Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space

Apoptosis-inducing factor (AIF) is a mitochondrial intermembrane flavoprotein that is translocated to the nucleus in response to proapoptotic stimuli, where it induces nuclear apoptosis. Here we show that AIF is synthesized as an approximately 67-kDa preprotein with an N-terminal extension and imported into mitochondria, where it is processed to the approximately 62-kDa mature form. Topology analysis revealed that mature AIF is a type-I inner membrane protein with the N-terminus exposed to the matrix and the C-terminal portion to the intermembrane space. Upon induction of apoptosis, processing of mature AIF to an approximately 57-kDa form occurred caspase-independently in the intermembrane space, releasing the processed form into the cytoplasm. Bcl-2 or Bcl-XL inhibited both these events. These findings indicate that AIF release from mitochondria occurs by a two-step process: detachment from the inner membrane by apoptosis-induced processing in the intermembrane space and translocation into the cytoplasm. The results also suggest the presence of a unique protease that is regulated by proapoptotic stimuli in caspase-independent cell death.     

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.     

Cathepsin D triggers Bax activation,resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis

Activated human T lymphocytes exposed to apoptotic stimuli targeting mitochondria (i.e. staurosporine), enter an early, caspase-independent phase of commitment to apoptosis characterized by cell shrinkage and peripheral chromatin condensation. We show that during this phase, AIF is selectively released from the intermembrane space of mitochondria, and that Bax undergo conformational change, relocation to mitochondria, and insertion into the outer mitochondrial membrane, in a Bid-independent manner. We analyzed the subcellular distribution of cathepsins (Cat) B, D, and L, in a search for caspase-independent factors responsible for Bax activation and AIF release. All were translocated from lysosomes to the cytosol, in correlation with limited destabilization of the lysosomes and release of lysosomal molecules in a size selective manner. However, only inhibition of Cat D activity by pepstatin A inhibited the early apoptotic events and delayed cell death, even in the presence of bafilomycin A1, an inhibitor of vacuolar type H+-ATPase, which inhibits acidification in lysosomes. Small interfering RNA-mediated gene silencing was used to inactivate Cat D, Bax, and AIF gene expression. This allowed us to define a novel sequence of events in which Cat D triggers Bax activation, Bax induces the selective release of mitochondrial AIF, and the latter is responsible for the early apoptotic phenotype.     

Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death.

Although much emphasis has been laid on the role of caspase in cell death, recent data indicate that, in many instances, mammalian cell death is caspase-independent. Thus, in many examples of mammalian cell death the 'decision' between death and life is upstream or independent of caspase activation. Similarly, it is unclear whether PCD of plants and fungi involves the activation of caspase-like enzymes, and no caspase-like gene has thus far been cloned in these phyla. Apoptosis inducing factor (AIF) is a new mammalian, caspase-independent death effector which, upon apoptosis induction, translocates from its normal localization, the mitochondrial intermembrane space, to the nucleus. Once in the nucleus, AIF causes chromatin condensation and large scale DNA fragmentation to fragments of approximately 50 kbp. The AIF cDNA from mouse and man codes for a protein which possesses three domains (i) an amino-terminal presequence which is removed upon import into the intermembrane space of mitochondria; (ii) a spacer sequence of approximately 27 amino acids; and (iii) a carboxyterminal 484 amino acid oxidoreductase domain with strong homology to oxidoreductases from other vertebrates (X. laevis), non-vertebrate animals (C. elegans, D. melanogaster), plants, fungi, eubacteria, and archaebacteria. Functionally important amino acids involved in the interaction with the prosthetic groups flavin adenine nucleotide and nicotinamide adenine nucleotide are strongly conserved between AIF and bacterial oxidoreductase. Several eukaryotes possess a similar domain organisation in their AIF homologs, making them candidates to be mitochondrial oxidoreductases as well as caspase-independent death effectors. The phylogenetic implications of these findings are discussed.     

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.     

