Emi Ogasawara

Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNALys gene

Significance We generated transmitochondrial mito-mice-tRNALys7731 as models for precise examination of the pathogenesis and transmission profiles of mtDNA mutations in the tRNALys genes and have obtained important information regarding primary prevention of the diseases caused by the mtDNA mutations. Although nuclear transplantation from oocytes of affected mothers into enucleated oocytes of unrelated women has been suggested, the methodology carries the technical risk of inducing nuclear abnormalities and prompts ethical concerns regarding the production of three-parent babies with normal mtDNA from unrelated oocyte donors. The current study suggests that the selection of oocytes with high proportions of normal mtDNA from affected mothers can be used to avoid these issues and therefore provides insights into mitochondrial genetics and medicine. We generated transmitochondrial mice (mito-mice) that carry a mutation in the tRNALys 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 tRNALys and tRNALeu(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 tRNALys or tRNALeu(UUR) genes in mtDNA of P29 cells, one had a potentially pathogenic mutation (G7731A) in the tRNALys 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-tRNALys7731.” Maternal inheritance and random segregation of G7731A mtDNA occurred in subsequent generations. Mito-mice-tRNALys7731 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 tRNALys 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 tRNALys-based mitochondrial diseases may be effective as a primary prevention for obtaining unaffected children.

Lactic acidemia in the pathogenesis of mice carrying mitochondrial DNA with a deletion

Lactic acidemia is one manifestation of the mitochondrial diseases caused by pathogenic mutant mitochondrial DNA (mtDNA). However, little is known about its chronic effects in the progression of mitochondrial disease phenotypes. To obtain experimental evidence on this point, we used trans-mitochondrial model mice (mito-mice) heteroplasmic for wild-type and deleted mtDNA (DeltamtDNA). Mito-mice carrying predominantly DeltamtDNA showed mitochondrial respiration defects and the resultant disease phenotypes, including lactic acidemia; they also showed a decrease in mitochondrial biogenesis regulated by the peroxisome proliferative activated receptor gamma, coactivator 1 alpha (PGC1alpha)-mediated pathway, such as the expression of mitochondrial transcription factor A and mtDNA-encoded gene products and the control of mtDNA content. When the accelerated lactate production of these mito-mice was pharmacologically inhibited by sodium dichloroacetate (DCA), the decrease in mitochondrial biogenesis improved, thus leading to the relaxation of mitochondrial respiration defects and extension of life span. These results showed that chronic overproduction of lactate caused by metabolic adaptation in mitochondrial diseases further deconditioned mitochondrial function. Mitochondrial respiration defects in mitochondrial diseases are therefore induced not only directly by the presence of mutant mtDNA, but also by the chronic lactic acidemia. Our in vivo study also suggested that inhibition of chronic lactic acidemia is a potential strategy for treating some mitochondrial diseases.

A Novel MitoNEET Ligand, TT01001, Improves Diabetes and Ameliorates Mitochondrial Function in db/db Mice

The mitochondrial outer membrane protein mitoNEET is a binding protein of the insulin sensitizer pioglitazone (5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione) and is considered a novel target for the treatment of type II diabetes. Several small-molecule compounds have been identified as mitoNEET ligands using structure-based design or virtual docking studies. However, there are no reports about their therapeutic potential in animal models. Recently, we synthesized a novel small molecule, TT01001 [ethyl-4-(3-(3,5-dichlorophenyl)thioureido)piperidine-1-carboxylate], designed on the basis of pioglitazone structure. In this study, we assessed the pharmacological properties of TT01001 in both in vitro and in vivo studies. We found that TT01001 bound to mitoNEET without peroxisome proliferator-activated receptor-γ activation effect. In type II diabetes model db/db mice, TT01001 improved hyperglycemia, hyperlipidemia, and glucose intolerance, and its efficacy was equivalent to that of pioglitazone, without the pioglitazone-associated weight gain. Mitochondrial complex II + III activity of the skeletal muscle was significantly increased in db/db mice. We found that TT01001 significantly suppressed the elevated activity of the complex II + III. These results suggest that TT01001 improved type II diabetes without causing weight gain and ameliorated mitochondrial function of db/db mice. This is the first study that demonstrates the effects of a mitoNEET ligand on glucose metabolism and mitochondrial function in an animal disease model. These findings support targeting mitoNEET as a potential therapeutic approach for the treatment of type II diabetes.

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 (∆mtDNA), but there is no direct experimental evidence for this. To determine whether ∆mtDNA has the pathologic potential to induce multiple mitochondrial disease phenotypes, we used trans-mitochondrial mice with a heteroplasmic state of wild-type mtDNA and ∆mtDNA (mito-mice∆). Late-stage embryos carrying ≥50% ∆mtDNA 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 ∆mtDNA load in the affected tissues, peripheral blood and liver, and they recovered from PS-like phenotypes. The proportion of ∆mtDNA in various tissues of the surviving mito-mice∆ 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 ∆mtDNA 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.

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.