In Vivo Investigations of AMPA receptor transport

项目概要/摘要： 这项提议的总体目标是调查控制长时间睡眠的知之甚少的机制。 AMPAR在传递和移除受体中的距离运输，用于突触维持和可塑性。 谷氨酸和AMPA亚型离子型谷氨酸受体介导的兴奋性神经传递 突触的AMPAR在认知中起着核心作用。严格控制这些组织的数量和功能 受体是必不可少的。由于突触通常远离神经元细胞体， 严重依赖于微管依赖性分子马达的长距离运输，以提供稳定的 供应AMPAR。兴奋性突触传递领域对细胞- 信号通路控制局部突触AMPAR运输，但几乎不了解这些突触 信令事件控制长距离AMPAR传输。造成这种缺乏的主要原因是技术性的：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。直接观察和提供信息 在脊椎动物中，体内转运的操作是极其困难的。我们开创了实时体内研究 利用透明模式生物C.优雅 在这里，我们将测试一个新的机制框架，调节细胞分布的AMPAR突触 集中在受体通过分子马达的长距离运输上。我们的模型预测驱动蛋白-1 支架（JIP 1和3）对于AMPAR转运是必需的，并且它们在驱动蛋白-1上的组装依赖于 神经元活性、钙和钙钙调蛋白依赖性激酶2（CaMKII）。此外，我们还确定了一个 一种转运调节剂，PTP-3A，可调节从细胞体的输出和突触传递，最终影响 记忆具体目标1将确定细胞体和树突处的突触输入如何改变钙离子， AMPAR运输。具体目标2测试突触活动导致AMPAR修饰的假设 传递复合物赋予不同的输出和突触传递特性。具体目标3测试假设 PTP-3A是酪氨酸磷酸酶PTP-3的最长亚型，调节AMPAR体细胞输出， 以及使用由神经元活性诱导的裂解释放的2个结构域的突触递送。实验 在这些目的中描述的将结合联合收割机遗传学，体内旋转盘双通道显微术，光遗传学， 光漂白和光转换，生物化学和行为分析，以阐明长期的机制， 通过突触信号传导调节AMPAR转运距离。我们的研究将：1）提供一个新的模式， 理解调节突触功能的细胞机制，2）对理解 通过适用于多种生物系统的分子马达的货物递送和移除机制，以及 3)提高对AMPAR转运的理解，这可能揭示新的治疗靶点， 兴奋性突触传递在人类疾病的背景下。

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.