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1.  Apoptosis-Inducing Factor: Structure, Function, and Redox Regulation 
Antioxidants & Redox Signaling  2011;14(12):2545-2579.
Abstract
Apoptosis-inducing factor (AIF) is a flavin adenine dinucleotide-containing, NADH-dependent oxidoreductase residing in the mitochondrial intermembrane space whose specific enzymatic activity remains unknown. Upon an apoptotic insult, AIF undergoes proteolysis and translocates to the nucleus, where it triggers chromatin condensation and large-scale DNA degradation in a caspase-independent manner. Besides playing a key role in execution of caspase-independent cell death, AIF has emerged as a protein critical for cell survival. Analysis of in vivo phenotypes associated with AIF deficiency and defects, and identification of its mitochondrial, cytoplasmic, and nuclear partners revealed the complexity and multilevel regulation of AIF-mediated signal transduction and suggested an important role of AIF in the maintenance of mitochondrial morphology and energy metabolism. The redox activity of AIF is essential for optimal oxidative phosphorylation. Additionally, the protein is proposed to regulate the respiratory chain indirectly, through assembly and/or stabilization of complexes I and III. This review discusses accumulated data with respect to the AIF structure and outlines evidence that supports the prevalent mechanistic view on the apoptogenic actions of the flavoprotein, as well as the emerging concept of AIF as a redox sensor capable of linking NAD(H)-dependent metabolic pathways to apoptosis. Antioxid. Redox Signal. 14, 2545–2579.
Introduction
Multiple Forms of AIF
AIF precursor
Membrane-tethered mature AIFΔ1–54
Soluble apoptogenic AIFΔ1–102/118
AIF associated with the outer mitochondrial membrane
Splice variants AIF2, AIFsh, AIFsh2, and AIFsh3
Transcriptional Regulation
Phylogenetic Roots
Redox Properties of Recombinant AIF
Refolded murine AIFΔ1–120
Refolded human AIFsh2
Naturally folded murine AIFΔ1–53 and Δ1–101
AIF Structure
X-ray structures of murine and human AIFΔ1–120
X-ray structure of murine AIFΔ1–77
X-ray structure of reduced NAD-bound murine AIFΔ1–101
Redox-linked changes in the active site
Reorganization in the C-terminal domain
Conformational changes in the 509–559 peptide
Role of AIF in PCD
Apoptogenic effects of AIF in cell free systems and live cells
Release of mitochondrial AIF
Proteolysis of mature AIF
Release of truncated AIF into the cytoplasm
Release of AIF associated with the outer mitochondrial membrane
Cytoplasmic interactions of apoptogenic AIF
Pro-survival partners of AIF
Heat shock protein Hsp70
X-linked inhibitor of apoptosis protein
Pro-death partners of AIF
Eukaryotic translation initiation factor 3 subunit p44
T-cell ubiquitin ligand
Cyclophilin A
Phospholipid scramblase
Scythe
Nuclear effects of apoptogenic AIF
Transport of AIF to the nucleus
Interaction of AIF with DNA
Nuclear partners of AIF
Endonuclease G
Cyclophilin A
Histone H2AX
Relocation of AIF in late apoptosis
Apoptogenic properties of the AIF homologs
D. melanogaster
D. discoideum
Tetrahymena thermophila
S. cerevisiae
Vital Functions of Mitochondrial AIF
Role of AIF in mitochondrial respiration
Hq mouse phenotype
Tissue-specific AIF defects
Role of AIF in neurodegeneration, neurogenesis, and neuroprotection
AIF deficiency in lower eukaryotes
AIF and mitochondrial morphology
Mitochondrial abnormalities in telencephalon-specific AIFΔ mice
Association of AIF with the optic atrophy 1 protein
AIF isoform-specific cristae morphology
Human mitochondrial encephalomyopathy linked to the AIFΔ201 mutation
D. Involvement of AIF in regulation of cytoplasmic stress granules
Possible Redox Sensing Role of AIF
Concluding Remarks
doi:10.1089/ars.2010.3445
PMCID: PMC3096518  PMID: 20868295
2.  The molecular archaeology of a mitochondrial death effector: AIF in Drosophila 
Cell death and differentiation  2008;15(6):1009-1018.
Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (ΔN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. ΔN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that ΔN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.
doi:10.1038/cdd.2008.24
PMCID: PMC2907157  PMID: 18309327
mitochondria; oxidative phosphorylation; Drosophila
3.  Poly (ADP-ribose) (PAR) Binding to Apoptosis-Inducing Factor Is Critical For PAR Polymerase-1-Dependent Cell Death (Parthanatos) 
Science signaling  2011;4(167):ra20.
The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. Here, we show that AIF is a high affinity poly(ADP-ribose) (PAR)–binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF’s DNA binding site and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH oxidase activity, its ability to bind FAD or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death following PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF’s bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially-derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide unique opportunities for preventing cell death following activation of PARP-1.
doi:10.1126/scisignal.2000902
PMCID: PMC3086524  PMID: 21467298
4.  Apoptosis-Inducing Factor Regulates Skeletal Muscle Progenitor Cell Number and Muscle Phenotype 
PLoS ONE  2011;6(11):e27283.
Apoptosis Inducing Factor (AIF) is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal and cardiomyocyte apoptosis induced by oxidative stress. Conversely in vitro, AIF has been demonstrated to have a pro-apoptotic role upon induction of the mitochondrial death pathway, once AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. Given that the aif hypomorphic harlequin (Hq) mutant mouse model displays severe sarcopenia, we examined skeletal muscle from the aif hypomorphic mice in more detail. Adult AIF-deficient skeletal myofibers display oxidative stress and a severe form of atrophy, associated with a loss of myonuclei and a fast to slow fiber type switch, both in “slow” muscles such as soleus, as well as in “fast” muscles such as extensor digitorum longus, most likely resulting from an increase of MEF2 activity. This fiber type switch was conserved in regenerated soleus and EDL muscles of Hq mice subjected to cardiotoxin injection. In addition, muscle regeneration in soleus and EDL muscles of Hq mice was severely delayed. Freshly cultured myofibers, soleus and EDL muscle sections from Hq mice displayed a decreased satellite cell pool, which could be rescued by pretreating aif hypomorphic mice with the manganese-salen free radical scavenger EUK-8. Satellite cell activation seems to be abnormally long in Hq primary culture compared to controls. However, AIF deficiency did not affect myoblast cell proliferation and differentiation. Thus, AIF protects skeletal muscles against oxidative stress-induced damage probably by protecting satellite cells against oxidative stress and maintaining skeletal muscle stem cell number and activation.
doi:10.1371/journal.pone.0027283
PMCID: PMC3208607  PMID: 22076146
5.  Control of AIF-mediated cell death by antagonistic functions of CHIP ubiquitin E3 ligase and USP2 deubiquitinating enzyme 
Cell Death and Differentiation  2011;18(8):1326-1336.
Apoptosis inducing factor (AIF) is a mitochondrial oxidoreductase that scavenges reactive oxygen species under normal conditions. Under certain stresses, such as exposure to N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG), AIF is truncated and released from the mitochondria and translocated into the nucleus, where the truncated AIF (tAIF) induces caspase-independent cell death. However, it is unknown how cells decide to kill themselves or operate ways to survive when they encounter stresses that induce the release of tAIF. Here, we demonstrated that USP2 and CHIP contribute to the control of tAIF stability. USP2 deubiquitinated and stabilized tAIF, thus promoting AIF-mediated cell death. In contrast, CHIP ubiquitinated and destabilized tAIF, thus preventing the cell death. Consistently, CHIP-deficient cells showed an increased sensitivity to MNNG. On the other hand, knockdown of USP2 attenuated MNNG-induced cell death. Moreover, exposure to MNNG caused a dramatic decrease in CHIP level, but not that of USP2, concurrent with cell shrinkage and chromatin condensation. These findings indicate that CHIP and USP2 show antagonistic functions in the control of AIF-mediated cell death, and implicate the role of the enzymes as a switch for cells to live or die under stresses that cause tAIF release.
doi:10.1038/cdd.2011.3
PMCID: PMC3172096  PMID: 21293491
apoptosis inducing factor; caspase-independent cell death; CHIP; USP2; ubiquitin
6.  Calcium Dysregulation Induces Apoptosis-inducing Factor Release: Cross-talk Between PARP-1- and Calpain- Signaling Pathways 
Experimental neurology  2009;218(2):213-220.
