Epigenetics and Incubation of Craving

描述(申请人提供)：到目前为止，还没有有效的治疗方法来治疗可卡因的渴望和复发，这可以通过重新暴露于以前与吸毒有关的环境刺激而在戒毒的人类成瘾者中加速。这种线索诱导的人类对药物的渴望在早期戒毒期间逐渐增加，并在长期戒毒期间保持较高水平。这一现象是用自我管理/恢复范式在大鼠身上模拟的，在这种范式中，对以前与可卡因配对的线索的反应性在强制戒毒的头两个月中逐渐增加(或“孵化”)。脑源性神经营养因子(BDNF)蛋白在中皮质边缘核的表达也以与可卡因渴求(ICC)孵育相似的时间进程增加，提示BDNF可能增强了ICC的持续表达。然而，调控ICC过程中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，在可卡因戒断过程中，中脑边缘BDNF mRNA的表达也会增加，其时间过程与ICC的表达相似。拟议的研究和培训计划的目标是通过有监督的动手培训和授课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，重点是阐明在ICC过程中调控BDNF表达的机制。K01应用的主要假设是，ICC过程中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需要进行蛋白质印迹、酶联免疫吸附试验和实时定量聚合酶链式反应方法的培训，以确定在ICC的发育和表达过程中，BDNF mRNA和蛋白在中皮质边缘核中的表达受到调控的确切时间进程。AIM 2将需要熟练的芯片技术，以检测与ICC期间BDNF mRNA表达增加有关的BDNF启动子的染色质重塑(即组蛋白乙酰化和/或甲基化)。为了确定在BDNF启动子上调节染色质结构的酶，在AIM 2中还将使用芯片方法来分离与BDNF启动子结合的转录因子[TF]。在目标3中，将需要进一步的分子生物学培训，以便使用病毒介导的siRNAs传递来击倒在ICC过程中与BDNF启动子结合的转录因子。病毒介导的Tf基因敲除后，ICC的表达以及组蛋白乙酰化和/或甲基化的改变将被检测，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。了解染色质重塑如何改变ICC过程中BDNF的表达，将有助于对新兴的表观遗传成瘾研究领域的重要见解，并可能为减少人类吸毒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.