Pearson syndrome-like anemia induced by accumulation of mutant mtDNA and anemia with imbalanced white blood cell lineages induced by Drp1 deletion in a murine model.

Regulation of mitochondrial respiration and morphology is important for maintaining steady-state hematopoiesis, yet few studies have comparatively evaluated the effects of abnormal mitochondrial respiration and dynamics on blood-cell differentiation in isolation or combination. This study sought to explore these effects in mouse models with one or both of the following deficits: a large-scale deletion of mitochondrial DNA (ΔmtDNA), accumulated to varying extents, or knockout of the mitochondrial fission factor Drp1. Each deficit was found to independently provoke anemia but with clearly different manifestations. The former showed signs of aberrant respiration, analogous to Pearson syndrome, while the latter showed signs of abnormal mitochondrial dynamics and was associated with changes in the relative proportions of leukocyte lineages. Combining these deficits acted to amplify abnormal iron metabolism in erythropoiesis, exacerbating anemia in an additive manner. Our results indicate that mitochondrial respiration and dynamics play distinct roles in different sets of processes and cell lineages in hematopoietic differentiation.

Aberrant RNA processing contributes to the pathogenesis of mitochondrial diseases in trans-mitochondrial mouse model carrying mitochondrial tRNALeu(UUR) with a pathogenic A2748G mutation.

Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.

Mitochondrial DNA deletion-dependent podocyte injuries in Mito-miceΔ, a murine model of mitochondrial disease.

Focal segmental glomerulosclerosis (FSGS) is a major renal complication of human mitochondrial disease. However, its pathogenesis has not been fully explained. In this study, we focused on the glomerular injury of mito-miceΔ and investigated the pathogenesis of their renal involvement. We analyzed biochemical data and histology in mito-miceΔ. The proteinuria began to show in some mito-miceΔ with around 80% of mitochondrial DNA deletion, then proteinuria developed dependent with higher mitochondrial DNA deletion, more than 90% deletion. Mito-miceΔ with proteinuria histologically revealed FSGS. Immunohistochemistry demonstrated extensive distal tubular casts due to abundant glomerular proteinuria. Additionally, the loss of podocyte-related protein and podocyte's number were found. Therefore, the podocyte injuries and its depletion had a temporal relationship with the development of proteinuria. This study suggested mitochondrial DNA deletion-dependent podocyte injuries as the pathogenesis of renal involvement in mito-miceΔ. The podocytes are the main target of mitochondrial dysfunction originated from the accumulation of mitochondrial DNA abnormality in the kidney.

TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication.

Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.

Mitochondrial DNA mutations are involved in the acquisition of cisplatin resistance in human lung cancer A549 cells.

The efficacy of cisplatin (CDDP) has been demonstrated in the treatment of various cancers as monotherapy and combination therapy with immunotherapy. However, acquired CDDP resistance is a major obstacle to successful treatment. In the present study, the mechanisms underlying acquired CDDP resistance were examined using ACR20 cells, which are CDDP­resistant cells derived from A549 lung cancer cells. CDDP induces cytotoxicity by binding nuclear DNA and generating reactive oxygen species (ROS). Contrary to our expectation, ROS levels were elevated in ACR20 cells not treated with CDDP. Pretreatment with an ROS inhibitor enhanced the sensitivity of ACR20 cells to CDDP and prevented the activation of nuclear factor (NF)­ÐºB signaling and upregulation of inhibitor of apoptosis proteins (IAPs). Notably, evaluation of the mitochondrial oxygen consumption rate and mitochondrial superoxide levels revealed a deterioration of mitochondrial function in ACR20 cells. Mitochondrial DNA PCR­RFLP analysis revealed four mutations with varying percentage levels in ACR20 cells. In addition, in cytoplasmic hybrids with mitochondria from ACR20 cells, intrinsic ROS levels were elevated, expression of IAPs was increased, and complex I activity and sensitivity to CDDP were decreased. Analysis of three­dimensional structure data indicated that a mutation (ND2 F40L) may impact the proton translocation pathway, thereby affecting mitochondrial complex I activity. Together, these findings suggest that intrinsic ROS levels were elevated by mitochondrial DNA mutations, which decreased the sensitivity to CDDP via activation of NF­κB signaling and induction of IAP expression in ACR20 cells. These findings indicate that newly identified mutations in mitochondrial DNA may lead to acquired cisplatin resistance in cancer.

A high mutation load of m.14597A>G in MT-ND6 causes Leigh syndrome.

