Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Acquired Expression of Mutant Mitofusin 2 Causes Progressive Neurodegeneration and Abnormal Behavior.

Neurons have high plasticity in developmental and juvenile stages that decreases in adulthood. Mitochondrial dynamics are highly important in neurons to maintain normal function. To compare dependency on mitochondrial dynamics in juvenile and adult stages, we generated a mouse model capable of selective timing of the expression of a mutant of the mitochondrial fusion factor Mitofusin 2 (MFN2). Mutant expression in the juvenile stage had lethal effects. Contrastingly, abnormalities did not manifest until 150 d after mutant expression during adulthood. After this silent 150 d period, progressive neurodegeneration, abnormal behaviors, and learning and memory deficits similar to those seen in human neurodegenerative diseases were observed. This indicates that abnormal neuronal mitochondrial dynamics seriously affect survival during early life stages and can also significantly damage brain function after maturation. Our findings highlight the need to consider the timing of disease onset in mimicking human neurodegenerative diseases.SIGNIFICANCE STATEMENT To compare the dependency on mitochondrial dynamics in neurons in juvenile and adult stages, we generated a mouse model expressing a mutant of the mitochondrial fusion factor MFN2 in an arbitrary timing. Juvenile expression of the mutant showed acute and severe phenotypes and had lethal effects; however, post-adult expression induced delayed but progressive phenotypes resembling those found in human neurodegenerative diseases. Our results indicate that abnormal neuronal mitochondrial dynamics seriously affect survival during early life stages and can also significantly damage brain function after maturation. This strongly suggests that the timing of expression should be considered when establishing an animal model that closely resembles human neurodegenerative diseases.

Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNA(Lys) gene.

We generated transmitochondrial mice (mito-mice) that carry a mutation in the tRNA(Lys) gene encoded by mtDNA for use in studies of its pathogenesis and transmission profiles. Because patients with mitochondrial diseases frequently carry mutations in the mitochondrial tRNA(Lys) and tRNA(Leu(UUR)) genes, we focused our efforts on identifying somatic mutations of these genes in mouse lung carcinoma P29 cells. Of the 43 clones of PCR products including the tRNA(Lys) or tRNA(Leu(UUR)) genes in mtDNA of P29 cells, one had a potentially pathogenic mutation (G7731A) in the tRNA(Lys) gene. P29 subclones with predominant amounts of G7731A mtDNA expressed respiration defects, thus suggesting the pathogenicity of this mutation. We then transferred G7731A mtDNA into mouse ES cells and obtained F0 chimeric mice. Mating these F0 mice with C57BL/6J (B6) male mice resulted in the generation of F1 mice with G7731A mtDNA, named "mito-mice-tRNA(Lys7731)." Maternal inheritance and random segregation of G7731A mtDNA occurred in subsequent generations. Mito-mice-tRNA(Lys7731) with high proportions of G7731A mtDNA exclusively expressed respiration defects and disease-related phenotypes and therefore are potential models for mitochondrial diseases due to mutations in the mitochondrial tRNA(Lys) gene. Moreover, the proportion of mutated mtDNA varied markedly among the pups born to each dam, suggesting that selecting oocytes with high proportions of normal mtDNA from affected mothers with tRNA(Lys)-based mitochondrial diseases may be effective as a primary prevention for obtaining unaffected children.

A somatic T15091C mutation in the Cytb gene of mouse mitochondrial DNA dominantly induces respiration defects.

