Leigh Syndrome Mouse Model Can Be Rescued by Interventions that Normalize Brain Hyperoxia, but Not HIF Activation

Abstract

Leigh syndrome is a devastating mitochondrial disease for which there are no proven therapies. We previously showed that breathing chronic, continuous hypoxia can prevent and even reverse neurological disease in the Ndufs4 knockout (KO) mouse model of complex I (CI) deficiency and Leigh syndrome. Here, we show that genetic activation of the hypoxia-inducible factor transcriptional program via any of four different strategies is insufficient to rescue disease. Rather, we observe an age-dependent decline in whole-body oxygen consumption. These mice exhibit brain tissue hyperoxia, which is normalized by hypoxic breathing. Alternative experimental strategies to reduce oxygen delivery, including breathing carbon monoxide (600 ppm in air) or severe anemia, can reverse neurological disease. Therefore, unused oxygen is the most likely culprit in the pathology of this disease. While pharmacologic activation of the hypoxia response is unlikely to alleviate disease in vivo, interventions that safely normalize brain tissue hyperoxia may hold therapeutic potential.

Keywords: Leigh syndrome; anemia; carbon monoxide; hemoglobin; hyperoxia; hypoxia; mitochondria; oxygen; therapy.

Figure 1.

Genetic Activation of the Hypoxia Response Is Insufficient to Prevent Leigh Disease (A) Kaplan-Meier curves for double KOs relative to single Ndufs4^−/− KO mice. Log-rank p values and sample size within insert. Single KOs are homozygous-null for Ndufs4 locus and WT or Het for other genetic locus of given cross. (B) MRI of vestibular nucleus (red arrow for Phd1, Phd2 and Phd3 crosses) of late-stage disease mice for each genotype. In Vhl cross, red arrow corresponds to lateral lesions (dentate nucleus) and midline lesion (central lobule, cerebellum). (C) qPCR for canonical HIF targets (mean ± SD, n = 4 per group). Mice are homozygous for given genotype and WT or Het for Ndufs4 genotype. (D) Hematocrit of control or Phd and Vhl genetic models (WT or Het for Ndufs4) (mean ± SEM, Ctrl [n=3], Vhl [n=6], Phd1 [n=3], Phd2 [n=4], Phd3 [n=5]). One-tailed unpaired t test p value shown for genetic crosses relative to control mice. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA, with Dunnett’s test for multiple comparison).

Figure 2.

Whole-Body and Brain Oxygenation of KO and WT Mice (A and B) Whole-body oxygen consumption (mean SEM), normalized for body weight, during (A) active hours or (B) inactive hours. Data smoothened over 70 collection points (~5.5 h). Values normalized to body weight at each collection point (n = 6 per group). Unpaired t test p value shown for each genotype comparison at each day. (C) Tissue PO₂ (mean ± SEM) measured stereotaxically in hypothalamus of WT and KO mice as a function of age using a Clark electrode (young = 36–41 days, old 50–54 days). n = 6 for WT, n = 5 for young KO and n = 5 for old KO. Dotted line represents PO₂ of WT mice breathing room air. Unpaired t test p value shown for comparison of KOs to WT breathing room air. (D) Tissue PO₂ (mean ± SD) measurement using an optical PO₂ probe stereotaxically placed near vestibular nuclei of WT and KO mice breathing varying oxygen tensions (n = 4–6 per group). Dotted line represents PO₂ of WT mice breathing room air. (E) Hydrogen peroxide released during complex I-mediated respiration by isolated mitochondria from WT and KO brains as a function of dissolved oxygen. (F) Median survival of KO mice as a function of inhaled oxygen, using data from this paper and from previously published studies (Ferrari et al., 2017; Jain et al., 2016).

Figure 3.

Breathing Carbon Monoxide Reduces Brain Hyperoxia and Reverses Disease in Ndufs4 KO Mice (A) Hemoglobin and (B) Hematocrit (mean ± SEM) of WT and KO mice exposed to air or ~600 ppm carbon monoxide in air for ~3 weeks. Unpaired t test p value shown within inserts. (C) Percentage of carboxyhemoglobin (mean ± SEM) of WT mice breathing 11% F_iO₂ versus 600 ppm CO. (D) Body weight trajectory of mice exposed to preventative (gray) hypoxia therapy, late-stage hypoxia therapy (black), or late-stage carbon monoxide therapy (red). (n = 8 for KO prevention in gray, n = 8 for KO disease reversal with hypoxia breathing in black, and n = 6 for KO disease reversal with CO breathing). Note: hypoxia body weight curves taken from historical data previously reported in (Ferrari et al., 2017) and adapted for this figure. (E) Survival of mice breathing room air (historical data from (Ferrari et al., 2017) versus 600 ppm CO in room air. Log-rank p value shown within insert. n = 12 for CO survival in red, n = 14 for mice breathing room air in black. (F) Reversal of T2-weighted MRI lesions in mice exposed to 600 ppm CO starting a late stage of disease. Pre-treatment scans on left, post-treatment of same mice after 2 to 3 weeks of CO on right. Three rows are three individual mice. (G) Brain (vestibular nucleus) PO₂ of WT and KO (mean ± SD) mice breathing 21% F_iO₂, 11% F_iO_2, or 600 ppm CO in 21% O₂. (data for WT taken from same data as Figure 2). (H) T2-weighted MRI scan sections through late-stage KO mouse breathing room air versus lesions observed in KO mouse chronically exposed to 600 ppm CO.From left to right in 3H, lesions correspond to top 1 (vestibular nucleus), bottom 2 (red nucleus), bottom 4 (caudoputamen) and top 5 (olfactory bulb). Typical disease lesions in vestibular nucleus shown with red arrow. New lesions in CO-treated KO mice shown with orange arrow.

Figure 4.

Severe Anemia Decreases Brain Oxygenation and Rescues Neurological Disease in the Ndufs4 KO Mouse (A) Schematic of variables affecting tissue oxygenation. Theoretical therapeutic interventions shown in red, physiological variables represented in blue or black. (B) Gradual decrease in hemoglobin following serial phlebotomy every 2 to 3 days for ~20 days, in combination with an Fe-deficient diet (mean ± SEM, n = 7). (C) Brain PO₂ (vestibular nuclei) of WT and KO mice that are untreated or made anemic using phlebotomy, in combination with an Fe-deficient diet (WT data same as Figure 2). (D) Survival of untreated (Ferrari et al., 2017) or anemic Ndufs4 KO mice. (E) MRI of anemic Ndufs4 KO mice at 80 days or 170 days of age. Note: Mice scanned in left and right panels are distinct. Rows correspond to different mice to show reproducibility. Red arrow depicts lesions in vestibular nuclei.