Abstract: Article4 November 2004free access AIF deficiency compromises oxidative phosphorylation Nicola Vahsen Nicola Vahsen CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Céline Candé Céline Candé CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Jean-Jacques Brière Jean-Jacques Brière INSERM U393, Service de Génétique, Hôpital Necker-Enfants Malades, France Search for more papers by this author Paule Bénit Paule Bénit INSERM U393, Service de Génétique, Hôpital Necker-Enfants Malades, France Search for more papers by this author Nicholas Joza Nicholas Joza IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Nathanael Larochette Nathanael Larochette CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Pier Giorgio Mastroberardino Pier Giorgio Mastroberardino Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Marie O Pequignot Marie O Pequignot CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Noelia Casares Noelia Casares CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Vladimir Lazar Vladimir Lazar Unité de Génomique Fonctionelle, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Olivier Feraud Olivier Feraud INSERM U362, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Najet Debili Najet Debili INSERM U362, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Silke Wissing Silke Wissing Physiologisch-chemisches Institut, Tübingen, Germany Search for more papers by this author Silvia Engelhardt Silvia Engelhardt Physiologisch-chemisches Institut, Tübingen, Germany Search for more papers by this author Frank Madeo Frank Madeo Physiologisch-chemisches Institut, Tübingen, Germany Search for more papers by this author Mauro Piacentini Mauro Piacentini Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Josef M Penninger Josef M Penninger IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Hermann Schägger Hermann Schägger Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Pierre Rustin Pierre Rustin INSERM U393, Service de Génétique, Hôpital Necker-Enfants Malades, France Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Nicola Vahsen Nicola Vahsen CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Céline Candé Céline Candé CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Jean-Jacques Brière Jean-Jacques Brière INSERM U393, Service de Génétique, Hôpital Necker-Enfants Malades, France Search for more papers by this author Paule Bénit Paule Bénit INSERM U393, Service de Génétique, Hôpital Necker-Enfants Malades, France Search for more papers by this author Nicholas Joza Nicholas Joza IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Nathanael Larochette Nathanael Larochette CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Pier Giorgio Mastroberardino Pier Giorgio Mastroberardino Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Marie O Pequignot Marie O Pequignot CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Noelia Casares Noelia Casares CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Vladimir Lazar Vladimir Lazar Unité de Génomique Fonctionelle, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Olivier Feraud Olivier Feraud INSERM U362, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Najet Debili Najet Debili INSERM U362, Institut Gustave Roussy, Villejuif, France Search for more papers by this author Silke Wissing Silke Wissing Physiologisch-chemisches Institut, Tübingen, Germany Search for more papers by this author Silvia Engelhardt Silvia Engelhardt Physiologisch-chemisches Institut, Tübingen, Germany Search for more papers by this author Frank Madeo Frank Madeo Physiologisch-chemisches Institut, Tübingen, Germany Search for more papers by this author Mauro Piacentini Mauro Piacentini Department of Biology, University of Rome Tor Vergata, Rome, Italy Search for more papers by this author Josef M Penninger Josef M Penninger IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria Search for more papers by this author Hermann Schägger Hermann Schägger Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Pierre Rustin Pierre Rustin INSERM U393, Service de Génétique, Hôpital Necker-Enfants Malades, France Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Author Information Nicola Vahsen1, Céline Candé1, Jean-Jacques Brière2, Paule Bénit2, Nicholas Joza3, Nathanael Larochette1, Pier Giorgio Mastroberardino4, Marie O Pequignot1, Noelia Casares1, Vladimir Lazar5, Olivier Feraud6, Najet Debili6, Silke Wissing7, Silvia Engelhardt7, Frank Madeo7, Mauro Piacentini4, Josef M Penninger3, Hermann Schägger8, Pierre Rustin2,8 and Guido Kroemer 1,8 1CNRS-UMR8125, Institut Gustave Roussy, Villejuif, France 2INSERM U393, Service de Génétique, Hôpital Necker-Enfants Malades, France 3IMBA, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria 4Department of Biology, University of Rome Tor Vergata, Rome, Italy 5Unité de Génomique Fonctionelle, Institut Gustave Roussy, Villejuif, France 6INSERM U362, Institut Gustave Roussy, Villejuif, France 7Physiologisch-chemisches Institut, Tübingen, Germany 8Institut für Biochemie I, Universitätsklinikum Frankfurt, Frankfurt am Main, Germany *Corresponding author. CNRS-UMR 8125, Institut Gustave Roussy, Pavillon de Recherche 1, 39, rue Camille Desmoulins, 94805 Villejuif, France. Tel.: +33 1 42 11 60 46; Fax: +33 1 42 11 52 44; E-mail: [email protected] The EMBO Journal (2004)23:4679-4689https://doi.org/10.1038/sj.emboj.7600461 These authors share senior co-authorship PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death. Introduction Mitochondria fulfill a dual role in our cells. As the cell's powerhouses, they generate ATP by oxidative phosphorylation (OXPHOS), and constitute a central node for a cornucopia of metabolic pathways. In addition, they play an important role as suicide organelles (Green and Reed, 1998; Kroemer and Reed, 2000; Wang, 2002). Indeed, the fate of a cell driven into apoptosis is sealed when the inner and/or the outer mitochondrial membranes are permeabilized, a process that leads to the release of potentially toxic proteins and ultimately disrupts the vital bioenergetic function of mitochondria. One of the mitochondrial proteins that contributes to the apoptotic process is cytochrome c (Cyt c), a heme protein normally involved in electron shuttling between complexes III and IV of the respiratory chain (Wang, 2002). Upon apoptosis induction, Cyt c is released from the mitochondria and allosterically activates Apaf-1, thereby stimulating the apoptosome caspase activation complex. The proapoptotic function of Cyt c does not depend on its redox activity, since exchange of Fe2+ by Cu2+ in the active center of the heme group (which abolishes electron transfer) does not destroy the capacity of Cyt c to activate caspases in cell-free systems (Budijardjo et al, 1999; Wang, 2002). Apoptosis-inducing factor (AIF) is another mitochondrial protein that can contribute to apoptosis (Cande et al, 2002; Lipton and Bossy-Wetzel, 2002). AIF is a flavoprotein with NADH oxidase activity normally contained in the mitochondrial intermembrane space (Susin et al, 1999) or loosely associated with the inner mitochondrial membrane (Arnoult et al, 2002). Upon apoptosis induction, AIF translocates from mitochondria to the cytosol and to the nucleus (Susin et al, 1999; Yu et al, 2002). In Caenorhabditis elegans, AIF cooperates with the endonuclease G (Wang et al, 2002), a DNAse that also undergoes a mitochondrio-nuclear translocation process (Li et al, 2001). In mammalian cells, AIF cooperates with cyclophilin A to become an active DNAse (Cande et al, 2004a). AIF carries negatively charged amino acids on the surface (Ye et al, 2002), allowing it to interact with DNA and to contribute to chromatin condensation and chromatinolysis, two hallmarks of apoptosis. Chemical inactivation of the flavine adenine nucleotide (FAD) moiety required for AIF redox activity does not block its apoptogenic function in cell-free systems (Miramar et al, 2001), and mutations that destroy the FAD binding site do not affect the apoptogenic function of AIF in transfection assays (Loeffler et al, 2001). Thus, the apoptogenic activity of AIF does not depend on its NADH oxidase activity. Conversely, mutations of amino-acid residues required for DNA binding (but not for redox activity) abrogate the apoptogenic potential of AIF evaluated on isolated nuclei as well as in intact cells (Ye et al, 2002). The physiological (nonapoptotic) function of AIF in the mitochondria has been elusive. With the hope to determine this role, AIF-deficient embryonic stem (ES) cells were constructed by homologous recombination of the single AIF gene found in male cells (AIF−/y). Unfortunately, attempts to generate mice from such AIF−/y ES cells failed, precluding the in vivo assessment of the general AIF knockout and restricting the analysis to in vitro cell culture models, in which a partial apoptosis defect was detected (Joza et al, 2001). A hypomorphic mutation of AIF has been reported in the Harlequin mouse strain, which develops neurodegeneration (with ataxia due to cerebellar atrophia as a hallmark) as well as blindness due to retinal degeneration (Klein et al, 2002), two among the most commonly observed symptoms associated with OXPHOS defects in mammals. In Harlequin mice, the expression of AIF is reduced to 10–20% of the normal value due to a retroviral insertion into the first intron of the AIF gene. Increased neuronal cell death in response to oxidative stress was found in such mice (Klein et al, 2002), leading to the speculation that AIF might act as an antioxidant enzyme (Lipton and Bossy-Wetzel, 2002; Klein and Ackerman, 2003). Driven by these premises and incognita, we decided to evaluate the potential role of AIF in OXPHOS. As shown here, the absence of AIF was found to compromise the composition and function of the respiratory chain, targeting mostly complex I, in both in vitro cell models (HeLa and mouse ES cells) and in vivo in Harlequin mice. Results Deficient OXPHOS in AIF-deficient cells AIF-deficient ES cells (AIF−/y) manifest a reduced in vitro growth (with a duplication interval of 17±2 h as compared to 13±1 h in AIF+/y controls; Figure 1A), yet cause an accelerated acidification of the culture medium (Figure 1B), correlating with enhanced lactate production (Figure 1C), as compared to AIF-expressing controls. This pointed to an altered oxidative metabolism in AIF−/y cells. Complex I-dependent substrate oxidation was assessed by polarographic measurements of oxygen consumption in digitonin-permeabilized cells, upon addition of pyruvate in the presence of malate (which both furnish NADH) and ADP (which can be phosphorylated to ATP). A significant decrease (∼40%) of this complex I-dependent substrate oxidation rate was observed between AIF-deficient ES cells and control cells (Figure 2A and D). In view of the fact that oxidation ratios are accurate indicators of partial respiratory chain deficiencies (Rustin et al, 1991), we compared the complex I-dependent substrate oxidation to succinate oxidation (determined upon addition of the complex II substrate succinate, in the presence of complex I-inhibitor rotenone, ATP, and an uncoupler, m-Cl-CCP), and we consistently detected a marked (∼50%) decrease in complex I activity in AIF−/y mouse ES cells as compared to control ES cells (Figure 2A and E). Thus, complex I was deficient both relative to succinate oxidation (Figure 2A and E) and in absolute terms (Figure 2A and D). Very similar results were found in a human cell line (HeLa) in which the expression of the AIF gene was downregulated by two different small interfering RNA (siRNA) oligonucleotides (data shown for siRNA AIF1; Figure 2B, D, and E). Figure 1.Increased lactate production and decreased complex I activity in AIF-deficient cells. (A) Number of divisions per day of AIF−/y as compared to AIF+/y ES cells. Values are given as mean±s.d. (n=12). (B) Acidification of media by AIF−/y and AIF+/y cells, as determined in nonconfluent cultures (2.5 × 105 cells/ml at day 0). (C) Lactate production by AIF−/y and AIF+/y ES cells, as evaluated at different densities (mean±s.e.m., n=3). Asterisks indicate significant (P<0.01, unpaired Student's t-test) differences between AIF−/y and AIF+/y cells. Download figure Download PowerPoint Figure 2.OXPHOS deficiency in AIF-negative cells. (A) AIF−/y or AIF+/y ES cells were introduced into a polarograph to monitor their oxygen consumption, permeabilized with digitonin, followed by addition of the indicated respiratory substrates and inhibitors. The results are representative of three independent experiments. (B) Control HeLa cells or cells manipulated by siRNA to lose AIF expression were analyzed as in (A), yielding similar results in three experiments. (C) Suppression of AIF expression by siRNA. Cells were mock treated or transfected with two different control siRNAs or an AIF-specific siRNA, and the abundance of AIF was determined by immunoblot 72 h later. (D) Reduced absolute O2 consumption after addition of malate, pyruvate, and ADP. (E) Reduced pyruvate plus malate oxidation (as compared to succinate oxidation) after AIF knockout in ES cells or AIF knockdown with siRNA in HeLa cells, as determined by respirometry. Asterisks in (D, E) indicate significant (P<0.01) differences between AIF-deficient cells and their controls. Download figure Download