Our previous studies provided evidence that mammalian mitochondrial DNA (mtDNA) mutations that cause mitochondrial respiration defects behave in a recessive manner, because the induction of respiration defects could be prevented with the help of a small proportion (10%-20%) of mtDNA without the mutations. However, subsequent studies found the induction of respiration defects by the accelerated accumulation of a small proportion of mtDNA with various somatic mutations, indicating the presence of mtDNA mutations that behave in a dominant manner. Here, to provide the evidence for the presence of dominant mutations in mtDNA, we used mouse lung carcinoma P29 cells and examined whether some mtDNA molecules possess somatic mutations that dominantly induce respiration defects. Cloning and sequence analysis of 40-48 mtDNA molecules from P29 cells was carried out to screen for somatic mutations in protein-coding genes, because mutations in these genes could dominantly regulate respiration defects by formation of abnormal polypeptides. We found 108 missense mutations existing in one or more of 40-48 mtDNA molecules. Of these missense mutations, a T15091C mutation in the Cytb gene was expected to be pathogenic due to the presence of its orthologous mutation in mtDNA from a patient with cardiomyopathy. After isolation of many subclones from parental P29 cells, we obtained subclones with various proportions of T15091C mtDNA, and showed that the respiration defects were induced in a subclone with only 49% T15091C mtDNA. Because the induction of respiration defects could not be prevented with the help of the remaining 51% mtDNA without the T15091C mutation, the results indicate that the T15091C mutation in mtDNA dominantly induced the respiration defects.

G7731A mutation in mouse mitochondrial tRNALys regulates late-onset disorders in transmitochondrial mice.

We previously generated mito-mice-tRNA(Lys7731) as a model for primary prevention of mitochondrial diseases. These mice harbour a G7731A mtDNA mutation in the tRNA(Lys) gene, but express only muscle weakness and short body length by four months. Here, we examined the effects of their aging on metabolic and histologic features. Unlike young mito-mice-tRNA(Lys7731), aged mito-mice-tRNA(Lys7731) developed muscle atrophy, renal failures, and various metabolic abnormalities, such as lactic acidosis and anemia, characteristic of patients with mitochondrial diseases. These observations provide convincing evidence that the respiration defects induced by high G7731A mtDNA levels cause these late-onset disorders that are relevant to mitochondrial diseases.

Transmitochondrial mito-miceΔ and mtDNA mutator mice, but not aged mice, share the same spectrum of musculoskeletal disorders.

The spectra of phenotypes associated with aging and mitochondrial diseases sometimes appear to overlap with each other. We used aged mice and a mouse model of mitochondrial diseases (transmitochondrial mito-miceΔ with deleted mtDNA) to study whether premature aging phenotypes observed in mtDNA mutator mice are associated with aging or mitochondrial diseases. Here, we provide convincing evidence that all the mice examined had musculoskeletal disorders of osteoporosis and muscle atrophy, which correspond to phenotypes prevalently observed in the elderly. However, precise investigation of musculoskeletal disorders revealed that the spectra of osteoporosis and muscle atrophy phenotypes in mtDNA mutator mice were very close to those in mito-miceΔ, but different from those of aged mice. Therefore, mtDNA mutator mice and mito-miceΔ, but not aged mice, share the spectra of musculoskeletal disorders.

Mouse somatic mutation orthologous to MELAS A3302G mutation in the mitochondrial tRNA(Leu(UUR)) gene confers respiration defects.

We searched for mtDNA harboring somatic mutations in mouse B82 cells, and found an A2748G mutation orthologous to the A3302G mutation in tRNA(Leu(UUR)) gene reported in a patient with MELAS, the most prevalent mitochondrial disease. We isolated subclones of B82 cells until we obtained one subclone harboring >95% A2748G mtDNA. Cytoplasmic transfer of A2748G mtDNA resulted in cotransfer of A2748G mtDNA and respiration defects into mouse ES cells. Thus, A2748G mtDNA is responsible for respiration defects, and the ES cells harboring A2748G mtDNA may be useful for generation of transmitochondrial mice harboring A2748G mtDNA as potential disease models of MELAS.

Mosaic dysfunction of mitophagy in mitochondrial muscle disease.