Recent discoveries show that caspase-independent cell death pathways are a pervasive mechanism in neurodegenerative diseases, and apoptosis-inducing factor (AIF) is an important effector of this mode of neuronal death. There are currently two known mechanisms underlying AIF release following excitotoxic stress, PARP-1 and calpain. To test whether there is an interaction between PARP-1 and calpain in triggering AIF release, we used the NMDA toxicity model in rat primary cortical neurons. Exposure to NMDA resulted in AIF truncation and nuclear translocation, and shRNA-mediated knock down of AIF resulted in neuroprotection. Both calpain and PARP-1 are involved with AIF processing as AIF truncation, nuclear translocation and neuronal death were attenuated by calpain inhibition using adeno-associated virus-mediated overexpression of the endogenous calpain inhibitor, calpastatin, or treatment with the PARP-1 inhibitor 3-ABA. Activation of PARP-1 is necessary for calpain activation as PARP-1 inhibition blocked mitochondrial calpain activation. Finally, NMDA toxicity induces mitochondrial Ca2+ dysregulation in a PARP-1 dependent manner. Thus, PARP-1 and mitochondrial calpain activation are linked via PARP-1-induced alterations in mitochondrial Ca2+ homeostasis. Collectively, these findings link the two seemingly independent mechanisms triggering AIF-induced neuronal death.
doi:10.1016/j.expneurol.2009.04.032
PMCID: PMC2710414  PMID: 19427306
NMDA toxicity; calpain; PARP-1; apoptosis-inducing factor; ischemia; mitochondria; calcium homeostasis
7.  Mitochondrial μ-calpain is not involved in the processing of apoptosis-inducing factor 
Experimental neurology  2009;218(2):221-227.
Caspase-independent cell death, an important death pathway in many cells including neurons, is executed via apoptosis-inducing factor (AIF), an oxidoreductase, localized to the mitochondrial intermembrane space. AIF is processed and released from mitochondria following mitochondrial permeability transition pore (mPTP) formation, and translocates to the nucleus to induce DNA fragmentation and cell death. The release of AIF requires cleavage of its N-terminus anchored in the inner mitochondrial membrane. The protease responsible for this AIF truncation has not been established, although there is considerable evidence suggesting a role for μ-calpain. We previously found that a pool of μ-calpain is localized to the mitochondrial intermembrane space, the submitochondrial compartment in which AIF truncation occurs. The close submitochondrial proximity of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be the protease responsible for processing AIF prior to its release. In the present study, AIF was released from rat liver mitochondria following mPTP induction by atractyloside. This release was inhibited by the cysteine protease inhibitor MDL28170, but not by more specific calpain inhibitors PD150606 and human erythrocyte calpastatin. Atractyloside caused swelling in rat brain mitochondria, but did not induce AIF release. In a mitochondrial fraction from SH-SY5Y neuroblastoma cells, incubation with 5 mM Ca2+ resulted in the activation of μ-calpain but not in AIF truncation. In summary, the localization of μ-calpain to the mitochondrial intermembrane space is suggestive of its possible involvement in AIF processing, but direct experimental evidence supporting such a role has been elusive.
doi:10.1016/j.expneurol.2009.04.013
PMCID: PMC2756010  PMID: 19393648
8.  Sequential Activation of Poly(ADP-Ribose) Polymerase 1, Calpains, and Bax Is Essential in Apoptosis-Inducing Factor-Mediated Programmed Necrosis▿  
Molecular and Cellular Biology  2007;27(13):4844-4862.
Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N′-nitro-N′-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.
doi:10.1128/MCB.02141-06
PMCID: PMC1951482  PMID: 17470554
9.  Combined inhibition of cell death induced by apoptosis inducing factor and caspases provides additive neuroprotection in experimental traumatic brain injury 
Neurobiology of Disease  2012;46(3):745-758.
