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/文摘