Oligodendrocyte Differentiation and Myelin Biogenesis

描述(申请人提供)：少突胶质细胞(OL)是一个特征明确的发育谱系的最终产物，在内在和外在因素的控制下，细胞经历不同的表型阶段。这一过程的最终结果是形态的精确重组和大量髓鞘的产生，这种髓鞘与神经元有着独特的联系，导致跳跃传导，并显著节省了能量和空间。髓磷脂是一种动态的、功能活跃的膜，其丢失或损伤会导致严重的神经功能障碍，如多发性硬化症。目的I.决定停止增殖并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，该模型认为神经鞘糖脂，特别是半乳糖脑苷脂(GalC)和/或硫脂，参与了OL分化的负调控。一个关键的发现是，针对GalC/硫脂的抗体阻止了OL进入终末分化。这种阻断是无毒的，可逆的，并在mRNA水平上起作用；因此，它为研究进入末端分化的调控机制提供了一个独特的工具。相反，与模型一致的是，在GalC/硫脂缺失小鼠中，末端分化增强。我们将研究细胞黏附分子、src家族激酶和成纤维细胞生长因子受体-3参与这一调节的可能性。目的II.当OL进入终末分化时，它们开始大量产生髓鞘膜，并开始与神经元轴突接触。我们研究了OL分选和运输髓鞘脂类和蛋白质从跨高尔基网络到新髓磷膜的机制，以产生具有多个亚膜区的高度极化的细胞。免疫分离和免疫成像的结合将被用来研究离散的运输囊泡群体的形成以及它们与质膜上假定的特定位置的调节通道、对接和融合。我们将从对sec6/8复合体的分析开始，研究被认为是将囊泡靶向膜插入的独特位置的系链复合体的作用。重要的实验工具包括：高度特征化的原代OL细胞培养系统；2D-PAGE/串联质谱学蛋白质组分析；糖鞘糖脂信号微域的分析；GalC和/或硫脂或FGFR3基因缺失的突变小鼠；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的基因修饰。这个项目的一个长期目标是详细了解OL分化和髓鞘生物发生的分子机制，以便在临床环境下进行知情干预，鼓励脱髓鞘疾病进行更多实质性的髓鞘再分化

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