2/2 NADIA U24 Epigenetic/Molecular Core

 描述(申请人提供)：酒精是青春期最广泛使用的成瘾药物之一，持续使用和滥用会导致成年后出现包括酗酒在内的精神障碍。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都在酒精中毒的发生发展中发挥作用。在酒精中毒的人类和动物模型中进行的遗传学研究已经确定了在酒精中毒的病理生理学中可能至关重要的基因。表观遗传机制涉及基因表达的调控，最近成为神经精神疾病研究的一个有前途的领域，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，有助于脑发育过程中基因表达的稳定性。NAIDA(成年期青少年饮酒的神经生物学)研究发现，青少年间歇性乙醇(AIE)暴露会改变关键大脑区域的几个基因和分子通路，这些基因和分子通路与神经炎症、突触可塑性和神经发生有关。然而，在AIE诱导的成年期病理的病因学中操作的表观遗传学机制仍未被充分探讨。表观遗传学/分子核心的主要目标是为NAIDA的每个研究成分提供一组基因靶标的实验分析资源，以了解在AIE诱导的分子和行为变化中起作用的表观遗传学机制。我们推测，AIE引起的表观遗传过程的扰动可能导致关键脑回路(前额叶皮质、杏仁核、海马体、下丘脑、隔核、腹侧被盖区和伏隔核)转录的动态变化，这些回路与成年后持续的行为和神经化学表型有关。以下特定目的将验证这一假说：1)使用实时定量聚合酶链式反应(QPCR)检测AIE诱导的NAIDA每个研究成分的选定靶基因的mRNA水平的变化。2)用染色质免疫沉淀(CHIP)和定量聚合酶链式反应(QPCR)检测AIE诱导的组蛋白修饰(组蛋白H3-K9乙酰化/甲基化)与每个项目的靶基因启动子相关。3)用甲基DNA免疫沉淀法(MeDIP)或甲基CpG结合域(MBD)结合定量聚合酶链式反应(QPCR)检测靶基因启动子的DNA甲基化状态。核心还将使用5-羟甲基胞嘧啶免疫沉淀(HMeDIP)分析来检测目标基因启动子的5-羟甲基半胱氨酸水平。这个核心将能够检查AIE介导的跨Nadia大脑不同区域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中的共同分子靶点，这些靶标可能会导致开发新的治疗策略，用于青少年酗酒引起的成人精神病理。

 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.