PowerPoint Noticeably, under the conditions used for succinate oxidation rate determination, oxygen uptake is known to be limited by complex II activity, rather than by complex III or IV (Rustin et al, 1994). The relative inhibition of complex III activity by antimycin was not significantly different (more than 85%) between AIF−/y and AIF+/y cell lines, with residual activity being due to the direct chemical reduction of Cyt c by quinol in solution (Chretien et al, 2004). No indication of significant mitochondrial ubiquinone depletion could be observed, as quinone-dependent activities such as succinate cytochrome c reductase or glycerol 3-phosphate cytochrome c reductase were neither found decreased nor stimulated by exogenous quinone derivatives (not shown). The quantitation of the enzymatic activity of complexes I–V in isolated mitochondria confirmed that complex I (but not complexes II, IV, and V) exhibited a significant (P<0.001) defect (more than 70%) in AIF−/y ES cells, as well as in HeLa cells treated with AIF siRNA. In addition, complex III activity was reduced to about 50% in ES AIF−/y cells but not in AIF siRNA HeLa cells (Figure 3A). Thus, the AIF defect entails an OXPHOS deficiency that mostly affects complex I activity. This complex I defect also affected the NADH-dependent ferricyanide reductase activity, taking place at the NADH oxidation site of complex I, which is not rotenone sensitive. Again, we found a similar 50% decrease for both AIF−/y ES cells and AIF siRNA-treated HeLa cells (Figure 3B), suggesting a general defect of complex I rather than a specific catalytic dysfunction. Figure 3.Respiratory chain complex activities in AIF-negative cells. (A) Measurements of isolated respiratory chain complexes. Mitochondrion-enriched fractions from AIF−/y, AIF+y ES cells or control siRNA and AIF siRNA HeLa cells were monitored for the activity of each of the respiratory chain complexes. Data (mean±s.d.) were ratioed to the AIF-positive controls, which were considered as 100% value. This experiment has been performed three times, in triplicate, with similar results. Asterisks denote a significant (P<0.01) AIF deficiency as compared to the control value (100%). (B) Reduced ferricyanide reduction in AIF-deficient ES and HeLa cells. As compared to ES control cells (trace a), AIF−/y ES cells (trace b) show a 50% decreased NADH-ferricyanide reductase activity. A 50% reduction of NADH-ferricyanide reductase activity was also observed in AIF siRNA HeLa cells (trace d) as compared to control HeLa cells (trace c), in three independent determinations. Arrows indicate addition of dodecylmaltoside. Download figure Download PowerPoint Reduced complex I and III expression in AIF-deficient cells Blue native (BN) polyacrylamide gel electrophoresis (PAGE) revealed that complex I and, to a less extent, complex III of AIF−/y ES were reduced in their abundance, as compared to AIF+/y controls (∼980 kDa) (Figure 4A). The in-gel activity of complex I was reduced, yet detectable, in AIF-deficient cells (Figure 4A). Two-dimensional gel electrophoresis (BN-PAGE followed by SDS–PAGE) revealed that all subunits of complex I from AIF−/y ES cells were reduced in their expression (Figure 4B). This applies also to complex III subunits, although the reduction is much lower (Figure 4B). Immunoblot experiments revealed that complex I subunits (e.g. the 75, 39, and 30 kDa subunits) migrated within complex I in BN-PAGE (Figure 4B), both in AIF-expressing and AIF-deficient mitochondria, although they were less abundant in the latter. AIF did not comigrate with complex I in BN-PAGE and rather migrated at a higher mobility than complex III (Supplementary Figure 1). Thus, AIF itself is not part of the complex I (nor of a higher order structure comprising complex I). SDS–PAGE followed by immunoblot (Figure 4C) revealed that the AIF knockout or knockdown resulted in the loss of complex I subunits (e.g. the 17, 20, and 39 kDa subunits, also called NDUFB6, NDUFS7, and NDUFA9, as well as the recently identified Grim19 subunit), and this was confirmed for distinct AIF−/y ES cell lines and AIF siRNA heteroduplexes (Supplementary Figure 2). We also found that subunits of complex III were deficient in AIF−/y ES cells, in particular the core subunits (gene numbers UQCRC1 and UQCRC2) and the iron–sulfur subunit (gene number UQCRFS1). A decreased expression of complex I (but not III) subunits was also detected in HeLa cells