Early mitochondrial alterations in ATRA-induced cell death

All-trans retinoic acid (ATRA) induces differentiation and subsequent apoptosis in a variety of cell lines. Using the myeloid cell line P39, we show that ATRA disturbs mitochondrial functional activity long before any detectable signs of apoptosis occur. These early changes include diminished mitochondrial oxygen consumption, decreased calcium uptake by mitochondria and as a result, a lower mitochondrial matrix calcium concentration. Granulocyte colony-stimulating factor (G-CSF) increases mitochondrial respiration and calcium accumulation capacity and subsequently blocks ATRA-induced apoptosis. Nifedipine, a plasma membrane calcium channel blocker, inhibits apoptosis-related changes, such as the loss of the mitochondrial membrane potential and activation of caspases. Thus, the properties of ATRA and G-CSF to modulate mitochondrial respiration and intracellular calcium control are novel findings, which give insight into their precise molecular mode of action.     

Methyl jasmonate induces production of reactive oxygen species and alterations in mitochondrial dynamics that precede photosynthetic dysfunction and subsequent cell death

Methyl jasmonate (MeJa) is a well-known plant stress hormone. Upon exposure to stress, MeJa is produced and causes activation of programmed cell death (PCD) and defense mechanisms in plants. However, the early events and the signaling mechanisms of MeJa-induced cell death have yet to be fully elucidated. To obtain some insights into the early events of this cell death process, we investigated mitochondrial dynamics, chloroplast morphology and function, production and localization of reactive oxygen species (ROS) at the single-cell level as well as photosynthetic capacity at the whole-seedling level under MeJa stimulation. Our results demonstrated that MeJa induction of ROS production, which first occurred in mitochondria after 1 h of MeJa treatment and subsequently in chloroplasts by 3 h of treatment, caused a series of alterations in mitochondrial dynamics including the cessation of mitochondrial movement, the loss of mitochondrial transmembrane potential (MPT), and the morphological transition and aberrant distribution of mitochondria. Thereafter, photochemical efficiency dramatically declined before obvious distortion in chloroplast morphology, which is prior to MeJa-induced cell death in protoplasts or intact seedlings. Moreover, treatment of protoplasts with ascorbic acid or catalase prevented ROS production, organelle change, photosynthetic dysfunction and subsequent cell death. The permeability transition pore inhibitor cyclosporin A gave significant protection against MPT loss, mitochondrial swelling and subsequent cell death. These results suggested that MeJa induces ROS production and alterations of mitochondrial dynamics as well as subsequent photosynthetic collapse, which occur upstream of cell death and are necessary components of the cell death process.     

The mitochondrial gateway to cell death

Mitochondria play a key role in death signaling. The intermembrane space of these organelles contains a number of proteins which promote cell death once they are redistributed to the cytosol. The formation of pores in the outer membrane of mitochondria defines a gateway through which the apoptogenic proteins pass during death signaling. Interactions between pro-apoptotic and pro-survival members of the Bcl-2 family of proteins are decisive in the initiation of pore opening. While the specific composition of the pore in molecular terms is still subject to debate and continuing investigation, it is recognized functionally as a passive channel which not only allows egress of proteins to cytosol but also entry in the reverse direction. A variety of constraints may restrict the release of proteins from the intermembrane space to the cytosol. These include trapping in the intercristal spaces formed by the convoluted invaginations of the inner membrane, binding of proteins to the inner membrane or to other soluble proteins of the intermembrane space, or insertion of proteins into the inner membrane. There is a corresponding variety of mechanisms that facilitate release of apoptogenic proteins from such entrapment. Morphological changes that expand the inner membrane enable proteins to be released from enclosure in intercristal spaces, allowing these proteins access to the mitochondrial gateway. Specific cases include cytochrome c molecules bound to inner membrane cardiolipin and released upon oxidation of that lipid component. Further, AIF that is embedded in the inner membrane is released by proteases (caspases or calpains), which enter from the cytosol once the outer membrane pore has opened. The facilitation (or restriction) of apoptogenic protein release through the mitochondrial gateway may provide new opportunities for regulating cell death.     

Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells

Survivin, a member of inhibitor of apoptosis family of proteins, plays important roles in both cell proliferation and cell death. We previously observed that Survivin is overexpressed in leukemic cell lines and blasts from patients with acute myelogenous leukemia (AML). To understand the roles of Survivin in AML and search for new approaches to the treatment of AML, we inhibited Survivin expression in HL-60 cells with a Survivin anti-sense oligonucleotide (sur-AS-ODN) (ISIS 23722). This blocked significant numbers of HL-60 cells in G2/M phase, and halted cell proliferation at 24 hrs and progressing over time. There was only a slight increase in the number of apoptotic cells at 24 hrs compared with cells treated with nonsense oligonucleotide (NS-ODN). At 48 hrs, however, there were significant increases in sub-G1 phase and annexin V+ cells, suggesting that cell division defects caused cell death. This was supported by the finding that a reduction in the Survivin protein by sur-AS-ODN in cells under serum-free medium did not induce G2/M block and cell death compared to cells treated with NS-ODN. The formation of polyploid cells was observed 48 hrs after sur-AS-ODN treatment, as was the activation of caspase 3, which suggested that apoptotic cell death had occurred. The mitochondrial release of cytochrome C and Smac and the nuclear translocation of the apoptosis-inducing factor were also detected. Our results suggest that Survivin is essential for cell cycle progression in leukemic cells. Reduced Survivin expression causes a cell-cycle defect that leads to cell death through a mitochondrial pathway. This finding has potential utility for therapy of patients with AML.     

Apoptosis: a mitochondrial perspective on cell death

Mitochondria play an important role in both the life and death of cells. The past 7-8 years have seen an intense surge in research devoted toward understanding the critical role of mitochondria in the regulation of cell death. Mitochondria have, next to their function in respiration, an important role in apoptotic signaling pathway. Apoptosis is a form of programmed cell death important in the development and tissue homeostasis of multicellular organisms. Apoptosis can be initiated by a wide array of stimuli, including multiple signaling pathways that, for the most part, converge at the mitochondria. Although classically considered the powerhouses of the cell, it is now understood that mitochondria are also "gatekeepers" that ultimately determine the fate of the cell. Malfunctioning at any level of the cell is eventually translated in the release of apoptogenic factors from the mitochondrial intermembrane space resulting in the organized demise of the cell. These mitochondrial factors may contribute to both caspase-dependent and caspase-independent processes in apoptotic cell death. In addition, several Bcl-2 family members and other upstream proteins also contribute to and regulate the apoptosis. In this review, we attempt to summarize our current view of the mechanism that leads to the influx and efflux of many proteins from/to mitochondria during apoptosis.     

Marked mitochondrial alterations upon starvation without cell death, caspases or Bcl-2 family members

Dictyostelium HMX44A cells can withstand starvation under monolayer conditions for a few days without dying. They die only when the differentiation factor DIF-1 is exogenously added. Still, when HMX44A were subjected to starvation without addition of DIF-1 they showed, by electron microscopy and electron tomography, gross mitochondrial lesions including marked cristae alterations with frequent "holes" probably originating from dilated cristae. Since these cells did not die as shown for instance by FACS analysis, these results showed unexpected resilience of cells bearing markedly altered mitochondria, and thus showed that apparently destructive mitochondrial alterations may not lead to cell death. Also, these marked mitochondrial lesions could not be caused by caspases or bcl-2 family members, which these cells do not encode.     

