Establishing the Cohort of Early Active Zone Proteins and their Role in Synaptic Strength and Maturation at the Drosophila Neuromuscular Junction

项目摘要/摘要 突触前活动区(AZs)聚集突触小泡(SV)融合机制穿过突触后受体 场，促进有效的神经信号传递。谷氨酸能突触和AZs的正常发育对 哺乳动物正常的大脑发育。这些突触参与学习和记忆，它们的 功能障碍会导致神经发育障碍，如智力残疾和自闭症谱系障碍。 然而，人们对这些区域的发展和成熟知之甚少。黑腹果蝇幼虫 运动神经元形成许多AZ，这些AZ在基因上和实验上都是哺乳动物的易驯化模型 谷氨酸能突触。此前在果蝇神经肌肉接头(NMJ)的研究已经证实，AZ 物质的积累分两个步骤：在AZ发育的早期，Syd-1、Liprin-a和UNC-13B等蛋白质形成 初步释放支架和BRP、CAC(果蝇电压门控钙通道)、RIM和UNC-13A到达 几个小时后。AZ年龄和晚期成分BRP和CAC的掺入与成熟和突触相关 单个AZ的囊泡释放概率(Pr)。早期支架对突触强度和突触的贡献 亚洲区的成熟是一个悬而未决的问题。此外，有助于AZ播种的早期蛋白质的完整队列 成熟度还不得而知。在目标1中，将评估各个自贸区的结构和功能成熟度。 在早期AZ支架耗尽和过度表达后。荧光标记谷氨酸的积累 受体(GluR)亚单位将在整个发育过程中使用高分辨率共聚焦成像进行测量 活着的动物。在成熟的AZs，GluRIIA和GluRIIB亚基分离成不同的环。突触前水平 个别AZ的BRP和CAC也将被量化，以评估结构成熟度。个体的功能成熟度 AZE将通过计算Pr来评估。使用连接到突触后的荧光钙传感器 膜，电刺激后的单个SV融合事件可通过钙离子进入通过 谷氨酸。在目标2中，有助于AZ形成和成熟的蛋白质将使用 基于CRISPR的单个神经元的筛选。目前发现的许多AZ蛋白具有脂结合结构域，这些结构域 可能与富含个别类脂物质的突触膜特定区域结合。脂蛋白激酶与磷酸酶 在使用Cas9的单个神经元中将被消除，以确定AZ形成和成熟的中断。 这些实验是通过只在果蝇身上可用的实验方法实现的，但将提供 与人类神经发育疾病和谷氨酸能突触发育相关的见解。所有的工作 实现这些目标的先决条件培训将在麻省理工学院进行 在特洛伊·利特尔顿博士的实验室里。

PROJECT SUMMARY/ABSTRACT Presynaptic active zones (AZs) cluster synaptic vesicle (SV) fusion machinery across from postsynaptic receptor fields, facilitating efficient neural signaling. Proper development of glutamatergic synapses and AZs is critical for normal mammalian brain development. These synapses are involved in learning and memory and their dysfunction causes neurodevelopmental disorders like intellectual disability and autism spectrum disorder. However, the development and maturation of these AZs is poorly understood. Drosophila melanogaster larval motor neurons form many AZs which serve as a genetically and experimentally tractable model for mammalian glutamatergic synapses. Previous work at the Drosophila neuromuscular junction (NMJ) has established that AZ material accumulates in two steps: early in AZ development, proteins such as Syd-1, Liprin-a, and Unc-13B form an initial release scaffold and Brp, Cac (Drosophila voltage-gated calcium channel), RIM and Unc-13A arrive hours later. AZ age and incorporation of the late components Brp and Cac correlate with maturation and synaptic vesicle release probability (Pr) at individual AZs. The contribution of the early scaffolds to synaptic strength and AZ maturation is an open question. In addition, the full cohort of early proteins that can contribute to AZ seeding and maturation is unknown. In Aim 1, structural and functional maturity of individual AZs will be assessed following depletion and overexpression of early AZ scaffolds. Accumulation of fluorescently tagged Glutamate receptor (GluR) subunits will be measured throughout development using high resolution confocal imaging of live animals. At mature AZs, GluRIIA and GluRIIB subunits segregate into distinct rings. Levels of presynaptic Brp and Cac at individual AZs will also be quantified to assess structural maturity. Functional maturity of individual AZs will be assessed by calculating Pr. Using a fluorescent calcium sensor attached to the postsynaptic membrane, individual SV fusion events following electrical stimulation are visualized by calcium entry through GluRs. In Aim 2, proteins which contribute to formation and maturation of the AZ will be identified using a CRISPR-based screen in single neurons. Many currently identified AZ proteins have lipid binding domains which may bind specific regions of synaptic membrane rich in individual lipid species. Lipid kinases and phosphatases will be eliminated in single neurons with Cas9 in order to identify disruptions in AZ formation and maturation. These experiments are made possible by experimental approaches only available in Drosophila, but will provide insights relevant to human neurodevelopmental disease and glutamatergic synapse development. All of the work and prerequisite training to accomplish these Aims will be performed at Massachusetts Institute of Technology in Dr. Troy Littleton’s lab.