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.

项目概要/摘要 突触前活性区 (AZ) 聚集突触后受体对面的突触小泡 (SV) 融合机制 场，促进有效的神经信号传导。谷氨酸能突触和 AZ 的正确发育对于 正常哺乳动物大脑发育。这些突触参与学习和记忆及其作用 功能障碍会导致神经发育障碍，例如智力障碍和自闭症谱系障碍。 然而，人们对这些可用区的发展和成熟却知之甚少。果蝇幼虫 运动神经元形成许多 AZ，作为哺乳动物的遗传和实验易处理模型 谷氨酸能突触。先前对果蝇神经肌肉接头 (NMJ) 的研究已证实 AZ 物质的积累分两个步骤：在 AZ 发育早期，Syd-1、Liprin-a 和 Unc-13B 等蛋白质形成 初始释放支架和 Brp、Cac（果蝇电压门控钙通道）、RIM 和 Unc-13A 抵达 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 各个 AZ 处的囊泡释放概率 (Pr)。早期支架对突触强度和突触强度的贡献 AZ 成熟度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 并且成熟度未知。在目标 1 中，将评估各个可用区的结构和功能成熟度 早期 AZ 支架的耗尽和过度表达后。荧光标记谷氨酸的积累 受体（GluR）亚基将在整个开发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 各个可用区的 Brp 和 Cac 也将被量化，以评估结构成熟度。个人功能成熟度 将通过计算 Pr 来评估 AZ。使用连接到突触后的荧光钙传感器 电刺激后的膜、个体 SV 融合事件通过钙离子进入进行可视化 GluR。在目标 2 中，将使用以下方法鉴定有助于 AZ 形成和成熟的蛋白质： 基于 CRISPR 的单个神经元筛选。目前鉴定的许多 AZ 蛋白都具有脂质结合域， 可能结合富含单个脂质种类的突触膜的特定区域。脂质激酶和磷酸酶 将用 Cas9 在单个神经元中消除，以便识别 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成为可能的，但将提供 与人类神经发育疾病和谷氨酸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 Troy Littleton 博士的实验室里。