Dephosphorylation by calcineurin regulates translocation of Drp1 to mitochondria.

Changes in mitochondrial morphology that occur during cell cycle, differentiation, and death are tightly regulated by the balance between fusion and fission processes. Excessive fragmentation can be caused by inhibition of the fusion machinery and is a common consequence of dysfunction of the organelle. Here, we show a role for calcineurin-dependent translocation of the profission dynamin related protein 1 (Drp1) to mitochondria in dysfunction-induced fragmentation. When mitochondrial depolarization is associated with sustained cytosolic Ca(2+) rise, it activates the cytosolic phosphatase calcineurin that normally interacts with Drp1. Calcineurin-dependent dephosphorylation of Drp1, and in particular of its conserved serine 637, regulates its translocation to mitochondria as substantiated by site directed mutagenesis. Thus, fragmentation of depolarized mitochondria depends on a loop involving sustained Ca(2+) rise, activation of calcineurin, and dephosphorylation of Drp1 and its translocation to the organelle.

A cut short to death: Parl and Opa1 in the regulation of mitochondrial morphology and apoptosis.

Mitochondria are crucial amplifiers of death signals. They release cytochrome c and other pro-apoptotic factors required to fully activate effector caspases. This release is accompanied by fragmentation of the mitochondrial reticulum and by remodelling of the internal structure of the organelle. Here we review data supporting the existence of a regulatory network in the inner mitochondrial membrane that includes optic atrophy 1 (Opa1), a dynamin-related protein, and presenilin-associated rhomboid-like (Parl), a rhomboid protease. Opa1 regulates remodelling of the cristae independent of its effect on fusion. Cristae remodelling conversely requires Parl, which participates in the production of a soluble form of Opa1 retrieved together with the integral membrane one in oligomers that are disrupted early during apoptosis. Parl itself is regulated by proteolysis to generate a cleaved form, which in turn modulates the shape of the mitochondrial reticulum. Cleavage of Parl depends on its phosphorylation state around the cleavage site, implicating mitochondrial kinases and phosphatases in the regulation of mitochondrial shape.

The many shapes of mitochondrial death.

Mitochondria integrate apoptotic signalling by releasing cytochrome c and other proapoptotic cofactors needed for activation of effector caspases. Previously overlooked morphological changes, mitochondrial fragmentation and cristae remodelling, emerged as subroutines of the mitochondrial programme of apoptosis in mammalian cells, as well as in developmental cell death of Caenorhabditis elegans. Mitochondrial morphology results from fusion and fission processes, controlled by a growing set of 'mitochondria-shaping' proteins. Their levels and function appear to influence mitochondrial pathways of cell death, but mechanisms are largely unknown. An emerging model implicates different signals converging on mitochondria-shaping proteins to activate or deactivate them during apoptosis. In turn, these proteins can orchestrate changes in mitochondrial shape to insure cytochrome c release and progression of the apoptotic cascade. These therefore appear an appealing novel therapeutic target to modulate cell death in cancer.

Targeting cell death.

Functional consequences of myocardial or cerebral infarction are the result of excessive cell death. It is patent that preventing cell death is the therapeutic goal in any ischemia-reperfusion setting. Mitochondria amplify apoptotic cascades and have emerged as crucial organelles in ischemia-reperfusion. Changes in mitochondrial inner membrane permeability and in the morphology of the organelle are regulated, perhaps interconnected processes that are starting to emerge as novel therapeutic targets for reducing cell death induced by ischemia-reperfusion.

Synaptic dysfunction, memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons.

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.

The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria.

Components of the death receptor-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIP(L)), a well-known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca(2+)-release as well as ER-mitochondria tethering was decreased in c-FLIP(-/-) mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIP(L) and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIP(L) emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4.

Early effects of the antineoplastic agent salinomycin on mitochondrial function.

Salinomycin, isolated from Streptomyces albus, displays antimicrobial activity. Recently, a large-scale screening approach identified salinomycin and nigericin as selective apoptosis inducers of cancer stem cells. Growing evidence suggests that salinomycin is able to kill different types of non-stem tumor cells that usually display resistance to common therapeutic approaches, but the mechanism of action of this molecule is still poorly understood. Since salinomycin has been suggested to act as a K(+) ionophore, we explored its impact on mitochondrial bioenergetic performance at an early time point following drug application. In contrast to the K(+) ionophore valinomycin, salinomycin induced a rapid hyperpolarization. In addition, mitochondrial matrix acidification and a significant decrease of respiration were observed in intact mouse embryonic fibroblasts (MEFs) and in cancer stem cell-like HMLE cells within tens of minutes, while increased production of reactive oxygen species was not detected. By comparing the chemical structures and cellular effects of this drug with those of valinomycin (K(+) ionophore) and nigericin (K(+)/H(+) exchanger), we conclude that salinomycin mediates K(+)/H(+) exchange across the inner mitochondrial membrane. Compatible with its direct modulation of mitochondrial function, salinomycin was able to induce cell death also in Bax/Bak-less double-knockout MEF cells. Since at the concentration range used in most studies (around 10 µM) salinomycin exerts its effect at the level of mitochondria and alters bioenergetic performance, the specificity of its action on pathologic B cells isolated from patients with chronic lymphocytic leukemia (CLL) versus B cells from healthy subjects was investigated. Mesenchymal stromal cells (MSCs), proposed to mimic the tumor environment, attenuated the apoptotic effect of salinomycin on B-CLL cells. Apoptosis occurred to a significant extent in healthy B cells as well as in MSCs and human primary fibroblasts. The results indicate that salinomycin, when used above µM concentrations, exerts direct, mitochondrial effects, thus compromising cell survival.

