GLYCOPROTEINS AND GLYCOLIPIDS ASSOCIATED WITH MYELIN IN DEVELOPMENT AND DISEASE

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，而其他糖缀合物和MAG本身上的唾液酸片段可以调节其功能。众所周知，大多数脑糖蛋白上的唾液酸键都是α 2-3，但我们发现小鼠和人类周围神经上的唾液酸键主要是α 2-6。此外，在一些遗传性小鼠和人类神经病变中，α - 2-3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，这可能通过干扰MAG的正常功能而导致病理。我们获得的证据表明，一些微管相关蛋白1B （MAP1B）在轴突表面以唾液化膜糖蛋白的形式表达，并与MAG相互作用。如果MAP1B是MAG的生理结合伙伴，那么它可能在MAG介导的信号传递中起作用，这些信号已知会影响有髓鞘轴突的细胞骨架结构（见下文）。MAG基因敲除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。外周神经系统中有髓鞘轴突的退化与细胞骨骼异常引起的轴突直径减少有关，包括神经丝的表达减少和磷酸化。这些观察结果表明，MAG参与了从雪旺细胞到有髓鞘轴突的信号通路，这对轴突细胞骨架的完整性至关重要。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。此外，mag介导的信号传导对细胞骨架元件表达和磷酸化的影响可以在体外与表达mag的细胞共培养或用MAG-Fc嵌合体处理的神经元中得到证实。这些体外模型为通过药理学方法进一步研究mag介导的信号传导机制提供了手段。对衰老mag敲除小鼠大脑的生化分析表明，致密髓磷脂（髓磷脂碱性蛋白和蛋白脂质蛋白）蛋白水平正常，但少突胶质蛋白（如2′，3′-环核苷酸3′-磷酸二酯酶）和120 kD神经细胞粘附分子异构体水平下降。这些发现表明少突胶质细胞病理，并与衰老MAG-null小鼠的“死背”少突胶质细胞病的形态学表现一致。我们之前对多发性硬化症（MS）的研究表明，与其他髓磷脂蛋白相比，MAG优先从一些活性MS斑块的边缘丢失。由于MAG仅局限于成人大脑少突胶质细胞轴周膜，MS中MAG的选择性丢失提示这些远端少突胶质膜受到损伤。这被解释为由于MS中濒死的少突胶质病变，而在MAG缺失的小鼠中出现类似的少突胶质病变表明，MS中MAG的缺失不仅是轴周膜损伤的标志，而且可能积极地促进了这种病理。

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, and that sialic acid moieties on other glycoconjugates and on MAG itself could modulate its function. Whereas most of the sialic acid linkages on brain glycoproteins are well known to be alpha 2-3, we have found that sialic acid linkages in mouse and human peripheral nerve are predominantly alpha 2-6. Furthermore, the 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 the normal functioning of MAG. Also, we obtained evidence indicating that some microtubule-associated protein 1B (MAP1B) is expressed as a sialylated membrane glycoprotein on the axonal surface and interacts with MAG. If MAP1B is a physiological binding partner for MAG, it could function in the transmission of MAG-mediated signals that are known to affect the cytoskeletal structure of myelinated axons (see below). MAG knockout mice myelinate relatively normally during early development, but neuropathological changes occur as the mice age. Myelinated axons in the peripheral nervous system degenerate in association with a reduction of axonal caliber caused by cytoskeletal abnormalities, including decreased expression and phosphorylation of neurofilaments. These observations suggest that MAG participates in a signaling pathway from Schwann cells to myelinated axons that is essential for integrity of the axonal cytoskeleton. We have shown that the 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). Furthermore, the effects of MAG-mediated signaling on the expression and phosphorylation of cytoskeletal elements can be demonstrated in vitro in neurons co-cultured with MAG-expressing cells or treated with a MAG-Fc chimera. These in vitro models provide a means for further investigation of the mechanism of MAG-mediated signaling by pharmacological approaches. Biochemical analyses of the brains of aging MAG-knockout mice demonstrated normal levels of the proteins of compact myelin (myelin basic protein and proteolipid protein), but decreased levels of oligodendroglial proteins such as 2',3'-cyclic nucleotide 3'-phosphodiesterase and the 120 kD isoform of neural cell adhesion molecule. These findings are indicative of oligodendroglial pathology and consistent with the morphological demonstration of a "dying back" oligodendrogliopathy in aging MAG-null mice. Our previous research on multiple sclerosis (MS) had shown that, in comparison to other myelin proteins, MAG is preferentially lost from the edge of some active MS plaques. Because MAG is localized exclusively in periaxonal membranes of oligodendroctyes in adult brain, its selective loss in MS suggests that there is damage to these distal oligodendroglial membranes. This was interpreted to be due to a dying back oligodendrogliopathy in MS, and the occurrence of a similar oligodendrogliopathy in MAG-null mice suggests that the loss of MAG in MS is not just a marker for damage to periaxonal membranes, but may actively contribute to this pathology.