after suppression of AIF expression (Figure 4C), correlating with the respirometric data. The decreased expression of complex I subunits was a post-transcriptional phenomenon as it was not accompanied by reduced mRNA levels (Figure 4D). Retransfection of AIF−/y ES cells with full-length mouse AIF (which is imported into mitochondria) restored the expression of complex I and III subunits, while transfection with an AIF mutant lacking the mitochondrial targeting sequence (Δ1–100) failed to do so (Figure 4E). Note that the AIF defect did not affect the expression of proteins contained in a variety of different mitochondrial multienzyme complexes, namely complexes IV and V (Figure 4A and B), as well as TOM40 of the TOM complex and the VDAC protein of the permeability transition pore complex (Figure 4C). Moreover, the AIF deficiency did not lead to the disappearance of mitochondrial DNA-encoded respiratory chain subunits such as COX1, 2, and 3 (in complex IV; Figure 4B) and cytochrome b (in complex III; Figure 4B and Supplementary Figure 3) at the protein level, and mitochondrial DNA was normally transcribed in AIF-deficient cells (Figure 4F), thus excluding a gross perturbation of mitochondrial biogenesis and/or a depletion of mitochondrial DNA. Figure 4.Reduced abundance of complexes I and III. (A) BN-PAGE and in-gel activity of complex I measured on isolated mitochondria from AIF−/y or AIF+/y ES cells. Dodecylmaltoside was used for solubilization and separation of the mitochondrial complexes I, V, III, and IV (I, V, III, IV) by BN-PAGE. Densitometry (normalized to complex V) revealed a relative deficiency in AIF−/y cells, as far as the abundance of complex I (<30% of control AIF+/y value) and its in-gel activity are concerned (14% of control value), a reduced abundance of complex III (54% of control), and no defect in complex IV (84%) and complex V (100%). (B) Two-dimensional resolution of OXPHOS from AIF−/y or AIF+/y ES cells. Silver-stained 2D gels, as well as their immunodecoration with antibodies specific for three complex I subunits are shown. Complex III subunits (black arrows) are core protein II, the mitochondrially encoded cytochrome b, cytochrome c1, and the 'Rieske' iron–sulfur protein, in the order of descending mass. Complex IV subunits (white arrows) are the mitochondrially encoded subunits COX I, II, and III. (C) SDS–PAGE determination of the subunit composition of complexes I and III using a number of monoclonal antibodies. Whole cell lysates from AIF−/y or AIF+/y ES and control or AIF siRNA (siRNA-AIF1) HeLa cells were subjected to immunoblot detection of the indicated antigens. This experiment has been reproduced five times. Data were confirmed for another ES knock-out cell line and additional siRNA controls (Supplementary Figure 2). (D) Expression of nuclear DNA-encoded complex II subunits in AIF-deficient cells, as determined by RT–PCR, normalized to 18S RNA. The values are given as percentage of the control (AIF+/y ES cells for AIF−/y ES cells and HeLa cells treated with emerin-specific siRNA for HeLa cells treated with AIF-specific siRNA). (E) Expression of AIF into AIF−/y ES restores complex I expression. Cells were transfected (efficiency 12±2%) with full-length (FL) AIF, Δ1–100 AIF (which lacks the mitochondrial targeting sequence), or empty vector, and 48 h later the expression of the indicated complex I and III subunits was monitored. (F) Expression of mitochondrial RNA in AIF-negative cells. Total cellular RNA was extracted from AIF+/y and AIF−/y ES cells, and the levels of the indicated RNA species (ND1, NADH dehydrogenase subunit 1; ND6, NADH dehydrogenase subunit 6; cytB, cytochrome b; cox1, Cyt c oxidase subunit 1; cox2, Cyt c oxidase subunit 2; ATP6, ATP synthase F0 subunit 6; 16S RNA; tRNA Leu-1, tRNA Leucine 1) encoded by mitochondrial DNA were quantified by RT–PCR. The results (mean±s.e.m., n=3) were expressed as percentage of control values (100% in AIF+/y ES cells). Download figure Download PowerPoint Absence of significant oxidative insult but enhanced glucose dependency of AIF-deficient cells AIF-deficient and AIF-sufficient ES cells exhibit similar capacities to oxidize the fluorescent indicator dehydrorhodamine 123 (which is nonfluorescent and can be oxidized by reactive oxygen species (ROS) to fluorescent rhodamine 123) and DCF-DA (which measures cellular H2O2 production). AIF-deficient cells contain normal (or slightly elevated) levels of