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(PTM)与包括成瘾在内的许多精神疾病有关。这 Project利用一种小鼠系来表达Teto调节的、HA标记的组蛋白H3.3蛋白，该蛋白仅在 前脑神经元。H3.3变异体的独特之处在于，H3.3独立于DNA并入染色质 复制，并优先在转录和转录因子结合的活性部位。因此，修改 H3.3和与含有H3.3的核小体相关的组蛋白特别可能参与 可塑性，例如在反复接触可卡因之后。这项提议是双方合作的成果 皮尔斯实验室，专注于成瘾的动物模型，以及加西亚实验室，专门研究尖端技术 基于质谱仪(MS)的组蛋白修饰定量。这两个具体目标都利用了新的MS- 基于蛋白质组学平台的综合询问组蛋白的PTM图谱 激发-(目标1)或线索-(目标2)可诱导可卡因寻求的恢复。所有的实验都将在 雄鼠和雌鼠都有。尽管某些组蛋白修饰已经被牵连到 成瘾，对可卡因自我给药反应的组合组蛋白修饰的变化仍然存在 人们对此知之甚少。定量MS提供了以无偏见的方式研究组蛋白修饰的能力。 具体目标1侧重于复吸的可卡因自我给药/灭绝/恢复模式。老鼠会 被允许自行注射可卡因，或将接受被动的生理盐水或可卡因输注。跟随 寻找灭绝的可卡因，小鼠将受到全身注射可卡因的挑战，以促进 可卡因恢复或将被注射生理盐水。注射可卡因或生理盐水一小时后， 小鼠将被处死，伏隔核，一个与可卡因诱导的神经元相关的大脑区域 可塑性，将被解剖并准备供MS量化急剧改变的组蛋白PTM。具体目标2 重点是与线索诱导的可卡因孵育相关的组合组蛋白修饰 复职。在自我注射可卡因后，小鼠将经历1至30天的强制戒断。 一些动物将被评估为线索诱导的恢复，然后在60分钟的训练后被处死； 对照伏隔组织将在没有恢复试验的情况下采集。组蛋白PTMS的改变 随后将通过目标1中的MS进行评估。我们假设组蛋白乙酰化和 神经元基因组活跃区域的磷酸化将增加，特别是在 在可卡因恢复后(如目标1和2所评估的，引爆或暗示诱导恢复， )。相反，我们希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 在目标2中，在可卡因自我给药后延长了戒断期。