Analysis of Mouse Models of Cytochrome c Oxidase Deficiency Owing to Mutations in Sco2

Analysis of mouse models of cytochrome c oxidasedeficiency owing to mutations in Sco2

Hua Yang1,{, Sonja Brosel2,{, Rebeca Acin-Perez3, Vesna Slavkovich4, Ichizo Nishino1,{,

Raffay Khan5, Ira J. Goldberg5, Joseph Graziano4, Giovanni Manfredi3 and Eric A. Schon1,2,�

1Department of Neurology and 2Department of Genetics and Development, Columbia University Medical Center, 1150

Saint Nicholas Avenue, Berrie-303A, New York, NY 10032, USA 3Department of Neurology and Neuroscience, Weill

Medical College of Cornell University, New York, NY, USA and 4Department of Environmental Health Sciences and5Department of Medicine, Columbia University Medical Center, New York, NY, USA

Received August 7, 2009; Revised and Accepted October 12, 2009

Mutations in SCO2, a protein required for the proper assembly and functioning of cytochrome c oxidase(COX; complex IV of the mitochondrial respiratory chain), cause a fatal infantile cardioencephalomyopathywith COX deficiency. We have generated mice harboring a Sco2 knock-out (KO) allele and a Sco2 knock-in(KI) allele expressing an E!K mutation at position 129 (E129K), corresponding to the E140K mutationfound in almost all human SCO2-mutated patients. Whereas homozygous KO mice were embryonic lethals,homozygous KI and compound heterozygous KI/KO mice were viable, but had muscle weakness; biochemi-cally, they had respiratory chain deficiencies as well as complex IV assembly defects in multiple tissues.There was a concomitant reduction in mitochondrial copper content, but the total amount of copper in exam-ined tissues was not reduced. These mouse models should be of use in further studies of Sco2 function, aswell as in testing therapeutic approaches to treat the human disorder.

INTRODUCTION

Mammalian cytochrome c oxidase (COX), also known ascomplex IV of the mitochondrial respiratory chain, is a multi-subunit holoprotein composed of three subunits encoded bymitochondrial DNA (mtDNA) and 10 subunits encoded bynuclear DNA, as well as two hemes (a and a3), threecoppers, one magnesium and one zinc as prosthetic groups(1). The mtDNA-encoded subunits (COX I–III) form the cat-alytic core of the enzyme, and contain three redox centers thatoxidize cytochrome c and reduce oxygen to form water, whileat the same time pumping protons from the matrix to the inter-membrane space. In this reaction, electrons from cytochrome care transferred first to the CuA site in subunit II (containingtwo copper atoms), then to the heme a site in subunit I, thento the CuB-heme a3 binuclear center, also in subunit I, andfinally to molecular oxygen (2).

The assembly of the COX holoprotein is a complicated andregulated process, requiring more than 20 ancillary assembly

factors, including proteins required for processing of the struc-tural subunits and insertion of the holoprotein into the mito-chondrial inner membrane, for heme biosynthesis andmaturation, and for metabolism and insertion of copper intothe holoprotein (2–7).

At least eight proteins are required for insertion of copperinto human COX, including CMC1, COX11, COX17,COX19, COX23, PET191, SCO1 and SCO2 (2). SCO1 andSCO2 (SCO stands for synthesis of cytochrome c oxidase)are homologous proteins related to bacterial prrC, a memberof the prrBCA operon associated with the photosynthetic regu-latory response in Rhodobacter sphaeroides (8,9). As such,SCO may function not only to transport copper to the CuA

site (10), but may also function as a redox sensor (9,11,12).While both SCO1 and SCO2 contain a highly conservedCXXXC motif that presumably binds copper, and thereforeare thought to be metallochaperones that are essential for theassembly of the catalytic core of COX (13,14), the two pro-teins apparently have non-overlapping functions (15,16) and

†The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.‡Present address: Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry,4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-8502, Japan.

�To whom correspondence should be addressed. Tel: þ1 2128515532; Fax: þ1 2123053986; Email: [email protected]

# The Author 2009. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2010, Vol. 19, No. 1 170–180doi:10.1093/hmg/ddp477Advance Access published on October 16, 2009

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may participate differentially in the regulation of cellularcopper homeostasis (16). To date, however, the precise func-tion of SCO proteins is still unclear.

