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. 2006 Nov 28;103(48):18314-9.
doi: 10.1073/pnas.0606528103. Epub 2006 Nov 20.

Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death

Seong-Woon Yu  1 , Shaida A Andrabi, Hongmin Wang, No Soo Kim, Guy G Poirier, Ted M Dawson, Valina L Dawson
Affiliations

Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death

Seong-Woon Yu et al. Proc Natl Acad Sci U S A. .

Abstract

Apoptosis-inducing factor (AIF), a mitochondrial oxidoreductase, is released into the cytoplasm to induce cell death in response to poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation. How PARP-1 activation leads to AIF release is not known. Here we identify PAR polymer as a cell death signal that induces release of AIF. PAR polymer induces mitochondrial AIF release and translocation to the nucleus. PAR glycohydrolase, which degrades PAR polymer, prevents PARP-1-dependent AIF release. Cells with reduced levels of AIF are resistant to PARP-1-dependent cell death and PAR polymer cytotoxicity. These results reveal PAR polymer as an AIF-releasing factor that plays important roles in PARP-1-dependent cell death.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Poly(ADP-ribosyl)ated extranuclear supernatant SN(PAR) induces AIF release from isolated mouse brain mitochondria. (A) Schematic diagram of in vitro PARP-1 activation and preparation of extranuclear supernatant (1). Isolated HeLa cell nuclei were incubated with various combinations of NAD+, ATP, and MNNG to induce DNA damage and PARP-1 activation. After centrifugation, the extranuclear supernatant [SN(PAR)] was separated from the nuclear pellet (2). (A–C) SN(PAR) (A), SN(PAR) + Pro K (B), or SN(PAR) + PARG or PD1 (C) was added to isolated brain mitochondria. After centrifugation, the extramitochondrial supernatant was separated and monitored for AIF release. (B) HeLa cell nuclei incubated with NAD+ alone or in combination with ATP and MNNG can synthesize PAR polymer in both the pellet (P) and supernatant (S). The nuclear supernatant containing poly (ADP-ribosyl)ated proteins after treatment of the nuclei with NAD+, ATP, and MNNG is designated as SN(PAR). SN(C) is the nuclear supernatant after incubation of the nuclei with buffer alone without reagents. (C) SN(PAR) can induce AIF release from isolated mouse brain mitochondria. The mitochondria were incubated for 10 min for all AIF release assays. M stands for the total mitochondria and is the input control (5 μg per lane). Subunit IV of cytochrome oxidase (COX IV) serves as a loading control to ensure the equal amount of mitochondria for each incubation. (D) Cortical neuron SN(PAR) can induce AIF release from isolated mouse brain mitochondria. (E) The AIF releasing activity of SN(PAR) is unaffected by Pro K pretreatment (Upper). SN(PAR) was treated with 20 μg of Pro K for 1 h and PMSF was added to inhibit Pro K activity. Most of the proteins of SN(PAR) are digested under this condition, as revealed by Coomassie blue protein staining (Lower). Arrow indicates Pro K. Pro K itself, which is inhibited by PMSF, has no AIF release activity. These experiments have been replicated in separate experiments at least three times with similar results.
Fig. 2.
Fig. 2.
PAR polymer induces AIF release from isolated mouse brain mitochondria. (A) Degradation of PAR moiety of SN(PAR) with PD1 (0.01 unit) or recombinant PARG (1 unit). Removal of poly(ADP-ribosyl)ation in SN(PAR) was verified using an anti-PAR antibody. (B) Treatment of SN(PAR) with PD1 (0.01 unit) or recombinant PARG (1 unit) for 1 h abolishes AIF releasing activity. (C) Purified PAR polymer (PAR, 100 nM) induces AIF release in isolated mouse brain mitochondria. AIF is not detected in the supernatant of the mitochondria incubated with buffer alone (B). (D) The AIF releasing activity of PAR polymer is unaffected by Pro K (20 μg, 1 h) pretreatment. (E) Purified PAR polymer (PAR, 100 nM) pretreated with PD1 (0.01 unit) or recombinant PARG (1 unit) for 1 h loses AIF releasing activity. (F) Different PAR polymer fractions with average polymer size (16-, 30-, and 60-mer) were delivered into the cortical neurons via the BioPorter delivery reagent at a final concentration of 80 nM and AIF release was monitored. B and M stand for buffer and total mitochondria protein (5 μg), respectively. (G) Dose-dependent AIF release by PAR polymer of 60 ADP-ribose units. These experiments have been replicated in separate experiments at least three times with similar results.
