Mitochondrial DNA dysfunction in oncocytic hepatocytes

Abstract: Hepatic oncocytes with abundant granular, eosinophilic cytoplasm due to mitochondrial hyperplasia are seen in various chronic liver diseases, particularly chronic hepatitis and cirrhosis. Increased mitochondria in oncocytes are thought to be a compensatory mechanism for deficiencies in the hepatocellular respiratory chain, although the pathogenesis of these deficiencies has been uncertain. We selected seven cases of cirrhosis (six with oncocytes, one without) for the following analysis: histoenzymatic and immunohistochemical staining of several mitochondrial DNA (mtDNA)‐ and nuclear DNA (nDNA)‐encoded respiratory chain enzymes; immuno‐staining using antibodies against double‐strand‐DNA (anti‐DNA) and against Ki‐67 (a cell proliferation marker); and Southern blot analysis for mtDNA and nDNA. Eighty percent of oncocytes showed histoenzymatic and immunohistochemical deficiencies of cytochrome c oxidase and the mtDNA‐encoded subunit I of complex IV, with preserved expression of nDNA‐encoded succinate dehydrogenase and the iron‐sulfur subunit of complex III (FeS). Cytoplasmic (but not nuclear) anti‐DNA staining was partially or completely absent in approximately 50% of oncocytes. Three cases with oncocytes studied by Southern blot showed mtDNA reductions of 66%, 71%, and 85%. In conclusion, hepatic oncocytes demonstrate significant deficiencies of mtDNA and mtDNA‐encoded respiratory chain enzymes. We propose that mtDNA depletion plays an important role in hepatocellular oncocytic transformation.     

DNA‐editing enzymes as potential treatments for heteroplasmic mtDNA diseases

Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and, in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild‐type and mutant mtDNA molecules co‐exist within the cell. Disease only manifests once the per cent of mutant mtDNA reaches a high threshold (usually >80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA‐editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes and discusses roadblocks of applying these to gene therapy in humans.     

Inheritance of mitochondrial DNA in humans: implications for rare and common diseases

The first draft human mitochondrial DNA (mtDNA) sequence was published in 1981, paving the way for two decades of discovery linking mtDNA variation with human disease. Severe pathogenic mutations cause sporadic and inherited rare disorders that often involve the nervous system. However, some mutations cause mild organ‐specific phenotypes that have a reduced clinical penetrance, and polymorphic variation of mtDNA is associated with an altered risk of developing several late‐onset common human diseases including Parkinson’s disease. mtDNA mutations also accumulate during human life and are enriched in affected organs in a number of age‐related diseases. Thus, mtDNA contributes to a wide range of human pathologies. For many decades, it has generally been accepted that mtDNA is inherited exclusively down the maternal line in humans. Although recent evidence has challenged this dogma, whole‐genome sequencing has identified nuclear‐encoded mitochondrial sequences (NUMTs) that can give the false impression of paternally inherited mtDNA. This provides a more likely explanation for recent reports of ‘bi‐parental inheritance’, where the paternal alleles are actually transmitted through the nuclear genome. The presence of both mutated and wild‐type variant alleles within the same individual (heteroplasmy) and rapid shifts in allele frequency can lead to offspring with variable severity of disease. In addition, there is emerging evidence that selection can act for and against specific mtDNA variants within the developing germ line, and possibly within developing tissues. Thus, understanding how mtDNA is inherited has far‐reaching implications across medicine. There is emerging evidence that this highly dynamic system is amenable to therapeutic manipulation, raising the possibility that we can harness new understanding to prevent and treat rare and common human diseases where mtDNA mutations play a key role.     

