Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene

Dillon W Leong,Roman Chrast,Estelle Arnaud,Gayle M Davey,René P Zahedi,Hamish S Scott,Matthew Mckenzie,Gordon K Smyth,Belinda Phipson,Albert Sickmann,David R Thorburn,Adrienne Laskowski,Anne K Voss,Melanie Bahlo,William R Heath,J.c Komen ,James Pitt ,Sarah Kinkel ,Michael Ryan ,Chelsee Hewitt

doi:10.1074/jbc.m111.327601

Abstract

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.

Highlights

Mitochondrial complex I deficiency is a common inherited metabolic disease
Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system
Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of complex I (CI) (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood

Summary

Conclusion

NDUFS4 subunit is required for complex I stability. Significance: Understanding the effects of oxidative phosphorylation defects is essential for the development of treatments. The only mouse models with CI deficiency were the spontaneous mutant “Harlequin” mouse with a proviral insertion in intron 1 of the Aif (apoptosis-inducing factor) gene, resulting in a reduced overall expression of AIF protein and the subsequently generated conditional Aif knockouts [7,8,9,10]. These models have some limitations because the exact role of AIF in CI maintenance and activity is still unclear. We demonstrate a clear CI assembly defect in Ndufs4fky/fky mice, which is similar to that found in patients with mutations in NDUFS4

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION