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Keywords:

  • AIF;
  • apoptosis;
  • BAX;
  • BCL-2;
  • calpains;
  • caspase-independent cell death;
  • DNA-damage;
  • mitochondria;
  • MNNG;
  • necroptosis;
  • PARP-1;
  • programmed necrosis

Abstract

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

Cell death has been initially divided into apoptosis, in which the cell plays an active role, and necrosis, which is considered a passive cell death program. Intense research performed in the last decades has concluded that “programmed” cell death (PCD) is a more complex physiological process than initially thought. Indeed, although in most cases the PCD process is achieved via a family of Cys proteases known as caspases, an important number of regulated PCD pathways do not involve this family of proteases. As a consequence, active forms of PCD are initially referred to as caspase-dependent and caspase-independent. More recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis). The existence of necroptotic forms of death was corroborated by the discovery of key executioners such as the kinase RIP1 or the mitochondrial protein apoptosis-inducing factor (AIF). AIF is a Janus protein with a redox activity in the mitochondria and a pro-apoptotic function in the nucleus. We have recently described a particular form of AIF-mediated caspase-independent necroptosis that also implicates other molecules such as PARP-1, calpains, Bax, Bcl-2, histone H2AX, and cyclophilin A. From a therapeutic point of view, the unraveling of this new form of PCD poses a question: is it possible to modulate this necroptotic pathway independently of the classical apoptotic paths? Because the answer is yes, a wider understanding of AIF-mediated necroptosis could theoretically pave the way for the development of new drugs that modulate PCD. To this end, we present here an overview of the current knowledge of AIF and AIF-mediated necroptosis. We also summarize the state of the art in some of the most interesting therapeutic strategies that could target AIF or the AIF-mediated necroptotic pathway. © 2011 IUBMB IUBMB Life, 63(4): 221–232, 2011


APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

Probably the result of a symbiosis between an ancestral eukaryotic cell and a eubacterion, mitochondria are important organelles with a dual/paradoxical function. Indeed, they are involved in life by their role in oxidative phosphorylation, and they are implicated in death by the release of apoptogenic factors. Among these factors, cytochrome c and AIF play a dual role: they are related to both the mitochondrial respiration machinery and to cell death. Interestingly, while cytochrome c is involved in caspase-dependent classical apoptosis, AIF is mostly associated with caspase-independent necroptosis (or programmed necrosis).

AIF is a phylogenetically old protein essential for life and death. It is essential for life because of its critical role as a mitochondrial oxydoreductase, and it is essential for death due to its proapoptotic nuclear activity. AIF is synthesized from a nuclear gene as a precursor form of 67 kDa (Fig. 1). It is imported to mitochondria by two mitochondrial localization sequences (MLS) that are placed within the N-terminal pro-domain of the protein. Upon being imported into mitochondria, this precursor is processed to a mature form of 62 kDa by proteolytic cleavage. On this configuration, AIF is an inner membrane-anchored protein whose N-terminus part is exposed to the mitochondrial matrix and whose C-terminal portion is exposed to the mitochondrial intermembrane space (1). The mature form of AIF comprises three structural domains: a FAD-binding domain, a NADH-binding domain, and a C-terminal domain (2–4). In the same way as other flavoproteins, the oxydoreductase part of AIF (composed by the NADH- and FAD-binding domains) adopts a typical Rossmann fold conferring an electron transfer activity to the protein. FAD-bound AIF can be reduced by NAD(P)H without accumulation of a semiquinone intermediate. Furthermore, reduced AIF can catalyze a NADH-dependent reduction of small molecules, such as cytochrome c (5). Despite important advances, the enzymatic function of AIF has not been totally unraveled. Recent in vitro studies on naturally folded AIF indicated that, in its reduced form, AIF is a dimer (6, 7). These works suggested that the balance and transition between the dimeric and the monomeric state could influence both AIF redox and apoptogenic activities.

