Mito-mice: animal models for mitochondrial DNA-based diseases

We have successfully produced "Mito-mice" harbouring a pathogenic mtDNA mutation. We generated the mice by introducing mitochondria with a 4696 base-pair mtDNA deletion (Delta mtDNA4696) into mouse embryos. This deletion encompasses nucleotides 7759-12 454 and includes six tRNA genes and seven structural genes. In Mito-mice, the Delta mtDNA4696 is transmitted maternally, and induces mitochondrial dysfunction in various tissues. Most of the Mito-mice with high proportions of the Delta mtDNA4696 died at about age 6 months due to renal failure. Mito-mice are the first animal model for mtDNA-based diseases and will be valuable for studying pathogenesis and for identifying effective drug and gene therapies.     

Mouse models of mitochondrial DNA defects and their relevance for human disease

Qualitative and quantitative changes in mitochondrial DNA (mtDNA) have been shown to be common causes of inherited neurodegenerative and muscular diseases, and have also been implicated in ageing. These diseases can be caused by primary mtDNA mutations, or by defects in nuclear‐encoded mtDNA maintenance proteins that cause secondary mtDNA mutagenesis or instability. Furthermore, it has been proposed that mtDNA copy number affects cellular tolerance to environmental stress. However, the mechanisms that regulate mtDNA copy number and the tissue‐specific consequences of mtDNA mutations are largely unknown. As post‐mitotic tissues differ greatly from proliferating cultured cells in their need for mtDNA maintenance, and as most mitochondrial diseases affect post‐mitotic cell types, the mouse is an important model in which to study mtDNA defects. Here, we review recently developed mouse models, and their contribution to our knowledge of mtDNA maintenance and its role in disease.     

线粒体疾病与核基因－线粒体基因的表达调控

线粒体与疾病是当前生物医学领域最前沿之一。本文简单介绍线粒体生物医学的基础知识、线粒体疾病的遗传模式,综述了近年来在线粒体DNA（mtDNA）突变和疾病、核基因突变和疾病等领域的研究进展,着重阐明核基因（特别是核修饰基因）调控mtDNA突变致病表达的分子机制。     

Mitochondrial genome maintenance in health and disease

Human mitochondria harbor an essential, high copy number, 16,569 base pair, circular DNA genome that encodes 13 gene products required for electron transport and oxidative phosphorylation. Mutation of this genome can compromise cellular respiration, ultimately resulting in a variety of progressive metabolic diseases collectively known as ‘mitochondrial diseases’. Mutagenesis of mtDNA and the persistence of mtDNA mutations in cells and tissues is a complex topic, involving the interplay of DNA replication, DNA damage and repair, purifying selection, organelle dynamics, mitophagy, and aging. We briefly review these general elements that affect maintenance of mtDNA, and we focus on nuclear genes encoding the mtDNA replication machinery that can perturb the genetic integrity of the mitochondrial genome.     

Beyond base excision repair: an evolving picture of mitochondrial DNA repair

Mitochondria are highly specialised organelles required for key cellular processes including ATP production through cellular respiration and controlling cell death via apoptosis. Unlike other organelles, mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function – deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular ageing and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear-encoded DNA repair proteins that are translocated into the mitochondria.Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.     

Mitochondrial diseases

By convention, the term "mitochondrial diseases" refers to disorders of the mitochondrial respiratory chain, which is the only metabolic pathway in the cell that is under the dual control of the mitochondrial genome (mtDNA) and the nuclear genome (nDNA). Therefore, a genetic classification of the mitochondrial diseases distinguishes disorders due to mutations in mtDNA, which are governed by the relatively lax rules of mitochondrial genetics, and disorders due to mutations in nDNA, which are governed by the stricter rules of mendelian genetics. Mutations in mtDNA can be divided into those that impair mitochondrial protein synthesis in toto and those that affect any one of the 13 respiratory chain subunits encoded by mtDNA. Essential clinical features for each group of diseases are reviewed. Disorders due to mutations in nDNA are more abundant not only because most respiratory chain subunits are nucleus-encoded but also because correct assembly and functioning of the respiratory chain require numerous steps, all of which are under the control of nDNA. These steps (and related diseases) include: (i) synthesis of assembly proteins; (ii) intergenomic signaling; (iii) mitochondrial importation of nDNA-encoded proteins; (iv) synthesis of inner mitochondrial membrane phospholipids; (v) mitochondrial motility and fission.     

