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Alternating metabolic pathways in NGF-deprived sympathetic neurons affect caspase-independent death.

Chang LK, Schmidt RE, Johnson EM - J. Cell Biol. (2003)

Bottom Line: However, the events themselves that culminate in caspase activation can have deleterious effects because caspase inhibitor-saved cells ultimately die in a caspase-independent manner.Third, permeability transition pore inhibition by cyclosporin A attenuates NGF deprivation-induced loss of mitochondrial proteins, suggesting that permeability transition pore opening may have a function in regulating the degradation of mitochondria after cytochrome c release.Identification of changes in caspase inhibitor-saved cells may provide the basis for rational strategies to augment the effectiveness of the therapeutic use of postmitochondrial interventions.

View Article: PubMed Central - PubMed

Affiliation: Washington University School of Medicine, Saint Louis, MO 63110, USA.

ABSTRACT
Mitochondrial release of cytochrome c in apoptotic cells activates caspases, which execute apoptotic cell death. However, the events themselves that culminate in caspase activation can have deleterious effects because caspase inhibitor-saved cells ultimately die in a caspase-independent manner. To determine what events may underlie this form of cell death, we examined bioenergetic changes in sympathetic neurons deprived of NGF in the presence of a broad-spectrum caspase inhibitor, boc-aspartyl-(OMe)-fluoromethylketone. Here, we report that NGF-deprived, boc-aspartyl-(OMe)-fluoromethylketone-saved neurons rely heavily on glycolysis for ATP generation and for survival. Second, the activity of F0F1 contributes to caspase-independent death, but has only a minor role in the maintenance of mitochondrial membrane potential, which is maintained primarily by electron transport. Third, permeability transition pore inhibition by cyclosporin A attenuates NGF deprivation-induced loss of mitochondrial proteins, suggesting that permeability transition pore opening may have a function in regulating the degradation of mitochondria after cytochrome c release. Identification of changes in caspase inhibitor-saved cells may provide the basis for rational strategies to augment the effectiveness of the therapeutic use of postmitochondrial interventions.

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Electron transport maintains ΔΨm in NGF-deprived, BAF-saved cells. To determine the mechanism by which ΔΨm is generated, cells were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. ΔΨm was determined by JC-1 staining before and after a 15-min exposure to CCCP (50 μM), rotenone, and antimycin A (Rot/AA, 2 μM each) to block electron transport, or oligomycin (5 μg/ml) to inhibit the F0F1 ATPase. Values are normalized to the JC-1 ratio of each culture before treatment. Values represent mean ± SD of 22–23 wells from four independent experiments. Asterisk indicates a statistically significant increase (P < 0.05) from CCCP treatment.
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fig5: Electron transport maintains ΔΨm in NGF-deprived, BAF-saved cells. To determine the mechanism by which ΔΨm is generated, cells were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. ΔΨm was determined by JC-1 staining before and after a 15-min exposure to CCCP (50 μM), rotenone, and antimycin A (Rot/AA, 2 μM each) to block electron transport, or oligomycin (5 μg/ml) to inhibit the F0F1 ATPase. Values are normalized to the JC-1 ratio of each culture before treatment. Values represent mean ± SD of 22–23 wells from four independent experiments. Asterisk indicates a statistically significant increase (P < 0.05) from CCCP treatment.

