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EMBO Molecular Medicine, 2013
Mitochondrial-dependent (intrinsic) programmed cell death (PCD) is an essential homoeostatic mechanism that selects bioenergetically proficient cells suitable for tissue/organ development. However, the link between mitochondrial dysfunction, intrinsic apoptosis and developmental anomalies has not been demonstrated to date. Now we provide the evidence that non-canonical mitochondrialdependent apoptosis explains the phenotype of microphthalmia with linear skin lesions (MLS), an X-linked developmental disorder caused by mutations in the holo-cytochrome c-type synthase (HCCS) gene. 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). We thus propose that HCCS plays a key role in central nervous system (CNS) development by modulating a novel non-canonical start-up of cell death and provide the first experimental evidence for a mechanistic link between mitochondrial dysfunction, intrinsic apoptosis and developmental disorders.
Cytochrome c Deficiency Causes Embryonic Lethality and Attenuates Stress-Induced Apoptosis
Cell, 2000
or caspase-9 (Hakem et al., 1998; Kuida et al., and R. Sanders Williams* § # 1998) results in prenatal or perinatal death of homozy-* Department of Internal Medicine gous null animals, which exhibit prominent overgrowth † Department of Biochemistry of neural structures based on a failure of developmen- ‡ Department of Pathology tally mediated apoptosis. Cells derived from these § Department of Molecular Biology knockout mice demonstrate defects in response to a Howard Hughes Medical Institute variety of apoptotic stimuli. However, T lymphocytes University of Texas Southwestern Medical Center from mice lacking Apaf-1 or caspase-9 undergo apo-Dallas, Texas 75390 ptosis normally in response to TNF␣ or antibodies that function as agonists for Fas receptor signaling (Hakem et al., 1998; Yoshida et al., 1998). These observations Summary have supported the notion of discrete apoptotic signaling pathways (reviewed in Green and Reed, 1998; Vaux Cytochrome c released from mitochondria has been and Korsmeyer, 1999): a "cellular stress" or "mitochonproposed to be an essential component of an apodrial" pathway dependent on cytochrome c, Apaf-1, and ptotic pathway responsive to DNA damage and other caspase-9; and a "death ligand" or "death receptor" forms of cell stress. Murine embryos devoid of cytopathway mediated by other signaling proteins such as chrome c die in utero by midgestation, but cell lines FADD and caspase-8. Both pathways converge on established from early cytochrome c null embryos are caspase-3 and other proteases and nucleases that drive viable under conditions that compensate for defective the terminal events of programmed cell death. The two oxidative phosphorylation. As compared to cell lines pathways are thought to function in parallel, although established from wild-type embryos, cells lacking cythe death receptor pathway is amplified by the mitotochrome c show reduced caspase-3 activation and chondrial pathway in certain cell types through the proare resistant to the proapoptotic effects of UV irradiatein Bid (Li et al., 1998; Luo et al., 1998). tion, serum withdrawal, or staurosporine. In contrast, Unlike Apaf-1, caspase-9, and caspase-3, the biocells lacking cytochrome c demonstrate increased chemical function of cytochrome c proposed in this sensitivity to cell death signals triggered by TNF␣. model has not been subjected to the tests provided by These results define the role of cytochrome c in differthe analysis of cells or animals bearing null mutations. ent apoptotic signaling cascades. Since cytochrome c is required for mitochondrial respiration, genetic approaches that have been applied suc-Introduction cessfully in model organisms to characterize the function of other apoptotic signaling intermediates have not Cytochrome c (Cyt c), the only water-soluble component been useful in assessing the proposed function of cytoof the electron transfer chain, was unexpectedly found chrome c in apoptosis. to promote the activation of apoptotic caspases in cell-Here, we employ genetically modified mice to investifree extracts or when introduced ectopically into the gate the importance of cytochrome c in apoptotic signalcytoplasm of intact cells (Liu et al., 1996; Li et al., 1997; ing pathways. Cell lines devoid of cytochrome c were Zhivotovsky et al., 1998). In response to a variety of established from early embryos under conditions that death-promoting stimuli, cytochrome c is released from compensate for their defect in mitochondrial respiration, its normal position within the intermembrane space of as defined previously for culture of cells lacking mitomitochondria, in association with changes in mitochonchondrial DNA (King and Attardi, 1989). In comparison drial permeability, membrane potential, and ultrastrucwith wild-type cells, cells lacking cytochrome c are resisture (Heiskanen et al., 1999). Once in the cytosol, cytotant to death induced by UV irradiation or proapoptotic chrome c binds Apaf-1 with high affinity, an event that drugs and are partially resistant to the apoptotic effects triggers oligomerization of Apaf-1/cytochrome c in comof serum withdrawal. In contrast, the apoptotic response plexes that activate procaspase-9 (Zou et al., 1999).
