Epigenetics and Incubation of Craving

表观遗传学和渴望的孵化

• 批准号: 8142895
• 负责人: HEATH D SCHMIDT
• 金额: $ 13.53万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142895
• 关键词: Abstinence; Acetylation; Addictive Behavior; Address; Animal Model; Area; Behavior; Behavioral; Binding; Brain-Derived Neurotrophic Factor; Cell Nucleus; Cells; Chromatin Structure; Chronic; Cocaine; Cocaine Dependence; Confocal Microscopy; Cues; Cyclic AMP-Responsive DNA-Binding Protein; DNA-Protein Interaction; Data; Development; Drug Delivery Systems; Enzyme-Linked Immunosorbent Assay; Enzymes; Epigenetic Process; Exposure to; Foundations; Genetic Transcription; Genetic Translation; Goals; Histone Acetylation; Human; Immunoassay; Immunohistochemistry; Incubated; Infusion procedures; Learning; Link; Literature; Measures; Medial; Mediating; Mediator of activation protein; Mentored Research Scientist Development Award; Mentors; Messenger RNA; Methods; Methyl-CpG-Binding Protein 2; Methylation; Modeling; Molecular; Molecular Biology; Molecular Probes; Neurosciences; Nucleus Accumbens; Pattern; Pharmaceutical Preparations; Play; Population; Prefrontal Cortex; Promoter Regions; Proteins; Rattus; Regulation; Relapse; Research; Research Training; Rodent; Role; Self Administration; Small Interfering RNA; Stimulus; Techniques; Therapeutic; Time; Tissues; Training; Transcript; Transcriptional Silencer Elements; Ventral Tegmental Area; Viral; Virus; Western Blotting; Withdrawal; Work; addiction; chromatin immunoprecipitation; chromatin remodeling; cocaine exposure; craving; drug abstinence; drug craving; drug development; drug relapse; drug seeking behavior; drug withdrawal; experience; insight; knock-down; mRNA Expression; mesolimbic system; myocyte-specific enhancer-binding factor 2; neuroadaptation; novel; preclinical study; prevent; promoter; protein expression; public health relevance; research study; response; skills; transcription factor

DESCRIPTION (provided by applicant): To date, there are no effective therapeutic treatments for cocaine craving and relapse, which can be precipitated in abstinent human addicts by re-exposure to environmental stimuli that were previously associated with drug taking. This cue-induced drug craving in humans progressively increases during early drug withdrawal and remains high throughout extended periods of drug abstinence. This phenomenon was modeled in rats using a self-administration/reinstatement paradigm in which responsiveness to cues previously paired with cocaine was shown to progressively increase (or "incubate") over the first two months of forced drug abstinence. Brain-derived neurotrophic factor (BDNF) protein expression also was increased in mesocorticolimbic nuclei according to a similar time course as the incubation of cocaine craving (ICC), which suggests that BDNF may potentiate the ongoing expression of ICC. However, the precise molecular mechanisms that regulate time-dependent changes in BDNF expression during ICC remain to be determined. Preliminary data from our lab indicate for the first time that mesolimbic BDNF mRNA expression also is increased during cocaine abstinence according to a similar time course to the expression of ICC. The goal of the proposed Research and Training Plans is to acquire molecular neuroscience techniques through supervised hands-on training and didactic coursework in order to incorporate molecular/epigenetic methods into Specific Aims focused on elucidating the mechanisms that regulate BDNF expression during ICC. The main hypothesis of this K01 application is that altered BDNF mRNA expression during ICC is mediated by chromatin remodeling within BDNF promoters. Aim 1 will require training in Western blot, ELISA, and real-time PCR methods in order to determine the precise time course that BDNF mRNA and protein expression is regulated within mesocorticolimbic nuclei during the development and expression of ICC. Aim 2 will require proficiency in ChIP techniques in order to examine chromatin remodeling (i.e. histone acetylation and/or methylation) at BDNF promoters associated with increased BDNF mRNA expression during ICC. In order to identify the enzymes that regulate chromatin structure at BDNF promoters, ChIP methods also will be used in Aim 2 to isolate transcription factors [TFs] bound to BDNF promoters. Further training in molecular biology, will be required in Aim 3 in order to use viral-mediated delivery of siRNAs to knock-down TFs that bind to BDNF promoters during ICC. Expression of ICC as well as altered histone acetylation and/or methylation will be examined following viral-mediated TF knock-down in order to demonstrate a causal association between DNA- protein interactions, chromatin remodeling, enhanced BDNF expression and ICC. Understanding how chromatin remodeling alters BDNF expression during ICC will contribute important insights into the emerging field of epigenetic addiction research and may provide novel drug targets aimed at reducing cocaine craving and relapse in human addicts. PUBLIC HEALTH RELEVANCE: These proposed experiments will investigate the molecular mechanisms (i.e., epigenetic/chromatin remodeling) that regulate mesolimbic brain-derived neurotrophic factor (BDNF) expression during the incubation of cocaine craving, an animal model of drug craving and relapse in human addicts. This proposal aims to identify the molecular substrates that regulate chromatin structure and transcription of BDNF promoter regions during the incubation of cocaine craving. Therefore, this proposal will provide insight into the epigenetic mechanisms underlying cocaine addiction as well as identify novel targets for treating drug craving and relapse.

