One means of treating the injured brain may be via regulation of expression of specific neurotrophic factors, in particular those synthesized by astrocytes. Our studies focus on (1) identification of novel neurotrophic factors; and (2) the analysis of the regulation of neurotrophic factor and neuropeptide gene expression during development and in response to injury. One novel neurotrophic factor under investigation is pigment epithelium-derived factor (PEDF). PEDF not only functions as a survival factor for cerebellar granule cell neurons but also can protect them against both glutamate toxicity and apoptotic cell death. A lentiviral construct carrying the PEDF cDNA provides sufficient PEDF to infected cerebellar granule cells for both survival and anti-glutamate protection. PEDFs ability to block glutamate toxicity involves modulation of the changes in intracellular calcium caused by glutamate. PEDF also activates microglia, which produce an as yet unidentified factor which inhibits astrocyte proliferation and may thus be useful when brain injury results in gliosis due to astrocyte division. The factor also inhibits proliferation of gliomas. Since astrocytes can synthesize a number of neurotrophic factors, primary cultures of astrocytes are being used to determine factors which regulate production of trophic factors such as nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF) in response to 6- OHDA lesion of rat substantia nigra, a Parkinsonian-like model. Reactive astrocytes are prepared from 6-OHDA-lesioned brain: monoclonal antibodies raised against epitopes expressed only by reactive astrocytes in vivo distinguish between normal adult and reactive astrocytes in culture. 6-OHDA lesions of the substantia nigra induce reactive astrocytes at the site of lesion as well as in the terminal fields in striatum and cortex. Whereas all these reactive astrocytes express significantly more glial fibrillary acidic protein, S-100-beta, and vimentin, a major difference is found in terms of expression of the adhesion molecule PSA-NCAM. PSA-NCAM expression is turned on in astrocytes in the lesioned SN, but not the contralateral side, nor even in the terminal fields: thus, PSA-NCAM expression can distinguish subtypes of reactive astrocytes. Reactive astrocytes in culture also do not express PSA-NCAM. Thus, although comparable differences are seen between what occurs in vivo and in the cultures of reactive astrocytes, the changes do not all parallel those of the classical markers of reactive gliosis. A second lesion model, systemic 3-acetylpyridine, damages mossy fiber input to the cerebellum, inducing reactive astrocytes, and at higher doses also induces reactive gliosis in the striatum. Cerebellar granule cell (CGC) neurons plated on reactive cerebellar astrocytes produce significantly longer and more fasciculated processes compared with those cultured on normal adult cerebellar astrocytes. Culture of the CGCs on striatal astrocytes produces a different set of growth patterns, again suggesting the existence of subsets of reactive astrocytes. We are currently testing the hypothesis that these differences are due to differential expression of PSA-NCAM. - CNS development, CNS injury, Parkinson's model, astrocytes, neurotrophic factors, PEDF, primary culture, rat, mouse
