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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 deprivation increases sensitivity to glucose deprivation. Cultures were deprived of NGF or maintained in NGF for 2 d in a standard medium, and then for an additional 8 d in a standard medium (con) or glucose-free medium supplemented with 0, 5, or 25 mM glucose as schematized in A. The cells were rescued with NGF for an additional 7 d and counted. Values shown in B are mean ± SD of three independent experiments performed in triplicate of the proportion of cells that could be rescued by NGF after this 10-d treatment. Asterisk indicates statistical significance of P < 0.05 compared with survival in a control medium.
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fig3: NGF deprivation increases sensitivity to glucose deprivation. Cultures were deprived of NGF or maintained in NGF for 2 d in a standard medium, and then for an additional 8 d in a standard medium (con) or glucose-free medium supplemented with 0, 5, or 25 mM glucose as schematized in A. The cells were rescued with NGF for an additional 7 d and counted. Values shown in B are mean ± SD of three independent experiments performed in triplicate of the proportion of cells that could be rescued by NGF after this 10-d treatment. Asterisk indicates statistical significance of P < 0.05 compared with survival in a control medium.

Mentions: One prediction from the finding that NGF-deprived, BAF-saved cells rely more heavily on glycolysis for ATP generation than NGF-maintained neurons (Fig. 2) is that NGF-deprived, BAF-saved cells should be more dependent on glycolysis for survival and more sensitive to glucose deprivation. To test this hypothesis, the effects of altering the concentration of glucose in the medium on NGF-maintained and NGF-deprived, BAF-saved cells on survival were determined. As schematized in Fig. 3 A, the latter was performed by first depriving cultures of NGF in the presence of BAF for 2 d, to generate a synchronized population of neurons that had released cytochrome c but had not committed to die. At this time, cells were switched from the standard medium, which contains 5 mM glucose, to a medium containing 0, 5, or 25 mM glucose for 8 d, after which the proportion that had committed-to-die was determined by readdition of a medium containing NGF and 5 mM glucose for 7 d. As seen in Fig. 3 B, survival in the presence of NGF was identical in a medium containing 0, 5, or 25 mM glucose medium. Because NGF-deprived, BAF-saved cells become committed-to-die during the course of the experiment, only roughly 40% of NGF-deprived, BAF-saved neurons incubated in 5 mM glucose after 10 d can be rescued by NGF (Chang and Johnson, 2002). In contrast, only 11% of NGF-deprived, BAF-saved cells maintained in a medium lacking glucose for the final 8 d of the 10-d period could be rescued. Thus, glucose deprivation decreases by roughly 75% the number of BAF-saved cells that are rescued by NGF. Increasing the glucose concentration to 25 mM did not increase the proportion of cells that could be rescued, suggesting that insufficient glucose in the standard culture medium does not underlie caspase-independent death of NGF-deprived sympathetic neurons. These data demonstrate that NGF-deprived, BAF-saved neurons were more sensitive to glucose deprivation than were NGF-maintained neurons, which were remarkably resistant to this insult.


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

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

NGF deprivation increases sensitivity to glucose deprivation. Cultures were deprived of NGF or maintained in NGF for 2 d in a standard medium, and then for an additional 8 d in a standard medium (con) or glucose-free medium supplemented with 0, 5, or 25 mM glucose as schematized in A. The cells were rescued with NGF for an additional 7 d and counted. Values shown in B are mean ± SD of three independent experiments performed in triplicate of the proportion of cells that could be rescued by NGF after this 10-d treatment. Asterisk indicates statistical significance of P < 0.05 compared with survival in a control medium.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: NGF deprivation increases sensitivity to glucose deprivation. Cultures were deprived of NGF or maintained in NGF for 2 d in a standard medium, and then for an additional 8 d in a standard medium (con) or glucose-free medium supplemented with 0, 5, or 25 mM glucose as schematized in A. The cells were rescued with NGF for an additional 7 d and counted. Values shown in B are mean ± SD of three independent experiments performed in triplicate of the proportion of cells that could be rescued by NGF after this 10-d treatment. Asterisk indicates statistical significance of P < 0.05 compared with survival in a control medium.
Mentions: One prediction from the finding that NGF-deprived, BAF-saved cells rely more heavily on glycolysis for ATP generation than NGF-maintained neurons (Fig. 2) is that NGF-deprived, BAF-saved cells should be more dependent on glycolysis for survival and more sensitive to glucose deprivation. To test this hypothesis, the effects of altering the concentration of glucose in the medium on NGF-maintained and NGF-deprived, BAF-saved cells on survival were determined. As schematized in Fig. 3 A, the latter was performed by first depriving cultures of NGF in the presence of BAF for 2 d, to generate a synchronized population of neurons that had released cytochrome c but had not committed to die. At this time, cells were switched from the standard medium, which contains 5 mM glucose, to a medium containing 0, 5, or 25 mM glucose for 8 d, after which the proportion that had committed-to-die was determined by readdition of a medium containing NGF and 5 mM glucose for 7 d. As seen in Fig. 3 B, survival in the presence of NGF was identical in a medium containing 0, 5, or 25 mM glucose medium. Because NGF-deprived, BAF-saved cells become committed-to-die during the course of the experiment, only roughly 40% of NGF-deprived, BAF-saved neurons incubated in 5 mM glucose after 10 d can be rescued by NGF (Chang and Johnson, 2002). In contrast, only 11% of NGF-deprived, BAF-saved cells maintained in a medium lacking glucose for the final 8 d of the 10-d period could be rescued. Thus, glucose deprivation decreases by roughly 75% the number of BAF-saved cells that are rescued by NGF. Increasing the glucose concentration to 25 mM did not increase the proportion of cells that could be rescued, suggesting that insufficient glucose in the standard culture medium does not underlie caspase-independent death of NGF-deprived sympathetic neurons. These data demonstrate that NGF-deprived, BAF-saved neurons were more sensitive to glucose deprivation than were NGF-maintained neurons, which were remarkably resistant to this insult.

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