Pathogenic mutations in human SCO1 and SCO2 cause mito-chondrial diseases associated with fatal infantile COXdeficiency. Although both proteins appear to be expressed inall tissues (17), pathogenic mutations in SCO1 lead to fatalinfantile hepatoencephalomyopathy (18), whereas those inSCO2 lead to hypertrophic cardiomyopathy, encephalopathyand myopathy soon after birth (17). Why mutations in the twogenes cause such different presentations is under investigation.

To date, over 25 patients with autosomal recessive COXdeficiency caused by mutations in SCO2 have been described(17,19–27). With but one exception (27), all patients harboreda missense mutation that converts Glu-140 to Lys (i.e. E140K)on at least one allele, at a position located only three residuesaway from the CXXXC copper-binding domain (9). Interest-ingly, patients homozygous for the E140K mutation had adelayed onset of symptoms and a more prolonged course ofthe disease, with death during mid- to late-infancy, when com-pared with compound heterozygotes who had E140K on oneallele and a null mutation (either a truncation or a frameshiftmutation) on the other allele (20).

In order to better understand the biological role of SCO2,and to test potential therapeutic approaches, we have createdmice harboring both a Sco2 knock-out (KO) allele and aSco2 knock-in (KI) E129K allele, corresponding to thecommon E140K mutation in humans. Although homozygousKO mice are embryonic lethals, both homozygous KI miceand compound heterozygous KI/KO mice show COXdeficiency in tissues affected in the human disease.

RESULTS

Generation of Sco2 mutant mice

Using a 5 kb KpnI fragment containing the entire Sco2 geneand portions of the flanking Tymp and Ncaph2 genes as a tar-geting vector, we generated Sco2 KO and E129K KI con-structs (Supplementary Material, Fig. S1). These weretransfected into ES cells, and positive clones were injectedinto the blastocysts from C57BL/6J mice. HeterozygousWT/KO and WT/KI mice were verified by polymerase chainreaction (PCR) analysis (Supplementary Material, Fig. S2)and sequencing of tail DNA. After germline transmissionwas obtained, we removed the NeoR cassette and one of theflanking LoxP sites by crossing the heterozygous mice withcre mice. The progeny were then backcrossed with WT129Sv/J mice. The resulting heterozygous mice were healthyand fertile, and did not show any apparent differences whencompared with their wild-type (WT) littermates.

Inbreeding of heterozygous KI (E129K) mice resulted in theproduction of 80 mice, harboring Sco2 E129/E129 (WT),homozygous K129/K129 and heterozygous E129/K129 allelesin the expected Mendelian ratio (20:38:22 animals, respect-ively). Adult weight, postnatal growth and development wereindistinguishable from their WT littermates (data not shown).

Further matings, including those with heterozygous KOmice, allowed us to generate homozygous KI/KI (hereaftercalled KIKI mice) and compound heterozygous KI/KO

(KIKO) mice, but we were unable to obtain viable homozy-gous KO/KO (KOKO) mice: upon inbreeding WT/KO hetero-zygotes, we observed a Mendelian ratio of progeny at 7.5 dpc(days postcoitum) (6 WT/WT, 14 WT/KO and 7 KO/KO), butobserved no viable KO homozygotes at birth (42 WT/WT, 87WT/KO and 0 KO/KO). The embryonic lethality in the KOKOmice occurred during early gestation; the latest KOKO embryothat we could obtain was at day 8.5 postcoitum, and had severemorphological abnormalities (Fig. 1). Histochemistry to detectCOX activity showed profound COX deficiency in the embryocompared with an age-matched WT embryo, even though bothWT and mutant embryos had mitochondria, as determined bythe positive staining for succinate dehydrogenase (SDH)activity (Fig. 1B).

In contrast to the KOKO mice, the KIKI and KIKO mice wereviable. They had longevities similar to that of WT littermates,and their body weights were similar to that of controls atevery age tested (not shown). The mice did not develop overtsymptoms of the cardioencephalomyopathy seen in thehuman disease, but upon further investigation, we found signifi-cant biochemical, morphological and behavioral differences inthe Sco2-mutant mice compared with their normal counterparts.

