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

miR-8 和 Ena 在突触结构和功能中的作用

• 批准号: 7807300
• 负责人: CARLOS MANUEL LOYA
• 金额: $ 2.65万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7807300
• 关键词: Accounting; Actins; Affect; Autistic Disorder; Behavior; Behavioral; Biochemical; Comprehension; Conserved Sequence; Cytoskeleton; Defect; Development; Disease; Dissection; Drug Delivery Systems; Electron Microscopy; Elements; Engineering; Event; Fragile X Syndrome; Gene Expression; Gene Targeting; Genetic; Growth; Learning; Logic; Maintenance; Memory; Mental Retardation; Methods; MicroRNAs; Molecular; Morphogenesis; Morphology; Motor; Muscle; Nerve Degeneration; Neuromuscular Junction; Neurons; Neurotransmitter Receptor; Neurotransmitters; Phosphorus; Physiological; Physiology; Play; Post-Transcriptional Regulation; Protein Biosynthesis; Regulation; Repression; Research; Reticulum; Role; Series; Specificity; Spinal Muscular Atrophy; Structure; Synapses; Technology; Testing; Therapeutic; Transgenes; Transgenic Organisms; Work; base; cytomatrix; gene function; in vivo; loss of function; molecular imaging; mutant; nervous system disorder; neural circuit; neuron apoptosis; neuropathology; neurotransmitter release; novel; overexpression; postsynaptic; presynaptic; synaptic function; synaptogenesis; targeted delivery; tool

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.

描述（由申请人提供）：鉴于突触发育和功能的破坏已被证明在从精神发育迟滞到神经变性的广泛的神经系统疾病中起作用，进一步了解参与建立适当突触接触的分子组分对于我们理解这些疾病状态以及潜在地发现药物靶点和治疗方法是重要的。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因子，并且已经成为调节和整合突触处的活性依赖性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在突触发育过程中microRNA控制细胞骨架动力学的结构，功能和细胞基础。作为第一步，我们将全面研究保守的microRNA，miR-8及其功能靶点Ena在突触中的作用。使用microRNA沉默P元件（miR-SP），一种新的和通用的基于转基因的技术，我们已经工程化，以消除microRNA的活性与空间和时间特异性，我们发现，miR-8促进神经肌肉接头（NMJ）的生长，肌肉特异性抑制Ena，一个完善的肌动蛋白动力学的调节。我们将使用广泛的遗传，生理，成像和分子方法来研究miR-8耗尽和Ena过表达在突触中的作用。 具体而言，我将1）使用电生理学分析评估神经递质（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查miR-8缺失和Ena过表达对突触后和突触前超微结构装置的影响;和3）测试一系列Ena突变转基因以确定哪些保守序列元件对于Ena在NMJ的定位和功能是关键的。这将使我们能够确定参与Ena和miR-8依赖性调节NMJ形态发生的可能效应子和细胞机制。 异常的突触形态和功能已被证明是神经病理学的基础，例如Fragije X精神发育迟滞、脊髓性肌萎缩和自闭症。了解适当的突触形成的关键基因的功能有可能产生的治疗进展，以改善或治愈这些神经元疾病，因此，我们提出的研究具有重大意义的生物医学。