Exploring nucleocytoplasmic IEG mRNA export in striatal neuron subpopulations

探索纹状体神经元亚群中核细胞质 IEG mRNA 输出

• 批准号: 9005845
• 负责人: KRISTEN A KEEFE
• 金额: $ 18.44万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9005845
• 关键词: Adult; Affect; American; Antibodies; Basal Ganglia; Behavior; Behavioral Paradigm; Brain; Cell Nucleus; Cell physiology; Centrifugation; Complex; Confocal Microscopy; Corpus striatum structure; Coupled; Cytoplasm; Data; Detection; Development; Dopamine; Dopamine D1 Receptor; Dopamine D2 Receptor; Dorsal; Drug Addiction; Drug Exposure; Drug abuse; Efferent Neurons; Extinction (Psychology); Flow Cytometry; Fluorescent in Situ Hybridization; Genes; Goals; Growth; Health; Immediate-Early Genes; Immunoprecipitation; Individual; Label; Literature; Mediating; Mediator of activation protein; Memory; Messenger RNA; Molecular; Mus; Neurons; Neurotransmitters; Nuclear; Nuclear Export; Outcome; Pathway interactions; Pharmaceutical Preparations; Population; Process; Protein Export Pathway; Proteins; Receptor Activation; Recording of previous events; Regulation; Response to stimulus physiology; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoproteins; Role; Seminal; Sorting - Cell Movement; Synaptic plasticity; System; Testing; Transcriptional Activation; Transcriptional Regulation; Translations; Treatment outcome; brain circuitry; drug seeking behavior; improved; in vivo; insight; long term memory; mRNA Export; mRNA Expression; new therapeutic target; novel; novel therapeutics; promoter; response; trafficking

 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.

 描述（由申请人提供）：截至 2012 年，大约有 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大脑回路变化的结果。突触可塑性过程是药物滥用和成瘾的许多方面的基础，包括药物线索的形成、行动结果和影响工具行为的刺激反应关联，以及寻求药物行为的消失。突触可塑性很大程度上取决于立即早期基因 (IEG) 的表达和功能，特别是活性调节基因、细胞骨架相关基因 (Arc) 和早期生长反应 1 (egr1/zif268) 基因。 这些基因关键介导长期记忆的巩固和重新巩固，包括与药物滥用和成瘾有关的记忆。因此，了解这些 IEG 的调节对于确定新的治疗靶点以破坏异常的潜力具有重要意义。 塑料变化导致药物滥用和成瘾。许多研究都集中于了解介导 IEG 转录调控的过程，以及 Arc mRNA 的树突运输和局部翻译。然而，迄今为止，现有文献中还没有研究检查成年哺乳动物大脑中 mRNA 核输出的基本细胞过程，更不用说 IEG RNA 了。我们的实验证据表明，这些 IEG mRNA 的核细胞质输出在背侧纹状体的纹状体黑质（直接途径）和纹状体苍白球（间接途径）神经元中不同。此外，我们最近的数据表明，纹状体黑质传出神经元中 IEG mRNA 的核输出可能受到多巴胺的调节。因此，拟议研究的总体目标是检验新的假设，即纹状体传出神经元亚群中 IEG mRNA 的核细胞质输出存在差异。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 FACS 将细胞核与纹状体黑质和纹状体苍白球神经元分离，然后进行核糖核蛋白免疫沉淀和 RT-PCR，以生化评估 Arc 和 zif268 与参与核输出的特定核糖核蛋白复合物的相互作用。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 IEG 核输出调节中的作用。鉴于缺乏关于成年哺乳动物大脑神经元中 mRNA 核输出调节的数据，以及包括 DA 在内的神经递质系统对该过程的潜在调节，拟议研究的成功完成将为了解这些与药物滥用和成瘾错综复杂的纹状体回路中与可塑性相关的 IEG 的调节提供开创性的见解。因此，这些发现有可能揭示调节纹状体可塑性的新治疗靶点，从而改善有药物滥用和成瘾史的个体的治疗结果。