Biochemistry

We assessed the activities of respiratory chain complexes I, IIIand IV (COX), normalized to that of citrate synthase (CS), incrude mitochondria isolated from the brain, heart, liver andmuscle of WT, KIKI and KIKO mice at 4 months of age. Inall cases, the normalized complex I activities in KIKI andKIKO mice were comparable to the values in WT controls(Fig. 2). However, complex IV activities were reduced in allexamined tissues from the KIKO mice (by approximately20–60%), with the lowest values in liver (by approximately60%; Fig. 2). Complex IV activity was also reduced in alltissues of the KIKI mice, but to a lesser extent (by approxi-mately 15–45%) (Fig. 2). Surprisingly, we also detectedreduced complex III activities in all tissues from the mutantmice (by approximately 15–45%; Fig. 2).

We analyzed the amount of copper in the same tissues andin crude mitochondria. There was essentially no difference inthe total copper content of the tissues (Fig. 3A and B), butthere was a noticeable trend towards reduced amounts ofcopper in the crude mitochondrial fractions isolated from allexamined tissues from the KIKI and KIKO mice when com-pared with WT animals (Fig. 3C).

While not strictly quantitative, histochemical detection ofCOX activity in muscle, heart, brain, liver and kidney fromthe mice showed a greater apparent degree of COX deficiencyin the KIKO mice than in the KIKI mice when compared withWT, with skeletal muscle and brain most noticeably affected(Supplementary Material, Fig. S3), similar to the two tissuesmost affected in human SCO2 deficiency (17). More detailedhistochemical examination of skeletal muscle showed that thepattern of COX activity was similar to the pattern of the Sco2immunostaining in both WT and mutant animals (Fig. 4).

We performed one-dimensional blue-native gel electrophor-esis (1D-BNGE) (28,29) of mitochondria from liver and brain,probing with representative antibodies to each of the fiveOxPhos complexes. We found reduced levels of fully

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assembled complex IV in both KIKO and KIKI mice, togetherwith the apparent disappearance of a band in KIKO mice corre-sponding to a supercomplex (SC) consisting of complexes IIIand IV that was present in WT and KIKI mice (Fig. 5A). Tofurther investigate complex IV assembly, we subjected thesamples to two-dimensional BNGE (2D-BNGE) (28) followedby western blotting to detect different subunits of complex IV.When antibodies to COX I and COX II were used, we observedthe appearance of a subcomplex, approximately 80–100 kDa insize, in KIKI and KIKO samples, in both liver and brain, thatwere absent in the WT controls (Fig. 5B). Using antibodies toCOX Va, we observed the appearance of a different subcom-plex, approximately 150 kDa in size, again in both liver andbrain mitochondria from KIKI and KIKO, but not WT, mice(Fig. 5B). We saw no evidence of free unassembled subunitsin the low-molecular weight portion of the gels (i.e. between40 and 66 kDa), but we cannot exclude the possibility that sub-units smaller than 40 kDa were present but ran off the gel. Onthe other hand, we did not observe any aberrant assembly inter-mediates when blotting with antibodies to COX IV (Fig. 5B).These results reveal a role for Sco2 not only in complex IV func-tion but also in its assembly. Finally, using a cocktail of fiveantibodies that detect one representative polypeptide fromeach of the five OxPhos complexes in western blotting of pro-teins from brain and liver crude mitochondria separated bysodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE), we found decreased amounts of COX I inKIKI and KIKO mice compared with WT (Fig. 5C). Thisdecrease did not appear to correlate with the amount of Sco2(or Sco1, for that matter) in these tissues (Fig. 5D).

