Molecular Dissection of Active Zone Functions in Neurotransmitter Release

神经递质释放中活性区功能的分子剖析

• 批准号: 9275552
• 负责人: Pascal Simon Kaeser
• 金额: $ 37.08万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9275552
• 关键词: Action Potentials; Address; Affect; Animal Model; Architecture; Autistic Disorder; Binding; Biochemical; Brain; Brain Diseases; Caenorhabditis elegans; Cognition; Communication; Complex; Data; Dissection; Docking; Electrophysiology (science); Elements; Exocytosis; Gene Family; Gene Structure; Genes; Genetic; Goals; Homologous Gene; Image; Impairment; Individual; Inhibitory Synapse; Knock-out; Knockout Mice; Knowledge; Lead; Learning; Measurement; Mediating; Modeling; Molecular; Morphology; Nerve; Nerve Degeneration; Neurons; Pathologic; Pathway interactions; Phenotype; Probability; Process; Proteins; Recruitment Activity; Regulation; Research; Role; Schizophrenia; Signal Transduction; Site; Speed; Structure; Synapses; Synaptic Transmission; Synaptic Vesicles; Testing; Vertebrates; Vesicle; Work; addiction; chemical release; controlled release; experimental study; genetic approach; inhibitor/antagonist; innovation; insight; mutant; nervous system disorder; neural circuit; neuronal circuitry; neurotransmission; neurotransmitter release; novel; presynaptic; presynaptic neurons; public health relevance; scaffold; synaptic function; synaptogenesis; tool; transmission process; vesicular release

DESCRIPTION (provided by applicant): Speed and precise regulation of synaptic transmission are critical for complex brain functions such as cognition and learning. Release of neurotransmitters from a presynaptic nerve terminal is often impaired in neurological disorders, including autism, schizophrenia, addiction and neurodegeneration. Exact knowledge of the molecular mechanisms for neurotransmitter release is thus critical for understanding brain disease. The active zone of a presynaptic nerve terminal is the site of neurotransmitter release. An active zone consists of a highly specialized network of proteins that organizes synaptic vesicles for fast Ca2+-triggering of release, a central requirement for speed and precision of synaptic transmission. It is our over-arching goal to understand how the protein machinery at the active zone operates. We approach this goal by dissecting the molecular functions of active zone components. ELKS proteins are highly enriched at active zones, indicating that ELKS functions in neuronal exocytosis at the active zone. Before release, active zones dock and prime synaptic vesicles for exocytosis close to presynaptic Ca2+-channels. How ELKS operates during these processes to control release is not understood, maybe in part because no systematic genetic approach has been taken in vertebrates to address ELKS function. We have now generated conditional knockout mice for both mammalian ELKS genes, ELKS1 and ELKS2. Ample preliminary data lead to our central hypothesis: ELKS proteins increase release probability though controlling presynaptic Ca2+-influx, and they modulate the size of the pool of readily releasable vesicles. We address separate components of this hypothesis in three specific aims, and we dissect the underlying molecular mechanisms. In aim 1, we hypothesize that ELKS1 and ELKS2 proteins have both shared and distinct functions. We determine how each ELKS gene contributes to the functions of active zones in neurotransmitter release by systematically studying presynaptic phenotypes in the newly generated conditional single knockout mice for ELKS1 and ELKS2, and in the ELKS1/2 double knockout mice. In preliminary experiments we find that ELKS proteins enhance presynaptic Ca2+-influx, and that individual and double ELKS deletions differentially affect the pool of readily releasable vesicles. In aim 2, we determine the mechanisms by which ELKS controls presynaptic Ca2+-influx. In aim 3, we propose a specific hypothesis that unifies effects on vesicle pools observed in ELKS mutants. We examine this hypothesis, determine the underlying molecular mechanisms and consider numerous alternative explanations. Our research is innovative because it addresses a novel hypothesis by a combination of genetic, biochemical and functional experiments of unique depth. Ultimately, this approach will lead to precise insights into the molecular control of neurotransmitter release, a key neuronal process that fails during various brain diseases.

描述（由申请人提供）：突触传递的速度和精确调节对于认知和学习等复杂的大脑功能至关重要。在神经系统疾病中，突触前神经末梢释放的神经递质通常会受到损害，包括自闭症、精神分裂症、成瘾和神经变性。因此，准确了解神经递质释放的分子机制对于理解脑部疾病至关重要。突触前神经末梢的活跃区是神经递质释放的部位。活性区由高度专业化的蛋白质网络组成，该网络组织突触小泡以快速触发 Ca2+ 释放，这是突触传递速度和精度的核心要求。我们的首要目标是了解活性区的蛋白质机器如何运作。我们通过剖析活性区成分的分子功能来实现这一目标。 ELKS 蛋白在活性区高度富集，表明 ELKS 在活性区的神经元胞吐作用中发挥作用。在释放之前，活性区对接并启动突触小泡，以进行靠近突触前 Ca2+ 通道的胞吐作用。 ELKS 在这些过程中如何运作以控制释放尚不清楚，部分原因可能是因为在脊椎动物中尚未采取系统的遗传方法来解决 ELKS 功能。我们现在已经培育出哺乳动物 ELKS 基因 ELKS1 和 ELKS2 的条件敲除小鼠。充足的初步数据引出了我们的中心假设：ELKS 蛋白通过控制突触前 Ca2+ 流入来增加释放概率，并且它们调节易于释放的囊泡池的大小。我们在三个特定目标中解决了该假设的不同组成部分，并剖析了潜在的分子机制。在目标 1 中，我们假设 ELKS1 和 ELKS2 蛋白具有共同且不同的功能。我们通过系统地研究新生成的 ELKS1 和 ELKS2 条件单敲除小鼠以及 ELKS1/2 双敲除小鼠的突触前表型，确定每个 ELKS 基因如何促进神经递质释放中活性区的功能。在初步实验中，我们发现 ELKS 蛋白增强突触前 Ca2+ 流入，并且单个和双 ELKS 缺失对易于释放的囊泡库有不同的影响。在目标 2 中，我们确定 ELKS 控制突触前 Ca2+ 流入的机制。在目标 3 中，我们提出了一个具体的假设，该假设统一了在 ELKS 突变体中观察到的对囊泡池的影响。我们研究了这一假设，确定了潜在的分子机制并考虑了许多替代解释。我们的研究具有创新性，因为它通过结合独特深度的遗传、生化和功能实验提出了新的假设。最终，这种方法将带来对神经递质释放的分子控制的精确见解，神经递质释放是在各种脑部疾病期间失败的关键神经元过程。