Molecular mechanisms of synapse assembly and function

描述(由申请人提供)：异常的突触功能是从自闭症到精神健康障碍再到神经退化的神经疾病的标志。突触活动区(Synaptic Active Zones，AZ)是调节神经递质释放的特殊部位。不同AZ之间的释放动力学有很大的不同，并受到神经活动的调节。尽管神经递质的调节释放对神经系统功能很重要，但我们对AZ是如何组织以达到精确的释放特性以及它们是如何根据活动进行重组的了解仍然有限。我们最近发现了Fife，一种无脊椎动物短笛的同源物。Piccolo以前被认为不存在于无脊椎动物基因组中，它是一种AZ蛋白，被认为通过与AZ蛋白的多重连接来调节释放动力学。果蝇是研究Fife/Piccolo功能的理想模型，因为它缺乏巴松管和相关的遗传冗余，这减缓了哺乳动物系统的进展。我们产生了零FIFE等位基因，并发现FIFE在AZ组织、神经递质中起着关键作用 释放和行为。在这里，我们测试了与我们的功能数据和脊椎动物生化数据一致的模型，即FIFE通过组织钙通道和突触小泡(SVS)促进神经递质释放，并在近距离实现可靠的钙依赖胞吐(目标1)。果蝇模型的一个关键优势是能够快速发展到基于蛋白质相互作用的机械模型的体内功能测试。我们已经在串联质谱仪(MS/MS)实验中确定了cAMP信号通路的组成部分为FIFE相互作用蛋白，并提议进行实验以从功能上测试这些相互作用在突触可塑性中的在体意义(目标2A和B)。最后，我们提出了一种新颖的、高效的遗传筛选，它利用FIFE突变体中神经起源的雌性不育来识别蛋白质组鉴定可能无法获得的功能相互作用因子(目标2C)。

DESCRIPTION (provided by applicant): Aberrant synaptic function is a hallmark of neurological diseases ranging from autism to mental health disorders to neurodegeneration. Synaptic active zones (AZs) are specialized sites for the regulated release of neurotransmitter. Release dynamics vary significantly between AZs and are modulated in response to neural activity. Despite the importance of regulated neurotransmitter release to nervous system function, our understanding of how AZs are organized to achieve precise release properties and how they are reorganized in response to activity remains limited. We recently identified Fife, an invertebrate Piccolo homolog. Piccolo, previously believed absent from invertebrate genomes, is an AZ protein hypothesized to regulate release dynamics through its multiple connections to AZ proteins. Drosophila is an ideal model for studying Fife/Piccolo function as it lacks Bassoon and the associated genetic redundancy that has slowed progress in mammalian systems. We generated null fife alleles and found a critical role for Fife in AZ organization, neurotransmitter release and behavior. Here, we test the model, consistent with our functional data and vertebrate biochemical data, that Fife promotes neurotransmitter release by organizing Ca2+ channels and synaptic vesicles (SVs) and in close proximity for reliable Ca2+- dependent exocytosis (Aim 1). A key advantage of the Drosophila model is the ability to rapidly progress to in vivo functional tests of mechanistic models based on protein interactions. We have identified components of the cAMP-signaling pathway as Fife interacting proteins in tandem mass spectrometry (MS/MS) experiments, and propose experiments to functionally test the in vivo significance of these interactions in synaptic plasticity (Aim 2A and B). Finally, we propose a novel, highly efficient genetic screen that takes advantage of neuronally derived female sterility in fife mutants to identify functional interactors that may be inaccessible to proteomic identification (Aim 2C).