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• W2155891625 abstract "Article17 August 2006free access Dissociating the dual roles of apoptosis-inducing factor in maintaining mitochondrial structure and apoptosis Eric CC Cheung Eric CC Cheung Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Nicholas Joza Nicholas Joza IMBA, Institute of Molecular Biotechnology of the Austria Academy of Sciences, Vienna, Austria Search for more papers by this author Nancy AE Steenaart Nancy AE Steenaart Gemin X Biotechnologies Inc., Montreal, Quebec, Canada Search for more papers by this author Kelly A McClellan Kelly A McClellan Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Margaret Neuspiel Margaret Neuspiel Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Stephen McNamara Stephen McNamara Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Jason G MacLaurin Jason G MacLaurin Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Peter Rippstein Peter Rippstein Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author David S Park David S Park Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Gordon C Shore Gordon C Shore Department of Biochemistry, McGill University, Montreal, Quebec, Canada Search for more papers by this author Heidi M McBride Heidi M McBride Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Josef M Penninger Corresponding Author Josef M Penninger IMBA, Institute of Molecular Biotechnology of the Austria Academy of Sciences, Vienna, Austria Search for more papers by this author Ruth S Slack Corresponding Author Ruth S Slack Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Eric CC Cheung Eric CC Cheung Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Nicholas Joza Nicholas Joza IMBA, Institute of Molecular Biotechnology of the Austria Academy of Sciences, Vienna, Austria Search for more papers by this author Nancy AE Steenaart Nancy AE Steenaart Gemin X Biotechnologies Inc., Montreal, Quebec, Canada Search for more papers by this author Kelly A McClellan Kelly A McClellan Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Margaret Neuspiel Margaret Neuspiel Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Stephen McNamara Stephen McNamara Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Jason G MacLaurin Jason G MacLaurin Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Peter Rippstein Peter Rippstein Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author David S Park David S Park Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Gordon C Shore Gordon C Shore Department of Biochemistry, McGill University, Montreal, Quebec, Canada Search for more papers by this author Heidi M McBride Heidi M McBride Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Josef M Penninger Corresponding Author Josef M Penninger IMBA, Institute of Molecular Biotechnology of the Austria Academy of Sciences, Vienna, Austria Search for more papers by this author Ruth S Slack Corresponding Author Ruth S Slack Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Search for more papers by this author Author Information Eric CC Cheung1, Nicholas Joza2, Nancy AE Steenaart3, Kelly A McClellan1, Margaret Neuspiel4, Stephen McNamara1, Jason G MacLaurin1, Peter Rippstein4, David S Park1, Gordon C Shore5, Heidi M McBride4, Josef M Penninger 2 and Ruth S Slack 1 1Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada 2IMBA, Institute of Molecular Biotechnology of the Austria Academy of Sciences, Vienna, Austria 3Gemin X Biotechnologies Inc., Montreal, Quebec, Canada 4Ottawa Heart Institute, University of Ottawa, Ottawa, Ontario, Canada 5Department of Biochemistry, McGill University, Montreal, Quebec, Canada *Corresponding authors: Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr Bohr-gasse 3, 1030 Vienna, Austria. Tel.: +43 (1) 790 44; Fax: +43 (1) 790 44-110; E-mail: [email protected] Ottawa Health Research Institute, Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Room 2452, Ottawa, Ontario, Canada K1H8M5. Tel.: +1 613 562 5800 ext 8459; Fax: +1 613 562 5403; E-mail: [email protected] The EMBO Journal (2006)25:4061-4073https://doi.org/10.1038/sj.emboj.7601276 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The mitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and induces apoptosis. Recent studies, however, have indicated the importance of AIF for survival in mitochondria. In the absence of a means to dissociate these two functions, the precise roles of AIF remain unclear. Here, we dissociate these dual roles using mitochondrially anchored AIF that cannot be released during apoptosis. Forebrain-specific AIF null (tel. AifΔ) mice have defective cortical development and reduced neuronal survival due