MOLECULAR MECHANISMS OF NEURONAL CELL MIGRATION

DESCRIPTION (Investigator's Abstract): This is an application for a FIRST award to study the molecular components of neuronal migration in the mammalian CNS. The identification of the molecules that drive selective neuronal cell displacement to specific cortical laminae or nuclear aggregates is only beginning to emerge. At present, data indicate that members of several distinct classes of cell adhesions proteins are likely to mediate many of the cell sorting events that characterize neuronal cell migration. The applicant and his postdoctoral advisor, Dr. Pasko Rakic have recently identified a monoclonal antibody that recognizes two unique polypeptides: a 48 kD antigen and a pair of antigens of 72 and 130 kD. The antibodies stain the junctional region formed between migrating neuronal cells and radial glial cell processes. The PI suggests that the peptides play a critical role in normal migration, and proposes to follow up on these observations with a series of studies consisting of five specific aims. The first aim is to further refine the tissue localization of the 72/130 kD antigen at the ultrastructural level. The model systems to be explored are the cerebral cortex, cerebellum and hippocampus of wild- type and neurological mutant mice (reeler, weaver and dreher). The chick optic tectum will also be examined. Pre-embedding staining will be used to determine the cell type specificity of the staining. Post embedding staining with colloidal gold will be used to explore the fine details of the subcellular localization of the antigen and its relationship to the junctional complex between glia and neuron. Second, the PI will determine whether monoclonal antibodies to the above antigen can perturb various aspects of nervous system cell behavior including, aggregation, adhesion, or migration in vitro. Three tests, a short-term aggregation assay, an adhesion assay (to a carpet of astroglial cells), and a bead/liposome assay with defined antigens of various (undescribed) flavors will be done by the applicant. A fourth approach will be to do high resolution time-lapse video microscopy. This last assay will be performed at Yale in the laboratory of the PI's postdoctoral mentor and current collaborator, Dr. Pasko Rakic. The third aim is to obtain primary structural information on the 72/130 antigen. A cDNA clone has been isolated from a neonatal rat library from Stratagene. Sequencing information was being derived as the grant was written and additional clones will be sought until a full length cDNA is obtained. The fourth specific aim is to develop monospecific antibodies against the smaller, 48 kD, antigen. This antigen is recognized by the initial monoclonal, called D4. But a subsequent series of fusions led only to new antibodies against the 72/130 antigen. The purpose of this aim is to facilitate the study of the temporal and spatial distribution of the 48 kD antigen and ultimately to allow for its cloning and characterization. The fifth specific aim is to return to the business of making monoclonal antibodies. Two directed strategies will be used: immunosuppression of common antigens using cyclophosphamide or induction of neonatal tolerance. Both procedures will select against a plasmalemma subfraction of cerebellar granule cells and then challenge with one of two primary immunogens, enriched for glycosylated forms of antigens isolated from the membranes of radial glial cells. A final section entitled "Future directions" presents a significant set of preliminary data that hint at the role of phosphotyrosine epitopes in the action (or at least regulation) of the 70/130 antigen in particular and neuronal migration in general.