OPA1 controls apoptotic cristae remodeling independently from mitochondrial fusion

Mitochondria amplify activation of caspases during apoptosis by releasing cytochrome c and other cofactors. This is accompanied by fragmentation of the organelle and remodeling of the cristae. Here we provide evidence that Optic Atrophy 1 (OPA1), a profusion dynamin-related protein of the inner mitochondrial membrane mutated in dominant optic atrophy, protects from apoptosis by preventing cytochrome c release independently from mitochondrial fusion. OPA1 does not interfere with activation of the mitochondrial "gatekeepers" BAX and BAK, but it controls the shape of mitochondrial cristae, keeping their junctions tight during apoptosis. Tightness of cristae junctions correlates with oligomerization of two forms of OPA1, a soluble, intermembrane space and an integral inner membrane one. The proapoptotic BCL-2 family member BID, which widens cristae junctions, also disrupts OPA1 oligomers. Thus, OPA1 has genetically and molecularly distinct functions in mitochondrial fusion and in cristae remodeling during apoptosis.     

Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity,leading to cytochrome c release and apoptosis

OPA1 encodes a large GTPase related to dynamins, anchored to the mitochondrial cristae inner membrane, facing the intermembrane space. OPA1 haplo-insufficiency is responsible for the most common form of autosomal dominant optic atrophy (ADOA, MIM165500), a neuropathy resulting from degeneration of the retinal ganglion cells and optic nerve atrophy. Here we show that down-regulation of OPA1 in HeLa cells using specific small interfering RNA (siRNA) leads to fragmentation of the mitochondrial network concomitantly to the dissipation of the mitochondrial membrane potential and to a drastic disorganization of the cristae. These events are followed by cytochrome c release and caspase-dependent apoptotic nuclear events. Similarly, in NIH-OVCAR-3 cells, the OPA1 siRNA induces mitochondrial fragmentation and apoptosis, the latter being inhibited by Bcl2 overexpression. These results suggest that OPA1 is a major organizer of the mitochondrial inner membrane from which the maintenance of the cristae integrity depends. As loss of OPA1 commits cells to apoptosis without any other stimulus, we propose that OPA1 is involved in the cytochrome c sequestration and might be a target for mitochondrial apoptotic effectors. Our results also suggest that abnormal apoptosis is a possible pathophysiological process leading to the retinal ganglion cells degeneration in ADOA patients.     

Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis

Most cell death stimuli trigger the mitochondrial release of cytochrome c and other cofactors that induce caspase activation and ensuing apoptosis. Apoptosis is also associated with massive mitochondrial fragmentation and cristae remodeling. Dynamin-related protein 1 (Drp1), a protein of the mitochondrial fission machinery, has been reported to participate in apoptotic mitochondrial fragmentation. Several theories explaining the mechanisms of cytochrome c release have been proposed. One suggests that it relies on the activation of Drp1-mediated mitochondrial fission. Here, we report that downregulation of Drp1 inhibits fragmentation of the mitochondrial network and partially prevents the release of cytochrome c but fails to prevent the release of other mitochondrial factors such as second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, Omi/HtrA2, adenylate kinase 2 and deafness dystonia peptide/TIMM8a. An explanation for the prevention of cytochrome c release is provided by our observation that inhibiting Drp1-mediated mitochondrial fission prevents the mitochondrial release of soluble OPA1 that was proposed to regulate cristae remodeling and complete cytochrome c release during apoptosis. Finally, we observed that downregulation of Drp1 delays but does not inhibit apoptosis, suggesting that mitochondrial fragmentation is not a prerequisite for apoptosis.     