Down-regulation of apoptosis-inducing factor protein by RNA interference inhibits UVA-induced cell death

Apoptosis-inducing factor (AIF) is a caspase-independent apoptosis effector. UVA-induced Raji cell death was not completely inhibited by pan-caspase inhibitor zVAD.fmk. Moreover, AIF translocated from its normal location, the mitochondrial intermembrane space, into the nucleus, and induced peripheral chromatin condensation during the early stage of UVA-inducing cell death. Enforced expression of AIF can induce Raji cell death in a caspase-independent manner. Down-regulation of AIF protein level by RNA interference (RNAi) can reduce UVA-induced Raji cell death, but the combination of down-regulation of AIF and zVAD.fmk almost completely inhibited UVA-induced Raji cell death. All these suggest that caspase and AIF are two independent pathways and that UVA-induced Raji cell death is dependent on caspase and AIF.     

Dissociating the dual roles of apoptosis-inducing factor in maintaining mitochondrial structure and apoptosis

The mitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and induces apoptosis. Recent studies, however, have indicated the importance of AIF for survival in mitochondria. In the absence of a means to dissociate these two functions, the precise roles of AIF remain unclear. Here, we dissociate these dual roles using mitochondrially anchored AIF that cannot be released during apoptosis. Forebrain-specific AIF null (tel. AifDelta) mice have defective cortical development and reduced neuronal survival due to defects in mitochondrial respiration. Mitochondria in AIF deficient neurons are fragmented with aberrant cristae, indicating a novel role of AIF in controlling mitochondrial structure. While tel. AifDelta Apaf1(-/-) neurons remain sensitive to DNA damage, mitochondrially anchored AIF expression in these cells significantly enhanced survival. AIF mutants that cannot translocate into nucleus failed to induce cell death. These results indicate that the proapoptotic role of AIF can be uncoupled from its physiological function. Cell death induced by AIF is through its proapoptotic activity once it is translocated to the nucleus, not due to the loss of AIF from the mitochondria.     

Mitochondrial regulation of cell death: Processing of apoptosis-inducing factor (AIF)

Apoptosis might proceed through the activation of both caspase-dependent and -independent pathways. Apoptosis-inducing factor (AIF) was discovered as the first protein that mediated caspase-independent cell death. Initially, it was regarded as a soluble protein residing in the intermembrane space of mitochondria, from where it could be exported to the nucleus to participate in large-scale DNA fragmentation and chromatin condensation. However, later it was demonstrated that AIF is N-terminally anchored to the inner mitochondrial membrane. Hence, AIF must be liberated from its membrane anchor prior to being released into the cytosol. The current knowledge about the molecular mechanisms regulating the processing and release of AIF from the mitochondria will be summarized and discussed in this review.     

Zinc induced apoptosis in HEP-2 cancer cells: the role of oxidative stress and mitochondria

Induction of apoptosis by zinc sulfate was investigated during 96 h exposure on the cancer Hep-2 cell line. During 48 h of exposure, zinc translocated into mitochondria and stimulated production of reactive oxygen species (ROS), affected cellular GSH management and induced moderate activation of p53 and dissipation of mitochondrial membrane potential. In Zn-exposed cells, mitochondria released cytochrome c and AIF, whose translocation to the cytoplasm or the nucleus coincided with the activation of apoptosis. The use of various pharmacological inhibitors inhibiting particular apoptotic targets (antioxidants such as N-acetyl-cysteine and coenzyme Q, the caspase inhibitors z-DEVD-fmk and z-VAD-fmk, cyclosporin A and bonkgrekic acid) proved that Zn acts both directly and indirectly on mitochondria and observed apoptosis is executed by caspase-dependent and caspase-independent pathways.     

Role of apoptosis-inducing factor in myocardial cell death by ischemia-reperfusion

Although apoptosis contributes to myocardial cell death in the ischemia-reperfused heart, the molecular basis of apoptosis is poorly understood. Apoptosis-inducing factor (AIF) has been characterized as a caspase-independent death effector. Upon the induction of apoptosis, mitochondrial AIF is released to the cytoplasm and then enters the nucleus, in which it induces chromatin condensation and 50 kbp DNA fragmentation. In the present study, we examined the role of AIF in ischemia-reperfusion injury in isolated rat hearts. AIF was detected in the cytosolic and nuclear fractions of hearts subjected to ischemia-reperfusion, whereas it was detected only in the mitochondria of control hearts. Moreover, AIF release increased in a reperfusion time-dependent manner. Pulse field gel electrophoresis revealed that 50 kbp DNA fragments were produced by ischemia/reperfusion. In contrast, cytochrome c release and the activation of caspase-3 did not occur to a significant extent. Moreover, ischemic preconditioning attenuated the AIF release and the 50 kbp DNA fragmentation. These results suggest that AIF-dependent apoptosis is likely to attribute to myocardial cell death in the ischemia-reperfused heart and that it is related with the protective effect of ischemic preconditioning.     

Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis.

Apoptosis inducing factor (AIF) is a novel apoptotic effector protein that induces chromatin condensation and large-scale ( approximately 50 kbp) DNA fragmentation when added to purified nuclei in vitro. Confocal and electron microscopy reveal that, in normal cells, AIF is strictly confined to mitochondria and thus colocalizes with heat shock protein 60 (hsp60). On induction of apoptosis by staurosporin, c-Myc, etoposide, or ceramide, AIF (but not hsp60) translocates to the nucleus. This suggests that only the outer mitochondrial membrane (which retains AIF in the intermembrane space) but not the inner membrane (which retains hsp60 in the matrix) becomes protein permeable. The mitochondrio-nuclear redistribution of AIF is prevented by a Bcl-2 protein specifically targeted to mitochondrial membranes. The pan-caspase inhibitor Z-VAD. fmk does not prevent the staurosporin-induced translocation of AIF, although it does inhibit oligonucleosomal DNA fragmentation and arrests chromatin condensation at an early stage. ATP depletion is sufficient to cause AIF translocation to the nucleus, and this phenomenon is accelerated by the apoptosis inducer staurosporin. However, in conditions in which both glycolytic and respiratory ATP generation is inhibited, cells fail to manifest any sign of chromatin condensation and advanced DNA fragmentation, thus manifesting a 'necrotic' phenotype. Both in the presence of Z-VAD. fmk and in conditions of ATP depletion, AIF translocation correlates with the appearance of large-scale DNA fragmentation. Altogether, these data are compatible with the hypothesis that AIF is a caspase-independent mitochondrial death effector responsible for partial chromatinolysis.     

Different patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells

Cisplatin (cisPt) is a chemotherapeutic drug used for several human malignancies. CisPt cytotoxicity is primarily mediated by its ability to cause DNA damage and subsequent apoptotic cell death. DNA is the primary target of cisPt; however, recent data have shown that cisPt may have important direct interactions with mitochondria, which can induce apoptosis and may account for a significant part of the clinical activity associated with this drug. We have previously demonstrated that in the rat neuronal cell line B50, at 20 h-treatment with cisPt activates apoptosis through an intrinsic pathway involving an alteration of mitochondrial membrane permeability and the release of cytochrome c. The present study investigates different death pathways induced in the same cell line by a prolonged treatment with 40 μM cisPt for 48 h. To address this issue, we focused on caspases-8 and -12, and on the mitochondrial apoptosis inducing factor (AIF), which translocates to the nucleus and induces cell death via caspase-independent pathway. We found that cisPt activates different forms of cell death, i.e. the receptor-mediated apoptotic extrinsic pathway and a death process mediated by endoplasmic reticulum stress. Moreover, we demonstrated that AIF-mediated death occurs, being characterized by the translocation of AIF from mitochondria to the nucleus. On the whole, we provided evidence that prolonged cisPt treatment is able to activate both caspase-dependent and caspase-independent apoptotic pathways in B50 rat neuronal cells.     

Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.     

Caspase-independent apoptotic pathways in T lymphocytes: a minireview

Cell death by apoptosis is involved in the maintenance of T cell receptor diversity, self tolerance, and T-cell number homeostasis. Until recently, apoptosis was thought to require caspase activation. Evidence is now accumulating that a caspase-independent pathway exists, shown by in vitro experiments with broad-range caspase inhibitors. Mature T lymphocytes readily undergo caspase-independent apoptosis in vitro, and recent data suggest that this type of apoptosis may be involved in the negative selection of thymocytes. Mitochondria likely release death triggers specific for both caspase-dependent and caspase-independent apoptotic pathways (cytochrome c and AIF respectively) in response to apoptotic stimuli. A caspase-independent pathway is triggered first in activated T lymphocytes subjected to apoptotic stimuli that do not rely on receptors with death domains. In this pathway, the early commitment phase to apoptosis involves cell shrinkage, peripheral DNA condensation and the translocation of mitochondrial AIF to the cytosol and nucleus. This process is reversible until mitochondrial cytochrome c is released and m dissipated. Only at this stage are caspases activated.     

