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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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Caspase-9 dependent cell death underlies microphthalmia in hccs-deficient embryosA–D. TUNEL assays on retinal sections. Embryos injected with control-MO (A, B) and with hccs-MO (C, D) at st24 and st30.E, F. Immunofluorescence analysis with an anti-active-caspase-3 antibody on control-MO (E) and hccs-MO injected embryos (F) at st38.G. Number of TUNEL positive cells/eye (n ≥ 5 embryos/stage; Error bars are SEM; p-values were calculated by ANOVA).H. Caspase activation in control and morphant embryos measured by cleavage of a synthetic substrate and release of luciferin (DEVD-aminoluciferin for caspase-3, LETD-aminoluciferin for caspase-8, and LEHD-aminoluciferin for caspase-9). Histograms show the relative levels of emitted signals displayed in arbitrary units of luminescence. Values represent means of four samples. Each sample represents a group of 20 embryos (error bars are SEM; p-values were calculated by unpaired, two-tailed Student's t-test).I–M. TUNEL assays on st38 embryos injected with control-MO (I) and hccs-MO alone (J), in association with the pan-caspase inhibitor (ZVAD) (K) or with the caspase-9 inhibitor (L), or with the caspase-1 inhibitor (M) (n = 100 embryos for each treatment). The pan-caspase inhibitor and the specific caspase-9 inhibitor rescue the increased levels of cell death (G, K, L). Scale bars: 20 µm.
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fig04: Caspase-9 dependent cell death underlies microphthalmia in hccs-deficient embryosA–D. TUNEL assays on retinal sections. Embryos injected with control-MO (A, B) and with hccs-MO (C, D) at st24 and st30.E, F. Immunofluorescence analysis with an anti-active-caspase-3 antibody on control-MO (E) and hccs-MO injected embryos (F) at st38.G. Number of TUNEL positive cells/eye (n ≥ 5 embryos/stage; Error bars are SEM; p-values were calculated by ANOVA).H. Caspase activation in control and morphant embryos measured by cleavage of a synthetic substrate and release of luciferin (DEVD-aminoluciferin for caspase-3, LETD-aminoluciferin for caspase-8, and LEHD-aminoluciferin for caspase-9). Histograms show the relative levels of emitted signals displayed in arbitrary units of luminescence. Values represent means of four samples. Each sample represents a group of 20 embryos (error bars are SEM; p-values were calculated by unpaired, two-tailed Student's t-test).I–M. TUNEL assays on st38 embryos injected with control-MO (I) and hccs-MO alone (J), in association with the pan-caspase inhibitor (ZVAD) (K) or with the caspase-9 inhibitor (L), or with the caspase-1 inhibitor (M) (n = 100 embryos for each treatment). The pan-caspase inhibitor and the specific caspase-9 inhibitor rescue the increased levels of cell death (G, K, L). Scale bars: 20 µm.

Mentions: The spatio-temporal distribution of developmental cell death has been well characterized in medaka CNS (Iijima & Yokoyama, 2007). In good agreement with this report, in the retina of control embryos we detected the highest peak of TUNEL-positive cells at st24, whereas only occasional apoptotic cells were detected thereafter (Fig 4A and G). In contrast, the retinas of hccs-morphants not only displayed a significant increase in the number of TUNEL-positive cells at st24, but were also characterized by a sustained apoptosis at later stages of development coinciding with the onset of a visible microphthalmia (Fig 4A–D and G).


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)

Caspase-9 dependent cell death underlies microphthalmia in hccs-deficient embryosA–D. TUNEL assays on retinal sections. Embryos injected with control-MO (A, B) and with hccs-MO (C, D) at st24 and st30.E, F. Immunofluorescence analysis with an anti-active-caspase-3 antibody on control-MO (E) and hccs-MO injected embryos (F) at st38.G. Number of TUNEL positive cells/eye (n ≥ 5 embryos/stage; Error bars are SEM; p-values were calculated by ANOVA).H. Caspase activation in control and morphant embryos measured by cleavage of a synthetic substrate and release of luciferin (DEVD-aminoluciferin for caspase-3, LETD-aminoluciferin for caspase-8, and LEHD-aminoluciferin for caspase-9). Histograms show the relative levels of emitted signals displayed in arbitrary units of luminescence. Values represent means of four samples. Each sample represents a group of 20 embryos (error bars are SEM; p-values were calculated by unpaired, two-tailed Student's t-test).I–M. TUNEL assays on st38 embryos injected with control-MO (I) and hccs-MO alone (J), in association with the pan-caspase inhibitor (ZVAD) (K) or with the caspase-9 inhibitor (L), or with the caspase-1 inhibitor (M) (n = 100 embryos for each treatment). The pan-caspase inhibitor and the specific caspase-9 inhibitor rescue the increased levels of cell death (G, K, L). Scale bars: 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig04: Caspase-9 dependent cell death underlies microphthalmia in hccs-deficient embryosA–D. TUNEL assays on retinal sections. Embryos injected with control-MO (A, B) and with hccs-MO (C, D) at st24 and st30.E, F. Immunofluorescence analysis with an anti-active-caspase-3 antibody on control-MO (E) and hccs-MO injected embryos (F) at st38.G. Number of TUNEL positive cells/eye (n ≥ 5 embryos/stage; Error bars are SEM; p-values were calculated by ANOVA).H. Caspase activation in control and morphant embryos measured by cleavage of a synthetic substrate and release of luciferin (DEVD-aminoluciferin for caspase-3, LETD-aminoluciferin for caspase-8, and LEHD-aminoluciferin for caspase-9). Histograms show the relative levels of emitted signals displayed in arbitrary units of luminescence. Values represent means of four samples. Each sample represents a group of 20 embryos (error bars are SEM; p-values were calculated by unpaired, two-tailed Student's t-test).I–M. TUNEL assays on st38 embryos injected with control-MO (I) and hccs-MO alone (J), in association with the pan-caspase inhibitor (ZVAD) (K) or with the caspase-9 inhibitor (L), or with the caspase-1 inhibitor (M) (n = 100 embryos for each treatment). The pan-caspase inhibitor and the specific caspase-9 inhibitor rescue the increased levels of cell death (G, K, L). Scale bars: 20 µm.
Mentions: The spatio-temporal distribution of developmental cell death has been well characterized in medaka CNS (Iijima & Yokoyama, 2007). In good agreement with this report, in the retina of control embryos we detected the highest peak of TUNEL-positive cells at st24, whereas only occasional apoptotic cells were detected thereafter (Fig 4A and G). In contrast, the retinas of hccs-morphants not only displayed a significant increase in the number of TUNEL-positive cells at st24, but were also characterized by a sustained apoptosis at later stages of development coinciding with the onset of a visible microphthalmia (Fig 4A–D and G).

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