Regulation of D1 Dopamine Receptor Expression by ncRNA in Cocaine Addiction

描述（由申请人提供）：此竞争性修订R03申请是对通知号（NOT-OD-10-032）和通知标题的响应：NIH宣布通过NIH基础行为和社会科学机会网络（OppNet）为竞争性修订申请（R01, R03, R15, R21， R21/R33和R37）提供恢复法案资金。神经递质多巴胺在大脑奖励过程中起着重要作用。多巴胺能系统的功能障碍与成瘾行为有关。多巴胺能系统与可卡因成瘾之间的关系已被很好地表征。大量研究表明，D1多巴胺受体亚型参与介导可卡因的作用。急性和慢性服用可卡因可改变中皮质边缘通路中D1受体的表达水平。在可卡因处理的动物中，介导D1受体表达变化的分子机制和细胞外因子在很大程度上是未知的；然而，一些报告表明，变化发生在转录后水平。在母体R03项目中，我们正在测试一个新的假设，即D1多巴胺受体表达的转录后调节是由microrna介导的，microrna结合D1受体mRNA 3‘非翻译区（3’ utr）的顺式作用元件。亲本R03项目的目标是确定可卡因诱导的D1受体表达的变化是否与特定microRNA的表达变化有关，并通过D1受体表达神经元的原代培养证明，改变这种microRNA的表达也会改变D1受体蛋白的表达。在这个项目进行了一年之后，我们已经确定了D1受体在幼年小鼠中以特定大脑区域的方式表现出转录后调控。尾状核的定量microRNA分析已经鉴定出139个microRNA，在可卡因治疗7天后表达变化超过3倍。这些microrna的一个子集（10）表达增加，并且在D1 3'UTR中有推测的结合位点，暗示它们参与D1受体转录后调控。在这一修订应用中，我们验证了一个新的假设，即在表现出可卡因诱导行为致敏的小鼠中，可卡因刺激会引起microrna介导的D1受体转录后调控的快速改变，增加D1蛋白的表达，并促进运动活动的增加。拟议实验的目的是暂时将microrna介导的转录后调控的潜在快速变化与可卡因诱导的运动行为的变化联系起来。修订应用程序符合OppNet和恢复法案的目标，因为它将行为范式纳入了母R03项目，请求为行为测试设备提供资金，并增加了新的人员来协助进行行为实验。该项目的结果将提高我们对调节可卡因成瘾中多巴胺受体表达变化的分子机制的理解，特别是对成瘾过程的理解。

DESCRIPTION (provided by applicant): This competitive revision R03 application is in response to Notice Number (NOT-OD-10-032) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications (R01, R03, R15, R21, R21/R33, and R37) through the NIH Basic Behavioral and Social Science Opportunity Network (OppNet). The neurotransmitter dopamine plays an important role in the brain reward process. Dysfunction of the dopaminergic system is implicated in addictive behaviors. The relationship between dopaminergic system and cocaine addiction has been well characterized. Numerous studies have shown that the D1 dopamine receptor subtype is involved in mediating the effects of cocaine. Acute and chronic administration of cocaine alters the expression levels of D1 receptors in the mesocorticolimbic pathway. The molecular mechanisms and extracellular factors that mediate the changes in D1 receptor expression in cocaine-treated animals are largely unknown; however some reports have indicated that changes occur at the post-transcriptional level. In the parent R03 project, we are testing a novel hypothesis that posttranscriptional modulation of D1 dopamine receptor expression is mediated by microRNAs that bind cis- acting elements in the 3' untranslated region (3'UTR) of the D1 receptor mRNA. The goal of the parent R03 project is to determine if cocaine-induced changes in D1 receptor expression can be related to changes in expression of a specific microRNA, and to demonstrate, using primary cultures of D1 receptor-expressing neurons, that altering the expression of this microRNA will also alter the expression of D1 receptor protein. One year into the project, we have determined that D1 receptor exhibits post transcriptional regulation in a brain region-specific manner in juvenile mice. Quantitative microRNA profiling in the caudate has identified 139 microRNAs that show a greater than 3-fold change in expression following 7 days of cocaine treatment. A subset of these microRNAs (10) show increased expression AND have putative binding sites in the D1 3'UTR, implicating them in D1 receptor posttranscriptional regulation. In this revision application, we test a new hypothesis that in mice that exhibit cocaine-induced behavior sensitization, a cocaine challenge will elicit a rapid alteration in microRNA-mediated posttranscriptional regulation of D1 receptors, increasing D1 protein expression and contributing to the increased locomotor activity. The goal of the proposed experiments is to temporally relate potential rapid changes in microRNA-mediated posttranscriptional regulation to changes in cocaine-induced locomotor behavior. The revision application meets the goals of OppNet and the Recovery Act, as it incorporates behavioral paradigms into the parent R03 project, requests funds for behavioral testing equipment and adds new personnel to assist with the behavioral experiments. The results from this project will improve our understanding of molecular mechanisms that regulate changes in dopamine receptor expression in cocaine addiction in particular, and addictive processes in general. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to determine the role of microRNAs in mediating regulation of D1 dopamine receptor expression in cocaine-induced behavior sensitization. The project will identify microRNAs that are involved in the sensitization process and determine if the levels of these microRNAs control the post- transcriptional regulation of D1 receptor expression following cocaine challenge. The results of this proposal will open a new area of research in dopamine receptor biology and lead to the development of potentially novel therapeutic methods for treating cocaine addiction.