Decreased phosphorylation of neurofilaments in mice lacking myelin-associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular-signal regulated kinases (ERK1/2) and cyclin-dependent kinase-5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG-mediated signaling pathway or secondary to a general disruption of the Schwann cell-axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS-7 cells stably transfected with MAG or with empty vector were co-cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF-M), microtubule-associated protein 1B (MAP1B), MAP2, and tau were up-regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF-M were increased significantly in PC12 neurons co-cultured with MAG-expressing COS cells or treated with a soluble MAG Fc-chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline-directed protein kinases.

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Complement cascade activation on peripheral nerve myelin can cause myelin destruction. Although terminal complement complexes (TCCs) are transiently detected on Schwann cells (SchCs) during inflammatory neuropathy, SchCs appear resistant to complement-mediated lysis, and little is known about the functional consequences of sublytic TCC deposition on SchCs. We studied the effects of sublytic complement in modulating myelin gene expression at the posttranscriptional and transcriptional levels. Cultured SchCs, stimulated to express protein zero (P 0 ), were treated with sensitizing antibody (Ab) and normal human serum (NHS) complement. P 0 mRNA content decreased by 71% during 12 h. In the presence of actinomycin D, P 0 mRNA levels declined 50% following incubation with Ab plus 10% NHS over 6 h, compared with control levels, suggesting enhanced P 0 mRNA degradation. The decreases, in part, reflected TCC formation because C7 reconstitution of Ab plus C7-depleted human serum (C7dHS) or TCCs assembled from purified components down-regulated P 0 mRNA 53 and 55% over that of Ab plus C7dHS or heat-activated components, respectively. Expression of a P 0 promoter/ luciferase reporter construct transiently transfected into SchCs was reduced 70% by sublytic TCCs at 6 h, demonstrating that P 0 gene transcription was also inhibited. c-jun mRNA was up-regulated within 30 min by sublytic TCCs, before the reduction in P 0 mRNA expression. Our data suggest that sublytic complement activation on SchCs may contribute to peripheral nerve demyelination by decreasing expression of genes important in myelin formation and compaction. Key Words: Schwann cellsComplement-C5b-9 -Protein zero-Myelin genes.

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia–axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal 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. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553–562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.