Translational and epigenetic profiling of cell types associated with addiction

描述(由申请者提供)：项目摘要/摘要：最近对成瘾的研究突出了大脑中被认为与目标导向和药物寻找行为有关的几个区域。参与调控这些行为的特定神经元类别正在开始被识别，并试图描述由于成瘾而发生的这些细胞类型的分子变化，已经取得了一些成功。虽然这些研究表明有可能发现与成瘾相关的细胞类型的变化，在某些情况下还会导致成瘾，但我们在这一领域的知识是零碎和不完整的。这是由于这一领域几十年来一直面临的技术障碍，最近我们实验室开发的新方法克服了这些障碍。这项研究的目的是确定所有基因表达的变化和伴随的表观遗传调节的变化，这些变化导致已知的在控制成瘾的神经回路中重要的细胞类型的成瘾状态。我们将在这个项目中采取的方法是：1)使用TRAP方法和bacTRAP转基因小鼠品系，全面分析15种小鼠CNS细胞类型的翻译mRNAs，这些细胞类型是控制成瘾的神经回路的组件；2)收集暴露于可卡因、哌甲酯和甲基苯丙胺的小鼠这些细胞类型中每一种细胞的翻译概况；3)对这些得到的微阵列数据集进行深入的比较分析，以确定每种细胞类型中伴随成瘾状态的基因表达变化；4)同时从每种细胞类型分离细胞核，并将MC和HMC修饰的细胞特定位置映射到神经元基因组；5)对前面确定的基因组基因座进行后续研究目的是通过使用H3K9m2和H3K27m3特异性抗体的芯片分析来定位其他表观遗传调控事件，以确定与常染色质基因表达抑制相关的基因组基因座。该项目将提供信息和实验动物，将在广泛的实验室中刺激成瘾研究，并为在其他实验条件下对这些相同的神经回路进行全面研究提供材料和范例。因此，这一计划将对现代分子神经生物学以及中枢神经系统疾病药物干预新靶点的开发产生巨大影响。 与公共健康相关：根据美国国家药物滥用研究所的数据，“据估计，美国每年因滥用药物而造成的总成本超过5000亿美元”。该项目的目标是全面描述与成瘾相关的区域特定细胞类型中发生的分子变化，并同时绘制这些细胞类型中发生的表观遗传变化图。它将刺激对导致成瘾状态建立的细胞特定分子事件的深入研究，并为药物成瘾新疗法的开发确定新的靶点。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Recent studies of addiction have highlighted several regions of the brain that are thought to be involved in goal directed and drug seeking behaviors. The specific neuronal classes involved in the regulation of these behaviors are beginning to be identified, and attempts to profile the molecular changes in these cell types occurring as a consequence of addiction have met with some success. While these studies demonstrate that it is possible to discover changes in cell types that are correlated with, and in some cases contribute to, addiction, our knowledge in this area is fragmentary and incomplete. This is due to technical obstacles that have faced this field for decades, and that have recently been overcome by novel methodologies developed in our laboratories. The objective of this program of research is to identify all of the changes in gene expression and accompanying alterations in epigenetic regulation that contribute to the addictive state in cell types known to be important in the neural circuitry controlling addiction. The approach we will take in this program is to: 1) employ TRAP methodology and bacTRAP transgenic mouse lines to comprehensively profile the translated mRNAs from fifteen mouse CNS cell types that are components of the neural circuitry controlling addiction; 2) to collect translational profiles from each of these cell types in mice exposed to cocaine, methylphenidate, and methamphetamine 3) perform in depth comparative analysis of these resulting microarray datasets to identify changes in gene expression that accompany the addictive state in each cell type; 4) to concurrently isolate nuclei from each cell type and map cell specific sites of mC and hmC modification to the neuronal genomes; 5) to conduct follow up studies on genomic loci identified in the preceding aims to map additional epigenetic regulatory events by ChIP assays using H3K9m2 and H3K27m3 specific antibodies to identify genomic loci associated with suppression of gene expression in euchromatin. This project will provide information and experimental animals that will stimulate addiction research in a broad spectrum of laboratories, and provide materials and a paradigm for comprehensive studies of these same neural circuits under other experimental conditions. As such, this program will have an enormous impact on modern molecular neurobiology, and on the development of novel targets for pharmacological interventions in CNS disorders. PUBLIC HEALTH RELEVANCE: According to the National Institute on Drug Abuse, "Estimates of the total overall costs of substance abuse in the United States ..... exceed half a trillion dollars annually". The goals of the project are to comprehensively profile molecular changes occurring in specific cell types in regions associated with addiction, and to concurrently map epigenetic changes occurring in these cell types. It will stimulate in depth research into cell specific molecular events that are responsible for establishment of the addictive state, and identify novel targets for the development of new therapies for drug addiction.