The mitochondrial permeability transition.

This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.

Optic atrophy 1 mediates mitochondria remodeling and dopaminergic neurodegeneration linked to complex I deficiency.

Mitochondrial complex I dysfunction has long been associated with Parkinson's disease (PD). Recent evidence suggests that mitochondrial involvement in PD may extend beyond a sole respiratory deficit and also include perturbations in mitochondrial fusion/fission or ultrastructure. Whether and how alterations in mitochondrial dynamics may relate to the known complex I defects in PD is unclear. Optic atrophy 1 (OPA1), a dynamin-related GTPase of the inner mitochondrial membrane, participates in mitochondrial fusion and apoptotic mitochondrial cristae remodeling. Here we show that complex I inhibition by parkinsonian neurotoxins leads to an oxidative-dependent disruption of OPA1 oligomeric complexes that normally keep mitochondrial cristae junctions tight. As a consequence, affected mitochondria exhibit major structural abnormalities, including cristae disintegration, loss of matrix density and swelling. These changes are not accompanied by mitochondrial fission but a mobilization of cytochrome c from cristae to intermembrane space, thereby lowering the threshold for activation of mitochondria-dependent apoptosis by cell death agonists in compromised neurons. All these pathogenic changes, including mitochondrial structural remodeling and dopaminergic neurodegeneration, are abrogated by OPA1 overexpression, both in vitro and in vivo. Our results identify OPA1 as molecular link between complex I deficiency and alterations in mitochondrial dynamics machinery and point to OPA1 as a novel therapeutic target for complex I cytopathies, such as PD.

Inhibition of Drp1-dependent mitochondrial fragmentation and apoptosis by a polypeptide antagonist of calcineurin.

During apoptosis, mitochondria lose their membrane potential and undergo fragmentation around the time of release of cytochrome c. Apoptotic fission is at least in part sustained by the translocation of dynamin-related protein 1 (Drp1), normally located in the cytosol, to mitochondria. This process depends on dephosphorylation of Drp1 by the phosphatase calcineurin. Here, we report the identification of a novel inhibitor of this process. A polypeptide (PPD1) from the immunophilin FKBP52 inhibits calcineurin activation triggered by mitochondrial dysfunction. PPD1 blocks Drp1 translocation to mitochondria and fragmentation of the organelle. PPD1 delays apoptosis by intrinsic stimuli by preventing fragmentation and release of cytochrome c. Cells expressing PPD1 display enhanced clonogenic ability after exposure to staurosporine. A genetic analysis revealed that the activity of PPD1 is independent of the BH3-only protein BAD, another target of calcineurin during apoptosis, and is not additive to inhibition of Drp1. Thus, PPD1 is a novel inhibitor of apoptosis that elucidates the function of calcineurin-dependent mitochondrial fragmentation in the amplification of cell death.

Endosome-mitochondria juxtaposition during apoptosis induced by H. pylori VacA.

The vacuolating cytotoxin (VacA) is an important virulence factor of Helicobacter pylori with pleiotropic effects on mammalian cells, including the ability to trigger mitochondria-dependent apoptosis. However, the mechanism by which VacA exerts its apoptotic function is unclear. Using a genetic approach, in this study we show that killing by VacA requires the proapoptotic Bcl-2 family members BAX and BAK at the mitochondrial level, but not adequate endoplasmic reticulum Ca²(+) levels, similarly controlled by BAX and BAK. A combination of subcellular fractionation and imaging shows that wild-type VacA, but not mutants in its channel-forming region, induces the accumulation of BAX on endosomes and endosome-mitochondria juxtaposition that precedes the retrieval of active BAX on mitochondria. It is noteworthy that in Bax- and Bak-deficient cells, VacA is unable to cause endosome-mitochondria juxtaposition and is not retrieved in mitochondria. Thus, VacA causes BAX/BAK-dependent juxtaposition of endosomes and mitochondria early in the process of cell death, revealing a new function for these proapoptotic proteins in the regulation of relative position of organelles.

