Luigi Ravagnan

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death

Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome—a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies—the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

Hsp27 negatively regulates cell death by interacting with cytochrome c

Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

Heat-shock protein 70 antagonizes apoptosis-inducing factor.

Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex. Here we show that overexpression of Hsp70 protects Apaf-1−/− cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.

Apoptosis‐inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis

Apoptosis‐inducing factor (AIF) is encoded by one single gene located on the X chromosome. AIF is ubiquitously expressed, both in normal tissues and in a variety of cancer cell lines. The AIF precursor is synthesized in the cytosol and is imported into mitochondria. The mature AIF protein, a flavoprotein (prosthetic group: flavine adenine dinucleotide) with significant homology to plant ascorbate reductases and bacterial NADH oxidases, is normally confined to the mitochondrial intermembrane space. In a variety of different apoptosis‐inducing conditions, AIF translocates through the outer mitochondrial membrane to the cytosol and to the nucleus. Ectopic (extra‐mitochondrial) AIF induces nuclear chromatin condensation, as well as large scale (∼50 kb) DNA fragmentation. Thus, similar to cytochrome c, AIF is a phylogenetically old, bifunctional protein with an electron acceptor/donor (oxidoreductase) function and a second apoptogenic function. In contrast to cytochrome c, however, AIF acts in a caspase‐independent fashion. The molecular mechanisms via which AIF induces apoptosis are discussed.

Mitochondria, the killer organelles and their weapons

Apoptosis is a cell‐autonomous mode of death that is activated to eradicate superfluous, damaged, mutated, or aged cells. In addition to their role as the cells powerhouse, mitochondria play a central role in the control of apoptosis. Thus, numerous pro‐apoptotic molecules act on mitochondria and provoke the permeabilization of mitochondrial membranes. Soluble proteins contained in the mitochondrial intermembrane space are released through the outer membrane and participate in the organized destruction of the cell. Several among these lethal proteins can activate caspases, a class of cysteine proteases specifically activated in apoptosis, whereas others act in a caspase‐independent fashion, by acting as nucleases (e.g., endonuclease G), nuclease activators (e.g., apoptosis‐inducing factor), or serine proteases (e.g., Omi/HtrA2). In addition, mitochondria can generate reactive oxygen species, following uncoupling and/or inhibition of the respiratory chain. The diversity of mitochondrial factors participating in apoptosis emphasizes the central role of these organelles in apoptosis control and unravels novel mechanisms of cell death execution.

Apoptosis-inducing factor (AIF): a novel caspase-independent death effector released from mitochondria.

Apoptosis-inducing factor (AIF) is a phylogenetically ancient mitochondrial intermembrane flavoprotein endowed with the unique capacity to induce caspase-independent peripheral chromatin condensation and large-scale DNA fragmentation when added to purified nuclei. In addition to its apoptogenic activity on nuclei, AIF can also participate in the regulation of apoptotic mitochondrial membrane permeabilization and exhibits an NADH oxidase activity. Under normal circumstances, AIF is secluded behind the outer mitochondrial membrane. However, upon apoptosis induction AIF translocates to the cytosol and the nucleus. Injection of anti-AIF antibodies or knockout of the AIF gene have demonstrated that AIF may be required for cell death occurring in response to some stimuli. In particular, inactivation of AIF renders embryonic stem cells resistant to cell death following growth factor withdrawal. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies, the very first wave of (caspase-independent) cell death indispensable for mouse morphogenesis. We have recently found that AIF is neutralized by heat-shock protein (HSP) 70, in a reaction that appears to be independent of ATP or the ATP-binding domain (ABD) of HSP70 and thus differs from the previously described Apaf-1/HSP70 interaction (which requires ATP and the HSP70 ABD). Intriguingly, HSP70 lacking ABD (HSP70 Delta ABD) inhibits apoptosis induced by serum withdrawal, staurosporin, and menadione, three models of apoptosis which are also affected by micro-injection of anti-AIF antibody or genetic ablation of AIF. Altogether, these data suggest that AIF plays a role in the regulation of caspase-independent cell death.

Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore.

