Microtubule-associated protein 1B: a neuronal binding partner for myelin-associated glycoprotein.

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.

Terminal complement complexes concomitantly stimulate proliferation and rescue of Schwann cells from apoptosis.

The consequences of sublytic terminal complement complex (TCC) assembly on Schwann cell proliferation and apoptosis were examined by using purified complement proteins (C5*-9) or antibody-sensitized Schwann cells in the presence of a serum that was depleted of the seventh component of complement (C7dHS) and reconstituted with purified C7. Stimulation of cultured Schwann cells with antibody plus 10% C7dHS and C7 or C5*-9 induced DNA synthesis over antibody plus 10% C7dHS alone or in Schwann cells in which C5*-9 insertion was inhibited by heat inactivation, respectively. Cell cycle analysis with propidium iodide showed that, at 24 h, viable Schwann cells in defined medium were synchronized in G1/G0 phase. C5*-9 shifted 64% of these cells into S or G2/M phases in a manner similar to beta-neuregulin (beta-NRG), a known Schwann cell mitogen. Furthermore, antibody with 10% C7dHS and C7 or purified C5*-9 induced proliferation of viable Schwann cells. These effects were mediated by signal-transduction pathways involving p44 ERK1 (extracellular-regulated kinase 1), Gi proteins, and protein kinase C. Culturing in defined medium for 24 h resulted in apoptosis of up to 50% of Schwann cells that was prevented by treatment with beta-NRG or TCC. Sublytic C5*-9 significantly inhibited apoptosis 41% by 24 h, as determined by a terminal deoxyuridine triphosphate-biotin nick end labeling assay, and also decreased annexin-V binding at 4 h. Collectively, these data suggest that sublytic TCC, like beta-NRG, is a potent Schwann cell trophic factor that is capable of stimulating mitogenesis and apoptotic rescue. TCC assembly on Schwann cells during inflammatory demyelination of peripheral nerves may promote survival of mature cells to enhance repair and remyelination processes.

Sublytic terminal complement complexes decrease P0 Gene expression in Schwann cells.

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 (P0), were treated with sensitizing antibody (Ab) and normal human serum (NHS) complement. P0 mRNA content decreased by 71% during 12 h. In the presence of actinomycin D, P0 mRNA levels declined 50% following incubation with Ab plus 10% NHS over 6 h, compared with control levels, suggesting enhanced P0 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 P0 mRNA 53 and 55% over that of Ab plus C7dHS or heat-activated components, respectively. Expression of a P0 promoter/luciferase reporter construct transiently transfected into SchCs was reduced 70% by sublytic TCCs at 6 h, demonstrating that P0 gene transcription was also inhibited. c-jun mRNA was up-regulated within 30 min by sublytic TCCs, before the reduction in P0 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.

Dibutyryl cyclic AMP and inflammatory cytokines mediate C3 expression in Schwann cells.

Schwann cells (SchC), the myelinating glia of the peripheral nervous system, are immunocompetent cells and secrete a variety of immune and inflammatory mediators. In this report, we show that rat SchC in vitro express both C3 mRNA and protein in response to dibutyryl cyclic AMP (dbcAMP) and the cytokines IFN-gamma, TNF-alpha, and IL-1beta. SchC in culture constitutively expressed low levels of C3 which were significantly upregulated upon stimulation with 1mM dbcAMP by 24 hours, and persisted up to 120 hours. This response was minimally enhanced by costimulation with 100 U/ml IFN-gamma, whereas costimulation with 100 U/ml IFN-gamma together with 150-450 ng/ml TNF-alpha induced a greatly increased C3 response. TNF-alpha alone did not induce C3 expression in SchC. Cycloheximide inhibited this dbcAMP-dependent delayed C3 production, thus implying an intermediary signal in the induction pathway requiring protein synthesis. Treatment with 0.1-10 ng/ml IL-1beta for 0-72 hours induced C3 mRNA and protein in a dose-dependent manner. C3 mRNA was detectable at 1 hour and mRNA and protein peaked by 6-12 hours on stimulation with 10 ng/ml IL-1beta, or at 48 hours with 1.0 ng/ml IL-1beta. Furthermore, IL-1beta mRNA was detected at 6 hours in dbcAMP-treated SchC, preceding the dbcAMP-induced C3 expression by 18 hours. Induction of C3 mRNA and protein by dbcAMP at 24 hours was inhibited >85% by a neutralizing anti-IL-1beta antibody and 76% with an IL-1 receptor antagonist. This suggests that dbcAMP-induced synthesis of IL-1beta mediates the C3 production by SchC in an autocrine/paracrine fashion by binding to a functional IL-1 receptor expressed on the surface of SchC. Endoneurial IL-1 and C3 production by SchC may therefore contribute to the inflammatory events associated with peripheral nerve demyelination.