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.
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