Functional assays

We conducted evaluations of motor function, coordination andneuromuscular strength. There was no difference in neuromus-

cular coordination between mutant and WT mice, as deter-mined by performance on a vertical pole test. However,when the mice were subjected to a running test on a treadmill,the endurance of 4-month-old KIKO male mice was signifi-cantly poorer than that of their WT littermates (Fig. 6B).The KIKO mice also had muscle weakness, as measured bythe hanging wire test (Fig. 6A). Interestingly, while bothmale and female mice developed muscle weakness, its onsetwas delayed in the females: whereas males had obvious lossof strength at 4 months, the females were normal at thatage, and did not show loss of strength until 8 months of age(Fig. 6A). We note, in this regard, that there was a genderdifference in the degree of response to upregulation of peroxi-some proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) of mice whose muscle lacked Cox10,another COX assembly protein (30).

We found no significant differences in cardiac functionbetween WT and Sco2-mutated mice, as measured by trans-thoracic M-mode and two-dimensional echocardiography(31) (not shown).

DISCUSSION

We describe here the first mouse models of COX deficiencyowing to mutations in Sco2, a COX-assembly protein. Whilehomozygous Sco2 KO mice were embryonic lethals, boththe homozygous E129K KIKI and compound heterozygousKIKO mice showed biochemical, morphological and func-tional defects reminiscent of, and compatible with, loss ofCOX activity in affected tissues in human SCO2 deficiency.However, while the Sco2-mutated mice recapitulated manyof the biochemical and functional features of the humandisease, they did not replicate all of them. In particular,whereas most SCO2-mutant patients die in infancy of a com-bined cardiopathy and myopathy, both the KIKI and KIKO

Figure 1. Morphological analysis of mouse embryos. (A) PCR analysis of WT and KOKO embryos. Notation as in Supplementary Material, Fig. S2. (B) His-tochemistry to detect COX and SDH activity in WT (top panels) and KOKO (bottom panels) 8.5 dpc embryos. Note the absence of COX activity but the presenceof SDH activity in the KOKO embryo.

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mice showed no evidence of cardiomyopathy and no reductionin lifespan.

The relatively mild phenotype observed in our Sco2 mousemodels when compared with that in the human disease standsin stark contrast to the embryonic lethal phenotype observed inthe homozygous Sco2 KO mice, which itself contrasts withmice lacking Surf1, another COX assembly gene, whichwere essentially normal (32,33). In particular, homozygousdisruption of the mouse Sco2 gene led to embryonic lethalityprior to 8.5 dpc, implying that Sco2 plays a crucial role duringearly development. Further analysis of cells derived fromthese embryos will be required to better determine thenature of the lethality.

Our data also support the view that although SCO1 andSCO2 are duplicated genes in both mammals and yeast (17),they probably have non-overlapping functions (15,34). Wenote that Sco1 mRNA has been detected in mouse embryos

as early as the 2-cell stage (Genbank AK139512.10), as wellas in 8-, 9-, and 10-day embryos (AK017794.1, AK083729.1and AK011251.1, respectively), implying that Sco1 proteinis present during early development. If so, our results implythat Sco1 was not able to rescue the loss of Sco2 function inhomozygous KO mouse embryos, supporting the idea thatSco2 is not only critical for embryonic viability, but alsothat Sco1 and Sco2 have at least partially non-overlappingfunctions at this stage of development (and possibly in post-natal life as well). The lack of complementation betweenSco1 and Sco2 has also been reported in cell culture, wherereduced copper levels and COX activity owing to mutationsin either SCO1 or SCO2 could be suppressed by overexpres-sion of SCO2, but not SCO1 (16).

The two viable mouse models created in this study carriedthe E129K mutation (the homolog of the human E140Kmutation), either as a homozygous KIKI or as a compound

Figure 2. Respiratory chain enzyme activities. All activities were normalized to that of citrate synthase. Values in the Sco2-mutant mice are presented as a %relative to those in WT littermates+SE. Asterisks (�) denote significant difference versus WT (P , 0.05); double asterisks (��) denote significant difference ofKIKO versus both WT and KIKI (P , 0.05).




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heterozygote, with one E129K allele and one KO allele(KIKO). Both mice were viable, fertile and appeared to liveas long as their WT littermates, and we found no differencebetween the mutant mice and their WT littermates in bodyweight, appearance, grooming, nesting and sleeping behaviors.This apparent good health of the KIKI and KIKO mice is instriking contrast to the analagous situation in human SCO2patients with E140K homozygous (20) and compound hetero-zygous (17) mutations, who die within approximately 1 yearof life, typically of cardioencephalomyopathy.

