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激酶1和2(ERK 1/2)和细胞周期蛋白依赖性激酶5(CDK5)活性降低。MAG与神经元相互作用的体外实验范式表明，MAG的存在导致ERK1/2和CDK5活性升高，并增加了磷酸化神经丝和其他细胞骨架元件的表达。今年完成的药理学实验表明，细胞骨架相关的ERK1/2的激活通过传统的Ras-Raf-MEK途径发生，并受到蛋白激酶A的负调控。众所周知，MAG作为神经元再生的白质抑制剂之一，也表明MAG能够影响轴突的特性，但目前尚不清楚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。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。我们正在进行的研究的一个主要目标是确定这些未成熟可塑性神经元的发现与有髓轴突周围区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。我们最近对培养神经元ERK1/2激活的实验表明，MAG信号的这种作用确实涉及Nogo受体。此外，我们通过免疫印迹显示，Nogo受体和p75神经营养素受体存在于白质分离的膜组分中，该膜组分富含在轴膜和轴突周围少突胶质细胞膜中。此外，通过免疫共沉淀证实了Nogo受体与MAG在该位置的相互作用。 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，以促进它们的分化和生存。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。对原代少突胶质细胞培养和少突胶质细胞系的实验正在进行中，以证明和表征MAG介导的促进这些细胞分化和/或存活的信号通路。我们正在探索在培养的少突胶质细胞中激活MAG介导的信号的两种方法，一种是与抗MAG抗体交联，另一种是在含有2，3-唾液酸糖蛋白的底物上生长。MAG的抗体交联会导致MAG重新分布到洗涤剂不溶的RAFT样复合体中，增加Fyn酪氨酸激酶的磷酸化，使其他一些蛋白质中的丝氨酸和苏氨酸残基去磷酸化，并导致α-fodrin的裂解，随后肌动蛋白短暂解聚。这些体外实验结果支持MAG在轴突-神经胶质细胞相互作用中的作用，激活影响少突胶质细胞特性的信号级联反应。

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. This 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). In vitro experimental paradigms of MAG interaction with neurons demonstrate that the presence of MAG causes elevated ERK 1/2 and cdk5 activities and increased expression of phosphorylated neurofilaments and other cytoskeletal elements. Pharmacological experiments completed this year demonstrate that activation of cytoskeleton-associated ERK 1/2 occurs through the traditional Ras-Raf-Mek pathway and is negatively regulated by protein kinase A. 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 ongoing research is to determine how these findings with immature plastic neurons relate to MAG-mediated signaling within the periaxonal compartment of myelinated axons that is essential for their normal maintenance. Our recent experiments described above on ERK 1/2 activation in cultured neurons indicated that this effect of MAG signaling does involve the Nogo receptor. In addition, we showed by western blotting that the Nogo receptor and the p75 neurotrophin receptor are present in a membrane fraction isolated from white matter that is enriched in axolemma and periaxonal oligodendroglial membranes. Furthermore, an interaction of the Nogo receptor with MAG at this location was demonstrated by co-immunoprecipitation. In contrast to the PNS, the most important function of MAG in the CNS appears to be signaling in the opposite direction from 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 these cells. Two approaches, which we are exploring to activate MAG-mediated signaling in cultured oligodendrocytes, are cross linking with anti-MAG antibodies and growth on a substratum containing a 2,3-sialylated glycoprotein. Antibody cross-linking of MAG leads to its redistribution into detergent insoluble, raft-like complexes, increased phosphorylation of Fyn tyrosine kinase, dephosphorylation of serine and threonine residues in some other proteins, and cleavage of alpha-fodrin followed by a transient depolymerization of actin. These in vitro results support the hypothesis MAG functions in axon-glia interactions to activate a signaling cascade affecting the properties of oligodendrocytes.