Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.

Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.

mitoTALEN reduces the mutant mtDNA load in neurons.

Mutations within mtDNA frequently give rise to severe encephalopathies. Given that a majority of these mtDNA defects exist in a heteroplasmic state, we harnessed the precision of mitochondrial-targeted TALEN (mitoTALEN) to selectively eliminate mutant mtDNA within the CNS of a murine model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T). This targeted approach was accomplished by the use of AAV-PHP.eB and a neuron-specific synapsin promoter for effective neuronal delivery and expression of mitoTALEN. We found that most CNS regions were effectively transduced and showed a significant reduction in mutant mtDNA. This reduction was accompanied by an increase in mitochondrial tRNA alanine levels, which are drastically reduced by the m.5024C>T mutation. These results showed that mitochondrial-targeted gene editing can be effective in reducing CNS-mutant mtDNA in vivo, paving the way for clinical trials in patients with mitochondrial encephalopathies.

Cultured lymphocytes' mitochondrial genome integrity is not altered by cladribine.

Cladribine tablets are a treatment for multiple sclerosis with effects on lymphocytes, yet its mode of action has not been fully established. Here, we analyzed the effects of cladribine on mitochondrial DNA integrity in lymphocytes. We treated cultured human T-cell lines (CCRF-CEM and Jurkat) with varying concentrations of cladribine to mimic the slow cell depletion observed in treated patients. The CCRF-CEM was more susceptible to cladribine than Jurkat cells. In both cells, mitochondrial protein synthesis, mitochondrial DNA copy number, and mitochondrial cytochrome-c oxidase-I mRNA mutagenesis was not affected by cladribine, while caspase-3 cleavage was detected in Jurkat cells at 100 nM concentration. Cladribine treatment at concentrations up to 10 nM in CCRF-CEM and 100 nM in Jurkat cells did not induce significant increase in mitochondrial DNA mutations. Peripheral blood mononuclear cells from eight multiple sclerosis patients and four controls were cultured with or without an effective dose of cladribine (5 nM). However, we did not find any differences in mitochondrial DNA somatic mutations in lymphocyte subpopulations (CD4+, CD8+, and CD19+) between treated versus nontreated cells. The overall mutation rate was similar in patients and controls. When different lymphocyte subpopulations were compared, greater mitochondrial DNA mutation levels were detected in CD8+ (P = 0.014) and CD4+ (P = 0.038) as compared to CD19+ cells, these differences were independent of cladribine treatment. We conclude that T cells have more detectable mitochondrial DNA mutations than B cells, and cladribine has no detectable mutagenic effect on lymphocyte mitochondrial genome nor does it impair mitochondrial function in human T-cell lines.

High-throughput single-cell analysis reveals progressive mitochondrial DNA mosaicism throughout life.

Heteroplasmic mitochondrial DNA (mtDNA) mutations are a major cause of inherited disease and contribute to common late-onset human disorders. The late onset and clinical progression of mtDNA-associated disease is thought to be due to changing heteroplasmy levels, but it is not known how and when this occurs. Performing high-throughput single-cell genotyping in two mouse models of human mtDNA disease, we saw unanticipated cell-to-cell differences in mtDNA heteroplasmy levels that emerged prenatally and progressively increased throughout life. Proliferating spleen cells and nondividing brain cells had a similar single-cell heteroplasmy variance, implicating mtDNA or organelle turnover as the major force determining cell heteroplasmy levels. The two different mtDNA mutations segregated at different rates with no evidence of selection, consistent with different rates of random genetic drift in vivo, leading to the accumulation of cells with a very high mutation burden at different rates. This provides an explanation for differences in severity seen in human diseases caused by similar mtDNA mutations.

Cell lineage-specific mitochondrial resilience during mammalian organogenesis.

Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.

Large dataset of octocoral mitochondrial genomes provides new insights into mt-mutS evolution and function.

