Electron Tomography of Axo-Glial Junctions

轴-胶质连接的电子断层扫描

• 批准号: 8033138
• 负责人: Andrea Nans
• 金额: $ 1.08万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-04 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8033138
• 关键词: Actins; Adherens Junction; Architecture; Area; Axon; Binding Sites; Brain; Cell Adhesion Molecules; Cell membrane; Cells; Coculture Techniques; Communication; Cytoplasm; Cytoskeletal Filaments; Cytoskeletal Proteins; Cytoskeleton; Degenerative Disorder; Demyelinating Diseases; Docking; Electron Microscopy; Electrons; Elements; Extracellular Matrix; Freeze Substitution; Freezing; Future; Homology Modeling; Human; Knock-out; Knockout Mice; Knowledge; Label; Link; Location; Maps; Membrane; Modeling; Multiple Sclerosis; Mus; Myelin; Myelinated nerve fiber; Neurofilament Proteins; Neuroglia; Neurologic; Oligodendroglia; Pathogenesis; Patients; Play; Proteins; Research; Resolution; Role; Sampling; Schwann Cells; Series; Signal Transduction; Site; Source; Spectrin; Speed; Spinal Ganglia; Structure; Techniques; Tissue Harvesting; Travel; axonal degeneration; brain tissue; contactin; density; electron tomography; human disease; insight; mouse model; neurofascin; neurofilament; pressure; research study; scaffold; three dimensional structure; tomography

DESCRIPTION (provided by applicant): Project Summary: Myelin, the outgrowth of glial cells, tightly associates with axons and increases the speed of electrical impulses that travel along myelinated nerve fibers. The axo-glial interface at the paranode is the site for a critical series of adhesive junctions that contain the cell-adhesion molecules, Caspr and contactin on the axonal membrane and Neurofascin 155 (NF155) on the glial cell membrane. Notably, Caspr is aberrantly located in the axons from patients with the demyelinating human disease, multiple sclerosis (MS), suggesting its mislocalization may contribute to myelin loss and axonal degeneration. Therefore, detailed information about the structural organization of the axo-glial junctions will be useful for future studies related to the pathogenesis of demyelinating diseases. The broad, long-term research objective of the proposed studies is to use electron tomography to reveal the structural organization of axo-glial interactions, thus contributing to our current knowledge of the axon and glial cell architecture as well as the resulting cellular substructure from demyelinating diseases. The specific aims for the proposed studies are as follows: 1. Electron tomography will be conducted on myelinated axons to obtain high-resolution density maps of the axo-glial junctions. Sample sources of myelinated axons include wildtype mouse brain tissue and myelinating co-cultures, all of which will be prepared for electron microscopy using the sophisticated preparative techniques of high-pressure freezing and freeze substitution. Immunogold labeling and crystal- structure homology modeling of Caspr, contactin, and NF155 will be used in conjunction with the tomography to produce a model of the junctions that will contribute to our understanding of axo-glial interactions. 2. Experiments are planned to investigate the relationship between the axo-glial junctions and the axon cytoskeleton using electron tomography and the above mentioned techniques. 3. Finally, electron tomography will be conducted on myelinated axons from various knock-out mice for comparisons with the wild-type 3D structures of the junctions and other cellular components. I will use mice deficient for the junctional proteins and cytoskeletal proteins such as Protein 4.1B, spectrin, and neurofilament subunits. Relevance: This research seeks to determine the various structural differences between axons from normal mouse brains and axons from various demyelinating mouse models. Ultimately, this information will provide insight into the ultrastructure of axons from patients with multiple sclerosis and will provide valuable information for future research involving human neurological degenerative diseases.

描述（由申请人提供）： 项目摘要：髓磷脂是神经胶质细胞的生长物，与轴突紧密相连，并增加沿着有髓神经纤维传播的电脉冲的速度。旁节点的轴-神经胶质界面是一系列关键粘附连接的位点，这些粘附连接包含细胞粘附分子、轴突膜上的 Caspr 和 contactin 以及神经胶质细胞膜上的 Neurofascin 155 (NF155)。值得注意的是，Caspr 异常定位于患有脱髓鞘人类疾病、多发性硬化症 (MS) 的患者的轴突中，表明其错误定位可能导致髓鞘质损失和轴突变性。因此，有关轴-胶质连接结构组织的详细信息将有助于未来与脱髓鞘疾病发病机制相关的研究。拟议研究的广泛、长期研究目标是使用电子断层扫描来揭示轴突-胶质细胞相互作用的结构组织，从而有助于我们目前对轴突和胶质细胞结构以及脱髓鞘所产生的细胞亚结构的了解疾病。本研究的具体目标如下： 1. 对有髓鞘轴突进行电子断层扫描，以获得轴突-胶质连接处的高分辨率密度图。有髓轴突的样品来源包括野生型小鼠脑组织和有髓鞘共培养物，所有这些都将使用高压冷冻和冷冻替代的复杂制备技术进行电子显微镜准备。 Caspr、contactin 和 NF155 的免疫金标记和晶体结构同源建模将与断层扫描结合使用，生成连接模型，这将有助于我们理解轴突-神经胶质相互作用。 2. 计划使用电子断层扫描和上述技术进行实验来研究轴胶质细胞连接和轴突细胞骨架之间的关系。 3. 最后，将对来自各种基因敲除小鼠的有髓轴突进行电子断层扫描，以与连接和其他细胞成分的野生型 3D 结构进行比较。我将使用缺乏连接蛋白和细胞骨架蛋白（例如蛋白 4.1B、血影蛋白和神经丝亚基）的小鼠。相关性：本研究旨在确定正常小鼠大脑的轴突与各种脱髓鞘小鼠模型的轴突之间的各种结构差异。最终，这些信息将提供对多发性硬化症患者轴突超微结构的深入了解，并将为涉及人类神经退行性疾病的未来研究提供有价值的信息。