Oligodendrocyte Differentiation and Myelin Biogenesis

描述（由申请人提供）：少突胶质细胞（OL）是一种充分表征的发育谱系的终产物，其中细胞在内在和外在因素的控制下经历不同的表型阶段。这一过程最终导致形态的精确重组和大量髓鞘与神经元的独特联系，导致跳跃式传导和能量和空间的巨大节省。髓磷脂是一种动态的功能活性膜，其损失或损伤导致严重的神经功能缺损，如多发性硬化症。AIM岛决定停止增殖和启动终末分化是细胞分化的关键阶段。我们研究了一个模型，提出鞘糖脂，特别是半乳糖苷（GalC）和/或硫苷脂，参与OL分化的负调控。一个关键的发现是针对GalC/硫苷脂的抗体阻断OL进入终末分化。这种阻断是无毒的、可逆的，并且在mRNA水平上起作用;因此，它为研究进入终末分化的调节机制提供了独特的工具。相反，与模型一致，在GalC/硫苷脂缺失小鼠中，终末分化增强。拟参与这种调节的细胞粘附分子，src家族激酶，和FGF受体-3将进行调查。AIM II.当OL进入终末分化时，它们开始以显著的规模产生髓鞘膜并开始与神经元轴突接触。我们调查的机制，其中OLs排序和运输髓鞘脂质和蛋白质从trans-Golgi网络的新髓鞘膜产生一个高度极化的细胞与多个亚膜结构域。将使用免疫分离和免疫成像的组合来研究运输囊泡的离散群体的形成以及它们与质膜中假定的特定位点的调节通过、对接和融合。拴系复合物的作用，认为目标运输囊泡膜插入的独特位点将进行调查，开始与分析的sec 6/8复杂。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;鞘糖脂信号微结构域分析; GalC和/或硫苷脂或FGFR 3缺失的突变小鼠;用于实时图像分析的高分辨率共聚焦成像和荧光标记的髓鞘和运输蛋白;用于功能分析的原代OL的遗传修饰。本项目的长期目标是详细了解OL分化和髓鞘生物发生的分子机制，以便在临床环境中进行明智的干预，鼓励脱髓鞘疾病中更多的髓鞘再生

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