to defects in mitochondrial respiration. Mitochondria in AIF deficient neurons are fragmented with aberrant cristae, indicating a novel role of AIF in controlling mitochondrial structure. While tel. AifΔ Apaf1−/− neurons remain sensitive to DNA damage, mitochondrially anchored AIF expression in these cells significantly enhanced survival. AIF mutants that cannot translocate into nucleus failed to induce cell death. These results indicate that the proapoptotic role of AIF can be uncoupled from its physiological function. Cell death induced by AIF is through its proapoptotic activity once it is translocated to the nucleus, not due to the loss of AIF from the mitochondria. Introduction Mitochondria are the central relaying stations for apoptotic signals. After the induction of apoptosis, cytochrome c is released from the mitochondria that interacts with Apaf1 and procaspase 9, which in turn activates the caspase cascade (reviewed in Yuan et al, 2003; Danial and Korsmeyer, 2004). Apart from the caspase-dependent pathway, mitochondrial factors also initiate a caspase-independent apoptotic signaling cascade (reviewed in Cregan et al, 2004; Hong et al, 2004). This pathway is initiated by the release of the mitochondrial protein, apoptosis-inducing factor (AIF), which translocates to the nucleus and induces DNA fragmentation through interactions with factors including EndoG in Caenorhabditis elegans, CypA in mice, and others such as FEN-1 (Susin et al, 1999; Daugas et al, 2000; Wang et al, 2002; Parrish and Xue, 2003; Cande et al, 2004). The significance of these interactions, however, are not yet clear, as EndoG and CypA null animals have no apparent defect in apoptosis (Colgan et al, 2000; Irvine et al, 2005). The role of AIF in neuronal cell death was first suggested from the observation that AIF translocates to nucleus after the induction of various types of acute neuronal injury in vitro and in vivo (Zhang et al, 2002; Cao et al, 2003; Plesnila et al, 2004; Wang et al, 2004). Mitochondrial release of AIF has been shown to depend on PARP activity (Yu et al, 2002; Wang et al, 2004). We have previously demonstrated that AIF translocation following neuronal injury is caspase independent (Cregan et al, 2002; Cheung et al, 2005). Using Apaf1−/− neurons, we have shown that AIF is translocated to the nucleus on induction of apoptosis, and this can be inhibited by microinjecting AIF neutralizing antibodies (Cregan et al, 2002). Depending on the cell type and death stimulus, the release of AIF may also be caspase dependent, as studies using C. elegans with BH-3 only protein EGL-1 (Wang et al, 2002), HeLa cells with staurosporine (Arnoult et al, 2003), and rat cortical neurons (Lang-Rollin et al, 2003) have previously shown. We have used Harlequin (Hq) mice, which exhibit only 20% AIF expression (Klein et al, 2002), to directly investigate the role of AIF in various models of neuronal cell death. Using Hq/Apaf1−/− double mutant mice we have shown that reduced levels of AIF, along with inactivation of caspase activity, can sustain neuronal survival after DNA damage and excitotoxic induced cell death. These results revealed that AIF is involved in both Bax dependent and Bax independent mechanisms of cell death (Cheung et al, 2005). In mammalian systems, therefore, AIF is a key death inducer that functions in multiple mechanisms of neuronal cell death; thus understanding its mechanism of action is crucial. Apart from the apoptotic role of AIF, studies with AIF depleted cells have indicated that AIF also has a physiological role in the mitochondria. Studies using Hq mice, which exhibit cerebellar degeneration and increased sensitivity to oxidative stress (Klein et al, 2002), suggesting that AIF acts as an oxidative radical scavenger in the mitochondria (Lipton and Bossy-Wetzel, 2002). A recent study, however, revealed that AIF depleted cells (Aif−/− ES and Hq cells) have defective oxidative phosphorylation and reduced expression of Complex I and III in the electron transport chain of the mitochondria. AIF, however, was not found to be associated with either Complex I or III (Vahsen et al, 2004); therefore, the mechanism by which AIF stabilizes complex I remains unknown. Since AIF has dual roles, dissociating its functions in each of these cellular events has been difficult. For example, it remains unknown whether cell death is triggered by the loss of AIF from mitochondria. This would argue that AIF's proapoptotic role is not essential to the induction of apoptosis. Here, we resolve this controversy by dissociating the physiological role and the apoptotic role of AIF. To this end, we have constructed a mitochondrial inner membrane anchored form of AIF (anchored AIF) that cannot be released from the mitochondria during apoptosis and thus maintains its physiological role. These constructs were then introduced in AIF deficient neurons from a telencephalon specific conditional mutant of AIF. Here, we show that: (a) AIF plays an important role in neuronal survival by maintaining mitochondrial structure; and (b) AIF has a major role in proapoptotic signaling following nuclear translocation. Expression of anchored AIF in cells with endogenous AIF can offer protection only during the initial stages of apoptosis by maintaining the pool of AIF in mitochondria. At longer time points, however, the cells still succumb to death even when AIF is present in the mitochondria. This demonstrates that reconstitution of mitochondrial AIF is not sufficient to rescue cell death and that AIF plays an active role in proapoptotic signaling in the nucleus. In conclusion, we dissociated the dual functions of AIF and directly demonstrate the importance of the proapoptotic role of AIF, apart from its novel role in maintaining mitochondrial structure. Results Generation of telencephalon conditional AifΔ mice AIF null embryos die around embryonic day (E) 12, and muscle-specific loss of AIF leads to mitochondrial dysfunction, skeletal muscle atrophy and dilated cardiomyopathy (Joza et al, 2005). To study the function of AIF in neurons, we generated telencephalon-specific AIF mutant mice (tel. AifΔ) by crossing Aifflox/flox mice with mice carrying Cre driven by the promoter Foxg1. Cre mediated excision of the floxed allele occurs in neuronal precursors of the telencephalon at E9 (Hebert and McConnell, 2000), resulting in deletion of the targeted gene in all cortical neurons. Deletion of the floxed AIF allele was assessed by PCR and Western blot analysis. In the absence of Cre, the floxed allele was present (Figure 1A, lanes 1 and 2). Cre expression resulted in deletion of AIF (Figure 1A, lane 4). Western blot analysis confirmed the absence of AIF only in the mutant telencephalon (Figure 1B). Figure 1.AIF is essential for neuronal survival during cortical development. (A) PCR analysis of E15.5 telencephalon tissue. Lane 1: Aif+/+cre+/+; lane 2: Aifflox/Ycre+/+; lane 3: Aifflox/+, cre+/−; lane 4: Aifflox/Y, cre+/− (tel. AifΔ). (B) Western blot analysis for AIF and control β-actin expression of various tissues from tel. AifΔ and wild-type littermates at E15.5. (C) Cresyl violet staining of control and tel. AifΔ mice coronal forebrain sections at E15.5. SVZ=subventricular zone, IZ=intermediate zone, CP=cortical plate. Bar=250 μm. n=3. (D) Active caspase 3 immunohistochemistry of control and tel. AifΔ coronal forebrain sections at E15.5. n=3. (E) PH3 immunohistochemistry of control and tel. AifΔ coronal forebrain sections at E15.5. n=3. *P<0.05 compared to wildtype. Download figure Download PowerPoint AIF is required for neuronal survival during cortical development We next examined the telencephalon of the conditional mutants to investigate the role of AIF in cortical development. Tel. AifΔ conditional mutants die by E17 (data not shown). The tel. AifΔ mice exhibited reduced cortical thickness at E15.5 compared to control littermates (Figure 1C). This reduction of thickness occurs mostly at the cortical plate (CP) and intermediate zone (IZ), and to a lesser extent at the subventricular zone (SVZ) (Figure 1C). To address whether this reduction in size is due to increased apoptosis or reduced numbers of progenitor cells, active caspase 3 and phospho-histone H3 (PH3) staining were used to assess cell death and progenitor proliferation, respectively (Ferguson et al, 2002). Active caspase 3 staining revealed a marked increase in cell death in the tel. AifΔ in regions of postmitotic cells (Figure 1D), whereas PH3 staining indicated similar numbers of proliferating progenitor cells along the ventricles in both the conditional mutants and their control littermates (Figure 1E). These data indicate that AIF is essential for survival of maturing neurons during cortical development, but AIF expression is dispensable for the proliferation of neuronal progenitors. To assess whether cell death due to AIF depletion was cell autonomous, primary neuronal cultures were examined. After 2 days of culture, primary neuronal cells from E14.5 tel. AifΔ cortices exhibited increased cell death relative to neuronal cells from wild-type controls (Figure 2A). Consistent with previous reports on Aif−/Y ES cells (Vahsen et al, 2004), we found that expression of respiratory chain complex I was abrogated in mutant neurons compared to control neurons (Figure 2E). Importantly, the reduced viability of tel. AifΔ neurons can be rescued