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: 10615711
• 负责人: Ellen Guss
• 金额: $ 1.87万
• 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 2023-04-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10615711
• 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; Drosophila genus; Drosophila melanogaster; Electric Stimulation; Endocytosis; Event; Excitatory Synapse; Functional disorder; Glutamate Receptor; Glutamates; Guide RNA; Hour; Human; Human Development; Image; Individual; Intellectual functioning disability; Learning; Lipid Binding; Lipids; Maps; 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; Site; Stretching; Synapses; Synaptic Membranes; Synaptic Receptors; Synaptic Transmission; Synaptic Vesicles; Technology; Training; Visualization; Work; active control; autism spectrum disorder; cohort; confocal imaging; experimental study; fluorescence imaging; genetic approach; insight; loss of function; neurotransmission; novel; overexpression; postsynaptic; presynaptic; presynaptic neurons; prevent; protein transport; scaffold; segregation; sensor; trafficking; vesicular release; voltage; 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; Drosophila genus; Drosophila melanogaster; Electric Stimulation; Endocytosis; Event; Excitatory Synapse; Functional disorder; Glutamate Receptor; Glutamates; Guide RNA; Hour; Human; Human Development; Image; Individual; Intellectual functioning disability; Learning; Lipid Binding; Lipids; Maps; 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; Site; Stretching; Synapses; Synaptic Membranes; Synaptic Receptors; Synaptic Transmission; Synaptic Vesicles; Technology; Training; Visualization; Work; active control; autism spectrum disorder; cohort; confocal imaging; experimental study; fluorescence imaging; genetic approach; insight; loss of function; neurotransmission; novel; overexpression; postsynaptic; presynaptic; presynaptic neurons; prevent; protein transport; scaffold; segregation; 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.

项目摘要/摘要 突触前活动区（AZS）簇突触囊泡（SV）融合机械从突触后接收器对面 场，支持有效的神经信号传导。正确开发谷氨酸能突触和AZS对于 正常的哺乳动物脑发育。这些突触涉及学习和记忆及其 功能障碍会引起神经发育障碍，例如智力障碍和自闭症谱系障碍。 但是，这些AZS的发展和成熟知之甚少。果蝇melanogaster幼虫 运动神经元形成了许多AZ，它们是哺乳动物的一般且实验性的模型 谷氨酸能突触。果蝇神经肌肉连接（NMJ）的先前工作已经确定 材料分为两个步骤：AZ开发早期，蛋白质，例如SYD-1，Liprin-A和UNC-13B形式 初始释放脚手架和BRP，CAC（果蝇电压门控钙通道），RIM和UNC-13A到达 几个小时后。 AZ年龄并结合晚期组件BRP和CAC与成熟和突触相关 在单个AZS处的囊泡释放概率（PR）。早期脚手架对突触强度和 AZ成熟是一个悬而未决的问题。另外，可以有助于AZ播种的早期蛋白质的全部队列 成熟是未知的。在AIM 1中，将评估单个AZ的结构和功能成熟度 耗尽和过表达早期AZ支架。荧光标记的谷氨酸的积累 使用高分辨率共聚焦成像，将在整个发育过程中测量受体（Glur）亚基 活动物。在成熟的AZS，Gluriia和Gluriib亚基分为不同的环。突触前的水平 在单个AZS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 AZS将通过计算PR进行评估。使用连接到突触后的荧光钙传感器 膜，电模拟后的单个SV融合事件通过通过钙进入可视化 gl。在AIM 2中，将使用A确定有助于AZ形成和成熟的蛋白质 基于CRISPR的单神经元中的屏幕。许多目前确定的AZ蛋白具有脂质结合域 可以结合富含单个脂质物种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，以确定AZ形成和成熟的中断。 这些实验是通过仅在果蝇中可用的实验方法才能实现的，但会提供 与人类神经发育疾病和谷氨酸能突触发育有关的见解。所有的工作 马萨诸塞州理工学院将进行实现这些目标的先决条件培训 在特洛伊·利特尔顿博士的实验室中。