Mitochondrial diseases in adults

Mitochondrial medicine is a field that expanded exponentially in the last 30 years. Individually rare, mitochondrial diseases as a whole are probably the most frequent genetic disorder in adults. The complexity of their genotype–phenotype correlation, in terms of penetrance and clinical expressivity, natural history and diagnostic algorithm derives from the dual genetic determination. In fact, in addition to the about 1.500 genes encoding mitochondrial proteins that reside in the nuclear genome (nDNA), we have the 13 proteins encoded by the mitochondrial genome (mtDNA), for which 22 specific tRNAs and 2 rRNAs are also needed. Thus, besides Mendelian genetics, we need to consider all peculiarities of how mtDNA is inherited, maintained and expressed to fully understand the pathogenic mechanisms of these disorders. Yet, from the initial restriction to the narrow field of oxidative phosphorylation dysfunction, the landscape of mitochondrial functions impinging on cellular homeostasis, driving life and death, is impressively enlarged. Finally, from the clinical standpoint, starting from the neuromuscular field, where brain and skeletal muscle were the primary targets of mitochondrial dysfunction as energy‐dependent tissues, after three decades virtually any subspecialty of medicine is now involved. We will summarize the key clinical pictures and pathogenic mechanisms of mitochondrial diseases in adults.     

Decline in skeletal muscle mitochondrial function with aging in humans

Cumulative mtDNA damage occurs in aging animals, and mtDNA mutations are reported to accelerate aging in mice. We determined whether aging results in increased DNA oxidative damage and reduced mtDNA abundance and mitochondrial function in skeletal muscle of human subjects. Studies performed in 146 healthy men and women aged 18-89 yr demonstrated that mtDNA and mRNA abundance and mitochondrial ATP production all declined with advancing age. Abundance of mtDNA was positively related to mitochondrial ATP production rate, which in turn, was closely associated with aerobic capacity and glucose tolerance. The content of several mitochondrial proteins was reduced in older muscles, whereas the level of the oxidative DNA lesion, 8-oxo-deoxyguanosine, was increased, supporting the oxidative damage theory of aging. These results demonstrate that age-related muscle mitochondrial dysfunction is related to reduced mtDNA and muscle functional changes that are common in the elderly.     

Effects of in utero antiretroviral exposure on mitochondrial DNA levels, mitochondrial function and oxidative stress

Objectives

HIV and antiretroviral (ART) exposure in utero may have deleterious effects on the infant, but uncertainty still exists. The objective of this study was to evaluate aspects of mitochondrial DNA (mtDNA) content, mitochondrial function and oxidative stress simultaneously in placenta, umbilical cord blood and infant blood in HIV/ART‐exposed infants compared with uninfected controls.

Methods

HIV‐1‐infected pregnant women and HIV‐1‐uninfected healthy pregnant controls were enrolled in the study prospectively. Placenta and umbilical cord blood were obtained at delivery and infant blood was obtained within 48 h of delivery. mtDNA content was determined for each specimen. Nuclear [subunit IV of cytochrome c‐oxidase (COX IV)]‐ and mitochondrial (COX II)‐encoded polypeptides of the oxidative phosphorylation enzyme cytochrome c‐oxidase were quantified in cord and infant blood. Placental mitochondria malondialdehyde (MDA) concentrations were measured as a marker of oxidative stress.

Results

Twenty HIV‐positive/HIV‐exposed and 26 control mother–infant pairs were enrolled in the study. All HIV‐infected women and their infants received ART. Placental MDA concentration and mtDNA content in placenta and cord blood were similar between groups. The cord blood COX II:IV ratio was lower in the HIV‐positive group than in the controls, whereas the infant peripheral blood mtDNA content was higher in the HIV‐exposed infants, but the infant peripheral blood COX II:IV ratio was similar. No infant had clinical evidence of mitochondrial disease or acquired HIV infection. In multivariable regression analyses, the significant findings in cord and infant blood were both most associated with HIV/ART exposure.

Conclusions

HIV‐exposed infants showed reduced umbilical cord blood mitochondrial enzyme expression with increased infant peripheral blood mitochondrial DNA levels, the latter possibly reflecting a compensatory mechanism to overcome HIV/ART‐associated mitochondrial toxicity.     

