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. 2017 Aug;12:558-570.
doi: 10.1016/j.redox.2017.03.007. Epub 2017 Mar 9.

BID Links Ferroptosis to Mitochondrial Cell Death Pathways

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Free PMC article

BID Links Ferroptosis to Mitochondrial Cell Death Pathways

Sandra Neitemeier et al. Redox Biol. .
Free PMC article

Abstract

Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the Xc- system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc- inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death.

Keywords: BID; CRISPR; Ferroptosis; Mitochondria; Neuronal death; Oxytosis.

Figures

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Fig. 1
Fig. 1
Bid KO does not inhibit glutathione depletion, but prevents ROS formation. a: Western blot verified knockout of Bid (22 kDa) in HT-22 Bid KO cells. b: Sequencing of the Bid CRISPR target region revealed different indel mutations in the alleles indicated as multiple peaks. c, d: Measurement of glutathione (GSH) depicted rapid decrease of GSH after glutamate (8 mM; c) or erastin (1 µM; d) exposure which was not restored by Bid KO (n=4/treatment condition). e: BODIPY 581/591 staining and subsequent FACS analysis for measurement of lipid peroxide formation showed time-dependent increase in the fluorescence after erastin (1 µM) exposure in HT-22 WT cells (n=3/treatment condition). f: MitoSOX staining and subsequent FACS analysis revealed time-dependent mitochondrial ROS formation after erastin (1 µM) exposure in HT-22 cells (n=3/treatment condition). g: Cells were stained with BODIPY 581/591 and changes of fluorescence were detected by FACS analysis after 10 h of glutamate (7.5 mM) or erastin (1 µM) treatment. Bid KO fully prevented the formation of lipid peroxides compared to WT control cells (n=4/treatment condition). h: MitoSOX staining and subsequent FACS analysis depicted reduced formation of mitochondrial ROS in HT-22 Bid KO cells compared to WT HT-22 cells after erastin (1 µM, 19 h) or glutamate (7 mM, 19 h) challenge (n=4/treatment condition). All data are given as mean + S.D. #p<0.05, ###p<0.001 compared to untreated HT-22 control; ***p<0.001 compared to erastin-/ glutamate-treated HT-22 control (ANOVA, Scheffé‘s test).
Fig. 2
Fig. 2
Bid KO preserves mitochondrial integrity. a: Representative images show mitochondrial morphology in HT-22 WT cells in the presence and absence of erastin (1 µM, 2–12 h) (63x objective). Scale bar: 50 µm. b: Quantification of 500 cells counted blind to treatment of conditions of 3 independent experiments revealed time-dependent mitochondrial fission in HT-22 cells after erastin (1 µM) exposure. c: Representative images show mitochondrial morphology in HT-22 WT and Bid KO cells in the presence and absence of glutamate (5 mM, 15 h) or erastin (0.5 µM, 15 h) (63x objective). Scale bar: 50 µm. d: Quantification of 500 cells counted blind to treatment of conditions of 3 independent experiments revealed reduction of glutamate/ erastin-induced mitochondrial fission in Bid KO cells. e: After 17 h of treatment with glutamate (7 mM) or erastin (1 µM) ATP levels were measured. Bid KO prevented ATP depletion compared to WT controls (n=8/treatment condition). f, g: Measurement of the oxygen consumption rate (OCR) revealed restored basal and maximal respiration in Bid KO cells compared to WT controls after 16 h glutamate (2 mM; e) or erastin (0.25 µM; f) exposure (n=6/treatment condition). h: HT-22 Bid KO cells exhibited restored MMP measured by TMRE fluorescence compared to WT HT-22 cells after glutamate (7 mM, 19 h) or erastin (1 µM, 19 h) exposure (n=4/treatment condition). All data are given as mean +S.D. or±S.D. ##p<0.01; ###p<0.001 compared to untreated HT-22 control, ***p<0.001 compared to erastin-/ glutamate-treated HT-22 control (ANOVA, Scheffé‘s test).
Fig. 3
Fig. 3
Bid KO prevents glutamate- and erastin induced cell death. a: Annexin V/PI double staining and subsequent FACS analysis revealed time-dependent cell death after erastin (1 µM) exposure in HT-22 cells (n=3/treatment condition). b: Representative plots of Annexin V/PI staining and subsequent FACS analysis showed increase in Annexin V (Green fluorescence) and PI (Red fluorescence) positive cells after glutamate (7 mM, 16 h) and erastin (1 µM, 16 h) exposure in HT-22 WT cells. In Bid KO cells, neither erastin nor glutamate altered the amount of Annexin V or PI positive cells. Numbers in the dot blots present percentage mean±S.D. of AV positive cells (lower right) and AV+PI positive cells (upper right) (n=4) c: MTT confirmed protection of HT-22 Bid KO cells against erastin (1 µM, 16 h) and glutamate (7 mM, 16 h) exposure. Furthermore, in HT-22 WT cells, the BID-inhibitor BI-6c9 (10 µM) fully prevented cell death in response to oxidative stress (n=8/treatment condition). d, e: Real-time impedance measurements revealed protective effect of Bid KO against both glutamate (7 mM; d) and erastin (1 µM; e) toxicity. Data were derived from the same experiment, but shown in separated graphs for better visualization; (n=6/treatment condition). f: Over-expression of tBid (0.75 µg plasmid-DNA/well) induced death of both WT and Bid KO HT-22 cells detected by xCELLigence recordings (n=6/treatment condition), representative experiment shown. All data are given as mean +S.D. or±S.D. ###p<0.001 compared to untreated control; ***p<0.001 compared to erastin-/ glutamate-treated HT-22 control (ANOVA, Scheffé‘s test).