Mitochondrial regulation of epigenetics and its role in human diseases

Most pathogenic mitochondrial DNA (mtDNA) mutations induce defects in mitochondrial oxidative phosphorylation (OXPHOS). However, phenotypic effects of these mutations show a large degree of variation depending on the tissue affected. These differences are difficult to reconcile with OXPHOS as the sole pathogenic factor suggesting that additional mechanisms contribute to lack of genotype and clinical phenotype correlationship. An increasing number of studies have identified a possible effect on the epigenetic landscape of the nuclear genome as a consequence of mitochondrial dysfunction. In particular, these studies demonstrate reversible or irreversible changes in genomic DNA methylation profiles of the nuclear genome. Here we review how mitochondria damage checkpoint (mitocheckpoint) induces epigenetic changes in the nucleus. Persistent pathogenic mutations in mtDNA may also lead to epigenetic changes causing genomic instability in the nuclear genome. We propose that “mitocheckpoint” mediated epigenetic and genetic changes may play key roles in phenotypic variation related to mitochondrial diseases or host of human diseases in which mitochondrial defect plays a primary role.     

Mitochondrial Genome Mutation in Cell Death and Aging

This article reviews the concept, molecular genetics, and pathology of cell death and agingin relation to mitochondrial genome mutation. Accumulating evidence emphasizes the role ofgenetic factors in the development of naturally occurring cell death and aging. The ATPrequired for a cell's biological activity is almost exclusively produced by mitochondria. Eachmitochondrion possesses its own DNA (mtDNA) that codes essential subunits of themitochondrial energy-transducing system. Recent studies confirm that mtDNA is unexpectedly fragileto hydroxyl radical damage, hence to the oxygen stress. Cellular mtDNA easily fragmentsinto over a hundred-types of deleted mtDNA during the life of an individual. Cumulativeaccumulation of these oxygen damages and deletions in mtDNA results in a defective energytransducing system and in bioenergetic crisis. The crisis leads cells to the collapse ofmitochondrial trans-membrane potential, to the release of the apoptotic protease activating factors intocytosol, to uncontrolled cell death, to tissue degeneration and atrophy, and to aging. Thetotal base sequencing of mtDNA among individuals revealed that germ-line point mutationstransmitted from ancestors accelerate the somatic oxygen damages and mutations in mtDNAleading to phenotypic expression of premature aging and degenerative diseases. A practicalsurvey of point mutations will be useful for genetic diagnosis in predicting the life-span ofan individual.     

Mitochondrial DNA diseases: Histological and cellular studies

Large-scale deletions and tRNA point mutations in mitochondrial DNA (mtDNA) are associated with a variety of different mitochondrial encephalomyopathies. Skeletal muscle in these patients shows a typical pathology, characterized by the focal accumulation of large numbers of morphologically and biochemically abnormal mitochondria (ragged-red fibers). Both mtDNA deletions and tRNA point mutations impair mitochondrial translation and produce deficiencies in oxidative phosphorylation. However, mutant and wild-type mtDNAs co-exist (mtDNA heteroplasmy) and the translation defect is not expressed until the ratio of mutant: wild-type mtDNAs exceeds a specific threshold. Below the threshold the phenotype can be rescued by intramitochondrial genetic complementation. The mosaic expression of the skeletal muscle pathology is thus determined by both the cellular and organellar distribution of mtDNA mutants.     

