Regulation of D1 Dopamine Receptor Expression by ncRNA in Cocaine Addiction

描述(由申请人提供)：本竞争修订R03申请是对通知编号(NOT-OD-10-032)和通知标题的回应：NIH宣布通过NIH基本行为和社会科学机会网络(OppNet)为竞争修订申请(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经有了很好的描述。大量研究表明，D1多巴胺受体亚型参与了可卡因的调节作用。急性和长期给予可卡因改变了中皮质边缘通路中D1受体的表达水平。在可卡因处理的动物中，介导D1R表达变化的分子机制和细胞外因素在很大程度上是未知的；然而，一些报告表明，这种变化发生在转录后水平。在父R03项目中，我们正在测试一个新的假设，即D1受体表达的转录后调控是由与D1受体mRNA 3‘非翻译区(3’UTR)中的顺式作用元件结合的microRNAs介导的。母公司R03项目的目标是确定可卡因引起的D1受体表达的变化是否与特定microRNA的表达变化有关，并利用表达d1受体的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。该项目实施一年后，我们已经确定，在幼年小鼠中，D1受体以脑区特异性的方式表现出转录后调节。尾状核中的定量microRNA图谱已经鉴定出139个microRNAs，它们在7天的可卡因治疗后表达变化超过3倍。这些microRNAs的一个子集(10)在D13‘非编码区表达增加，并具有假定的结合部位，暗示它们参与了D1R的转录后调节。在这一修订应用中，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，可卡因的挑战将导致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，并有助于增强运动活性。这项实验的目的是在时间上将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。修订申请符合OppNet和恢复法案的目标，因为它将行为范例整合到父R03项目中，请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控可卡因成瘾尤其是成瘾过程中多巴胺受体表达变化的分子机制的理解。 公共卫生相关性：这项建议的目标是确定在可卡因诱导的行为敏化中，microRNAs在调节D1多巴胺受体表达方面的作用。该项目将识别参与致敏过程的microRNAs，并确定这些microRNAs的水平是否控制可卡因攻击后D1受体表达的转录后调控。这项提议的结果将开启多巴胺受体生物学的新研究领域，并导致潜在的治疗可卡因成瘾的新疗法的开发。

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.