Neuroglial interactions at nodes of Ranvier

Ranvier 节点的神经胶质细胞相互作用

基本信息

批准号：
9817002
负责人：
MATTHEW N RASBAND
金额：
$ 49.14万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-09-30 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9817002
关键词：
Action Potentials Affinity Chromatography Alleles Ankyrins Axon Behavior Binding Biotin Biotinylation Brain Brain region Cell Adhesion Molecules Cells Cytoskeleton Diffusion Disease Extracellular Matrix Goals Guillain-Barré Syndrome Immunoprecipitation In Vitro Injury Ion Channel Knock-in Mouse Knockout Mice Ligase LoxP-flanked allele Maintenance Mass Spectrum Analysis Membrane Proteins Molecular Molecular Genetics Multiple Sclerosis Mus Natural regeneration Nervous System Physiology Nervous system structure Neuroglia Neurons Neuropathy Nodal Phenotype Physiological Physiology Positioning Attribute Proteins Proteome Proteomics Ranvier&apos s Nodes Research Role Spectrin Spinal cord injury Streptavidin Therapeutic Work base betaIV spectrin cell type experimental study in vivo innovative technologies loss of function mouse model nervous system disorder neurofascin repaired

项目摘要

Project Summary: Ion channel clustering in myelinated axons is essential for proper nervous system function. Ion channels are clustered at nodes of Ranvier through neuron-glia interactions. However, the mechanisms responsible for channel clustering and neuron-glia interactions remain poorly understood. Our work shows that multiple glia-dependent mechanisms contribute to node formation. Furthermore, these mechanisms converge on axonal and glial ankyrin and spectrin cytoskeletons. In this project, we will determine the importance of nodal spectrin cytoskeletons by analyzing conditional knockout mice lacking βI and βIV spectrins, singly and in combination. We also discovered that AnkyrinR (AnkR) can function as secondary Na+ channel clustering mechanism. However, the primary functions of AnkR in the nervous system are unknown. We generated a conditional allele for AnkR and will use this in loss-of-function studies to determine the role of AnkR in various cells throughout the nervous system. We performed differential proteomics on WT and AnkR-deficient brains together with immunoprecipitation to identify AnkR interacting proteins. We will investigate these interactions to further define the functions of AnkR in the nervous system. Finally, in a discovery aim, we will use a newly generated mouse (Nfasc-BioID) to perform in vivo proximity biotinylation at nodes and paranodes. We will then capture these proteins using streptavidin affinity purification, followed by mass spectrometry to identify biotinylated proteins. This will begin to uncover the node and paranode interactomes. We will validate and investigate these biotinylated proteins through localization, and gain and loss of function studies both in vitro and in vivo.

项目概要：有髓轴突中的离子通道聚集对于神经系统的正常功能至关重要。通道通过神经元-胶质细胞相互作用聚集在郎飞叶节点。负责通道聚类和神经元-胶质细胞相互作用的机制仍然很差我们的工作表明多种神经胶质细胞依赖性机制有助于节点。此外，这些机制集中在轴突和神经胶质锚蛋白和血影蛋白上。在这个项目中，我们将确定节点血影蛋白细胞骨架的重要性。通过分析单独和组合缺乏 βI 和 βIV 血影蛋白的条件敲除小鼠。我们还发现 AnkyrinR (AnkR) 可以作为次级 Na+ 通道聚集然而，AnkR 在神经系统中的主要功能尚不清楚。生成了 AnkR 的条件等位基因，并将在功能丧失研究中使用它来确定我们对 AnkR 在整个神经系统的各种细胞中的作用进行了差异分析。对 WT 和 AnkR 缺陷大脑进行蛋白质组学结合免疫沉淀来鉴定 AnkR 我们将研究这些相互作用以进一步定义蛋白质的功能。最后，为了发现目标，我们将使用新生成的小鼠。（Nfasc-BioID）在节点和旁节点进行体内邻近生物素化。使用链霉亲和素亲和纯化捕获这些蛋白质，然后进行质谱分析识别生物素化蛋白质，这将开始揭示节点和副节点相互作用组。我们将通过定位来验证和研究这些生物素化蛋白质，并获得和体外和体内功能丧失研究。