OPA1, a molecular regulator of dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a disease with no specific treatment, poor prognosis and high mortality. During DCM development, there is apoptosis, mitochondrial dynamics imbalance and changes in cristae structure. Optic atrophy 1 (OPA1) appears at high frequency in these three aspects. DCM LMNA (LaminA/C) gene mutation can activate TP53, and the study of P53 shows that P53 affects OPA1 through Bak/Bax and OMA1 (a metalloprotease). OPA1 can be considered the missing link between DCMp53 and DCM apoptosis, mitochondrial dynamics imbalance and changes in cristae structure. OPA1 regulates apoptosis by regulating the release of cytochrome c from the mitochondrial matrix through CJs (crisp linkages, located in the inner mitochondrial membrane) and unbalances mitochondrial fusion and fission by affecting mitochondrial inner membrane (IM) fusion. OPA1 is also associated with the formation and maintenance of mitochondrial cristae. OPA1 is not the root cause of DCM, but it is an essential mediator in P53 mediating the occurrence and development of DCM, so OPA1 also becomes a molecular regulator of DCM. This review discusses the implication of OPA1 for DCM from three aspects: apoptosis, mitochondrial dynamics and ridge structure.     

Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling

Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis. This phenotype in MiD49/51-KO cells, but not Drp1-KO cells, was completely abolished by treatments disrupting cristae structure such as OPA1 depletion. Unexpectedly, OPA1 oligomers generally thought to resist cytochrome c release by stabilizing the cristae structure were similarly disassembled in Drp1-KO and MiD49/51-KO cells, indicating that disassembly of OPA1 oligomers is not directly linked to cristae remodeling for cytochrome c release. Together, these results indicate that Drp1-dependent mitochondrial fission through MiD49/MiD51 regulates cristae remodeling during intrinsic apoptosis.     

Identification of a novel protein MICS1 that is involved in maintenance of mitochondrial morphology and apoptotic release of cytochrome c

Mitochondrial morphology dynamically changes in a balance of membrane fusion and fission in response to the environment, cell cycle, and apoptotic stimuli. Here, we report that a novel mitochondrial protein, MICS1, is involved in mitochondrial morphology in specific cristae structures and the apoptotic release of cytochrome c from the mitochondria. MICS1 is an inner membrane protein with a cleavable presequence and multiple transmembrane segments and belongs to the Bi-1 super family. MICS1 down-regulation causes mitochondrial fragmentation and cristae disorganization and stimulates the release of proapoptotic proteins. Expression of the anti-apoptotic protein Bcl-XL does not prevent morphological changes of mitochondria caused by MICS1 down-regulation, indicating that MICS1 plays a role in maintaining mitochondrial morphology separately from the function in apoptotic pathways. MICS1 overproduction induces mitochondrial aggregation and partially inhibits cytochrome c release during apoptosis, regardless of the occurrence of Bax targeting. MICS1 is cross-linked to cytochrome c without disrupting membrane integrity. Thus, MICS1 facilitates the tight association of cytochrome c with the inner membrane. Furthermore, under low-serum condition, the delay in apoptotic release of cytochrome c correlates with MICS1 up-regulation without significant changes in mitochondrial morphology, suggesting that MICS1 individually functions in mitochondrial morphology and cytochrome c release.     

The mitochondrial permeability transition regulates cytochrome c release for apoptosis during endoplasmic reticulum stress by remodeling the cristae junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.     

The antiapoptotic OPA1/Parl couple participates in mitochondrial adaptation to heat shock

The mitochondria-shaping protein optic atrophy 1 (OPA1) has genetically distinguishable roles in mitochondrial morphology and apoptosis. The latter depends on the presenilin associated rhomboid like (PARL) protease, essential for the accumulation of a soluble intermembrane space form of OPA1 (IMS-OPA1). Here we show that OPA1 and PARL participate in the heat shock response, a stereotypical cellular process of adaptation to thermal stress. Upon heat shock, long forms of OPA1 are lost and mitochondria fragment. However, mitochondrial fusion is dispensable to maintain viability, whereas IMS-OPA1 is required. Upon conditioning-a process of mild heat shock and recovery-IMS-OPA1 accumulates, OPA1 oligomers increase and mitochondria release less cytochrome c, ultimately resulting in cellular resistance to subsequent apoptotic inducers. In Parl(-/-) cells accumulation of IMS-OPA1 is blunted and conditioning fails to protect from cytochrome c release and apoptosis. Thus, the OPA1/PARL dependent pathway of cristae remodeling is implicated in heat shock. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

OPA1 and PARL keep a lid on apoptosis

A change in the shape of mitochondrial cristae must take place to attain rapid and complete release of cytochrome c during apoptosis. In this issue of Cell, Cipolat et al. and Frezza et al. (2006) show that a rhomboid intramembrane protease PARL and a dynamin-related protein OPA1 are critical regulators of cristae remodeling.     