when cells were cultured in media enriched with pyruvate, uridine, and additional glucose (PU media) to bypass defects in mitochondrial respiration (Figure 2A). These supplements were used previously to culture cells lacking cytochrome c (Li et al, 2000) or mtDNA (King and Attardi, 1989), which exhibit defective mitochondrial respiration. These results show that AIF is required for neuronal cell survival and normal mitochondrial respiration in neurons. Figure 2.Mitochondrially anchored AIF rescues reduced survival of tel. AifΔ neurons. (A–C) Cortical neurons were isolated from E15.5 tel. AifΔ and wild-type littermates and cultured in normal media or enriched PU media containing 50 mg/l pyruvate, 110 mg/l uridine and 5 mM glucose. (A) Quantitative analysis of cell death of tel. AifΔ and wild-type neurons cultured in normal and PU media (n=3). (B) Wild-type cortical neurons were infected with recombinant adenoviral vector containing GFP-tagged wild-type AIF (AIF-GFP) or GFP-tagged N-AIF (N-AIF-GFP) and were treated with or without campthothecin. After 36 h, cells were fixed and stained with Hoechst to visualize nuclei and an anti-Tom20 antibody (red) to detect mitochondria. Green GFP fluorescence indicates AIF localization. (C) Western analysis on subcellular fractionation of neurons infected with GFP-tagged wildtype AIF, N-AIF and D-AIF. Upper panel: no camptothecin, lower panel: 12 h after camptothecin treatment. m=mitochondrial fraction, n=nuclear fraction, and c=cytoplasmic fraction. (D) Quantitative analysis of spontaneous cell death of cortical neurons isolated from tel. AifΔ and wild-type littermates infected with control virus, wild-type AIF (wt AIF), or mitchondrially anchored AIF (N-AIF and D-AIF) at 50 MOI in normal media. Cell death was quantified by apoptotic nuclear morphology using Hoechst (n=3). *P<0.05 compared to tel. AifΔ in normal media. (E) Western analysis of complex I (39 kDa subunit) expression in tel. AifΔ neurons expressing N-AIF and D-AIF compared to wild-type and control tel. AifΔ neurons. (F) ATP production of the tel. AifΔ neurons expressing either N-AIF, D-AIF, or GFP as control (n=3). *P<0.05 compared to tel. AifΔ neurons with GFP control. (G) Oxygen consumption of the tel. AifΔ neurons expressing either N-AIF, D-AIF, or GFP as control (n=3). *P<0.05 compared to tel. AifΔ neurons with GFP control. Download figure Download PowerPoint Construction of AIF anchored to the inner membrane of the mitochondria During apoptosis, AIF is released from mitochondria and translocates to the nucleus, inducing chromatin condensation and degradation. Since AIF depletion causes early lethality in neurons, the dual roles of AIF in mediating apoptosis and cellular homeostasis are difficult to resolve. In order to dissect the potential role of AIF in apoptosis from its role in mitochondria, we reconstituted AIF deficient neurons with mitochondrially anchored AIF constructs such that AIF was permanently tethered to the inner mitochondrial membrane. To this end, we exchanged the mitochondrial localization sequence (MLS) of AIF with the MLS of two proteins (D-lactate dehydrogenase (Rojo et al, 1998; Flick and Konieczny, 2002) for D-AIF, and a modified form of pOSA-141I4 for N-AIF (Steenaart and Shore, 1997)) that are anchored to the outer leaflet of the inner membrane of mitochondria (Supplementary Figure 1A). Quantification of GFP fluorescence and Western blot analysis of infected cells revealed similar expression of these constructs, comparable to endogenous level (Supplementary Figure 1B and C). Western analysis of digitonin gradient treatment on isolated mitochondria with anchored AIF mutant, as well as immunocytochemical analysis on digitonin treated neurons with anchored AIF mutant, showed that the anchored AIF mutant is located in the intermembrane space, similar to wild-type AIF (Supplementary Figure 1D and Figure 1E). These results indicate that the anchored AIF constructs maintained the normal orientation of AIF in the mitochondria. GFP fluorescence from these anchored AIF constructs showed that both D-AIF and N-AIF were localized in mitochondria. Importantly, in contrast to wild-type AIF, both D-AIF and N-AIF remained associated with mitochondria after an apoptotic insult at all time points, as shown by immunocytochemistry and subcellular fractionation followed by Western blot analysis (Figure 2B and C). Expression of respiratory chain complex I (Figure 2E) was restored in AIF deficient neurons expressing mitochondrially anchored AIF, indicating that these constructs are functional. We next assessed survival of tel. AifΔ neurons expressing mitochondrially anchored AIF. After 2 days under normal nonenriched