Localization of mRNAs at Synaptic Sites on Dendrites

描述(申请人提供)：本方案的实验将确定神经元树突上突触位置的信使核糖核酸定位和局部翻译的机制和功能作用。突触关键蛋白的局部合成对于包括长时程增强(LTP)和长时程抑制(LTD)在内的长时间突触可塑性以及记忆的巩固是必要的。最近的研究表明，脆性X智力低下综合征(FXS)是一种常见的神经系统疾病，其核心神经功能缺陷可能与FXS中Fmr1基因缺陷产物--Fmr1基因缺失导致突触蛋白质合成障碍有关。因此，对信使核糖核酸的转运、定位和翻译的研究可能为这种重要而普遍的神经系统疾病的治疗提供新的靶点。我们以前的研究已经通过研究独特的即刻早期基因(IEG)Arc，即活性调节的细胞骨架相关蛋白，揭示了选择性将mRNA靶向活性突触的机制。ARC是由生理活动强烈诱导的，其mRNA根据mRNA序列中的靶向信号迅速传递到树突。Arc mRNA选择性地定位于活性突触，并介导Arc蛋白的局部合成。核内Arc mRNA的转录和新合成的转录物的靶向活性突触都是由ERK1/2的NMDA受体激活和MAP激酶途径触发的。我们的研究也揭示了其他mRNAs以不同的方式定位。本提案的实验将继续识别和表征定位在树突中的mRNAs，并使用体内和体外准备进一步表征mRNAs对接在活性突触的机制。我们将测试这一假设，即对接机制涉及突触下或突触后特化内的分子支架，突触后特化被强烈突触活动产生的信号所改变。我们将通过评估含有部分Arc mRNA的外源表达转录本的靶向性，来检验Arc mRNA包含的序列决定mRNA将被运输到树突(树突运输序列)和导致mRNA选择性停靠在活性突触(突触靶向序列)的序列(S)的假设。我们以前的研究表明，其他树突状mRNAs不能重新定位到活性突触上，因此我们将确定这些mRNAs在树突中是不活动的，还是缺乏必要的靶向序列。总之，我们的研究将揭示调节突触蛋白质合成的机制的关键特征，从而为深入了解依赖蛋白质合成的突触修改的基本机制，包括FXS和其他影响mRNA运输和定位的神经疾病的机制提供洞察力。这项研究计划将确定生物机制，使神经细胞有可能通过控制突触接触部位突触的蛋白质构建块的合成来改变它们彼此之间的连接(突触)。这些研究将为大脑中信息存储的基本机制以及破坏这一基本机制的神经疾病提供新的见解，包括脆性X智力低下综合症。

DESCRIPTION (provided by applicant): The experiments of this proposal will define the mechanisms and functional role of mRNA localization and local translation at synaptic sites on neuronal dendrites. Local synthesis of critical proteins at synapses is necessary for long-lasting forms of synaptic plasticity including long-term potentiation (LTP) and long-term depression (LTD), and for the consolidation of memory. Recent studies indicate that the core neurological defect in a common neurological disorder Fragile X Mental Retardation Syndrome (FXS), may involve a disruption of protein synthesis at synapses due to the loss of Fragile X Mental Retardation Protein, the product of the Fmr1 gene which is defective in FXS. Thus, studies of mRNA transport, localization and translation may reveal new targets for therapy for this important and prevalent neurological disorder. Our previous studies have revealed aspects of the mechanisms underlying the selective targeting of mRNA to active synapses through studies of the unique immediate early gene (IEG) Arc, activity-regulated cytoskeleton-associated protein. Arc is strongly induced by physiological activity and its mRNA is rapidly delivered throughout dendrites based on a targeting signal in the mRNA sequence. Arc mRNA localizes selectively at active synapses, and mediates a local synthesis of Arc protein. Both the transcription of Arc mRNA in the nucleus, and the targeting of the newly synthesized transcript to active synapses are triggered by NMDA receptor activation of ERK1/2 and the MAP kinase pathway. Our studies have also revealed that other mRNAs are localized in different ways. The experiments of the present proposal will continue to identify and characterize mRNAs that are localized in dendrites, and further characterize the mechanisms underlying the docking of mRNAs at active synapses using in vivo and in vitro preparations. We will test the hypothesis that the docking mechanism involves a molecular scaffold beneath synapses or within the postsynaptic specialization that is modified by signals generated by intense synaptic activity. We will test the hypothesis that Arc mRNA contains sequences that determine that the mRNA will be transported into dendrites (a dendritic transport sequence) and sequence(s) that cause the mRNA to dock selectively at active synapses (a synaptic targeting sequence) by assessing targeting of exogenously expressed transcripts containing portions of Arc mRNA. Our previous studies indicate that other dendritic mRNAs do not re-localize to active synapses, and so we will determine whether these mRNAs are immobile in dendrites or instead lack the necessary targeting sequences. Together, our studies will reveal key features of the mechanism through which protein synthesis at synapses is regulated, thus providing insights into the fundamental mechanisms that underlie protein synthesis-dependent synaptic modifications, including the mechanisms that are disrupted in FXS and other neurological disorders that affect mRNA transport and localization. This research program will define biological mechanisms that make it possible for nerve cells to modify their connections with one-another (synapses) by controlling the synthesis of the protein building blocks of synapses at the synaptic contact site. These studies will provide novel insights into the basic mechanisms underlying information storage in the brain and neurological disorders that disrupt this fundamental mechanism, including Fragile-X Mental Retardation Syndrome.