All studied octocoral mitochondrial genomes (mt-genomes) contain a homologue of the Escherichia coli mutS gene, a member of a gene family encoding proteins involved in DNA mismatch repair, other types of DNA repair, meiotic recombination, and other functions. Although mutS homologues are found in all domains of life, as well as viruses, octocoral mt-mutS is the only such gene found in an organellar genome. While the function of mtMutS is not known, its domain architecture, conserved sequence, and presence of several characteristic residues suggest its involvement in mitochondrial DNA repair. This inference is supported by exceptionally low rates of mt-sequence evolution observed in octocorals. Previous studies of mt-mutS have been limited by the small number of octocoral mt-genomes available. We utilized sequence-capture data from the recent Quattrini et al. 2020 study [Nature Ecology & Evolution 4:1531-1538] to assemble complete mt-genomes for 94 species of octocorals. Combined with sequences publicly available in GenBank, this resulted in a dataset of 184 complete mt-genomes, which we used to re-analyze the conservation and evolution of mt-mutS. In our analysis, we discovered the first case of mt-mutS loss among octocorals in one of the two Pseudoanthomastus spp. assembled from Quattrini et al. data. This species displayed accelerated rate and changed patterns of nucleotide substitutions in mt-genome, which we argue provide additional evidence for the role of mtMutS in DNA repair. In addition, we found accelerated mt-sequence evolution in the presence of mt-mutS in several octocoral lineages. This accelerated evolution did not appear to be the result of relaxed selection pressure and did not entail changes in patterns of nucleotide substitutions. Overall, our results support previously reported patterns of conservation in mt-mutS and suggest that mtMutS is involved in DNA repair in octocoral mitochondria. They also indicate that the presence of mt-mutS contributes to, but does not fully explain, the low rates of sequence evolution in octocorals.

Mitochondrial DNA heteroplasmy is modulated during oocyte development propagating mutation transmission.

Heteroplasmic mitochondrial DNA (mtDNA) mutations are a common cause of inherited disease, but a few recurrent mutations account for the vast majority of new families. The reasons for this are not known. We studied heteroplasmic mice transmitting m.5024C>T corresponding to a human pathogenic mutation. Analyzing 1167 mother-pup pairs, we show that m.5024C>T is preferentially transmitted from low to higher levels but does not reach homoplasmy. Single-cell analysis of the developing mouse oocytes showed the preferential increase in mutant over wild-type mtDNA in the absence of cell division. A similar inheritance pattern is seen in human pedigrees transmitting several pathogenic mtDNA mutations. In m.5024C>T mice, this can be explained by the preferential propagation of mtDNA during oocyte maturation, counterbalanced by purifying selection against high heteroplasmy levels. This could explain how a disadvantageous mutation in a carrier increases to levels that cause disease but fails to fixate, causing multigenerational heteroplasmic mtDNA disorders.

Extreme heterogeneity of human mitochondrial DNA from organelles to populations.

Contrary to the long-held view that most humans harbour only identical mitochondrial genomes, deep resequencing has uncovered unanticipated extreme genetic variation within mitochondrial DNA (mtDNA). Most, if not all, humans contain multiple mtDNA genotypes (heteroplasmy); specific patterns of variants accumulate in different tissues, including cancers, over time; and some variants are preferentially passed down or suppressed in the maternal germ line. These findings cast light on the origin and spread of mtDNA mutations at multiple scales, from the organelle to the human population, and challenge the conventional view that high percentages of a mutation are required before a new variant has functional consequences.

Accurate mapping of mitochondrial DNA deletions and duplications using deep sequencing.

Deletions and duplications in mitochondrial DNA (mtDNA) cause mitochondrial disease and accumulate in conditions such as cancer and age-related disorders, but validated high-throughput methodology that can readily detect and discriminate between these two types of events is lacking. Here we establish a computational method, MitoSAlt, for accurate identification, quantification and visualization of mtDNA deletions and duplications from genomic sequencing data. Our method was tested on simulated sequencing reads and human patient samples with single deletions and duplications to verify its accuracy. Application to mouse models of mtDNA maintenance disease demonstrated the ability to detect deletions and duplications even at low levels of heteroplasmy.

Characterization of E-cigarette coil temperature and toxic metal analysis by infrared temperature sensing and scanning electron microscopy - energy-dispersive X-ray.

INTRODUCTION: Electronic cigarettes (e-cigarettes) have rapidly evolved since their introduction to the U.S. market. The rebuildable atomizer (RBA) offers user-driven modification to the heating element (coil) and wicking systems. Different coil materials can be chosen based on user needs and preferences. However, the heating element of an e-cigarette is believed to be one-source for toxic metal exposure. METHODS: E-cigarette coils from Kanthal and nichrome wires were constructed in a contact and non-contact configuration and heated at four voltages. The maximum temperatures of the coils were measured by infrared temperature sensing when dry and when saturated with 100% vegetable glycerin or 100% propylene glycol. The metal composition of each coil was analyzed with Scanning Electron Microscopy-Energy-Dispersive X-Ray (SEM-EDX) when new, and subsequently after 1, 50, and 150 heat cycles when dry. RESULTS: The coils reached temperatures above 1000 °C when dry, but were below 300 °C in both liquid-saturated mediums. Metal analysis showed a decrease of 9-19% chromium and 39-58% iron in Kanthal wire and a decrease of 12-14% iron and 39-43% nickel in nichrome wire after 150 heat cycles. Significant metal loss was observed after one heat cycle for both coil alloys and configurations. CONCLUSIONS: The loss of metals from these heat cycles further suggests that the metals from the coils are potentially entering the aerosol of the e-cigarette, which can be inhaled by the user.

