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Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

Schüll S, Günther SD, Brodesser S, Seeger JM, Tosetti B, Wiegmann K, Pongratz C, Diaz F, Witt A, Andree M, Brinkmann K, Krönke M, Wiesner RJ, Kashkar H - Cell Death Dis (2015)

Bottom Line: Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown.The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress.Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne (CMMC) and Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), University of Cologne, Cologne, Germany.

ABSTRACT
Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

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COX deficiency enhances susceptibility to H2O2-induced cell death. (a) List of utilized chemical inhibitors of the mitochondrial RC. Inhibition of individual RC complexes in HeLa cells was quantified by a reduction in oxygen consumption (left panel). Cytotoxicity was quantified by trypan-blue exclusion after 48 h (right panel). (b) Analysis of cell death by trypan-blue exclusion in HeLa cells treated for 24 h with individual RC complex inhibitors as indicated and subjected to H2O2 for further 24 h. (c) Analysis of cell death by trypan-blue exclusion in 143BΔCOX cybrid cells and COX10−/− fibroblasts subjected to H2O2 for 20 h. The error bars in panel a and c represent the mean±S.D. (n=3), the error bars in panel b represent the mean±S.D. (n=6), two-tailed unpaired t-test. *P <0.05, ***P<0.001
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fig1: COX deficiency enhances susceptibility to H2O2-induced cell death. (a) List of utilized chemical inhibitors of the mitochondrial RC. Inhibition of individual RC complexes in HeLa cells was quantified by a reduction in oxygen consumption (left panel). Cytotoxicity was quantified by trypan-blue exclusion after 48 h (right panel). (b) Analysis of cell death by trypan-blue exclusion in HeLa cells treated for 24 h with individual RC complex inhibitors as indicated and subjected to H2O2 for further 24 h. (c) Analysis of cell death by trypan-blue exclusion in 143BΔCOX cybrid cells and COX10−/− fibroblasts subjected to H2O2 for 20 h. The error bars in panel a and c represent the mean±S.D. (n=3), the error bars in panel b represent the mean±S.D. (n=6), two-tailed unpaired t-test. *P <0.05, ***P<0.001

Mentions: We first investigated whether the alteration of mitochondrial respiration in general or the specific inhibition of one of the mitochondrial respiratory chain (RC) complexes directly impacts on cellular viability. Different RC complexes were chemically inhibited in HeLa cells and oxygen consumption was monitored to examine the efficacy of the inhibition (Figure 1a). Although efficiently reducing oxygen consumption, none of the tested chemical inhibitors of RC exhibited significant cytotoxic activity within 48 h, suggesting that the inhibition of RC per se does not directly activate the cellular death program in highly glycolytic HeLa cells.9 The elevated occurrence of apoptosis within a tissue upon mitochondrial dysfunction may additionally arise from an increased susceptibility of cells toward environmental cues such as oxidative stress. Indeed, physiologic levels of reactive oxygen species (ROS) are able to promote oxidative stress and cause lethality when cellular integrity or fitness is altered, for example, upon aging.10, 11 Therefore, we examined whether RC inhibition impacts on cellular sensitivity to ROS. Accordingly, HeLa cells were first exposed to different inhibitors of RC complexes (24 h) and subsequently treated with H2O2 (24 h). In striking contrast to complex I, II, III and V, the inhibition of the respiratory complex IV (COX) by KCN markedly increased the susceptibility of cells toward H2O2 treatment (Figure 1b and Supplementary Figure 1).


Cytochrome c oxidase deficiency accelerates mitochondrial apoptosis by activating ceramide synthase 6.

