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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).

View Article: PubMed Central - PubMed

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

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Knock-down of hccs in medaka recapitulates the phenotypic features of MLS syndromeA–E. Bright-field dorsal (A–C) and lateral (D, E) views of wt (A), hccs-MO (B, E) and control-MO (C, D) -injected embryos at st38. hccs-morphants display microphthalmia (vertical dashed line in B), microcephaly (horizontal dashed line in B) and cardiac defects including failure of heart loop formation and pericardial oedema (red arrow in E). In a significant number of the microphthalmic embryos (50%), RPE layering and closure of ventral optic fissure were also impaired resulting in coloboma formation (black arrow in E; see also Supporting Information Table S1). Embryos injected with control-MO did not show any abnormal phenotype (C, D). a, atrium; v, ventricle. Scale bars: 100 µm.F. Analysis of the eye size at st24 and st38 (error bars are SEM; n ≥ 19 eyes, p-values were calculated by two-tailed Student's t-test).G. Western blotting analysis revealed decreased levels of total Cytc in morphants (hccs-MO) at st19 and st24 compared to control embryos (control-MO). GAPDH was used as loading control.
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fig02: Knock-down of hccs in medaka recapitulates the phenotypic features of MLS syndromeA–E. Bright-field dorsal (A–C) and lateral (D, E) views of wt (A), hccs-MO (B, E) and control-MO (C, D) -injected embryos at st38. hccs-morphants display microphthalmia (vertical dashed line in B), microcephaly (horizontal dashed line in B) and cardiac defects including failure of heart loop formation and pericardial oedema (red arrow in E). In a significant number of the microphthalmic embryos (50%), RPE layering and closure of ventral optic fissure were also impaired resulting in coloboma formation (black arrow in E; see also Supporting Information Table S1). Embryos injected with control-MO did not show any abnormal phenotype (C, D). a, atrium; v, ventricle. Scale bars: 100 µm.F. Analysis of the eye size at st24 and st38 (error bars are SEM; n ≥ 19 eyes, p-values were calculated by two-tailed Student's t-test).G. Western blotting analysis revealed decreased levels of total Cytc in morphants (hccs-MO) at st19 and st24 compared to control embryos (control-MO). GAPDH was used as loading control.

Mentions: To determine the function of hccs during embryonic development and its role in the onset and progression of MLS syndrome, we designed two specific morpholinos (MO), one directed against the ATG region of hccs (hccs-MO), the second against the third splice donor site (hccs-MO2). Embryos injected with hccs-MO developed normally until optic-cup stage, when they begun to display a very frequent (70 ± 5% of 3000 injected embryos) and morphologically recognizable microphthalmia (vertical dashed line in Fig 2B and E). These defects were associated with microcephaly (horizontal dashed line in Fig 2B) and cardiovascular abnormalities including failure of heart loop formation and pericardial oedema (red arrow in Fig 2E) and death at hatching stage (st39). Furthermore, RPE layering and closure of ventral optic fissure were impaired resulting in coloboma formation in 50% of microphthalmic embryos (black arrow in Fig 2E). A small fraction of embryos (20 ± 5%) displayed a more severe phenotype with anophthalmia and/or microphthalmia associated with severe trunk defects, whereas the remaining embryos (10 ± 5%) died before gastrulation (see Supporting Information Table S1). Embryos injected with hccs-MO2 showed the same phenotype (Supporting Information Fig S3B and Table S1). Since no phenotypic differences were observed between embryos injected with hccs-MO or hccs-MO2, subsequent studies were performed exclusively with the hccs-MO. The efficiency and specificity of the selected MO were further verified using a series of recommended controls (Eisen & Smith, 2008; Robu et al, 2007; see Supporting Information Materials and Methods section, Supporting Information Fig S3 and Table S1). A mutated form of the hccs-MO (control-MO, see Supporting Information Materials and Methods section and Supporting Information Table S1) was used as control.