Mitophagy is a quality control mechanism that eliminates damaged mitochondria, yet its significance in mammalian pathophysiology and aging has remained unclear. Here, we report that mitophagy contributes to mitochondrial dysfunction in skeletal muscle of aged mice and human patients. The early disease stage is characterized by muscle fibers with central nuclei, with enhanced mitophagy around these nuclei. However, progressive mitochondrial dysfunction halts mitophagy and disrupts lysosomal homeostasis. Interestingly, activated or halted mitophagy occur in a mosaic manner even in adjacent muscle fibers, indicating cell-autonomous regulation. Rapamycin restores mitochondrial turnover, indicating mTOR-dependence of mitochondrial recycling in advanced disease stage. Our evidence suggests that (1) mitophagy is a hallmark of age-related mitochondrial pathology in mammalian muscle, (2) mosaic halting of mitophagy is a mechanism explaining mosaic respiratory chain deficiency and accumulation of pathogenic mtDNA variants in adult-onset mitochondrial diseases and normal aging, and (3) augmenting mitophagy is a promising therapeutic approach for muscle mitochondrial dysfunction.

Disruption of the mouse Shmt2 gene confers embryonic anaemia via foetal liver-specific metabolomic disorders.

In a previous study, we proposed that age-related mitochondrial respiration defects observed in elderly subjects are partially due to age-associated downregulation of nuclear-encoded genes, including serine hydroxymethyltransferase 2 (SHMT2), which is involved in mitochondrial one-carbon (1C) metabolism. This assertion is supported by evidence that the disruption of mouse Shmt2 induces mitochondrial respiration defects in mouse embryonic fibroblasts generated from Shmt2-knockout E13.5 embryos experiencing anaemia and lethality. Here, we elucidated the potential mechanisms by which the disruption of this gene induces mitochondrial respiration defects and embryonic anaemia using Shmt2-knockout E13.5 embryos. The livers but not the brains of Shmt2-knockout E13.5 embryos presented mitochondrial respiration defects and growth retardation. Metabolomic profiling revealed that Shmt2 deficiency induced foetal liver-specific downregulation of 1C-metabolic pathways that create taurine and nucleotides required for mitochondrial respiratory function and cell division, respectively, resulting in the manifestation of mitochondrial respiration defects and growth retardation. Given that foetal livers function to produce erythroblasts in mouse embryos, growth retardation in foetal livers directly induced depletion of erythroblasts. By contrast, mitochondrial respiration defects in foetal livers also induced depletion of erythroblasts as a consequence of the inhibition of erythroblast differentiation, resulting in the manifestation of anaemia in Shmt2-knockout E13.5 embryos.

Mito-mice∆ and mitochondrial DNA mutator mice as models of human osteoporosis caused not by aging but by hyperparathyroidism.

Mitochondrial DNA (mtDNA) mutator mice showing accelerated accumulation of mtDNA with somatic mutations are potentially useful models of human aging, whereas mito-miceΔ showing accelerated accumulation of mtDNA with a deletion mutation (ΔmtDNA) are potentially useful models of mitochondrial diseases but not human aging, even though both models express an age-associated decrease in mitochondrial respiration. Because osteoporosis is the only premature aging phenotype observed in mtDNA mutator mice with the C57BL/6J nuclear genetic background, our previous study precisely examined its expression spectra and reported that both mtDNA mutator mice and mito-miceΔ, but not aged mice, developed decreased cortical bone thickness. Moreover, decreased cortical bone thickness is usually not seen in aged humans but is commonly seen in the patients with hyperparathyroidism caused by oversecretion of parathyroid hormone (PTH). In the present study, we showed higher concentrations of blood PTH in mtDNA mutator mice and mito-miceΔ than in aged mice. We also found that both models developed decreased mitochondrial respiration in the duodenum or renal tubules, which would lead to hypocalcemia, oversecretion of PTH, and ultimately osteoporosis. Thus, mtDNA mutator mice and mito-miceΔ may be useful models of human osteoporosis caused not by aging but by hyperparathyroidism.

Mice deficient in the Shmt2 gene have mitochondrial respiration defects and are embryonic lethal.

Accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for human aging and age-associated mitochondrial respiration defects. However, our previous findings suggested an alternative hypothesis of human aging-that epigenetic changes but not mutations regulate age-associated mitochondrial respiration defects, and that epigenetic downregulation of nuclear-coded genes responsible for mitochondrial translation [e.g., glycine C-acetyltransferase (GCAT), serine hydroxymethyltransferase 2 (SHMT2)] is related to age-associated respiration defects. To examine our hypothesis, here we generated mice deficient in Gcat or Shmt2 and investigated whether they have respiration defects and premature aging phenotypes. Gcat-deficient mice showed no macroscopic abnormalities including premature aging phenotypes for up to 9 months after birth. In contrast, Shmt2-deficient mice showed embryonic lethality after 13.5 days post coitum (dpc), and fibroblasts obtained from 12.5-dpc Shmt2-deficient embryos had respiration defects and retardation of cell growth. Because Shmt2 substantially controls production of N-formylmethionine-tRNA (fMet-tRNA) in mitochondria, its suppression would reduce mitochondrial translation, resulting in expression of the respiration defects in fibroblasts from Shmt2-deficient embryos. These findings support our hypothesis that age-associated respiration defects in fibroblasts of elderly humans are caused not by mtDNA mutations but by epigenetic regulation of nuclear genes including SHMT2.

Epigenetic regulation of the nuclear-coded GCAT and SHMT2 genes confers human age-associated mitochondrial respiration defects.

Age-associated accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been proposed to be responsible for the age-associated mitochondrial respiration defects found in elderly human subjects. We carried out reprogramming of human fibroblast lines derived from elderly subjects by generating their induced pluripotent stem cells (iPSCs), and examined another possibility, namely that these aging phenotypes are controlled not by mutations but by epigenetic regulation. Here, we show that reprogramming of elderly fibroblasts restores age-associated mitochondrial respiration defects, indicating that these aging phenotypes are reversible and are similar to differentiation phenotypes in that both are controlled by epigenetic regulation, not by mutations in either the nuclear or the mitochondrial genome. Microarray screening revealed that epigenetic downregulation of the nuclear-coded GCAT gene, which is involved in glycine production in mitochondria, is partly responsible for these aging phenotypes. Treatment of elderly fibroblasts with glycine effectively prevented the expression of these aging phenotypes.

Mitochondrial DNA with a large-scale deletion causes two distinct mitochondrial disease phenotypes in mice.

Studies in patients have suggested that the clinical phenotypes of some mitochondrial diseases might transit from one disease to another (e.g., Pearson syndrome [PS] to Kearns-Sayre syndrome) in single individuals carrying mitochondrial (mt) DNA with a common deletion (ΔmtDNA), but there is no direct experimental evidence for this. To determine whether ΔmtDNA has the pathologic potential to induce multiple mitochondrial disease phenotypes, we used trans-mitochondrial mice with a heteroplasmic state of wild-type mtDNA and ΔmtDNA (mito-miceΔ). Late-stage embryos carrying ≥50% ΔmtDNA showed abnormal hematopoiesis and iron metabolism in livers that were partly similar to PS (PS-like phenotypes), although they did not express sideroblastic anemia that is a typical symptom of PS. More than half of the neonates with PS-like phenotypes died by 1 month after birth, whereas the rest showed a decrease of ΔmtDNA load in the affected tissues, peripheral blood and liver, and they recovered from PS-like phenotypes. The proportion of ΔmtDNA in various tissues of the surviving mito-miceΔ increased with time, and Kearns-Sayre syndrome-like phenotypes were expressed when the proportion of mtDNA in various tissues reached >70-80%. Our model mouse study clearly showed that a single ΔmtDNA was responsible for at least two distinct disease phenotypes at different ages and suggested that the level and dynamics of mtDNA load in affected tissues would be important for the onset and transition of mitochondrial disease phenotypes in mice.

Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0)) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.