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 (DeltaPsiM) 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 DeltaPsiM. 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 DeltaPsiM. 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 DeltaPsiM 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 DeltaPsiM. 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 DeltaPsiM. (-)-Deprenyl appeared to alter the relationship between intramitochondrial Ca2+ levels and DeltaPsiM, possibly through its reported capacity to increase the synthesis of proteins such as BCL-2.

Susceptibility of HIV-1-TAT transfected cells to undergo apoptosis. Biochemical mechanisms

“…Serum withdrawal can induce lipid peroxidation and the hydrolysis of sphingomyeline which increase the levels of endogenous ceramide (PenÄ a et al, 1997) and mitochondrial PT (Wadia et al, 1998). Nevertheless, K562 cells that express the bcrabl gene are protected against this type of apoptotic stimuli and we show here that HIV-Tat may bypass this protection and induce apoptosis.…”

Mitochondrial ATP-sensitive potassium channels enhance angiotensin-induced oxidative damage and dopaminergic neuron degeneration. Relevance for aging-associated susceptibility to Parkinson’s disease

“…CMTMR binds irreversibly to mitochondrial matrix thiols and can be fixed for immunocytochemical localization of proteins in the same cells that have previously been exposed to CMTMR. As a result of the thiol binding, CMTMR fluorescence represents the highest level of negativity difference in the mitochondria during exposure to the dye before fixation (Sugrue et al 1999;Wadia et al 1998). MitoTracker Green FM (MTGFM) is a mitochondrion-selective probe that becomes fluorescent in the lipid environment of mitochondria.…”

Multiple death pathways in retina-derived 661W cells following growth factor deprivation: crosstalk between caspases and calpains

“…As serum depletion has been described as a mitochondrial pathway-triggering stimulus, 33 we initially performed Western blot (WB) analyses of caspase-9 and -3 in 661W whole-cell extracts. Upregulation of caspase-9 (49 kilo Dalton (kDa)) and processing into 39/37-kDa fragments was detected from 24 h of treatment, providing evidence for a caspase-dependent death pathway ( Figure 2a).…”