Mitochondrial stress response triggered by defects in protein synthesis quality control.

Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.

Author Correction: MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation.

In the version of this article originally published, there was an error in Fig. 1a. The m.5024C>T mutation, shown as a green T, was displaced by one base. The error has been corrected in the print, HTML and PDF versions of this article.

Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin.

MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation.

Mutations in the mitochondrial DNA (mtDNA) are responsible for several metabolic disorders, commonly involving muscle and the central nervous system1. Because of the critical role of mtDNA in oxidative phosphorylation, the majority of pathogenic mtDNA mutations are heteroplasmic, co-existing with wild-type molecules1. Using a mouse model with a heteroplasmic mtDNA mutation2, we tested whether mitochondrial-targeted TALENs (mitoTALENs)3,4 could reduce the mutant mtDNA load in muscle and heart. AAV9-mitoTALEN was administered via intramuscular, intravenous, and intraperitoneal injections. Muscle and heart were efficiently transduced and showed a robust reduction in mutant mtDNA, which was stable over time. The molecular defect, namely a decrease in transfer RNAAla levels, was restored by the treatment. These results showed that mitoTALENs, when expressed in affected tissues, could revert disease-related phenotypes in mice.

Delivery of mtZFNs into Early Mouse Embryos.

Mitochondrial diseases often result from mutations in the mitochondrial genome (mtDNA). In most cases, mutant mtDNA coexists with wild-type mtDNA, resulting in heteroplasmy. One potential future approach to treat heteroplasmic mtDNA diseases is the specific elimination of pathogenic mtDNA mutations, lowering the level of mutant mtDNA below pathogenic thresholds. Mitochondrially targeted zinc-finger nucleases (mtZFNs) have been demonstrated to specifically target and introduce double-strand breaks in mutant mtDNA, facilitating substantial shifts in heteroplasmy. One application of mtZFN technology, in the context of heteroplasmic mtDNA disease, is delivery into the heteroplasmic oocyte or early embryo to eliminate mutant mtDNA, preventing transmission of mitochondrial diseases through the germline. Here we describe a protocol for efficient production of mtZFN mRNA in vitro, and delivery of these into 0.5 dpc mouse embryos to elicit shifts of mtDNA heteroplasmy.

Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice.

Mitochondrial DNA (mtDNA) mutations become more prevalent with age and are postulated to contribute to the ageing process. Point mutations of mtDNA have been suggested to originate from two main sources, i.e. replicative errors and oxidative damage, but the contribution of each of these processes is much discussed. To elucidate the origin of mtDNA mutations, we measured point mutation load in mice with deficient mitochondrial base-excision repair (BER) caused by knockout alleles preventing mitochondrial import of the DNA repair glycosylases OGG1 and MUTYH (Ogg1 dMTS, Mutyh dMTS). Surprisingly, we detected no increase in the mtDNA mutation load in old Ogg1 dMTS mice. As DNA repair is especially important in the germ line, we bred the BER deficient mice for five consecutive generations but found no increase in the mtDNA mutation load in these maternal lineages. To increase reactive oxygen species (ROS) levels and oxidative damage, we bred the Ogg1 dMTS mice with tissue specific Sod2 knockout mice. Although increased superoxide levels caused a plethora of changes in mitochondrial function, we did not detect any changes in the mutation load of mtDNA or mtRNA. Our results show that the importance of oxidative damage as a contributor of mtDNA mutations should be re-evaluated.

A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency.

Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age-related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh-frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase-deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels - this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX-deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell-by-cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria.

Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.

A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease.

Mutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials.

Hierarchical RNA Processing Is Required for Mitochondrial Ribosome Assembly.

The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-seq enabled us to identify that in vivo 5' tRNA cleavage precedes 3' tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome.