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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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CsA alters ultrastructure of NGF- deprived, BAF-saved cells. Electron micrographs of neurons that were maintained in NGF (A and B), deprived of NGF in the presence of BAF (C and D), or deprived of NGF in the presence of BAF and CsA (E and F) for 10 d. (A) This representative NGF-maintained neuron has a large, round nucleus (N) that contains a nucleolus. (B) At a higher magnification, abundant mitochondria (m) and occasional electron-dense, membrane-limited autophagic vesicles (white arrow) can be seen. (C) NGF- deprived, BAF-saved cells display decreased cytoplasmic volume, nuclear irregularity, and chromatin margination, but retain a prominent nucleolus. Note the numerous electron-dense lipid droplets throughout the cytosol. (D) At higher magnification, lipid droplets (black arrows) lack limiting membranes and internal structure, whereas late autophagosomes (white arrow) are membrane limited. (E) CsA-treated neurons appear similar to NGF-deprived, BAF-saved cells. Note the absence of lipid droplets and the abundant multilamellar structures (black arrowheads), which are likely to be autophagosomes. (F) At higher magnification, the multilamellar structure of these autophagic vesicles (black arrowheads) is evident. Some of these vesicles contain electron-dense material (white arrowheads). Normal mitochondria (m) can be seen in this section. Boxes in low power views in left column indicate the area of magnification in high power views shown in the right column. Bars: 1 μm; bar in B also applies to D and F.
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fig7: CsA alters ultrastructure of NGF- deprived, BAF-saved cells. Electron micrographs of neurons that were maintained in NGF (A and B), deprived of NGF in the presence of BAF (C and D), or deprived of NGF in the presence of BAF and CsA (E and F) for 10 d. (A) This representative NGF-maintained neuron has a large, round nucleus (N) that contains a nucleolus. (B) At a higher magnification, abundant mitochondria (m) and occasional electron-dense, membrane-limited autophagic vesicles (white arrow) can be seen. (C) NGF- deprived, BAF-saved cells display decreased cytoplasmic volume, nuclear irregularity, and chromatin margination, but retain a prominent nucleolus. Note the numerous electron-dense lipid droplets throughout the cytosol. (D) At higher magnification, lipid droplets (black arrows) lack limiting membranes and internal structure, whereas late autophagosomes (white arrow) are membrane limited. (E) CsA-treated neurons appear similar to NGF-deprived, BAF-saved cells. Note the absence of lipid droplets and the abundant multilamellar structures (black arrowheads), which are likely to be autophagosomes. (F) At higher magnification, the multilamellar structure of these autophagic vesicles (black arrowheads) is evident. Some of these vesicles contain electron-dense material (white arrowheads). Normal mitochondria (m) can be seen in this section. Boxes in low power views in left column indicate the area of magnification in high power views shown in the right column. Bars: 1 μm; bar in B also applies to D and F.

Mentions: Control neurons maintained in NGF (Fig. 7 A) had large, round nuclei (N) with prominent nucleoli. The cytosol contained many mitochondria (Fig. 7 B, m) and occasional electron-dense, membrane-limited late autophagic vesicles. Neurons deprived of NGF in the presence of BAF for 10 d displayed extensive atrophy (Fig. 7 C), as expected from their light microscopic appearance. The most prominent change was the appearance of numerous electron-dense bodies in the cytosol (Fig. 7 D, black arrows), which may represent lipid droplets (Martin et al., 1988) or autolysosomes that have engulfed a large amount of lipid membranes or other electron-dense material (Xue et al., 1999). Similar structures were also occasionally seen in NGF-maintained neurons, although these were often smaller and had limiting membranes (Fig. 7 B, white arrows). These structures were present at a much greater frequency in sections of NGF-deprived, BAF-saved neurons (0.51 ± 0.08 per μm2 of cytosol, ± SEM, in 11 sections from different neurons), than in NGF-maintained neurons (0.07 ± 0.04 per μm2, n = 6). Cells deprived of NGF in the presence of BAF and CsA also displayed cytoplasmic atrophy and convolution of the nuclear membrane (Fig. 7 E). However, two key differences were observed between the appearance of these cells and those treated with BAF alone. First, these electron-dense bodies were much less abundant in cells treated with BAF and CsA (0.15 ± 0.04 per μm2, n = 11) than in neurons saved with BAF only (Fig. 7 E). Second, abundant multilamellar vesicles were present throughout the cytosol of these cells (Fig. 7, E and F, black arrowheads). These multilamellar vesicles were abundant throughout multiple sections of NGF-deprived neurons treated with BAF and CsA (0.59 ± 0.11 per μm2, n = 11), but rarely present in NGF-deprived, BAF-saved cells (0.09 ± 0.03 per μm2, n = 11) and completely absent in NGF-maintained neurons (n = 6). Although the precise nature of these structures is uncertain, they resemble autolysosomes seen in NGF-deprived, caspase inhibitor–saved neurons (Xue et al., 1999). When normalized to surface area, NGF-deprived, BAF-saved cells with (0.22 ± 0.08, ± SEM) or without CsA treatment (0.17 ± 0.08) had slightly fewer mitochondria than NGF-maintained neurons (0.33 ± 0.09), but this difference was not statistically significant. In NGF-deprived, BAF-saved cells, there was a trend toward more mitochondria in CsA-treated cells, but this difference was not statistically significant.


