Proc. Natl. Acad. Sci. USA Vol. 96, pp. 846–851, February 1999 Biochemistry Mitochondrial disease in superoxide dismutase 2 mutant mice SIMON MELOV†,PINAR COSKUN†,MANISHA PATEL‡,ROBBYN TUINSTRA§,BARBARA COTTRELL†,ALBERT S. JUN†, i TOMSZ H. ZASTAWNY¶,MIRAL DIZDAROGLU¶,STEPHEN I. GOODMAN ,TING-TING HUANG††,HENRY MIZIORKO§, CHARLES J. EPSTEIN††, AND DOUGLAS C. WALLACE†‡‡ †Center for Molecular Medicine, Emory University, Atlanta, GA 30322; ‡National Jewish Medical and Research Center, Denver, CO 80206; §Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226; ¶National Institute of Standards and Technology, Gaithersburg, MD 20899; iDepartment of Pediatrics, University of Colorado School of Medicine, Denver, CO 80262; and ††Division of Medical Genetics, University of California at San Francisco, San Francisco, CA 94143-0748 Contributed by Douglas C. Wallace, December 4, 1998 ABSTRACT Oxidative stress has been implicated in many mitochondrial defects can affect many different types of diseases. The chief source of reactive oxygen species within the metabolic pathways. In addition to mitochondria being the cell is the mitochondrion. We have characterized a variety of main source of ATP within the cell, they are also responsible the biochemical and metabolic effects of inactivation of the for fatty acid oxidation and are a major source of metabolic mouse gene for the mitochondrial superoxide dismutase intermediates for cytosolic processes. (CD1-Sod2tm1Cje). The Sod2 mutant mice exhibit a tissue- A number of inherited diseases disrupt metabolic pathways specific inhibition of the respiratory chain enzymes NADH- in the mitochondria. One such disease results in a 3-methyl- dehydrogenase (complex I) and succinate dehydrogenase glutaconic aciduria (3-MGC) and can be caused by defects in (complex II), inactivation of the tricarboxylic acid cycle 3-hydroxy-3-methylglutaryl (HMG)-CoA lyase. HMG-CoA enzyme aconitase, development of a urine organic aciduria in lyase is a mitochondrial matrix protein that catalyzes cleavage conjunction with a partial defect in 3-hydroxy-3-methylglu- of HMG-CoA into acetyl-CoA and acetoacetate. This enzyme taryl-CoA lyase, and accumulation of oxidative DNA damage. is a critical enzyme in ketogenesis and is required in the last These results indicate that the increase in mitochondrial step of leucine catabolism, and mutations in the HMG-CoA reactive oxygen species can result in biochemical aberrations gene have been associated with some forms of 3-MGC aciduria with features reminiscent of mitochondrial myopathy, Fried- (18, 19). In urine, 3-MGC is detectable by gas chromatogra- reich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase defi- y y ciency. phy mass spectrometry (GC MS), and patients with 3-MGC aciduria show a marked clinical heterogeneity that can en- compass cardiomyopathy, bilateral optic atrophy, movement The pathophysiology of mitochondrial diseases has been at- disorders, disorders of the respiratory chain, and hepatomeg- tributed to decreased mitochondrial ATP production and aly (20–24). toxicity resulting from increased mitochondrial reactive oxy- All three genes for superoxide dismutase (Sod1, Sod2, and gen species (ROS) generation. Mammalian mitochondria gen- Sod3) have been inactivated genetically in mice through ho- erate most of the ATP for cells by the process of oxidative phosphorylation (OXPHOS). During OXPHOS, between 0.4 mologous recombination (25–27). However, inactivation of the and 4% of the oxygen consumed is reduced to form superoxide mitochondrial gene Sod2 has resulted in the most severe anion (O.) (1–5). Under normal circumstances, superoxide is phenotype (25, 28). This indicates that the toxicity of the 2 mitochondrial ROS is particularly deleterious to health. Mice reduced to H2O2 by the mitochondrial form of superoxide lacking SOD2 have been generated in two genetic back- dismutase (SOD2). Within the mitochondria, the H2O2 then either is converted to water by mitochondrial glutathione grounds, an outbred background, CD1 (25), and a hybrid peroxidase or can participate in Fenton type chemistry, giving background (28). In both cases, the phenotype is neonatal rise to further ROS such as the hydroxyl radical (6). lethal, with the most severely affected tissues, such as the heart ROS have been implicated in a wide variety of disorders, and brain, being postmitotic and having high-energy demands including Alzheimer disease, Parkinson disease, ischemic (25, 28, 29). In mice mutant for Sod2 on the CD1 background, heart disease, and many mitochondrial diseases (7–12). More- dilated cardiomyopathy develops within the first week of life, over, the pathogenic role of ROS has been strongly implicated accompanied by a profound accumulation of fat within the in familial amyotrophic lateral sclerosis and Friedreich ataxia. liver. A metabolic acidosis and a dramatic reduction in succi- Specifically, certain cases of familial amyotrophic lateral scle- nate dehydrogenase (complex II) histochemical staining have rosis have been shown to be caused by mutations in the been reported (25). To