Mitochondrial dysfunction as a cause of ageing

Mitochondrial dysfunction is heavily implicated in the ageing process. Increasing age in mammals correlates with accumulation of somatic mitochondrial DNA (mtDNA) mutations and decline in respiratory chain function. The age-associated respiratory chain deficiency is typically unevenly distributed and affects only a subset of cells in various human tissues, such as heart, skeletal muscle, colonic crypts and neurons. Studies of mtDNA mutator mice has shown that increased levels of somatic mtDNA mutations directly can cause a variety of ageing phenotypes, such as osteoporosis, hair loss, greying of the hair, weight reduction and decreased fertility. Respiratory-chain-deficient cells are apoptosis prone and increased cell loss is therefore likely an important consequence of age-associated mitochondrial dysfunction. There is a tendency to automatically link mitochondrial dysfunction to increased generation of reactive oxygen species (ROS), however, the experimental support for this concept is rather weak. In fact, respiratory-chain-deficient mice with tissue-specific mtDNA depletion or massive increase of point mutations in mtDNA typically have minor or no increase of oxidative stress. Mitochondrial dysfunction is clearly involved in the human ageing process, but its relative importance for mammalian ageing remains to be established.     

Mitochondrial heteroplasmy and the evolution of insecticide resistance: Non-Mendelian inheritance in action

Genes encoded by mitochondrial DNA (mtDNA) exist in large numbers per cell but can be selected very rapidly as a result of unequal partitioning of mtDNA between germ cells during embryogenesis. However, empirical studies of this “bottlenecking” effect are rare because of the apparent scarcity of heteroplasmic individuals possessing more than one mtDNA haplotype. Here, we report an example of insecticide resistance in an arthropod pest (Tetranychus urticae) being controlled by mtDNA and on its inheritance in a heteroplasmic mite strain. Resistance to the insecticide bifenazate is highly correlated with remarkable mutations in cytochrome b, a mitochondrially encoded protein in the respiratory pathway. Four sites in the Q_o site that are absolutely conserved across fungi, protozoa, plants, and animals are mutated in resistant mite strains. Despite the unusual nature of these mutations, resistant mites showed no fitness costs in the absence of insecticide. Partially resistant strains, consisting of heteroplasmic individuals, transmit their resistant and susceptible haplotypes to progeny in highly variable ratios consistent with a sampling bottleneck of ≈180 copies. Insecticide selection on heteroplasmic individuals favors those carrying resistant haplotypes at a frequency of 60% or more. This combination of factors enables very rapid evolution and accounts for mutations being fixed in most field-collected resistant strains. The results provide a rare insight into non-Mendelian mechanisms of mitochondrial inheritance and evolution, relevant to anticipating and understanding the development of other mitochondrially encoded adaptations in arthropods. They also provide strong evidence of cytochrome b being the target site for bifenazate in spider mites.     

Somatic alterations in mitochondrial DNA and mitochondrial dysfunction in gastric cancer progression

Energy metabolism reprogramming was recently identified as one of the cancer hallmarks.One of the underlying mechanisms of energy metabolism reprogramming is mitochondrial dysfunction caused by mutations in nuclear genes or mitochondrial DNA(mtDNA).In the past decades,several types of somatic mtDNA alterations have been identified in gastric cancer.However,the role of these mtDNA alterations in gastric cancer progression remains unclear.In this review,we summarize recently identified somatic mtDNA alterations in gastric cancers as well as the relationship between these alterations and the clinicopathological features of gastric cancer.The causative factors and potential roles of the somatic mtDNA alterations in cancer progression are also discussed.We suggest that point mutations and mtDNA copy number decreases are the two most common mtDNA alterations that result in mitochondrial dysfunction in gastric cancers.The two primary mutation types(transition mutations and mononucleotide or dinucleotide repeat instability)imply potential causative factors.Mitochondrial dysfunction-generated reactive oxygen species may be involved in the malignant changes of gastric cancer.The search for strategies to prevent mtDNA alterations and inhibit the mitochondrial retrograde signaling will benefit the development of novel treatments for gastric cancer and other malignancies.     

