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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

CsA attenuates NGF deprivation–induced loss of mitochondrial proteins. Mitochondrial proteins in NGF deprivation in the presence of BAF or BAF + 10 μM CsA were examined by Western blot. Blots were simultaneously probed with antibodies directed against tubulin, VDAC, and cytoCOX IV. (B) Levels of VDAC and COX IV after 9 d of NGF deprivation were normalized to the amount of tubulin in each sample. Data shown are mean ± SD of three independent experiments. Asterisks denote that differences between BAF and BAF + CsA are statistically significant (P < 0.05).
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fig6: CsA attenuates NGF deprivation–induced loss of mitochondrial proteins. Mitochondrial proteins in NGF deprivation in the presence of BAF or BAF + 10 μM CsA were examined by Western blot. Blots were simultaneously probed with antibodies directed against tubulin, VDAC, and cytoCOX IV. (B) Levels of VDAC and COX IV after 9 d of NGF deprivation were normalized to the amount of tubulin in each sample. Data shown are mean ± SD of three independent experiments. Asterisks denote that differences between BAF and BAF + CsA are statistically significant (P < 0.05).

Mentions: As seen in Fig. 2, CsA did not preserve the ability of mitochondria in NGF-deprived, BAF-saved neurons to generate ATP. Therefore, to investigate alternate mechanisms by which PTP inhibition by CsA might block Commitment 2, we asked whether CsA attenuated the loss of mitochondria observed in caspase inhibitor–saved neurons that may correlate with commitment-to-die (Xue et al., 2001). Western blots (Fig. 6 A) demonstrate that the levels of two mitochondrial proteins, voltage-dependent anion channel (VDAC) and cytochrome oxidase subunit IV (COX IV) decreased over time in NGF-deprived, BAF-saved cells, but less dramatically in cells also treated with CsA. When normalized to tubulin levels as a loading control, cultures in the presence of CsA had nearly twice the amount of VDAC and COX IV of cultures treated with BAF alone (Fig. 6 B). These findings demonstrate that CsA prevents the loss of mitochondrial proteins that occurs in NGF-deprived, BAF-saved neurons.


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

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

CsA attenuates NGF deprivation–induced loss of mitochondrial proteins. Mitochondrial proteins in NGF deprivation in the presence of BAF or BAF + 10 μM CsA were examined by Western blot. Blots were simultaneously probed with antibodies directed against tubulin, VDAC, and cytoCOX IV. (B) Levels of VDAC and COX IV after 9 d of NGF deprivation were normalized to the amount of tubulin in each sample. Data shown are mean ± SD of three independent experiments. Asterisks denote that differences between BAF and BAF + CsA are statistically significant (P < 0.05).
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Related In: Results  -  Collection

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

fig6: CsA attenuates NGF deprivation–induced loss of mitochondrial proteins. Mitochondrial proteins in NGF deprivation in the presence of BAF or BAF + 10 μM CsA were examined by Western blot. Blots were simultaneously probed with antibodies directed against tubulin, VDAC, and cytoCOX IV. (B) Levels of VDAC and COX IV after 9 d of NGF deprivation were normalized to the amount of tubulin in each sample. Data shown are mean ± SD of three independent experiments. Asterisks denote that differences between BAF and BAF + CsA are statistically significant (P < 0.05).
Mentions: As seen in Fig. 2, CsA did not preserve the ability of mitochondria in NGF-deprived, BAF-saved neurons to generate ATP. Therefore, to investigate alternate mechanisms by which PTP inhibition by CsA might block Commitment 2, we asked whether CsA attenuated the loss of mitochondria observed in caspase inhibitor–saved neurons that may correlate with commitment-to-die (Xue et al., 2001). Western blots (Fig. 6 A) demonstrate that the levels of two mitochondrial proteins, voltage-dependent anion channel (VDAC) and cytochrome oxidase subunit IV (COX IV) decreased over time in NGF-deprived, BAF-saved cells, but less dramatically in cells also treated with CsA. When normalized to tubulin levels as a loading control, cultures in the presence of CsA had nearly twice the amount of VDAC and COX IV of cultures treated with BAF alone (Fig. 6 B). These findings demonstrate that CsA prevents the loss of mitochondrial proteins that occurs in NGF-deprived, BAF-saved 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