Faculty Bibliography

Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact-dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the alpha subunit of the sodium channel and against ankyrin(G), a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells

Rapid conduction in myelinated axons depends on the generation of specialized subcellular domains to which different sets of ion channels are localized. Here, we describe the identification of Caspr2, a mammalian homolog of Drosophila Neurexin IV (Nrx-IV), and show that this neurexin-like protein and the closely related molecule Caspr/Paranodin demarcate distinct subdomains in myelinated axons. While contactin-associated protein (Caspr) is present at the paranodal junctions, Caspr2 is precisely colocalized with Shaker-like K+ channels in the juxtaparanodal region. We further show that Caspr2 specifically associates with Kv1.1, Kv1.2, and their Kvbeta2 subunit. This association involves the C-terminal sequence of Caspr2, which contains a putative PDZ binding site. These results suggest a role for Caspr family members in the local differentiation of the axon into distinct functional subdomains

Neurotrimin (Ntm) together with the limbic system-associated membrane protein (LAMP) and the opioid-binding cell adhesion molecule (OBCAM) comprise the IgLON family of neural cell adhesion molecules. These glycosylphosphatidylinositol (GPI)-anchored proteins are expressed in distinct neuronal systems. In the case of Ntm, its expression pattern suggests a role in the development of thalamocortical and pontocerebellar projections (Struyket al., 1995). We have now characterized Ntm's function in cell adhesion and in neurite outgrowth. Cross-linking studies of transfected cells show that Ntm forms noncovalent homodimers and multimers at the cell surface. Ntm mediates homophilic adhesion, as evidenced by the reaggregation of the transfected cells and the specific binding of an Ntm-Fc chimera to these cells. Consistent with these results, Ntm-Fc binds to neurons that express Ntm at high levels, e.g., dorsal root ganglion (DRG) and hippocampal neurons. It does not bind to DRG neurons treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or to sympathetic neurons that do not express Ntm or other members of the IgLON family at significant levels. Ntm promotes the outgrowth of DRG neurons, even after PI-PLC treatment, suggesting that its effects on outgrowth are mediated by heterophilic interactions. Of particular note, both membrane-bound and soluble Ntm inhibit the outgrowth of sympathetic neurons. These results strongly suggest that Ntm, and other members of the IgLON family, regulate the development of neuronal projections via attractive and repulsive mechanisms that are cell type specific and are mediated by homophilic and heterophilic interactions

Cell interactions in the nervous system are frequently mediated by surface proteins that are attached to the membrane by a glycosyl phosphatidylinositol (GPI) anchor. In this study, we have characterized the expression of such proteins on glial cells. We have detected a major GPI-anchored protein on astrocytes and Schwann cells, with a molecular weight of 140 kD. When Schwann cells were treated with forskolin to promote a myelinating phenotype, expression of this 140-kD protein dramatically decreased, whereas another GPI-anchored protein of 80 kD was strongly induced; expression of other integral membrane proteins were likewise dramatically altered. The size and pattern of expression of the 140-kD protein suggested that it might correspond to the Ran-2 antigen, a glial lineage marker. This notion was confirmed by immunoprecipitating this 140-kD protein with the Ran-2 monoclonal antibody. The Ran-2 antigen is expressed over the entire Schwann cell surface in a punctate fashion; it is removed by phosphatidylinositol phospholipase C treatment, thereby confirming that it is GPI-anchored. When Schwann cells are cocultured with neurons, the Ran-2 antigen initially concentrates at sites of Schwann cell contact with neurons, suggesting that it may play a role in early Schwann cell-neuron interactions; it is then downregulated. Protein sequencing of the Ran-2 antigen immunopurified from rat brain membranes showed complete identity over two extended segments with the copper binding protein ceruloplasmin. These findings indicate that astrocytes and Schwann cells express a novel GPI-anchored form of ceruloplasmin and suggest that this GPI form plays a role in axonal-glial interactions

alpha-Dystroglycan (alpha-DG) is a component of the dystroglycan complex, which is involved in early development and morphogenesis and in the pathogenesis of muscular dystrophies. Here, alpha-DG was shown to serve as a Schwann cell receptor for Mycobacterium leprae, the causative organism of leprosy. Mycobacterium leprae specifically bound to alpha-DG only in the presence of the G domain of the alpha2 chain of laminin-2. Native alpha-DG competitively inhibited the laminin-2-mediated M. leprae binding to primary Schwann cells. Thus, M. leprae may use linkage between the extracellular matrix and cytoskeleton through laminin-2 and alpha-DG for its interaction with Schwann cells

The mammalian CNS does not regenerate after injury due largely to myelin-specific inhibitors of axonal growth. The PNS, however, does regenerate once myelin is cleared and myelin proteins are down-regulated by Schwann cells. Myelin-associated glycoprotein (MAG), a sialic acid binding protein, is a potent inhibitor of neurite outgrowth when presented to neurons in culture. Here, we present additional evidence that strongly supports the suggestion that MAG contributes to the overall inhibitory properties of myelin. When myelin from MAG-/- mice is used as a substrate, axonal length is 100 and 60% longer for neonatal cerebellar and older DRG neurons, respectively, compared to MAG+/+ myelin. The converse is true for neurites from neonatal DRG neurons, which are twice as long on MAG+/+ relative to MAG-/- myelin, consistent with MAG's dual role of promoting axonal growth from neonatal DRG neurons but inhibiting growth in older DRG and all other postnatal neurons examined. Furthermore, desialylating neurons reverses inhibition by CNS myelin by 45%. Contrary to previous reports, under these conditions PNS myelin is also inhibitory for axonal regeneration. Importantly, results using PNS MAG-/- myelin as a substrate suggest that MAG contributes to this inhibition. Finally, when Schwann cells not expressing MAG and permissive for axonal growth are induced to express MAG by retroviral infection, not only is axonal outgrowth greatly inhibited by these cells but so also is neurite branching. This suggests for the first time that MAG not only affects axonal regeneration but may also play a role in the control of axonal sprouting.