Localization of mRNAs at Synaptic Sites on Dendrites

描述（由申请人提供）：本提案的实验将定义神经元树突突触位点mRNA定位和局部翻译的机制和功能作用。突触中关键蛋白的局部合成对于突触可塑性的持久形式（包括长期增强（LTP）和长期抑制（LTD））和记忆的巩固是必要的。最近的研究表明，脆性X智力发育迟滞综合征（脆性X智力发育迟滞综合征，FXS）常见神经系统疾病的核心神经缺陷可能涉及脆性X智力发育迟滞蛋白（脆性X智力发育迟滞蛋白是FXS缺陷基因的产物）的缺失，导致突触蛋白质合成中断。因此，对mRNA转运、定位和翻译的研究可能会为这种重要和普遍的神经系统疾病的治疗提供新的靶点。我们之前的研究通过研究独特的即时早期基因（IEG） Arc，活性调节的细胞骨架相关蛋白，揭示了mRNA选择性靶向活性突触的机制。Arc受到生理活动的强烈诱导，其mRNA基于mRNA序列中的靶向信号在树突中快速传递。Arc mRNA选择性地定位于活跃突触，并介导Arc蛋白的局部合成。Arc mRNA在细胞核内的转录和新合成的转录物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。我们的研究还揭示了其他mrna以不同的方式定位。本研究的实验将继续鉴定和表征树突中定位的mrna，并利用体内和体外制剂进一步表征mrna在活性突触上对接的机制。我们将测试对接机制涉及突触下或突触后特化的分子支架的假设，该分子支架被强烈的突触活动产生的信号所修改。我们将通过评估含有部分Arc mRNA的外源表达转录物的靶向性来验证Arc mRNA包含决定mRNA转运到树突的序列（树突转运序列）和导致mRNA选择性停靠在活跃突触的序列（突触靶向序列）的假设。我们之前的研究表明，其他树突mrna不会重新定位到活跃的突触，因此我们将确定这些mrna是在树突中不移动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中蛋白质合成机制的关键特征，从而深入了解蛋白质合成依赖性突触修饰的基本机制，包括FXS和其他影响mRNA运输和定位的神经系统疾病中被破坏的机制。该研究计划将定义生物学机制，使神经细胞通过控制突触接触部位突触蛋白质构建块的合成来改变它们彼此之间的连接（突触）成为可能。这些研究将为大脑信息存储的基本机制和破坏这一基本机制的神经系统疾病（包括脆性x智力发育迟滞综合征）提供新的见解。

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.