Oligodendrocyte Differentiation and Myelin Biogenesis

描述（由申请人提供）：少突胶质细胞（OLs）是一个具有良好特征的发育谱系的最终产物，在这个谱系中，细胞在内在和外在因素的控制下经历了不同的表型阶段。这一过程在形态的精确重组和髓磷脂的产生中达到高潮，髓磷脂与神经元有着独特的联系，导致了跳跃式传导和能量和空间的显著节省。髓磷脂是一种动态的、功能活跃的膜，它的丢失或损伤会导致严重的神经功能缺陷，如多发性硬化症。停止增殖和启动终末分化的决定是细胞分化的关键阶段。我们研究了一个模型，该模型提出鞘糖脂，特别是半乳糖脑苷（GalC）和/或硫脂，参与OL分化的负调控。一个关键的发现是抗GalC/硫脂的抗体，阻止OLs进入终端分化。该阻断剂无毒，可逆，在mRNA水平上起作用；因此，它为研究进入终末分化的调控机制提供了一个独特的工具。相反，与模型一致，在GalC/硫脂缺失小鼠中，终末分化增强。研究人员将对细胞粘附分子、src家族激酶和FGF受体-3的调控进行研究。目的二世。当OLs进入末梢分化时，它们开始大规模地产生髓鞘膜，并开始与神经元轴突接触。我们研究了OLs将髓磷脂脂质和蛋白质从反式高尔基网络转运到新髓鞘膜，从而产生具有多个亚膜结构域的高度极化细胞的机制。免疫分离和免疫成像的结合将用于研究转运囊泡离散种群的形成及其与质膜中假定的特定位点的调节通道，对接和融合。将从分析sec6/8复合物开始，研究被认为将运输囊泡定位到膜插入的独特位点的栓系复合物的作用。重要的实验工具包括高度表征的原代OL细胞培养系统；2D-PAGE/串联质谱蛋白质组学分析；鞘糖脂信号微域分析突变小鼠GalC和/或硫脂或FGFR3缺失；高分辨率共聚焦成像和荧光标记髓磷脂和运输蛋白实时图像分析；用于功能分析的初级ol基因修饰。该项目的长期目标是详细了解OL分化和髓磷脂生物发生的分子机制，以便为临床环境中的知情干预做出贡献，从而鼓励脱髓鞘疾病中更多的实质髓鞘再生

DESCRIPTION (provided by applicant): Oligodendrocytes (OLs) are the end product of a well-characterized developmental lineage in which the cells pass through distinct phenotypic stages under the control of both intrinsic and extrinsic factors. This process culminates in a precise reorganization of morphology and the production of dramatic amounts of myelin in a unique association with neurons, leading to saltatory conduction and dramatic savings in energy and space. Myelin is a dynamic, functionally active membrane whose loss or damage results in serious neurological deficits such as occur in Multiple Sclerosis. AIM I. The decision to cease proliferation and initiate terminal differentiation is a critical stage in cellular differentiation. We investigated a MODEL that proposes that glycosphingolipids, in particular galactocerebroside (GalC) and/or sulfatide, are involved in negative regulation of OL differentiation. A key finding is that antibodies against GalC/sulfatide, block entry of OLs into terminal differentiation. This block is non-toxic, reversible and operates at the level of mRNA; thus, it offers a unique tool for studying the mechanism of regulation of entry into terminal differentiation. In contrast, and consistent with the model, in GalC/sulfatide null mice, terminal differentiation is enhanced. The proposed participation in this regulation of cell adhesion molecules, src-family kinases, and FGF receptor-3 will be investigated. AIM II. As OLs enter terminal differentiation, they begin to produce myelin membrane on a remarkable scale and initiate contact with neuronal axons. We investigate the mechanism by which OLs sort and transport myelin lipids and proteins from the trans-Golgi network to the neo-myelin membrane to produce a highly polarized cell with multiple submembrane domains. A combination of immuno-isolation and immuno-imaging will be used to study the formation of discrete populations of transport vesicles and their regulated passage, docking and fusion with presumed specific sites in the plasma membrane. The roles of tethering complexes thought to target transport vesicles to unique sites of membrane insertion will be investigated, beginning with an analysis of the sec6/8 complex. Important experimental tools include a highly characterized primary OL cell culture system; 2D-PAGE/tandem mass spectrometric proteomic analysis; analysis of glycosphingolipid signaling microdomains; mutant mice null for GalC and/or sulfatide or FGFR3; high resolution confocal imaging and fluorescently-tagged myelin and trafficking proteins for real-time image analysis; genetic modification of primary OLs for functional analyses. A long-term goal of this project is to develop a detailed understanding of the molecular mechanisms of OL differentiation and myelin biogenesis in order to contribute to an informed intervention in clinical settings that will encourage more substantial remyelination in demyelinating diseases