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Mitochondrial Membrane Potential and Nuclear Changes in Apoptosis Caused by Serum and Nerve Growth Factor Withdrawal: Time Course and Modification by (−)-Deprenyl

Studies in non-neural cells have suggested that a fall in mitochondrial membrane potential (ΔΨM) is one of the earliest events in apoptosis. It is not known whether neural apoptosis caused by nerve growth factor (NGF) and serum withdrawal involves a decrease in ΔΨM. We used epifluorescence and laser confocal microscopy with the mitochondrial potentiometric dyes chloromethyl-tetramethylrosamine methyl ester and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethybenzimidazol carbocyanine iodide to estimate ΔΨM. PC12 cells were differentiated in media containing serum and NGF for 6 d before withdrawal of trophic support. After washing, the cells were incubated with media containing serum and NGF (M/S+N), media without serum and NGF, or media with the “trophic-like” monoamine oxidase B inhibitor, (−)-deprenyl. Mitochondria in cells without trophic support underwent a progressive shift to lower ΔΨM values that was significant by 3 hr after washing. The percentages of cells with nuclear chromatin condensation or nuclear DNA fragmentation were not significantly increased above those for cells in M/S+N until 6 hr after washing. Replacement of cells into M/S+N or treatment with (−)-deprenyl markedly reduced the proportion of mitochondria with decreased ΔΨM. Measurements of cytoplasmic peroxyl radical levels with 2′,7′-dihydrodichlorofluorescein fluorescence and intramitochondrial Ca2+ with dihydro-rhodamine-2-acetylmethyl ester indicated that cytoplasmic peroxyl radical levels were not increased until after 6 hr, whereas increases in intramitochondrial Ca2+ paralleled the decreases in ΔΨM. (−)-Deprenyl appeared to alter the relationship between intramitochondrial Ca2+ levels and ΔΨM, possibly through its reported capacity to increase the synthesis of proteins such as BCL-2.