Exploring nucleocytoplasmic IEG mRNA export in striatal neuron subpopulations

 描述(申请人提供)：截至2012年，约有2200万美国人因药物依赖而需要接受治疗。药物成瘾是药物暴露引起的大脑回路改变以及由此产生的多巴胺释放的结果。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、行为结果和刺激反应联系的形成，以及药物寻找行为的消亡。突触的可塑性在很大程度上依赖于即刻早期基因(IEGs)的表达和功能，特别是活性调节、细胞骨架相关(Arc)和早期生长反应-1(Egr1/Zif268)基因。这些基因关键地调节长期记忆的巩固和再巩固，包括与药物滥用和成瘾有关的记忆。因此，了解这些IEG的调节对于确定干扰异常的新的治疗靶点的潜力具有重要意义 塑料变化会导致药物滥用和成瘾。许多研究集中在了解介导IEGs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，到目前为止，在现有的文献中还没有研究一般情况下mRNAs核输出的基本细胞过程，更不用说成年哺乳动物脑中的IEG RNAs了。我们的实验证据表明，这些IEG mRNAs在背侧纹状体的纹状体(直接通路)和纹状体(间接通路)神经元中的核质输出是不同的。此外，我们最近的数据表明，纹状体黑质传出神经元中IEG mRNAs的核输出可能受多巴胺的调节。因此，拟议研究的总体目标是检验这一新的假设，即在纹状体传出神经元亚群中存在核质输出IEG mRNAs的差异。在特定目标1中，我们将确定已知的核质mRNA输出介体的表型分布，以及它们与IEG mRNAs在纹状体传出神经元亚群中的共存。在具体目标2中，我们将区别离心法和 FACS分离纹状体黑质和纹状体苍白质神经元的细胞核，然后进行核糖核蛋白免疫沉淀和RT-PCR，以生化方法评估Arc和Zif268与参与核输出的特定核糖核蛋白复合体的相互作用。在特定的目标3中，我们将确定多巴胺D1受体激活在调节纹状体黑质传出神经元IEGs核输出中的作用。由于缺乏关于成年哺乳动物大脑神经元中mRNAs核输出的调控以及包括DA在内的神经递质系统对这一过程的潜在调控的数据，拟议中的研究的成功完成将为调控这些复杂地参与药物滥用和成瘾的纹状体回路中与可塑性相关的关键IEG提供开创性的见解。因此，这些发现有可能揭示调节纹状体可塑性的新的治疗靶点，从而改善有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.