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• W1988565273 abstract "To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until ∼5 weeks of age, when ataxic signs began, progressing to death at ∼7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters. To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until ∼5 weeks of age, when ataxic signs began, progressing to death at ∼7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters. Mitochondria play vital roles in energy production, Ca2+ buffering, apoptotic events, and production of reactive oxygen species (ROS) (Nemoto et al., 2000Nemoto S. Takeda K. Yu Z.X. Ferrans V.J. Finkel T. Role for mitochondrial oxidants as regulators of cellular metabolism.Mol. Cell. Biol. 2000; 20: 7311-7318Crossref PubMed Scopus (322) Google Scholar, Sayer, 2002Sayer R.J. Intracellular Ca2+ handling.Adv. Exp. Med. Biol. 2002; 513: 183-196Crossref PubMed Google Scholar, Schapira, 1996Schapira A.H. Oxidative stress and mitochondrial dysfunction in neurodegeneration.Curr. Opin. Neurol. 1996; 9: 260-264Crossref PubMed Scopus (102) Google Scholar, Smith et al., 2000Smith M.A. Rottkamp C.A. Nunomura A. Raina A.K. Perry G. Oxidative stress in Alzheimer's disease.Biochim. Biophys. Acta. 2000; 1502: 139-144Crossref PubMed Scopus (636) Google Scholar, Wallace, 1999Wallace D.C. Mitochondrial diseases in man and mouse.Science. 1999; 283: 1482-1488Crossref PubMed Scopus (2507) Google Scholar). Given these essential functions, it is not surprising that mitochondrial failure is implicated in a wide variety of diseases, generally involving tissues with high-energy demand (Coskun et al., 2004Coskun P.E. Beal M.F. Wallace D.C. Alzheimer's brains harbor somatic mtDNA control-region mutations that suppress mitochondrial transcription and replication.Proc. Natl. Acad. Sci. USA. 2004; 101: 10726-10731Crossref PubMed Scopus (405) Google Scholar, Finsterer, 2006Finsterer J. Central nervous system manifestations of mitochondrial disorders.Acta Neurol. Scand. 2006; 114: 217-238Crossref PubMed Scopus (127) Google Scholar, Kim et al., 2001Kim S.H. Vlkolinsky R. Cairns N. Fountoulakis M. Lubec G. The reduction of NADH ubiquinone oxidoreductase 24- and 75-kDa subunits in brains of patients with Down syndrome and Alzheimer's disease.Life Sci. 2001; 68: 2741-2750Crossref PubMed Scopus (115) Google Scholar, Orth and Schapira, 2002Orth M. Schapira A.H. Mitochondrial involvement in Parkinson's disease.Neurochem. Int. 2002; 40: 533-541Crossref PubMed Scopus (174) Google Scholar, Zeviani et al., 1998Zeviani M. Tiranti V. Piantadosi C. Mitochondrial disorders.Medicine (Baltimore). 1998; 77: 59-72Crossref PubMed Scopus (91) Google Scholar). Neural, muscular, and cardiac pathologies frequently result from dysfunctional mitochondria, and progressive encephalomyopathy is a common clinical phenotype. Mitochondrial complex I (CI)-associated defects are the most common mitochondrial disorders (Benit et al., 2003Benit P. Steffann J. Lebon S. Chretien D. Kadhom N. de Lonlay P. Goldenberg A. Dumez Y. Dommergues M. Rustin P. et al.Genotyping microsatellite DNA markers at putative disease loci in inbred/multiplex families with respiratory chain complex I deficiency allows rapid identification of a novel nonsense mutation (IVS1nt −1) in the NDUFS4 gene in Leigh syndrome.Hum. Genet. 2003; 112: 563-566Crossref PubMed Scopus (47) Google Scholar, Kirby et al., 1999Kirby D.M. Crawford M. Cleary M.A. Dahl H.H. Dennett X. Thorburn D.R. Respiratory chain complex I deficiency: an under diagnosed energy generation disorder.Neurology. 1999; 52: 1255-1264Crossref PubMed Google Scholar, Loeffen et al., 2000Loeffen J.L. Smeitink J.A. Trijbels J.M. Janssen A.J. Triepels R.H. Sengers R.C. van den Heuvel L.P. Isolated complex I deficiency in children: clinical, biochemical and genetic aspects.Hum. Mutat. 