Fig. 3.
Fig. 3.
Cytosolic and mitochondrial PAR polymer after PARP-1 activation. (A) Representative confocal images of cortical neurons 15, 30, and 60 min after NMDA receptor stimulation. DIC images are provided to illustrate neurons. Immunocytochemical staining indicates that PAR polymer (red) not only exists in the nucleus (blue), but also in the cytoplasm in the NMDA (500 μM) treated neurons. (B) Representative images of high-power examination of individual neurons after 60 min of NMDA treatment, showing that PAR polymer is present as a thin cytoplasmic rim around the nucleus of cortical neurons. Nuclear PAR localization appears as pink staining and cytoplasmic localization appears as red staining. (Scale bar, 10 μm.) (C) Subcellular fractionations of cortical neurons indicate that PAR polymer can be found in nuclear (N), cytoplasmic (C), and mitochondrial (M) fractions. Western blotting with antibodies against histones, GAPDH, and MnSOD confirms the integrity of nuclear, cytosolic, and mitochondrial fractions, respectively. (D) Mitochondrial location of PAR polymer. Cortical neurons were immunostained with the mitochondrial protein, cytochrome oxidase 1 (COX-1, green), and PAR polymer (red); nuclei were counterstained with DAPI (blue, 300 nM in PBS) in NMDA (500 μM for 5 min)-treated neurons. PAR polymer colocalizes to mitochondria (merged image, Lower) 60 min after NMDA-treatment, whereas no PAR formation is seen in CSS-treated neurons (Upper, control). Yellow indicates overlap between COX-1 and PAR staining. A single neuron under higher magnification (magnified) is shown (Right). These experiments have been replicated in separate experiments at least three times with similar results. (Scale bar, 10 μm.)
Fig. 4.
Fig. 4.
Delivery of purified PAR polymer into neurons induces AIF translocation and nuclear condensation. (A) Representative confocal images of AIF translocation and nuclear condensation after BioPorter mediated delivery of purified PAR polymer into wild-type (WT) cortical cultures with and without pretreatment with PARG. AIF (red) is seen in well defined mitochondria like structures in the neuronal cell body as well as long axonal processes and no AIF is seen in the nucleus (blue) in control or PAR + PARG-treated cultures, whereas a clear nuclear translocation and shrinkage of AIF (Pink) is seen in PAR treated cultures. (Scale bar, 20 μm.) (B) Quantitative analysis of AIF translocation and nuclear condensation in the neurons treated with PAR polymer with and without pretreatment with PARG or PD1. BioPorter mediated delivery of poly(A) serves as a control. ∗, P < 0.05, Student's t test. Data represent the mean ± SD; n = 3. (C) Western blots of subcellular fractions of WT cultures, showing nuclear AIF translocation after PAR delivery. Synthetic polymer, poly(A), or enzymatic digestion of PAR with PARG or PD1 did not induce any nuclear AIF translocation. Integrity of mitochondrial fraction (M) and nuclear fraction is shown with antibodies against MnSOD or histone, respectively. (D) Nuclear translocation of AIF accompanies decrease of AIF in mitochondrial fraction. PAR polymer was delivered into cortical neurons using BioPorter for 6 h and subjected to subcellular fractionation into nuclear and mitochondrial fractions. MnSOD and histone were probed as mitochondrial and nuclear markers, respectively. PAR polymer induces the nuclear translocation of AIF, concurrent with loss of AIF in the mitochondrial fraction. (E) Representative confocal images of AIF translocation and nuclear condensation after BioPorter mediated delivery of purified PAR polymer into PARP-1 knockout (KO) cultures with and without pretreatment with PARG. (Scale bar, 20 μm.) (F) Western blots of subcellular fractions of PARP-1 KO cultures, showing nuclear AIF translocation after PAR delivery. Synthetic polymer poly(A) or enzymatic digestion of PAR with PARG or PD1 did not induce any nuclear AIF translocation. These experiments have been replicated in separate experiments at least three times with similar results.