线粒体DNA突变与相关人类疾病

在过去的20年里, 人们发现线粒体DNA(mitochondrial DNA, mtDNA)突变与多种人类疾病相关, 其致病范围从单器官组织损害到多系统受累。文章目的在于探讨mtDNA突变与人类疾病的关系。文章重点论述: (1)线粒体遗传学特征; (2) mtDNA突变与人类遗传性疾病; (3)体细胞mtDNA突变在衰老和肿瘤中的作用; (4)mtDNA疾病的诊断和治疗。     

Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA

Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.     

Mitochondrial regulation of epigenetics and its role in human diseases

Most pathogenic mitochondrial DNA (mtDNA) mutations induce defects in mitochondrial oxidative phosphorylation (OXPHOS). However, phenotypic effects of these mutations show a large degree of variation depending on the tissue affected. These differences are difficult to reconcile with OXPHOS as the sole pathogenic factor suggesting that additional mechanisms contribute to lack of genotype and clinical phenotype correlationship. An increasing number of studies have identified a possible effect on the epigenetic landscape of the nuclear genome as a consequence of mitochondrial dysfunction. In particular, these studies demonstrate reversible or irreversible changes in genomic DNA methylation profiles of the nuclear genome. Here we review how mitochondria damage checkpoint (mitocheckpoint) induces epigenetic changes in the nucleus. Persistent pathogenic mutations in mtDNA may also lead to epigenetic changes causing genomic instability in the nuclear genome. We propose that “mitocheckpoint” mediated epigenetic and genetic changes may play key roles in phenotypic variation related to mitochondrial diseases or host of human diseases in which mitochondrial defect plays a primary role.     

Visualizing,quantifying, and manipulating mitochondrial DNA in vivo

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.     

Disorders of Nuclear-Mitochondrial Intergenomic Signalling

In addition to sporadic or maternally-inherited mutations of the mitochondrial genome, abnormalities of mtDNA can be transmitted as mendelian traits. The latter are believed to be caused by mutations in still unknown nuclear genes, which deleteriously interact with the mitochondrial genome. Two groups of mtDNA-related mendelian disorders are known: those associated with mtDNA large-scale rearrangements and those characterized by severe reduction of the mtDNA copy number. The most frequent presentation of the first group of disorders is an adult-onset encephalomyopathy, defined clinically by the syndrome of progressive external ophthalmoplegia plus, genetically by autosomal dominant transmission of the trait, and molecularly by the presence of multiple deletions of mtDNA. The second group of disorders comprises early-onset, organ-specific syndromes, associated with mtDNA depletion, that are presumably transmitted as autosomal recessive traits. Linkage analysis and search for candidate genes are two complementary strategies to clarify the molecular basis of these disorders of the nuclear-mitochondrial intergenomic signalling.     

Analysis of nuclear copies of mitochondrial sequences in honeybee (Apis mellifera) genome

At least 0.08% of the Apis mellifera nuclear genome contains sequences that originated from mitochondria. These nuclear copies of mitochondrial sequences (numts) are scattered all over the honeybee chromosomes and have originated by multiple independent insertions of mitochondrial DNA (mtDNA) as evident by phylogenetic analysis. Apart from original insertions, moderate duplications of numts also contributed to the present pattern and distribution of mitochondrial sequences in honeybee chromosomes. Assimilation of mitochondrial genes in the nuclear genome is mediated by extensive fragmentations of the original inserts. Replication slippage seems to be a major mechanism by which small sequences are inserted or deleted from mtDNA destined to nucleus. Most of the honeybee numts (84%) are located in the nongenic regions. The majority (94%) of the numts that are located in predicted nuclear genes have originated from mitochondrial genes coding for cytochrome oxidase and NADH dehydrogenase subunits. On the other hand, the mitochondrial rRNA or tRNA gene sequences are predominantly (88%) located in nongenic regions of the genome. Evidences also support for exertion of purifying selection on numts located in specific genes. Comparative analysis of numts of European, African, and Africanized honeybees suggests that numt evolution in A. mellifera is probably not demarked by speciation time frame but may be a continuous and dynamic process.     

