Molecular mechanisms of synapse assembly and function

描述（由申请人提供）：异常突触功能是从自闭症到精神健康障碍再到神经退行性疾病的神经系统疾病的标志。突触活动区是调节神经递质释放的专门场所。释放动力学在AZ之间变化显著，并且响应于神经活动而被调制。尽管调节神经递质释放对神经系统功能的重要性，但我们对AZ如何组织以实现精确的释放特性以及它们如何响应于活动而重组的理解仍然有限。我们最近发现了法夫，一种无脊椎短笛的同源物。Piccolo，以前被认为是无脊椎动物基因组中不存在的，是一种AZ蛋白，假设通过其与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。我们产生了无效的fife等位基因，发现Fife在AZ组织，神经递质 释放和行为。在这里，我们测试的模型，符合我们的功能数据和脊椎动物的生化数据，法夫促进神经递质的释放，通过组织Ca 2+通道和突触囊泡（SV）和紧密接近可靠的Ca 2+依赖性胞吐（目的1）。果蝇模型的一个关键优势是能够快速进行基于蛋白质相互作用的机制模型的体内功能测试。我们已经确定的cAMP信号通路的组件Fife相互作用蛋白质的串联质谱（MS/MS）实验，并提出实验功能测试这些相互作用在突触可塑性的体内意义（目的2A和B）。最后，我们提出了一种新的，高效的遗传筛选，利用神经源性的女性不育的fife突变体，以确定功能的相互作用，可能无法访问的蛋白质组学鉴定（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).