Schüll S, Günther SD, Brodesser S, Seeger JM, Tosetti B, Wiegmann K, Pongratz C, Diaz F, Witt A, Andree M, Brinkmann K, Krönke M, Wiesner RJ, Kashkar H - Cell Death Dis (2015)

COX deficiency enhances susceptibility to H2O2-induced cell death. (a) List of utilized chemical inhibitors of the mitochondrial RC. Inhibition of individual RC complexes in HeLa cells was quantified by a reduction in oxygen consumption (left panel). Cytotoxicity was quantified by trypan-blue exclusion after 48 h (right panel). (b) Analysis of cell death by trypan-blue exclusion in HeLa cells treated for 24 h with individual RC complex inhibitors as indicated and subjected to H2O2 for further 24 h. (c) Analysis of cell death by trypan-blue exclusion in 143BΔCOX cybrid cells and COX10−/− fibroblasts subjected to H2O2 for 20 h. The error bars in panel a and c represent the mean±S.D. (n=3), the error bars in panel b represent the mean±S.D. (n=6), two-tailed unpaired t-test. *P <0.05, ***P<0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385940&req=5

fig1: COX deficiency enhances susceptibility to H2O2-induced cell death. (a) List of utilized chemical inhibitors of the mitochondrial RC. Inhibition of individual RC complexes in HeLa cells was quantified by a reduction in oxygen consumption (left panel). Cytotoxicity was quantified by trypan-blue exclusion after 48 h (right panel). (b) Analysis of cell death by trypan-blue exclusion in HeLa cells treated for 24 h with individual RC complex inhibitors as indicated and subjected to H2O2 for further 24 h. (c) Analysis of cell death by trypan-blue exclusion in 143BΔCOX cybrid cells and COX10−/− fibroblasts subjected to H2O2 for 20 h. The error bars in panel a and c represent the mean±S.D. (n=3), the error bars in panel b represent the mean±S.D. (n=6), two-tailed unpaired t-test. *P <0.05, ***P<0.001
Mentions: We first investigated whether the alteration of mitochondrial respiration in general or the specific inhibition of one of the mitochondrial respiratory chain (RC) complexes directly impacts on cellular viability. Different RC complexes were chemically inhibited in HeLa cells and oxygen consumption was monitored to examine the efficacy of the inhibition (Figure 1a). Although efficiently reducing oxygen consumption, none of the tested chemical inhibitors of RC exhibited significant cytotoxic activity within 48 h, suggesting that the inhibition of RC per se does not directly activate the cellular death program in highly glycolytic HeLa cells.9 The elevated occurrence of apoptosis within a tissue upon mitochondrial dysfunction may additionally arise from an increased susceptibility of cells toward environmental cues such as oxidative stress. Indeed, physiologic levels of reactive oxygen species (ROS) are able to promote oxidative stress and cause lethality when cellular integrity or fitness is altered, for example, upon aging.10, 11 Therefore, we examined whether RC inhibition impacts on cellular sensitivity to ROS. Accordingly, HeLa cells were first exposed to different inhibitors of RC complexes (24 h) and subsequently treated with H2O2 (24 h). In striking contrast to complex I, II, III and V, the inhibition of the respiratory complex IV (COX) by KCN markedly increased the susceptibility of cells toward H2O2 treatment (Figure 1b and Supplementary Figure 1).

Bottom Line: Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown.The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress.Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Medicine Cologne (CMMC) and Institute for Medical Microbiology, Immunology and Hygiene (IMMIH), University of Cologne, Cologne, Germany.

ABSTRACT
Although numerous pathogenic changes within the mitochondrial respiratory chain (RC) have been associated with an elevated occurrence of apoptosis within the affected tissues, the mechanistic insight into how mitochondrial dysfunction initiates apoptotic cell death is still unknown. In this study, we show that the specific alteration of the cytochrome c oxidase (COX), representing a common defect found in mitochondrial diseases, facilitates mitochondrial apoptosis in response to oxidative stress. Our data identified an increased ceramide synthase 6 (CerS6) activity as an important pro-apoptotic response to COX dysfunction induced either by chemical or genetic approaches. The elevated CerS6 activity resulted in accumulation of the pro-apoptotic C16 : 0 ceramide, which facilitates the mitochondrial apoptosis in response to oxidative stress. Accordingly, inhibition of CerS6 or its specific knockdown diminished the increased susceptibility of COX-deficient cells to oxidative stress. Our results provide new insights into how mitochondrial RC dysfunction mechanistically interferes with the apoptotic machinery. On the basis of its pivotal role in regulating cell death upon COX dysfunction, CerS6 might potentially represent a novel target for therapeutic intervention in mitochondrial diseases caused by COX dysfunction.

Show MeSH
Related in: MedlinePlus