Glycoproteins And Glycolipids Associated With Myelin In

本项目主要研究雪旺细胞和少突胶质细胞在髓鞘形成和脱髓鞘过程中的糖复合物。这项研究的一个主要方面涉及髓鞘相关糖蛋白（MAG），它位于有髓纤维的轴突周围神经胶质膜中，在轴突和髓鞘形成细胞之间传递信号。MAG属于免疫球蛋白超家族的siglec亚组，并与含有末端α 2 -3-连接的唾液酸的糖缀合物结合，表明其轴突受体或配体可能是糖蛋白或神经节苷脂。在过去的一年中，有四个报告的潜在的生理结合伙伴的MAG，其中包括我们自己的发现的一种新的糖基化亚型的微管相关蛋白1B的神经元表面上，GD 1a和GT 1b神经节苷脂，p75神经营养因子受体/GT 1b神经节苷脂复合物和Nogo受体。本实验室和其他实验室对MAG缺失小鼠的研究表明，MAG在CNS和PNS中最重要的功能是不同的。在CNS中，其主要作用似乎是在从轴突到少突胶质细胞的信号传导中促进髓鞘形成和少突胶质细胞健康。然而，在PNS中，它对于从雪旺细胞到轴突的相反方向的信号传导是必不可少的，这是有髓鞘轴突正常维持所需的。在没有MAG的情况下，PNS中的病理学特征在于与由细胞骨架异常引起的轴突口径减小相关的轴突变性，所述细胞骨架异常包括神经丝的表达和磷酸化降低。我们已经表明，这种减少的MAG-null小鼠的神经丝磷酸化是由于细胞外信号调节激酶（ERK）和细胞周期蛋白依赖性激酶5（cdk 5）的活性降低。这种体内PNS病理学的解释可能是MAG本身是轴突维持所必需的信号转导途径的一部分，或者在MAG不存在的情况下，雪旺细胞-轴突连接普遍破裂，破坏了其他分子对轴突的信号传导。因此，MAG与神经元相互作用的体外实验范例用于确定MAG是否直接影响细胞骨架蛋白及其相关激酶的表达和磷酸化。在这些实验中，将神经元与用MAG稳定转染或用可溶性MAG Fc嵌合体处理的COS-7细胞共培养。MAG的存在导致ERK 1/2和cdk 5活性升高，神经丝亚基和其他细胞骨架蛋白的表达和磷酸化增加，从而支持MAG直接参与信号通路。假设MAG作为抑制轴突生长的突出白色物质组分的众所周知的作用是由于生理学上重要的MAG介导的信号传导，其促进成熟有髓鞘轴突的稳定性，被体内再生轴突或体外发育中的神经突不适当地接收。此外，我们继续使用这些体外模型的药理学研究的分子机制的MAG介导的信号转导的神经元。这些研究表明，腺苷酸环化酶的抑制剂增强MAG引起的细胞骨架元件的磷酸化增加，表明cAMP下调MAG的作用。此外，MAG的作用被磷脂酰肌醇-3-激酶（PI 3 K）的抑制所消除，表明信号通路涉及或由PI 3 K调节。我们还使用这些体外模型来确定上述任何潜在的MAG结合配偶体是否充当影响轴突细胞骨架的信号传导的MAG受体。 以往的形态学研究表明，对MAG-null小鼠的中枢神经系统的异常或多余的髓鞘环，多余的髓鞘，异常的结旁结构和髓鞘形成的显着延迟。因此，在没有MAG的情况下，一些少突胶质细胞似乎不能有效地决定何时、何地以及形成多少髓鞘。此外，有一个损失的少突胶质细胞蛋白和变性的轴周少突胶质细胞的过程中老化的MAG-空小鼠，符合一个垂死的回少突胶质细胞病变类似的发生在一些脱髓鞘病变的多发性硬化症。这些发现表明，MAG作为轴突信号的受体，增强髓鞘形成过程和少突胶质细胞的活力。实验正在进行中，与原代少突胶质细胞培养物和少突胶质细胞系，以测试和表征推定的MAG介导的信号通路，促进少突胶质细胞的分化。

This project focuses on glycoconjugates of Schwann cells and oligodendrocytes during myelination and demyelination. A major aspect of the research concerns the myelin-associated glycoprotein (MAG), which is localized in periaxonal glial membranes of myelinated fibers and functions in transmitting signals between axons and myelin-forming cells. MAG is in the siglec subgroup of the immunoglobulin superfamily and binds to glycoconjugates containing terminal alpha2-3-linked sialic acid, suggesting that its axonal receptor or ligand could be a glycoprotein or ganglioside. In the past year, there have four reports of potential physiological binding partners for MAG, which include our own finding of a novel glycosylated isoform of microtubule associated protein 1B on the neuronal surface, GD1a and GT1b gangliosides, a p75 neurotrophin receptor/GT1b ganglioside complex and the Nogo receptor. Previous studies from our laboratory and others on MAG-null mice indicate that the most important functions of MAG are different in the CNS and PNS. In the CNS, its primary role appears to be in signaling from axons to oligodendrocytes to promote myelin formation and oligodendroglial health. However, in the PNS, it is essential for signaling in the opposite direction from Schwann cells to axons that is needed for the normal maintenance of myelinated axons. In the absence of MAG, the pathology in the PNS is characterized by axonal degeneration in association with a reduction of axonal caliber caused by cytoskeletal abnormalities including decreased expression