. The following Specific Aims will test this hypothesis: 1) To examine AIE-induced changes in mRNA levels of selected target genes for each research component of NADIA using real-time quantitative PCR (qPCR). 2) To examine AIE-induced histone modifications (histone H3-K9 acetylation/methylation) associated with target gene promoters for each project using chromatin immunoprecipitation (ChIP) followed by qPCR. 3) To examine DNA methylation status of promoters of target genes using methyl DNA immunoprecipitation (MeDIP) or methyl-CpG binding domain (MBD) based- assays (MethylMiner(tm)) followed by qPCR. The Core will also examine levels of 5-hydroxymethylcystosine of target genes promoters using 5- hydroxymethylcytosine immunoprecipitation (hMeDIP) assays. This core will be able to examine epigenetic mechanisms underlying AIE-mediated changes in gene expression in various brain regions across NADIA. This effort will identify common molecular targets within the epigenome that may lead to the development of novel treatment strategies for adult psychopathologies resulting from adolescent binge drinking.