Neuronal programmed cell death (PCD) contributes to delayed tissue damage after traumatic brain injury (TBI). Both caspase-dependent and caspase-independent mechanisms have been implicated, with the latter including apoptosis inducing factor (AIF). The peptidyl-proplyl isomerase Cyclophilin A (CypA) transports AIF from the cytosol to the nucleus, a key step for AIF-dependent cell death. We compared the effects of single versus combined inhibition of caspase and AIF pathways in a mouse controlled cortical impact (CCI) model, by examining the effects of CypA gene knockout (CypA−/−), caspase inhibition with a pan-caspase inhibitor (boc-aspartyl(OMe)-fluoromethylketone, BAF), or combined modulation. TBI caused caspase activation as well as translocation of AIF to the nucleus. Markers of caspase activation including caspase-specific fodrin cleavage fragments and number of FLIVO positive cells were reduced in BAF-treated CypA+/+ mice, whereas markers of AIF activation including AIF/H2AX interaction and AIF translocation to the nucleus were attenuated in CypA−/− mice. Each single intervention, (CypA−/− or BAF-treated CypA+/+) reduced the number of apoptotic cells (TUNEL-positive) in the cortex and improved long-term sensorimotor function; CypA−/− also attenuated microglial activation after injury. Importantly, BAF-treated CypA−/− mice, showed greater effects than either intervention alone on multiple outcomes including: reduction in TUNEL-positive cells, decrease in neuroinflammation, improved motor and cognitive recovery, and attenuation of lesion volume and neuronal loss in the hippocampus. Using two in vitro neuronal cell death models known to induce AIF-mediated PCD, we also showed that neurons from CypA−/− animals were protected and that effects were unrelated to caspase activation. These data indicate that AIF-mediated and caspase-dependent pathways contribute independently and in parallel to secondary injury after TBI, and suggest that combined therapeutic strategies directed at multiple PCD pathways may provide superior neuroprotection than those directed at single mechanisms.
doi:10.1016/j.nbd.2012.03.018
PMCID: PMC3352990  PMID: 22426396
Traumatic Brain Injury; Apoptosis Inducing Factor; Caspase; Cyclophilin A
10.  Pneumococcal pneumolysin and H2O2 mediate brain cell apoptosis during meningitis 
Pneumococcus is the most common and aggressive cause of bacterial meningitis and induces a novel apoptosis-inducing factor–dependent (AIF–dependent) form of brain cell apoptosis. Loss of production of two pneumococcal toxins, pneumolysin and H2O2, eliminated mitochondrial damage and apoptosis. Purified pneumolysin or H2O2 induced microglial and neuronal apoptosis in vitro. Both toxins induced increases of intracellular Ca2+ and triggered the release of AIF from mitochondria. Chelating Ca2+ effectively blocked AIF release and cell death. In experimental pneumococcal meningitis, pneumolysin colocalized with apoptotic neurons of the hippocampus, and infection with pneumococci unable to produce pneumolysin and H2O2 significantly reduced damage. Two bacterial toxins, pneumolysin and, to a lesser extent, H2O2, induce apoptosis by translocation of AIF, suggesting new neuroprotective strategies for pneumococcal meningitis.
doi:10.1172/JCI12035
PMCID: PMC150815  PMID: 11781347
11.  Bid-induced release of AIF from mitochondria causes immediate neuronal cell death 
Cell death and differentiation  2008;15(10):1553-1563.
Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8–10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.
doi:10.1038/cdd.2008.78
PMCID: PMC2862690  PMID: 18535584
HT-22 cells; glutamate; apoptosis; mitochondrial membrane potential; caspase-independent cell death
12.  Influence of Duration of Focal Cerebral Ischemia and Neuronal Nitric Oxide Synthase on Translocation of Apoptosis-Inducing Factor to the Nucleus 
Neuroscience  2006;144(1):56-65.
Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 hour of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 hours of permanent ischemia. However, as little as 20 minutes of reperfusion after 1 hour of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 hours of reperfusion. In contrast, 20 minutes of reperfusion after 2 hours of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 hours of reperfusion. With brief occlusion of 30 minute duration, nuclear AIF remained undetectable at both 20 minutes and 6 hours and became evident only after 24 hours of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 hour), but is markedly delayed after brief ischemia (30 minutes). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.
doi:10.1016/j.neuroscience.2006.08.065
PMCID: PMC1876769  PMID: 17049179
apoptosis; middle cerebral artery; mouse; poly(ADP-ribose); reperfusion; stroke
13.  Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death 
The Journal of Cell Biology  2002;158(3):507-517.