and phosphorylation of neurofilaments. We have shown that this decreased phosphorylation of neurofilaments in MAG-null mice is due in part to decreased activities of extracellular signal regulated kinases (ERKs) and cyclin dependent kinase 5 (cdk5). The explanation for this PNS pathology in vivo could be either that MAG itself is part of a signal transduction pathway that is necessary for axonal maintenance or that there is a general breakdown of the Schwann cell-axon junction in the absence of MAG that disrupts signaling to the axon by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. In these experiments, neurons were co-cultured with COS-7 cells stably transfected with MAG or treated with a soluble MAG Fc-chimera. The presence of MAG caused elevated ERK 1/2 and cdk5 activities and increased expression and phosphorylation of neurofilament subunits and other cytoskeletal proteins, thereby supporting the direct involvement of MAG in the signaling pathway. It is hypothesized that the well-known role of MAG as a prominent white matter component that inhibits axonal growth is due to the physiologically important MAG-mediated signaling, which promotes the stability of mature myelinated axons, being received inappropriately by plastic regenerating axons in vivo or developing neurites in vitro. In addition, we have continued to use these in vitro models for pharmacological investigation of molecular mechanisms of MAG-mediated signal transduction in neurons. These studies demonstrate that inhibitors of adenylyl cyclase enhance the increased phosphorylation of cytoskeletal elements caused by MAG, suggesting that c-AMP down regulates the effect of MAG. Also, the effects of MAG were abolished by inhibition of phosphatidylinositol-3-kinase (PI3K), indicating that the signaling pathway involves or is modulated by PI3K. We are also using these in vitro models to determine if any of the potential MAG binding partners described above serves as the MAG receptor for the signaling that affects the axonal cytoskeleton. Previous morphological studies on the CNS of MAG-null mice revealed aberrant or redundant myelin loops, supernumerary myelin sheaths, abnormal paranodal structures and a significant delay of myelination. Thus in the absence of MAG, some oligodendrocytes do not seem to be efficient at determining when, where and how much myelin to form. Furthermore, there are a loss of oligodendroglial proteins and degeneration of periaxonal oligodendroglial processes in aging MAG-null mice, consistent with a dying back oligodendrogliopathy similar to that occurring in some demyelinating lesions of multiple sclerosis. These findings suggest that MAG functions as the receptor for an axonal signal that enhances the process of myelination and the vitality of oligodendrocytes. Experiments are in progress with primary oligodendrocyte cultures and oligodendroglial cell lines to test for and characterize the putative MAG-mediated signaling pathway that promotes the differentiation of oligodendrocytes.