Role of presynaptic calcium stores

描述(申请人提供)：细胞内钙信号是神经元电路信息处理的一个组成部分，促进突触前和突触后的功能，包括突触传递的长时程增强(LTP)和抑制(LTD)，长期突触可塑性的电生理关联。由于细胞内钙离子的微调变化可调节多种细胞内功能，因此信号特异性需要对胞内钙信号进行严格的空间和时间控制。这对快速释放神经递质的需求最为明显，电压依赖的钙信号在亚毫秒级触发突触前终末突触小泡的融合。内质网既可以作为钙存储/汇聚，也可以作为钙源，可能在突触前终末平衡这两种双重作用，以满足突触对递质释放的特定需求。然而，内质网在突触前钙信号和神经递质释放中的作用尚不清楚。我们假设内质网在Fly NMJ起主要的突触前钙离子汇聚的作用。如果是这样的话，突触前内质网可能不仅通过SERCA泵暂时缓冲胞内钙离子，还可能通过将管腔钙离子“隧道”输送到轴突中而永久地从突触终末移走钙离子。内质网钙离子在终末的有限释放可能调节神经递质的释放，但主要服务于线粒体钙摄取，激活线粒体能量产生。我们将利用基因操纵的果蝇和图像去极化诱导的内质网管腔、线粒体和幼虫NMJ胞浆突触前钙水平的变化来验证这一假说。这一建议的核心是开发能够真实地报告内质网和线粒体管腔内钙变化的转基因钙指示剂，我们预计这也将被广泛用于任何果蝇细胞中线粒体和/或内质网钙动态的遗传分析。具体地说，我们将确定内质网介导的钙摄取，钙释放和/或钙扩散(隧道)在重复神经刺激果蝇幼虫NMJ(目标1)，并确定内质网-线粒体相互作用对幼虫NMJ突触前钙稳态的作用(目标2)。通过这一系统的分析，将为更好地理解内质网在突触前钙信号转导/突触前终末动态平衡中的作用提供一个基本框架，扩大我们对突触传递的重要调控机制的理解，以及它们与神经系统功能可塑性和人类健康的关系。

DESCRIPTION (provided by applicant): Intracellular Ca2+ signals are an integral part of information processing by neuronal circuits facilitating pre- and postsynaptic functions including long-term potentiation (LTP) and depression (LTD) of synaptic transmission, the electrophysiological correlates of long-term synaptic plasticity. Since finely tuned changes in Ca2+ modulate a variety of intracellular functions, signal specificity requires a tight spatial and temporal control of the cytosolic Ca2+ signal. This need is most apparent for fast neurotransmitter release where voltage-dependent Ca2+ signaling triggers the fusion of synaptic vesicles at presynaptic terminals on a sub-millisecond scale. The endoplasmic reticulum (ER) can act as a Ca2+ store/sink or as a Ca2+ source and is likely to balance these dual roles at presynaptic terminals to meet the specific needs of the synapse for transmitter release. However, the contributions of ER to presynaptic Ca2+ signaling and neurotransmitter release are not well understood. We hypothesize that the ER acts as the primary presynaptic Ca2+ sink at fly NMJs. If so, the presynaptic ER may not only temporarily buffer cytosolic Ca2+ by SERCA pumping but also permanently remove Ca from synaptic terminals by "tunneling" luminal Ca2+ into axons. Limited ER Ca2+ release within terminals may modulate neurotransmitter release but mainly serve mitochondrial Ca2+ uptake activating mitochondrial energy production. We will test this hypothesis by exploiting genetically manipulated Drosophila and image depolarization-induced changes in presynaptic Ca levels of the ER lumen, mitochondria and the cytosol of larval NMJs. At the center of this proposal is the development of transgenic Ca2+ indicators that can faithfully report Ca2+ changes in the lumen of the ER and mitochondria, which we expect will also be of wide use for the genetic analysis of mitochondrial and/or ER Ca2+ dynamics in any cell of Drosophila. Specifically, we will determine the dynamics of ER-mediated Ca2+ uptake, Ca2+ release and/or Ca2+ diffusion (tunneling) upon repetitive nerve stimulation at presynaptic terminals of larval Drosophila NMJs (Aim 1) and determine the role of ER-mitochondria interactions for presynaptic Ca2+ homeostasis at larval Drosophila NMJs (Aim 2). From this systematic analysis a basic framework will emerge for better understanding the role of the ER in presynaptic Ca2+ signaling/homeostasis at presynaptic terminals expanding our understanding of important regulatory mechanisms of synaptic transmission and their relation to the functional plasticity of the nervous system and human health.