Cytochrome c release from mitochondria: all or nothing.

During the process of apoptosis, cytochrome c is released from mitochondria into the cytosol where it helps to activate the caspases, a family of killer proteases. The release of cytochrome c is a rapid, complete and kinetically invariant event.

Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in mitochondrial membranes.

Activation of Bax or Bak is essential for the completion of many apoptotic programmes. Under cytotoxic conditions, these proteins undergo a series of conformational rearrangements that end up with their oligomerization. We found that unlike inactive monomeric Bax, active oligomerized Bax is partially resistant to trypsin digestion, providing a convenient read out to monitor Bax activation. Using this assay, we studied how the lipid composition of membranes affects tBid-induced Bax activation in vitro with pure liposomes. We report that Bax activation is inhibited by cholesterol and by decreases in membrane fluidity. This observation was further tested in vivo using the drug U18666A, which we found increases mitochondrial cholesterol levels. When incubated with tBid, mitochondria isolated from U18666A-treated cells showed a delay in the release of Smac/Diablo and Cytochrome c, as well as in Bax oligomerization. Moreover, pre-incubation with U18666A partially protected cells from stress-induced apoptosis. As many tumours display high mitochondrial cholesterol content, inefficient Bax oligomerization might contribute to their resistance to apoptosis-inducing agents.

Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane.

Under many apoptotic conditions, Bax undergoes conformational rearrangements, leading to its insertion in the mitochondrial outer membrane as a transmembrane oligomer. At the same time, mitochondria undergo fragmentation and activated Bax was reported to localize to fission sites. We studied how lipid composition and membrane curvature regulate Bax activation. When isolated mitochondria were incubated with phospholipase A2, which led to phosphatidylethanolamine and cardiolipin hydrolysis, tBid and Bax insertion were hindered. We thus studied in liposomes how phosphatidylethanolamine, cardiolipin, and its hydrolysis products affect Bax activation. Whereas phosphatidylethanolamine, a lipid with negative curvature, did not affect Bax insertion, it inhibited Bax oligomerization. Conversely, Bax insertion required cardiolipin, and was not blocked by cardiolipin hydrolysis products. These experiments support a direct role for cardiolipin in the recruitment and activation of Bax. To examine if the increase in membrane curvature that accompanies mitochondrial fission participates in Bax activation, we studied how liposome size affects the process, and observed that it was inhibited in small liposomes (

TCTP protects from apoptotic cell death by antagonizing bax function.

Translationally controlled tumor protein (TCTP) is a potential target for cancer therapy. It functions as a growth regulating protein implicated in the TSC1-TSC2 -mTOR pathway or a guanine nucleotide dissociation inhibitor for the elongation factors EF1A and EF1Bbeta. Accumulating evidence indicates that TCTP also functions as an antiapoptotic protein, through a hitherto unknown mechanism. In keeping with this, we show here that loss of tctp expression in mice leads to increased spontaneous apoptosis during embryogenesis and causes lethality between E6.5 and E9.5. To gain further mechanistic insights into this apoptotic function, we solved and refined the crystal structure of human TCTP at 2.0 A resolution. We found a structural similarity between the H2-H3 helices of TCTP and the H5-H6 helices of Bax, which have been previously implicated in regulating the mitochondrial membrane permeability during apoptosis. By site-directed mutagenesis we establish the relevance of the H2-H3 helices in TCTP's antiapoptotic function. Finally, we show that TCTP antagonizes apoptosis by inserting into the mitochondrial membrane and inhibiting Bax dimerization. Together, these data therefore further confirm the antiapoptotic role of TCTP in vivo and provide new mechanistic insights into this key function of TCTP.

Mitochondria as the central control point of apoptosis.

Mitochondria play a major role in apoptosis triggered by many stimuli. They integrate death signals through Bcl-2 family members and coordinate caspase activation through the release of cytochrome c as a result of the outer mitochondrial membrane becoming permeable. The mechanisms that lead to this permeability are not yet completely understood. Here, we attempt to summarize our current view of the mechanisms that lead to the efflux of many proteins from mitochondria during apoptosis and the role played by Bcl-2 family proteins in the control of this event.

Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.

To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.

The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis.

