Role of miR-8 and Ena in Synaptic Structure and Function

描述(申请人提供)：鉴于突触发育和功能的中断已被证明在从智力低下到神经退化的一系列神经疾病中发挥作用，进一步了解建立适当的突触联系所涉及的分子成分对于我们理解这些疾病状态非常重要，并可能发现药物靶点和治疗方法。最近的研究表明，蛋白质合成的转录后调节和肌动蛋白-细胞骨架动力学的调节在神经元连接的形成和生理中起着关键作用。MicroRNA是转录后基因表达的精细调节者，已成为调节和整合突触上活性依赖的蛋白质合成和肌动蛋白动力学的优秀候选者。我的长期目标是确定microRNA在突触发育过程中控制细胞骨架动力学的结构、功能和细胞基础。作为第一步，我们将对保守的microRNA miR-8及其功能靶标EnA在突触中的作用进行全面的研究。利用microRNA沉默P元件(miR-SP)，这是一种新颖而通用的转基因技术，我们设计了一种消除具有时空特异性的microRNA活性的技术，我们发现miR-8通过肌肉特异性地抑制Ena促进神经肌肉接头(NMJ)的生长，Ena是一种公认的肌动蛋白动力学调节因子。我们将使用广泛的遗传学、生理学、成像和分子方法来研究miR-8缺失和突触ENA过度表达的作用。具体地说，我将1)使用电生理学分析来评估神经递质(NT)释放装置或突触后NT受体的敏感性是否受到普遍存在的和/或肌肉特异性的miR-8缺失的影响；2)在电子显微镜水平上检测miR-8的缺失和Ena的过度表达对突触后和突触前超微结构的影响；以及3)检测一系列ena突变的转基因，以确定哪些保守的序列元件对Ena在NMJ的定位和功能至关重要。这将使我们能够确定ENA和miR-8依赖的NMJ形态发生调节所涉及的可能的效应器和细胞机制。突触形态和功能的异常被证明是诸如Fragije X智力低下、脊髓肌萎缩和自闭症等神经病理的基础。了解对适当突触形成至关重要的基因的功能，有可能在治疗方面取得进展，以改善或治愈这些神经元疾病，因此，我们提出的研究对生物医学具有重要意义。

DESCRIPTION (provided by applicant): Given that disruption of synapse development and function has been show to play a role in a broad range of neurological disorders from mental retardation to neurodegeneration, further understanding of the molecular components involved in the establishment of appropriate synaptic contacts is important for our comprehension of these disease states, and potentially for the discovery of drug targets and therapeutics. Recent studies have demonstrated that post-transcriptional regulation of protein synthesis and the modulation of actin-cytoskeleton dynamics play a critical role in the formation and physiology of neuronal connections. MicroRNA are exquisite regulators of post-transcriptional gene expression and have emerged as excellent candidates to regulate and integrate activity-dependent protein synthesis and actin dynamics at the synapse. My long-term objective is to define the structural, functional, and cellular basis by which microRNA control cytoskeletal dynamics during synaptic development. As an initial step, we will perform a comprehensive study of the role of the conserved microRNA, miR-8, and its functional target Ena, at the synapse. Using microRNA silencing P elements (miR-SP), a novel and versatile transgenic-based technology we have engineered to eliminate microRNA activity with spatial and temporal specificity, we have discovered that miR-8 promotes neuromuscular junction (NMJ) growth by muscle-specific repression of Ena, a well-established regulator of actin dynamics. We will use a broad array of genetic, physiological, imaging and molecular approaches to study the role of miR-8 depletion and Ena overexpression at the synapse. Specifically I will 1) assess whether the neurotransmitter (NT) release apparatus or sensitivity of postsynaptic NT receptors is affected by ubiquitous and/or muscle-specific depletion of miR-8 using electrophysiological analysis; 2) examine the effect of miR-8 depletion and Ena overexpression to the postsynaptic and presynaptic ultrastructural apparatus at the electron microscopy level; and 3) test a series of Ena mutant transgenes to determine which conserved sequence elements are critical for the localization and function of Ena at the NMJ. This will allow us to determine the possible effectors and cellular mechanisms involved in Ena and miR-8-dependent regulation of NMJ morphogenesis. Abnormal synapse morphology and function has been shown to underlie neuropathologies such as Fragije X Mental Retardation, Spinal Muscular Atrophy, and autism. Understanding the function of genes critical for appropriate synapse formation has the potential to yield advances in therapeutics to ameliorate or cure these neuronal disorders, and hence, our proposed research has great significance to biomedicine.