Restoration of mitochondrial function reverses developmental neuronal death in vitro
The Journal of Comparative Neurology, 2001
In a previous study characterizing morphological and functional features of cell death in trophically deprived chick ciliary ganglion neurons (Pena and Pilar [2000] J. Comp. Neurol. 424:377-396), we hypothesized that early cell death events might be targets for reversal, allowing for rescue of dying neurons. To test this hypothesis, ciliary ganglion (CG) neurons were cultured with or without trophic support (choroid, iris, and pigment epithelium soluble extract [CIPE]), or without trophic support for 11 or 18 hours and then exposed to trophic support. Prior to and at the onset of cell death commitment (11 hours) CIPE-deprived neurons exhibited increased membrane permeability, blebbing, cytoplasmic vacuolization, swollen mitochondria, low adenosine triphosphate levels, and release of cytochrome c from mitochondria. CIPE readdition at 11 hours reversed these changes. Between 11 and 18 hours, irreversible DNA fragmentation increased in CIPE-deprived neurons. Cyclosporin A and bongkrekic acid (inhibitors of mitochondrial transition permeability pores) prevented membrane permeability increases and delayed the progression to death in trophically deprived neurons by 12 hours; however, by 48 hours all neurons had died. BOC-Asp-CH2F (BAF), a pan-caspase inhibitor, did not prevent early events of cell death including increased membrane permeability and Cyto c release, but it inhibited DNA fragmentation and prolonged neuronal survival to 48 hours. We conclude that mitochondria changes occur early, prior to commitment and that the suppression of these changes can prevent all the downstream events of death, whereas caspase inhibitors have no effect on the early mitochondria/plasma membrane changes. Mitochondria thus play a critical role in the transition from reversible to irreversible commitment to developmental neuronal death. Furthermore, neuronal death is brought about by activation of one of two distinct pathways, one localized in mitochondria and the other dependent on activation of caspases.
The American Journal of Human Genetics, 2012
Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possible candidates, we analyzed the X-linked COX7B and found deleterious de novo mutations in two simplex cases and a nonsense mutation, which segregates with the disease, in a familial case. COX7B encodes a poorly characterized structural subunit of cytochrome c oxidase (COX), the MRC complex IV. We demonstrated that COX7B is indispensable for COX assembly, COX activity, and mitochondrial respiration. Downregulation of the COX7B ortholog (cox7B) in medaka (Oryzias latipes) resulted in microcephaly and microphthalmia that recapitulated the MLS phenotype and demonstrated an essential function of complex IV activity in vertebrate CNS development. Our results indicate an evolutionary conserved role of the MRC complexes III and IV for the proper development of the CNS in vertebrates and uncover a group of mitochondrial diseases hallmarked by a developmental phenotype.