2000; 15: 123-134Crossref PubMed Scopus (245) Google Scholar, Petruzzella and Papa, 2002Petruzzella V. Papa S. Mutations in human nuclear genes encoding for subunits of mitochondrial respiratory complex I: the NDUFS4 gene.Gene. 2002; 286: 149-154Crossref PubMed Scopus (48) Google Scholar, Smeitink et al., 2001Smeitink J. Sengers R. Trijbels F. van den Heuvel L. Human NADH:ubiquinone oxidoreductase.J. Bioenerg. Biomembr. 2001; 33: 259-266Crossref PubMed Scopus (103) Google Scholar). CI is the primary entry point for electrons into the electron transport chain (ETC) and is comprised of at least 45 different proteins (Carroll et al., 2006Carroll J. Fearnley I.M. Skehel J.M. Shannon R.J. Hirst J. Walker J.E. Bovine complex I is a complex of 45 different subunits.J. Biol. Chem. 2006; 281: 32724-32727Crossref PubMed Scopus (381) Google Scholar). Defects in the nonenzymatic, nuclear-encoded CI protein NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4, 18 kDa) cause a Leigh-like phenotype in humans that results in death within 3–16 months after birth (Budde et al., 2000Budde S.M. van den Heuvel L.P. Janssen A.J. Smeets R.J. Buskens C.A. DeMeirleir L. Van Coster R. Baethmann M. Voit T. Trijbels J.M. Smeitink J.A. Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene.Biochem. Biophys. Res. Commun. 2000; 275: 63-68Crossref PubMed Scopus (154) Google Scholar, Budde et al., 2003Budde S.M. van den Heuvel L.P. Smeets R.J. Skladal D. Mayr J.A. Boelen C. Petruzzella V. Papa S. Smeitink J.A. Clinical heterogeneity in patients with mutations in the NDUFS4 gene of mitochondrial complex I.J. Inherit. Metab. Dis. 2003; 26: 813-815Crossref PubMed Scopus (47) Google Scholar, Petruzzella et al., 2001Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome.Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (112) Google Scholar, van den Heuvel et al., 1998van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit.Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Findings in patients with NDUFS4 mutations include retarded growth, developmental delay, visual defects, muscular hypotonia, encephalomyopathy, cardiomyopathy, lethargy, and failure to thrive (Budde et al., 2003Budde S.M. van den Heuvel L.P. Smeets R.J. Skladal D. Mayr J.A. Boelen C. Petruzzella V. Papa S. Smeitink J.A. Clinical heterogeneity in patients with mutations in the NDUFS4 gene of mitochondrial complex I.J. Inherit. Metab. Dis. 2003; 26: 813-815Crossref PubMed Scopus (47) Google Scholar, Petruzzella et al., 2001Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome.Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (112) Google Scholar, Scacco et al., 2003Scacco S. Petruzzella V. Budde S. Vergari R. Tamborra R. Panelli D. van den Heuvel L.P. Smeitink J.A. Papa S. Pathological mutations of the human NDUFS4 gene of the 18-kDa (AQDQ) subunit of complex I affect the expression of the protein and the assembly and function of the complex.J. Biol. Chem. 2003; 278: 44161-44167Crossref PubMed Scopus (104) Google Scholar, Ugalde et al., 2004Ugalde C. Janssen R.J. van den Heuvel L.P. Smeitink J.A. Nijtmans L.G. Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency.Hum. Mol. Genet. 2004; 13: 659-667Crossref PubMed Scopus (167) Google Scholar, van den Heuvel et al., 1998van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit.Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). In humans, NDUFS4 appears to be essential for proper assembly of CI (Petruzzella et al., 2001Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome.Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (112) Google Scholar, Scacco et al., 2003Scacco S. Petruzzella V. Budde S. Vergari R. Tamborra R. Panelli D. van den Heuvel L.P. Smeitink J.A. Papa S. Pathological mutations of the human NDUFS4 gene of the 18-kDa (AQDQ) subunit of complex I affect the expression of the protein and the assembly and function of the complex.J. Biol. Chem. 2003; 278: 44161-44167Crossref PubMed Scopus (104) Google Scholar, Vogel et al., 2007Vogel R.O. van den Brand M.A. Rodenburg R.J. van den Heuvel L.P. Tsuneoka M. Smeitink J.A. Nijtmans L.G. Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients.Mol. Genet. Metab. 2007; 91: 176-182Crossref PubMed Scopus (55) Google Scholar). Despite the importance of mitochondria and the many disorders that result from cellular respiratory dysfunction, there are few genetic models of mitochondria-associated disease. Although other mouse models indirectly affect CI activity (Larsson et al., 1998Larsson N.G. Wang J. Wilhelmsson H. Oldfors A. Rustin P. Lewandoski M. Barsh G.S. Clayton D.A. Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.Nat. Genet. 1998; 18: 231-236Crossref PubMed Scopus (1099) Google Scholar, Park et al., 2007Park C.B. Asin-Cayuela J. Cámara Y. Shi Y. Pellegrini M. Gaspari M. Wibom R. Hultenby K. Erdjument-Bromage H. Tempst P. et al.MTERF3 is a negative regulator of mammalian mtDNA transcription.Cell. 2007; 130: 273-285Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, Vahsen et al., 2004Vahsen N. Cande C. Briere J.J. Benit P. Joza N. Larochette N. Mastroberardino P.G. Pequignot M.O. Casares N. Lazar V. et al.AIF deficiency compromises oxidative phosphorylation.EMBO J. 2004; 23: 4679-4689Crossref PubMed Scopus (510) Google Scholar), none involve a CI-specific mutation. We created a mutation of a CI subunit gene (Ndufs4). In this paper, we describe the mitochondrial encephalomyopathy phenotype of these Ndufs4 knockout (KO) mice. Mice with a conditional allele of the Ndufs4 gene (exon 2 flanked by loxP sites) were generated (see Figures S1A and S1B available online). Homozygous Ndufs4lox/lox mice appear normal; however, deletion of exon 2 in all cells (Ndufs4Δ/Δ = KO) caused the severe phenotype described below. Exon 2 encodes the last part of a mitochondrial targeting sequence and the first 17 amino acids of the mature NDUFS4 protein. Excision of exon 2 produces a frameshift that precludes synthesis of mature NDUFS4 protein. NDUFS4 was undetectable in protein extracts from KO mice by western blot (Figure S1C). Mice heterozygous (HET) for the deleted Ndufs4 allele were indistinguishable from wild-type (WT) mice. By postnatal day 21 (P21), most KO mice were smaller than normal and had begun to lose their body hair; however, their hair grew back during the next hair-growth cycle (Figure S2). KO mice reached a maximum body weight of ∼15 g at ∼P30 (Figure 1A). Prior to P30, the KO mice groomed, fed, responded to novel objects, socialized, and generally behaved similarly to control mice. They had normal locomotor activity during both day and night until ∼P30, after which they became lethargic (Figure 1B). Total oxygen consumption (Figure 1C), CO2 clearance, and food consumption by KO mice during both day and night and during a fast were within normal ranges when measured over 2 days in metabolic chambers (data not shown), but body temperature was ∼2°C lower than controls after P30 (Figure 1D). KO mice were blind, which manifested as an absent B wave in an electroretinogram (Figure 1E) and failure to recognize a visual cliff (data not shown). As early as P20, cataracts sometimes appeared in one or both eyes; after P35, KO mice were often unable to open their eyes completely. The acoustic startle response of KO mice was similar to (and often exceeded) that of controls until ∼P35 (Figure 1F). After P35, but with variable time of onset, there was a rapid decline in startle response (Figure 1F, inset), such that some older KO mice (n = 4) no longer responded tones up to 120 dB. Starting at ∼P35, the KO mice developed severe ataxia: they had splayed legs, became unresponsive to a firm nudge, were slow and awkward at righting themselves, and sometimes would lose their balance and fall over. These older KO mice were unable to maintain balance