The interactions of primary rat CNS neurons with a variety of glial cell lines will be studied to examine the molecular cues which affect neurite growth in the central nervous system. A ready source of CNS cell lines has been generated by transformation of CNS progenitor cells by retroviral infection. Individual subclones of these cells are known to display antigenic and morphologic diversity; these subclones are probably also variable in their ability to support neuronal outgrowth. Therefore, distinct subclones of these glial cells which promote or inhibit neurite outgrowth in an in vitro neurite outgrowth assay will be selected and expanded. The cell surface determinants responsible for the effects on neurite growth will then be studied using monoclonal antibodies. Panels of monoclonal antibodies will be generated using protocols of -subtractive immunization:- these panels will be screened to find antibodies which distinguish between those cell lines which promote versus those that inhibit neurite outgrowth. A variation of the in vitro bioassay Will then be used to determine whether any of these antibodies perturb neurite outgrowth of primary neurons by binding to a cell surface antigen. If an antibody can block the capacity of a cell line either to support or to inhibit neurite outgrowth, it is likely that the antigen recognized by the antibody has an important function in mediating this activity. Thus, using the transformed CNS cell lines as -factories- of neurite promoting and inhibiting substances, and specific monoclonal antibodies with function-- blocking activity as reagents in biochemical and histochemical analysis, this approach will allow production, immunopurification, and characterization of molecules involved in permitting or restricting neurite outgrowth. A better understanding of the biological and biochemical aspects of these molecules offers hope for future clinical strategies of maximizing regeneration and repair after CNS injury in humans.
| Madsen, J R; MacDonald, P; Irwin, N et al. (1998) Tacrolimus (FK506) increases neuronal expression of GAP-43 and improves functional recovery after spinal cord injury in rats. Exp Neurol 154:673-83 |
