Hypoxia treatment reverses neurodegenerative disease in a mouse model of Leigh syndrome Michele Ferrari a,1 , Isha H. Jain b,c,d,e,1 , Olga Goldberger b,c,d,e , Emanuele Rezoagli a , Robrecht Thoonen f , Kai-Hung Cheng f,g , David E. Sosnovik f,h , Marielle Scherrer-Crosbie f,g , Vamsi K. Mootha b,c,d,e,2,3 , and Warren M. Zapol a,2,3 a Department of Anesthesia, Critical Care, and Pain Medicine, Massachusetts General Hospital, Boston, MA 02114; b Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114; c Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114; d Department of Systems Biology, Harvard Medical School, Boston, MA 02114; e Broad Institute of Harvard and MIT, Cambridge, MA 02142; f Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA 02114; g Cardiac Ultrasound Laboratory, Cardiology Division, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114; and h Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Charlestown, MA 02129 Contributed by Vamsi K. Mootha, March 27, 2017 (sent for review January 5, 2017; reviewed by Marni J. Falk and Matt Kaeberlein) The most common pediatric mitochondrial disease is Leigh syn- drome, an episodic, subacute neurodegeneration that can lead to death within the first few years of life, for which there are no proven general therapies. Mice lacking the complex I subunit, Ndufs4, develop a fatal progressive encephalopathy resembling Leigh syn- drome and die at ≈60 d of age. We previously reported that contin- uously breathing normobaric 11% O 2 from an early age prevents neurological disease and dramatically improves survival in these mice. Here, we report three advances. First, we report updated sur- vival curves and organ pathology in Ndufs4 KO mice exposed to hypoxia or hyperoxia. Whereas normoxia-treated KO mice die from neurodegeneration at about 60 d, hypoxia-treated mice eventually die at about 270 d, likely from cardiac disease, and hyperoxia-treated mice die within days from acute pulmonary edema. Second, we report that more conservative hypoxia regimens, such as contin- uous normobaric 17% O 2 or intermittent hypoxia, are ineffective in preventing neuropathology. Finally, we show that breathing normobaric 11% O 2 in mice with late-stage encephalopathy re- verses their established neurological disease, evidenced by im- proved behavior, circulating disease biomarkers, and survival rates. Importantly, the pathognomonic MRI brain lesions and neurohistopathologic findings are reversed after 4 wk of hyp- oxia. Upon return to normoxia, Ndufs4 KO mice die within days. Future work is required to determine if hypoxia can be used to prevent and reverse neurodegeneration in other animal models, and to determine if it can be provided in a safe and practical manner to allow in-hospital human therapeutic trials. hypoxia | mitochondria | Leigh syndrome | neurodegeneration | oxygen D efects in mitochondrial function, owing to mutations either in the nuclear genome or in the mitochondrial DNA, result in severe diseases that can present at any point from infancy through adulthood (1). The most common biochemical class of mitochondrial disorders arises from genetic mutations affecting the mitochondrial respiratory chain, with an incidence of ∼1 in 4,300 live births (2). The management of these disorders is challenging in part because of their clinical and genetic hetero- geneity. Virtually any organ system can be impacted, and more than 250 genes encoding mitochondrial proteins are known to be disease-causing (3). Of these, at least 150 can underlie disorders of oxidative phosphorylation. The mainstay of therapy involves using vitamin mixtures with little or no proven efficacy, as well as avoiding drugs known to be mitochondrial toxins. Leigh syndrome, the most common pediatric manifestation of mitochondrial disease, is characterized by bilaterally symmet- ric lesions in the gray matter of the brainstem, basal ganglia, or cerebellum. It affects ∼1 in 40,000 live births and can be due to mutations in any of 75 different genes (4). Children with this disorder often become hypotonic and may develop vision and hearing loss. Although the course of disease can vary widely among individuals, severely affected children can experience de- velopmental delay and ultimately succumb to respiratory failure within the first few years of life. These patients are typically healthy at birth but then experience a neurometabolic crisis, often in the context of a febrile illness, resulting in psychomotor re- gression and death within the first few years of life. Diagnosis is based on clinical presentation and classic findings on T2-weighted brain magnetic resonance imaging (MRI). A small subset of these disorders are related to defects in vitamin transport and alleviated with dietary supplementation, such as in the case of riboflavin, thiamine, and biotin deficiencies (5, 6); however, there are no proven therapies for the vast majority of these disorders. Several new experimental strategies are currently under investigation in preclinical and clinical settings, including agents that target oxi- dative stress, the mechanistic target of rapamycin pathway, NAD + pool sizes, and complex I bypass (idebenone) (7–10). We recently demonstrated the therapeutic potential of hyp- oxia in preventing mitochondrial disease. We reported that chronically exposing the Ndufs4 mouse model of Leigh syndrome (11, 12) to breathing 11% O 2 at normobaria before disease onset markedly extended the lifespan, improved behavior, and prevented neurodegeneration (13). Furthermore, we demon- strated that breathing 55% O 2 , a level that is well tolerated by WT mice, led to rapid death of the diseased mice. Significance Inherited or acquired defects in mitochondria lead to devas- tating disorders for which we have no effective general ther- apies. We recently reported that breathing normobaric 11% O 2 prevents neurodegeneration in a mouse model of a pediatric mitochondrial disease, Leigh syndrome. Here we provide updated survival curves of mice treated with varying doses of oxygen and explore eventual causes of death. We explore al- ternative hypoxia regimens and report that neither intermittent nor moderate hypoxia regimens suffice to prevent neurological disease. Finally, we show that hypoxia can not only prevent, but also reverses the brain lesions in mice with advanced neuropa- thology. Our preclinical studies will help guide future clinical studies aimed at harnessing hypoxia as a safe and practical therapy. Author contributions: M.F., I.H.J., V.K.M., and W.M.Z. designed research; M.F., I.H.J., O.G., E.R., R.T., K.-H.C., D.E.S., and M.S.-C. performed research; M.F., I.H.J., V.K.M., and W.M.Z. contributed new reagents/analytic tools; M.F., I.H.J., E.R., R.T., K.-H.C., D.E.S., M.S.-C., V.K.M., and W.M.Z. analyzed data; and M.F., I.H.J., V.K.M., and W.M.Z. wrote the paper. Reviewers: M.J.F., University of Pennsylvania; and M.K., University of Washington. Conflict of interest statement: V.K.M., W.M.Z., and I.H.J. are listed as coinventors on a patent application submitted by Massachusetts General Hospital on the therapeutic uses of hypoxia for mitochondrial disease. Freely available online through the PNAS open access option. 1 M.F. and I.H.J. contributed equally to this work. 2 V.K.M. and W.M.Z. contributed equally to this work. 3 To whom correspondence may be addressed. Email: [email protected] or wzapol@ mgh.harvard.edu. www.pnas.org/cgi/doi/10.1073/pnas.1621511114 PNAS | Published online May 8, 2017 | E4241–E4250 MEDICAL SCIENCES PNAS PLUS SEE CORRECTION FOR THIS ARTICLE SEE CORRECTION FOR THIS ARTICLE Downloaded from https://www.pnas.org by 117.3.248.167 on June 21, 2023 from IP address 117.3.248.167.
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