Fig. 5.
Fig. 5.
NMDA-induced AIF mitochondrial nuclear translocation is mediated by PAR polymer. (A) Confocal images of neurons of AIF translocation in cortical neurons that were treated with NMDA (500 μM) with or without PARG overexpression. Cortical neurons were infected with Av PARG WT, Av PARG Mut virus, or Av GFP virus as a control or treated with PBS, followed by NMDA-stimulation (500 μM). In control cultures (CSS), the neurons were incubated in CSS alone for 5 min. Mitochondria-like AIF staining is seen in CSS treated cultures (red), whereas in NMDA-treated cultures (PBS), a robust nuclear translocation of AIF is seen (pink). PARG overexpression (Av PARG WT) protects the neurons against NMDA-induced AIF translocation, whereas overexpression of catalytically inactive PARG (Av PARG Mut) fails to prevent AIF translocation. In control adenoviral experiments, the neurons show a robust nuclear AIF translocation (Av GFP) similar to NMDA-treated (PBS) cultures. (B) Overexpression of cytosolic, WT PARG reduces AIF translocation induced by NMDA treatment in cortical neurons. Neurons were infected with adenoviral constructs as in A. Six hours after NMDA treatment, neurons were harvested for subcellular fractionation and monitoring of AIF translocation. MnSOD and histone serve as mitochondria and nuclear fraction markers, respectively. These experiments have been replicated in separate experiments at least three times with similar results.
Fig. 6.
Fig. 6.
AIF mediates NMDA and PAR polymer-induced cytotoxicity. (A) Cortical neurons from Hq mice and their WT littermates were subjected to NMDA-excitotoxicity (100 μM, 5 min) on day in vitro (DIV) 14. After 18–24 h of NMDA application, the neurons were stained with PI (red) and Hoechst (blue). PI-positive cells are considered dead. (B) Quantification of the NMDA-induced cell death in Hq neurons. Hq neurons that have reduced expressions of AIF are significantly protected against NMDA-toxicity than their WT littermates. Control cultures were treated with CSS alone. Data are the mean ± SEM; n = 6; ∗, P < 0.05 vs. control. (C) PAR polymer in neurons from Hq mice and their WT littermates that were subjected to NMDA-stimulation on DIV 14. Samples were harvested 1 h after NMDA-stimulation and immunoblotted for PAR polymer using anti-PAR antibody. Hq mice that are resistant to NMDA-toxicity have the same PAR polymer levels as compared with their WT littermates. Equal protein loading is shown with the blots for β-actin. These experiments have been replicated in separate experiments at least three times with similar results. (D) Cortical neurons from Hq mouse embryos or their wild type litter mates were infected with adenoviral AIF-GFP (Av-AIF) or GFP-adenoviral control construct (Av-GFP). Forty-eight hours later, the cells were harvested, subjected to SDS/PAGE, and probed for AIF using an AIF-specific antibody. A GFP-AIF fusion protein band is detected in Hq-neurons that were treated with Av AIF-GFP adenovirus; whereas reduced levels of endogenous AIF are seen in PBS or Adeno-GFP-infected cultures. (E) Compensation of reduced AIF expression in Hq-neurons by AIF-expressing adenovirus makes Hq neurons equally susceptible to NMDA as their WT littermate neurons. Neurons (12 DIV) were infected with Av AIF-GFP or GFP-adenoviral control construct (Adeno-GFP). Control cultures were treated with an equal volume of PBS. Forty-eight hours later, the neurons were treated with NMDA (500 μM for 5 min) and cells were stained with Hoechst and PI to assess cell death, 18–24 h after NMDA administration. Data are the mean ± SEM; n = 6; ∗, P < 0.05. (F) Hq neurons are protected against PAR polymer-induced cell death as compared with their WT littermates. Representative pictures of propidium iodide (red), Hoechst (blue)-stained Hq neurons that were treated with purified PAR polymer delivered via the BioPorter reagent. The PI-positive cells were considered dead. (G) Quantification of the data shown in F. Hq neurons are significantly protected against PAR-polymer-induced cell death as compared with their WT littermates. Data are the mean ± SEM; n = 6; ∗, P < 0.05 vs. control.
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