mtDNA与冠心病相关性的研究进展

Mitochondria is the body''s energy factory, and it have their own separate genome--mtDNA. Myocardium is a kind of energy-consuming tissue, the normal energy supply of mitochondria is important, and mtDNA can affect the energy supply of mitochondria to a certain extent. According to the latest Global Burden of Disease study, cardiovascular disease (CVD) is the leading cause of death, and coronary heart disease (CAD) is one of the leading causes of death in patients with CVD. As a newly discovered biomarker, mtDNA has a strong correlation with the pathogenesis, potential therapeutic targets and prognosis of coronary heart disease. This article reviews the research progress of the correlation between mtDNA and coronary heart disease.     

Amplification of mitochondrial DNA in acute myeloid leukaemia

There is a long-standing interest in the possible role of mitochondria in malignancy. We sought to discover whether amplification of mitochondrial DNA (mtDNA) occurred in leukaemia, and found it was often remarkably amplified in the blast cells of acute myeloid leukaemia (AML).
We used gene dosage experiments to quantify the amount of mtDNA relative to nuclear DNA. DNA extracted from peripheral blood leucocytes or bone marrow of healthy individuals or patients was simultaneously hybridized with a probe for the mitochondrial genome and a control probe for the renin gene on human chromosome 1. Comparative densitometric ratios of approximately 1 were obtained between the two signals in 20 normal control peripheral blood samples. In contrast, comparative ratios in the range of 2–50 were observed in 25 AML samples and 13 of these showed 8-fold or greater amplification of mtDNA relative to normal peripheral blood controls. An additional four cases of AML were investigated at both presentation and remission and showed 3–10-fold amplification of mtDNA at presentation, but no amplification when in clinical remission. 18 cases of chronic granulocytic leukaemia (CGL) were also studied in chronic phase and showed mtDNA dosage levels equivalent to normal peripheral blood controls. However, 8/9 CGL patients showed mtDNA amplification during transformation from chronic phase. We conclude that amplification of mtDNA is an invariable feature of acute myeloid leukaemia and that it may be a useful marker for detecting transformation of CGL.     

Mitochondrial ageing

Mitochondria in largely postmitotic cells (e.g. cardiomyocytes, neurons or skeletal muscle myotubes) have a limited life span of a few weeks. Their replacement during normal turnover requires an intergenomic coordination between the mitochondrial genome (mtDNA, encoding for 13 protein subunits of the respiratory chain, two mitochondrial rRNAs and the 22 mitochondrial tRNAs) and the nuclear genome (encoding for more than 99 % of the mitochondrial proteins). The mtDNA contains only a very small non-coding region, it is exposed to radicals generated by the respiratory chain during aerobic ATP formation, and mitochondrial DNA repair capacity is rather low. Therefore, oxidative damage of mtDNA, accumulating with age, should affect mitochondrially encoded proteins, but the high number of mitochondrial genomes (roughly 10 per mitochondrion) allows a certain degree of heteroplasmy (different genomes within a mitochondrion) without effects on phenotype. Therefore, age-associated increments in mtDNA damage are to a major extent an epiphenomenon. On the other hand, however, there are clonal accumulations of damaged/mutated mtDNA within individual cells up to homoplasmy of mutant mtDNA, which are either neutral with regard to phenotype or which cause substantial phenotype alterations: hyporespiratory phenotype (less radicals and less ATP!) or a phenotype with a dysproportionate respiratory chain, i.e. partial defects within the chain with enhanced radical formation proximal to this defect and with enhanced susceptibility to oxidative stress-triggered apoptosis, probably explaining the progressive loss of cardiomyocytes with advanced age. Thus, a minority of age-associated alterations in mtDNA may explain important features of the ageing heart: myocyte losses and myocyte heterogeneity. However, documentation of definite proof for this possibility is lacking and may be difficult. Correspondence to: M. Szibor     

Quantitative analysis of human mitochondrial DNA using a real-time PCR assay

Objectives

Known for their ability to inhibit the human DNA polymerase‐γ, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase‐γ ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real‐time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria.