Huntingtin-interacting protein 1-mediated neuronal cell death occurs through intrinsic apoptotic pathways and mitochondrial alterations

Huntingtin interacting protein-1 (Hip1) is known to be associated with the N-terminal domain of huntingtin. Although Hip1 can induce apoptosis, the exact upstream signal transduction pathways have not been clarified yet. In the present study, we examined whether activation of intrinsic and/or extrinsic apoptotic pathways occurs during Hip1-mediated neuronal cell death. Overexpression of Hip1 induced cell death through caspase-3 activation in immortalized hippocampal neuroprogenitor cells. Interestingly, proteolytic processing of Hip1 into partial fragments was observed in response to Hip1 transfection and apoptosis-inducing drugs. Moreover, Hip1 was found to directly bind to and activate caspase-9. This promoted cytosolic release of cytochrome c and apoptosis-inducing factor via mitochondrial membrane perturbation. Furthermore, Hip1 could directly bind to Apaf-1, suggesting that the neurotoxic signals of Hip1 transmit through the intrinsic mitochondrial apoptotic pathways and the formation of apoptosome complex.     

Yeast as a tool to study Bax/mitochondrial interactions in cell death

The budding yeast Saccharomyces cerevisiae has proven to be a powerful tool in investigations of the molecular aspects of the events involved in apoptosis, particularly the steps implicating mitochondria. Yeast does not have obvious homologs of the proteins involved in the regulation of apoptosis, and provides a simplified model system in which the function of these proteins can be unraveled. This review focuses on the interactions of two of the major pro-apoptotic Bcl-2 family members, Bax and Bid, with mitochondria. It is shown that yeast has allowed questioning of several crucial aspects of the function of these two proteins, namely the molecular mechanisms driving their insertion into the mitochondrial outer membrane and those leading to the permeabilization to cytochrome c. More recently, signaling pathways leading to Bax-induced cell death, as well as other forms of cell death, have been identified in yeast. Both 'apoptosis-like' and autophagy-related forms of cell degradation are involved, and mitochondria play a central role in these two signaling pathways.     

Alteration of mitochondrial function and cell sensitization to death

Stimulation of cell death is a powerful instrument in the organism’s struggle with cancer. Apoptosis represents one mode of cell death. However, in a variety of tumor cells proapoptotic mechanisms are downregulated, or not properly activated, whereas antiapoptotic mechanisms are upregulated. Mitochondria are known as key players in the regulation of apoptotic pathways. Specifically, permeabilization of the mitochondrial outer membrane and subsequent release of proapoptotic proteins from the intermembrane space are viewed as decisive events in the initiation and/or execution of apoptosis. Disruption of mitochondrial functions by anticancer drugs, which induce oxidative stress, inhibit mitochondrial respiration, or uncouple oxidative phosphorylation, can sensitize mitochondria in these cells and facilitate outer membrane permeabilization.     

Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1–3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1–NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1–NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.     

Scleral calcification and photoreceptor cell death during aging and exposure to chronic stress

Male and female Fischer 344 rats of three different ages (12, 18, and 25 months) have been examined for the presence of photoreceptor (PR) cell loss and for occurrence of scleral cartilage and bone formation. In addition, male and female rats, aged 11 months at the beginning of the experiments, were exposed to chronic stress for either 0.5, 2, 4, or 6 months. Photoreceptor cell death gradually increases during the aging process and is exacerbated by exposure to chronic stress. It is more severe in the peripheral than the central retina and exposure to stress increases this pattern of cell loss. The superior retina is more severely affected than the inferior hemisphere in aging and during stress. The incidence of scleral cartilage or bone formation increases with age in male and female rats, but with stress exposure an increase is seen in males only. Bone formations occur more frequently in male than in female animals and are almost always (97%) located in the superior hemisphere of the eye. Although there appears to be a direct relationship between photoreceptor cell death and the occurrence of scleral ossifications in group data, in individual eyes the bone formations are not always associated with severity of PR cell loss. The relationship of PR cell death and incidence of scleral ossification to gender and to exposure to stress supports a hypothesis for an endocrine basis of ocular aging.     

