In Vivo Investigations of AMPA receptor transport

项目摘要/摘要： 这项提议的总体目标是调查鲜为人知的控制多头的机制。 AMPAR在传递和移除受体中的距离运输，以维持突触和可塑性。 AMPA亚型谷氨酸和离子型谷氨酸受体介导的兴奋性神经传递 (Ampar)At突触在认知过程中起着核心作用。对这些组织的数量和功能进行严格监管 因此，受体是必不可少的。由于突触通常远离神经元细胞体，所以它们 严重依赖于微管依赖的分子马达的长距离运输，以提供稳定的 AMPAR的供应。兴奋性突触传递领域对细胞如何- 信号通路控制局部突触AMPAR的运输，但几乎不了解这些突触是如何 信令事件控制远距离AMPAR传输。造成这种不足的主要原因是技术上的：运输 研究需要在完整的神经元回路中使用强大的高速显微镜。观察直观，信息量大 脊椎动物体内转运的操纵是极其困难的。我们开创了实时活体研究的先河 使用透明模式生物秀丽线虫，研究AMPAR在完整神经元回路中的转运。 在这里，我们将测试一种新的机制框架，用于调节AMPAR到突触的细胞分布 以分子马达对受体的长距离运输为中心。我们的模型预测Kinesin-1 支架(JIP1和3)是AMPAR转运所必需的，它们在Kinesin-1上的组装依赖于 神经元活性、钙及钙调素依赖的激酶2(CaMKII)。此外，我们还确定了一个 运输调节剂，PTP-3A，改变细胞体的输出和突触传递，最终影响 记忆。具体目标1将确定突触在细胞体和树突的输入如何改变钙和 安帕尔运输公司。《特殊目的2》验证了突触活动导致AMPAR改变的假设 具有不同输出和突触传递特性的运输复合体。《特定目标3》检验了这一假设 受体酪氨酸磷酸酶PTP-3的最长异构体PTP-3A调节AMPAR体细胞输出 利用神经元活动引起的分裂释放的2个结构域传递突触。这些实验 在这些目标中描述将结合遗传学、活体旋转盘双通道显微镜、光遗传学、 光漂白和光转化、生化和行为分析，以阐明长时间脱色的机理。 突触信号对距离AMPAR转运的调节。我们的研究将：1)提供一个新的模型 理解调节突触功能的细胞机制，2)对理解有广泛的影响 适用于多种生物系统的分子马达的货物运送和搬运机制，以及 3)提高对AMPAR转运的理解，可能揭示调节的新治疗靶点 人类疾病背景下的兴奋性突触传递。

Project Summary/Abstract: The overall goal of this proposal is to investigate the poorly understood mechanisms controlling long distance AMPAR transport in delivery and removal of receptors for synaptic maintenance and plasticity. Excitatory neurotransmission mediated by glutamate and ionotropic glutamate receptors of the AMPA subtype (AMPAR) at synapses plays a central role in cognition. Tight regulation of the number and function of these receptors is, therefore, essential. Since synapses are often far away from the neuronal cell body, they are critically dependent on long-distance transport by microtubule-dependent molecular motors to provide a steady supply of AMPARs. The field of excitatory synaptic transmission has a detailed understanding of how cell- signaling pathways control local synaptic AMPAR trafficking but almost no understanding of how these synaptic signaling events control long-distance AMPAR transport. The major reason for this lack is technical: transport studies require powerful, high-speed microscopy in intact neuronal circuits. Direct observation and informative manipulation of transport in vivo is extremely difficult in vertebrates. We have pioneered real-time in vivo studies of AMPAR transport in intact neuronal circuits using the transparent model organism, C. elegans. Here we will test a new mechanistic framework for the regulated cellular distribution of AMPARs to synapses centered on the long-distance transport of receptors by molecular motors. Our model predicts that Kinesin-1 scaffolds (JIP1 and 3) are necessary for AMPAR transport and their assembly onto Kinesin-1 is dependent on neuronal activity, calcium and calcium calmodulin-dependent kinase 2 (CaMKII). In addition, we identify a modulator of transport, PTP-3A, that modifies export from the cell body and synaptic delivery ultimately affecting memory. Specific Aim 1 will determine how synaptic inputs at cell bodies and at dendrites modify calcium and AMPAR transport. Specific Aim 2 tests the hypothesis that synaptic activity leads to modification of the AMPAR transport complex conferring different export and synaptic delivery properties. Specific Aim 3 tests the hypothesis that PTP-3A the longest isoform of the receptor tyrosine phosphatase PTP-3, regulates AMPAR somatic export and synaptic delivery using 2 domains released by cleavage induced by neuronal activity. The experiments described in these aims will combine genetics, in vivo spinning disk dual channel microscopy, optogenetics, photobleaching and photoconversion, biochemistry and behavior analyses to elucidate the mechanisms of long- distance AMPAR transport regulation by synaptic signaling. Our studies will: 1) provide a new model for understanding the cellular mechanisms regulating synaptic function, 2) have broad impact on the understanding of cargo delivery and removal mechanisms by molecular motors applicable to multiple biological systems, and 3) improve understanding of AMPAR transport that could reveal novel therapeutic targets for modulating excitatory synaptic transmission in the context of human disease.