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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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Oligomycin kills NGF-maintained neurons, but protects NGF-deprived, BAF-saved neurons. (A) To determine the effect of oligomycin on Commitment 2, oligomycin (OL) was added to cultures after they were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. (B) After 8 d of exposure to oligomycin, cells were rescued with NGF for 7 d and counted. At the time of oligomycin addition, nearly all cells had released cytochrome c, because <5% of NGF-deprived cells could be rescued. Asterisk indicates statistical significance of P < 10−4 compared with −NGF +BAF. For comparison, cyclosporin A (CsA) added after 2 d of deprivation had a similar effect on Commitment 2 at 10 d as oligomycin. This value was not different from that of the −NGF +BAF + OL condition. Values shown in B are mean ± SD of three independent experiments performed in quadruplicate for each condition.
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fig4: Oligomycin kills NGF-maintained neurons, but protects NGF-deprived, BAF-saved neurons. (A) To determine the effect of oligomycin on Commitment 2, oligomycin (OL) was added to cultures after they were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. (B) After 8 d of exposure to oligomycin, cells were rescued with NGF for 7 d and counted. At the time of oligomycin addition, nearly all cells had released cytochrome c, because <5% of NGF-deprived cells could be rescued. Asterisk indicates statistical significance of P < 10−4 compared with −NGF +BAF. For comparison, cyclosporin A (CsA) added after 2 d of deprivation had a similar effect on Commitment 2 at 10 d as oligomycin. This value was not different from that of the −NGF +BAF + OL condition. Values shown in B are mean ± SD of three independent experiments performed in quadruplicate for each condition.

Mentions: Because NGF-maintained neurons, but not NGF-deprived, BAF-saved cells, require oxidative phosphorylation to maintain maximal ATP levels, a second prediction from the data in Fig. 2 is that inhibition of oxidative phosphorylation should be more detrimental to NGF-maintained neurons than to NGF-deprived, BAF-saved neurons. To examine the effect of inhibiting oxidative phosphorylation on Commitment 2, cells were deprived of NGF in the presence of BAF for 2 d and maintained for an additional 8 d in the presence of 5 μg/ml oligomycin to block oxidative phosphorylation, and subsequently rescued with NGF (Fig. 4 A). Consistent with our previous report (Chang and Johnson, 2002) and data in Fig. 3, the number of NGF-deprived, BAF-saved cells that could be rescued after 2 and 10 d of NGF deprivation decreased from roughly 85% of control to 35%, indicating that caspase-independent death occurred during this period. After 8 d of treatment with oligomycin, only 10% of NGF-maintained neurons survived (Fig. 4 B). Surprisingly, treatment with oligomycin increased the number of BAF-saved neurons that could be rescued by NGF, from 35 to roughly 55% of control. Because only 85% of NGF-deprived, BAF-saved cells could be rescued at the time of oligomycin treatment, almost half of the cells that could be saved were protected by oligomycin. To illustrate the magnitude of this effect, CsA, a robust inhibitor of Commitment 2 (Chang and Johnson, 2002), increased the proportion of cells that were rescued to a similar degree when added after 2 d of NGF deprivation (Fig. 4 B). Thus, although oligomycin is toxic to NGF-maintained sympathetic neurons, it protects NGF-deprived, BAF-saved cells. These findings strongly suggest that activity of the F0F1 ATPase contributes to caspase-independent death of sympathetic neurons.


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

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

Oligomycin kills NGF-maintained neurons, but protects NGF-deprived, BAF-saved neurons. (A) To determine the effect of oligomycin on Commitment 2, oligomycin (OL) was added to cultures after they were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. (B) After 8 d of exposure to oligomycin, cells were rescued with NGF for 7 d and counted. At the time of oligomycin addition, nearly all cells had released cytochrome c, because <5% of NGF-deprived cells could be rescued. Asterisk indicates statistical significance of P < 10−4 compared with −NGF +BAF. For comparison, cyclosporin A (CsA) added after 2 d of deprivation had a similar effect on Commitment 2 at 10 d as oligomycin. This value was not different from that of the −NGF +BAF + OL condition. Values shown in B are mean ± SD of three independent experiments performed in quadruplicate for each condition.
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Related In: Results  -  Collection

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

fig4: Oligomycin kills NGF-maintained neurons, but protects NGF-deprived, BAF-saved neurons. (A) To determine the effect of oligomycin on Commitment 2, oligomycin (OL) was added to cultures after they were maintained in NGF or deprived of NGF in the presence of BAF for 2 d. (B) After 8 d of exposure to oligomycin, cells were rescued with NGF for 7 d and counted. At the time of oligomycin addition, nearly all cells had released cytochrome c, because <5% of NGF-deprived cells could be rescued. Asterisk indicates statistical significance of P < 10−4 compared with −NGF +BAF. For comparison, cyclosporin A (CsA) added after 2 d of deprivation had a similar effect on Commitment 2 at 10 d as oligomycin. This value was not different from that of the −NGF +BAF + OL condition. Values shown in B are mean ± SD of three independent experiments performed in quadruplicate for each condition.
Mentions: Because NGF-maintained neurons, but not NGF-deprived, BAF-saved cells, require oxidative phosphorylation to maintain maximal ATP levels, a second prediction from the data in Fig. 2 is that inhibition of oxidative phosphorylation should be more detrimental to NGF-maintained neurons than to NGF-deprived, BAF-saved neurons. To examine the effect of inhibiting oxidative phosphorylation on Commitment 2, cells were deprived of NGF in the presence of BAF for 2 d and maintained for an additional 8 d in the presence of 5 μg/ml oligomycin to block oxidative phosphorylation, and subsequently rescued with NGF (Fig. 4 A). Consistent with our previous report (Chang and Johnson, 2002) and data in Fig. 3, the number of NGF-deprived, BAF-saved cells that could be rescued after 2 and 10 d of NGF deprivation decreased from roughly 85% of control to 35%, indicating that caspase-independent death occurred during this period. After 8 d of treatment with oligomycin, only 10% of NGF-maintained neurons survived (Fig. 4 B). Surprisingly, treatment with oligomycin increased the number of BAF-saved neurons that could be rescued by NGF, from 35 to roughly 55% of control. Because only 85% of NGF-deprived, BAF-saved cells could be rescued at the time of oligomycin treatment, almost half of the cells that could be saved were protected by oligomycin. To illustrate the magnitude of this effect, CsA, a robust inhibitor of Commitment 2 (Chang and Johnson, 2002), increased the proportion of cells that were rescued to a similar degree when added after 2 d of NGF deprivation (Fig. 4 B). Thus, although oligomycin is toxic to NGF-maintained sympathetic neurons, it protects NGF-deprived, BAF-saved cells. These findings strongly suggest that activity of the F0F1 ATPase contributes to caspase-independent death of 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