PKG activity causes photoreceptor cell death in two retinitis pigmentosa models

Photoreceptor degeneration in retinitis pigmentosa is one of the leading causes of hereditary blindness in the developed world. Although causative genetic mutations have been elucidated in many cases, the underlying neuronal degeneration mechanisms are still unknown. Here, we show that activation of cGMP-dependent protein kinase (PKG) hallmarks photoreceptor degeneration in rd1 and rd2 human homologous mouse models. When induced in wild-type retinae, PKG activity was both necessary and sufficient to trigger cGMP-mediated photoreceptor cell death. Target-specific, pharmacological inhibition of PKG activity in both rd1 and rd2 retinae strongly reduced photoreceptor cell death in organotypic retinal explants. Likewise, inhibition of PKG in vivo, using three different application paradigms, resulted in robust photoreceptor protection in the rd1 retina. These findings suggest a pivotal role for PKG activity in cGMP-mediated photoreceptor degeneration mechanisms and highlight the importance of PKG as a novel target for the pharmacological intervention in RP.     

Lysosomal membrane permeabilization and autophagy blockade contribute to photoreceptor cell death in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a group of hereditary retinal dystrophies that normally result in photoreceptor cell death and vision loss both in animal models and in affected patients. The rd10 mouse, which carries a missense mutation in the Pde6b gene, has been used to characterize the underlying pathophysiology and develop therapies for this devastating and incurable disease. Here we show that increased photoreceptor cell death in the rd10 mouse retina is associated with calcium overload and calpain activation, both of which are observed before the appearance of signs of cell degeneration. These changes are accompanied by an increase in the activity of the lysosomal protease cathepsin B in the cytoplasm of photoreceptor cells, and a reduced colocalization of cathepsin B with lysosomal markers, suggesting that lysosomal membrane permeabilization occurs before the peak of cell death. Moreover, expression of the autophagosomal marker LC3-II (lipidated form of LC3) is reduced and autophagy flux is blocked in rd10 retinas before the onset of photoreceptor cell death. Interestingly, we found that cell death is increased by the induction of autophagy with rapamycin and inhibited by calpain and cathepsin inhibitors, both ex vivo and in vivo. Taken together, these data suggest that calpain-mediated lysosomal membrane permeabilization underlies the lysosomal dysfunction and downregulation of autophagy associated with photoreceptor cell death.Autophagy is a cellular self-degradative pathway that mediates the recycling of damaged or disposable cell components and is activated in situations of nutritional, oxidative and other forms of stress.¹ This process begins with the formation of the autophagosome, an intracellular double-membrane organelle that surrounds parts of the cytoplasm containing organelles and protein aggregates. Autophagosomes subsequently fuse with lysosomes to initiate the degradation of the engulfed cellular components. Autophagy dysfunction has been implicated in many pathological conditions including infections, cancer and muscular and degenerative diseases.² In the nervous system, autophagy has a key role in preventing intracellular accumulation of misfolded and/or aggregated proteins, and its pharmacological upregulation through the administration of rapamycin and other drugs exerts protective effects against a wide range of proteinopathies.³ Moreover, defects in different stages of the autophagy pathway, including autophagosome formation, cargo recognition and lysosomal fusion and degradation, have been often implicated in neurodegeneration.⁴In addition to their degradative role, lysosomes are emerging as key regulators of cellular homeostasis, acting as nutritional sensors or actively participating in cell death.