culture conditions, tel. AifΔ neurons with either one of the anchored AIF constructs maintained the same level of cell survival as wild-type neurons, which was not rescued by GFP control vector (Figure 2D). ATP production and oxygen consumption were also restored in AIF deficient neurons expressing the anchored AIF constructs compared to GFP control vector (Figure 2F and G). These experiments demonstrate that expression of mitochondrially anchored AIF rescues the cell death of tel. AifΔ neurons. AIF is required for maintaining mitochondrial morphology and cristae structure We next asked whether there was perturbation of mitochondrial morphology in tel. AifΔ neurons. The mitochondrial membrane potential sensitive dye TMRE was used to visualize mitochondria in cells cultured in enriched media. Wild-type neurons have elongated and tubular mitochondria that often spread along neurites (Figure 3A). In contrast, tel. AifΔ neurons exhibited short and fragmented mitochondria that are often perinuclear (Figure 3A). Few mitochondria were observed in the neurites of tel. AifΔ neurons. Mitochondrial membrane potential of tel. AifΔ neurons was hyperpolarized relative to wild-type cells (Supplementary Figure 2A), which can be dissipated using FCCP, the mitochondrial potential uncoupler (Supplementary Figure 2B). The hyperpolarized membrane potential as well as altered mitochondrial morphology were restored to normal by the expression of either anchored AIF or wild-type AIF in tel. AifΔ neurons (Figure 3A and Supplementary Figure 2). Interestingly, expression of anchored AIF in wild-type cells resulted in increased mitochondrial length compared to controls (Figure 3A and B), suggesting a role for AIF in maintaining mitochondrial morphology. Next, we asked if the mitochondrial ultrastructure is also disrupted in these cells. We used transmission electron microscopy to visualize mitochondria from neurons cultured in enriched PU media to eliminate secondary effects due to reduced survival in tel. AifΔ neurons. In wild-type cells, mitochondrial cristae are shaped as compact tubules in an orderly fashion (Figure 4A). Tel. AifΔ neurons, on the other hand, displayed aberrant cristae morphology. The cristae are dilated and do not orient in an orderly fashion (Figure 4A). The cross-sectional distance of tel. AifΔ mitochondrial cristae is ∼2.5 times wider than in control wild-type mitochondria (Figure 4B). Expression of anchored AIF in wild-type cells again reduced intracristae distances from 20 to 17 nm (Figure 4B), further supporting the notion that the defect seen in tel. AifΔ neurons does not result from secondary effects. A detailed analysis of intracristal cross-sectional distance is shown in Supplementary Figure 3A and B. These studies demonstrate a novel role for AIF in regulating mitochondrial structure and cristae morphology. Figure 3.AIF controls mitochondrial structure. Cortical neurons from E15.5 tel. AifΔ and wild-type littermates were infected at time of plating with mitochondrially anchored N-AIF and D-AIF and a GFP control virus at 50 MOI in enriched PU media. (A, B) After 36 h, 50 nm TMRE was added to media and live cell images were taken. (A) Representative images of tel. AifΔ and wild-type mitochondria infected with the indicated constructs. Bars=1 μm. (B) Average length of mitochondria of neurons. The cell types and treatments are as indicated (n=4). *P<0.05 compared to wild type with no virus; +P<0.05 compared to tel. AifΔ infected with the GFP control virus; **P<0.05 compared to wild type with GFP control. Download figure Download PowerPoint Figure 4.AIF depleted neurons have perturbed mitochondrial cristae structure. (A, B) Transmission electron microscopy of mitochondria. (A) Representative images of mitochondria of tel. AifΔ and wild-type mitochondria infected with the indicated constructs. Bars=100 nm. (B) Quantification of the intracristal cross-sectional distances (n=4). *P<0.05 compared to tel. AifΔ infected with the GFP control virus; **P<0.05 compared to wild-type neurons infected with the GFP control virus. Download figure Download PowerPoint Mitochondrially anchored AIF revealed critical role of AIF during apoptosis Defining the proapoptotic function of AIF has been confounded by recent findings demonstrating an essential physiological role for AIF in the mitochondria. Presently, it is unknown if mitochondrial release of AIF in itself induces apoptosis due to loss of AIF mitochondrial function, or whether AIF plays a proapoptotic role following nuclear translocation. First, we generated double tel. AifΔ Apaf1−/− animals in which both caspase dependent and independent pathways have been inactivated. Double mutant embryos