Exploring nucleocytoplasmic IEG mRNA export in striatal neuron subpopulations

 描述（由申请人提供）：截至2012年，约有2200万美国人需要治疗药物依赖。药物成瘾是由于药物暴露和由此产生的多巴胺释放引起的脑回路变化而产生的。突触可塑性过程是药物滥用和成瘾的许多方面的基础，包括影响工具性行为的药物线索，行动结果和刺激反应协会的形成，以及寻求药物行为的消退。突触可塑性在很大程度上依赖于立即早期基因（IEGs）的表达和功能，特别是活性调节的细胞内相关（Arc）和早期生长反应-1（egr 1/zif268）基因。 这些基因对长期记忆的巩固和再巩固起着关键性的调节作用，包括与药物滥用和成瘾有关的记忆。因此，了解这些IEGs的调节对于鉴定新的治疗靶点以破坏异常的免疫调节具有重要意义。 导致药物滥用和成瘾的塑料变化。许多研究都集中在了解介导IEG转录调控的过程，以及树突状细胞的运输和Arc mRNA的局部翻译。然而，迄今为止，在现存的文献中没有研究检查一般mRNA的核输出的基本细胞过程，更不用说IEG RNA，特别是在成年哺乳动物脑中。我们的实验证据表明，这些IEG mRNA的核质出口不同的纹状体黑质（直接途径）与striatopallidal（间接途径）在背侧纹状体的神经元。此外，我们最近的数据表明，纹状体黑质传出神经元的IEG mRNA的核输出可能受到多巴胺的调节。因此，所提出的研究的总体目标是测试新的假设，即纹状体传出神经元亚群中IEG mRNA的核质输出存在差异。在具体目标1中，我们将确定已知的核质mRNA输出介质的表型分布及其与IEG mRNA在纹状体传出神经元亚群中的共定位。在具体目标2中，我们将差速离心和 流式细胞术分离细胞核从纹状体黑质和纹状体苍白球神经元，然后通过核糖核蛋白免疫沉淀和RT-PCR生化评估的相互作用的Arc和zif268与特定的核糖核蛋白复合物参与核输出。在具体目标3中，我们将确定多巴胺D1受体激活在纹状体黑质传出神经元IEGs核输出调节中的作用。由于缺乏有关成年哺乳动物大脑神经元中mRNA核输出调节的数据，以及神经递质系统（包括DA）对这一过程的潜在调节，因此成功完成拟议研究将为这些关键可塑性相关IEGs在纹状体回路中的调节提供开创性的见解，这些纹状体回路复杂地参与药物滥用和成瘾。因此，这些发现有可能揭示新的治疗靶点，以调节纹状体可塑性，从而改善药物滥用和成瘾史的个人的治疗结果。

 DESCRIPTION (provided by applicant): As of 2012, approximately 22 million Americans required treatment for drug dependence. Drug addiction arises as a consequence of changes in brain circuitry induced by drug exposure and resultant dopamine release. Synaptic plasticity processes underlie numerous aspects of drug abuse and addiction, including the formation of drug-clue, action-outcome, and stimulus-response associations affecting instrumental behavior, as well as extinction of drug-seeking behavior. Synaptic plasticity is heavily dependent on the expression and function of immediate early genes (IEGs), in particular activity-regulated, cytoskeletal-associated (Arc) and early growth response-1 (egr1/zif268) genes. These genes critically mediate consolidation and reconsolidation of long-term memories, including memories implicated in aspects of drug abuse and addiction. Understanding the regulation of these IEGs is therefore significant in its potential to identify novel therapeutic targets to disrupt aberrant plastic changes contributing to drug abuse and addiction. Many studies have focused on understanding processes mediating transcriptional regulation of IEGs, as well as on dendritic trafficking and local translation of Arc mRNA. However, to date there are no studies in the extant literature examining the basic cellular process of nuclear export of mRNAs in general, much less IEG RNAs in particular, in the adult mammalian brain. Our experimental evidence suggests that nucleocytoplasmic export of these IEG mRNAs differs in striatonigral (direct pathway) vs. striatopallidal (indirect pathway) neurons in dorsal striatum. Further, our recent data suggest that the nuclear export of IEG mRNAs in striatonigral efferent neurons may be regulated by dopamine. The overall goal of the proposed studies is thus to test the novel hypothesis that there are differences in nucleocytoplasmic export of IEG mRNAs in striatal efferent neuron subpopulations. In Specific Aim 1, we will determine the phenotypic distribution of known mediators of nucleocytoplasmic mRNA export and their co-localization with IEG mRNAs in striatal efferent neuron subpopulations. In Specific Aim 2, we will differential centrifugation and FACS to separate nuclei from striatonigral and striatopallidal neurons, followed by ribonucleoprotein immunoprecipitation and RT-PCR to biochemically assess the interaction of Arc and zif268 with specific ribonucleoprotein complexes involved in nuclear export. In Specific Aim 3, we will determine the role of dopamine D1 receptor activation in the regulation of nuclear export of IEGs in striatonigral efferent neurons. Given the lack of data regarding the regulation of nuclear export of mRNAs in neurons of the adult mammalian brain and potential regulation of this process by neurotransmitter systems, including DA, successful completion of the proposed studies will provide seminal insight into the regulation of these critical plasticity-related IEGs n striatal circuitry intricately involved in drug abuse and addiction. These findings thus have the potential to reveal novel therapeutic targets to modulate striatal plasticity so as to improve treatment outcomes in individuals with histories of drug abuse and addiction.