SECOND MESSENGERS IN ASTROCYTES AND PRECURSOR CELLS

The goal of this proposal is to understand the role of second messenger systems in the regulation of glial fibrillary acidic protein(GFAP) and in the maturation of bipotential precursor cells from the nervous system in vitro. GFAP, the astrocyte specific intermediate filament (IF), is an major component of the astrocyte cytoskeleton. It is developmentally regulated and is elevated in most neurologic disease and brain traumas. Defining GFAP regulation in cultured cells will permit a clearer understanding of how GFAP expression is regulated in normal astrocytes and in disease states. The effects of agents which modulate cAMP or protein kinase C on GFAP RNA in primary astrocyte cultures and in the human astrocytoma cell line HTB-17 are being studies. Northern blot analysis of steady-state RNA levels, together with nuclear run-off experiments to assess relative transcription rates, indicate that cAMP, stimulated by treatment of cells with forskolin plus isobutylmethylxanthine (IMX) positively regulates GFAP mRNA at the post- transcriptional level. In contrast, GFAP steady-state mRNA is dramatically reduced in astrocytes and HTB-17 cultures treated with the phorbol ester PMA (10nM). This regulation is also post-transcriptional. Recently serum has been found to inhibit the effect of cAMP-stimulatory agents. Therefore we plan to refine our model system through the use of chemically defined (CD) medium, minus fetal calf serum. The exact nature of post-transcriptional up-regulation and down-regulation of GFAP mRNA will be determined. To investigate a possible role for second messengers in the in vitro maturation of bipotential precursor cells from the nervous system, two types of precursors will be grown in CD medium containing agents which modulate cAMP, protein kinase C and/ or calcium. One type of precursor, the AC-36A cell line, has the capacity to express either glial or neuronal characteristics; while the other, a bipotential precursor derived from neonatal rat forebrain, can become either astrocytic or oligodendrocytic, depending on culture conditions. We will follow the fate of these precursors in the presence of the various agents using immunocytochemical and DNA markers, as well as morphologic criteria. These studies will provide insight into the molecular mechanisms by which extracellular stimuli, such as cell-cell contact and growth factors, induce commitment of nervous system precursors to particular lineages.