ND5 Subunit Of Complex Research Articles

Cellular DNA effects of radiation and cancer risk assessment in cells with mitochondrial defects

Mitochondria play a key role in mediating non-targeted effects (NTE) post-radiation exposure. Mitochondrial DNA (mtDNA), due to its less efficient repair as compared to nuclear DNA, is more severely affected by radiation exposure and mutations in essential mtDNA genes can further exacerbate oxidative stress. Our objective was to evaluate the impact of mitochondrial defects on radiation-induced DDR and genomic instability, biomarkers that can foreshadow cancer development. To better understand this relationship, we used commercially available lymphoblastoid cell lines containing characterized mitochondrial mutations and studied the kinetics of DNA damage and relative telomere length (RTL) changes over time post 0.5 Gy X-ray exposure. Our work suggests that cells containing different mitochondrial mutations exhibit unique DNA damage and telomere length effects following radiation exposure. For example, a cell line containing a mitochondrial mutation in the ND4 subunit of complex 1 showed decreased growth rate, higher levels of persistent DNA damage, and telomere instability as compared to wild type. In contrast, a cell line with a mutation in the ATPase 6 mitochondrial protein showed more subtle negative alterations. These findings suggest mitochondrial integrity may play an important role in cellular changes that promote cancer. In total results indicate mitochondrial mutations can influence DNA damage kinetics and genomic instability and have long-term consequences in the ability to regain homeostasis following radiation exposure. These results may help explain, at least in part, the rationale for the persistent genomic instability often observed following a low dose of radiation and how it may promote cancer.

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness.

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased β-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson’s Disease.

Longterm Reversal of Severe Visual Loss by Mitochondrial Gene Transfer in a Mouse Model of Leber Hereditary Optic Neuropathy

In many human disorders mitochondrial dysfunction is central to degeneration of retinal ganglion cells. As these cells do not regenerate, vision is irreversibly lost. Here we show reversal of visual dysfunction by a mitochondrially targeted adeno associated virus in transgenic mice harboring a G11778A mutation in the ND4 subunit of complex I persists longterm and it is associated with reduced loss of RGCs and their axons, improved oxidative phosphorylation, persistence of transferred ND4 DNA and transcription of ND4 mRNA.

Potentially adaptive mitochondrial haplotypes as a tool to identify divergent nuclear loci

Summary Genetic tools are commonly used for conservation and management of at‐risk species. Individuals are often sampled from mixtures that are composed of many populations, which creates a need to assign individuals to their source. This can be problematic when the genetic divergence among source populations is weak but can be improved using adaptive genetic loci, which should show stronger levels of divergence. We previously reported a signature of positive selection in the mitochondrial‐encoded ND5 subunit of complex I in diverse taxa. The respiratory machinery of the mitochondria in salmonids is composed of more than 80 nuclear genes and there is substantial interaction between nuclear and mitochondrial expressed gene products. Recent studies report adaptive variation in mitochondrial function as well as co‐evolution between mitochondrial and nuclear genomes. We used potentially adaptive ND5‐based mitochondrial haplotypes to identify nuclear loci that would display increased levels of genetic divergence compared to neutral nuclear loci in chum salmon (Oncorhynchus keta). Populations in a geographic area the size of France have previously demonstrated weak genetic divergence even after substantial discovery efforts by multiple laboratories for allozymes, microsatellites and SNPs over the last two decades. We used RAD‐based next‐generation sequencing and identified a nuclear‐encoded subunit of mitochondrial complex I that was a significant FST outlier and 14 other divergent nuclear markers that improve genetic assignment of individuals to their population of origin relative to assignments based on neutral markers alone. This work demonstrates how a known adaptive marker can be leveraged to increase the probability of identifying divergent markers for applied genetics tools that may be biologically linked to it.

Leigh Syndrome Caused by the MT-ND5 m.13513G>A Mutation: A Case Presenting with WPW-Like Conduction Defect, Cardiomyopathy, Hypertension and Hyponatraemia.

Mitochondrial disease can present with a wide range of clinical phenotypes, and knowledge of the clinical spectrum of mitochondrial DNA mutation is constantly expanding. Leigh syndrome (LS) has been reported to be caused by the m.13513G>A mutation in the ND5 subunit of complex I (MT-ND5 m.13513G>A). We present a case of a 12-month-old infant initially diagnosed with tachyarrhythmia requiring defibrillation, subsequent presentation with hypertension and hyponatraemia secondary to renal salt loss and presumed inappropriate ADH secretion. Complex I activity in the muscle tissue was 54%, and mutation load in the muscle and lymphocytes was 50%. This case of Leigh syndrome caused by the m.13513G>A mutation in the ND5 gene illustrates that hyponatraemia due to renal sodium loss and inappropriate ADH secretion and hypertension can be features of this entity in addition to the previously reported cardiomyopathy and WPW-like conduction pattern and that they present additional challenges in diagnosis and management.