描述（由申请人提供）：迄今为止，还没有针对可卡因渴望和复发的有效治疗方法，戒断的人类成瘾者可能会因重新暴露于先前与吸毒相关的环境刺激而加剧这种渴望和复发。这种由提示引起的人类对药物的渴望在戒毒早期逐渐增加，并在长期戒毒期间保持较高水平。这种现象是在大鼠中使用自我给药/恢复范例进行建模的，其中对先前与可卡因配对的线索的反应在强制戒毒的前两个月中显示出逐渐增加（或“潜伏”）。中皮质边缘核中的脑源性神经营养因子 (BDNF) 蛋白表达也有所增加，其时间过程与可卡因渴望 (ICC) 的潜伏期相似，这表明 BDNF 可能会增强 ICC 的持续表达。然而，调节 ICC 期间 BDNF 表达随时间变化的精确分子机制仍有待确定。 我们实验室的初步数据首次表明，中脑边缘 BDNF mRNA 表达在可卡因戒断期间也会增加，其时间过程与 ICC 表达相似。拟议的研究和培训计划的目标是通过监督的实践培训和教学课程获得分子神经科学技术，以便将分子/表观遗传学方法纳入具体目标中，重点是阐明 ICC 期间调节 BDNF 表达的机制。 K01 应用的主要假设是 ICC 期间 BDNF mRNA 表达的改变是由 BDNF 启动子内的染色质重塑介导的。目标 1 需要进行蛋白质印迹、ELISA 和实时 PCR 方法方面的培训，以确定 ICC 发育和表达过程中中皮质边缘核内 BDNF mRNA 和蛋白表达受到调节的精确时间过程。目标 2 需要熟练掌握 ChIP 技术，以便检查 BDNF 启动子处的染色质重塑（即组蛋白乙酰化和/或甲基化）与 ICC 期间 BDNF mRNA 表达增加相关。为了鉴定调节 BDNF 启动子处染色质结构的酶，目标 2 中还将使用 ChIP 方法来分离与 BDNF 启动子结合的转录因子 [TF]。目标 3 需要进一步进行分子生物学培训，以便使用病毒介导的 siRNA 递送来敲低 ICC 期间与 BDNF 启动子结合的 TF。在病毒介导的 TF 敲低后，将检查 ICC 的表达以及组蛋白乙酰化和/或甲基化的改变，以证明 DNA-蛋白质相互作用、染色质重塑、增强的 BDNF 表达和 ICC 之间的因果关系。了解 ICC 期间染色质重塑如何改变 BDNF 表达将为表观遗传成瘾研究的新兴领域提供重要见解，并可能提供旨在减少人类成瘾者对可卡因的渴望和复吸的新药物靶点。 公共健康相关性：这些拟议的实验将研究可卡因渴望潜伏期间调节中脑边缘脑源性神经营养因子（BDNF）表达的分子机制（即表观遗传/染色质重塑），可卡因渴望是人类成瘾者药物渴望和复发的动物模型。该提案旨在确定在可卡因渴望孵化过程中调节染色质结构和 BDNF 启动子区域转录的分子底物。因此，该提案将深入了解可卡因成瘾的表观遗传机制，并确定治疗药物渴望和复发的新靶标。