Astrocytes have been prepared from adult rat cortex, cerebellum, and striatum, using a modification of the McCarthy-DeVellis (J Cell Bio 85:890, 1980) method. The cultures consist of 99% type 1 polygonal astrocytes, which divide more slowly than cells from newborn animals. One day after preparing the cultures, 90% of the cells are glial fibrillary acidic protein (GFAP)-positive and 80% are vimentin-positive by immunohistochemical staining, suggesting that they are present de novo and not derived from precursor cells. The astrocytes from adult brain respond to an elevation of intracellular cyclic AMP, following treatment with forskolin, by becoming more stellate in shape and putting out fine ramified processes. They contain the same amount of GFAP per mg protein, measured by immunoblot, as cells from newborn animals. These cultures thus offer the possibility of comparing the biochemical properties of astrocytes derived from adult animals with those from newborn animals, or with cultures of reactive astrocytes isolated from lesioned brain.
Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cells (1-3 days in vitro)against induced apoptosis and mature cells (5؉ days in vitro) against glutamate toxicity, but its precise mechanism is still unknown. Because the transcription factor NFB blocks cell death, including neuronal apoptosis, we have investigated the ability of PEDF to exert its effects via NFB activation. PEDF induced an increased phosphorylation of IB␣, decreased levels of IB proteins, and translocation of p65 (RelA) to the nucleus followed by a time-dependent increase of NFB-DNA binding activity in both immature and mature neurons. The protective effects of PEDF against both induced apoptosis and glutamate toxicity were blocked by the addition of either the IB kinase inhibitor BAY 11-7082, which inhibits the phosphorylation of IB, or N-acetylLeu-Leu-norleucinal, which blocks proteosome degradation of IB, demonstrating that NFB is required for the neuroprotective effects of PEDF. Reverse transcription-polymerase chain reaction analysis revealed that up-regulation of the anti-apoptotic genes for Bcl-2, Bcl-x, and manganese superoxide dismutase was observed in PEDF-treated immature but not mature neurons. Up-regulation of nerve growth factor, brainderived neurotrophic factor, and glial cell-derived neurotrophic factor mRNA was long-lasting in mature neurons. These results suggest that PEDF promotes neuronal survival through activation of NFB, which in turn induces expression of anti-apoptotic and/or neurotrophic factor genes.
One of the functions of glial receptors is to regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn act on neurons or other glia. Because of the potential importance of these interactions in injured brain, we have examined the role of two different receptors in the regulation of astrocyte neuropeptide synthesis. Stimulation of beta-adrenergic receptors on type 1 astrocytes resulted in increased mRNA and protein for the proenkephalin (PE) and somatostatin genes. This receptor also increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The potential role of opiate receptors was examined in several ways. Treatment of newborn rats for 7 days with the opiate antagonist naltrexone, prior to preparation of astrocytes, had no effect on PE mRNA or met-enkephalin content but resulted in a significant increase in NGF content. However, treatment of astrocytes in culture with met-enkephalin, morphine, or naltrexone had no effect on any of these parameters. No opiate binding could be detected, using either etorphine or bremazocine, to membranes of astrocytes prepared from cortex, cerebellum, striatum, or hippocampus of 1-day, 7-day, or 14-day postnatal rats. Thus we conclude that type 1 astrocytes do not express opiate receptors and that the in vivo effects of naltrexone are mediated indirectly via some other cell type/receptor.
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