on a 0.7 mm-wide ledge, failed a negative geotaxis test, attempted to clasp a hind leg when suspended by their tail, and could not remain on a rotarod as long as controls (Figure 1G). Light microscopic studies of sections from cerebrum, brainstem, cerebellum, and eye did not reveal any significant differences between KO and WT mice at P35. In particular, infarcts, neuronal loss, and gliosis were not evident in sections stained with either cresyl violet or hematoxylin and eosin (data not shown). Between P35 and P50, the KO mice stopped gaining weight, displayed worsened ataxia, ceased grooming, and died. Enriched mitochondrial samples were prepared from liver to measure respiratory capacity. Rotenone-sensitive CI activity was detected in submitochondrial particles (SMPs) isolated from WT and HET samples, but not from KO samples (Figure 2A). The maximal enzymatic activity of respiratory complex II (CII) of KO mice was significantly higher. Activities of complex III (CIII) and complex IV (CIV) of KO mice were comparable to control samples (Figure 2A), although CIII activity of KO mice tended to be lower. In contrast to the results obtained with SMPs, CI activity was detectable in intact KO cells (Figures 2B and 2C; Figure S3). The activities of CI, CII, and CIV were measured by monitoring oxygen consumption using a Clarke electrode. Complex activity measured in this manner implies coupling of a functional respiratory chain with oxygen as the final electron acceptor. Amounts of oxygen consumption by tissue samples from KO and control mice were always measured in parallel. CI activity of tissues from KO mice was about half that of controls, whereas CII and CIV activities were equivalent or slightly higher than controls (Figure 2C). Detection of rotenone-sensitive oxygen consumption in intact tissue but lack of CI activity in SMPs suggests that functional CI is unstable during preparation of SMPs in the absence of NDUFS4. The abundance of CI in mitochondria from liver and brain was assessed by blue native gel electrophoresis (BNGE). Equal amounts of mitochondria were loaded, electrophoresed, blotted, and then probed with antibodies to three different CI subunits (NDUFS3, NDUFB6, and NDUFA9) located in different parts of the complex (Figure S4A). The abundance of intact CI was reduced in mitochondria from liver and brain of KO mice compared to controls (Figure 2D). However, SDS-PAGE analysis of the same samples with the three antibodies gave equivalent signals from WT and KO tissue (Figure 2E). These results indicate that although individual subunits of CI are present at comparable amounts, the amount of intact CI is reduced in mitochondria from KO mice, which could account for the lower oxygen consumption by KO tissue. We used 31P-NMR spectroscopy to measure ATP turnover reactions and ATP, phosphocreatine (PCr), and inorganic phosphate (Pi) concentrations during hindlimb ischemia reperfusion in vivo. Resting O2 consumption rate was measured using optical spectroscopy of hemoglobin and myoglobin saturations in vivo. During ischemia, PCr was depleted while Pi increased (Figure 3A); when oxygen flow was restored, PCr returned to normal levels. The rate of PCr recovery following ischemia was used to determine the mitochondrial phosphorylation capacity (maximal mitochondrial ATP production). Resting ATP demand (Figure 3B), phosphorylation capacity (Figure 3C), and resting O2 consumption (data not shown) were not different between control and KO mice. At the end of the experiment, total muscle ATP and PCr were measured. Muscle from P30 KO mice had ATP levels at the low end of the normal range, but PCr, Cr, and Pi were normal (Table 1).Table 1Concentrations of Metabolites in Muscle and BloodGenotypeATPPiPCrCrGlucoseLactateWT or CT9.13 ± 0.504.04 ± 0.6826.89 ± 1.9813.31 ± 2.017.98 ± 0.491.26 ± 0.06KO7.85 ± 0.593.09 ± 0.4025.77 ± 2.2815.08 ± 1.427.48 ± 1.32†1.63 ± 0.18∗ATP, inorganic phosphate (Pi), phosphocreatine (PCr), and creatine (Cr) levels from muscle (given in μmol/g wet tissue weight) were measured in P30 mice (n = 6 WT; n = 5 KO) by high-pressure liquid chromatography using a photodiode array detector. Blood glucose (given in mM) was measured in P22–72 mice (n = 22 CT [WT and HET]; n = 13 KO). Blood lactate (given in mM) was measured in P18–56 CT mice (n = 24) and >P40 KO mice (n = 8). Values are mean ± SEM. ∗p = 0.03; †p = 0.12 versus CT by Student's two-tailed t test. Open table in a new tab ATP, inorganic phosphate (Pi), phosphocreatine (PCr), and creatine (Cr) levels from muscle (given in μmol/g wet tissue weight) were measured in P30 mice (n = 6 WT; n = 5 KO) by high-pressure liquid chromatography using a photodiode array detector. Blood glucose (given in mM) was measured in P22–72 mice (n = 22 CT [WT and HET]; n = 13 KO). Blood lactate (given in mM) was measured in P18–56 CT mice (n = 24) and >P40 KO mice (n = 8). Values are mean ± SEM. ∗p = 0.03; †p = 0.12 versus CT by Student's two-tailed t test. Fasting blood glucose concentration was not significantly different between KO mice and controls. Old (>P40) KO mice had slightly elevated serum lactate levels (1.63 ± 0.18 mM; Table 1), whereas young (∼P18) KO mice had lactate levels similar to WT mice (data not shown). The relative mitochondrial genome number was quantified by Southern blot analysis of total DNA isolated from brain, heart, kidney, liver, and soleus muscle of >P35 control and KO mice using a mouse mitochondrial DNA probe. The intensity of the mitochondrial DNA band relative to a nuclear gene from KO tissues was the same as controls (Figure S5). Electron microscopy was used to examine mitochondrial morphology and density in muscle tissue. Mitochondrial ultrastructure appeared normal, although large subsarcolemmal clusters of mitochondria were present in the soleus but not extensor digitorum longus (EDL) muscle fibers of KO mice (Figure 4A). Both glycolytic and oxidative muscles of >P30 KO mice had normal polygonal morphology with peripheral nuclei (Figure 4B). Muscle of control and KO mice appeared normal after staining using the Gomori trichrome method, and there was no evidence of a “ragged-red” fiber phenotype. All muscle fiber types were present as shown by myofibrillar ATPase staining in various muscles including tibialis anterioris and soleus muscle. Succinate dehydrogenase (SDH) stain intensity often appeared slightly darker for KO mice, although quantitative analysis revealed no difference. There was a marked decrease in NADH oxidase activity in KO muscle fibers, while cytochrome c oxidase (COX) activity was comparable in both control and KO muscle (Figure 4B). We have described the phenotype of a mouse model of CI deficiency caused by mutating one of its 45 subunits. Respiratory CI activity was impaired in the absence of NDUFS4 protein and a fatal phenotype developed, similar to the Leigh-like encephalomyopathy observed in humans with mutations in NDUFS4. In the absence of NDUFS4, the abundance of CI and the rate of CI-driven oxygen consumption by intact cells were significantly reduced. Nevertheless, ATP, PCr, glucose, and lactate levels were close to normal, and total oxygen consumption in KO mice was unaffected. Although their growth was retarded, the activity and behavior of KO mice were similar to WT mice during the first 4 weeks; this was followed by increasing lethargy, although oxygen consumption remained constant. After P35, KO mice suffered from rapid deterioration of motor ability, lost weight, and usually died by P50. We confirmed CI dysfunction in Ndufs4 KO mice. It was surprising that SMPs isolated from mitochondria of KO mice had no detectable CI activity when reduction of a ubiquinone derivative was measured, yet the KO mice lived 5 weeks before displaying severe