Epigenetics and Incubation of Craving

描述（由申请人提供）：到目前为止，没有有效的治疗方法来治疗可卡因的渴求和复发，这可以通过重新暴露于先前与吸毒有关的环境刺激而在戒断的人类成瘾者中沉淀。在人类中，这种线索诱导的药物渴望在早期停药期间逐渐增加，并在长期戒断药物期间保持高水平。这一现象在使用自我给药/恢复模式的大鼠中进行了模拟，在强制戒毒的前两个月，对先前与可卡因配对的线索的反应显示出逐渐增加（或“孵化”）。脑源性神经营养因子（BDNF）蛋白在中皮质边缘核中的表达也增加，其时间过程与可卡因渴求（ICC）的潜伏期相似，这表明BDNF可能增强了ICC的持续表达。然而，调控ICC期间BDNF表达随时间变化的确切分子机制仍有待确定。我们实验室的初步数据首次表明，在可卡因戒断期间，中边缘BDNF mRNA的表达也根据与ICC表达相似的时间过程而增加。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特定目标，重点阐明在ICC期间调节BDNF表达的机制。这项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。Aim 1将需要Western blot、ELISA和real-time PCR方法的培训，以确定ICC发育和表达过程中中皮质边缘核内BDNF mRNA和蛋白表达受到调控的精确时间过程。目标2将要求熟练掌握ChIP技术，以便在ICC期间检查与BDNF mRNA表达增加相关的BDNF启动子的染色质重塑（即组蛋白乙酰化和/或甲基化）。为了鉴定调节BDNF启动子染色质结构的酶，在Aim 2中也将使用ChIP方法分离与BDNF启动子结合的转录因子[tf]。Aim 3需要进一步的分子生物学培训，以便在ICC期间使用病毒介导的sirna递送来敲除结合BDNF启动子的tf。在病毒介导的TF敲除后，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、BDNF表达增强与ICC之间的因果关系。了解ICC过程中染色质重塑如何改变BDNF的表达，将为表观遗传成瘾研究的新兴领域提供重要见解，并可能为减少人类成瘾者对可卡因的渴望和复发提供新的药物靶点。

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.