Sulfide catabolism ameliorates hypoxic brain injury

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain’s sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.


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nature research | reporting summary
April 2020 A minimum sample size of 5 was used per group for in the majority of in vivo and in vitro experiments, as is standard and comparable to prior published experiments using similar techniques. The sample size is sufficient to detect a difference between two groups with coefficient of variation of 0.5 (ratio of standard deviation to mean difference) and two tailed level of significance (alpha) of 0.05 with a power of 80%. A smaller sample size of 3 was used in a limited number of experiments when the availability of sample is scarce and or the difference is expected to be large with small variation (e.g., brain samples of 13LG squirrels and brain SQOR levels in control mice). In these cases, the sample size is sufficient to detect a difference between two groups with coefficient of variation of 0.8 (ratio of standard deviation to mean difference) and two tailed level of significance (alpha) of 0.05 with a power of 80%. All experiments were performed at least twice and results were reliably reproduced. Independent biologic replicates are shown in all figures.
We used randomized paired (a.k.a. matched pairs) design. We paired animals to two or more treatment groups on the basis of similar weight, age, delivery date, and when possible holding cage.
In all in vivo assessment of survival and neurofunction, investigators who performed surgery and/or determined the outcome was blinded to the treatment (sulfide preconditioning, AAV-mediated SQOR knockdown or overexpression, sulfide scavengers), gender, or genotype of the animals. In all ex vivo and in vitro experiments in which levels of metabolites, mitochondrial function, viable or dead cell numbers, proteins, and mRNA were determined in tissue extracts or brain sections obtained from animals or in cultured cells or cell lysates, investigators who conducted measurements and data analysis were blinded to the treatment, gender, or genotype of the animals. We purchased SH-SY5Y cells from ATCC. This cell line was authenticated by ATCC using ampule passage number, population doubling level (PDL), total cells/ampule, post-freeze viability, growth properties, morphology, test for mycoplasma contamination, species determination: COI assay (interspecies), species determination: STR analysis (intraspecies), sterility test (BacT/ALERT 3D), and human pathogenic virus testing (PCR-based assay for HIV, HepB, HPV, EBV, and CMV).
SH-SY5Y cells were tested negative for mycoplasma by ATCC.
No commonly misidentified cell lines were used in this study.

nature research | reporting summary
April 2020

Wild animals
Field-collected samples

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This checklist template is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 4 The study did not involve wild animals.
The study did not involve the samples collected in the field.
All animal protocols were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee. Experimental procedures related to generation and initial characterization of Sqor dN/dN mice conformed to the Regulations for Animal Experiments and Related Activities at Tohoku University, were reviewed by the Institutional Laboratory Animal Care and Use Committee of Tohoku University, and were approved by the President of Tohoku University. Mice were housed under 12h light/12h dark cycle with room temperature and humidity at 21-23 degree Celsius and 40-60%, respectively.

nature research | reporting summary
April 2020