动物线粒体遗传系统理论与应用研究进展

随着线粒体与人类疾病之间关系研究的不断深入,线粒体DNA及其编码的13个多肽的功能和进化引起了众多研究者的关注。作为有氧呼吸的后生动物主要的产能系统——线粒体电子传递系统(ETS)由线粒体和核基因组共同编码,自然选择被认为偏爱那些增强ETS功能的突变,此类突变可发生在线粒体或核编码的ETS蛋白中并引起积极的基因组间的相互作用,即"共适应"。线粒体DNA进化通常被认为遵循一种稳定的突变速率平衡中性模型,但有证据表明该假设可能并不可靠。对线粒体遗传系统研究的最新进展进行了综述,这对科学和合理地使用线粒体DNA分析技术具重要意义。     

Defects of intergenomic communication: autosomal disorders that cause multiple deletions and depletion of mitochondrial DNA

Depletion and multiple deletions of mitochondrial DNA (mtDNA) have been associated with a growing number of autosomal diseases that have been classified as defects of intergenomic communication. MNGIE, an autosomal recessive disorder associated with mtDNA alterations is due to mutations in thymidine phosphorylase that may cause imbalance of the mitochondrial nucleotide pool. Subsequently, mutations in the mitochondrial proteins adenine nucleotide translocator 1, Twinkle, and polymerase gamma have been found to cause autosomal dominant progressive external ophthalmoplegia with multiple deletions of mtDNA. Uncovering the molecular bases of intergenomic communication defects will enhance our understanding of the mechanisms responsible for maintaining mtDNA integrity.     

Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection

Laser-capture microdissection was coupled with PCR to define the mitochondrial genotype of aged muscle fibers exhibiting mitochondrial enzymatic abnormalities. These electron transport system (ETS) abnormalities accumulate with age, are localized segmentally along muscle fibers, are associated with fiber atrophy and may contribute to age-related fiber loss. DNA extracted from single, 10 µm thick, ETS abnormal muscle fibers, as well as sections from normal fibers, served as templates for PCR-based deletion analysis. Large mitochondrial (mt) DNA deletion mutations (4.4–9.7 kb) were detected in all 29 ETS abnormal fibers analyzed. Deleted mtDNA genomes were detected only in the regions of the fibers with ETS abnormalities; adjacent phenotypically normal portions of the same fiber contained wild-type mtDNA. In addition, identical mtDNA deletion mutations were found within different sections of the same abnormal region. These findings demonstrate that large deletion mutations are associated with ETS abnormalities in aged rat muscle and that, within a fiber, deletion mutations are clonal. The displacement of wild-type mtDNAs with mutant mtDNAs results in concomitant mitochondrial enzymatic abnormalities, fiber atrophy and fiber breakage.     

Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys

Mitochondrial DNA (mtDNA) deletion mutations co-localize with electron transport system (ETS) abnormalities in rhesus monkey skeletal muscle fibers. Using laser capture microdissection in conjunction with PCR and DNA sequence analysis, mitochondrial genomes from single sections of ETS abnormal fibers were characterized. All ETS abnormal fibers contained mtDNA deletion mutations. Deletions were large, removing 20-78% of the genome, with some to nearly all of the functional genes lost. In one-third of the deleted genomes, the light strand origin was deleted, whereas the heavy strand origin of replication was conserved in all fibers. A majority (27/39) of the deletion mutations had direct repeat sequences at their breakpoints and most (36/39) had one breakpoint within or in close proximity to the cytochrome b gene. Several pieces of evidence support the clonality of the mtDNA deletion mutation within an ETS abnormal region of a fiber: (a) only single, smaller than wild-type, PCR products were obtained from each ETS abnormal region; (b) the amplification of mtDNA from two regions of the same ETS abnormal fiber identified identical deletion mutations, and (c) a polymorphism was observed at nucleotide position 16103 (A and G) in the wild-type mtDNA of one animal (sequence analysis of an ETS abnormal region revealed that mtDNA deletion mutations contained only A or G at this position). Species-specific differences in the regions of the genomes lost as well as the presence of direct repeat sequences at the breakpoints suggest mechanistic differences in deletion mutation formation between rodents and primates.     

