Cocaine-induced histone post-translational modifications

可卡因诱导的组蛋白翻译后修饰

• 批准号: 9304987
• 负责人: Benjamin A Garcia
• 金额: $ 20万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9304987
• 关键词: Abstinence; Acetylation; Active Sites; Acute; Animal Model; Animals; Binding; Brain; Brain region; Brain-Derived Neurotrophic Factor; Chromatin; Circadian Rhythms; Clinical; Cocaine; Cues; DNA biosynthesis; Epigenetic Process; Exons; Extinction (Psychology); Female; Gene Expression; Genetic Transcription; Genome; Goals; Histone Acetylation; Histone H3; Histones; Home environment; Hour; Infusion procedures; Injectable; Injection of therapeutic agent; Mass Spectrum Analysis; Mental disorders; Methylation; Modeling; Modification; Mus; Neuronal Plasticity; Neurons; Nucleosomes; Nucleus Accumbens; Phosphorylation; Post-Translational Protein Processing; Prosencephalon; Proteins; Proteomics; Relapse; Rodent; Saline; Sampling; Self Administration; Self-Administered; Techniques; Testing; Time; Tissues; Transcript; Variant; addiction; base; cell type; chromatin remodeling; cocaine exposure; cocaine relapse; combinatorial; craving; drug abstinence; drug craving; drug development; drug relapse; experimental study; histone methylation; histone modification; killings; mRNA Expression; male; novel; promoter; response; therapy development; transcription factor

A growing body of evidence indicates that epigenetic mechanisms, including post-translational modifications (PTMs) of histone proteins, are associated with a number of psychiatric disorders including addiction. This project utilizes a mouse line expressing a tetO-regulated, HA-tagged histone H3.3 protein exclusively in forebrain neurons. The H3.3 variant is unique in that H3.3 incorporates into chromatin independently of DNA replication and preferentially at active sites of transcription and transcription factor binding. Thus, modifications of H3.3 and histones associated with H3.3-containing nucleosomes are particularly likely to be involved in plasticity such as following repeated exposure to cocaine. This proposal is a collaborative effort between the Pierce lab, which focuses on animal models of addiction, and the Garcia lab, which specializes in cutting-edge mass spectrometry (MS)-based quantification of histone modifications. Both specific aims utilize novel MS- based proteomics platforms to comprehensively interrogate the PTM profiles of histones during and following priming- (Aim 1) or cue- (Aim 2) induced reinstatement of cocaine seeking. All experiments will be performed in both male and female mice. Although certain histone modifications have been implicated in models of addiction, changes in combinatorial histone modifications in response to cocaine self-administration remain poorly understood. Quantitative MS offers the ability to study histone modifications in an unbiased fashion. Specific Aim 1 focuses on the cocaine self-administration/extinction/reinstatement model of relapse. Mice will be allowed to self-administer cocaine or will receive passive, yoked infusions of saline or cocaine. Following the extinction of cocaine seeking, mice will be challenged with a systemic injection of cocaine that promotes cocaine reinstatement or will be injected with saline. One hour following the cocaine or saline injection, the mice will be killed and the nucleus accumbens, a brain region associated with cocaine-induced neuronal plasticity, will be dissected and prepared for MS to quantify acutely altered histone PTMs. Specific Aim 2 focuses on combinatorial histone modifications associated with the incubation of cue-induced cocaine reinstatement. Following cocaine self-administration, the mice will undergo forced abstinence for 1 or 30 days. Some animals will be assessed for cue-induced reinstatement and then killed after a 60-minute session; control accumbens tissue will be collected in the absence of the reinstatement test. Alterations in histone PTMs subsequently will be assessed via MS as in Aim 1. We hypothesize that histone acetylation and phosphorylation will increase in active regions of the neuronal genome particularly during and immediately following cocaine reinstatement (priming or cue-induced reinstatement, as assessed in Aims 1 and 2, respectively). In contrast, we expect to observe increases in histone methylation associated with reduced gene expression and decreases in histone methylation in regions of active transcription that persist through the extended abstinence period following cocaine self-administration in Aim 2.

越来越多的证据表明表观遗传机制，包括翻译后修饰 组蛋白的翻译后修饰（PTM）与包括成瘾在内的许多精神疾病有关。这 该项目利用仅在小鼠中表达 tetO 调节的 HA 标记组蛋白 H3.3 蛋白的小鼠品系 前脑神经元。 H3.3 变体的独特之处在于 H3.3 独立于 DNA 融入染色质 复制并优先在转录和转录因子结合的活性位点。因此，修改 H3.3 和与含 H3.3 核小体相关的组蛋白特别可能参与 可塑性，例如反复接触可卡因后。该提案是双方的共同努力 皮尔斯实验室专注于成瘾动物模型，加西亚实验室专注于尖端研究 基于质谱 (MS) 的组蛋白修饰定量。这两个具体目标都利用了新颖的 MS- 基于蛋白质组学平台，可在过程中和之后全面询问组蛋白的 PTM 图谱 启动-（目标 1）或提示-（目标 2）诱导恢复可卡因寻求。所有实验将在 雄性和雌性小鼠。尽管某些组蛋白修饰与模型有关 成瘾，响应可卡因自我给药的组合组蛋白修饰的变化仍然存在 不太了解。定量 MS 能够以公正的方式研究组蛋白修饰。 具体目标 1 侧重于可卡因自我给药/戒断/复吸模型。老鼠会 允许自行注射可卡因或接受被动、结合的盐水或可卡因输注。下列的 为了消除对可卡因的渴求，小鼠将面临全身注射可卡因的挑战，从而促进 可卡因恢复或将注射生理盐水。注射可卡因或盐水一小时后， 小鼠将被杀死，伏隔核（与可卡因诱导的神经元相关的大脑区域） 可塑性，将被解剖并准备用于 MS 以量化急剧改变的组蛋白 PTM。具体目标2 专注于与提示诱导可卡因孵育相关的组合组蛋白修饰 复职。自我给予可卡因后，小鼠将强制戒酒 1 或 30 天。 一些动物将接受提示诱导的恢复评估，然后在 60 分钟的训练后被杀死； 将在没有恢复测试的情况下收集对照伏隔核组织。组蛋白 PTM 的改变 随后将通过 MS 进行评估，如目标 1 所示。我们假设组蛋白乙酰化和 神经元基因组活性区域的磷酸化将增加，特别是在期间和立即 可卡因恢复后（如目标 1 和 2 中评估的启动或提示诱导恢复， 分别）。相反，我们期望观察到与基因减少相关的组蛋白甲基化增加 活性转录区域中组蛋白甲基化的表达和减少持续存在 目标 2 中自我服用可卡因后延长戒断期。