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. 2006 Sep 6;25(17):4061-73.
doi: 10.1038/sj.emboj.7601276. Epub 2006 Aug 17.

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

Eric C C Cheung  1 , Nicholas Joza, Nancy A E Steenaart, Kelly A McClellan, Margaret Neuspiel, Stephen McNamara, Jason G MacLaurin, Peter Rippstein, David S Park, Gordon C Shore, Heidi M McBride, Josef M Penninger, Ruth S Slack
Affiliations

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

Eric C C Cheung et al. EMBO J. .

Abstract

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

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Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
Figure 5
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.
Figure 6
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.
Figure 7
Figure 7
Anchored AIF can transiently protect wild-type neurons against DNA damage induced apoptosis. Cortical neurons from E14.5 wild-type mice were infected at time of plating with the anchored D-AIF and N-AIF constructs and a GFP only control at 50 MOI. Camptothecin were then added. (A) Survival of the cells was measured by nuclear morphology revealed using Hoechst staining (n=5), *P<0.05. (B) Mitochondrial membrane potential was measured by TMRE intensity (n=5), *P<0.05. (C) Percentages of cells with cytochome c retained in the mitochondria (n=5). Cytochrome c retention in mitochondria was determined using anti cyt-c immunohistochemistry. *P<0.05. (D) Western analysis on subcellular fractionation of neurons infected with N-AIF, D-AIF, and GFP control, to show cytochrome c release. Upper panel: no camptothecin, lower panel: 12 h after camptothecin treatment. m=mitochondrial fraction, n=nuclear fraction, and c=cytoplasmic fraction. (E) ATP concentration of the neurons expressing either N-AIF, D-AIF, or GFP as control. *P<0.05 compared GFP control. (F) Oxygen consumption of neurons expressing either N-AIF, D-AIF, or GFP as control. *P<0.05 compared to GFP control.
Figure 8
Figure 8
Endogenous AIF can still execute cell death in the presence of anchored AIF mutants in the mitochondria. Cortical neurons cultured (in conventional media) from E14.5 wild type, tel. AifΔ, Apaf1−/− and tel. AifΔ Apaf1−/− double mutant embryos were infected at the time of plating with the anchored D-AIF and N-AIF constructs. These cells were then treated with camptothecin and cell death was assessed at the indicated time points. (A) N-AIF (n=3) and (B) D-AIF (n=3). *P<0.05; **P<0.05 compared to wildtype at 12 h.
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