Mitochondrial medicine

After reviewing the history of mitochondrial diseases, I follow a genetic classification to discuss new developments and old conundrums. In the field of mitochondrial DNA (mtDNA) mutations, I argue that we are not yet scraping the bottom of the barrel because: (i) new mtDNA mutations are still being discovered, especially in protein-coding genes; (ii) the pathogenicity of homoplasmic mutations is being revisited; (iii) some genetic dogmas are chipped but not broken; (iv) mtDNA haplotypes are gaining interest in human pathology; (v) pathogenesis is still largely enigmatic. In the field of nuclear DNA (nDNA) mutations, there has been good progress in our understanding of disorders due to faulty intergenomic communication. Of the genes responsible for multiple deletions and depletion of mtDNA, mutations in POLG have been associated with a great variety of clinical phenotypes in humans and to precocious aging in mice. Novel pathogenetic mechanisms include alterations in the lipid milieu of the inner mitochondrial membrane and mutations in genes controlling mitochondrial motility, fission, and fusion.     

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.     

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.     

Disruption of mitochondrial function in interpopulation hybrids of Tigriopus californicus

Electron transport system (ETS) function in mitochondria is essential for the aerobic production of energy. Because ETS function requires extensive interactions between mitochondrial and nuclear gene products, coadaptation between mitochondrial and nuclear genomes may evolve within populations. Hybridization between allopatric populations may then expose functional incompatibilities between genomes that have not coevolved. The intertidal copepod Tigriopus californicus has high levels of nucleotide divergence among populations at mitochondrial loci and suffers F2 hybrid breakdown in interpopulation hybrids. We hypothesize that hybridization results in incompatibilities among subunits in ETS enzyme complexes and that these incompatibilities result in diminished mitochondrial function and fitness. To test this hypothesis, we measured fitness, mitochondrial function, and ETS enzyme activity in inbred recombinant hybrid lines of Tigriopus californicus. We found that (1) both fitness and mitochondrial function are reduced in hybrid lines, (2) only those ETS enzymes with both nuclear and mitochondrial subunits show a loss of activity in hybrid lines, and (3) positive relationships exist between ETS enzyme activity and mitochondrial function and between mitochondrial function and fitness. We also present evidence that hybrid lines harboring mitochondrial DNA (mtDNA) and mitochondrial RNA polymerase (mtRPOL) from the same parental source population have higher fitness than those with mtDNA and mtRPOL from different populations, suggesting that mitochondrial gene regulation may play a role in disruption of mitochondrial performance and fitness of hybrids. These results suggest that disruption of coadaptation between nuclear and mitochondrial genes contributes to the phenomenon of hybrid breakdown.     

Modeling pathogenic mutations of human twinkle in Drosophila suggests an apoptosis role in response to mitochondrial defects

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations.     

Evolution of interacting proteins in the mitochondrial electron transport system in a marine copepod

The extensive interaction between mitochondrial-encoded and nuclear-encoded subunits of electron transport system (ETS) enzymes in mitochondria is expected to lead to intergenomic coadaptation. Whether this coadaptation results from adaptation to the environment or from fixation of deleterious mtDNA mutations followed by compensatory nuclear gene evolution is unknown. The intertidal copepod Tigriopus californicus shows extreme divergence in mtDNA sequence and provides an excellent model system for study of intergenomic coadaptation. Here, we examine genes encoding subunits of complex III of the ETS, including the mtDNA-encoded cytochrome b (CYTB), the nuclear-encoded rieske iron-sulfur protein (RISP), and cytochrome c(1) (CYC1). We compare levels of polymorphism within populations and divergence between populations in these genes to begin to untangle the selective forces that have shaped evolution in these genes. CYTB displays dramatic divergence between populations, but sequence analysis shows no evidence for positive selection driving this divergence. CYC1 and RISP have lower levels of sequence divergence between populations than CYTB, but, again, sequence analysis gives no evidence for positive selection acting on them. However, an examination of variation at cytochrome c (CYC), a nuclear-encoded protein that transfers electrons between complex III and complex IV provides evidence for selective divergence. Hence, it appears that rapid evolution in mitochondrial-encoded subunits is not always associated with rapid divergence in interacting subunits (CYC1 and RISP), but can be in some cases (CYC). Finally, a comparison of nuclear-encoded and mitochondrial-encoded genes from T. californicus suggests that substitution rates in the mitochondrial-encoded genes are dramatically increased relative to nuclear genes.     