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Figure 1. Schematic representation of AIF-mediated caspase-independent PCD. AIF is transcribed and translated in a 67 kDa precursor protein carrying a N-terminal MLS (amino acids 1–100), a spacer sequence (aa 101–121), a FAD-binding domain (aa 122–262 and 400–477), a NADH-binding domain (aa 263–399), and a C-terminal domain (aa 478–613). Upon being imported into mitochondria, the MLS sequence is removed by a mitochondrial endopeptidase and AIF changes its configuration by incorporating FAD. The mature protein (62 kDa) is then anchored to the inner mitochondrial membrane by its transmembrane domain. In this form, AIF plays a vital function related to the respiratory chain stability and/or to maintenance of the mitochondrial structure. Upon various stimuli, AIF is cleaved and released from mitochondria to the cytosol. Cystein proteases such as calpains (cytosolic and/or mitochondrial) and cathepsins could cleave AIF, yielding the truncated form of the protein, tAIF (57 kDa). Different proteins, such as the proapoptotic Bcl-2 member Bax, regulate tAIF release through the formation of the outer mitochondrial membrane pores. Once in the cytosol, tAIF interacts with different targets. For instance, AIF may inhibit protein translation through its association with eIF3g and it could stimulate the activity of the lipid translocase scramblase 1 (SCRM1), which is responsible for classical PCD phosphatidyl serine exposure. In PCD, AIF is particularly known for translocating from the cytosol to the nucleus, where it induces chromatinolysis. This relocalization is positively regulated by cyclophilin A (CypA) and negatively regulated by heat shock protein 70 (Hsp70). Once in the nucleus, tAIF associates with phosphorylated histone H2AX (γH2AX), CypA, or endonuclease G (EndoG) to provoke chromatin condensation and DNA degradation. In glutamate excitotoxicity-induced parthanatos in brain, a 62 kDa form of AIF is associated to the outer mitochondrial membrane. When the PAR polymers generated by the nuclear protein PARP-1 are present, this AIF pool is released from the mitochondria and reaches the nucleus to provoke chromatinolysis via an unknown mechanism. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The C-terminal domain (amino acids 508–612) has become the most captivating part of AIF after recent data confirmed that this region encloses its pro-apoptotic function (8–11) (Fig. 1). These data indicate that the vital (mitochondrial oxydoreductase) and lethal (nuclear proapoptotic) function of AIF could be dissociated. The C-terminal domain displays a particular folding consisting of five anti-parallel β-strands, two α-helices and a large loop, exclusive to AIF (amino acids 509–559). This insertion includes a PEST motif followed by a Proline-rich module (PPSAPAVPQVP), usually involved in protein-protein interactions. As we have recently demonstrated, this Proline-rich domain constitutes a key element in the chromatinolytic/pro-apoptotic AIF function (11).

AIF ACROSS THE SPECIES

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

AIF is a highly conserved protein present in all primary kingdoms. It shares a highly significant homology with different families of oxydoreductases, from Archaea and Bacteria to invertebrates and vertebrates (4, 12) (Table 1). From ciliated protozoan to mammals, AIF loss is associated with growth retardation at different stages of animal development (14–17). Therefore, AIF is an important element of the apoptotic machinery that follows the rules of standard evolution (25). This means that the AIF gene is inherited from the last universal common ancestor and follows the tree topology with the primary radiation of the archaeo-eukaryotic and bacterial clades. Thus, if the implication of AIF orthologs in death processes is confirmed in the future, AIF could represent one of the oldest tool to die and/or, in ancestral microspecies, to kill.

Table 1. AIF knockout experimental models. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Studies on invertebrates have made it possible to identify some downstream effectors of AIF. The works on C. elegans, S.cereviseae, and D. melanogaster are of particular interest. In the case of C. elegans, the AIF ortholog WAH-1 has been demonstrated to interact with CSP-6 (the mammalian ortholog of the Endonuclease G), leading to nuclear DNA degradation (15). In the same species, AIF (WAH-1) may also associate with and stimulate the phospholipid scramblase Scrm-1 to promote externalization of phosphatidylserine (PS) on the surface of apoptotic cells (26). In S. cereviseae, it has been demonstrated that CypA is also essential for AIF-proapoptotic activity (13). In this eukaryote, Kim et al. (27) have discovered an interaction between the N-terminus of eIF3g and the AIF C-terminal region. Through this link, AIF could regulate protein synthesis during “programmed” cell death (PCD). In D. melanogaster, it has been found that the downregulation of the redox protein thioredoxin-2 (DmTrx-2) suppresses AIF-mediated PCD (16). Finally, the full deletion of AIF in mice induces a premature arrest in development (at larvae or embryo stages), demonstrating an important role of AIF in embryogenesis. Efforts to produce Aif-null mice were however unsuccessful (23, 24). An initial study indicating that disruption of the AIF gene prevents the first wave of caspase-independent-PCD was contradicted by a more recent work demonstrating that the AIF function is not required for cell death in early mouse embryos. Nevertheless, in this latter study, loss of AIF caused abnormal cell death on embryonic day 9 (E9) (Table 1). Unfortunately, complete deletion of AIF in these experimental mouse models induces loss of both pro-apoptotic and oxidoreductase functions. Thus, it is not clear whether the lethality associated to these models is due to the lack of caspase-independent-PCD or to the alteration of the mitochondrial function (see below).

