Somatostatin Receptor Type 2 and Thyroid-Stimulating Hormone Receptor Expression in Oncocytic Thyroid Neoplasms: Implications for Prognosis and Treatment.

Somatostatin receptor type 2 (SSTR2) and thyroid-stimulating hormone receptor (TSHR) display variable expression in primary thyroid tumors and have been implicated as theranostic targets. This study was designed to explore the differential expression of SSTR2 and TSHR in oncocytic (Hurthle cell) carcinoma (OC) vs oncocytic adenoma (OA). We performed a retrospective review for oncocytic neoplasms treated at our institution from 2012 to 2019. Formalin-fixed paraffin-embedded tissue blocks were used for tissue microarray construction. Tissue microarray blocks were cut into 5-µm sections and stained with anti-SSTR2 and anti-TSHR antibodies. Immunostains were analyzed by 3 independent pathologists. χ2 and logistic regression analysis were used to analyze clinical and pathologic variables. Sixty-seven specimens were analyzed with 15 OA and 52 OC. The mean age was 57 years, 61.2% were women, and 70% were White. SSTR2 positivity was noted in 2 OA (13%) and 15 OC (28%; 10 primary, 4 recurrent, and 1 metastatic) (P = .22). TSHR positivity was noted in 11 OA (73%) and 32 OC (62%; 31 primary and 1 metastatic) (P = .40). Those who presented with or developed clinical recurrence/metastasis were more likely to be SSTR2-positive (50% vs 21%; P = .04) and TSHR-negative (64.3% vs 28.9%; P = .02) than primary OC patients. Widely invasive OC was more likely to be SSTR2-positive compared to all other OC subtypes (minimally invasive and angioinvasive) (P = .003). For all patients with OC, TSHR positivity was inversely correlated with SSTR2 positivity (odds ratio, 0.12; CI, 0.03-0.43; P = .006). This relationship was not seen in the patients with OA (odds ratio, 0.30; CI, 0.01-9.14; P = .440). Our results show that recurrent/metastatic OC was more likely to be SSTR2-positive and TSHR-negative than primary OC. Patients with OC displayed a significant inverse relationship between SSTR2 and TSHR expression that was not seen in patients with OA. This may be a key relationship that can be used to prognosticate and treat OCs.

Living high - training low model applied to C57BL/6J mice: Effects on physiological parameters related to aerobic fitness and acid-base balance.

There is a scarcity of data regarding the acclimation to high altitude (hypoxic environment) accompanied by training at low altitude (normoxic conditions), the so-called "living high-training low" (LHTL) model in rodents. We aimed to investigate the effects of aerobic training on C57BL/6J mice living in normoxic (NOR) or hypoxic (HYP) environments on several parameters, including critical velocity (CV), a parameter regarded as a measure of aerobic capacity, on monocarboxylate transporters (MCTs) in muscles and hypothalamus, as well as on hematological parameters and body temperature. In each environment, mice were divided into non-trained (N) and trained (T). Forty rodents were distributed into the following experimental groups (N-NOR; T-NOR; N-HYP and T-HYP). HYP groups were in a normobaric tent where oxygen-depleted air was pumped from a hypoxia generator set an inspired oxygen fraction [FiO2] of 14.5 %. The HYP-groups were kept (18 h per day) in a normobaric tent for consecutive 8-weeks. Training sessions were conducted in normoxic conditions ([FiO2] = 19.5 %), 5 times per week (40 min per session) at intensity equivalent to 80 % of CV. In summary, eight weeks of LHTL did not promote a greater improvement in the CV, protein expression of MCTs in different tissues when compared to the application of training alone. The LHTL model increased red blood cells count, but reduced hemoglobin per erythrocyte was found in mice exposed to LHTL. Although the LHTL did not have a major effect on thermographic records, exercise-induced hyperthermia (in the head) was attenuated in HYP groups when compared to NOR groups.

SIRT3 deficiency decreases oxidative metabolism capacity but increases lifespan in male mice under caloric restriction.

Mitochondrial NAD+ -dependent protein deacetylase Sirtuin3 (SIRT3) has been proposed to mediate calorie restriction (CR)-dependent metabolic regulation and lifespan extension. Here, we investigated the role of SIRT3 in CR-mediated longevity, mitochondrial function, and aerobic fitness. We report that SIRT3 is required for whole-body aerobic capacity but is dispensable for CR-dependent lifespan extension. Under CR, loss of SIRT3 (Sirt3-/- ) yielded a longer overall and maximum lifespan as compared to Sirt3+/+ mice. This unexpected lifespan extension was associated with altered mitochondrial protein acetylation in oxidative metabolic pathways, reduced mitochondrial respiration, and reduced aerobic exercise capacity. Also, Sirt3-/- CR mice exhibit lower spontaneous activity and a trend favoring fatty acid oxidation during the postprandial period. This study shows the uncoupling of lifespan and healthspan parameters (aerobic fitness and spontaneous activity) and provides new insights into SIRT3 function in CR adaptation, fuel utilization, and aging.

Aerobic training associated with an active lifestyle exerts a protective effect against oxidative damage in hypothalamus and liver: The involvement of energy metabolism.