The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal effects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing effect of LND is amplified by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective effect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All effects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the effect of LND on the purified PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This effect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosis-inhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct effect on the mitochondrial PT pore.

GD3 ganglioside directly targets mitochondria in a bcl-2-controlled fashion

Lipid and glycolipid diffusible mediators are involved in the intracellular progression and amplification of apoptotic signals. GD3 ganglioside is rapidly synthesized from accumulated ceramide after the clustering of death‐inducing receptors and triggers apoptosis. Here we show that GD3 induces dissipation of ΔΨm and swelling of isolated mitochondria, which results in the mitochondrial release of cytochrome c, apoptosis inducing factor, and caspase 9. Soluble factors released from GD3‐treated mitochondria are sufficient to trigger DNA fragmentation in isolated nuclei. All these effects can be blocked by cyclosporin A, suggesting that GD3 is acting at the level of the permeability transition pore complex. We found that endogenous GD3 accumulates within mitochondria of cells undergoing apoptosis after ceramide exposure. Accordingly, suppression of GD3 synthase (ST8) expression in intact cells substantially prevents ceramide‐induced ΔΨm dissipation, indicating that endogenously synthesized GD3 induces mitochondrial changes in vivo. Finally, enforced expression of bcl‐2 significantly prevents GD3‐induced mitochondrial changes, caspase 9 activation, and apoptosis. These results show that mitochondria are a key destination for apoptogenic GD3 ganglioside along the lipid pathway to programmed cell death and indicate that relevant GD3 targets are under bcl‐2 control.—Rippo, M. R., Malisan, F., Ravagnan, L., Tomassini, B., Condo, I., Costantini, P., Susin, S. A., Rufini, A., Todaro, M., Kroemer, G., Testi, R. GD3 ganglioside directly targets mitochondria in a bcl‐2‐controlled fashion. FASEB J. 14, 2047–2054 (2000)

Caspases disrupt mitochondrial membrane barrier function

Mitochondrial intermembrane proteins including cytochrome c are known to activate caspases. Accordingly, a disruption of the mitochondrial membrane barrier function with release of cytochrome into the cytosol has been shown to precede caspase activation in a number of different models of apoptosis. Here, we addressed the question of whether caspases themselves can affect mitochondrial membrane function. Recombinant caspases were added to purified mitochondria and were found to affect the permeability of both mitochondrial membranes. Thus, caspases cause a dissipation of the mitochondrial inner transmembrane potential. In addition, caspases cause intermembrane proteins including cytochrome c and AIF (apoptosis‐inducing factor) to be released through the outer mitochondrial membrane. These observations suggest that caspases and mitochondria can engage in a circular self‐amplification loop. An increase in mitochondrial membrane permeability would cause the release of caspase activators, and caspases, once activated, would in turn increase the mitochondrial membrane permeability. Such a self‐amplifying system could accelerate the apoptotic process and/or coordinate the apoptotic response between different mitochondria within the same cell.

Pre-processed caspase-9 contained in mitochondria participates in apoptosis

As shown here, mitochondria purified from different organs (liver, brain, kidney, spleen and heart) contain both pro-caspase-9 and the processed, mature form of caspase-9. Purified liver mitochondria release mature caspase-9 upon induction of permeability transition in vitro. This is accompanied by a discrete increase in the enzymatic cleavage of pro-caspase-9 substrates. We found that SHEP neuroblastoma cells constitutively contain pre-processed caspase-9 in their mitochondria, using a combination of subcellular fractionation and immunofluorescence with an antibody specific for the processed caspase. This is a cell type-specific phenomenon since HeLa cells mitochondria mainly contain pro-caspase-9 and comparatively little processed caspase-9. Upon introduction of apoptosis, mitochondrial pro-caspase-9 translocates to the cytosol and to the nucleus. This phenomenon is inhibited by transfection with Bcl-2. In synthesis, we report the unexpected finding that mitochondria can contain a pre-processed caspase isoform in non-apoptotic cells. Bcl-2-mediated regulation of mitochondrial membrane permeabilization may contribute to apoptosis control by preventing mitochondrial, pre-processed caspase-9 from interacting with its cytosolic activators.