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结合。髓鞘相关糖蛋白（myelin-associated glycoprotein， MAG）是研究的一个主要方面，它定位于髓鞘纤维的轴突周围胶质膜，并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α 2-3链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。我们实验室和其他人对MAG-null小鼠的研究表明，MAG在PNS和CNS中最重要的功能是不同的。在PNS中，MAG对于雪旺细胞向轴突传递信号至关重要，这是有髓鞘轴突正常维持所必需的。在没有MAG的情况下，PNS的病理特征是髓鞘轴突变性，在此之前，由细胞骨骼异常引起的轴突直径减少，包括神经丝的表达和磷酸化减少，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）的活性降低。对体内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-轴突连接的普遍破坏，从而破坏了其他分子向轴突发出的信号。为了解决这一问题，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ERK 1/2和cdk5活性升高，磷酸化神经丝和其他细胞骨架元件的表达增加，从而支持MAG直接参与信号通路。我们正在继续使用这些体外模型对mag介导的神经元信号转导的分子机制进行药理学研究。到目前为止，我们的研究结果表明，细胞骨架相关erk1 /2的激活是通过传统的Ras-Raf-Mek途径发生的，并可能受到蛋白激酶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通路涉及或由磷脂酰肌醇-3激酶（PI3K）途径调节。众所周知，MAG作为神经元再生的几种白质抑制剂之一，也表明MAG能够影响轴突的特性，但尚不清楚这种抑制神经突生长的能力与髓鞘轴突轴突周围区域内胶质-轴突相互作用的正常功能之间的关系。抑制可塑性再生神经突生长的能力可能是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，以便在成熟的髓鞘轴突中快速传导动作电位。在过去的几年中，许多实验室对神经突生长抑制的研究提供了许多关于MAG神经元受体的新信息，该受体似乎涉及Nogo受体，p75神经营养因子受体和神经节苷类的复合物。我们未来研究的主要目标将是确定这些未成熟可塑性神经突的发现与髓鞘轴突轴周室中mag介导的信号传导之间的关系，这对髓鞘轴突的正常维持至关重要。

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.