We found that cerebellar granule cells (CGC) undergo spontaneous cell death after culturing in vitro for about 17 days. This neuronal death is characterized by hallmarks of apoptosis. This age-induced apoptosis is accelerated by N-methyl-D-aspartate (NMDA) but delayed by aurintric- arboxylic acid (ATA), tetrahydroaminoacri-dine (THA), antioxidants, and NMDA receptor antagonists. Over production of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and protein precedes the cell death. This over production is hastened by NMDA but diminished or prevented by ATA, THA, actinomycin-D, or cycloheximide. Antisense oligodeoxyribonucleotides to GAPDH effectively protect against apoptosis, while the corresponding sense oligonucleotides are ineffective. Thus, GAPDH is linked to neuronal apoptosis. Cerebral cortical neurons also undergo age-induced apoptosis in culture and this form of cell death also involves GAPDH over expression. Low potassium-induced death of CGC includes both apoptosis and necrosis and is blocked by cycloheximide and partially protected by actinomycin D, THA, and antisense oligonucleotides to GAPDH. GAPDH antisense oligonucleotides block the apoptotic, but not necrotic, component of CGC death. Moreover, BDNF and bFGF provide efficient protection, while NT-3 is inactive. The neuroprotective effect of THA could be related to its therapeutic action in the treatment of Alzheimer's disease. Apoptotic death of CGC induced by exposure to cytosine arabinoside (AraC) also involves GAPDH over production and is protected by GAPDH antisense oligonucleotides which are more effective than classical inhibitors of apoptosis. Subcellular fractionation studies show that the over expressed GAPDH immunoreactive proteins are predominantly in the crude nuclear mitochondrial and golgi fractions with little change in the crude microsomal fractions. GAPDH glycolytic activity is not changed in parallel with the levels of GAPDH protein. AraC-induced apoptosis is markedly protected by BDNF and NT-4/5, but not NT-3.
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