Inhibition of neuronal ferroptosis protects hemorrhagic brain
Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang
Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang
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Research Article Neuroscience

Inhibition of neuronal ferroptosis protects hemorrhagic brain

Abstract

Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of PTGS2 and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell–derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies.

Authors

Qian Li, Xiaoning Han, Xi Lan, Yufeng Gao, Jieru Wan, Frederick Durham, Tian Cheng, Jie Yang, Zhongyu Wang, Chao Jiang, Mingyao Ying, Raymond C. Koehler, Brent R. Stockwell, Jian Wang

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Figure 6

Inhibiting multiple forms of cell death optimizes neuronal rescue in vitro.

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Inhibiting multiple forms of cell death optimizes neuronal rescue in vit...
(A–C) Male C57BL/6 mice (6–8 weeks old) underwent collagenase injection or sham procedure. Brain slices were stained with 2% uranyl acetate and lead citrate. (A) Ultrastructure of neuron somas and axons. n, nuclei; c, cytoplasm; m, mitochondria. Red arrows show representative mitochondria in somas and axons. ICH, intracerebral hemorrhage. (B) Mitochondrial area frequency in somas and axons. Arrows indicate increased frequency of shrunken mitochondria in ICH groups. (C) Ultrastructure of (a) a healthy neuronal soma and (b) normal axon in sham group; (c) arrows indicate chromatin condensation in 2 cells undergoing apoptosis; (d, g) double arrows indicate organelle swelling in a cell undergoing necrosis with classical nuclear membrane rupture; (e, f) arrowhead indicates formation of double-membrane vesicles in a cell undergoing autophagy. n = 3 per time point. Number of soma mitochondria, sham: n = 272; ICH 3 days: n = 414; ICH 6 days: n = 312. Number of axon mitochondria, sham: n = 152; ICH 3 days: n = 306; ICH 6 days: n = 296. (D and E) Organotypic hippocampal slice cultures were treated as indicated for 16 hours (D) or 48 hours (E) before cell death was measured by propidium iodide (PI) staining. **P < 0.01, ***P < 0.001 versus control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus hemoglobin (Hb). (F–H) Human induced pluripotent stem cell–derived neurons were treated as indicated for 16 hours before cell viability/death was measured with the MTT assay (F) or PI staining (H). MAP2 staining shows the morphology of Hb-injured neurons (G). *P < 0.05, **P < 0.01, ***P < 0.001 versus control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus Hb. Results are shown as bar graphs or box-and-whisker plots (the middle horizontal line within the box represents the median, boxes extend from the 25th to the 75th percentile, and the whiskers represent 95% confidence intervals). n = 3 independent experiments; 1-way ANOVA with Bonferroni post hoc analysis. Scale bars: (A) upper panel, 2 μm and bottom panel, 500 nm; (C) 2 μm; (G) left panel, 100 μm and right panel, 50 μm. Original magnification for insets (A and G), ×600.