Involvement of hydroperoxide in mitochondria in the induction of apoptosis by the eicosapentaenoic acid

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 μM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.     

Wild-type herpes simplex virus 1 blocks programmed cell death and release of cytochrome c but not the translocation of mitochondrial apoptosis-inducing factor to the nuclei of human embryonic lung fibroblasts

Programmed cell death activated by herpes simplex virus 1 mutants can be caspase dependent or independent depending on the nature of the infected cell. The recently discovered mitochondrial apoptosis-inducing factor (AIF) on activation is translocated to the nucleus and induces programmed cell death that is caspase independent. To assess the role of AIF and also to assay apoptosis-related events in primary human embryonic lung (HEL) fibroblasts, cells were mock infected or infected with wild-type virus previously shown not to induce apoptosis in continuous lines of primate cells or with the d120 mutant lacking infected cell protein no. 4 (ICP4) and were shown to induce apoptosis in all cell lines tested. Cells exposed to dexamethasone or osmotic shock induced by sorbitol were the positive controls. The results were as follows: (i) AIF was translocated to the nucleus in all infected cell cultures and in cells treated with dexamethasone or sorbitol, but cells infected with the wild type-virus showed no evidence of undergoing programmed death. (ii) Cytochrome c was released from mitochondria of cells infected with the d120 mutant or exposed to dexamethasone or sorbitol but not from mitochondria in cells treated with sorbitol and infected with the wild-type virus. (iii) Poly(ADP-ribose) polymerase was cleaved in mock-infected cells exposed to sorbitol or dexamethasone and in cells infected with the d120 mutant but not in either untreated cells infected with wild-type virus or cells exposed to sorbitol and then infected with wild-type virus. In contrast to HEp-2 cells, neither d120 infection nor treatment with dexamethasone or sorbitol caused fragmentation of DNA in HEL fibroblasts. Electron microscopic examination showed chromatin condensation and vacuolization in a fraction of cells infected with d120 but not in wild-type virus-infected cells or cells treated with dexamethasone or sorbitol. We conclude that AIF is translocated to the nucleus in infected cells but apoptosis does not ensue in wild-type-infected cells. HEL fibroblasts infected with the d120 virus exhibit symptoms of classical apoptosis, such as cytochrome c release and cleavage of poly(ADP-ribose) polymerase observed also in cells undergoing caspase 3-dependent programmed cell death in which AIF is either not involved or not a contributory factor.     

Rapid reactive oxygen species (ROS) generation induced by curcumin leads to caspase-dependent and -independent apoptosis in L929 cells

Evidence that curcumin may have anticancer activities has renewed interest in its potential to prevent and treat disease. In this study, we show that curcumin-mediated rapid generation of reactive oxygen species (ROS) leads to apoptosis by modulating different apoptotic pathways in mouse fibroblast L929 cells. We show for the first time that curcumin-induced rapid ROS generation causes the release of apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus, hence, leading to caspase 3-independent apoptosis. However, our studies also show that curcumin induces the release of cytochrome c from mitochondria, causing activation of caspase 3, and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Furthermore, curcumin-induced ROS generation leads to the induction of the proapoptotic protein p53 and its effector protein p21 and down-regulation of cell cycle regulatory proteins such as Rb and cyclin D1 and D3. Both glutathione (GSH) and N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of curcumin-induced ROS generation, AIF release from mitochondria, and caspase activation. Additionally, pretreatment of L929 cells with these antioxidants completely blocked the induction of p53-dependent p21 accumulation. In conclusion, our data show that in addition to caspase 3 activation, curcumin-induced rapid ROS generation leads to AIF release, and the activation of the caspase-independent apoptotic pathway.     

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid-mediated apoptosis through Bak activation in ovarian cancer cells

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.