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.
doi:10.1083/jcb.200202130
PMCID: PMC2173837  PMID: 12147675
neurodegeneration; neurons; apoptosis; p53; Bax
14.  AIF, reactive oxygen species, and neurodegeneration: a “complex” problem 
Neurochemistry international  2012;62(5):695-702.
Apoptosis-inducing factor (AIF) is a flavin-binding mitochondrial intermembrane space protein that is implicated in diverse but intertwined processes that include maintenance of electron transport chain function, reactive oxygen species regulation, cell death, and neurodegeneration. In acute brain injury, AIF acquires a pro-death role upon translocation from the mitochondria to the nucleus, where it initiates chromatin condensation and large-scale DNA fragmentation. Although harlequin mice exhibiting an 80–90% global reduction in AIF protein are resistant to numerous forms of acute brain injury, they paradoxically undergo slow, progressive neurodegeneration beginning at three months of age. Brain deterioration, accompanied by markers of oxidative stress, is most pronounced in the cerebellum and retina, although it also occurs in the cortex, striatum, and thalamus. Loss of an AIF pro-survival function linked to assembly or stabilization of electron transport chain complex I underlies chronic neurodegeneration. To date, most studies of neurodegeneration have failed to adequately separate the relative importance of the mitochondrial and nuclear functions of AIF in determining the extent of injury, or whether oxidative stress plays a causative role. This review explores the complicated relationship among AIF, complex I, and the regulation of mitochondrial reactive oxygen species levels. It also discusses the controversial role of complex I deficiency in Parkinson’s disease, and what can be learned from the AIF- and complex I-depleted harlequin mouse.
doi:10.1016/j.neuint.2012.12.002
PMCID: PMC3610861  PMID: 23246553
mitochondria; electron transport; complex I; harlequin; oxidative stress; Parkinson’s disease
15.  Role of apoptosis-inducing factor, proline dehydrogenase, and NADPH oxidase in apoptosis and oxidative stress 
Cell health and cytoskeleton  2012;2012(4):11-27.
Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), proline dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of proline oxidation. Upregulation of proline dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of different pathways regulating cell proliferation and cell death. Potential therapeutic strategies for each enzyme are also highlighted.
doi:10.2147/CHC.S4955
PMCID: PMC3351110  PMID: 22593641
apoptosis; flavoproteins; apoptosis-inducing factor; NADPH oxidase; proline dehydrogenase
16.  Increased nuclear apoptosis-inducing factor after transient focal ischemia: a 12/15-lipoxygenasedependent organelle damage pathway 
12/15-lipoxygenase (12/15-LOX) contributes to acute neuronal injury and edema formation in mouse models of middle cerebral artery occlusion (MCAO). The apoptosis-inducing factor (AIF) is implicated in caspase-independent forms of apoptosis, and has been linked to ischemic neuronal cell death. We show here that increased AIF in the peri-ischemic cortex of mouse colocalizes with 12/15-LOX after 2 h of MCAO. The 12/15-LOX inhibitor baicalein prevents the increase and nuclear localization of AIF, suggesting this pathway may be partially responsible for the neuroprotective qualities of baicalein. Using an established cell line model of neuronal oxidative stress, we show that 12/15-LOX activated after glutathione depletion leads to AIF translocation to the nucleus, which is abrogated by the 12/15-LOX inhibitor baicalein (control: 19.3%±6.8% versus Glutamate: 64.0%±8.2% versus glutamate plus baicalein: 11.4%±2.2%). Concomitantly, resident proteins of the ER are dispersed throughout the cell (control: 31.0%±8.4% versus glutamate: 70.0%±5.5% versus glutamate plus baicalein: 8.0%±2.7%), suggesting cell death through organelle damage. Taken together, these findings show that 12/15-LOX and AIF are sequential actors in a common cell death pathway that may contribute to stroke-induced brain damage.