VITAL FUNCTION OF AIF

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

How AIF exerts its vital properties in mitochondria is still a matter of intense debate (7). However, it seems that the AIF redox function is indispensable for cell life. Studies in the harlequin (Hq) AIF-mutant mouse, which has an 80% reduction in AIF expression, have suggested that this protein acts as a free radical scavenger (17, 20) (Table 1). Apart from oxidative stress sensitivity, complementary observations have demonstrated that AIF-deficiency compromises oxidative phosphorylation. On the one hand, it seems that AIF is required for the assembly and stability of complex I and III of the mitochondrial respiratory chain (28, 29). On the other hand, AIF deletion impairs the function of the respiratory chain via its role on maintaining mitochondrial morphology (8). Consequently, patients affected by a mitochondrial encephalopathy linked to an AIF mutation exhibit abnormal mitochondria (30).

REGULATION OF MITOCHONDRIAL AIF RELEASE

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

As indicated above, because AIF is imbedded into the inner mitochondrial membrane, the release from mitochondria requires a caspase-independent proteolytic cleavage (Fig. 1). As described recently (1, 31, 32), the 62 kDa AIF-mitochondrial form is cleaved at position G102/L103 (in mouse) to yield a soluble proapoptotic protein (truncated AIF, tAIF) with an apparent molecular weight of 57 kDa.

Although several factors are involved in AIF release mechanisms (see next paragraph), the current status of knowledge establishes that two major elements take part in the AIF proteolytic process: (i) two families of cysteine proteases: calpains and cathepsins. Calpains regulate AIF cleavage in a Ca2+-dependentcontext. In contrast, calcium is not involved in the cathepsin-mediated control of AIF activation; (ii) the activation of proapoptotic molecules from the Bcl-2 family, such as Bax or Bid (32–37) (Fig. 1). Both systems are not necessarily independent, since some pro-apoptotic Bcl-2-like proteins can be activated by cysteine proteases. For example, Bidere et al. (38) demonstrated that, upon staurosporine treatment, cathepsin D was released from lysosomes in T cells to induce a Bax conformational change, relocation to mitochondria, and insertion into the outer mitochondrial membrane. Other works in DNA-damage models reported that mitochondrial AIF-release requires a cooperative upstream action of calpains and Bax. In this case, calpains cleave AIF into tAIF whereas Bax facilitates the mitochondrial outer membrane permeabilization required for AIF release (33, 39). This is in line with other findings showing a relationship between AIF-release and Bax activation in camptothecin-mediated PCD (40). It has been suggested that cytosolic calpains would require Bax pores to reach AIF in mitochondria. Recent studies identifying mitochondrial calpains tend to rule out this hypothesis in favor of a role of Bcl-2 proteins in AIF release after cleavage. As in caspase-dependent apoptotic pathways, the exact mechanisms underlying mitochondria permeabilization leading to AIF release remain poorly defined. Ozaki et al. (41) recently described VDAC cleavage by mitochondrial calpains and subsequent Bax binding to VDAC. They suspect these two molecules form a sufficiently large channel for the release of AIF. Nonetheless, this might not be the sole way to let AIF join the cytosol. Further studies should contribute to a deeper understanding of the functions of Bax and the other Bcl-2 proteins in the regulation of AIF release from mitochondria.

Once released from mitochondria to the cytosol tAIF translocates to the nucleus in a process positively regulated by cyclophilin A (CypA) and negatively by Hsp70 (42, 43) (Fig. 1). The relationship between these two proteins and AIF was confirmed by systematic deletions. This approach confirms that the Hsp70 and CypA binding domains localize respectively at amino acids 150–228 and 367–399 of human AIF (42, 44).

LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

Once it has arrived at the nucleus, AIF could theoretically interact with DNA and/or RNA to cause caspase-independent chromatinolysis (44–48). Interestingly, given that AIF does not display any intrinsic endonuclease activity by itself, the AIF-mediated DNA degradation depends on the recruitment of downstream nucleases. Studies on C. elegans have demonstrated the cooperation between AIF and Endonuclease G, a mitochondrial DNAse also released in a caspase-independent manner (15). In mammals, this cooperation has been initially reported in caspase-independent mitotic death (49, 50). Moreover, it has been demonstrated that the DNA-degrading capacity of AIF, at least in alkylating DNA damage-induced necroptosis (see below), is related to the capacity of AIF to organize a DNA-degrading complex with histone H2AX and CypA (11, 51). In other PCD systems, AIF could also interact directly with CypA to induce death (43, 44) (Fig. 1).

TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

As explained above, given that the translocation of AIF from mitochondria to the nucleus occurs in a large variety of caspase-independent PCD systems, AIF is considered as a main effector of this mode of PCD (47, 48, 52).

Strong evidence of the role of AIF in caspase-independent PCD has been shown in studies performed with excitotoxins or alkylating DNA damage agents (33, 53, 54). Here, the relationship between AIF and PARP-1 has been broadly established, and PARP-1 inhibitors, PARP-1 genetic ablation, or neutralizing anti-AIF antibodies have been demonstrated to inhibit excitotoxins or alkylating DNA damage-induced PCD (33, 53, 55). In spite of that, there are significant differences between these two modes of AIF-mediated death. In NMDA and glutamate excitotoxicity-induced death in brain, the uncleaved AIF form (62 kDa) is released from mitochondria by a mechanism implicating the PAR polymers generated by PARP-1 (56–58). An AIF pool localized in the outer mitochondrial membrane controls this type of PCD, named parthanatos (59). On the contrary, in alkylating DNA damage mediated necroptosis (see section below), AIF needs to be cleaved by the calpain Cys proteases into tAIF (57 kDa) to be released from mitochondria and to provoke PCD (33) (Fig. 1). Thus, even if parthanatos and alkylating DNA damage mediated necroptosis implicate PARP-1 and AIF they represent two alternative caspase-independent PCD pathways. This is substantiated by the fact that: (i) in parthanatos the calpain cys proteases are dispensable enzymes (58), (ii) in alkylating DNA damage-mediated necroptosis the PAR polymers do not play a relevant role. These polymers, which are efficiently generated by PARP-1 in calpain, Bax, or H2AX-deficient cells, are unable to induce AIF-release and PCD (11, 33).

AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

As a counterpart to uncontrolled necrosis, the term “programmed necrosis” (further named necroptosis) was introduced to define the forms of PCD with necrotic morphology (60–62). The first described pathway leading to necroptosis is initiated by ligation of TNFR1 (63, 64). It was first observed that, depending on the cell status, TNF administration result in caspase-dependent or caspase-independent PCD (64). Further studies of the TNFR1-mediated necroptotic pathway identified necrostatin-1, a blocker of the receptor-interacting protein 1 (RIP1) kinase activity that inhibits necroptosis (61). Necrostatin-1 abolishes the assembly of the RIP1/RIP3 complex, suggesting that the activities of these kinases are required in this necrotic form of death (65, 66).

