Genetic Analysis of Neuromuscular Synaptic Structure and Function

神经肌肉突触结构和功能的遗传分析

• 批准号: RGPIN-2020-05852
• 负责人: Stewart, Bryan
• 金额: $ 2.33万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740872
• 关键词: Genetic; Analysis; Neuromuscular; Synaptic; Structure

The central questions that guide my research are: How do synapses function? How do they develop? We are motivated to study these questions to better understand how the nervous system and the brain give rise to behaviors in both health and disease. To address our questions my research team combines classical and molecular genetics in Drosophila melanogaster with developmental analysis and measurements of synaptic physiology. Due to the high degree of conservation at the cellular, genetic and molecular level, the information we generate studying flies translates to a more general understanding of how neurons develop and function. Our basic approach is to use perturb genes in a systematic way and then determine how the synapses respond. The main technical approaches we use are fly genetics electrophysiology of synaptic transmission, imaging technologies, including confocal, live-cell, and electron microscopy; we also do molecular biology and biochemistry as required. NSERC Discovery Grants have continuously supported our exploration within this framework. In this grant, using newly available genetic tools, we will examine the role of developmental competition and co-operation on synaptic physiology. At mammalian NMJs, synaptic competition occurs when more than one neuron innervates a target muscle and subsequent interactions between neurons leads to one `winner' and the withdrawal of the other nerve terminals. However, at arthropod NMJs, including Drosophila, it is normal for two or more functionally specialized motor neurons to innervate the same muscle cell, often in very close proximity; they appear to co-operate to innervate the target cell. We now have the ability to selectively drive gene expression in one or the other of these MNs and ask how the NMJ responds physiologically and developmentally. As one example, we will kill one of the neurons by driving apoptosis-inducing genes and determine how the remaining motor neuron develops and functions in the absence of the partner neuron. In a second set of experiments, we will control electrical excitability using K+ channel expression. Finally, we will target synaptic activity by reducing synaptic transmission in one of the two motor neurons by expressing well known regulators, of the vesicle cycle. In each case, we will characterize the function and morphology of the untreated motor neuron, in the absence of its partner. Based upon preliminary data, we will design a forward screen, to harness the real advantage of Drosophila genetics, to uncover molecules crucial to determining communication between two innervating neurons. Since errors in synaptic development and function are associated with a wide variety of neurological, cognitive and behavioural diseases the proposed experiments will yield new data that will help us understand the basic development and function of synapses; the knowledge gained will further inform our concept of neurodevelopmental and neurophysiological disorders.

指导我研究的核心问题是：突触如何发挥作用？它们是如何发展的？我们有动力研究这些问题，以更好地了解神经系统和大脑如何引起健康和疾病的行为。为了解决我们的问题，我的研究团队将果蝇的经典和分子遗传学与突触生理学的发育分析和测量相结合。由于在细胞、遗传和分子水平上的高度保守性，我们研究果蝇产生的信息转化为对神经元如何发育和功能的更普遍的理解。我们的基本方法是以系统的方式使用干扰基因，然后确定突触如何反应。我们使用的主要技术方法是突触传递的果蝇遗传学电生理学，成像技术，包括共聚焦，活细胞和电子显微镜;我们还根据需要进行分子生物学和生物化学。 NSERC发现赠款一直支持我们在这个框架内的探索。在这项资助中，我们将利用最新的遗传工具，研究发育竞争与合作对突触生理学的作用。在哺乳动物的NMJ中，当一个以上的神经元支配目标肌肉时，就会发生突触竞争，随后神经元之间的相互作用会导致一个“赢家”和其他神经末梢的撤回。然而，在节肢动物的NMJ，包括果蝇，这是正常的两个或两个以上的功能专门的运动神经元支配同一个肌肉细胞，往往在非常接近;他们似乎合作，以支配靶细胞。我们现在有能力选择性地驱动这些MN中的一个或另一个的基因表达，并询问NMJ如何在生理和发育上做出反应。 举个例子，我们将通过驱动一个运动诱导基因来杀死其中一个神经元，并确定在没有伙伴神经元的情况下，剩余的运动神经元如何发育和发挥功能。在第二组实验中，我们将使用K+通道表达来控制电兴奋性。最后，我们将通过表达囊泡周期的众所周知的调节因子来减少两个运动神经元之一的突触传递，从而靶向突触活动。在每种情况下，我们将描述未处理的运动神经元的功能和形态，在没有它的伴侣。基于初步数据，我们将设计一个前向筛选，利用果蝇遗传学的真实的优势，揭示决定两个神经元之间通信的关键分子。由于突触发育和功能的错误与各种神经，认知和行为疾病有关，因此拟议的实验将产生新的数据，帮助我们了解突触的基本发育和功能;所获得的知识将进一步告知我们神经发育和神经生理障碍的概念。