Sequence analysis of the entire mitochondrial genome in Parkinson's disease

The pathogenesis of Parkinson's disease (PD) is largely unknown. Indirect evidence suggests that mutations in mitochondrial DNA (mtDNA) might play a role, but previous studies have not consistently associated any specific mutations with PD. However, these studies have generally been confined to limited areas of the mitochondrial genome. We therefore sequenced the entire mitochondrial genome from substantia nigra of 8 PD and 9 control subjects. Several sequence variants were distributed differently between PD and control subjects, but all were previously reported polymorphisms. Several secondary LHON mutations were found, as well as a number of novel missense mutations, but all were rare and did not differ between PD and control subjects. Finally, PD and control subjects did not differ in the total number of all mutations, nor the total number of missense mutations. Thus, mtDNA involvement in PD, if any, is likely to be complex and should be reconsidered carefully.     

Mitochondrial DNA integrity is not dependent on DNA polymerase-beta activity

Mutations in mitochondrial DNA (mtDNA) are involved in a variety of pathologies, including cancer and neurodegenerative diseases, as well as in aging. mtDNA mutations result predominantly from damage by reactive oxygen species (ROS) that is not repaired prior to replication. Repair of ROS-damaged bases occurs mainly via base excision repair (BER) in mitochondria and nuclei. In nuclear BER, the two penultimate steps are carried out by DNA polymerase-beta (Polbeta), which exhibits both 5'-deoxyribose-5-phosphate (5'-dRP) lyase and DNA polymerase activities. In mitochondria, DNA polymerase-gamma (Polgamma) is believed to be the sole polymerase and is therefore assumed to function in mitochondrial BER. However, a recent report suggested the presence of Polbeta or a "Polbeta-like" enzyme in bovine mitochondria. Consequently, in the present work, we tested the hypothesis that Polbeta is present and functions in mammalian mitochondria. Initially we identified two DNA polymerase activities, one corresponding to Polgamma and the other to Polbeta, in mitochondrial preparations obtained by differential centrifugation and discontinuous sucrose density gradient centrifugation. However, upon further fractionation in linear Percoll gradients, we were able to separate Polbeta from mitochondria and to show that intact mitochondria, identified by electron microscopy, lacked Polbeta activity. In a functional test for the presence of Polbeta function in mitochondria, we used a new assay for detection of random (i.e., non-clonal) mutations in single mtDNA molecules. We did not detect enhanced mutation frequency in mtDNA from Polbeta null cells. In contrast, mtDNA from cells harboring mutations in the Polgamma exonuclease domain that abolish proofreading displayed a >or=17-fold increase in mutation frequency. We conclude that Polbeta is not an essential component of the machinery that maintains mtDNA integrity.     

Mitochondrial haplotype does not affect sperm velocity in the zebra finch Taeniopygia guttata

Mitochondrial DNA (mtDNA) variation has been suggested as a possible cause of variation in male fertility because sperm activity is tightly coupled to mitochondrial oxidative phosphorylation and ATP production, both of which are sensitive to mtDNA mutations. Since male‐specific phenotypes such as sperm have no fitness consequences for mitochondria due to maternal mitochondrial (and mtDNA) inheritance, mtDNA mutations that are deleterious in males but which have negligible or no fitness effect in females can persist in populations. How often such mutations arise and persist is virtually unknown. To test whether there were associations between mtDNA variation and sperm performance, we haplotyped 250 zebra finches Taeniopygia guttata from a large pedigreed‐population and measured sperm velocity using computer‐assisted sperm analysis. Using quantitative genetic ‘animal’ models, we found no effect of mtDNA haplotype on sperm velocity. Therefore, there is no evidence that in this system mitochondrial mutations have asymmetric fitness effects on males and females, leading to genetic variation in male fertility that is blind to natural selection.