characterize further the biochemical cytosolic CuyZnSOD (13), and the mutant protein in Fried- and physiological basis of the lethal effects of the Sod2 reich ataxia has been associated with high levels of mitochon- mutation, we have extensively analyzed the biochemistry of the drial iron and a reduction in mitochondrial complexes I, II, and mutant mouse mitochondria. We have confirmed that the mice III and aconitase (7, 9), presumably because of oxidative have severe deficiencies in some iron-sulfur cluster-containing damage. enzymes of the respiratory chain and tricarboxylic acid cycle, Mitochondrial diseases genetically can be classified into two a partial defect in HMG-CoA lyase, and extensive ROS types: disease that result from mutations of the mitochondrial damage to the DNA. Hence, these animals have many of the genome (14) and those that are caused by mutations in nuclear genes encoding mitochondrial proteins (15). Recent examples Abbreviations: ROS, reactive oxygen species; OXPHOS, oxidative of nuclear mitochondrial disease include Friedreich ataxia, phosphorylation; SOD, superoxide dismutase; 3-MGC, 3-methyl- Wilson disease, and spastic paraplegia (7, 16, 17). Nuclear glutaconic aciduria; HMG, 3-hydroxy-3-methylglutaryl; GC, gas chro- matography; MS, mass spectrometry; IDMS, isotope-dilution MS; Ip, The publication costs of this article were defrayed in part by page charge iron–sulfur cluster-containing protein; SDH, succinate dehydroge- nase; IRE, iron-regulatory elements. payment. This article must therefore be hereby marked ‘‘advertisement’’ in ‡‡To whom reprint requests should be addressed at: Center For accordance with 18 U.S.C. §1734 solely to indicate this fact. Molecular Medicine, 1462 Clifton Road, Emory University, Atlanta, PNAS is available online at www.pnas.org. GA 30322. e-mail: [email protected]. 846 Downloaded by guest on September 30, 2021 Biochemistry: Melov et al. Proc. Natl. Acad. Sci. USA 96 (1999) 847 biochemical features of mitochondrial disease associated with Organic Acids Analysis and Mitochondrial HMG-CoA ROS toxicity. Lyase Assessment. Urine was collected daily from Sod2 mutant mice, as well as from wild-type or heterozygous littermates at ' MATERIALS AND METHODS 3 days of age. Because a minimum of 0.5 ml of urine is required for urine organic acids analysis, we pooled urine from Mice Husbandry, Breeding, and Genotyping. The Sod2 each individual animal up until the day of sacrifice in a 1.5-ml mutant allele was maintained on a CD1 background by back- Eppendorf tube. Urine was stored at 220°C between daily crossing to CD1 male mice obtained from Charles River collections. Organic acids in urine were analyzed by GCyMS Breeding Laboratories. Heterozygous crosses were set up, and as described (33), except that dimethylmalonic acid was used genotyping of the resultant pups was carried out at 2–3 days of as an internal standard instead of malonic, and chromatogra- age as described (29). After mating, pregnant dams were phy was carried out on a 30-m column of DB-17 (J & W housed singly and were fed a diet of Rodent laboratory chow Scientific, Folsom, CA). 5001 (PMI feeds, St. Louis) under standard conditions. All For HMG-CoA lyase activity, mitochondria were isolated procedures with animals were carried out under Emory Uni- from liver by homogenization and differential centrifugation m versity Institutional Animal Use and Care Committee ethical as described above. A 15- l sample of mitochondria suspended m guidelines (Institutional Animal Use and Care Committee in H-buffer was diluted into 300- l of cold 20 mM sodium 255-97). phosphate (pH 7.2) containing 1 mM EDTA and 1 mM DTT. Mitochondrial Isolation and OXPHOS Analysis. Pups of This sample was sonicated twice (15-second periods), and the 3 the indicated age and genotypes were killed by decapitation, resulting lysate was centrifuged at 12,000 g for 30 min (4°C). and the tissues were rapidly removed and placed on ice in The supernatant was used for spectrophotometric assays of H-Buffer (210 mM mannitoly70 mM sucrosey1 mM EGTAy5 HMG-CoA lyase, which is coupled to NADH production (340 y nm). A 1-ml reaction mixture contained 0.2 M TriszCl (pH 8.2), mM Hepes 0.5% BSA, pH 7.2) (30). Four to six hearts or 1 hind-limb skeletal muscles from each genotype were pooled 2.5 mM malate, 1.5 mM NAD , 10 mM MgCl2,5mMDTT, and homogenized on ice in 9 volumes of H-buffer by using a 0.05 mM NADH, 2 units of malate dehydrogenase, 2 units of 1-ml sintered glass homogenizer (Wheaton Scientific). Before citrate synthase, and an aliquot of the soluble mitochondrial homogenization, the hind-limb muscles were ‘‘sliced’’ into extract. The background rate of change in 340 nm absorbance was measured for 6 min before initiation of the reaction by smaller pieces by using a sterile razor blade. For brain, the m cerebellum, cortex, striatum, and brainstem were dissected out addition of HMG-CoA (100 M final concentration). A linear by using a dissecting microscope, and the tissues were trans- increase in 340-nm absorbance ensued because each cleavage ferred to a cold Eppendorf tube.