Leigh syndrome (LS) is an early-onset progressive neurodegenerative disorder associated with mitochondrial deficiency. m.14597A>G (p.Ile26Thr) in the MT-ND6 gene was reported to cause Leber's hereditary optic neuropathy (LHON) or dementia/dysarthria. In previous reports, less than 90% heteroplasmy was shown to result in adult-onset disease. Here, by whole mitochondrial sequencing, we identified m.14597A>G mutation of a patient with LS. PCR-RFLP analysis on fibroblasts from the patient revealed a high mutation load (> 90% heteroplasmy). We performed functional assays using cybrid cell models generated by fusing mtDNA-less rho0 HeLa cells with enucleated cells from patient fibroblasts carrying the m.14597A>G variant. Cybrid cell lines bearing the m.14597A>G variant exhibited severe effects on mitochondrial complex I activity. Additionally, impairment of cell proliferation, decreased ATP production and reduced oxygen consumption rate were observed in the cybrid cell lines bearing the m.14597A>G variant when the cells were metabolically stressed in medium containing galactose, indicating mitochondrial respiratory chain defects. These results suggest that a high mutation load of m.14597A>G leads to LS via a mitochondrial complex I defect, rather than LHON or dementia/dysarthria.

Attempts to understand the mechanisms of mitochondrial diseases: The reverse genetics of mouse models for mitochondrial disease.

BACKGROUND: Mitochondrial disease is a general term for a disease caused by a decline in mitochondrial function. The pathology of this disease is extremely diverse and complex, and the mechanism of its pathogenesis is still unknown. Using mouse models that develop the disease via the same processes as in humans is the easiest path to understanding the underlying mechanism. However, creating a mouse model is extremely difficult due to the lack of technologies that enable editing of mitochondrial DNA (mtDNA). SCOPE OF REVIEW: This paper outlines the complex pathogenesis of mitochondrial disease, and the difficulties in producing relevant mouse models. Then, the paper provides a detailed discussion on several mice created with mutations in mtDNA. The paper also introduces the pathology of mouse models with mutations including knockouts of nuclear genes that directly affect mitochondrial function. MAJOR CONCLUSIONS: Several mice with mtDNA mutations and those with nuclear DNA mutations have been established. Although these models help elucidate the pathological mechanism of mitochondrial disease, they lack sufficient diversity to enable a complete understanding. Considering the variety of factors that affect the cause and mechanism of mitochondrial disease, it is necessary to account for this background diversity in mouse models as well. GENERAL SIGNIFICANCE: Mouse models are indispensable for understanding the pathological mechanism of mitochondrial disease, as well as for searching new treatments. There is a need for the creation and examination of mouse models with more diverse mutations and altered nuclear backgrounds and breeding environments.

Neuronal degeneration and cognitive impairment can be prevented via the normalization of mitochondrial dynamics.

Neuronal cells possess a certain degree of plasticity to recover from cell damage. When the stress levels are higher than their plasticity capabilities, neuronal degeneration is triggered. However, the factors correlated to the plasticity capabilities need to be investigated. In this study, we generated a novel mouse model that able to express in an inducible manner a dominant-negative form of MFN2, a mitochondrial fusion factor. We then compared the phenotype of the mice continuously expressing the mutated MFN2 with that of the mice only transiently expressing it. Remarkably, the phenotypes of the group transiently expressing mutant MFN2 could be divided into 3 types: equivalent to what was observed in the continuous expression group, intermediate between the continuous expression group and the control group, and equivalent to the control group. In particular, in the continuous expression group, we observed remarkable hyperactivity and marked cognitive impairments, which were not seen, or were very mild in the transient expression group. These results indicate that abnormal mitochondrial dynamics lead to stress, triggering neuron degeneration; therefore, the neurodegeneration progression can be prevented via the normalization of the mitochondrial dynamics. Since the availability of mouse models suitable for the reproduction of both neurodegeneration and recovery at least partially is very limited, our mouse model can be a useful tool to investigate neuronal plasticity mechanisms and neurodegeneration.

Distal control of mitochondrial biogenesis and respiratory activity by extracellular lactate caused by large-scale deletion of mitochondrial DNA.

Lactate is highly produced under conditions of respiratory dysfunction such as anaerobic respiration and various types of mitochondrial diseases, and it was also known as an active molecule that plays various roles both within and between cells. High levels of extracellular lactate may lead to lactic acidosis, which has been related to pathology of the mitochondrial diseases with mutated mitochondrial DNA (mtDNA). In this study, to elucidate the poorly understood molecular roles of extracellular lactate in mitochondrial regulation, we analyzed mouse B82 cells and their cybrid cells carrying mutated mtDNA with a large-scale deletion (ΔmtDNA). Inhibition of lactate production by sodium dichloroacetate (DCA) treatment improved mitochondrial respiration in cells carrying ΔmtDNA through the activation of mitochondrial biogenesis. Chronic exposure to extracellular lactate (more than 3 days) repressed mitochondrial respiration in healthy cells via calcium and CaMK signaling, leading to a decrease in PGC1α-mediated mitochondrial biogenesis. These mitochondrial dysfunctions induced by the lactate treatment were repressed by pH buffering of the medium. These results suggest that lactate, produced in respiration-deficient cells, acts not only as an intracellular source of energy through the TCA cycle, but also as an extracellular messenger molecule regulating the respiratory activity of both cells carrying ΔmtDNA and the surrounding cells, which could cause whole-body repression of respiratory activity.

