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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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NGF-deprived, BAF-saved cells rely on glycolysis for ATP production. Cells were maintained in NGF or deprived of NGF in the presence of BAF or BAF and CsA for 3 d. ATP was measured after 2-h exposure to control the medium (gluc), glucose-free medium with 2-deoxyglucose and pyruvate (2-DG + pyr) to inhibit glycolysis, or oligomycin (5 μg/ml) to inhibit oxidative phosphorylation. Values are mean ± SD of the control level from three independent experiments performed in quadruplicate. Asterisk indicates statistical significance (P < 0.05) versus ATP in control medium for that culture condition.
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fig2: NGF-deprived, BAF-saved cells rely on glycolysis for ATP production. Cells were maintained in NGF or deprived of NGF in the presence of BAF or BAF and CsA for 3 d. ATP was measured after 2-h exposure to control the medium (gluc), glucose-free medium with 2-deoxyglucose and pyruvate (2-DG + pyr) to inhibit glycolysis, or oligomycin (5 μg/ml) to inhibit oxidative phosphorylation. Values are mean ± SD of the control level from three independent experiments performed in quadruplicate. Asterisk indicates statistical significance (P < 0.05) versus ATP in control medium for that culture condition.

Mentions: To identify which bioenergetic pathways are used to generate ATP within these cells, we determined whether acutely inhibiting either glycolysis or oxidative phosphorylation altered cellular ATP levels in NGF-maintained and NGF-deprived, BAF-saved neurons. We used total ATP, which is only a gross measure of cellular energy charge, to identify large perturbations in energy balance that were likely to affect cell survival. As seen in Fig. 2, inhibition of glycolysis, achieved by incubating cells in glucose-free medium supplemented with 5 mM 2-deoxyglucose and 1 mM pyruvate, for 2 h in NGF-maintained cells decreased total ATP to 66% of control. Treatment of cells with the F0F1 ATPase inhibitor, oligomycin, in standard, glucose-containing medium was used to inhibit ATP production by oxidative phosphorylation. In NGF-maintained cells, 5 μg/ml oligomycin caused a decrease in ATP levels to 75% of the control. These data indicate that both glycolysis and oxidative phosphorylation contribute to ATP production in NGF-maintained neurons. To determine the contributions of these pathways to ATP production in NGF-deprived, BAF-saved cells, cultures were synchronized by depriving them of NGF in the presence of BAF for 3 d, such that essentially all cells have released cytochrome c but few cells have become committed to die (Chang and Johnson, 2002). In NGF-deprived, BAF-saved cells, inhibiting glycolysis for 2 h caused a decrease in ATP levels to 32% of control, which is much greater than in NGF-maintained neurons (P < 10−7). In contrast, inhibiting oxidative phosphorylation in NGF-deprived, BAF-saved cells had no effect on cellular ATP (Fig. 2). Thus, unlike NGF-maintained neurons, NGF-deprived, BAF-saved neurons do not require oxidative phosphorylation to maintain maximal ATP levels, but instead rely more heavily, if not solely, on glycolysis for ATP production.


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

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

NGF-deprived, BAF-saved cells rely on glycolysis for ATP production. Cells were maintained in NGF or deprived of NGF in the presence of BAF or BAF and CsA for 3 d. ATP was measured after 2-h exposure to control the medium (gluc), glucose-free medium with 2-deoxyglucose and pyruvate (2-DG + pyr) to inhibit glycolysis, or oligomycin (5 μg/ml) to inhibit oxidative phosphorylation. Values are mean ± SD of the control level from three independent experiments performed in quadruplicate. Asterisk indicates statistical significance (P < 0.05) versus ATP in control medium for that culture condition.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172806&req=5

fig2: NGF-deprived, BAF-saved cells rely on glycolysis for ATP production. Cells were maintained in NGF or deprived of NGF in the presence of BAF or BAF and CsA for 3 d. ATP was measured after 2-h exposure to control the medium (gluc), glucose-free medium with 2-deoxyglucose and pyruvate (2-DG + pyr) to inhibit glycolysis, or oligomycin (5 μg/ml) to inhibit oxidative phosphorylation. Values are mean ± SD of the control level from three independent experiments performed in quadruplicate. Asterisk indicates statistical significance (P < 0.05) versus ATP in control medium for that culture condition.
Mentions: To identify which bioenergetic pathways are used to generate ATP within these cells, we determined whether acutely inhibiting either glycolysis or oxidative phosphorylation altered cellular ATP levels in NGF-maintained and NGF-deprived, BAF-saved neurons. We used total ATP, which is only a gross measure of cellular energy charge, to identify large perturbations in energy balance that were likely to affect cell survival. As seen in Fig. 2, inhibition of glycolysis, achieved by incubating cells in glucose-free medium supplemented with 5 mM 2-deoxyglucose and 1 mM pyruvate, for 2 h in NGF-maintained cells decreased total ATP to 66% of control. Treatment of cells with the F0F1 ATPase inhibitor, oligomycin, in standard, glucose-containing medium was used to inhibit ATP production by oxidative phosphorylation. In NGF-maintained cells, 5 μg/ml oligomycin caused a decrease in ATP levels to 75% of the control. These data indicate that both glycolysis and oxidative phosphorylation contribute to ATP production in NGF-maintained neurons. To determine the contributions of these pathways to ATP production in NGF-deprived, BAF-saved cells, cultures were synchronized by depriving them of NGF in the presence of BAF for 3 d, such that essentially all cells have released cytochrome c but few cells have become committed to die (Chang and Johnson, 2002). In NGF-deprived, BAF-saved cells, inhibiting glycolysis for 2 h caused a decrease in ATP levels to 32% of control, which is much greater than in NGF-maintained neurons (P < 10−7). In contrast, inhibiting oxidative phosphorylation in NGF-deprived, BAF-saved cells had no effect on cellular ATP (Fig. 2). Thus, unlike NGF-maintained neurons, NGF-deprived, BAF-saved neurons do not require oxidative phosphorylation to maintain maximal ATP levels, but instead rely more heavily, if not solely, on glycolysis for ATP production.

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