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

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

CsA alters ultrastructure of NGF- deprived, BAF-saved cells. Electron micrographs of neurons that were maintained in NGF (A and B), deprived of NGF in the presence of BAF (C and D), or deprived of NGF in the presence of BAF and CsA (E and F) for 10 d. (A) This representative NGF-maintained neuron has a large, round nucleus (N) that contains a nucleolus. (B) At a higher magnification, abundant mitochondria (m) and occasional electron-dense, membrane-limited autophagic vesicles (white arrow) can be seen. (C) NGF- deprived, BAF-saved cells display decreased cytoplasmic volume, nuclear irregularity, and chromatin margination, but retain a prominent nucleolus. Note the numerous electron-dense lipid droplets throughout the cytosol. (D) At higher magnification, lipid droplets (black arrows) lack limiting membranes and internal structure, whereas late autophagosomes (white arrow) are membrane limited. (E) CsA-treated neurons appear similar to NGF-deprived, BAF-saved cells. Note the absence of lipid droplets and the abundant multilamellar structures (black arrowheads), which are likely to be autophagosomes. (F) At higher magnification, the multilamellar structure of these autophagic vesicles (black arrowheads) is evident. Some of these vesicles contain electron-dense material (white arrowheads). Normal mitochondria (m) can be seen in this section. Boxes in low power views in left column indicate the area of magnification in high power views shown in the right column. Bars: 1 μm; bar in B also applies to D and F.
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fig7: CsA alters ultrastructure of NGF- deprived, BAF-saved cells. Electron micrographs of neurons that were maintained in NGF (A and B), deprived of NGF in the presence of BAF (C and D), or deprived of NGF in the presence of BAF and CsA (E and F) for 10 d. (A) This representative NGF-maintained neuron has a large, round nucleus (N) that contains a nucleolus. (B) At a higher magnification, abundant mitochondria (m) and occasional electron-dense, membrane-limited autophagic vesicles (white arrow) can be seen. (C) NGF- deprived, BAF-saved cells display decreased cytoplasmic volume, nuclear irregularity, and chromatin margination, but retain a prominent nucleolus. Note the numerous electron-dense lipid droplets throughout the cytosol. (D) At higher magnification, lipid droplets (black arrows) lack limiting membranes and internal structure, whereas late autophagosomes (white arrow) are membrane limited. (E) CsA-treated neurons appear similar to NGF-deprived, BAF-saved cells. Note the absence of lipid droplets and the abundant multilamellar structures (black arrowheads), which are likely to be autophagosomes. (F) At higher magnification, the multilamellar structure of these autophagic vesicles (black arrowheads) is evident. Some of these vesicles contain electron-dense material (white arrowheads). Normal mitochondria (m) can be seen in this section. Boxes in low power views in left column indicate the area of magnification in high power views shown in the right column. Bars: 1 μm; bar in B also applies to D and F.
Mentions: Control neurons maintained in NGF (Fig. 7 A) had large, round nuclei (N) with prominent nucleoli. The cytosol contained many mitochondria (Fig. 7 B, m) and occasional electron-dense, membrane-limited late autophagic vesicles. Neurons deprived of NGF in the presence of BAF for 10 d displayed extensive atrophy (Fig. 7 C), as expected from their light microscopic appearance. The most prominent change was the appearance of numerous electron-dense bodies in the cytosol (Fig. 7 D, black arrows), which may represent lipid droplets (Martin et al., 1988) or autolysosomes that have engulfed a large amount of lipid membranes or other electron-dense material (Xue et al., 1999). Similar structures were also occasionally seen in NGF-maintained neurons, although these were often smaller and had limiting membranes (Fig. 7 B, white arrows). These structures were present at a much greater frequency in sections of NGF-deprived, BAF-saved neurons (0.51 ± 0.08 per μm2 of cytosol, ± SEM, in 11 sections from different neurons), than in NGF-maintained neurons (0.07 ± 0.04 per μm2, n = 6). Cells deprived of NGF in the presence of BAF and CsA also displayed cytoplasmic atrophy and convolution of the nuclear membrane (Fig. 7 E). However, two key differences were observed between the appearance of these cells and those treated with BAF alone. First, these electron-dense bodies were much less abundant in cells treated with BAF and CsA (0.15 ± 0.04 per μm2, n = 11) than in neurons saved with BAF only (Fig. 7 E). Second, abundant multilamellar vesicles were present throughout the cytosol of these cells (Fig. 7, E and F, black arrowheads). These multilamellar vesicles were abundant throughout multiple sections of NGF-deprived neurons treated with BAF and CsA (0.59 ± 0.11 per μm2, n = 11), but rarely present in NGF-deprived, BAF-saved cells (0.09 ± 0.03 per μm2, n = 11) and completely absent in NGF-maintained neurons (n = 6). Although the precise nature of these structures is uncertain, they resemble autolysosomes seen in NGF-deprived, caspase inhibitor–saved neurons (Xue et al., 1999). When normalized to surface area, NGF-deprived, BAF-saved cells with (0.22 ± 0.08, ± SEM) or without CsA treatment (0.17 ± 0.08) had slightly fewer mitochondria than NGF-maintained neurons (0.33 ± 0.09), but this difference was not statistically significant. In NGF-deprived, BAF-saved cells, there was a trend toward more mitochondria in CsA-treated cells, but this difference was not statistically significant.

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