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)

Knock-down of hccs in medaka recapitulates the phenotypic features of MLS syndromeA–E. Bright-field dorsal (A–C) and lateral (D, E) views of wt (A), hccs-MO (B, E) and control-MO (C, D) -injected embryos at st38. hccs-morphants display microphthalmia (vertical dashed line in B), microcephaly (horizontal dashed line in B) and cardiac defects including failure of heart loop formation and pericardial oedema (red arrow in E). In a significant number of the microphthalmic embryos (50%), RPE layering and closure of ventral optic fissure were also impaired resulting in coloboma formation (black arrow in E; see also Supporting Information Table S1). Embryos injected with control-MO did not show any abnormal phenotype (C, D). a, atrium; v, ventricle. Scale bars: 100 µm.F. Analysis of the eye size at st24 and st38 (error bars are SEM; n ≥ 19 eyes, p-values were calculated by two-tailed Student's t-test).G. Western blotting analysis revealed decreased levels of total Cytc in morphants (hccs-MO) at st19 and st24 compared to control embryos (control-MO). GAPDH was used as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3569643&req=5

fig02: Knock-down of hccs in medaka recapitulates the phenotypic features of MLS syndromeA–E. Bright-field dorsal (A–C) and lateral (D, E) views of wt (A), hccs-MO (B, E) and control-MO (C, D) -injected embryos at st38. hccs-morphants display microphthalmia (vertical dashed line in B), microcephaly (horizontal dashed line in B) and cardiac defects including failure of heart loop formation and pericardial oedema (red arrow in E). In a significant number of the microphthalmic embryos (50%), RPE layering and closure of ventral optic fissure were also impaired resulting in coloboma formation (black arrow in E; see also Supporting Information Table S1). Embryos injected with control-MO did not show any abnormal phenotype (C, D). a, atrium; v, ventricle. Scale bars: 100 µm.F. Analysis of the eye size at st24 and st38 (error bars are SEM; n ≥ 19 eyes, p-values were calculated by two-tailed Student's t-test).G. Western blotting analysis revealed decreased levels of total Cytc in morphants (hccs-MO) at st19 and st24 compared to control embryos (control-MO). GAPDH was used as loading control.
Mentions: To determine the function of hccs during embryonic development and its role in the onset and progression of MLS syndrome, we designed two specific morpholinos (MO), one directed against the ATG region of hccs (hccs-MO), the second against the third splice donor site (hccs-MO2). Embryos injected with hccs-MO developed normally until optic-cup stage, when they begun to display a very frequent (70 ± 5% of 3000 injected embryos) and morphologically recognizable microphthalmia (vertical dashed line in Fig 2B and E). These defects were associated with microcephaly (horizontal dashed line in Fig 2B) and cardiovascular abnormalities including failure of heart loop formation and pericardial oedema (red arrow in Fig 2E) and death at hatching stage (st39). Furthermore, RPE layering and closure of ventral optic fissure were impaired resulting in coloboma formation in 50% of microphthalmic embryos (black arrow in Fig 2E). A small fraction of embryos (20 ± 5%) displayed a more severe phenotype with anophthalmia and/or microphthalmia associated with severe trunk defects, whereas the remaining embryos (10 ± 5%) died before gastrulation (see Supporting Information Table S1). Embryos injected with hccs-MO2 showed the same phenotype (Supporting Information Fig S3B and Table S1). Since no phenotypic differences were observed between embryos injected with hccs-MO or hccs-MO2, subsequent studies were performed exclusively with the hccs-MO. The efficiency and specificity of the selected MO were further verified using a series of recommended controls (Eisen & Smith, 2008; Robu et al, 2007; see Supporting Information Materials and Methods section, Supporting Information Fig S3 and Table S1). A mutated form of the hccs-MO (control-MO, see Supporting Information Materials and Methods section and Supporting Information Table S1) was used as control.

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