^{5, 6, 7} Lysosomal alterations including increases in lysosomal pH and lysosomal membrane permeabilization (LMP) have been demonstrated in Alzheimer''s and Parkinson''s diseases,^{8, 9} and mutations in lysosomal enzymes cause defects in autophagy, inducing a marked neurodegenerative phenotype in patients with lysosomal storage disorders.¹⁰ LMP induces the selective translocation of cathepsins to the cytoplasm, triggering caspase-dependent and -independent cell death.^{11, 12, 13} LMP has been implicated in mammary gland involution in physiological conditions,¹⁴ indicating that lysosomal-mediated cell death is not merely a consequence of accidental lysosomal damage. As in vivo administration of cathepsin inhibitors attenuates cell death in this model, a similar approach could hold therapeutic potential for the treatment of diseases associated with LMP, including Parkinson''s disease, Niemann–Pick disease type A and stroke.^{7, 10, 15} Oxidative stress and calpain activation are some of the many stimuli that can induce LMP, and have been observed both in vitro and in vivo.⁷ Several pathological processes in the nervous system associated with cell death, including excitotoxicity and ischaemia–reperfusion, have been linked to increased calpain activation.¹⁶ Calpains have also been shown to cleave many intracellular substrates including autophagy and lysosomal proteins,^{17, 18} suggesting links between calcium levels, calpain activation, lysosomal damage and autophagy blockade.Recent findings have begun to shed light on the role of autophagy in the retina. We previously reported decreased autophagy flux in the retinas of aged mice,¹⁹ and demonstrated photoreceptor cell death and decreased dim-light vision in the neuronal-specific Atg5-deficient mouse, a phenotype that closely resembles that observed during physiological aging.¹⁹ We have also demonstrated the essential cytoprotective role of autophagy in vivo in response to retinal ganglion cell damage in experimental models of glaucoma.²⁰ A recent study described lysosomal basification and decreased autophagic flux in travecular meshwork cells in response to chronic oxidative stress, with important implications for the pathogenesis of glaucoma.²¹ Furthermore, specific Atg5 deletion in pigment epithelium leads to reduced levels of visual pigments and vision alterations,²² indicating that autophagy has an important role in sustaining retinal pigment epithelium function.Retinitis pigmentosa is a large group of genetic disorders that normally involves photoreceptor cell death and leads to vision loss in both animal models and affected patients. To date, no treatment for this devastating disease has been developed to clinic. The study of animal models is thus essential to unravel the mechanisms of photoreceptor degeneration involved in these disorders and to identify therapeutic targets. The rd10 mouse, which harbours a mutation in the rod-specific phosphodiesterase gene Pde6b, is a suitable model of human retinitis pigmentosa.^{23, 24} This mutation results in reduced enzymatic function leading to increased cGMP and rod cell death, peaking around postnatal day 25 (P25), with only residual vision remaining after P30.^{24, 25} Here we show that rd10 mice exhibit massive intracellular calcium accumulation and m-calpain (calpain-2) activation at early ages, before the peak of photoreceptor cell death, that correlate with the blockade of autophagic flux. Moreover, we demonstrate an increase in cathepsin B activity in the cytoplasm of rd10 photoreceptors that correlates with the activation of DNAse II-dependent cell death. Induced calcium overload in wild-type (Wt) retinal explants phenocopies the degenerative features seen in rd10 retinas: lysosomal damage, cathepsin translocation and cell death. Finally, we show that calpain and cathepsin inhibitors attenuate cell death both in vitro, ex vivo and in vivo. Taken together, these data suggest that calpain-mediated LMP underlies the lysosomal dysfunction and downregulation of autophagy associated with photoreceptor cell death.     

