Background & Significance of the Research.
The correct formation and maintenance of the myelin sheath are pre-requisites for assuring its faultless function during the whole life of an individual and depend upon the maintenance of close contact between the axon and the myelin-producing cells: oligodendrocytes in the central nervous system (CNS) and Schwann cells (SC) in the peripheral nervous system (PNS). This contact involves both glycolipids (e.g. gangliosides and galactocerebrosides (Gal-CB)), and proteins (e.g. connexins, N-CAM and the myelin-associated glycoprotein (MAG)). Myelin allows the fast, targeted transmission of the nervous impulse. This role is crucial for the efficient functioning of the nervous system, as evidenced by the devastating effects of myelin loss in CNS and PNS diseases, for which the development of diagnostic and effective therapeutic strategies has been frustratingly slow.
Many diseases affecting the myelin sheath are long-term, progressive, severely handicapping, sometimes fatal, conditions that can start during childhood, or early adulthood. Multiple sclerosis (MS) affects an average of 0.05-0.1‰ of individuals in North America and Europe. Women develop MS twice as often as men. Of unknown cause, MS presents as an autoimmune disease directed against one or more CNS myelin antigens. Myelin basic protein (MBP) is the major target autoantigen, but antibodies to other proteins, including tubulin and MAG, are also found. Peripheral neuropathies are also common neurological diseases. Monoclonal gammopathy occurs in 10% of patients with idiopathic polyneuropathy. 50-60% of patients with IgM monoclonal gammopathy present anti-MAG antibodies, whose appearance predicts the onset of a neuropathy in asymptomatic patients. MAG immunoreactivity also parallels that of the glial cell marker S100 in some benign sheath tumours and several types of sarcoma, and has also been reported in several other types of tumour. While it is unlikely to be the primary cause of these pathologies, MAG could contribute to their evolution and severity.
MAG is a bitopic (i.e. has a single transmembrane domain) cell adhesion molecule (CAM) existing as two isoforms (L-MAG and S-MAG) that differ only by their respective cytoplasmic domains. The MAG extracellular domain regulates neurite outgrowth during nerve regeneration, and is believed to participate in the establishment and maintenance of the glial cell-axon contact. The colocalization of MAG with glial cell cytoskeletal proteins also suggests roles in the maintenance of myelin structure. L-MAG may play a role in glial cell signal transduction, as it interacts with several protein kinases. The subcellular localizations of MAG are compatible with all of the proposed functions. However, few studies have addressed the possible differential distributions and functions of the two MAG isoforms, and the mechanisms by which they fulfill their putative roles are unknown. MAG is one of the first myelin-specific proteins to be expressed during myelination, commencing when the SC contacts an axon. Cultured SCs cannot correctly interact with axons, nor initiate myelination, in the absence of MAG. Studies with several knock-out mice also reveal that MAG plays essential roles in both early and late stages of the myelination process, but have been unable to identify these roles; the mice synthesize morphologically normal myelin, but demyelination develops in the young adults. No human diseases have been correlated with the Mag gene.
The Delta-like protein (dlk) is involved in several differentiation processes, including adipogenesis, hematopoiesis, neuroendocrine differentiation and intercellular interactions. Recently, it was identified as a potentially important player in the initial phases of myelination; its expression appears to be required during the early stages of SC differentiation towards a myelinating phenotype, but must then be down-regulated for the myelination program to be completed.
Recombinant cytoplasmic domains (CT) of rat sciatic nerve S-MAG (S-MAGCT) and L-MAG (L-MAGCT) have been produced as GST- and His-tagged fusion proteins, as well as mutated GST fusion proteins, in which different regions of the S- and L-MAG-specific C-termini are deleted. Polyclonal antibodies against the MAGCT domains have been raised and used to purify native total MAG and L-MAG from human and rat nervous tissue. Purified human MAG is highly susceptible to degradation, characterized by the loss of the entire cytoplasmic domain. We showed that a trans-acting factor is involved, although not exclusively and, in contradiction with earlier reports, the degradation is not mediated by a cysteine protease activity. We established conditions for stabilizing the intact native protein from most brain samples, allowing its use for the analysis of native MAG functional properties. We have set up a myelinating SC/dorsal root ganglion neuronal (SC/DRGN) coculture system: the model is being employed for the analysis of MAG and MAG-ligand expression during PNS myelination.
We identified S100b as the first non-kinase, intracellular ligand for L-MAG. The interaction is divalent cation-dependent and the S100b-binding site has been localized. Like MAG, S100b may participate in numerous cellular events including the regulation of the cytoskeleton and signal transduction. We showed that both S-and L-MAG are phosphorylated by protein kinase A. Only the phosphorylation of L-MAG is regulated by the presence of S100b, suggesting a signalling role for the L-MAG/S100b interaction. Both MAGCTs regulate the autophosphorylation activity of casein kinase II. We showed that the L-MAGCT forms homodimers mediated by the L-MAG-specific C-terminal portion of the protein, and our recent analysis of purified human brain MAG and L-MAG revealed that most of the protein is recovered as oligomers, suggesting a receptor-like dimerization mechanism for L-MAG’s putative role in signal transduction.
We
demonstrated that the S-MAG-specific C-terminal binds tubulin, and
regulates
the polymerization of tubulin in vitro. We demonstrated the partial
colocalization
of S-MAG and tubulin at cell-cell contact zones and along the processes
emitted by differentiating SCs. However, total segregation of S-MAG and
tubulin occurs in growth cones that have not contacted other cells,
suggesting
the existence of other ligands in these structures. Taxol provokes the
redistribution of microtubules and S-MAG in cultured SCs, and
disorganizes
intercellular contacts. These results support a role for S-MAG in
intercellular
adhesion, and suggest its participation in intercellular recognition in
the growth cones. We demonstrated that the S-MAG-specific C-terminal
binds
Zn2+, inducing a conformational
change
of the S-MAGCT, which
appears
to induce S-MAG dimerization. We demonstrated a nucleotide (G/C) and
amino
acid (Arginine579/Proline579)
dimorphism in the rat S-MAG-specific C-terminal. No associated
phenotype
is apparent: both subtypes bind tubulin, but SPro-MAG
is significantly less stable than SArg-MAG.
Mice only express S Arg -MAG.
We have just found more candidate ligands for S-MAG, and we will tell
you more about them later.
Methods, Techniques & Approaches