signs of illness, and their total oxygen consumption appeared normal. We suspected that CI was at least partially functional in KO tissue and confirmed this by monitoring oxygen uptake in intact mitochondria. CI-dependent (rotenone-sensitive) oxygen consumption was evident in tissue from KO mice, although at less than half the rate of control tissue. The residual CI activity in intact mitochondria from KO mice had the same rotenone sensitivity as controls (data not shown). The presence of some intact CI explains why ATP levels of skeletal muscle in vivo were normal and ATP was regenerated after ischemia at a rate equivalent to WT. The activities of the other respiratory complexes were normal or slightly elevated, in agreement with studies using human fibroblasts and skeletal muscle with NDUFS4 mutations (Budde et al., 2000Budde S.M. van den Heuvel L.P. Janssen A.J. Smeets R.J. Buskens C.A. DeMeirleir L. Van Coster R. Baethmann M. Voit T. Trijbels J.M. Smeitink J.A. Combined enzymatic complex I and III deficiency associated with mutations in the nuclear encoded NDUFS4 gene.Biochem. Biophys. Res. Commun. 2000; 275: 63-68Crossref PubMed Scopus (154) Google Scholar, van den Heuvel et al., 1998van den Heuvel L. Ruitenbeek W. Smeets R. Gelman-Kohan Z. Elpeleg O. Loeffen J. Trijbels F. Mariman E. de Bruijn D. Smeitink J. Demonstration of a new pathogenic mutation in human complex I deficiency: a 5-bp duplication in the nuclear gene encoding the 18-kD (AQDQ) subunit.Am. J. Hum. Genet. 1998; 62: 262-268Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). Mutations in human NDUFS4 also abolish CI activity in SMPs, and this loss of activity is associated with the appearance of large subcomplexes, suggesting that the stability or assembly of intact CI is affected (Petruzzella et al., 2001Petruzzella V. Vergari R. Puzziferri I. Boffoli D. Lamantea E. Zeviani M. Papa S. A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome.Hum. Mol. Genet. 2001; 10: 529-535Crossref PubMed Scopus (112) Google Scholar, Scacco et al., 2003Scacco S. Petruzzella V. Budde S. Vergari R. Tamborra R. Panelli D. van den Heuvel L.P. Smeitink J.A. Papa S. Pathological mutations of the human NDUFS4 gene of the 18-kDa (AQDQ) subunit of complex I affect the expression of the protein and the assembly and function of the complex.J. Biol. Chem. 2003; 278: 44161-44167Crossref PubMed Scopus (104) Google Scholar, Vogel et al., 2007Vogel R.O. van den Brand M.A. Rodenburg R.J. van den Heuvel L.P. Tsuneoka M. Smeitink J.A. Nijtmans L.G. Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients.Mol. Genet. Metab. 2007; 91: 176-182Crossref PubMed Scopus (55) Google Scholar). When CI from KO mice was examined by BNGE, the results indicated that the abundance of intact CI was reduced, in agreement with reduced CI activity. There was no evidence of subcomplexes as reported for NDUFS4 mutations in human fibroblasts. The difference between our results and those reported for human NDUFS4 mutations could reflect differential effects of the native gel electrophoresis methods used to analyze CI integrity or different stability of the remaining CI components in the absence of NDUFS4 in the two species. Both species exhibit an absence of CI activity in SMPs. The failure of the spectrophotometric methods to record measurable CI activity may be due to the preparation of SMPs. Sonication, freeze-thaw, or detergents are used to fracture mitochondria and thereby allow access of substrates to the inner mitochondrial membrane. We hypothesize that these manipulations disrupt an already compromised complex, resulting in complete loss of activity. The nonfunctional subcomplex of human NDUFS4-deficient fibroblasts may represent an intermediate of an unstable complex under these conditions. Because humans with complete loss of NDUFS4 survive for several months, we suspect that they too retain some