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The impairment of HCCS leads to MLS syndrome by activating a non-canonical cell death pathway in the brain and eyes.

Indrieri A, Conte I, Chesi G, Romano A, Quartararo J, Tatè R, Ghezzi D, Zeviani M, Goffrini P, Ferrero I, Bovolenta P, Franco B - EMBO Mol Med (2013)

Bottom Line: Mitochondrial-dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development.By taking advantage of a medaka model that recapitulates the MLS phenotype we demonstrate that downregulation of hccs, an essential player of the mitochondrial respiratory chain (MRC), causes increased cell death via an apoptosome-independent caspase-9 activation in brain and eyes.We also show that the unconventional activation of caspase-9 occurs in the mitochondria and is triggered by MRC impairment and overproduction of reactive oxygen species (ROS).

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Affiliation: Telethon Institute of Genetics and Medicine, Naples, Italy.

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Overproduction of ROS triggers mitochondrial caspase-9 activation ultimately leading to the MLS phenotypeA. Determination of ROS levels in hccs-MO injected fish. Accumulation of the CM-H2DCFDA dye used as indicator of ROS levels, in hccs-MO and control-MO injected embryos at st24. Values represent means of 10 samples. Each sample represents a group of three embryos. Error bars are SEM.B–D. TUNEL assays on retinal sections of st30 embryos injected with control-MO (B), hccs-MO (C) and hccs-MO treated with NAC (D). Note how NAC treatment blocks cell death in morphant embryos. Scale bars: 20 µm.E. Caspase-9 activation in mitochondrial and cytoplasmic fractions of control and morphant embryos. Histograms show the fold changes in the levels of emitted luminescence signals. Values represent means of n samples. Each sample represents a group of 80 embryos. Caspase-9 activation was significantly increased in the mitochondrial fractions of morphants (n = 4, p = 0.01, one-tailed Student's t-test) whereas the differences in cytosolic fractions were smaller with lower statistical significance (n = 5, p = 0.1, one-tailed Student's t-test). Note how NAC treatment significantly blocks mitochondrial caspase-9 activation (n = 3, p = 0.04, one-tailed Student's t-test).F–H. Bright-field stereomicroscopy images of hccs-MO (F) and hccs-MO injected embryos at st38 treated with NAC (G, H). NAC treatment is able to improve microphthalmia (vertical dashed lines in G compared to F) and microcephaly (horizontal dashed lines in G compared to F) in 60% of morphant embryos (H). Scale bars: 100 µm.
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fig06: Overproduction of ROS triggers mitochondrial caspase-9 activation ultimately leading to the MLS phenotypeA. Determination of ROS levels in hccs-MO injected fish. Accumulation of the CM-H2DCFDA dye used as indicator of ROS levels, in hccs-MO and control-MO injected embryos at st24. Values represent means of 10 samples. Each sample represents a group of three embryos. Error bars are SEM.B–D. TUNEL assays on retinal sections of st30 embryos injected with control-MO (B), hccs-MO (C) and hccs-MO treated with NAC (D). Note how NAC treatment blocks cell death in morphant embryos. Scale bars: 20 µm.E. Caspase-9 activation in mitochondrial and cytoplasmic fractions of control and morphant embryos. Histograms show the fold changes in the levels of emitted luminescence signals. Values represent means of n samples. Each sample represents a group of 80 embryos. Caspase-9 activation was significantly increased in the mitochondrial fractions of morphants (n = 4, p = 0.01, one-tailed Student's t-test) whereas the differences in cytosolic fractions were smaller with lower statistical significance (n = 5, p = 0.1, one-tailed Student's t-test). Note how NAC treatment significantly blocks mitochondrial caspase-9 activation (n = 3, p = 0.04, one-tailed Student's t-test).F–H. Bright-field stereomicroscopy images of hccs-MO (F) and hccs-MO injected embryos at st38 treated with NAC (G, H). NAC treatment is able to improve microphthalmia (vertical dashed lines in G compared to F) and microcephaly (horizontal dashed lines in G compared to F) in 60% of morphant embryos (H). Scale bars: 100 µm.

Mentions: When the respiratory chain is inhibited downstream of complex III, electrons coming from succinate oxidation can lead to ROS generation by reverse electron transport from complex II to complex I (Lambert & Brand, 2004; St-Pierre et al, 2002). ROS production can also increase when electron transport is reduced as a consequence of low respiratory rates (Korshunov et al, 1997) or in pathological situations associated to MRC defects (Wallace, 2005). Accordingly, H2O2-induced growth inhibition was markedly increased in the B-8025-Δcyc3 strain defective for heme lyase function (Supporting Information Fig S8), as were ROS levels in hccs-morphants as determined by accumulation of oxidized CM-H2DCFDA (Fig 6A). Notably, in vitro experimental evidence suggested that ROS could directly mediate mitochondrial caspase-9 auto-activation, inducing cell death via an apoptosome-independent pathway (Katoh et al, 2008, 2004). We thus hypothesized that caspase-dependent cell death induced by hccs deficiency could be linked to MRC impairment and ROS overproduction. Indeed, detection of mitochondrial superoxide by MitoSOX staining revealed a specific increase of mitochondrial ROS levels in the CNS of alive morphants compared to controls (Supporting Information Fig S9).