nonoxidized cardiolipin (measured with the fluorescent probe nonylacridine orange (NAO)), normal levels of glutathione (GSH, measured with the fluorochrome monochlorobiman), as well as normal (or slightly elevated) levels of NAD(P)H (measured by assessing its specific autofluorescence; Supplementary Figure 4A). Superoxide dismutase (SOD) activity, which can be considered as an inducible marker for increased superoxide production (Geromel et al, 2002), was found similar in AIF-deficient cells and control cells, in both ES cells and siRNA-treated HeLa cells (Supplementary Figure 4B). Thus, no evidence for oxidative insult in AIF−/y cells could be obtained, suggesting that AIF does not behave as an antioxidant protein in these cells, at least in baseline conditions. Addition of 2-D-deoxyglucose, an inhibitor of glycolysis, did not affect the viability of control ES cells, yet killed a significant fraction of AIF-deficient cells indicative of a high glucose dependency of the latter cells (Figure 5A). Adherent (nonapoptotic) AIF−/y and AIF+/y ES cells exhibited similar base line ATP levels. Upon glucose withdrawal, AIF−/y (but not AIF+/y) manifested a strong decrease in intracellular ATP levels (Figure 5B). Accordingly, glucose withdrawal compromised the growth of AIF-negative cells, yet had no effect on AIF+/y controls (Figure 5C). Fructose, known to readily fuel anaerobic glycolysis, but not galactose or sucrose, which poorly enter glycolysis (Kim et al, 2003; Mazzio and Soliman, 2003), relieved the glucose dependence of AIF−/y cells. Neither glutamine nor respiratory substrates (succinate or pyruvate) could substitute for the need of glucose (Figure 5C). Figure 5.Increased dependence of AIF-deficient cells on glycolysis. (A) Increased death of AIF-deficient cells after inhibition of glycolysis by 2-D-deoxyglucose. Cells (AIF−/y or AIF+/y) were cultured for 72 h in the absence or presence of 6 mM deoxyglucose, followed by staining with propidium iodide (PI) to determine the frequency of dead cells. (B) Reduced ATP production in AIF−/y cells upon glucose withdrawal. Cells were cultured for 36 h in the absence or presence of glucose, and ATP was determined among the adherent fraction of cells. (C) Reduced growth of AIF−/y cells in the absence of glucose. AIF−/y and AIF+/y cells were cultured for 3 days in the presence (5 g/l in C) or absence (Glu−) of glucose, in the presence or absence of the indicated sugars or respiratory chain substrates (all at 5 mM), and the exact number of viable (DAPI−) cells was determined by adding FITC-labeled beads as an internal standard of the FACS analysis. The results are means of three independent determinations (mean±s.d.) and asterisks mark significant (P<0.01) effects of the AIF deficiency. Download figure Download PowerPoint In response to arsenate, AIF+/y cells exhibited a higher NAD(P)H depletion than AIF−/y ES cells (Cande et al, 2004b), and this effect was abolished by exogenous supplementation of GSH ester or the SOD mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin (MnTBAP) (Figure 6A). These antioxidant agents (as well as tocopherol and the mitochondrion-targeted antioxidant decylubiquinone) failed to cause the re-expression of complex I (as exemplified for the 20 kDa subunit) in AIF−/y ES cells (Figure 6B), although, as an additional control of their antioxidant potential, they did reduce the mitochondrial generation of ROS induced by menadione, both in AIF−/y and AIF+/y cells (Figure 6C). Supplementation with exogenous GSH was also unable to restore the growth of glucose-depleted AIF−/y cells, although it did restore the growth of menadione-treated cells (Figure 6D). Thus, although AIF is involved in NAD(P)H metabolism, this redox function is unlikely to be relevant to the OXPHOS defect that accompanies AIF deficiency. Figure 6.Modulation of the redox consequences of the AIF defect. (A) Enhanced NAD(P)H depletion in AIF+/y as compared to AIF−/y cells in response to arsenate. Cells were treated with 1 mM arsenate for 6 h, in the presence or absence of GSH ester (5 mM) or MnTBAP (50 μM), followed by cytofluorometric determination of cellular NAD(P)H levels. (B, C) Failure of antioxidants to cause re-expression of the 20 kDa complex I subunit. AIF−/y and AIF+/y cells were cultured for 72 h in the presence of tocopherol (200 μM), decylubiquinone (50 μM), GSH ester (10 mM), or MnTBAP (50 μM), all re-added every 12 h, followed by immunoblot (B).