Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1, or SURF1

Cytochrome c oxidase contains two redox-active copper centers (Cu(A) and Cu(B)) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the Cu(A) center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1.COX4.COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1.COX4.COX5A subassembly, suggesting that MTCO2 associates with the MTCO1.COX4.COX5A subassembly after the Cu(A) center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1.COX4.COX5A subassembly

Correction: Somatic mtDNA Mutation Spectra in the Aging Human Putamen.

The accumulation of heteroplasmic mitochondrial DNA (mtDNA) deletions and single nucleotide variants (SNVs) is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq) to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the "common" deletion and other "major arc" deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ(-) mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states

Characteristic landscape of mtDNA rearrangements in putamen.

<p>(A–C) 5′ and 3′ breakpoint position frequencies from representative young (A) and aged (B) specimens, and (C) detail of control region from (B). mtDNA map below (A) and (B) depicts rRNA genes (blue), tRNA genes (black bars), protein coding genes (white) and the control region (red). Map uses alternate numbering with a contiguous control region and m.1 as the 5′ base of <i>MT-TF</i>. In the map below (C), the top bar depicts the control region (light shading) with features indicated left to right (dark shading): termination associated sequence; conserved sequence boxes I, II (CSBII (red)), and III; light-strand promoter and heavy strand promoter-1. Middle bar shows the 7S DNA with an arrow at the 3′ end and lower bar marks heavy strand origin of replication (OH). The first base of conventional numbering is indicated (m.1). Where present (A–C), i = common deletion, ii = 3′ clustered breakpoints, iii = CRMs, iv = CRDs. (D) Dot-plot of mtDNA breakpoint distribution from (A) and (E) breakpoint distribution from (B) with axes colored accordingly and data normalized for coverage. Equivalent data to that in panels A–E for all samples is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003990#pgen.1003990.s001" target="_blank">Figs. S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003990#pgen.1003990.s003" target="_blank">S3</a> & <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003990#pgen.1003990.s004" target="_blank">S4</a>. (F) Upper panel, resolution of large amplicons from PCR of CRMs using inverted primers; Lower panel, PCR of CRDs using a breakpoint-specific primer. Sample order for both panels as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003990#pgen.1003990.s010" target="_blank">Table S1</a>. Molecular weight markers indicated (Kb).</p

A Novel Mutation in SURF1 Causes Skipping of Exon 8 in a Patient with Cytochrome c Oxidase-Deficient Leigh Syndrome and Hypertrichosis

Leigh syndrome is a rare pediatric neurodegenerative disorder attributed to impaired mitochondrial energy metabolism. Mutations in SURF1 have been described in several patients with Leigh syndrome associated with cytochrome c oxidase deficiency. We report a new 18-bp deletion (821del18), spanning the splice donor junction of exon 8 of SURF1, in an infant presenting with cytochrome c oxidase-deficient Leigh syndrome and hypertrichosis. cDNA sequencing demonstrated that this deletion results in a messenger lacking exon 8. RT-PCR experiments suggested a rapid degradation of the aberrant mRNA species from the 5′-end

Strand bias for transitions.

<p>(A) Average frequencies for each base change in the coding region and (B) the control region of the young and aged cohorts, (± SD). (C) Magnitude of [G>A]-[C>T] bias (ΔSNV bp⁻¹, the difference between G>A and C>T frequencies) and (D) [T>C]-[A>G] bias in the coding region. (E) Magnitude of [G>A]-[C>T] bias and (F) [T>C]-[A>G] bias in the control region. (G) Frequency of m.64C>T and m.16148C>T. Bars indicate cohort medians.</p

Frequencies of specific re-arrangements in putamen mtDNA.

<p>(A) Frequency of the common deletion per mtDNA (mtDNA⁻¹), (B) Major-arc deletions, (C) CRMs, (D) CRDs. Bars indicate cohort medians. (E) Frequency of the common deletion (Common) versus major arc deletions excluding the common deletion (Major arc), linear regression shown with R². (F) Changes in the levels of the common deletion as a proportion of cumulative major arc deletions excluding the common deletion (CD/MA) relative to age.</p

Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations

Increased mitochondrial biogenesis in muscle improves aging phenotypes in the mtDNA mutator mouse

Aging is an intricate process that increases susceptibility to sarcopenia and cardiovascular diseases. The accumulation of mitochondrial DNA (mtDNA) mutations is believed to contribute to mitochondrial dysfunction, potentially shortening lifespan. The mtDNA mutator mouse, a mouse model with a proofreading-deficient mtDNA polymerase γ, was shown to develop a premature aging phenotype, including sarcopenia, cardiomyopathy and decreased lifespan. This phenotype was associated with an accumulation of mtDNA mutations and mitochondrial dysfunction. We found that increased expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a crucial regulator of mitochondrial biogenesis and function, in the muscle of mutator mice increased mitochondrial biogenesis and function and also improved the skeletal muscle and heart phenotypes of the mice. Deep sequencing analysis of their mtDNA showed that the increased mitochondrial biogenesis did not reduce the accumulation of mtDNA mutations but rather caused a small increase. These results indicate that increased muscle PGC-1α expression is able to improve some premature aging phenotypes in the mutator mice without reverting the accumulation of mtDNA mutations

The mtDNA Mutation Spectrum of the Progeroid Polg Mutator Mouse Includes Abundant Control Region Multimers

Polg mtDNA mutator mice are important models for investigating the role of acquired mtDNA mutations in aging. Despite extensive study, there remains little consensus on either the etiology of the progeroid phenotype or the mtDNA mutation spectrum induced by disrupted polymerase-γ function. To investigate the latter, we have developed a novel, pragmatic approach we term “Mito-seq,” applying next-generation sequencing to enriched, native mtDNA. Regardless of detection parameters we observed an increase of at least two orders of magnitude in the number of mtDNA single nucleotide variants in Polg mutator mice compared to controls. We found no evidence for the accumulation of canonical mtDNA deletions but multimers of the mtDNA control region were identified in brain and heart. These control region multimers (CRMs) contained heterogeneous breakpoints and formed species that excluded the majority of mtDNA genes. CRMs demonstrate that polymerase-γ 3′-5′ exonuclease activity is required for preserving mtDNA integrity. ► Mito-Seq provides a sensitive, pragmatic approach to mtDNA mutation detection ► Polg D257A/D257A mice accumulate control region multimers (CRMS) distinct from mtDNA ► CRMs are associated with a mild mtDNA depletion and increased mtDNA gene expressio