Bcl-2 is a mitochondrial- and perinuclear-associated protein that prolongs the lifespan of a variety of cell types by interfering with programmed cell death (apoptosis). Bcl-2 seems to function in an antioxidant pathway, and it is believed that membrane attachment mediated by a COOH-terminal hydrophobic tail is required for its full activity. To identify critical regions in bcl-2 alpha for subcellular localization, activity, and/or interaction with other proteins, we created, by site-directed mutagenesis, various deletion, truncation, and point mutations. We show here that membrane attachment is not required for the survival activity of bcl-2 alpha. A truncation mutant of bcl-2 alpha lacking the last 33 amino acids (T3.1) including the hydrophobic COOH terminus shows full activity in blocking apoptosis of nerve growth factor-deprived sympathetic neurons or TNF-alpha-treated L929 fibroblasts. Confocal microscopy reveals that the T3 mutant departs into the extremities of neurites in neurons and filopodias in fibroblasts. Consistently, T3 is predominantly detected in the soluble fraction by Western blotting, and is not inserted into microsomes after in vitro transcription/translation. We further provide evidence for motifs (S-N and S-II) at the NH2 and COOH terminus of bcl-2, which are crucial for its activity.

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis.

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.

The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event.

During apoptosis induced by various stimuli, cytochrome c is released from mitochondria into the cytosol where it participates in caspase activation. This process has been proposed to be an irreversible consequence of mitochondrial permeability transition pore opening, which leads to mitochondrial swelling and rupture of the outer mitochondrial membrane. Here we present data demonstrating that NGF-deprived sympathetic neurons protected from apoptosis by caspase inhibitors possess mitochondria which, though depleted of cytochrome c and reduced in size, remained structurally intact as viewed by electron microscopy. After re-exposure of neurons to NGF, mitochondria recovered their normal size and their cytochrome c content, by a process requiring de novo protein synthesis. Altogether, these data suggest that depletion of cytochrome c from mitochondria is a controlled process compatible with function recovery. The ability of sympathetic neurons to recover fully from trophic factor deprivation provided irreversible caspase inhibitors have been present during the insult period, has therapeutical implications for a number of acute neuropathologies.

Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis.

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.

Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions.

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A-sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.

Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene.

Approximately half of the neurons produced during embryogenesis normally die before adulthood. Although target-derived neurotrophic factors are known to be major determinants of programmed cell death--apoptosis--the molecular mechanisms by which trophic factors interfere with cell death regulation are largely unknown. Overexpression of the bcl-2 proto-oncogene in cultured sympathetic neurons has now been shown to prevent apoptosis normally induced by deprivation of nerve growth factor. This finding, together with the previous demonstration of bcl-2 expression in the nervous system, suggests that the Bcl-2 protein may be a major mediator of the effects of neurotrophic factors on neuronal survival.

Inhibition of Bax channel-forming activity by Bcl-2.

Proteins of the Bcl-2 family are intracellular membrane-associated proteins that regulate programmed cell death (apoptosis) either positively or negatively by as yet unknown mechanisms. Bax, a pro-apoptotic member of the Bcl-2 family, was shown to form channels in lipid membranes. Bax triggered the release of liposome-encapsulated carboxyfluorescein at both neutral and acidic pH. At physiological pH, release could be blocked by Bcl-2. Bcl-2, in contrast, triggered carboxyfluorescein release at acidic pH only. In planar lipid bilayers, Bax formed pH- and voltage-dependent ion-conducting channels. Thus, the pro-apoptotic effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2.

Cholinergic differentiation factor (CDF/LIF) promotes survival of isolated rat embryonic motoneurons in vitro.

We present evidence that the cholinergic differentiation factor (CDF), originally purified from cardiac and skeletal muscle cell-conditioned medium and found to be identical to leukemia inhibitory factor (LIF), promotes survival of embryonic day 14 rat motoneurons in vitro. These neurons were retrogradely labeled with the fluorescent tracer Dil and enriched on a density gradient or purified to homogeneity by fluorescence-activated cell sorting. Subnanomolar concentrations of CDF/LIF supported the survival of 85% of the motoneurons that would have died between days 1 and 4 of culture. The enhanced survival was accompanied by a 4-fold increase in choline acetyltransferase (ChAT) activity per culture. CDF/LIF also increased ChAT activity in dorsal spinal cord cultures, but had no detectable effect on ChAT levels in septal or striatal neuronal cultures. For comparison, other neurotrophic molecules were tested on motoneuron cultures. Ciliary neurotrophic factor had effects on motoneuron survival similar to those of CDF/LIF, whereas basic fibroblast growth factor was somewhat less effective. Nerve growth factor had no effect on the survival of rat motoneurons.