Mentions: Inhibition of F0F1 does not decrease ATP levels in cells that have released cytochrome c (Fig. 2), arguing that it is not generating ATP at the expense of the mitochondrial proton gradient. However, the F0F1 ATPase inhibitor, oligomycin, prevents caspase-independent death, suggesting that the activity of this enzyme complex has a role in the death of these cells. As its name suggests, the F0F1 ATPase can hydrolyze ATP to transport protons actively against the electrochemical gradient. In this mode of operation, F0F1 activity would contribute to ΔΨm, which is maintained, albeit to a lesser degree, in NGF-deprived, BAF-saved neurons after cytochrome c has been released (Chang and Johnson, 2002). Because cytochrome c mediates electron transport from complexes III to IV, the loss of mitochondrial cytochrome c would be expected to impair electron transport chain–mediated maintenance of ΔΨm. To determine the mechanism by which caspase inhibitor–saved cells maintain ΔΨm, we examined the effect of inhibiting either electron transport or F0F1 on ΔΨm in NGF-deprived, BAF-saved neurons by using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraetylbenzimidazolylcarbocyanine iodide (JC-1), a cell-permeable, ΔΨm-sensitive dye (Smiley et al., 1991). JC-1 exists as a fluorescent monomer, but reversibly forms aggregates, which have different spectral properties than the monomeric form, in the matrix of polarized mitochondria (Nicholls and Ward, 2000). Sympathetic neurons that were either maintained in NGF or deprived of NGF in the presence of BAF for 3 d were loaded with JC-1 and treated with the 2 μM rotenone and 2 μM antimycin A , to block electron transport at sites I and II, respectively, and/or 5 μg/ml oligomycin, to block both forward and reverse operation of F0F1. The effects of these treatments on ΔΨm were determined by comparing the JC-1 ratios obtained immediately before and 15 min after drug or vehicle addition. Exposure to the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), decreased the JC-1 ratio in NGF-maintained cells, reflecting complete mitochondrial depolarization (Fig. 5). Oligomycin had very little effect on ΔΨm in NGF-maintained neurons after oligomycin treatment. Treatment of NGF-maintained neurons with rotenone and antimycin A caused complete mitochondrial depolarization, demonstrating that electron transport is responsible for maintenance of ΔΨm in NGF-maintained neurons. Inhibition of electron transport largely, but not completely, depolarized mitochondria in NGF-deprived, BAF-saved neurons because the degree of mitochondrial depolarization achieved by treatment with antimycin and rotenone was less than that with CCCP. This electron transport inhibitor–insensitive contribution to ΔΨm persisted for at least 45 min after drug addition, arguing against the possibility that inhibition of electron transport dissipates ΔΨm more slowly in NGF-deprived, BAF-saved cells (unpublished data). A combination of rotenone, antimycin A, and oligomycin completely depolarized mitochondria in NGF-deprived, BAF-saved cells, suggesting that the residual ΔΨm after inhibition of electron transport was maintained by reversal of F0F1 and hydrolysis of ATP (Fig. 5). Thus, although reversal of F0F1 has a minor function in the maintenance of ΔΨm in NGF-deprived, BAF-saved, but not NGF-maintained, neurons, electron transport is the primary mechanism by which ΔΨm is maintained before and after cytochrome c release in sympathetic neurons.


Alternating metabolic pathways in NGF-deprived sympathetic neurons affect caspase-independent death.

Chang LK, Schmidt RE, Johnson EM - J. Cell Biol. (2003)

Electron transport maintains ΔΨm in NGF-deprived, BAF-saved cells. To determine the mechanism by which ΔΨm is generated, cells were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. ΔΨm was determined by JC-1 staining before and after a 15-min exposure to CCCP (50 μM), rotenone, and antimycin A (Rot/AA, 2 μM each) to block electron transport, or oligomycin (5 μg/ml) to inhibit the F0F1 ATPase. Values are normalized to the JC-1 ratio of each culture before treatment. Values represent mean ± SD of 22–23 wells from four independent experiments. Asterisk indicates a statistically significant increase (P < 0.05) from CCCP treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172806&req=5

