APOPTOTIC CELL-DEATH OF CULTURED CEREBRAL CORTICAL-NEURONS INDUCED BYWITHDRAWAL OF ASTROGLIAL TROPHIC SUPPORT

Peripheral neurons which depend on NGF for their survival undergo apop tosis after NGF deprivation. However, a convenient in vitro method for assessing the programmed cell death of the central neurons has not be en established, because the dependence of particular central neurons o n neurotrophic factors has been clarified only for small populations o f neurons. Based on the fact that cortical neurons survive in culture for many weeks in the presence of astroglial cells, we have establishe d an in vitro cell death model in which the neurons die through apopto sis. Cortical neurons were maintained on a cover slip for 1 week on to p of astroglial cells, and then cell death was induced by separation o f the neurons from the astroglial cells. The cortical neurons died wit hin 2-4 days. Nuclei of the dying neurons showed the morphological fea tures of apoptosis, and DNA fragmentation was observed by the TUNEL me thod and by in situ nick translation (ISNT) staining. The cell death w as significantly suppressed by neurotrophic factors, NT-3, NT-4, BDNF and GDNF, but not by NGF. The neuronal survival was prolonged, as in t he case of peripheral neurons, by bFGF, elevated potassium, cAMP, fors kolin, and metabotropic glutamate receptor agonist. The cell death was inhibited by inhibitors of interleukin-1 beta-converting enzyme and C PP32. CPP32-like proteolytic activity was increased prior to the appea rance of apoptotic cells. These results suggest that cortical neurons die after separation from glial cells through apoptosis caused by depr ivation of neurotrophic factors produced by the astroglial cells. (C) 1998 Academic Press.