Epigenetics and Incubation of Craving

描述（由申请人提供）：迄今为止，没有有效的治疗可卡因渴望和复发的治疗方法，这可以通过重新暴露于先前与吸毒相关的环境刺激而在戒断的人类成瘾者中沉淀。这种线索诱导的人类药物渴求在早期药物戒断过程中逐渐增加，并在长期药物戒断期间保持较高水平。使用自我给药/恢复范例在大鼠中对这种现象进行建模，其中对先前与可卡因配对的线索的反应性显示出在强制药物戒断的前两个月内逐渐增加（或“潜伏”）。脑源性神经营养因子（BDNF）蛋白表达也增加中皮质边缘核根据一个类似的时间过程的可卡因渴求（ICC）的孵化，这表明，BDNF可能会加强正在进行的表达ICC。然而，精确的分子机制，调节时间依赖性变化的BDNF表达在ICC仍有待确定。 我们实验室的初步数据首次表明，根据与ICC表达相似的时间过程，在可卡因戒断期间，中脑边缘BDNF mRNA表达也增加。拟议的研究和培训计划的目标是通过监督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，重点是阐明ICC期间调节BDNF表达的机制。K 01申请的主要假设是，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。目的1将需要培训的Western印迹，ELISA和实时PCR方法，以确定精确的时间过程中，BDNF的mRNA和蛋白质的表达，在中皮质边缘核的发展和表达的ICC内调节。目标2将需要熟练掌握ChIP技术，以检查与ICC期间BDNF mRNA表达增加相关的BDNF启动子处的染色质重塑（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，ChIP方法也将用于Aim 2中以分离与BDNF启动子结合的转录因子[TF]。在目标3中需要进一步的分子生物学培训，以便使用病毒介导的siRNA递送来敲低在ICC期间与BDNF启动子结合的TF。将在病毒介导的TF敲低后检查ICC的表达以及改变的组蛋白乙酰化和/或甲基化，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。了解染色质重塑如何改变脑源性神经营养因子的表达在ICC将有助于重要的见解新兴领域的表观遗传成瘾研究，并可能提供新的药物靶点，旨在减少可卡因的渴望和复发的人类成瘾者。

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.