Methods

Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt‐b recombinant plasmid as standard control. We analysed eight HIV‐infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non‐infected donors.

Results

The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV‐infected patients were 2.94, 2.78 and 1.93 log HIV‐1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited.

Conclusions

The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL.

     

Prevalence of mitochondrial DNA haplogroups in an Australian population

Mitochondrial DNA (mtDNA) haplogroups are 'neutral polymorphisms' in the mtDNA genome, which have accumulated and persisted along maternal lineages as the human population has migrated worldwide. Three ethnically distinct lineages of human mtDNA populations have been identified: European, characterized by nine haplogroups H, I, J, K, T, U, V, W and X; African, characterized by superhaplogroup L and Asian, characterized by superhaplogroup M. We studied the prevalence of mtDNA haplogroups in participants of the Blue Mountains Eye Study, a large population-based survey of vision conducted between 1991 and 2000 of non-institutionalized permanent residents aged 49 years or older from two suburban postcode areas, west of Sydney, Australia. Total DNA isolated from either hair follicles or blood was available for 3377 of the 3509 participants (96.2%) to determine mtDNA haplogroups by polymerase chain reaction/restriction fragment length polymorphism analysis. Approximately 94.2% of samples could be assigned to one of the nine major European haplogroups, whereas a further 1.2% included the African (L) and Asian (M) superhaplogroups. The five principal haplogroups represented were H (42.9%), U (14.1%), J (10.7%), T (9.2%) and K (8.1%), which together included 85% of this population.     

A transgenic model to study the pathogenesis of somatic mtDNA mutation accumulation in β-cells

Low levels of somatic mutations accumulate in mitochondrial DNA (mtDNA) as we age; however, the pathogenic nature of these mutations is unknown. In contrast, mutational loads of >30% of mtDNA are associated with electron transport chain defects that result in mitochondrial diseases such as mitochondrial encephalopathy lactic acidosis and stroke-like episodes. Pancreatic β-cells may be extremely sensitive to the accumulation of mtDNA mutations, as insulin secretion requires the mitochondrial oxidation of glucose to CO₂. Type 2 diabetes arises when β-cells fail to compensate for the increased demand for insulin, and many type 2 diabetics progress to insulin dependence because of a loss of β-cell function or β-cell death. This loss of β-cell function/β-cell death has been attributed to the toxic effects of elevated levels of lipids and glucose resulting in the enhanced production of free radicals in β-cells. mtDNA, localized in close proximity to one of the major cellular sites of free radical production, comprises more than 95% coding sequences such that mutations result in changes in the coding sequence. It has long been known that mtDNA mutations accumulate with age; however, only recently have studies examined the influence of somatic mtDNA mutation accumulation on disease pathogenesis. This article will focus on the effects of low-level somatic mtDNA mutation accumulation on ageing, cardiovascular disease and diabetes.     

增龄大鼠心肌线粒体DNA损伤及DNA修复酶γ表达的变化

目的:探讨大鼠增龄过程中心肌组织DNA损伤和DNA修复酶表达的变化。方法:从不同月龄的大鼠心肌组织中提取DNA、RNA及蛋白。采用定量多聚酶链反应(Q-PCR)检测心肌DNA损伤;用RT-PCR和Western blot检测DNA修复酶γ(Polγ)表达的变化。用ELISA法检测DNA内8羟基脱氧鸟苷(8-OHdG)的水平。结果:随着鼠龄的增长,大鼠心肌组织中核DNA(nDNA)损伤不明显,线粒体DNA(mtDNA)损伤严重,DNA内8-OHdG的水平增加,Polγ的表达下降。结论:随着鼠龄的增长,大鼠心肌组织中DNA修复酶Polγ的表达下降,mtDNA损伤增加。DNA损伤与DNA修复能力下降间会引起恶性循环,最终可导致DNA损伤的增加加快心脏衰老。