Fig. 4
Fig. 4
BI-6c9 prevents erastin-induced cell death and mitochondrial demise. a: MTT assay revealed protection of BI-6c9 (10 µM) and ferrostatin-1 (2 µM) against erastin (1 µM, 16 h) toxicity (n=8). b: Cells were stained with BODIPY 581/591 and lipid peroxides were measured by FACS analysis after 16 h of erastin treatment (1 µM). BI-6c9 significantly reduced the lipid peroxide production (n=4/treatment condition). c: Mitochondrial ROS production was detected by MitoSOX staining and following FACS analysis. Erastin increased ROS production, which was blocked by BI-6c9 (n=4/treatment condition). d: Quantification of mitochondrial morphology in cells exposed to erastin (1 µM, 16 h) showed an increase in cells with mitochondria of category III. This erastin-induced fragmentation was fully prevented by BI-6c9. Mean values were pooled from 4 independent experiments, where mitochondrial morphology was determined from at least 500 cells per condition without knowledge of treatment history. ##p<0.01 compared to Cat III of untreated control; *p<0.05 compared to Cat III of erastin-treated cells (ANOVA, Scheffé’s test). e: Quantification of TMRE fluorescence of n=4 independent experiments showed that MMP was fully restored by BI-6c9 (10 µM) after erastin exposure (1 µM, 16 h). f: After 16 h of treatment with erastin (1 µM) ATP levels were measured. BI-6c9 prevented ATP depletion compared to erastin-treated controls (n=8). Data are shown as mean + SD. ###p<0.001 compared to untreated control; ***p<0.001 compared to erastin-treated control. (ANOVA, Scheffé‘s test).
Fig. 5
Fig. 5
Ferrostatin-1 inhibits glutamate-induced oxytosis and preserves mitochondrial integrity. a: MTT assay revealed protection of BI-6c9 (10 µM) and ferrostatin-1 (2 µM) against glutamate (4 mM, 16 h) toxicity (n=8). b: Cells were stained with BODIPY 581/591 and lipid peroxides were measured by FACS analysis after 16 h of glutamate treatment (3 mM). Ferrostatin-1 significantly reduced the lipid peroxide production (n=4). c: Glutamate treatment (3 mM, 16 h) increased mitochondrial ROS production, which was fully blocked by co-treatment with 2 µM ferrostatin-1 (n=4). d: Quantification of mitochondrial morphology in cells exposed to glutamate showed an increase in cells with mitochondria of category III. This glutamate-induced fragmentation was fully prevented by ferrostatin-1. ##p<0.01 compared to Cat I of glutamate-treated control; ***p<0.001 compared to Cat III of glutamate-treated cells (ANOVA, Scheffé’s test). Mean values were pooled from 4 independent experiments, where mitochondrial morphology was determined from at least 500 cells per condition without knowledge of treatment history. e: Quantification of TMRE fluorescence of n=4 independent experiments showed that MMP was fully restored by ferrostatin-1 (2 µM) after glutamate exposure (3 mM, 16 h). f: After 16 h of treatment with glutamate (3 mM) ATP levels were measured. Ferrostatin-1 prevented ATP depletion compared to glutamate-treated controls (n=8). All results are given as mean + S.D. ###p<0.001 compared to untreated control; ***p<0.001 compared to glutamate-treated control (ANOVA, Scheffé‘s test).
Fig. 6
Fig. 6
Bid links ferroptosis to mitochondrial cell death. a: Confocal microscopy pictures revealed translocation of BID (Bid-dsRed) to the mitochondria (MitoGFP) after erastin (1 µM, 16 h) or glutamate (3 mM, 16 h) exposure, which was fully blocked by BI-6c9 or ferrostatin-1 co-treatment. Scale bar: 20 µM. b: tBID-induced cell death (2 µg plasmid/24-well, 24 h) in HT-22 cells was analyzed by co-staining of cells with Annexin V (Green fluorescence) and PI (Red fluorescence) and subsequent FACS-analysis. Pre-incubation for 1 h with 10 µM BI-6c9 prevented pIRES-tBid-induced cell death, while ferrostatin-1 did not (representative dot plots). The empty vector pcDNA3.1 was used as a control plasmid. Numbers in the representative dot blots show percentage of AV positive cells (lower right) and AV+PI positive cells (upper right) (n=4) c: Quantification of dot plots shown in b (n=4). Data are shown as mean + S.D. ###p<0.001compared to cDNA 3.1; ***p<0.001compared to pIRES-tBid-transfected control (ANOVA, Scheffé‘s test).
Fig. 7
Fig. 7
Bid links ferroptosis to neuronal oxytosis. Glutamate and erastin inhibit the Xc--antiporter in paradigms of oxytosis and ferroptosis, respectively. Blocking the cellular cystine import results in decreased GSH levels and reduced Gpx4 activity, and the subsequent activation of 12/15 LOX mediates significant formation of reactive oxygen species (ROS). In erastin-induced ferroptosis cell death is induced through oxidative stress and independently of mitochondrial demise. In neuronal cells, ROS-induced transactivation of BID to the mitochondria links both pathways of oxytosis and ferroptosis, and causes mitochondrial ROS formation that is associated with irreversible morphological and functional damage, e.g. loss of MMP, decline of ATP levels and release of apoptosis inducing factor (AIF). The BID-inhibitor BI-6c9 and the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 are able to block these fatal pathways upstream of mitochondrial impairments. BI-6c9 directly inhibits BID and its detrimental effects at the level of mitochondria while ferrostatin-1 acts upstream of BID preventing ROS formation through 12/15 LOX.

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