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R 03申请是对通知编号（NOT-OD-10-032）和通知标题的回应：NIH通过NIH基本行为和社会科学机会网络（OppNet）宣布恢复法案资金可用于竞争性修订申请（R 01，R 03，R15，R21，R21/R33和R37）。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。多巴胺能系统和可卡因成瘾之间的关系已得到很好的表征。许多研究表明，D1多巴胺受体亚型参与介导可卡因的作用。可卡因的急性和慢性给药改变了中皮质边缘通路中D1受体的表达水平。介导可卡因处理动物中D1受体表达变化的分子机制和细胞外因子在很大程度上尚不清楚;然而，一些报告表明变化发生在转录后水平。在父R 03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结合D1受体mRNA的3'非翻译区（3' UTR）中的顺式作用元件的microRNA介导的。R 03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表达的变化有关，并使用表达D1受体的神经元的原代培养物证明，改变这种microRNA的表达也会改变D1受体蛋白的表达。一年的项目，我们已经确定，D1受体表现出转录后调节在幼年小鼠脑区域特异性的方式。尾状核中的定量microRNA分析已经鉴定出139种microRNA，其在可卡因处理7天后显示出大于3倍的表达变化。这些microRNA的一个子集（10）显示出增加的表达，并在D1 3 'UTR中具有推定的结合位点，表明它们参与D1受体的转录后调节。在本修订申请中，我们测试了一个新的假设，即在表现出可卡因诱导的行为敏化的小鼠中，可卡因激发将引起microRNA介导的D1受体转录后调控的快速改变，增加D1蛋白表达并有助于增加运动活性。拟议的实验的目标是在时间上将microRNA介导的转录后调节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。修订申请符合OppNet和恢复法案的目标，因为它将行为范式纳入父R 03项目，要求为行为测试设备提供资金，并增加新的人员来协助行为实验。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，特别是成瘾过程。 公共卫生相关性：该提案的目的是确定microRNA在可卡因诱导的行为敏化中介导D1多巴胺受体表达调节的作用。该项目将确定参与致敏过程的microRNA，并确定这些microRNA的水平是否控制可卡因刺激后D1受体表达的转录后调节。该提案的结果将开辟多巴胺受体生物学研究的新领域，并导致开发治疗可卡因成瘾的潜在新治疗方法。

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.