MOLECULAR MECHANISMS OF NEURONAL CELL MIGRATION

Project: Research project

Project Details

Description

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.
StatusNot started

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