Institution:
Burnham Institute for Medical Research, La Jolla, California

Introduction:
The rapid propagation of nerve impulses is achieved by the insulation the axons with myelin, a lipid-rich membrane sheath formed by oligodendrocytes in the central and by Schwann cells in the peripheral nervous system. The function of myelin depends on its segregation into specialized membrane domains: Myelin segments along the nerve are interspersed by the unmyelinated nodes of Ranvier that contain clusters of sodium channels and serve to regenerate the action potential. On both sides of the node, septate-like junctions tightly attach the terminal myelin loops to the axon membrane. These paranodal junctions physically segregate the sodium channel clusters at the node from potassium channel clusters at the juxtaparanode just underneath the myelin. The host laboratory has established an important function for the immunoglobulin cell adhesion molecule Contactin in the formation of the paranodal junctions in both the PNS (Boyle et al., 2001 Neuron 30:385-397) and more recently in the CNS. The formation of paranodal junctions requires the association of Contactin with Caspr (Contactin-associated Protein), a transmembrane molecule that permits Caspr trafficking to the axon membrane where the complex engages in junctional adhesion. However, little is known about how the expression of the Contactin-Caspr complex is regulated at central paranodes and if Contactin on oligodendrocytes contributes to the formation of the junctional complex in the CNS.

Preliminary results:

New probes for studying the paranodal complex:
To investigate posttranslational modifications and molecular associations of paranodal Caspr, reliable probes were needed. Two human recombinant Fab molecules were isolated from the huCalTM Gold phage display library (http://www.morphosys.com) by their ability to bind recombinant Caspr protein. The antibodies were tested by Western blot and immunohistochemistry to optimize working conditions. Both antibodies reacted with the 190kD Caspr protein in lysates from postnatal day 18 (P18) wild type brain and immunoprecipitated Caspr. Immunohistochemistry revealed the characteristic Caspr expression pattern at paranodal junctions flanking the nodes of Ranvier (Fig.1). These data demonstrate that the HuCal antibodies are exellent probes for studying the paranodal assembly.

Contactin in oligodendrocytes:
In central myelin, Contactin is not only expressed by neurons but also by oligodendrocytes. To begin understanding the contribution of oligdendrocyte-derived Contactin to the myelination process, we expressed GFP-tagged Contactin in cerebellar slice cultures via virus-mediated gene transfer. GFP- Contactin was expressed by oligodendrocytes and localized to the outer myelin membrane and to the paranodal region demarcated by staining with anti-Caspr antibodies. These observations suggest that Contactin both on neurons and on oligodendrocytes contributes to myelin formation or function. This hypothesis is now being further explored in the host laboratory.