The most prominent response to central nervous system (CNS) injury and disease is fibrous gliosis, characterized by astrocytic hyperplasia and hypertrophy with accompanying increase in glial filaments (glial fibrillary acidic protein, GFAP). Understanding the mechanisms and control of fibrous gliosis is essential for rational treatment and maintenance of CNS trauma and disease. If reactive fibrous gliosis could be inhibited or delayed in trauma and disease, the other cell types might have the opportunity to re- establish a more normal, functional CNS environment; conversely, a highly anaplastic astrocytoma might be induced to differentiate. Our working hypothesis is that one possible function of glial filaments is a structural role in maintaining cell shape and structural integrity of the CNS. This is beneficial in the normal state but can be detrimental for regeneration if fibrous gliosis inhibits or prevents remyelination or axonal regeneration by virtue of occupying potential sites for regeneration. Reactive astrocytes could also produce growth or inhibitory growth factors. The rationale for our study is that certain reactive properties of the stellate astrocyte in culture are more likely to be similar to the mature astrocyte in vivo and different than the flat, polygonal astrocyte. We propose to study astrocyte activation and reactive gliosis in cultured astrocytes employing state of the art immunologic and molecular probes. The cytoskeletal proteins that will be studied are GFAP, vimentin, alpha and beta tubulin and actin. We will compare the membrane properties and regulation of cytoskeletal proteins in cultured polygonal astroblast, astroblasts undergoing morphological maturation, and the mature stellate astrocyte obtained by treatment with specific agents such as cyclic AMP, by culturing on substrates such as nitrocellulose, and by employing serum-depleted or chemically defined medium. While this study will test both mitogenic and hypertrophic factors, the main focus is on the agents and mechanisms which activate the differentiated astrocyte to produce GFAP in its response to injury. This study will employ a correlated morphological and biochemical approach. Morphological studies will employ histological, immunocytochemical, and cDNA in situ hybridization methods. Biochemical procedures will determine total protein and individual cytoskeletal proteins by ELISA and by slab gel electrophoresis followed by transblotting and immunohistology; phosphorylation of vimentin and GFAP by 32-P incorporation; astrocyte proliferation by incorporation of 5-bromo-2- deoxyuridine; and mRNA by dot, Northern and Western blots.
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