The impairment of HCCS leads to MLS syndrome by activating a non-canonical cell death pathway in the brain and eyes.

Indrieri A, Conte I, Chesi G, Romano A, Quartararo J, Tatè R, Ghezzi D, Zeviani M, Goffrini P, Ferrero I, Bovolenta P, Franco B - EMBO Mol Med (2013)

Overproduction of ROS triggers mitochondrial caspase-9 activation ultimately leading to the MLS phenotypeA. Determination of ROS levels in hccs-MO injected fish. Accumulation of the CM-H2DCFDA dye used as indicator of ROS levels, in hccs-MO and control-MO injected embryos at st24. Values represent means of 10 samples. Each sample represents a group of three embryos. Error bars are SEM.B–D. TUNEL assays on retinal sections of st30 embryos injected with control-MO (B), hccs-MO (C) and hccs-MO treated with NAC (D). Note how NAC treatment blocks cell death in morphant embryos. Scale bars: 20 µm.E. Caspase-9 activation in mitochondrial and cytoplasmic fractions of control and morphant embryos. Histograms show the fold changes in the levels of emitted luminescence signals. Values represent means of n samples. Each sample represents a group of 80 embryos. Caspase-9 activation was significantly increased in the mitochondrial fractions of morphants (n = 4, p = 0.01, one-tailed Student's t-test) whereas the differences in cytosolic fractions were smaller with lower statistical significance (n = 5, p = 0.1, one-tailed Student's t-test). Note how NAC treatment significantly blocks mitochondrial caspase-9 activation (n = 3, p = 0.04, one-tailed Student's t-test).F–H. Bright-field stereomicroscopy images of hccs-MO (F) and hccs-MO injected embryos at st38 treated with NAC (G, H). NAC treatment is able to improve microphthalmia (vertical dashed lines in G compared to F) and microcephaly (horizontal dashed lines in G compared to F) in 60% of morphant embryos (H). Scale bars: 100 µm.
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fig06: Overproduction of ROS triggers mitochondrial caspase-9 activation ultimately leading to the MLS phenotypeA. Determination of ROS levels in hccs-MO injected fish. Accumulation of the CM-H2DCFDA dye used as indicator of ROS levels, in hccs-MO and control-MO injected embryos at st24. Values represent means of 10 samples. Each sample represents a group of three embryos. Error bars are SEM.B–D. TUNEL assays on retinal sections of st30 embryos injected with control-MO (B), hccs-MO (C) and hccs-MO treated with NAC (D). Note how NAC treatment blocks cell death in morphant embryos. Scale bars: 20 µm.E. Caspase-9 activation in mitochondrial and cytoplasmic fractions of control and morphant embryos. Histograms show the fold changes in the levels of emitted luminescence signals. Values represent means of n samples. Each sample represents a group of 80 embryos. Caspase-9 activation was significantly increased in the mitochondrial fractions of morphants (n = 4, p = 0.01, one-tailed Student's t-test) whereas the differences in cytosolic fractions were smaller with lower statistical significance (n = 5, p = 0.1, one-tailed Student's t-test). Note how NAC treatment significantly blocks mitochondrial caspase-9 activation (n = 3, p = 0.04, one-tailed Student's t-test).F–H. Bright-field stereomicroscopy images of hccs-MO (F) and hccs-MO injected embryos at st38 treated with NAC (G, H). NAC treatment is able to improve microphthalmia (vertical dashed lines in G compared to F) and microcephaly (horizontal dashed lines in G compared to F) in 60% of morphant embryos (H). Scale bars: 100 µm.
Mentions: When the respiratory chain is inhibited downstream of complex III, electrons coming from succinate oxidation can lead to ROS generation by reverse electron transport from complex II to complex I (Lambert & Brand, 2004; St-Pierre et al, 2002). ROS production can also increase when electron transport is reduced as a consequence of low respiratory rates (Korshunov et al, 1997) or in pathological situations associated to MRC defects (Wallace, 2005). Accordingly, H2O2-induced growth inhibition was markedly increased in the B-8025-Δcyc3 strain defective for heme lyase function (Supporting Information Fig S8), as were ROS levels in hccs-morphants as determined by accumulation of oxidized CM-H2DCFDA (Fig 6A). Notably, in vitro experimental evidence suggested that ROS could directly mediate mitochondrial caspase-9 auto-activation, inducing cell death via an apoptosome-independent pathway (Katoh et al, 2008, 2004). We thus hypothesized that caspase-dependent cell death induced by hccs deficiency could be linked to MRC impairment and ROS overproduction. Indeed, detection of mitochondrial superoxide by MitoSOX staining revealed a specific increase of mitochondrial ROS levels in the CNS of alive morphants compared to controls (Supporting Information Fig S9).

Bottom Line: Mitochondrial-dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development.By taking advantage of a medaka model that recapitulates the MLS phenotype we demonstrate that downregulation of hccs, an essential player of the mitochondrial respiratory chain (MRC), causes increased cell death via an apoptosome-independent caspase-9 activation in brain and eyes.We also show that the unconventional activation of caspase-9 occurs in the mitochondria and is triggered by MRC impairment and overproduction of reactive oxygen species (ROS).

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute of Genetics and Medicine, Naples, Italy.

Show MeSH
Related in: MedlinePlus