doi:10.1038/jcbfm.2009.281
PMCID: PMC2915762  PMID: 20068575
apoptosis; apoptosis-inducing factor; lipoxygenase; mitochondria; oxidative stress; stroke
17.  Increased nuclear apoptosis-inducing factor after transient focal ischemia: a 12/15-lipoxygenase-dependent organelle damage pathway 
12/15-lipoxygenase (12/15-LOX) contributes to acute neuronal injury and edema formation in mouse models of middle cerebral artery occlusion (MCAO). The apoptosis-inducing factor (AIF) is implicated in caspase-independent forms of apoptosis, and has been linked to ischemic neuronal cell death. We show here that increased AIF in the peri-ischemic cortex of mouse colocalizes with 12/15-LOX after 2 h of MCAO. The 12/15-LOX inhibitor baicalein prevents the increase and nuclear localization of AIF, suggesting this pathway may be partially responsible for the neuroprotective qualities of baicalein. Using an established cell line model of neuronal oxidative stress, we show that 12/15-LOX activated after glutathione depletion leads to AIF translocation to the nucleus, which is abrogated by the 12/15-LOX inhibitor baicalein (control: 19.3%±6.8% versus Glutamate: 64.0%±8.2% versus glutamate plus baicalein: 11.4%±2.2%). Concomitantly, resident proteins of the ER are dispersed throughout the cell (control: 31.0%±8.4% versus glutamate: 70.0%±5.5% versus glutamate plus baicalein: 8.0%±2.7%), suggesting cell death through organelle damage. Taken together, these findings show that 12/15-LOX and AIF are sequential actors in a common cell death pathway that may contribute to stroke-induced brain damage.
doi:10.1038/jcbfm.2009.281
PMCID: PMC2915762  PMID: 20068575
apoptosis; apoptosis-inducing factor; lipoxygenase; mitochondria; oxidative stress; stroke
18.  Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens 
BMC Neuroscience  2006;7:49.
Background
Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell death at high concentrations. Our previous studies have shown that depending on the neuronal cell type, glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/or caspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have investigated the role of calpain, an important calcium-dependent protease thought to be involved in apoptosis along with mitochondrial apoptosis inducing factor (AIF) and caspase-3 in primary cortical cells and a mouse hippocampal cell line HT22.
Results
Glutamate-induced apoptotic cell death in neuronal cells was associated with characteristic DNA fragmentation, morphological changes, activation of calpain and caspase-3 as well as the upregulation and/or translocation of AIF from mitochondria into cytosol and nuclei. Our results reveal that primary cortical cells and HT22 cells display different patterns of regulation of these genes/proteins. In primary cortical cells, glutamate induces activation of calpain, caspase-3 and translocation of AIF from mitochondria to cytosol and nuclei. In contrast, in HT22 cells, only the activation of calpain and upregulation and translocation of AIF occurred. In both cell types, these processes were inhibited/reversed by 17β-estradiol and Δ8,17β-estradiol with the latter being more potent.
Conclusion
Depending upon the neuronal cell type, at least two mechanisms are involved in glutamate-induced apoptosis: a caspase-3-dependent pathway and a caspase-independent pathway involving calpain and AIF. Since HT22 cells lack caspase-3, glutamate-induced apoptosis is mediated via the caspase-independent pathway in this cell line. Kinetics of this apoptotic pathway further indicate that calpain rather than caspase-3, plays a critical role in the glutamate-induced apoptosis. Our studies further indicate that glutamate- induced changes of these proteins can be inhibited by estrogens, with Δ8,17β-estradiol, a novel equine estrogen being more potent than 17β-estradiol. To our knowledge, this is the first demonstration that glutamate-induced apoptosis involves regulation of multiple apoptotic effectors that can be inhibited by estrogens. Whether these observations can help in the development of novel therapeutic approaches for the prevention of neurodegenerative diseases with estrogens and calpain inhibitors remains to be investigated.
doi:10.1186/1471-2202-7-49
PMCID: PMC1526740  PMID: 16776830
19.  An AIF orthologue regulates apoptosis in yeast 
The Journal of Cell Biology  2004;166(7):969-974.
Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).
doi:10.1083/jcb.200404138
PMCID: PMC2172025  PMID: 15381687
apoptosis-inducing factor; YNR074C; YCA1; cyclophilin A; aging
20.  Apoptosis Control in Syncytia Induced by the HIV Type 1–Envelope Glycoprotein Complex 
The Journal of Experimental Medicine  2000;192(8):1081-1092.