fig5: Electron transport maintains ΔΨm in NGF-deprived, BAF-saved cells. To determine the mechanism by which ΔΨm is generated, cells were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. ΔΨm was determined by JC-1 staining before and after a 15-min exposure to CCCP (50 μM), rotenone, and antimycin A (Rot/AA, 2 μM each) to block electron transport, or oligomycin (5 μg/ml) to inhibit the F0F1 ATPase. Values are normalized to the JC-1 ratio of each culture before treatment. Values represent mean ± SD of 22–23 wells from four independent experiments. Asterisk indicates a statistically significant increase (P < 0.05) from CCCP treatment.
Mentions: Inhibition of F0F1 does not decrease ATP levels in cells that have released cytochrome c (Fig. 2), arguing that it is not generating ATP at the expense of the mitochondrial proton gradient. However, the F0F1 ATPase inhibitor, oligomycin, prevents caspase-independent death, suggesting that the activity of this enzyme complex has a role in the death of these cells. As its name suggests, the F0F1 ATPase can hydrolyze ATP to transport protons actively against the electrochemical gradient. In this mode of operation, F0F1 activity would contribute to ΔΨm, which is maintained, albeit to a lesser degree, in NGF-deprived, BAF-saved neurons after cytochrome c has been released (Chang and Johnson, 2002). Because cytochrome c mediates electron transport from complexes III to IV, the loss of mitochondrial cytochrome c would be expected to impair electron transport chain–mediated maintenance of ΔΨm. To determine the mechanism by which caspase inhibitor–saved cells maintain ΔΨm, we examined the effect of inhibiting either electron transport or F0F1 on ΔΨm in NGF-deprived, BAF-saved neurons by using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraetylbenzimidazolylcarbocyanine iodide (JC-1), a cell-permeable, ΔΨm-sensitive dye (Smiley et al., 1991). JC-1 exists as a fluorescent monomer, but reversibly forms aggregates, which have different spectral properties than the monomeric form, in the matrix of polarized mitochondria (Nicholls and Ward, 2000). Sympathetic neurons that were either maintained in NGF or deprived of NGF in the presence of BAF for 3 d were loaded with JC-1 and treated with the 2 μM rotenone and 2 μM antimycin A , to block electron transport at sites I and II, respectively, and/or 5 μg/ml oligomycin, to block both forward and reverse operation of F0F1. The effects of these treatments on ΔΨm were determined by comparing the JC-1 ratios obtained immediately before and 15 min after drug or vehicle addition. Exposure to the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), decreased the JC-1 ratio in NGF-maintained cells, reflecting complete mitochondrial depolarization (Fig. 5). Oligomycin had very little effect on ΔΨm in NGF-maintained neurons after oligomycin treatment. Treatment of NGF-maintained neurons with rotenone and antimycin A caused complete mitochondrial depolarization, demonstrating that electron transport is responsible for maintenance of ΔΨm in NGF-maintained neurons. Inhibition of electron transport largely, but not completely, depolarized mitochondria in NGF-deprived, BAF-saved neurons because the degree of mitochondrial depolarization achieved by treatment with antimycin and rotenone was less than that with CCCP. This electron transport inhibitor–insensitive contribution to ΔΨm persisted for at least 45 min after drug addition, arguing against the possibility that inhibition of electron transport dissipates ΔΨm more slowly in NGF-deprived, BAF-saved cells (unpublished data). A combination of rotenone, antimycin A, and oligomycin completely depolarized mitochondria in NGF-deprived, BAF-saved cells, suggesting that the residual ΔΨm after inhibition of electron transport was maintained by reversal of F0F1 and hydrolysis of ATP (Fig. 5). Thus, although reversal of F0F1 has a minor function in the maintenance of ΔΨm in NGF-deprived, BAF-saved, but not NGF-maintained, neurons, electron transport is the primary mechanism by which ΔΨm is maintained before and after cytochrome c release in sympathetic neurons.

Bottom Line: However, the events themselves that culminate in caspase activation can have deleterious effects because caspase inhibitor-saved cells ultimately die in a caspase-independent manner.Third, permeability transition pore inhibition by cyclosporin A attenuates NGF deprivation-induced loss of mitochondrial proteins, suggesting that permeability transition pore opening may have a function in regulating the degradation of mitochondria after cytochrome c release.Identification of changes in caspase inhibitor-saved cells may provide the basis for rational strategies to augment the effectiveness of the therapeutic use of postmitochondrial interventions.

View Article: PubMed Central - PubMed

Affiliation: Washington University School of Medicine, Saint Louis, MO 63110, USA.

ABSTRACT
Mitochondrial release of cytochrome c in apoptotic cells activates caspases, which execute apoptotic cell death. However, the events themselves that culminate in caspase activation can have deleterious effects because caspase inhibitor-saved cells ultimately die in a caspase-independent manner. To determine what events may underlie this form of cell death, we examined bioenergetic changes in sympathetic neurons deprived of NGF in the presence of a broad-spectrum caspase inhibitor, boc-aspartyl-(OMe)-fluoromethylketone. Here, we report that NGF-deprived, boc-aspartyl-(OMe)-fluoromethylketone-saved neurons rely heavily on glycolysis for ATP generation and for survival. Second, the activity of F0F1 contributes to caspase-independent death, but has only a minor role in the maintenance of mitochondrial membrane potential, which is maintained primarily by electron transport. Third, permeability transition pore inhibition by cyclosporin A attenuates NGF deprivation-induced loss of mitochondrial proteins, suggesting that permeability transition pore opening may have a function in regulating the degradation of mitochondria after cytochrome c release. Identification of changes in caspase inhibitor-saved cells may provide the basis for rational strategies to augment the effectiveness of the therapeutic use of postmitochondrial interventions.

Show MeSH
Related in: MedlinePlus