Loss of OMA1 delays neurodegeneration by preventing stress-induced OPA1 processing in mitochondria

Proteolytic cleavage of the dynamin-like guanosine triphosphatase OPA1 in mitochondria is emerging as a central regulatory hub that determines mitochondrial morphology under stress and in disease. Stress-induced OPA1 processing by OMA1 triggersmitochondrial fragmentation, which is associated with mitophagy and apoptosis in vitro. Here, we identify OMA1 as a critical regulator of neuronal survival in vivo and demonstrate that stress-induced OPA1 processing by OMA1 promotes neuronal death and neuroinflammatory responses. Using mice lacking prohibitin membrane scaffolds as a model of neurodegeneration, we demonstrate that additional ablation of Oma1 delays neuronal loss and prolongs lifespan. This is accompanied by the accumulation of fusion-active, long OPA1 forms, which stabilize the mitochondrial genome but do not preserve mitochondrial cristae or respiratory chain supercomplex assembly in prohibitin-depleted neurons. Thus, long OPA1 forms can promote neuronal survival independently of cristae shape, whereas stress-induced OMA1 activation and OPA1 cleavage limit mitochondrial fusion and promote neuronal death.     

OPA1 alternate splicing uncouples an evolutionary conserved function in mitochondrial fusion from a vertebrate restricted function in apoptosis

In most eucaryote cells, release of apoptotic proteins from mitochondria involves fission of the mitochondrial network and drastic remodelling of the cristae structures. The intramitochondrial dynamin OPA1, as a potential central actor of these processes, exists as eight isoforms resulting from the alternate splicing combinations of exons (Ex) 4, 4b and 5b, which functions remain undetermined. Here, we show that Ex4 that is conserved throughout evolution confers functions to OPA1 involved in the maintenance of the DeltaPsi(m) and in the fusion of the mitochondrial network. Conversely, Ex4b and Ex5b, which are vertebrate specific, define a function involved in cytochrome c release, an apoptotic process also restricted to vertebrates. The drastic changes of OPA1 variant abundance in different organs suggest that nuclear splicing can control mitochondrial dynamic fate and susceptibility to apoptosis and pathologies.     

Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.     

Proteins That Fuse and Fragment Mitochondria in Apoptosis: Con-Fissing a Deadly Con-Fusion?

During apoptosis, mitochondria undergo multiple changes that culminate in the release of cytochrome c and other proapoptotic cofactors. Recently, a role for previously overlooked morphological changes, fission of the mitochondrial reticulum and remodeling of mitochondrial cristae, has been suggested in mammalian cells and in developmental apoptosis of C. elegans. Mitochondrial morphology is determined by fusion and fission processes, controlled by a growing set of “mitochondria-shaping” proteins, whose levels and function appear to regulate the mitochondrial pathways of cell death. Expression of pro-fusion proteins, as well as of inhibition of pro-fission molecules reduces apoptosis, suggesting a linear relationship between fragmentation and death. Mechanisms by which mitochondrial fragmentation promotes apoptosis and interactions between fragmentation and remodeling of the inner membrane are largely unclear. A tempting, unifying hypothesis suggests that fission is coupled to cristae remodeling to maximize cytochrome c release.     

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.     

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.     

Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis

During apoptosis, the mitochondrial membrane potential (MMP) decreases, but it is not known how this relates to the apoptotic process. It was recently suggested that cytochrome c is compartmentalized in closed cristal regions and therefore, matrix remodeling is required to attain complete cytochrome c release from the mitochondria. In this work we show that, at the onset of apoptosis, changes in MMP control matrix remodeling prior to cytochrome c release. Early after growth factor withdrawal the MMP declines and the matrix condenses. Both phenomena are reversed by adding oxidizable substrates. In mitochondria isolated from healthy cells, matrix condensation can be induced by either denying oxidizable substrates or by protonophores that dissipate the membrane potential. Matrix remodeling to the condensed state results in cristal unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria during apoptosis. In contrast, when a transmembrane potential is generated due to either electron transport or a pH gradient formed by acidifying the medium, mitochondria maintain an orthodox configuration in which most cytochrome c is sequestered in the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane.     

Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain

Mitochondrial outer membrane permeabilization and cytochrome c release promote caspase activation and execution of apoptosis through cleavage of specific caspase substrates in the cell. Among the first targets of activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (DeltaPsim), decline in ATP levels, production of reactive oxygen species (ROS), and loss of mitochondrial structural integrity. Here, we identify NDUFS1, the 75 kDa subunit of respiratory complex I, as a critical caspase substrate in the mitochondria. Cells expressing a noncleavable mutant of p75 sustain DeltaPsim and ATP levels during apoptosis, and ROS production in response to apoptotic stimuli is dampened. While cytochrome c release and DNA fragmentation are unaffected by the noncleavable p75 mutant, mitochondrial morphology of dying cells is maintained, and loss of plasma membrane integrity is delayed. Therefore, caspase cleavage of NDUFS1 is required for several mitochondrial changes associated with apoptosis.     

The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER–mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.     

Ischemia-induced cleavage of OPA1 at S1 site aggravates mitochondrial fragmentation and reperfusion injury in neurons

Neuronal mitochondrial dynamics are disturbed after ischemic stroke. Optic atrophy 1 (OPA1) and its GTPase activity are involved in maintaining mitochondrial cristae and inner membrane fusion. This study aimed to explore the role of OMA1-mediated OPA1 cleavage (S1-OPA1) in neurons exposed to cerebral ischemia and reperfusion. After oxygen-glucose deprivation (OGD) for 60 min, we found that mitochondrial fragmentation occurred successively in the axon and soma of neurons, accompanied by an increase in S1-OPA1. In addition, S1-OPA1 overexpression significantly aggravated mitochondrial damage in neurons exposed to OGD for 60 min and 24 h after OGD/R, characterized by mitochondrial fragmentation, decreased mitochondrial membrane potential, mitochondrial cristae ultrastructural damage, increased superoxide production, decreased ATP production and increased mitochondrial apoptosis, which was inhibited by the lysine 301 to alanine mutation (K301A). Furthermore, we performed neuron-specific overexpression of S1-OPA1 in the cerebral cortex around ischemia of middle cerebral artery occlusion/reperfusion (MCAO/R) mice. The results further demonstrated in vivo that S1-OPA1 exacerbated neuronal mitochondrial ultrastructural destruction and injury induced by cerebral ischemia-reperfusion, while S1-OPA1-K301 overexpression had no effect. In conclusion, ischemia induced neuronal OMA1-mediated cleavage of OPA1 at the S1 site. S1-OPA1 aggravated neuronal mitochondrial fragmentation and damage in a GTPase-dependent manner, and participated in neuronal ischemia-reperfusion injury.Subject terms: Stroke, Cell death in the nervous system     

A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis.

The mechanism during apoptosis by which cytochrome c is rapidly and completely released in the absence of mitochondrial swelling is uncertain. Here, we show that two distinct pathways are involved. One mediates release of cytochrome c across the outer mitochondrial membrane, and another, characterized in this study, is responsible for the redistribution of cytochrome c stored in intramitochondrial cristae. We have found that the "BH3-only" molecule tBID induces a striking remodeling of mitochondrial structure with mobilization of the cytochrome c stores (approximately 85%) in cristae. This reorganization does not require tBID's BH3 domain and is independent of BAK, but is inhibited by CsA. During this process, individual cristae become fused and the junctions between the cristae and the intermembrane space are opened.