Oxidative stress, mitochondrial DNA mutation, and apoptosis in aging

A wide spectrum of alterations in mitochondria and mitochondrial DNA (mtDNA) with aging has been observed in animals and humans. These include (i) decline in mitochondrial respiratory function; (ii) increase in mitochondrial production of reactive oxygen species (ROS) and the extent of oxidative damage to DNA, proteins, and lipids; (iii) accumulation of point mutations and large-scale deletions of mtDNA; and (iv) enhanced apoptosis. Recent studies have provided abundant evidence to substantiate the importance of mitochondrial production of ROS in aging. On the other hand, somatic mtDNA mutations can cause premature aging without increasing ROS production. In this review, we focus on the roles that ROS play in the aging-associated decline of mitochondrial respiratory function, accumulation of mtDNA mutations, apoptosis, and alteration of gene expression profiles. Taking these findings together, we suggest that mitochondrial dysfunction, enhanced oxidative stress, subsequent accumulation of mtDNA mutations, altered expression of a few clusters of genes, and apoptosis are important contributors to human aging.     

Molecular Evolution of Cytochrome c Oxidase in High-Performance Fish (Teleostei: Scombroidei)

The 13 peptides encoded by vertebrate mitochondrial DNA (mtDNA) are essential subunits of oxidative phosphorylation (OXPHOS) enzymes. These genes normally experience purifying selection and also coevolve with nuclear-encoded subunits of OXPHOS complexes. However, the role of positive selection on mtDNA evolution is still unclear, as most examples of intergenomic coevolution appear to be the result of compensation by nuclear-encoded genes for mildly deleterious mtDNA mutations, and not simultaneous positive selection in both genomes. Organisms that have experienced strong selective pressures to increase aerobic capacity or adapt to changes in thermal environment may be better candidates in which to examine the impact of positively selected changes on mtDNA evolution. The tuna (suborder Scombroidei, family Scombridae) and billfish (suborder Scombroidei, families Xiphiidae and Istiophoridae) are highly aerobic fish with multiple specializations in muscle energetics, including a high mitochondrial content and regional endothermy. We examined the role of positively selected mtDNA substitutions in the production of these unique phenotypes. Focusing on a catalytic subunit of cytochrome c oxidase (COX II), we found that the rate ratio of nonsynonymous (d(N); amino acid changing)-to-synonymous (d(S); silent) substitutions was not increased in lineages leading to the tuna but was significantly increased in the lineage preceding the billfish. Furthermore, there are a number of individual positively selected sites that, when mapped onto the COX crystal structure, appear to interact with other COX subunits and may affect OXPHOS function and regulation in billfish.     

Defects in mitochondrial DNA replication and human disease

Mitochondrial DNA (mtDNA) is replicated by the DNA polymerase g in concert with accessory proteins such as the mtDNA helicase, single stranded DNA binding protein, topoisomerase, and initiating factors. Nucleotide precursors for mtDNA replication arise from the mitochondrial salvage pathway originating from transport of nucleosides, or alternatively from cytoplasmic reduction of ribonucleotides. Defects in mtDNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mtDNA deletions, point mutations, or depletion which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders, such as progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). This review focuses on our current knowledge of genetic defects of mtDNA replication (POLG, POLG2, C10orf2) and nucleotide metabolism (TYMP, TK2, DGOUK, and RRM2B) that cause instability of mtDNA and mitochondrial disease.     