On the other hand, the KIKO mice performed poorly on atreadmill endurance test, and they were more susceptible tofatigue when compared with their WT littermates. They alsohad impaired motor function, as measured by the hanging

wire grip test. The defects in motor activity and muscle endur-ance were consistent with, and were likely due to, a significantdeficiency in COX activity in muscle. In fact, we found areduction in COX activity in all tissues examined, includingbrain, heart, liver and skeletal muscle, with the lowestvalues in liver. The reduction in COX activity as measuredbiochemically was also seen in the COX histochemistry ofthese tissues. However, the reduction in COX biochemicaland histochemical activity in the mice was less severe inmuscle than in, for example, liver or heart, and stands in con-trast to what has been observed in human SCO2 patients,where the COX deficiency appears to be most severe incardiac and skeletal muscle (17). We do not have an expla-nation for these differences, but they likely explain the rela-tively mild phenotypes that we observed.

It has been proposed that independent of its roles in COXassembly, SCO2 acts upstream of SCO1 to help maintain cel-lular copper homeostasis via regulation of the rate of copperefflux from the cell (16), which would explain the reductionin total cellular Cu found in SCO-mutant patient cells andtissues (16,35). However, while the amounts of copper foundin the crude mitochondrial fractions in the KIKI and KIKOmice were lower than in WT (consistent with the reductionin COX activity), the total amount of copper in the tissuesthat we examined did not decline in the Sco2-mutantanimals. We do not know why the data in the mice are discre-pant from those from human tissues.

A number of studies have shown that high residual levels ofCOX assembly intermediates are present in SCO2-mutantpatient tissues (23,36). The SCO2 mutations in patients arethought to reduce the efficiency by which the CuA center ofCOX II is formed (15,36), implying that the presence ofcopper in the CuA site is required for the assembly or stabilityof COX II (37). This idea is supported by studies in whichCOX activity was rescued in SCO2-mutant cells by theaddition of copper (34,36,38,39). However, the direct transferof copper to COX has still not been demonstrated and is stillspeculative, and nothing is known regarding the spatialarrangement of SCO2 vis-a-vis COX II.

We investigated the defect of COX in our mice by2D-BNGE, which is a sensitive way to detect assembly inter-mediates of the respiratory chain complexes. In brain and livermitochondria from the KIKI and KIKO mice, we found anaccumulation of aberrant assembly intermediates containingCOX I and COX II, implying that in addition to any defectof copper transport to the CuA site in COX II (10), theE129K mutation might also impair indirectly the transport ofcopper to COX I (40), thereby compromising COX assemblyand reducing COX activity in these tissues. We also detectedan aberrant assembly intermediate containing COX Va. Thedefects in COX assembly observed in our mice had similaritiesto those observed in cells and tissues from human SCOpatients, but it is noteworthy that the composition of these sub-assemblies has varied among patients. Taanman’s groupdescribed two sub-assemblies in one SCO1 patient, onecontaining COX I, IV and Va, and another containing justCOX I (41). Houstek’s group described a sub-assembly in aSCO1 patient containing both COX I and II but neitherCOX IV nor Va (35). They also found a SCO2 patient withaltered sub-assemblies containing COX I, IV and Va, but

Figure 3. Copper content of tissues shown in Figure 2. (A) Copper content oftotal tissue, mg/g wet weight basis. (B) Copper content of total tissue, mg/gprotein basis. (C) Copper content of crude mitochondrial fraction, mg/gcrude mitochondrial protein basis. As there was no apparent difference incopper values between male and female mice, the data were combined forboth sexes to increase statistical power; all values are averages+SE(number of samples analyzed indicated in parentheses in C). Asterisk (�) indi-cates significant difference versus WT (P , 0.05).




