INFLUENCE OF NEURONS ON ASTROGLIAL DIFFERENTIATION

Previous studies from our laboratory have shown that granule neurons inhibit astroglial proliferation by a membrane-mediated mechanism. The primary goal of this research is to define the molecular mechanism of neuronal inhibition of astroglial growth. Three projects are planned. Firs, we will purify the inhibitory activity from C17A cell membranes by preparing detergent extracts of the membranes, fractionating the extracts by gel filtration, lectin affinity chromatography or FPLC, coupling partially purified material to Covaspheres MX beads and assaying for inhibition of astroglial[3H]-thymidine incorporation. The identity of the activity will then be confirmed in primary granule neurons by competition and immunoprecipitation experiments. In addition, we will analyze whether antibodies against neuron-glia binding molecules (astrotactin, NILE, AMOG,N-cadherin, integrin-B1) block the inhibitory activity purified from neuronal membranes. Second, we will analyze whether neuronal membranes, or purified inhibitory material, affect growth factor receptor levels on the astroglial cell surface, by studying the binding and rate of removal from the cell surface of radiolabeled EGF, FGF, PDGF and insulin. Other studies from our laboratory indicate that whereas neuronal membranes arrest glial growth, living neurons are required to induce cerebellar astroglial process extension in vitro. In a third project, we will arrest glial proliferation with neuronal membranes, or purified inhibitory activity, and then assay the effect of growth factors (EGF, bFGF, PDGF and TGF-B), lymphokines (II-1, II-2, II-3) and of the culture substratum (polyamines, lectins, fibronectin, laminin, Matrigel) on glial process extension in vitro.