exhibit some increase in cortical thickness relative to tel. AifΔ animals (Figure 5A and B), possibly due to the increased survival of cells in Apaf1−/− background. As reported previously (Cozzolino et al, 2004), Apaf1−/− mice have increased numbers of progenitor cells compared to wild type and tel. AifΔ mice (Figure 5A and D). Next, we asked if the double-mutant neurons exhibit protection against DNA damage induced cell death. After inducing cell death with camptothecin, the tel. AifΔ Apaf1−/− double mutant neurons cultured in pyruvate supplemented media, exhibited increased survival relative to single mutants and wild-type control neurons (Figure 6A). AIF deficiency alone can offer transient but significant protection at 12 h (Figure 6A, ∼45% for tel. AifΔ versus ∼55% for wild type), and at later time points the percentage of cell death of tel. AifΔ cells becomes similar to wild-type cells due to the presence of caspases. This is in agreement with Hq neurons, which also showed a transient delay in chromatin condensation compared to wildtype during cell death (Cheung et al, 2005). Cytochrome c release in these mutants is not affected, suggesting that mitochondrial permeablization is not affected by the deletion of Apaf1 and AIF (Supplementary Figure 4). AIF release in Apaf1 neurons is similar to wild-type cells (Supplementary Figure 5A and B), which is in agreement with our previous results showing AIF release in Apaf1 neurons is similar to wild type after p53 and camptothecin induced cell death (Cregan et al 2002). Caspase activity was not detected in Apaf1−/− and tel. AifΔ Apaf1−/− compared to wild type and tel. AifΔ (Supplementary Figure 6A). Figure 5.Apaf1 deficiency can partially compensate neuronal loss due to AIF deficiency during development. (A) Cresyl violet staining, active caspase 3 staining, and PH3 staining of coronal telencephalon sections from control wild type, tel. AifΔ, Apaf1−/−, and tel. AifΔ Apaf1−/− double mutant mice (E14.5). Bar=250 μm. (B–D) Quantitative analysis of (B) cortical thickness (n=3), (C) active caspase 3 positive cells (n=3) and (D) PH3 positive cells (n=3); *P<0.05 compared to wild type; **P<0.05 compared to tel. AifΔ. Download figure Download PowerPoint Figure 6.Dissociation of the dual roles of AIF in tel. AifΔ Apaf1−/− neurons revealed AIF's proapoptotic role at the nucleus apart from its physiological role in the mitochondria. (A) Cortical neurons cultured from E14.5 wild type, tel. AifΔ, Apaf1−/− and tel. AifΔ Apaf1−/− double mutant embryos were treated with camptothecin in enriched media and cell death were assessed at the indicated time points (n=3). (B) Cortical neurons from E14.5 tel. AifΔ Apaf1−/− double mutant embryos were infected at the time of plating with mitochondrially anchored D-AIF and N-AIF and a GFP control virus at 50 MOI in enriched media. Campthothecin was then added and cell death was assessed by Hoechst staining (n=3). (C) Oxygen consumption of tel. AifΔ Apaf1−/− neurons expressing D-AIF and N-AIF after camptothecin treatment (n=3). *P<0.05 compared to tel. AifΔ Apaf1−/− neurons expressing GFP control. (D) Cortical neurons from tel. AifΔ Apaf1−/− double mutant were infected at the time of plating with AIF, NES-AIF or a GFP control at 50 MOI in enriched media. Camptothecin was then added, and cell death was assessed at the indicated time points (n=3). *P<0.05 compared to GFP control. Download figure Download PowerPoint Using the anchored AIF mutants and tel. AifΔ Apaf1−/− neurons, we asked whether AIF is indeed an apoptotic effector after induction of cell death. We first addressed if anchored AIF can provide further protection against cell death in tel. AifΔ Apaf1−/− neurons. Western analysis revealed that anchored AIF is not released during cell death but is retained in the mitochondria (Supplementary Figure 7), and similar to tel. AifΔ Apaf1−/− neurons, caspase was not activated (Supplementary Figure 6B). Strikingly, expression of either of the anchored AIF constructs in the double mutant neurons in enriched media could provide further protection against cell death for an extended time after insult (Figure 6B). Anchored mutants could also maintain oxygen consumption following camptothecin treatment (Figure 6C). This indicates that by retaining AIF in the mitochondria during cell death, survival can be sustained" @default.
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• W2155891625 date "2006-08-17" @default.
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• W2155891625 title "Dissociating the dual roles of apoptosis-inducing factor in maintaining mitochondrial structure and apoptosis" @default.
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