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not COX II; interestingly, the assembly defects varied amongtissues (37). Finally, Shoubridge’s group found a SCO1 patientwith a sub-assembly containing COX I and IV, but not COX II(COX Va was not examined) (15). Thus, the aberrant sub-assemblies found in the Sco2-mutant mice—containing CoxI, II and Va, but not Cox IV—mimic closely, but not

exactly, those found in the human patients. It may be thatmutations in SCO1 cause different assembly defects thanthose in SCO2, or that SCO mutations perturb the COX assem-bly pathway in a tissue-specific manner, or that differentspecific SCO mutations have different effects on the COXassembly process. Finally, it is also possible that some of

Figure 4. Muscle histochemistry and immunohistochemistry. Histochemistry to detect COX and SDH, and immunohistochemistry to detect Sco2, in muscle fromrepresentative male and female 4-month-old mice. Note the reduction in the intensity and distribution of the COX stain, especially in the KIKO mice (�20).




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Figure 5. (A) Western blot analyses of isolated mouse mitochondria separated by 1D-BNGE. Note the SC that is present in WT and KIKI mice but is apparentlyabsent in KIKO mice. (B) Western blot analyses of Complex IV assembly intermediates separated by 2D-BNGE. Probing with the indicated subunits of ComplexIV revealed intermediate subcomplexes in both liver and brain mitochondria from KIKI and KIKO mice using antibodies to COX I, COX II and COX Va (aster-isks), but not with antibodies to COX IV. Molecular weight markers (in kilodaltons) are at top. (C) Western blot analyses of proteins from isolated mouse mito-chondria separated by SDS–PAGE. Mitochondrial proteins from the indicated tissues were separated by SDS–PAGE and probed with a cocktail of antibodies tothe indicated subunits. The loading control was anti-cytochrome c. (D) Western blot analysis to detect Sco1 and Sco2, as in (C). The loading control was anti-porin.




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the subassemblies that we (and perhaps others) have observedare degradation products rather than authentic assembly inter-mediates, since the COX holocomplex is more unstable whenSCO2 is mutated.

According to the current model of COX assembly, theprocess starts with the interaction of COX I with eitherCOX IV or COX Va (29,41,42). The intermediates containingCOX I in our 2D-BNGE analysis indicate that the E129Kmutation impairs COX assembly at a very early step in theassembly pathway, and is consistent with observations ofCOX deficiency in patients with mutations in COX I whohave defective assembly of the COX holoenzyme (43,44).Similarly, immunoblot analysis of cells harboring a mutationin COX II also revealed decreased amounts of both COX Iand COX II, again accompanied by a reduction in COXactivity (45,46). In spite of the fact that SCO1 and SCO2may have non-overlapping functions, their roles in the COXassembly pathway appear to be similar, as assembly inter-mediates containing COX I and COX II, but not COX IV,were found both in our Sco2-deficient mice and in cellsfrom a SCO1-deficient patient (35).

We also detected reduced complex III activity in both theKIKO and KIKI mice. This finding was somewhat unexpected,as complex III deficiency had not been observed in the initialreports on human patients with SCO2 mutations (17,19,20),but we note that reduced complex III activity in a SCO2patient was reported recently (27). While patients with mito-chondrial diseases in which there is combined complex I þIII (47) and I þ III þ IV (48) deficiency have been reported,III þIV deficiency is relatively rare, although we note thatsome SCO2 patients have been reported with I þ III deficiency(23,27). Of course, since the respiratory chain can exist as aSC (49), it is possible that a mutation in a complex IV assem-bly protein, such as SCO2, could also affect the activity ofcomplex III.

An equally intriguing result was the quantitative nature ofthe COX deficiency observed in the KIKI versus KIKOmice. The phenotype of the KIKI mice, with two E129Kalleles, was less severe than that of the KIKO mice, withone E129K allele and one null allele. This result parallelswhat has been observed with the corresponding E140K

mutation in humans, where patients carrying two E140Kalleles survive longer than those with one E140K allele andone null allele (20). The quantitative behavior of this missensemutation in both mice and humans implies that it is a hypo-morph that still allows for residual function, either in coppertransport, or more speculatively, in redox sensing (9). IfE140K is indeed hypomorphic, unaffected heterozygousparents carrying this mutation ought to have more than half-normal SCO2 function (and hence .50% COX activity),and thereby might have a selective advantage over those car-riers with more deleterious alleles, thereby explaining why theE140K mutation is so common in the population. Thus, whileour KIKI and KIKO mice reproduce only some aspects of thehuman disease, they model quite nicely the quantitative natureof the common E140K mutation.