Mitochondria-mediated apoptosis in mammals
Protein & cell, 2014
The mitochondria-mediated caspase activation pathway is a major apoptotic pathway characterized by mitochondrial outer membrane permeabilization (MOMP) and subsequent release of cytochrome c into the cytoplasm to activate caspases. MOMP is regulated by the Bcl-2 family of proteins. This pathway plays important roles not only in normal development, maintenance of tissue homeostasis and the regulation of immune system, but also in human diseases such as immune disorders, neurodegeneration and cancer. In the past decades the molecular basis of this pathway and the regulatory mechanism have been comprehensively studied, yet a great deal of new evidence indicates that cytochrome c release from mitochondria does not always lead to irreversible cell death, and that caspase activation can also have non-death functions. Thus, many unsolved questions and new challenges are still remaining. Furthermore, the dysfunction of this pathway involved in cancer development is obvious, and targeting th...
Maternally-inherited Leigh syndrome-related mutations bolster mitochondrial-mediated apoptosis
Journal of Neurochemistry, 2004
The key role of mitochondria in the apoptotic process is well understood, but not many data are available regarding the specific role of mitochondrial DNA mutations in determining cell fate. We investigated whether two mitochondrial DNA mutations (L217R and L156R) associated with maternallyinherited Leigh syndrome may play a specific role in triggering the apoptotic cascade. Considering that different nuclear genetic factors may influence the expression of mtDNA mutations, we used a 143BTKosteosarcoma cell line deprived from its own mtDNA in order to insert mutated mtDNAs. Analysis of mitochondrial features in these cybrids indicated that both mitochondrial DNA mutations produced evidence of biochemical, functional and ultrastructural modifications of mitochondria, and that these modifications were associated with an increased apoptotic proneness. Cybrids were highly susceptible to two different apoptotic stimuli, tumour necrosis factor-a and Staurosporin. The mechanism involved was the mitochondrial 'intrinsic' pathway, i.e. the caspase 9-driven cascade. More importantly, our results also indicated that the polarization state of the mitochondrial membrane, i.e. a constitutive hyperpolarization detected in cybrid clones, played a specific role. Interestingly, the different effects of the two mutations in terms of susceptibility to apoptosis probably reflect the deeper bioenergetic defect associated with the L217R mutation. This work provides the first evidence that hyperpolarization of mitochondria may be a 'risk factor' for cells with a deep ATPase dysfunction, such as cells from patients with maternally-inherited Leigh syndrome.
Lack of apoptosis in mitochondrial encephalomyopathies
Neurology, 2001
Article abstract-Background/Objective: Apoptosis, or programmed cell death, is an evolutionary conserved mechanism essential for morphogenesis and tissue homeostasis, but it plays an important role also in pathologic conditions, including neurologic disorders. Its execution pathway is critically regulated at the mitochondrial level. Evidence of apoptosis in muscle specimens was investigated in patients with genetically defined mitochondrial encephalomyopathies. Methods: Thirty-three muscle biopsies from patients with genotypically different mitochondrial diseases (single and multiple deletions, A3243G/A8344G point mutations of the mitochondrial DNA) were studied. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction was used as a marker of nuclear DNA fragmentation, as well as antibodies against pro-(Fas) or anti-(Bcl-2) apoptotic factors. Also, because one hallmark of apoptosis is morphologic, ultrastructural studies were performed on skeletal muscle from 18 of 33 patients, examining both phenotypically normal and ragged red fibers. Results: In all muscle biopsies, no significant expression of either pro (Fas) and inhibiting (Bcl-2) apoptosis-related proteins was found, nor TUNEL positivity. This latter finding is confirmed by lack of morphologic evidence of apoptosis in all the fibers examined at the ultrastructural level. Conclusion: The authors' findings suggest that genetically determined defects of oxidative phosphorylation do not induce the apoptotic process and that apoptosis is not involved in the pathogenesis of mitochondrial disorders.
Role of mitochondria in apoptotic and necroptotic cell death in the developing brain
Clinica Chimica Acta, 2015
Hypoxic-ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short-and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia-ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia-ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome (composed of RIP1, RIP3 and MLKL), both of which converge at the mitochondria.