The accessory subunit of human DNA polymerase γ is required for mitochondrial DNA maintenance and is able to stabilize the catalytic subunit.

Human DNA polymerase γ (POLG) is a mitochondria-specific replicative DNA polymerase consisting of a single catalytic subunit, POLGα, and a dimeric accessory subunit, POLGß. To gain a deeper understanding of the role of POLGß, we knocked out this protein in cultured human cybrid cells and established numerous knockout clones. POLGß-knockout clones presented a clear phenotype of mitochondrial DNA loss, indicating that POLGß is necessary for mitochondrial DNA replication. Moreover, POLGß-knockout cells showed a severe decrease in POLGα levels and acute suppression of POLGß expression efficiently down-regulated POLGα levels. These results suggest that, in addition to its role as the processivity factor of POLG, POLGß acts as a POLGα stabilizer, an important role for POLGß in mitochondrial DNA maintenance.

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.

Concentration of mitochondrial DNA mutations by cytoplasmic transfer from platelets to cultured mouse cells.

Accumulation of mutations in mitochondrial DNA (mtDNA) is thought to be responsible for mitochondrial, and other, diseases and biological phenomena, such as diabetes, cancer, neurodegenerative diseases, and aging. Mouse models may elucidate the relationship between mutations in mtDNA and these abnormalities. However, because of the difficulty of mtDNA manipulation, generation of mouse models has not sufficiently progressed to enable such studies. To overcome this difficulty and to establish a source of diverse mtDNA mutations, we here generated cultured mouse cells containing mtDNA derived from an mtDNA mutator mouse that accumulates random mtDNA mutations with age. Mutation analysis of the obtained transmitochondrial cytoplasmic hybrid cells (cybrids) revealed that the cells harbored diverse mtDNA mutations occurring at a higher frequency than in mouse tissues, and exhibited severe respiration defects that would be lethal in tissues or organs. Abnormal respiratory complex formation and high stress on the mitochondrial protein quality control system appeared to be involved in these severe respiration defects. The mutation rates of the majority of highly accumulated mutations converged to either approximately 5%, 10%, or 40%, suggesting that these mutations are linked on the respective mtDNA molecules, and mtDNA in cybrid cells likely consisted of mtDNA molecules clonally expanded from the small population of introduced mtDNAs. Thus, the linked mutations in these cybrid cells cannot be evaluated individually. In addition, mtDNA mutations homologous to confirmed pathogenic mutations in human were rarely observed in our generated cybrids. However, the transmitochondrial cybrids constitute a useful tool for concentrating pathogenic mtDNA mutations and as a source of diverse mtDNA mutations to elucidate the relationship between mtDNA mutations and diseases.

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.

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.

Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29H(sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29H(sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice.

RLR-mediated antiviral innate immunity requires oxidative phosphorylation activity.

Mitochondria act as a platform for antiviral innate immunity, and the immune system depends on activation of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) signaling pathway via an adaptor molecule, mitochondrial antiviral signaling. We report that RLR-mediated antiviral innate immunity requires oxidative phosphorylation (OXPHOS) activity, a prominent physiologic function of mitochondria. Cells lacking mitochondrial DNA or mutant cells with respiratory defects exhibited severely impaired virus-induced induction of interferons and proinflammatory cytokines. Recovery of the OXPHOS activity in these mutants, however, re-established RLR-mediated signal transduction. Using in vivo approaches, we found that mice with OXPHOS defects were highly susceptible to viral infection and exhibited significant lung inflammation. Studies to elucidate the molecular mechanism of OXPHOS-coupled immune activity revealed that optic atrophy 1, a mediator of mitochondrial fusion, contributes to regulate the antiviral immune response. Our findings provide evidence for functional coordination between RLR-mediated antiviral innate immunity and the mitochondrial energy-generating system in mammals.

Mitochonic Acid 5 (MA-5) Facilitates ATP Synthase Oligomerization and Cell Survival in Various Mitochondrial Diseases.