In summary, mutations in mouse Sco2 decrease COXactivity through impairment of the stability of the COX holo-protein, with an unexpected impact on the activity of complexIII as well. Since these Sco2-mutant mice also recapitulatemany of the functional features of the human disease, includ-ing muscle weakness, and yet are viable and fertile, they willbe of value in testing the effects of potential therapies (30) forMendelian-inherited COX deficiency, a group of currentlyincurable and fatal disorders.

MATERIAL AND METHODS

Animal care

All experiments were performed according to a protocolapproved by the Institutional Animal Care and Use Committeeof the Columbia University Medical Center, which is consist-ent with the National Institutes of Health Guide for the Careand Use of Laboratory Animals. Mice were housed and bredaccording to international standard conditions, with a 12-hlight, 12-h dark cycle.

Generation of Sco2 mouse models

The detailed procedures to generate and analyze the Sco2-mutant mice are described in the Supplementary Material.

Figure 6. Functional tests. (A) Mice were tested for their ability to hang from a suspended wire. Numbers in parentheses denote the number of mice analyzed. (B)Endurance on a treadmill; Asterisks and double asterisks are as indicated in Figure 2.




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Enzyme activities of respiratory complexes

Crude mitochondria were obtained by homogenization anddifferential centrifugation of brain, heart, liver and muscletissues. Tissues were homogenized in four volumes of SETHbuffer (250 mM sucrose, 2 mM EDTA, 10 mM Tris–HCl,50 U/ml heparin, pH 7.4) in a glass-glass homogenizer (5–8strokes) on ice. The homogenate was centifugated at 600gfor 5 min at 48C. The supernatant was centrifugated asecond time at 8000g for 15 min at 48C. The pellet containingthe crude mitochondria was resuspended in SETH buffer(approximately 10–25 mg/ml). Protein concentration wasdetermined by the method of Bradford using bovine serumalbumin as a standard.

We used spectrophotometric assays to measure CS (50),complex I, complex III (51) and complex IV (52) activities incrude mitochondria isolated from mouse tissues, as described.

Histochemical and immunohistochemical analyses

For histochemistry, brain, heart, liver and muscle tissues werefrozen in isopentane cooled in liquid nitrogen. The tissueswere cryo-sectioned (8 mm thick) and stained for SDH andCOX activities, as described (53). For immunohistochemistry,tissue sections were fixed for 10 min at room temperature (RT)in 4% paraformaldehyde in phosphate-buffered saline (PBS).Tissues were rinsed in PBS followed by an antigen retrievalstep in citrate buffer (10 mM citric acid, pH 6.0, at 1008Cfor 45 min). Slides were then incubated in 0.4% TritonX-100 in PBS for 20 min at RT and blocked for 1 h at RTin SuperBlock blocking buffer (Pierce) containing 160 ml/mlAvidin Block (Vector Labs). A cross-reacting rabbit polyclo-nal antibody to human SCO2 (see below) was diluted 1:100in SuperBlock blocking buffer containing 160 ml/ml BiotinBlock (Vector Labs) and was added to the tissue sections over-night at 48C. Secondary biotinylated rabbit antibody (Amer-sham) was added for 1 h at RT, followed by incubation withstreptavidin fluorescein (Amersham) for 1 h at RT.

Copper analyses

We assayed for copper using the inductively coupled plasma-mass spectrometer (ICP-MS) method. We digested tissuehomogenates in concentrated HNO3 for 20 h at RT, dilutedto a final acid concentration of 10%, and analyzed for Cu con-centration using a Perkin-Elmer Elan Dynamic Reaction Cell(DRC) II ICP-MS equipped with an AS 93þ autosampleralong with appropriate calibration standards. We correctedfor matrix-induced interferences by using an internal standardmatched to the mass and ionization properties of copper,namely gallium. We suppressed polyatomic interferences inthe instrument’s DRC using ammonia as a second gas. Theintraprecision coefficient of variation for standard serumsamples with known Cu concentrations was 2.8%, and forselected replicate tissue samples was 1.6%.

BNGE and western blotting

For western blotting of proteins separated by SDS–PAGE,15 mg of crude mitochondria were electrophoresed through a

12% polyacrylamide gel and electroblotted onto polyvinyli-dene difluoride (PVDF) filters (Bio-Rad). For protein detec-tion, we used the MitoProfile Total OxPhos rodent antibodycocktail (Mitosciences MS604). Anti-cytochrome c (Invitro-gen no. 456100; 1:1000 dilution) was used as a loadingcontrol. For western blotting of Sco proteins, 30 mg ofmouse crude mitochondrial proteins were loaded onto a 15%polyacrylamide gel, separated by SDS–PAGE, transferred toa nitrocellulose membrane, and probed with rabbit antiserato human SCO1 or SCO2 that cross-react with the correspond-ing mouse proteins. Antisera were raised against amino acidsnos 272–285 (EFLDYFGQNKRKGE) of human SCO1 andamino acids nos 236–249 (LFTDYYGRSRSAEQ) of humanSCO2 (Zymed Laboratories, Invitrogen). After affinity purifi-cation of the antisera, the antibodies were diluted 1:400 forimmunoblotting. Anti-porin (Abcam no. ab15895; 1:500dilution) was used as a loading control.

For BNGE, 50–75 mg of mitochondrial proteins that hadbeen extracted with lauryl maltoside were applied on a 5–13% gradient BN gel followed, in the case of 2D-BNGE, byseparation through a 12.5% denaturing gel (54). After electo-phoresis, proteins were electroblotted onto PVDF filters andsequentially probed with specific antibodies. For protein detec-tion, we used antibodies against NDUFA9 (complex I), Fp(complex II), core 2 (complex III), COX I (complex IV),COX II (complex IV), COX IV (complex IV), COX Va(complex IV) and ATPase-b (complex V). All antibodieswere from Invitrogen.

Mouse functional assays

To measure endurance capacity, the mice were run on a tread-mill (Columbus Instruments) at a speed of 22 m/min until theyreached fatigue. Fatigue was defined as the inability of themouse to maintain an appropriate pace despite continuoushand prodding for 1 min, at which time the mouse wasremoved from the treadmill and its run time recorded.

The coordination and balance of the mice were measured bya vertical pole test. A plastic pole, 2 cm in diameter and 40 cmlong, was wrapped with cloth tape for improved traction. Themice were placed in the center of the pole, in a horizontal pos-ition. The pole was then gradually lifted to a vertical position.Normal coordination and balance was defined as the ability ofthe mouse to stay on the pole beyond a 458 angle.

Muscular abnormalities in the mice were detected by a stan-dard hanging wire test (55). The mice were placed on a wirecage lid, and the lid was slowly inverted. The lid was heldat a height approximately 20 cm above the bench. Maskingtape placed around the perimeter of the lid prevented themice walking off the edge. The time till the mice fell off thecage lid was recorded, with a 2-min cut-off time. The testwas repeated five times, with a 20 s rest period between trials.

Cardiac function was measured by transthoracic M-modeand two-dimensional echocardiography, as described (31).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.




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ACKNOWLEDGEMENTS

We thank Victor C.S. Lin (Columbia Transgenic/Gene-Targeting Facility) for assistance with obtaining germlinetransmission of the Sco2 constructs, and Virginia Papaioannou(Columbia Department of Genetics and Development) forhelpful advice and discussions.

Conflict of Interest statement. The authors have no conflicts ofinterest.

FUNDING

This work was supported by grants from the National Insti-tutes of Health (HD83062, NS11766 and AG08702 [toE.A.S.], K02NS047306 [to G.M.], HL73029 [to I.J.G.],T3207343 [to R.K.] and P42ES10340 and P30ES09089 [toJ.G.]), the Muscular Dystrophy Association (to E.A.S. andG.M.), the United Mitochondrial Disease Foundation (toR.A.-P.), and the Marriott Foundation (to E.A.S.).

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Analysis of Mouse Models of Cytochrome c Oxidase Deficiency Owing to Mutations in Sco2