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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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Related in: MedlinePlus

NGF-deprived, BAF-saved cells maintain high levels of ATP. (A) ATP was measured in NGF-maintained and -deprived, BAF-saved sister cultures at the times indicated on the x axis by a luciferase-based assay. (B) ATP levels after 3 d of treatment were normalized to the total amount of protein in each sample. Values were normalized to the time 0 value for each experiment and represent mean ± SD from three independent experiments performed in quadruplicate.
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fig1: NGF-deprived, BAF-saved cells maintain high levels of ATP. (A) ATP was measured in NGF-maintained and -deprived, BAF-saved sister cultures at the times indicated on the x axis by a luciferase-based assay. (B) ATP levels after 3 d of treatment were normalized to the total amount of protein in each sample. Values were normalized to the time 0 value for each experiment and represent mean ± SD from three independent experiments performed in quadruplicate.

Mentions: NGF-deprived sympathetic neurons that are prevented from completing apoptosis by caspase inhibition exist in a depressed metabolic state, as determined by a number of morphological and biochemical parameters (Deshmukh et al., 1996; Chang and Johnson, 2002). To determine the effect of caspase inhibition on the energetic status of neurons, we measured total cellular ATP by the luciferase/luciferin method in sympathetic neuronal cultures that were either maintained in NGF or deprived of NGF in the presence of BAF. As seen in Fig. 1 A, the total amount of ATP increased in NGF-maintained cultures over 9 d, whereas it decreased in NGF-deprived, BAF-saved cells. Most of the decrease in ATP in NGF-deprived cells occurred within the first 3 d, because total ATP was 79% of that of cells at the time of deprivation after 3 d and 65% after 9 d. However, cells maintained in NGF increased in size during this time, whereas NGF-deprived, BAF-saved neurons atrophied and had a decreased rate of protein synthesis (Deshmukh et al., 1996). When normalized to total protein of each sample, NGF-deprived, BAF-saved cells actually had higher levels of ATP than NGF-maintained neurons (Fig. 1 B). Total cellular ATP is only one measure of overall energy balance. However, maintenance of relatively normal amounts of intracellular ATP suggests ongoing metabolism in NGF-deprived, BAF-saved cells consistent with their ability to survive long periods of NGF deprivation (Chang and Johnson, 2002).


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 maintain high levels of ATP. (A) ATP was measured in NGF-maintained and -deprived, BAF-saved sister cultures at the times indicated on the x axis by a luciferase-based assay. (B) ATP levels after 3 d of treatment were normalized to the total amount of protein in each sample. Values were normalized to the time 0 value for each experiment and represent mean ± SD from three independent experiments performed in quadruplicate.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: NGF-deprived, BAF-saved cells maintain high levels of ATP. (A) ATP was measured in NGF-maintained and -deprived, BAF-saved sister cultures at the times indicated on the x axis by a luciferase-based assay. (B) ATP levels after 3 d of treatment were normalized to the total amount of protein in each sample. Values were normalized to the time 0 value for each experiment and represent mean ± SD from three independent experiments performed in quadruplicate.
Mentions: NGF-deprived sympathetic neurons that are prevented from completing apoptosis by caspase inhibition exist in a depressed metabolic state, as determined by a number of morphological and biochemical parameters (Deshmukh et al., 1996; Chang and Johnson, 2002). To determine the effect of caspase inhibition on the energetic status of neurons, we measured total cellular ATP by the luciferase/luciferin method in sympathetic neuronal cultures that were either maintained in NGF or deprived of NGF in the presence of BAF. As seen in Fig. 1 A, the total amount of ATP increased in NGF-maintained cultures over 9 d, whereas it decreased in NGF-deprived, BAF-saved cells. Most of the decrease in ATP in NGF-deprived cells occurred within the first 3 d, because total ATP was 79% of that of cells at the time of deprivation after 3 d and 65% after 9 d. However, cells maintained in NGF increased in size during this time, whereas NGF-deprived, BAF-saved neurons atrophied and had a decreased rate of protein synthesis (Deshmukh et al., 1996). When normalized to total protein of each sample, NGF-deprived, BAF-saved cells actually had higher levels of ATP than NGF-maintained neurons (Fig. 1 B). Total cellular ATP is only one measure of overall energy balance. However, maintenance of relatively normal amounts of intracellular ATP suggests ongoing metabolism in NGF-deprived, BAF-saved cells consistent with their ability to survive long periods of NGF deprivation (Chang and Johnson, 2002).

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