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

Abstract

Studies in non-neural cells have suggested that a fall in mitochondrial membrane potential (ΔΨ M ) 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 ΔΨ M . 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 ΔΨ M . 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 ΔΨ M 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 ΔΨ M . Measurements of cytoplasmic peroxyl radical levels with 2′,7′-dihydrodichlorofluorescein fluorescence and intramitochondrial Ca 2+ with dihydro-rhodamine-2-acetylmethyl ester indicated that cytoplasmic peroxyl radical levels were not increased until after 6 hr, whereas increases in intramitochondrial Ca 2+ paralleled the decreases in ΔΨ M . (−)-Deprenyl appeared to alter the relationship between intramitochondrial Ca 2+ levels and ΔΨ M , possibly through its reported capacity to increase the synthesis of proteins such as BCL-2.