More recently, new forms of necroptosis have been unveiled (67–71). One of them is the pathway scrutinized in our laboratory: alkylating DNA damage (MNNG)-mediated PCD (11, 33, 39, 51). In response to high levels of DNA alkylation, PARP-1 activation stimulates the release of AIF from the mitochondrial intermembrane space, a process that depends on calpain Cys proteases and the proapoptotic Bcl-2 member Bax. Once in the cytosol, AIF rapidly relocalizes to the nuclear compartment where, in cooperation with histone H2AX and cyclophilin A, provokes DNA degradation and PCD (Fig. 1). As indicated above, to induce chromatinolysis, AIF interacts with phosphorylated histone H2AX and CypA (11, 51). This interaction involves the C-terminal Proline-rich module of AIF. This multi-protein complex constitutes an authentic “DNA degradosome,” where CypA should disclose the endonuclease activity. Of note, AIF interacts with H2AX even in the absence of CypA, while the inverse is not the case, hence suggesting a sequential interaction between the three partners (11). In MNNG-mediated PCD induced in mouse embryonic fibroblasts (MEFs), the kinase RIP1 plays a critical role. Indeed, RIP1 genetic ablation precludes the mitochondrial alterations characterizing this type of necroptosis (e.g., ΔΨm loss and AIF release). Consequently, RIP1-deficient MEFs are resistant to MNNG-induced PCD (55). The relationship between RIP1 and AIF has been fully confirmed, by using necrostatin-1 inhibitor in other necroptotic systems, such as retinal detachment-induced photoreceptor necrosis or glutamate-induced oxytosis in hippocampal HT-22 cells (71, 72).

RIP1 is therefore a milestone in both TNFR1- and MNNG-mediated necroptosis. However, TNFR1-mediated necroptosis cannot be blocked by inhibition of calpains and PARP-1 or by downregulation of AIF (61), whereas MNNG-mediated necroptosis is not abolished in TNFR1-deficient MEFs (55). This indicates that these two necroptotic models represent alternate outcomes of a RIP1 and mitochondrial-controlled mode of PCD.

THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

Regardless of the form of death involved, the misunderstanding of PCD rules has pathological repercussions. It is well known that apoptosis defects entail cell proliferation, causing cancer or autoimmune diseases. Contrariwise, too much apoptosis could damage vital organs or tissues, as in the case of degenerative diseases. Therefore, therapeutic control over cell life or death can provide attractive and powerful tools for the treatment of a large panoply of pathologies.

In the past few years, therapeutic strategies based on the control of death have essentially targeted the caspases, the main regulators of caspase-dependent PCD. However, at present it is clear that the apoptosis/necroptosis dichotomy does not necessarily involve a mutual exclusion of both mechanisms. Caspases are neither alone nor unique in the cell death scene. In line with this view, emerging protocols targeting caspase-independent systems are being developed for clinical trials. These strategies follow two major approaches: (i) the inhibition of AIF-mediated PCD, precluding the excessive PCD observed in ischemic or degenerative processes; (ii) the activation of the AIF caspase-independent necrotic cell death pathway to treat uncontrolled proliferate disorders such as cancer. Representative examples of the current therapeutic strategies discussed below are provided in Table 2.

Table 2. Therapeutics involving AIF-mediated programmed cell death
CompoundTreatmentPhasePrincipleEffectsAssociationSponsor
  1. Sponsor abbreviations: MDACC, MD Anderson Cancer Center; UUWSM, Urologische Universitatätsklinik im Waldkrankenhaus St. Marien; MIC, Marseille Institute of Cancer; MSKCC, Memorial Sloan-Kettering Cancer Center; NCI-SB, National Cancer Institute Surgery Branch; CALGB, Cancer and Leukemia Group B; GOG-UCh, Gynecologic Oncology Group, University of Chicago; NCI-MB, National Cancer Institute Medicine Branch; DFCI, Dana-Farber Cancer Institute; MGH, Massachusetts General Hospital; NIH-F, Foundation for the National Institutes of Health; NCI-RTO, Radiation Therapy Oncology Group/NCI; BRI, Beckman Research Institute; CR-UK, Cancer Research UK.

