Mitochondrial dysfunction in an Opa1(Q285STOP) mouse model of dominant optic atrophy results from Opa1 haploinsufficiency.

Mutations in the opa1 (optic atrophy 1) gene lead to autosomal dominant optic atrophy (ADOA), a hereditary eye disease. This gene encodes the Opa1 protein, a mitochondrial dynamin-related GTPase required for mitochondrial fusion and the maintenance of normal crista structure. The majority of opa1 mutations encode truncated forms of the protein, lacking a complete GTPase domain. It is unclear whether the phenotype results from haploinsufficiency or rather a deleterious effect of truncated Opa1 protein. We studied a heterozygous Opa1 mutant mouse carrying a defective allele with a stop codon in the beginning of the GTPase domain at residue 285, a mutation that mimics human pathological mutations. Using an antibody raised against an N-terminal portion of Opa1, we found that the level of wild-type protein was decreased in the mutant mice, as predicted. However, no truncated Opa1 protein was expressed. In embryonic fibroblasts isolated from the mutant mice, this partial loss of Opa1 caused mitochondrial respiratory deficiency and a selective loss of respiratory Complex IV subunits. Furthermore, partial Opa1 deficiency resulted in a substantial resistance to endoplasmic reticulum stress-induced death. On the other hand, the enforced expression of truncated Opa1 protein in cells containing normal levels of wild-type protein did not cause mitochondrial defects. Moreover, cells expressing the truncated Opa1 protein showed reduced Bax activation in response to apoptotic stimuli. Taken together, our results exclude deleterious dominant-negative or gain-of-function mechanisms for this type of Opa1 mutation and affirm haploinsufficiency as the mechanism underlying mitochondrial dysfunction in ADOA.

The rheostat in the membrane: BCL-2 family proteins and apoptosis.

Apoptosis, a mechanism for programmed cell death, has key roles in human health and disease. Many signals for cellular life and death are regulated by the BCL-2 family proteins and converge at mitochondria, where cell fate is ultimately decided. The BCL-2 family includes both pro-life (e.g. BCL-XL) and pro-death (e.g. BAX, BAK) proteins. Previously, it was thought that a balance between these opposing proteins, like a simple 'rheostat', could control the sensitivity of cells to apoptotic stresses. Later, this rheostat concept had to be extended, when it became clear that BCL-2 family proteins regulate each other through a complex network of bimolecular interactions, some transient and some relatively stable. Now, studies have shown that the apoptotic circuitry is even more sophisticated, in that BCL-2 family interactions are spatially dynamic, even in nonapoptotic cells. For example, BAX and BCL-XL can shuttle between the cytoplasm and the mitochondrial outer membrane (MOM). Upstream signaling pathways can regulate the cytoplasmic-MOM equilibrium of BAX and thereby adjust the sensitivity of cells to apoptotic stimuli. Thus, we can view the MOM as the central locale of a dynamic life-death rheostat. BAX invariably forms extensive homo-oligomers after activation in membranes. However, recent studies, showing that activated BAX monomers determine the kinetics of MOM permeabilization (MOMP), perturb the lipid bilayer and form nanometer size pores, pose questions about the role of the oligomerization. Other lingering questions concern the molecular mechanisms of BAX redistribution between MOM and cytoplasm and the details of BAX/BAK-membrane assemblies. Future studies need to delineate how BCL-2 family proteins regulate MOMP, in concert with auxiliary MOM proteins, in a dynamic membrane environment. Technologies aimed at elucidating the structure and function of the full-length proteins in membranes are needed to illuminate some of these critical issues.

Mitochondria frozen with trehalose retain a number of biological functions and preserve outer membrane integrity.

In apoptosis, Bcl-2-family proteins regulate the barrier function of the mitochondrial outer membrane (MOM), controlling the release of proapoptotic proteins from the intermembrane space into the cytoplasm. This process can be studied in vitro with freshly isolated mouse liver mitochondria. Unfortunately, mitochondria frozen/thawed in standard sucrose-mannitol buffers become leaky and useless for apoptosis research. However, here we show that mitochondria frozen in buffer containing the sugar, trehalose, maintained MOM integrity and responsiveness to Bcl-2-family proteins, much like fresh mitochondria. Trehalose also preserved ultrastructure, as well as biological functions such as ATP synthesis, calcium-induced swelling, transmembrane potential, and the import and processing of protein precursors. However, bioenergetic function was somewhat reduced. Thus, trehalose-frozen mitochondria retained most of the biological features of mitochondria including MOM integrity. Although not ideal for studies involving bioenergetics, this method will facilitate research on apoptosis and other mitochondrial functions that rely on an intact MOM.

A distinct pathway of cell-mediated apoptosis initiated by granulysin.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway.

Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B and perforin. Granzyme B, a serine proteinase with substrate specificity similar to the caspase family of apoptotic cysteine proteinases, is capable of cleaving and activating a number of death proteins in target cells. Despite the ability to engage the death pathway at multiple entry points, the preferred mechanism for rapid induction of apoptosis by granzyme B has yet to be clearly established. Here we use time lapse confocal microscopy to demonstrate that mitochondrial cytochrome c release is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event. Caspase activation is not required for cytochrome c release, an activity that correlates with cleavage and activation of Bid, which we have found to be cleaved more readily by granzyme B than either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of targets by granzyme B by blocking mitochondrial involvement in the process.

Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release.

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1-mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events.

Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation.

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.

Bcl-xL does not inhibit the function of Apaf-1.

Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9.

The 'harmless' release of cytochrome c.

Release of cytochrome c from the mitochondria plays an integral role in apoptosis; however, the mechanism by which cytochrome c is released remains one of the conundrums that has occupied the field. Recently, evidence has emerged that the commitment to death may be regulated downstream of cytochrome c release; therefore the mechanism of release must be subtle enough for the cell to recover from this event. In this review, we discuss the evidence that cytochrome c release is mediated by Bcl-2 family proteins in a process that involves only outer membrane permeability but leaves inner membrane energization, protein import function and the ultrastructure of mitochondria intact. Cell Death and Differentiation (2000) 7, 1192 - 1199.

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol.

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome c.

Apoptosis often involves the release of cytochrome c from mitochondria, leading to caspase activation. However, in apoptosis mediated by CD95 (Fas/APO-1), caspase-8 (FLICE/MACH/Mch5) is immediately activated and, in principle, could process other caspases directly. To investigate whether caspase-8 could also act through mitochondria, we added active caspase-8 to a Xenopus cell-free system requiring these organelles. Caspase-8 rapidly promoted the apoptotic program, culminating in fragmentation of chromatin and the nuclear membrane. In extracts devoid of mitochondria, caspase-8 produced DNA degradation, but left nuclear membranes intact. Thus, mitochondria were required for complete engagement of the apoptotic machinery. In the absence of mitochondria, high concentrations of caspase-8 were required to activate downstream caspases. However, when mitochondria were present, the effects of low concentrations of caspase-8 were vastly amplified through cytochrome c-dependent caspase activation. Caspase-8 promoted cytochrome c release indirectly, by cleaving at least one cytosolic substrate. Bcl-2 blocked apoptosis only at the lowest caspase-8 concentrations, potentially explaining why CD95-induced apoptosis can often evade inhibition by Bcl-2.

Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization.

Mitochondrial cytochrome c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in the activation of DEVD-specific caspases. The apoptosis inhibitors Bcl-2 or Bcl-xL prevent the efflux of cytochrome c from mitochondria. The mechanism responsible for the release of cytochrome c from mitochondria during apoptosis is unknown. Here, we report that cytochrome c release from mitochondria is an early event in the apoptotic process induced by UVB irradiation or staurosporine treatment in CEM or HeLa cells, preceding or at the time of DEVD-specific caspase activation and substrate cleavage. A reduction in mitochondrial transmembrane potential (Deltapsim) occurred considerably later than cytochrome c translocation and caspase activation, and was not necessary for DNA fragmentation. Although zVAD-fmk substantially blocked caspase activity, a reduction in Deltapsim and cell death, it failed to prevent the passage of cytochrome c from mitochondria to the cytosol. Thus the translocation of cytochrome c from mitochondria to cytosol does not require a mitochondrial transmembrane depolarization.

Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.

Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-1beta converting enzyme family proteases, and a dense membrane fraction.

We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or placed in these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in apoptosis of proteins containing Src homology 2 (SH2) domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a number of signaling proteins, including Lck, Src, and Abl, inhibited apoptosis when present at concentrations of 10-100 nM. The inhibition was dependent on specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined three phases in the apoptotic process occurring in this cell-free system. SH2 domains and ceramide act throughout the first 60-90 min of the process (the "initiation" phase). Next, Bcl-2, interleukin-1beta converting enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring approximately 90-120 min after the start of incubation (the "sentencing" phase). In the final phase ("execution"), the process of active nuclear destruction ensues.

The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.

In a cell-free apoptosis system, mitochondria spontaneously released cytochrome c, which activated DEVD-specific caspases, leading to fodrin cleavage and apoptotic nuclear morphology. Bcl-2 acted in situ on mitochondria to prevent the release of cytochrome c and thus caspase activation. During apoptosis in intact cells, cytochrome c translocation was similarly blocked by Bcl-2 but not by a caspase inhibitor, zVAD-fmk. In vitro, exogenous cytochrome c bypassed the inhibitory effect of Bcl-2. Cytochrome c release was unaccompanied by changes in mitochondrial membrane potential. Thus, Bcl-2 acts to inhibit cytochrome c translocation, thereby blocking caspase activation and the apoptotic process.