Mitochondrial dysfunction increases oxidative stress and depletes ATP in a variety of disorders. Several antioxidant therapies and drugs affecting mitochondrial biogenesis are undergoing investigation, although not all of them have demonstrated favorable effects in the clinic. We recently reported a therapeutic mitochondrial drug mitochonic acid MA-5 (Tohoku J. Exp. Med., 2015). MA-5 increased ATP, rescued mitochondrial disease fibroblasts and prolonged the life span of the disease model "Mitomouse" (JASN, 2016). To investigate the potential of MA-5 on various mitochondrial diseases, we collected 25 cases of fibroblasts from various genetic mutations and cell protective effect of MA-5 and the ATP producing mechanism was examined. 24 out of the 25 patient fibroblasts (96%) were responded to MA-5. Under oxidative stress condition, the GDF-15 was increased and this increase was significantly abrogated by MA-5. The serum GDF-15 elevated in Mitomouse was likewise reduced by MA-5. MA-5 facilitates mitochondrial ATP production and reduces ROS independent of ETC by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin/Mic60. MA-5 reduced mitochondria fragmentation, restores crista shape and dynamics. MA-5 has potential as a drug for the treatment of various mitochondrial diseases. The diagnostic use of GDF-15 will be also useful in a forthcoming MA-5 clinical trial.

A novel mutation in TAZ causes mitochondrial respiratory chain disorder without cardiomyopathy.

Tafazzin, encoded by the TAZ gene, is a mitochondrial membrane-associated protein that remodels cardiolipin (CL), an important mitochondrial phospholipid. TAZ mutations are associated with Barth syndrome (BTHS). BTHS is an X-linked multisystemic disorder affecting usually male patients. Through sequence analysis of TAZ, we found one novel mutation c.39_60del p.(Pro14Alafs*19) by whole-exome sequencing and a reported missense mutation c.280C>T p.(Arg94Cys) by Sanger sequencing in two male patients (Pt1 and Pt2). Patient with c.280C>T mutation had dilated cardiomyopathy, while another patient with c.39_60del mutation had no feature of cardiomyopathy. A reported m.1555A>G homoplasmic variant was also identified in the patient having mutation c.39_60del by whole mitochondrial DNA sequencing method. This variant was not considered to be the main cause of mitochondrial dysfunction based on a cytoplasmic hybrid (cybrid) assay. Tafazzin expression was absent in both patient-derived fibroblast cells. Complementation of TAZ expression in fibroblasts from the patient with the novel mutation c.39_60del restored mitochondrial respiratory complex assembly. High-performance liquid chromatography-tandem mass spectrometry-based metabolic analysis revealed the decline of CL and the accumulation of monolysocardiolipin, indicating the loss of tafazzin activity. Owing to phenotypic variability, it is difficult to diagnose BTHS based on clinical features only. We conclude that genetic analysis should be performed to avoid underdiagnosis of this potentially life-threatening inborn error of metabolism.

FGF21 is a biomarker for mitochondrial translation and mtDNA maintenance disorders.

OBJECTIVE: To validate new mitochondrial myopathy serum biomarkers for diagnostic use. METHODS: We analyzed serum FGF21 (S-FGF21) and GDF15 from patients with (1) mitochondrial diseases and (2) nonmitochondrial disorders partially overlapping with mitochondrial disorder phenotypes. We (3) did a meta-analysis of S-FGF21 in mitochondrial disease and (4) analyzed S-Fgf21 and skeletal muscle Fgf21 expression in 6 mouse models with different muscle-manifesting mitochondrial dysfunctions. RESULTS: We report that S-FGF21 consistently increases in primary mitochondrial myopathy, especially in patients with mitochondrial translation defects or mitochondrial DNA (mtDNA) deletions (675 and 347 pg/mL, respectively; controls: 66 pg/mL, p < 0.0001 for both). This is corroborated in mice (mtDNA deletions 1,163 vs 379 pg/mL, p < 0.0001). However, patients and mice with structural respiratory chain subunit or assembly factor defects showed low induction (human 335 pg/mL, p < 0.05; mice 335 pg/mL, not significant). Overall specificities of FGF21 and GDF15 to find patients with mitochondrial myopathy were 89.3% vs 86.4%, and sensitivities 67.3% and 76.0%, respectively. However, GDF15 was increased also in a wide range of nonmitochondrial conditions. CONCLUSIONS: S-FGF21 is a specific biomarker for muscle-manifesting defects of mitochondrial translation, including mitochondrial transfer-RNA mutations and primary and secondary mtDNA deletions, the most common causes of mitochondrial disease. However, normal S-FGF21 does not exclude structural respiratory chain complex or assembly factor defects, important to acknowledge in diagnostics. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that elevated S-FGF21 accurately distinguishes patients with mitochondrial myopathies from patients with other conditions, and FGF21 and GDF15 mitochondrial myopathy from other myopathies.