CI activity and that fibroblast cell lines derived from patients may not represent the situation in vivo. Alternatively, mice and humans may respond differently to NDUFS4 deficiency. Our observation that some intact CI can form in the absence of NDUFS4 suggests that this subunit plays an important role in the assembly or stability of the complex but that the intact complex can form without it, perhaps because of compensation by other proteins. We examined whether metabolite levels were affected by CI deficiency. Serum lactate was slightly higher in the older mice, but not enough to suggest toxic acidosis. Lowered CI activity may result in accumulation of NADH, which would negatively affect energy flux, and reduce lactate dehydrogenase activity. Lactic acidosis occurs in humans with dysfunctional mitochondria, including patients with Leigh syndrome, although not usually in patients with NDUFS4 mutations, in agreement with our data (Loeffen et al., 2000Loeffen J.L. Smeitink J.A. Trijbels J.M. Janssen A.J. Triepels R.H. Sengers R.C. van den Heuvel L.P. Isolated complex I deficiency in children: clinical, biochemical and genetic aspects.Hum. Mutat. 2000; 15: 123-134Crossref PubMed Scopus (245) Google Scholar). Blood glucose levels were also normal. NADH oxidase was reduced in muscle from KO mice, substantiating a CI deficiency. Despite the decrease of NADH oxidase activity, muscle morphology appeared normal, supporting the idea that some functional CI is retained in muscle from KO mice. COX staining of KO muscle was comparable but sometimes appeared slightly less intense than controls in highly oxidative muscle such as the soleus. Lower COX activity has been observed when SDH activity is increased (Lopez et al., 2000Lopez M.E. Van Zeelanda N.L. Dahlb D.B. Weindruchc R. Aiken J.M. Cellular phenotypes of age-associated skeletal muscle mitochondrial abnormalities in rhesus monkeys.Mutat. Res. 2000; 452: 123-138Crossref PubMed Scopus (54) Google Scholar, Rifai et al., 1995Rifai Z. Welle S. Kamp C. Thornton C.A. Ragged red fibers in normal aging and inflammatory myopathy.Ann. Neurol. 1995; 37: 24-29Crossref PubMed Scopus (142) Google Scholar). SDH staining in oxidative muscle of KO mice is sometimes more intense than controls, and CII-driven O2 consumption is often slightly higher than controls. Furthermore, mitochondria isolated from KO mice are capable of supporting significantly higher CII activity, suggesting that CII substrate delivered in vivo could have therapeutic properties. Although dysfunctional mitochondria sometimes accumulate to extraordinary quantities in muscle from individuals with mitochondrial dysfunction (Schmiedel et al., 2003Schmiedel J. Jackson S. Schäfer J. Reichmann H. Mitochondrial cytopathies.J. Neurol. 2003; 250: 267-277Crossref PubMed Scopus (130) Google Scholar), Southern blot analysis indicated that the mitochondrial genome numbers from muscle and three other organs of KO mice were normal. Electron microscopy revealed subsarcolemmal accumulation of mitochondria in a subset of highly oxidative KO muscle fibers. In spite of this, ragged-red muscle fibers, which reflect accumulation of dysfunctional mitochondria in the subsarcolemmal region, were not detectable by the Gomori trichrome method. Muscle fiber morphology of KO mice was normal," @default.
• W1988565273 created "2016-06-24" @default.
• W1988565273 creator A5007946034 @default.
• W1988565273 creator A5016661046 @default.
• W1988565273 creator A5046458049 @default.
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• W1988565273 creator A5065405147 @default.
• W1988565273 creator A5079450341 @default.
• W1988565273 date "2008-04-01" @default.
• W1988565273 modified "2023-10-14" @default.
• W1988565273 title "Mice with Mitochondrial Complex I Deficiency Develop a Fatal Encephalomyopathy" @default.
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