Anti-apoptotic actions of R-2HMP in cerebellar granule cells : changes of mitochondrial membrane potential and sub-cellular GAPDH protein
(R)-'N'-(2-heptyl)-'N'-methylpropargylamine (R-2HMP) has been shown to reduce neuronal death through an action at the same site as R-deprenyl: this site has been proposed to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using cultured cerebellar granule cells, two modes of apoptosis were induced by adding cytosine arabinoside (Ara-c) (p53-dependent) or by lowering the extracellular concentration of potassium (p53-independent). A fluorescent probe, chloromethyl-tetramethylrhodamine methyl ester (CMTMR), was introduced to the medium 0, 4, 6, 12, 18, or 24 hours after the induction of apoptosis. Chromatin condensation was visualized with bisbenzamide. Treated and untreated cells at 6, 12 or 24 hours were collected, subcellular fractions obtained and Western blotting of GAPDH performed. Northern Blot analysis was done to examine the effect of R-2HMP on the expression of GAPDH. The results indicate that: (1) A decrease in mitochondrial membrane potential is an early event, occurring between 4 and 6 hours after Ara-c induced apoptosis; (2) Increased nuclear and mitochondrial GAPDH protein is a later event, occurring between 6 and 12 hours after Ara-c induced apoptosis; (3) GAPDH mRNA is increased 1 hour following Ara-c; (4) There was no decrease of mitochondrial membrane potential or subcellular changes of GAPDH associated with low K+ induced apoptosis; (5) R-2HW prevents Ara-c induced apoptosis, prevents mitochondrial membrane potential changes and GAPDH protein and mRNA changes, but has no effect on low K + induced apoptosis. Taken together, these results provide good evidence that some forms of apoptosis involve a change of mitochondrial membrane potential followed by altered subcellular GAPDH. R-2HMP can prevent the changes associated with Ara-c induced apoptosis, and appears to act very early in the apoptotic cascade.