Overexpression of BCL-2 in transgenic mice protects neurons from naturally occurring cell death and experimental ischemia.

Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.

Mitochondria and cancer: is there a morphological connection?

Mitochondria are key players in several cellular functions including growth, division, energy metabolism, and apoptosis. The mitochondrial network undergoes constant remodelling and these morphological changes are of direct relevance for the role of this organelle in cell physiology. Mitochondrial dysfunction contributes to a number of human disorders and may aid cancer progression. Here, we summarize the recent contributions made in the field of mitochondrial dynamics and discuss their impact on our understanding of cell function and tumorigenesis.

Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane.

In many types of apoptosis, the proapoptotic protein Bax undergoes a change in conformation at the level of the mitochondria. This event always precedes the release of mitochondrial cytochrome c, which, in the cytosol, activates caspases through binding to Apaf-1. The mechanisms by which Bax triggers cytochrome c release are unknown. Here we show that following binding to the BH3-domain-only proapoptotic protein Bid, Bax oligomerizes and then integrates in the outer mitochondrial membrane, where it triggers cytochrome c release. Bax mitochondrial membrane insertion triggered by Bid may represent a key step in pathways leading to apoptosis.

Impairing the bioenergetic status and the biogenesis of mitochondria triggers mitophagy in yeast.

Autophagy, a highly regulated programme found in almost all eukaryotes, is mainly viewed as a catabolic process that degrades nonessential cellular components into molecular building blocks, subsequently available for biosynthesis at a lesser expense than de novo synthesis. Autophagy is largely known to be regulated by nutritional conditions. Here we show that, in yeast cells grown under nonstarving conditions, autophagy can be induced by mitochondrial dysfunction. Electron micrographs and biochemical studies show that an autophagic activity can result from impairing the mitochondrial electrochemical transmembrane potential. Furthermore, mitochondrial damage-induced autophagy results in the preferential degradation of impaired mitochondria (mitophagy), before leading to cell death. Mitophagy appears to rely on classical macroautophagy machinery while being independent of cellular ATP collapse. These results suggest that in this case, autophagy can be envisioned either as a process of mitochondrial quality control, or as an ultimate cellular response triggered when cells are overwhelmed with damaged mitochondria.

tBid interaction with cardiolipin primarily orchestrates mitochondrial dysfunctions and subsequently activates Bax and Bak.

TNFR1/Fas engagement results in the cleavage of cytosolic Bid to truncated Bid (tBid), which translocates to mitochondria. We demonstrate that recombinant tBid induces in vitro immediate destabilization of the mitochondrial bioenergetic homeostasis. These alterations result in mild uncoupling of mitochondrial state-4 respiration, associated with an inhibition the adenosine diphosphate (ADP)-stimulated respiration and phosphorylation rate. tBid disruption of mitochondrial homeostasis was inhibited in mitochondria overexpressing Bcl-2 and Bcl-XL. The inhibition of state-3 respiration is mediated by the reorganization of cardiolipin within the mitochondrial membranes, which indirectly affects the activity of the ADP/ATP translocator. Cardiolipin-deficient yeast mitochondria did not exhibit any respiratory inhibition by tBid, proving the absolute requirement for cardiolipin for tBid binding and activity. In contrast, the wild-type yeast mitochondria underwent a similar inhibition of ADP-stimulated respiration associated with reduced ATP synthesis. These events suggest that mitochondrial lipids rather than proteins are the key determinants of tBid-induced destabilization of mitochondrial bioenergetics.

ICE-like proteases execute the neuronal death program.

The past year has witnessed significant advances in our understanding of the mechanisms that kill neurons during programmed cell death. The executioners are members of a family of proteases founded by ced-3, the product of a gene that is required for programmed cell death in the nematode Caenorhabditis elegans, and by mammalian interleukin-1 beta-converting enzyme. These proteases represent interesting novel targets for the therapy of acute and chronic pathologies of the nervous system associated with neuronal death.