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共轭。这项研究的一个主要方面涉及髓鞘相关糖蛋白(MAG)，它定位于有髓纤维的轴突周围神经胶膜，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亚群，与含有末端α2-3连接的唾液酸的糖共轭结合，提示其轴突受体或配体可能是糖蛋白或神经节苷脂。我们实验室和其他实验室以前对MAG缺失小鼠的研究表明，MAG在三叉神经节和中枢神经系统中最重要的功能是不同的。在三叉神经核，MAG是雪旺细胞向轴突发出信号所必需的，而有髓轴突的正常维持是必需的。在没有MAG的情况下，PNS的病理特征是有髓轴突的变性，在此之前，细胞骨架异常导致轴突口径减小，包括神经细丝表达和磷酸化减少，部分原因是细胞外信号调节蛋白1和2(ERK 1/2)和细胞周期蛋白依赖蛋白依赖性激酶5(CDK5)的活性降低。对这种体内PNS病理的解释可能是MAG本身是轴突维持所必需的信号转导途径的一部分，或者在没有MAG的情况下雪旺细胞-轴突连接普遍破裂，从而扰乱了其他分子向轴突发送的信号。为了解决这个问题，我们使用MAG与神经元相互作用的体外实验范式来证明MAG的存在导致ERK1/2和CDK5活性升高，以及磷酸化神经丝和其他细胞骨架元件表达增加，从而支持MAG直接参与信号通路。我们将继续使用这些体外模型来研究MAG介导的神经元信号转导的分子机制。到目前为止，我们的发现表明，细胞骨架相关的ERK1/2的激活是通过传统的Ras-Raf-MEK途径进行的，并且可能受到蛋白激酶A(PKA)的负调控。其他初步研究结果表明，MAG介导的影响轴突的信号涉及磷脂酰肌醇-3-激酶(PI3K)途径或受其调节。MAG作为神经元再生的几种白质抑制剂之一的广为人知的作用也表明MAG能够影响轴突的特性，但这种抑制轴突生长的能力与其在有髓轴突周围区域的神经胶质-轴突相互作用中的正常功能之间的关系尚不清楚。这种抑制再生神经突起生长的能力可能是MAG介导的信号系统的早期表现，该信号系统促进轴突成熟，最终优化其结构，以便在成熟的有髓轴突中快速传导动作电位。在过去的几年里，许多实验室对抑制轴突生长的研究提供了许多关于MAG的神经元受体的新信息，它似乎涉及Nogo受体、p75神经营养素受体和神经节苷脂的复合体。我们未来研究的一个主要目标将是确定这些未成熟的塑料神经突起与有髓轴突周围区MAG介导的信号之间的关系，这对它们的正常维持是必不可少的。 与三叉神经节相反，MAG在中枢神经系统中最重要的功能似乎是从轴突到少突胶质细胞的相反方向的信号传递，以促进其分化和存活。对MAG基因敲除小鼠中枢神经系统的形态研究显示，髓鞘形成明显延迟，髓鞘环异常或多余，结旁结构异常。因此，在缺少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，在MAG基因缺失的老年小鼠中，存在少突胶质蛋白的丢失和轴突周围少突胶质细胞的退化，这与多发性硬化症的一些脱髓鞘病变中发生的“垂死性”少突胶质病相一致。这些发现表明，MAG作为轴突信号的受体，增强了髓鞘形成过程和少突胶质细胞的活力。原代培养的少突胶质细胞和少突胶质细胞系正在进行实验，以证明和表征MAG介导的促进少突胶质细胞分化和/或存活的信号通路。过去一年的研究结果表明，少突胶质细胞中的MAG信号可以通过与抗MAG抗体交联或生长在含有2，3-唾液酸化糖蛋白的底物上而被激活。

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. Previous studies from our laboratory and others on MAG-null mice indicate that the most important functions of MAG are different in the PNS and CNS. In the PNS, MAG is essential for signaling 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 degeneration of myelinated axons that is preceded by a reduction of axonal caliber caused by cytoskeletal abnormalities including decreased expression and phosphorylation of neurofilaments, due in part to decreased activities of extracellular signal regulated kinases 1 & 2 (ERK 1/2) 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. To address this issue, we used in vitro experimental paradigms of MAG interaction with neurons to demonstrate that the presence of MAG causes elevated ERK 1/2 and cdk5 activities and increased expression of phosphorylated neurofilaments and other cytoskeletal elements, thereby supporting the direct involvement of MAG in the signaling pathway. We are continuing to use these in vitro models for pharmacological investigation of the molecular mechanisms of MAG-mediated signal transduction in neurons. Our findings so far indicate that activation of cytoskeleton-associated ERK 1/2 occurs through the traditional Ras-Raf-Mek pathway and may be negatively regulated by protein kinase A (PKA). Other preliminary findings suggest that MAG-mediated signaling affecting the axon involves or is modulated by the phosphatidylinositol-3-kinase (PI3K) pathway. The well-known role of MAG as one of several white matter inhibitors of neuronal regeneration also shows that MAG is able to influence the properties of axons, but it is unclear how this capacity to inhibit neurite outgrowth relates to its normal function in glia-axon interactions within the periaxonal region of myelinated axons. The capacity to inhibit outgrowth of plastic regenerating neurites may be an early manifestation of a MAG-mediated signaling system that promotes axonal maturation to eventually optimize their structure for rapid conduction of action potentials in mature myelinated axons. In the past few years, research in many laboratories on the inhibition of neurite outgrowth has provided much new information about a neuronal receptor for MAG, which appears to involve a complex of the Nogo receptor, the p75 neurotrophin receptor and gangliosides. A principal goal of our future research will be to determine how these findings with immature plastic neurites relate to MAG-mediated signaling within the periaxonal compartment of myelinated axons that is essential for their normal maintenance. In contrast to the PNS, the most important function of MAG in the CNS appears to be signaling in the opposite direction from the axons to oligodendrocytes to promote their differentiation and survival. Morphological studies on the CNS of MAG-null mice revealed a significant delay of myelination, aberrant or redundant myelin loops and abnormal paranodal structures. 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 is 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 demonstrate and characterize a MAG-mediated signaling pathway that promotes the differentiation and/or survival of oligodendrocytes. Results obtained in the past year indicate that MAG signaling in oligodendrocytes can be activated by cross linking with anti-MAG antibodies or growth on a substratum containing a 2,3-sialylated glycoprotein.