Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase isoformsduring neuronal apoptosis

Treatment with cytosine beta-D-arabinoside (AraC; 300 mu M) induced a time- dependent accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in nuclei purified from cultured cerebellar granule cells, with a c oncomitant degradation of lamin B1, a nuclear membrane protein and a substr ate of CPP32/caspase-3. Moreover, Asp-Glu-Val-Asp-fluoromethyl ketone (DEVD -fmk), a CPP32-selective antagonist, dose-dependently suppressed AraC-induc ed apoptosis of these neurons. Nuclear accumulation of GAPDH protein was as sociated with a progressive decrease in the activity of uracil-DNA glycosyl ase (UDG), one of the nuclear functions of GAPDH, The nuclear dehydrogenase activity of GAPDH was initially increased after treatment and then decreas ed parallel to UDG activity. Six GAPDH isoforms were detected in the nuclei of AraC-treated cells. The more alkaline isoforms, 1-3, constituted the bu lk of the nuclear GAPDH, and the remaining isoforms, 4-6, were the minor sp ecies. Levels of all six isoforms were increased after treatment with AraC for 16 h; a 4-h treatment increased levels of only isoforms 4 and 5. Thus, it appears that various GAPDH isoforms are differentially regulated and may have distinct apoptotic roles. Pretreatment with GAPDH antisense oligonucl eotide blocked the nuclear translocation of GAPDH isoforms, and the latter process occurred concurrently with a decrease in cytosolic GAPDH isoforms, Sodium nitroprusside-induced NAD labeling of nuclear GAPDH showed a 60% los s of GAPDH labeling after AraC treatment, suggesting that the active site o f GAPDH may be covalently modified, denatured, or improperly folded. The un folded protein response elicited by denatured GAPDH may contribute to AraC- induced neuronal death.