Granzyme A cleaves a mitochondrial complex I protein to initiate caspase-independent cell death

The killer lymphocyte protease granzyme A (GzmA) triggers caspase-independent target cell death with morphological features of apoptosis. We previously showed that GzmA acts directly on mitochondria to generate reactive oxygen species (ROS) and disrupt the transmembrane potential (DeltaPsi(m)) but does not permeabilize the mitochondrial outer membrane. Mitochondrial damage is critical to GzmA-induced cell death since cells treated with superoxide scavengers are resistant to GzmA. Here we find that GzmA accesses the mitochondrial matrix to cleave the complex I protein NDUFS3, an iron-sulfur subunit of the NADH:ubiquinone oxidoreductase complex I, after Lys56 to interfere with NADH oxidation and generate superoxide anions. Target cells expressing a cleavage site mutant of NDUFS3 are resistant to GzmA-mediated cell death but remain sensitive to GzmB.     

Early neural cell death: dying to become neurons

The importance of programmed cell death (PCD) during vertebrate development has been well established. During the development of the nervous system in particular, neurotrophic cell death in innervating neurons matches the number of neurons to the size of their target field. However, PCD also occurs during earlier stages of neural development, within populations of proliferating neural precursors and newly postmitotic neuroblasts, all of which are not yet fully differentiated. This review addresses early neural PCD, which is distinct from neurotrophic death in differentiated neurons. Although early neural PCD is observed in a range of organisms, from Caenorhabditis elegans to mouse, the role and the regulation of early neural PCD are not well understood. The regulation of early neural PCD can be inferred from the function of factors such as bone morphogenetic proteins (BMPs), Wnts, fibroblast growth factors (FGFs), and Sonic Hedgehog (Shh), which regulate both early neural development and PCD occurring in other developmental processes. Cell number control, removal of damaged or misspecified cells (spatially or temporally), and selection are the proposed roles early neural PCDs play during neural development. Data from developmental PCD in C. elegans and Drosophila provide insights into the possible signaling pathways integrating PCD with other processes during early neural development and the roles they might play.     

Ceramide triggers metacaspase-independent mitochondrial cell death in yeast

The activation of ceramide-generating enzymes, the blockade of ceramide degradation, or the addition of ceramide analogues can trigger apoptosis or necrosis in human cancer cells. Moreover, endogenous ceramide plays a decisive role in the killing of neoplastic cells by conventional anticancer chemotherapeutics. Here, we explored the possibility that membrane-permeable C2-ceramide might kill budding yeast (Saccharomyces cerevisiae) cells under fermentative conditions, where they exhibit rapid proliferation and a Warburg-like metabolism that is reminiscent of cancer cells. C2-ceramide efficiently induced the generation of reactive oxygen species (ROS), as well as apoptotic and necrotic cell death, and this effect was not influenced by deletion of the sole yeast metacaspase. However, C2-ceramide largely failed to cause ROS hypergeneration and cell death upon deletion of the mitochondrial genome. Thus, mitochondrial function is strictly required for C2-ceramide-induced yeast lethality. Accordingly, mitochondria from C2-ceramide-treated yeast cells exhibited major morphological alterations including organelle fragmentation and aggregation. Altogether, our results point to a pivotal role of mitochondria in ceramide-induced yeast cell death.     

Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death

Combination of retinoic acids (RAs) and interferons (IFNs) has synergistic apoptotic effects and is used in cancer treatment. However, the underlying mechanisms remain unknown. Here, we demonstrate that mitochondrial respiratory chain (MRC) plays an essential role in the IFN-beta/RA-induced cancer cell death. We found that IFN-beta/RA upregulates the expression of MRC complex subunits. Mitochondrial-nuclear translocation of these subunits was not observed, but overproduction of reactive oxygen species (ROS), which causes loss of mitochondrial function, was detected upon IFN-beta/RA treatment. Knockdown of GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) and NDUFS3 (NADH dehydrogenase (ubiquinone) Fe-S protein 3), two subunits of MRC complex I, by siRNA in two cancer cell lines conferred resistance to IFN-beta/RA-induced apoptosis and reduced ROS production. In parallel, expression of late genes induced by IFN-beta/RA that are directly involved in growth inhibition and cell death was also repressed in the knockdown cells. Our data suggest that the MRC regulates IFN-beta/RA-induced cell death by modulating ROS production and late gene expression.     

The role of caspases in photoreceptor cell death of the retinoschisin-deficient mouse

Early schisis cavities in the retinal bipolar cell layer accompanied by progressive loss of cone and rod photoreceptor cells are the hallmark of the retinoschisin-deficient (Rs1h(-/Y)) murine retina. With this study we aimed at elucidating the molecular events underlying the photoreceptor cell death in this established murine model of X-linked juvenile retinoschisis. We show that photoreceptor degeneration in the Rs1h(-/Y) mouse is due to apoptotic events peaking around postnatal day 18. Cell death is accompanied by increased expression of initiator and inflammatory caspases but not by downstream effector caspases. The strong induction of caspase-1 (Casp1) prompted us to explore its involvement in the apoptotic process. We therefore generated double knock-out mice deficient for both retinoschisin and Casp1. No direct influence of the Casp1 genotype on apoptosis could be identified although striking differences in the overall number of resident microglia were observed independent of the Rs1h genotype.