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经得到了很好的描述。大量研究表明，D1多巴胺受体亚型参与介导可卡因的作用。据报道，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。利用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和脑源性神经营养因子治疗期间在转录后水平受到调节。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R03项目中，我们验证了一个新的假设，即急性和慢性可卡因给药对D1多巴胺受体表达的转录后调节是由microrna介导的，这些microrna结合D1受体基因3'非翻译区域的顺式作用元件。为了验证这一假设，我们将使用由NINDS基因表达神经系统图谱（GENSAT）项目产生的Drd1a-EGFP报告基因转基因小鼠。在急性和慢性可卡因治疗Drd1a-EGFP报告基因转基因小鼠后，我们将评估D1受体mRNA和D1受体蛋白在伏隔核、尾状核、皮层、海马和腹侧被盖区的表达。此外，我们还将检测来自不同脑区的可卡因治疗动物中针对D1受体基因的推测microrna的表达。这个R03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。到目前为止，还没有关于microrna调节多巴胺受体表达的报道。该项目的研究结果将为多巴胺受体生物学开辟一个新的研究领域，并提高我们对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): 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.