Bezielle (BZL101)Metastatic breast cancerIIStrong ROS induction, DNA damageAIF release Bionovo
Bobel-24Pancreatic cancerI   Bionovo
Pancreatic cancerPreclinicalROS induction, Cathepsins releaseAIF release Bionovo
AK275, AK295, MDL 28170, SNJ-1945, DY-9760eIschemiaPreclinicalCalpain inbibitors   
OSU-03012Multiple myelome gliomaPreclinicalCathepsin B activationAIF release  
AtiprimodNeuroendocrine carcinomaIIActivation JNK Bax/Bad upregulationAIF release Callisto
Multiple myelomeII   Callisto
Advanced cancerI/II   Callisto + MDACC
SorafenibHepatocellular carcinomaIVRaf inhibitionAIF release Bayer
Unresectable melanomaIII   Bayer
Renal cell carcinomaIII   Bayer
Renal cell carcinomaIII  SunitinibIomedico AG UUWSM
Pancreas adenocarcinomaIII  GemcitabineMIC
Non-small cell lung cancerIII   Bayer
FlavopiridolEsophageal cancerIICDK inhibitionAIF releasePaclitaxelMSKCC, NCI-SB
ChronicII   CALGB
Lymphocytic leukemia     
Endometrial carcinomaII   GOG-UCh
Squamous cell carcinomaII   NCI-MB
Breast cancerI/II  DocetaxelNCI-MB
Olaparib (AZD2281)Ovarian cancerIIPARP-1 inbibitor CediranibDFCI
Breast cancer    MGH
Gastric cancerII  PaclitaxelAstraZeneca
Colorectal cancerII   AstraZeneca
Ovarian cancerII  Paclitaxel, CarboplatinAstraZeneca
INO-1001Acute myocardial infarctionII Blockage of AIF release Inotek
Heart diseases postoperative complicationsII   Inotek
Veliparib (ABT-888)Ovarian cancerII  liposomal Doxorubicin/TemozolomideAbbott
Solid tumorsII  TemozolomideAbbott
Colorectal cancerII  TemozolomideAbbott
MelanomaII  TemozolomideAbbott
Breast cancerII   NIH-F
Ovarian cancerI/II   Mayo Clinic
Brain tumorsI/II  TemozolomideNCI-RTO
AG-014699Breast cancerII   CR-UK
Solid tumorsI   Pfizer

a) General therapeutics targeting the AIF PCD pathway. A first interesting therapeutic approach consists in controlling AIF release in necroptosis via the inhibition of the kinase RIP1 by necrostatin-1. For example, necroptosis, AIF, and RIP1 have been implicated in neuronal excitotoxicity. Note that excitotoxicity is involved in the pathologies associated with chronic neurodegeneration such as Alzheimer or Parkinson, and also in the acute neurodegeneration resulting from stroke. In this way, necrostatin-1 decreases infarct size in a mouse stroke model (61) and protects against glutamate-induced oxytosis in hippocampal HT-22 cells (71), NMDA-induced excitotoxicity in rat cortical neurons (73), and retinal detachment-induced photoreceptor necrosis (72). Alternatively, necrostatin-1 treatment decreases damage in neonatal hypoxia-ischemia (74), protects against myocardial ischemia-reperfusion injury (75), or ameliorates symptoms of Huntington's disease (76). Consequently, necrostatin-1 provides an exciting opportunity for developing new therapeutics targeting RIP1, AIF release, and necroptosis.

An alternative general therapeutic approach is the control of AIF release via the production of reactive oxygen species (ROS). It is very well known that cancer cells produce high levels of ROS (77). In contrast, ROS levels are low and detoxifying oxidants systems are well regulated in normal cells (78). Taking this premise as a starting-point, BioNovo has developed BZL101 (registered as Bezielle®), an extract of the plant Scutellaria barbatae that specifically provokes strong ROS induction, DNA-damage, AIF translocation from mitochondria to the nucleus, chromatinolysis, and PCD. Importantly, BZL101 kills specifically malignant but not normal cells, following a non-classical apoptotic model with a very limited activation of caspases. Recent works have reported complementary mechanisms related to BZL101-induced PCD (79) and have demonstrated that intraperitoneal injection of BZL101 inhibits tumor growth in mice (80). Importantly, results from the phase I trial, performed in patients with advanced breast cancer, proved an efficient anti-tumor activity of this drug (81). Given these promising results, a phase II clinical trial is currently underway.

Atiprimod belongs to the azaspirane class of cationic amphiphilic molecules, which is known for its anti-inflammatory, anti-neoplasic, and anti-angiogenic properties. Atiprimod activates JNK kinases in MCL cells and up-regulates the level of Bax, Bad, and phosphorylated Bcl-2, resulting in the release of AIF from mitochondria (82). AIF seems to be the major PCD regulator in this model, since inhibition of caspases with z-VAD.fmk barely affects Atiprimod-induced PCD. In contrast, preincubation in MCL cells with AIF inhibitor N-phenylmaleimide completely prevents nucleosomal DNA fragmentation (82). These results indicate that Atiprimod triggers apoptosis of MCL cells through a caspase-independent, AIF-mediated pathway. Hamasaki et al. (83) have shown that Atiprimod can also induce cell death in other multiple myeloma cell lines (MM.1S, U266, and RPMI8226) via activation of caspases. This compound is being tested for metastatic carcinoid tumors treatment (phase II) and also in refractive multiple myelomas (phase II).