Nitric oxide inhibition of Drp1-mediated mitochondrial fission is critical for myogenic differentiation.

During myogenic differentiation the short mitochondria of myoblasts change into the extensively elongated network observed in myotubes. The functional relevance and the molecular mechanisms driving the formation of this mitochondrial network are unknown. We now show that mitochondrial elongation is required for myogenesis to occur and that this event depends on the cellular generation of nitric oxide (NO). Inhibition of NO synthesis in myogenic precursor cells leads to inhibition of mitochondrial elongation and of myogenic differentiation. This is due to the enhanced activity, translocation and docking of the pro-fission GTPase dynamin-related protein-1 (Drp1) to mitochondria, leading also to a latent mitochondrial dysfunction that increased sensitivity to apoptotic stimuli. These effects of NO inhibition were not observed in myogenic precursor cells containing a dominant-negative form of Drp1. Both NO-dependent repression of Drp1 action and maintenance of mitochondrial integrity and function were mediated through the soluble guanylate cyclase. These data uncover a novel level of regulation of differentiation linking mitochondrial morphology and function to myogenic differentiation.

MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer.

Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a approximately 62 kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in approximately 5 and approximately 11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

On the voltage dependence of the mitochondrial permeability transition pore. A critical appraisal.

The mitochondrial permeability transition pore, a cyclosporin A-sensitive channel, can be opened by the addition of protonophoric uncouplers such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) after energy-dependent accumulation of Ca2+. We have proposed that the relevant effect of FCCP on the pore is membrane depolarization, suggesting that this channel is voltage-dependent (Bernardi, P. (1992) J. Biol. Chem. 267, 8334-8339). Here, we reconsider this hypothesis in the light of recent observations suggesting that increased production of reactive oxygen species and/or direct effects of FCCP, rather than membrane depolarization, could be the actual triggers of the FCCP-dependent permeability transition. We show that although reactive oxygen species can contribute to the permeability transition, pore opening by FCCP can still be observed under strict anaerobiosis after ATP-dependent Ca2+ accumulation and that the permeability transition can be induced by the addition of valinomycin to respiring mitochondria treated with nigericin in low potassium medium. In this system, pore opening in increasing fractions of mitochondria depends on the concentration of valinomycin, i.e. on the magnitude of the potassium current that determines the extent of membrane depolarization. We conclude that the permeability transition pore is directly modulated by the membrane potential in intact isolated rat liver mitochondria.

Commitment to apoptosis by GD3 ganglioside depends on opening of the mitochondrial permeability transition pore.

We have studied the effects of GD3 ganglioside on mitochondrial function in isolated mitochondria and intact cells. In isolated mitochondria, GD3 ganglioside induces complex changes of respiration that depend on the substrate being oxidized. However, these effects are secondary to opening of the cyclosporin A-sensitive permeability transition pore and to the ensuing swelling and cytochrome c depletion rather than to an interaction with the respiratory chain complexes. By using a novel in situ assay based on the fluorescence changes of mitochondrially entrapped calcein (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725-734), we unequivocally show that GD3 ganglioside also induces the mitochondrial permeability transition in intact cells and that this event precedes apoptosis. The mitochondrial effects of GD3 ganglioside are selective, in that they cannot be mimicked by either GD1a or GM3 gangliosides, and they are fully sensitive to cyclosporin A, which inhibits both the mitochondrial permeability transition in situ and the onset of apoptosis induced by GD3 ganglioside. These results provide compelling evidence that opening of the permeability transition pore is causally related to apoptosis.

Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-alpha aopototic signaling.

We have investigated the effects of arachidonic and palmitic acids in isolated rat liver mitochondria and in rat hepatoma MH1C1 cells. We show that both compounds induce the mitochondrial permeability transition (PT). At variance from palmitic acid, however, arachidonic acid causes a PT at concentrations that do not cause PT-independent depolarization or respiratory inhibition, suggesting a specific effect on the PT pore. When added to intact MH1C1 cells, arachidonic acid but not palmitic acid caused a mitochondrial PT in situ that was accompanied by cytochrome c release and rapidly followed by cell death. All these effects of arachidonic acid could be prevented by cyclosporin A but not by the phospholipase A(2) inhibitor aristolochic acid. In contrast, tumor necrosis factor alpha caused phospholipid hydrolysis, induction of the PT, cytochrome c release, and cell death that could be inhibited by both cyclosporin A and aristolochic acid. These findings suggest that arachidonic acid produced by cytosolic phospholipase A(2) may be a mediator of tumor necrosis factor alpha cytotoxicity in situ through induction of the mitochondrial PT.