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.     

Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers

Skeletal muscle-mass loss with age has severe health consequences, yet the molecular basis of the loss remains obscure. Although mitochondrial DNA (mtDNA)-deletion mutations have been shown to accumulate with age, for these aberrant genomes to be physiologically relevant, they must accumulate to high levels intracellularly and be present in a significant number of cells. We examined mtDNA-deletion mutations in vastus lateralis (VL) muscle of human subjects aged 49-93 years, using both histologic and polymerase-chain-reaction (PCR) analyses, to determine the physiological and genomic integrity of mitochondria in aging human muscle. The number of VL muscle fibers exhibiting mitochondrial electron-transport-system (ETS) abnormalities increased from an estimated 6% at age 49 years to 31% at age 92 years. We analyzed the mitochondrial genotype of 48 single ETS-abnormal, cytochrome c oxidase-negative/succinate dehydrogenase-hyperreactive (COX-/SDH++) fibers from normal aging human subjects and identified mtDNA-deletion mutations in all abnormal fibers. Deletion mutations were clonal within a fiber and concomitant to the COX-/SDH++ region. Quantitative PCR analysis of wild-type and deletion-containing mtDNA genomes within ETS-abnormal regions of single fibers demonstrated that these deletion mutations accumulate to detrimental levels (>90% of the total mtDNA).     

Review: can diet influence the selective advantage of mitochondrial DNA haplotypes?

This review explores the potential for changes in dietary macronutrients to differentially influence mitochondrial bioenergetics and thereby the frequency of mtDNA haplotypes in natural populations. Such dietary modification may be seasonal or result from biogeographic or demographic shifts. Mechanistically, mtDNA haplotypes may influence the activity of the electron transport system (ETS), retrograde signalling to the nuclear genome and affect epigenetic modifications. Thus, differential provisioning by macronutrients may lead to selection through changes in the levels of ATP production, modulation of metabolites (including AMP, reactive oxygen species (ROS) and the NAD⁺/NADH ratio) and potentially complex epigenetic effects. The exquisite complexity of dietary influence on haplotype frequency is further illustrated by the fact that macronutrients may differentially influence the selective advantage of specific mutations in different life-history stages. In Drosophila, complex I mutations may affect larval growth because dietary nutrients are fed through this complex in immaturity. In contrast, the majority of electrons are provided to complex III in adult flies. We conclude the review with a case study that considers specific interactions between diet and complex I of the ETS. Complex I is the first enzyme of the mitochondrial ETS and co-ordinates in the oxidation of NADH and transfer of electrons to ubiquinone. Although the supposition that mtDNA variants may be selected upon by dietary macronutrients could be intuitively consistent to some and counter intuitive to others, it must face a multitude of scientific hurdles before it can be recognized.     

Mitochondrial DNA replication and repair defects: Clinical phenotypes and therapeutic interventions

Mitochondria is a unique cellular organelle involved in multiple cellular processes and is critical for maintaining cellular homeostasis. This semi-autonomous organelle contains its circular genome – mtDNA (mitochondrial DNA), that undergoes continuous cycles of replication and repair to maintain the mitochondrial genome integrity. The majority of the mitochondrial genes, including mitochondrial replisome and repair genes, are nuclear-encoded. Although the repair machinery of mitochondria is quite efficient, the mitochondrial genome is highly susceptible to oxidative damage and other types of exogenous and endogenous agent-induced DNA damage, due to the absence of protective histones and their proximity to the main ROS production sites. Mutations in replication and repair genes of mitochondria can result in mtDNA depletion and deletions subsequently leading to mitochondrial genome instability. The combined action of mutations and deletions can result in compromised mitochondrial genome maintenance and lead to various mitochondrial disorders. Here, we review the mechanism of mitochondrial DNA replication and repair process, key proteins involved, and their altered function in mitochondrial disorders. The focus of this review will be on the key genes of mitochondrial DNA replication and repair machinery and the clinical phenotypes associated with mutations in these genes.