Flavopiridol (Alvocidib) is a synthetic N-methylpiperidinyl chlorophenyl flavone. As an inhibitor of cyclin-dependent kinase, flavopiridol induces cell cycle arrest by preventing phosphorylation of cyclin-dependent kinases and by down-regulating cyclin D1 and D3 expression, resulting in G1 cell cycle arrest and PCD. This agent is also a competitive inhibitor of adenosine triphosphate activity provoking down-expression of Bcl-2, AIF release and caspase-independent PCD. Flavopiridol has demonstrated a potent anti-tumor activity in chronic lymphocytic leukemia and solid tumors (84–86). A large number of clinical trials using this synthetic flavone are currently in progress.

Sorafenib is a synthetic compound targeting growth signaling and angiogenesis. Sorafenib blocks the RAF kinase-signaling which controls cell division and proliferation. In addition, Sorafenib inhibits the VEGFR-2/PDGFR-beta signaling cascade, thereby blocking tumor angiogenesis. Although the tumor growth inhibition produced can involve caspase activation (87), some works consider the release of AIF as the principal way of death (88). Sorafenib is being widely used in clinical trials mostly for the treatment of hepatocarcinomas, renal cell carcinoma or melanomes, many of them in advanced phase of study.

As described above, PARP-1 is a key regulator of AIF-mediated caspase-independent necroptosis and therefore represents a potential target to modulate this type of PCD. Accordingly, inhibition of PARP-1 enhances the efficiency of radiotherapy and sensitizes tumor cells to chemotherapeutic agents. A large spectrum of PARP inhibitors is being tested in clinical trials, including: Olaparib (KU-59436 or AZD2281), tested for the treatment of solid tumors; Veliparib (ABT-888), in leukemias, lymphomas, and adult solid tumors; and AG-014699 in advanced or metastatic breast tumors and advanced ovarian cancer.

Numerous studies have signaled oxidative stress as a pathological factor in ischemic injuries. One of the key links between oxidative stress and cell death is an excessive activation of PARP-1 and further caspase-independent PCD. Minocycline, a semisynthetic tetracycline, is also a potent PARP-1 inhibitor that exhibits strong antiapoptotic activity in ischemia and neurodegeneration (89). It is being tested in patients affected by ischemic stroke, multiple sclerosis, acute kidney insufficiency, amyotrophic lateral sclerosis (phase III) or Huntington's disease.

INO1-1001, developed by Inotek Pharmaceuticals, is an isoindolinone derivative and potent inhibitor of PARP-1. Xiao and colleagues have demonstrated that PARP-1 inhibition by INO-1001 reduces AIF-release, apoptosis, fibrosis, and heart hypertrophy of diabetic rats (90). INO-1001 is currently tested for the treatment of myocardial ischemia (phase II) and in patients undergoing heart surgery that involves heart-lung bypass (phase II).

b) Therapeutics targeting proteases implied in AIF release. Another PCD-control strategy consists in controlling AIF release more directly via modulation of proteases upstream of mitochondria. Since proteolytic processing of AIF is essential for its proapoptotic function, calpains and cathepsins constitute main targets to block AIF cleavage and further release from mitochondria. A double regulation of both calpains and cathepsins is possible in some cases. In this sense, Tsubokawa et al. (91) reported that E64d, a dual inhibitor of cathepsin B and calpains, provides neuroprotection after experimental focal cerebral ischemia in rats. A wide number of other works reported promising results with more specific molecules:

  • (i)
    Calpain regulation. To date, the in vivo use of calpains inhibitors is limited to preclinical studies. The first promising results came from the inhibitors AK275, AK295, and MDL 28170, which significantly decreased the infarct size in rat ischemia models (92, 93). It has also been shown that SNJ-1945, an optimized aqueous-soluble calpain inhibitor, provides neuroprotection or cardioprotection after ischemic injuries (94, 95). Another interesting approach to modulating AIF-mediated PCD via calpain regulation relies on the control of calpastatin, an endogenous inhibitor of calpains. DY-9760e can rescue neurons and cardiomyocytes from ischemic injury by inhibiting proteolysis of calpastatin (96), and can also provide neuroprotection in cerebral ischemia through the inhibition of the calpain-induced fodrin breakdown (97). More recent works have demonstrated the beneficial effects of calpain inhibitors such as MDL-28170 in a rat model of Parkinson's disease (98) or PD150606 in photoreceptors of a retinitis pigmentosa rat model (99). In these models, calpain inhibitors blocked the release of AIF.
  • (ii)
    Cathepsin regulation. It is well documented that the leakage of lysosomal cathepsins involves selective mitochondrial membrane permeabilization and release of mitochondrial pro-apoptotic molecules (100). Thus, the positive or negative management of lysosomal permeability is of evident therapeutic interest. For example, paclitaxel, epothilone B, discodermolide and lipopolysaccharide trigger disruption of the lysosomal integrity, followed by release and activation of cathepsin B and PCD (101, 102). Additionally, an important number of conventional anti-cancer agents (including etoposide, cisplatin or 5-fluorouracil) provoke lysosomal destabilization, cathepsin release, and PCD (103). Of special interest is OSU-03012, a celecoxib derivative, inducing PCD in a large variety of cancer cells (e.g., multiple myeloma or glioma cells). OSU-03012- mediated death is dependent on ER stress, lysosomal dysfunction, Bid-activation and release of AIF (37, 104). The synthetic drug Bobel-24 (2,4,6-triiodophenol) triggers caspase-independent lysosomal and mitochondrial death in human pancreatic cancer lines. In this case, cell death is associated with ROS production, lysosomal cathepsin release, mitochondrial depolarization, AIF mitochondrial release and AIF nuclear translocation (105).

c) Cytotoxic therapies that target AIF directly. The use of recombinant forms of AIF has been recently proposed. Some studies have demonstrated the feasibility of the Ig-AIF chimeras as a novel approach to treat cancers that overexpress Erb2/HER2. As an example, a fusion protein including a HER2 antibody fragment linked to the C-terminal domain of AIF has been capable of specifically killing malignant cells (106, 107).

CONCLUDING REMARKS

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

PCD has been definitively accepted as a crucial phenomenon in life and evolution. We have just started to realize that there is more than one way for a cell to die. AIF represents one of the alternatives to classical apoptosis, which can be targeted in cancers and in neuronal death. Therefore, an understanding of the caspase-independent AIF-mediated necroptotic mechanisms in normal and pathological situations should provide conceptual progress for therapeutic interventions in deficient or excessive PCD. In this sense, the encouraging results in clinical trials described above fully support the potentiality of AIF and the AIF-mediated caspase-independent necroptotic pathway as therapeutic targets. Additionally, the unraveling of the mechanisms governing alternative necrotic PCD pathways will lead to a better comprehension of cellular homeostasis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES

We are grateful to Marcela Segade for proofreading. Research in the laboratory of Santos A Susin is supported by grants from Agence Nationale de la Recherche (ANR; contract ANR-09-BLAN-0247), Association pour la Recherche sur le Cancer (ARC; contracts 5104 and 7987), and Fondation de France. Lauriane Cabon receives a PhD fellowship from École Normale Supérieure de Cachan (ENS-Cachan).

REFERENCES

  1. Top of page
  2. Abstract
  3. APOPTOSIS-INDUCING FACTOR (AIF): THE PROTEIN
  4. AIF ACROSS THE SPECIES
  5. VITAL FUNCTION OF AIF
  6. REGULATION OF MITOCHONDRIAL AIF RELEASE
  7. LETHAL FUNCTION OF AIF: A CASPASE-INDEPENDENT PCD PROTEIN
  8. TWO EXAMPLES OF AIF-MEDIATED CASPASE-INDEPENDENT PCD: PARTHANATOS AND NECROPTOSIS
  9. AIF-MEDIATED CASPASE-INDEPENDENT NECROPTOSIS
  10. THERAPEUTIC STRATEGIES TARGETING AIF-MEDIATED CASPASE-INDEPENDENT PCD AND AIF ITSELF
  11. CONCLUDING REMARKS
  12. Acknowledgements
  13. REFERENCES
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