Regulation of D1 Dopamine Receptor Expression by ncRNA in Cocaine Addiction

描述（由申请人提供）：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经得到了很好的表征。许多研究表明，D1多巴胺受体亚型参与介导可卡因的作用。据报道，可卡因的急性和慢性给药可改变中皮质边缘通路中D1受体的表达水平。介导可卡因处理动物多巴胺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。我们最近确定了一些调节D1多巴胺受体基因表达的细胞外因子和分子机制。使用一个儿茶酚胺能神经元细胞系，我们确定，内源性D1多巴胺受体在体外分化过程中的转录后水平进行调节，也脑源性神经营养因子治疗。我们的初步研究还表明，D1多巴胺受体表达的转录后调控是由基因3'非翻译区的顺式作用元件介导的。在这个R 03项目中，我们测试了一个新的假设，即通过急性和慢性给予可卡因对D1多巴胺受体表达的转录后调节是由结合D1受体基因3'非翻译区顺式作用元件的microRNA介导的。为了验证这一假设，我们将使用NINDS基因表达神经系统图谱（GENSAT）项目产生的Drd 1a-EGFP报告基因转基因小鼠。在急性和慢性可卡因处理的Drd 1a-EGFP报告基因转基因小鼠，我们将评估D1受体mRNA和D1受体蛋白的表达的延髓核，尾状核，皮质，海马和腹侧被盖区。此外，我们还将检测可卡因治疗动物不同脑区中靶向D1受体基因的推定microRNA的表达。这个R 03项目的目标是确定可卡因诱导的D1多巴胺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。迄今为止，还没有关于microRNA调节多巴胺受体表达的报道。该项目的结果将开辟多巴胺受体生物学研究的新领域，并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，以及一般成瘾过程。 公共卫生相关性：这项研究的目的是确定microRNA在可卡因成瘾中介导D1多巴胺受体表达调节中的作用。该项目将确定所涉及的microRNA，并证明它如何介导可卡因处理动物中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): 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 particularly 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 has been reported to alter the expression levels of D1 receptors in the mesocorticolimbic pathway. The molecular mechanisms and extra cellular factors that mediate the changes in dopamine receptor expression in cocaine-treated animals are largely unknown; however some reports have indicated that changes occur at the post transcriptional level. We have recently determined some of the extra cellular factors and molecular mechanisms that regulate the expression of the D1 dopamine receptor gene. Using a catecholaminergic neuronal cell line we determined that the endogenous D1 dopamine receptor is regulated at the post-transcriptional level during in vitro differentiation, and also following brain-derived neurotrophic factor treatment. Our preliminary studies also show that the post-transcriptional regulation of D1 dopamine receptor expression is mediated by cis-acting elements in the 3' untranslated region of the gene. In this R03 project, we test a novel hypothesis that posttranscriptional modulation of D1 dopamine receptor expression by acute and chronic administration of cocaine is mediated by microRNAs that bind cis-acting elements in the 3' untranslated region of the D1 receptor gene. To test this hypothesis, we will use the Drd1a-EGFP reporter transgenic mice generated by the NINDS Gene Expression Nervous System Atlas (GENSAT) project. Following acute and chronic cocaine treatment of Drd1a-EGFP reporter transgenic mice, we will evaluate the expression of D1 receptor mRNA and D1 receptor protein the nucleus accumbens, caudate, cortex, hippocampus and ventral tegmental area. In addition, we will also assay the expression of putative microRNAs that target the D1 receptor gene in the cocaine-treated animals from various brain regions. The goal of this R03 project is to determine if cocaine-induced changes in D1 dopamine 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. To date, there have been no reports of microRNAs regulating dopamine receptor expression. The results from this project would open a new area of research in dopamine receptor biology and 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 addiction. The project will identify the microRNA that is involved and demonstrate how it mediates the post-transcriptional regulation of D1 receptor expression in cocaine-treated animals. 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.