Syncytia arising from the fusion of cells expressing a lymphotropic HIV type 1–encoded envelope glycoprotein complex (Env) with cells expressing the CD4/CXC chemokine receptor 4 complex spontaneously undergo cell death. Here we show that this process is accompanied by caspase activation and signs of mitochondrial membrane permeabilization (MMP), including the release of intermembrane proteins such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF) from mitochondria. In Env-induced syncytia, caspase inhibition did not suppress AIF- and Cyt-c translocation, yet it prevented all signs of nuclear apoptosis. Translocation of Bax to mitochondria led to MMP, which was inhibited by microinjected Bcl-2 protein or bcl-2 transfection. Bcl-2 also prevented the subsequent nuclear chromatin condensation and DNA fragmentation. The release of AIF occurred before that of Cyt-c and before caspase activation. Microinjection of AIF into syncytia sufficed to trigger rapid, caspase-independent Cyt-c release. Neutralization of endogenous AIF by injection of an antibody prevented all signs of spontaneous apoptosis occurring in syncytia, including the Cyt-c release and nuclear apoptosis. In contrast, Cyt-c neutralization only prevented nuclear apoptosis, and did not affect AIF release. Our results establish that the following molecular sequence governs apoptosis of Env-induced syncytia: Bax-mediated/Bcl-2–inhibited MMP → AIF release → Cyt-c release → caspase activation → nuclear apoptosis.
PMCID: PMC2195869  PMID: 11034598
apoptosis-inducing factor; Bcl-2; cell death; cytochrome c; glycoprotein 120
21.  On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial Release of an Apoptosis-inducing Factor during Dictyostelium discoideum Cell Death 
Molecular Biology of the Cell  2001;12(10):3016-3030.
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 (ΔΨm) 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.
PMCID: PMC60152  PMID: 11598188
22.  CALPAIN ACTIVATION IS NOT REQUIRED FOR AIF TRANSLOCATION IN PARP-1-DEPENDENT CELL DEATH (PARTHANATOS) 
Journal of neurochemistry  2009;110(2):687-696.
Apoptosis-inducing factor (AIF) is critical for poly(ADP-ribose) polymerase-1 (PARP-1)-dependent cell death (parthanatos). The molecular mechanism of mitochondrial AIF release to the nucleus remains obscure, although a possible role of calpain I has been suggested. Here we show that calpain is not required for mitochondrial AIF release in parthanatos. Although calpain I cleaved recombinant AIF in a cell free system, in intact cells under conditions where endogenous calpain was activated by either NMDA or MNNG administration, AIF was not cleaved, and it was released from mitochondria to the nucleus in its 62 kDa uncleaved form. Moreover, NMDA administration under conditions that failed to activate calpain still robustly induced AIF nuclear translocation. Inhibition of calpain with calpastatin or genetic knockout of the regulatory subunit of calpain failed to prevent NMDA- or MNNG-induced AIF nuclear translocation and subsequent cell death, respectively, which was markedly prevented by the PARP-1 inhibitor DPQ. Our study clearly shows that calpain activation is not required for AIF release during parthanatos, suggesting that other mechanisms rather than calpain are involved in mitochondrial AIF release in parthanatos.
doi:10.1111/j.1471-4159.2009.06167.x
PMCID: PMC2819224  PMID: 19457082
Apoptosis-inducing factor; calpain; parthanatos; poly(ADP-ribose) polymerase-1
23.  Two Distinct Pathways Leading to Nuclear Apoptosis 
Apaf-1−/− or caspase-3−/− cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1−/− or caspase-3−/− cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1−/− or caspase-3−/− cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation.
PMCID: PMC2193229  PMID: 10952727
apoptosis-inducing factor; Apaf-1; chromatin condensation; caspases; caspase-activated DNase
24.  AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis 
PLoS ONE  2012;7(10):e47328.
Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.
doi:10.1371/journal.pone.0047328
PMCID: PMC3471829  PMID: 23077592
25.  Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid mediated apoptosis through Bak activation in ovarian cancer cells 
Biochimica et Biophysica Acta  2012;1823(8):1378-1388.
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.
doi:10.1016/j.bbamcr.2012.05.031
PMCID: PMC3389262  PMID: 22683989
IFN-α; apoptosis; mitochondria; AIF; Bid; Bak

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