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。本实验室和其他实验室对MAG缺失小鼠的研究表明，MAG在PNS和CNS中最重要的功能是不同的。在PNS中，从雪旺细胞到轴突的MAG信号传导对于有髓轴突的正常维持是必不可少的。在没有MAG的情况下，PNS的病理学特征是有髓鞘轴突的变性。这之前是由细胞骨架异常引起的轴突口径减小，包括神经丝的表达和磷酸化降低，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（cdk 5）的活性降低。MAG与神经元相互作用的体外实验范例表明，MAG的存在导致ERK 1/2和cdk 5活性升高，磷酸化神经丝和其他细胞骨架元件的表达增加。因此，MAG本身是影响轴突细胞骨架的信号传导途径的一部分，并且MAG缺失小鼠中的细胞骨架变化似乎不仅仅是由于雪旺细胞-轴突连接的一般破坏而破坏了其他分子的信号传导。MAG作为神经元再生的几种白色物质抑制剂之一的众所周知的作用也表明MAG能够影响轴突的性质，但尚不清楚这种抑制神经突生长的能力如何与其在有髓鞘轴突的轴周区域内的胶质-轴突相互作用中的正常功能相关。抑制可塑性再生神经突生长的能力可能是MAG介导的信号传导系统的早期表现，该信号传导系统促进轴突成熟以最终优化它们的结构，从而在成熟的有髓鞘轴突中快速传导动作电位。在过去的几年中，在许多实验室中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，其似乎涉及Nogo受体（NgR）、p75神经营养因子受体（p75 NtR）、其他蛋白质和神经节苷脂的复合物。我们正在进行的研究的一个主要目标是确定这些未成熟的可塑性神经元的发现如何与有髓轴突的轴周隔室内的MAG介导的信号传导相关，这对它们的正常维护至关重要。最近的实验涉及蛋白质印迹与分离的膜馏分从白色物质，是丰富的轴膜和轴周少突胶质细胞膜。MAG在该级分中富集，而NgR和p75 NtR存在，但不富集。NogoR和p75 NtR在活性髓鞘形成时的水平高于成人脑。证明NgR和/或p75 NtR与MAG在这些膜内相互作用的免疫共沉淀实验正在进行中。 与PNS相反，MAG在CNS中最重要的功能似乎是在从轴突到少突胶质细胞的相反方向上发出信号，以促进它们的分化和存活。对MAG基因敲除小鼠中枢神经系统的形态学研究显示，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突周少突胶质细胞的过程中老化的MAG-空小鼠一致的“垂死回”少突胶质细胞病变，类似于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，以证明和表征MAG介导的信号通路，促进这些细胞的分化和/或存活。在培养的少突胶质细胞中激活MAG介导的信号传导的一种方法是与抗MAG抗体交联。这导致少突胶质细胞中的许多变化，包括Fyn酪氨酸激酶的磷酸化，一些其他蛋白质中丝氨酸和苏氨酸残基的去磷酸化，以及α-胞衬蛋白的切割，随后是肌动蛋白的瞬时解聚。Fyn的磷酸化导致其活化，并且可能是引起少突胶质细胞中这些和其他变化的细胞内信号传导中的关键步骤。这些在体外的结果支持的假设，MAG功能作为受体的轴突神经胶质细胞的相互作用，激活信号级联影响少突胶质细胞的特性。

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 signaling from Schwann cells to axons is essential 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 demonstrated that the presence of MAG causes elevated ERK 1/2 and cdk5 activities and increased expression of phosphorylated neurofilaments and other cytoskeletal elements. Therefore, MAG itself is part of a signaling pathway that affects the axonal cytoskeleton, and the cytoskeletal changes in MAG-null mice do not appear to be due only to a general breakdown of the Schwann cell-axon junction that disrupts signaling by other molecules. 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 (NgR), the p75 neurotrophin receptor (p75NtR), other proteins 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. Recent experiments involve Western blotting with a membrane fraction isolated from white matter that is enriched in axolemma and periaxonal oligodendroglial membranes. MAG is enriched in this fraction, whereas the NgR and p75NtR are present, but not enriched. The levels of the NogoR and p75NtR in the fractions are higher at the time of active myelination than in adult brain. Co-immunoprecipitation experiments to demonstrate an interaction of the NgR and/or p75NtR with MAG within these membranes are in progress. 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. One approach for activating MAG-mediated signaling in cultured oligodendrocytes is cross-linking with anti-MAG antibodies. This results in a number of changes in the oligodendroctyes, including 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. The phosphorylation of Fyn leads to its activation and is probably a key step in the intracellular signaling that causes these and other changes in the oligodendrocytes. These in vitro results support the hypothesis that MAG functions as a receptor in axon-glia interactions that activates a signaling cascade affecting the properties of oligodendrocytes.