Establishing the cohort of early active zone proteins and their role in synaptic strength and maturation at the Drosophila neuromuscular junction.

建立早期活性区蛋白群体及其在果蝇神经肌肉接头突触强度和成熟中的作用。

• 批准号: 10462313
• 负责人: Ellen Guss
• 金额: $ 4.68万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-07 至 2025-09-06
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462313
• 关键词: Action Potentials; Age; Amino Acids; Animals; Automobile Driving; Autophagocytosis; Binding; Biological Assay; Biological Models; Brain; C2 Domain; CRISPR screen; Calcium; Calcium Channel; Cell physiology; Charge; Coiled-Coil Domain; Complement; Confocal Microscopy; Data; Defect; Deposition; Development; Disease; Drosophila genus; Drosophila melanogaster; Electric Stimulation; Endocytosis; Event; Excitatory Synapse; Functional disorder; Glutamate Receptor; Glutamates; Guide RNA; Hour; Human; Human Development; Image; Individual; Institutes; Intellectual functioning disability; Lead; Learning; Lipid Binding; Lipids; Massachusetts; Measures; Mediating; Membrane; Memory; Modeling; Molecular; Motor Neurons; Neurodevelopmental Disorder; Neuromuscular Junction; Neurons; Optics; Output; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphotransferases; Play; Postsynaptic Membrane; Probability; Process; Proteins; Research; Resolution; Role; Scaffolding Protein; Seeds; Site; Stretching; Synapses; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; Technology; Training; Work; active control; autism spectrum disorder; brain dysfunction; cohort; confocal imaging; density; experimental study; fluorescence imaging; genetic approach; insight; loss of function; neurotransmission; novel; overexpression; postsynaptic; presynaptic; presynaptic neurons; prevent; protein transport; quantum; receptor; scaffold; sensor; trafficking; vesicular release; voltage

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.

项目总结/摘要 突触前活动区（AZs）聚集突触小泡（SV）与突触后受体的融合机制 场，促进有效的神经信号。神经元能突触和AZ的适当发育对于 正常的哺乳动物大脑发育这些突触参与学习和记忆， 功能障碍导致神经发育障碍，如智力残疾和自闭症谱系障碍。 然而，这些AZ的发育和成熟知之甚少。黑腹果蝇幼虫 运动神经元形成许多AZ，其作为哺乳动物的遗传和实验上易处理的模型， 神经元突触。以前在果蝇神经肌肉接头（NMJ）的工作已经确定，AZ 材料的积累分两步：在AZ发育的早期，蛋白质如Syd-1、Liprin-a和Unc-13 B形成 初始释放支架和Brp、Cac（果蝇电压门控钙通道）、RIM和Unc-13 A到达 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。 囊泡释放概率（Pr）。早期支架对突触强度的贡献， AZ成熟是一个开放的问题。此外，可以有助于AZ播种的早期蛋白质的完整队列 成熟度未知。在目标1中，将评估单个AZ的结构和功能成熟度 在早期AZ支架的耗尽和过度表达之后。荧光标记谷氨酸的蓄积 受体（GluR）亚基将在整个发育过程中使用高分辨率共聚焦成像进行测量， 活的动物在成熟的AZ中，GluRIIA和GluRIIB亚基分离成不同的环。突触前水平 还将量化各个AZ的Brp和Cac，以评估结构成熟度。个人功能成熟度 AZ将通过使用连接到突触后的荧光钙传感器计算Pr来评估。 膜，电刺激后的单个SV融合事件通过钙进入而可视化。 GluRs。在目标2中，将使用免疫组织化学方法鉴定有助于AZ形成和成熟的蛋白质。 基于CRISPR的单个神经元筛选许多目前鉴定的AZ蛋白具有脂质结合结构域， 可以结合富含单个脂质物质的突触膜的特定区域。脂质激酶和磷酸酶 将在具有Cas9的单个神经元中消除，以鉴定AZ形成和成熟的破坏。 这些实验是通过仅在果蝇中可用的实验方法实现的，但将提供 与人类神经发育疾病和突触发育相关的见解。所有的工作 为实现这些目标，将在马萨诸塞州理工学院进行必要的培训 在特洛伊利特尔顿博士的实验室里