Disease-Causing Mitochondrial Heteroplasmy Segregated Within Induced Pluripotent Stem Cell Clones Derived from a Patient with MELAS

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Uric acid and anti-TNF antibody improve mitochondrial dysfunction in ob/ob mice

The mechanisms responsible for low mitochondrial respiratory chain (MRC) activity in the liver of patients with nonalcoholic steatohepatitis are unknown. In this study, we examined the cause of this dysfunction in ob/ob mice. Forty-six mice were distributed in six groups: group I: C57BL/6J mice; group II: C57BL/6J Lep(-/-) mice (ob/ob); group III, ob/ob mice treated with manganese [III] tetrakis (5,10,15,20 benzoic acid) porphyrin (MnTBAP); group IV, ob/ob mice treated with IgG1 immunoglobulin; group V, ob/ob mice treated with anti-TNF antibody; group VI: ob/ob mice treated with uric acid. In liver tissue, we measured MRC activity, fatty acid beta-oxidation, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), 3-tyrosine-nitrated proteins, 3-tyrosine-nitrated mitochondrial proteins, including cytochrome c and ND4 subunit of complex I. MRC activity was decreased in ob/ob mice. TNF levels, iNOS protein expression, and tyrosine nitrated proteins were markedly increased in the liver of ob/ob mice. In these animals, mitochondrial proteins were markedly tyrosine nitrated, particularly the ND4 subunit of complex I and cytochrome c. Treatment of these animals with uric acid, a peroxynitrite scavenger, anti-TNF antibody, or MnTBAP decreased tyrosine nitrated proteins, improved the activity of MRC complexes, and led to a marked regression of hepatic steatosis and inflammation. In conclusion, MRC dysfunction and liver lesions found in ob/ob mice are likely to reflect the tyrosine nitration of mitochondrial proteins by peroxynitrite or a peroxynitrite-derivate radical. Increased hepatic TNF and iNOS expression might enhance peroxynitrite formation and inhibition of MRC complexes.

New variants in the mitochondrial genomes of schizophrenic patients

The impaired mitochondrial function hypothesis in schizophrenia is based on evidence of altered brain metabolism, morphology, biochemistry and gene expression. Mitochondria have their own genome, which is needed to synthesize some of the subunits of the respiratory chain enzymes. Mitochondrial DNA (mtDNA) is maternally inherited and we observed an excess of maternal transmission of schizophrenia in a set of parent-offspring affected pairs. We therefore hypothesized that mutations in the mtDNA may contribute to the complex genetic basis of schizophrenia. The entire mtDNA of six schizophrenic patients with an apparent maternal transmission of the disease was sequenced and compared to the reference sequence. We have identified 50 variants and among these six have not been previously reported. Three of them were missense variants: MTCO2 7750C>A, MTATP6 8857G>A and MTND4 12096T>A. These were maternally inherited because they were also present in the mtDNA of their respective schizophrenic mothers and none of them were found in 95 control individuals. The MTND4 12096T>A (Leu446His) is a heteroplasmic variant present in five of the six mother-offspring patient pairs that triggers a non-conservative substitution in the ND4 subunit of complex I. Sequence alignment of 110 ND4 peptides from all eukaryotic kingdoms shows that only hydrophobic amino acids are found in this position. Moreover, leucine was conserved or substituted by an isoleucine in all mammalian species. This indicates that the presence of histidine could affect complex I activity in patients with schizophrenia.

212. AAV-Mediated Gene Transfer Protects Against Mitochondrial Optic Neuropathy in Mice

Mutations affecting mitochondrial function lead to a variety of disorders affecting the heart, skeletal muscles, endocrine glands and central nervous system. These diseases have been hard to replicate in animals, because of the difficulty in manipulating mitochondrial DNA and the lethality of nuclear mutations affecting mitochondrial respiration. We have used an AAV-mediated RNA knockdown strategy to produce two mouse models of Leber Hereditary Optic Neuropathy (LHON). Patients with LHON typically contain mutations in one of the subunits of Complex I of the mitochondrial respiratory chain (NADH-ubiquinone oxidoreductase) and experience bilateral loss of vision beginning around age 20. In mice, blocking synthesis of subunits of this enzyme leads to the pathologic hallmarks of the disease—apoptotic death of retinal ganglion cells, demyelination and loss of axons in the optic nerve. Using these mouse models, we have tested two approaches for gene therapy of LHON. In the first strategy, we delivered the gene for mitochondrial manganese superoxide dismutase (SOD2) to limit the accumulation of reactive oxygen species that result from Complex I defects. Delivery of SOD2 caused an 18% increase (p=0.001) in the survival of retinal ganglion cells and a proportional increase in the thickness of the optic nerve. In the second therapeutic strategy, we re-coded the mitochondrial gene for the ND4 subunit of Complex I, attached a mitochondrial transport sequence to its amino terminus and expressed it as a nuclear gene (allotopic expression). Treatment of the optic nerve with AAV expressing this protein led to a 13% increase (p<0.01) in diameter of the optic nerve relative to contralateral eyes treated with a control virus. Thus both approaches prevented tissue injury resulting from defects to Complex I of the respiratory chain. These provide the first evidence in animals that gene therapy for mitochondrial disease may be possible.