Study on Intracellular Mechanism in Neuronal Apoptosis

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When embryonic rat hippocampal neurons were cultured in a 50% oxygen atmosphere, neurons gradually died after 20 hr in culture. This death pattern was found to be mediated by an intracellular active death program, so called apoptosis, as follows : 1) Cycloheximide and actinomycin-D,protein and RNA synthesis inhibitors, respectively, prevented cell death, indicating that cell death requied new protein biosynthesis. 2) DNA fragmentation (called a "DNA ladder" ), a specific biochemical marker of apoptosis, was detected during the course of cell death. 3) Depolarization with high K+ medium (26-50mM) prevented cell death. This effect was suppressed by some dihydropyridine derivatives, L-type Ca channel blockers, such as nifedipine and nicardipine. These results suggested that disruption of intracellular oxygen metabolism activated the cell suicide program in the cultured hippocampal neurons, and that neuronal activity playd an important role in saving neurons from oxidative damage during th … More eir long life without division.Cerebellar granule neurons obtained from 7-9 day-old rats were grown in vitro for 4-5 days in high K+ (26 mM) medium. The culture medium was then with that containing low K+ (5 mM) which caused a large number of granule neurons to die. The death of granule neurons has been characterized as apoptosis. In this study, we investigated the effects of various neurotrophins on neuronal survival using the above system. We found that brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) but not nerve growth factor (NGF) can protect these neurons from apoptosis in low K+. Neurotrophin-3 (NT-3) had a small effect on neuronal survival as reported. To determinewhether the granule neurons respond directly to BDNF,we analyzed the induction of the Fos protein in these neurons. Individual cells that synhtesize Fos protein after exposure to neurotrpohin can be recognized using antibodies to Fos. Immunocytochemical staining of the cultures demonstrated that a relatively large number of cerebellar granule neurons showed immunoreactivity in response to BDNF,but few of them were immunoreactive in the absence of BDNF or in the presence of NGF.Our results suggested that BDNF has a direct effect on mature cerebellar granule neurons and can protect these neurons from apoptosis in low K+. Less