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。这表明其轴突受体或配体可能是糖蛋白或神经节苷脂。此外，其他轴突或胶质糖缀合物和MAG本身上的唾液酸片段可以调节其功能，因为已知其他siglec家族成员的功能受顺式和反式唾液酸片段的表达调节。在一些遗传性小鼠和人类神经病变中，α - 2-唾液酸在MAG和其他神经糖蛋白上的表达增加，我们的研究结果表明，这通过干扰MAG介导的信号传导来促进病理。然而，MAG的功能结合伙伴的身份尚不清楚。我们的Western blot覆盖和共免疫沉淀实验表明，MAG与背根神经节神经元（DRGNs）中表达的微管相关蛋白1B （MAP1B）磷酸化异构体结合，而不是与神经胶质细胞表达的MAP1B异构体结合。这些结果，加上我们之前的证明，一些MAP1B以神经元质膜糖蛋白的形式表达，与它在轴突表面作为MAG的结合伙伴是一致的。MAG- fc嵌合体在DRGNs上的结合位点与MAP1B在神经元多样性上共定位，MAG和MAP1B也在髓鞘轴突的轴周区域共定位。此外，当DRGNs与mag转染的COS细胞共培养时，MAP1B磷酸化异构体的表达显著增加。基于这些发现，我们假设MAG/MAP1B相互作用可以在髓鞘形成细胞的轴周膜和轴突细胞骨架之间提供结构联系，从而有助于髓鞘影响髓鞘轴突结构和稳定性的已知能力（见下文）。mag缺失小鼠在早期发育过程中髓鞘形成相对正常，但随着小鼠年龄的增长，神经病理改变发生。对1岁以上的mag缺失小鼠进行生化和形态学研究，以更详细地表征这些病理变化。正如去年报道的那样，中枢神经系统的结果表明少突胶质病变，并且与“死背”少突胶质病变的形态学表现一致，类似于多发性硬化症中某些脱髓鞘病变的发生。然而，PNS的病理是完全不同的，其特征是轴突变性与轴突直径减少有关，这是由细胞骨骼异常引起的，包括神经丝的表达减少和磷酸化。我们已经证明，在MAG-null小鼠中，神经丝磷酸化的降低部分是由于细胞外信号调节激酶（ERKs）和细胞周期蛋白依赖性激酶5 （cdk5）的活性降低。虽然以前的研究没有发现年轻的mag缺失小鼠的电生理异常，但我们对这些一岁突变体的电生理评估显示，传导速度和复合肌肉动作电位幅度轻微降低。这些发现表明了一种类似于人类2型夏科-玛丽-图斯病的轴突病，并表明雪旺细胞蛋白的突变可导致功能显著的轴突变性。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，破坏了其他分子向轴突发出的信号。我们的体外实验支持了MAG在信号通路中的直接参与，实验表明，在表达MAG的细胞或MAG- fc嵌合体中培养神经元会导致ERK 1/2和cdk5活性升高，并增加神经丝的表达和磷酸化。此外，体外模型为通过药理学方法进一步研究mag介导的信号转导的分子机制提供了手段。

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. This suggests that its axonal receptor or ligand could be a glycoprotein or ganglioside. Furthermore, the sialic acid moieties on other axonal or glial glycoconjugates and on MAG itself could modulate its function, because it is known that the function of other siglec family members are regulated by the expression of cis and trans sialic acid moieties. Expression of alpha 2-3 sialic acid on MAG and other glycoproteins of nerve is increased in some inherited mouse and human neuropathies, and our results suggest that this contributes to pathology by interfering with MAG-mediated signaling. However, the identity of the functional binding-partner(s) for MAG is not known. Our experiments involving Western blot overlay and co-immunoprecipitation demonstrated that MAG binds to a phosphorylated isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. These results, plus our previous demonstration that some MAP1B is expressed as a neuronal plasma membrane glycoprotein, is consistent with its being a binding-partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs co-localized with MAP1B on neuronal varicosities, and MAG and MAP1B also co-localized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was significantly increased when DRGNs were co-cultured with MAG-transfected COS cells. Based on these findings, we hypothesize that a MAG/MAP1B interaction could provide a structural link between the periaxonal membrane of the myelin-forming cell and the axonal cytoskeleton, thereby contributing to the known capacity of myelin to affect the structure and stability of myelinated axons (see below). MAG-null mice myelinate relatively normally during early development, but neuropathological changes occur as the mice age. Biochemical and morphological studies on MAG-null mice over one-year old were undertaken to characterize these pathological changes in greater detail. As reported last year, the results on the CNS were indicative of oligodendroglial pathology and consistent with the morphological demonstration of a "dying back" oligodendrogliopathy, similar to that occurring in some demyelinating lesions in multiple sclerosis. However, the pathology in the PNS is quite different, being 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). Although previous studies had not revealed electrophysiological abnormalities in younger MAG-null mice, our electrophysiological evaluation of these one year-old mutants showed mild reductions in conduction velocity and compound muscle action potential amplitudes. These findings are indicative of an axonopathy resembling type 2 Charcot-Marie-Tooth disease in humans and demonstrate that mutation of a Schwann cell protein can cause functionally significant axonal degeneration. The explanation 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. The direct involvement of MAG in the signaling pathway is supported by our in vitro experiments showing that culturing neurons in the presence of MAG-expressing cells or a MAG-Fc chimera caused elevated ERK 1/2 and cdk5 activities and increased expression and phosphorylation of neurofilaments. Furthermore, the in vitro models provide a means for further investigation of the molecular mechanisms of MAG-mediated signaling by pharmacological approaches.