BACKGROUND: Oxidation resistance protein 1 (OXR1) is of scientific interest due its role in protecting tissues against oxidative stress, DNA mutations and tumorigenesis, but little is known regarding strategies to increase OXR1 in different tissues. As an improved antioxidant defense may result from a high total amount of physical activity, the present study was designed to determine whether an active lifestyle including aerobic training exercise and spontaneous physical activity (SPA) can increase OXR1. We have built a large cage (LC) that allows animals to move freely, promoting an increase in SPA in comparison to a small cage (SC). METHODS: We examined the effects of aerobic training applied for 8 weeks on SPA and OXR1 of C57BL/6 J mice living in two types of housing (SC and LC). OXR1 protein was studied in hypothalamus, muscle and liver, which were chosen due to their important role in energy and metabolic homeostasis. RESULTS: LC-mice were more active than SC-mice as determined by SPA values. Despite both trained groups exhibiting similar gains in aerobic capacity, only trained mice kept in a large cage (but not for trained mice housed in SC) exhibited high OXR1 in the hypothalamus and liver. Trained mice housed in LC that exhibited an up-regulation of OXR1 also were those who exhibited an energy-expensive metabolism (based on metabolic parameters). CONCLUSIONS: These results suggest that aerobic training associated with a more active lifestyle exerts a protective effect against oxidative damage and may be induced by changes in energy metabolism.

Establishment of Quantitative PCR Assays for Active Long Interspersed Nuclear Element-1 Subfamilies in Mice and Applications to the Analysis of Aging-Associated Retrotransposition.

The retrotransposon long interspersed nuclear element-1 (LINE-1) can autonomously increase its copy number within a host genome through the retrotransposition process. LINE-1 is active in the germline and in neural progenitor cells, and its somatic retrotransposition activity has a broad impact on neural development and susceptibility to neuropsychiatric disorders. The method to quantify the genomic copy number of LINE-1 would be important in unraveling the role of retrotransposition, especially in the brain. However, because of the species-specific evolution of LINE-1 sequences, methods for quantifying the copy number should be independently developed. Here, we developed a quantitative PCR (qPCR) assay to measure the copy number of active LINE-1 subfamilies in mice. Using the assay, we investigated aging-associated alterations of LINE-1 copy number in several brain regions in wild-type mice and Polg+/D257A mice as a model for accelerated aging. We found that aged Polg+/D257A mice showed higher levels of the type GfII LINE-1 in the basal ganglia than the wild-type mice did, highlighting the importance of assays that focus on an individual active LINE-1 subfamily.

A Novel Micronutrient Blend Mimics Calorie Restriction Transcriptomics in Multiple Tissues of Mice and Increases Lifespan and Mobility in C. elegans.

BACKGROUND: We previously described a novel micronutrient blend that behaves like a putative calorie restriction mimetic. The aim of this paper was to analyze the beneficial effects of our micronutrient blend in mice and C. elegans, and compare them with calorie restriction. METHODS: Whole transcriptomic analysis was performed in the brain cortex, skeletal muscle and heart in three groups of mice: old controls (30 months), old + calorie restriction and old + novel micronutrient blend. Longevity and vitality were tested in C. elegans. RESULTS: The micronutrient blend elicited transcriptomic changes in a manner similar to those in the calorie-restricted group and different from those in the control group. Subgroup analysis revealed that nuclear hormone receptor, proteasome complex and angiotensinogen genes, all of which are known to be directly related to aging, were the most affected. Furthermore, a functional analysis in C. elegans was used. We found that feeding C. elegans the micronutrient blend increased longevity as well as vitality. CONCLUSIONS: We describe a micronutrient supplement that causes similar changes (transcriptomic and promoting longevity and vitality) as a calorie restriction in mice and C. elegans, respectively, but further studies are required to confirm these effects in humans.

Prematurely aging mitochondrial DNA mutator mice display subchondral osteopenia and chondrocyte hypertrophy without further osteoarthritis features.

Mitochondrial mutations and dysfunction have been demonstrated in several age-related disorders including osteoarthritis, yet its relative contribution to pathogenesis remains unknown. Here we evaluated whether premature aging caused by accumulation of mitochondrial DNA mutations in PolgD275A mice predisposes to the development of knee osteoarthritis. Compared with wild type animals, homozygous PolgD275A mice displayed a specific bone phenotype characterized by osteopenia of epiphyseal trabecular bone and subchondral cortical plate. Trabecular thickness was significantly associated with osteocyte apoptosis rates and osteoclasts numbers were increased in subchondral bone tissues. While chondrocyte apoptosis rates in articular and growth plate cartilage were similar between groups, homozygous mitochondrial DNA mutator mice displayed elevated numbers of hypertrophic chondrocytes in articular calcified cartilage. Low grade cartilage degeneration, predominantly loss of proteoglycans, was present in all genotypes and the development of osteoarthritis features was not found accelerated in premature aging. Somatically acquired mitochondrial DNA mutations predispose to elevated subchondral bone turnover and hypertrophy in calcified cartilage, yet additional mechanical or metabolic stimuli would seem required for induction and accelerated progression of aging-associated osteoarthritis.

Housing conditions modulate spontaneous physical activity, feeding behavior, aerobic running capacity and adiposity in C57BL/6J mice.

There is evidence of reduced adiposity in rodents living in a large cages (LC) as compared to animals housed in small cages (SC). Because spontaneous physical activity (SPA) provides an important portion of the total daily energy expenditure, an increase of SPA in rodents kept in LC could explain their reduced body fat accumulation. The relationship between SPA and components of physical fitness (i.e. aerobic and anaerobic fitness and body leanness) has not been previously determined. We examined the effects of eight weeks of LC exposure on SPA, body composition, feeding behavior, as well as aerobic and anaerobic running capacity in adult C57BL/6J mice. Male mice were housed in cages of two different sizes for 8 weeks: a small (SC, n = 10) and large (LC n = 10) cages with 1320 cm2 and 4800 cm2 floor space, respectively. SPA was measured gravimetrically, and food and water intake were recorded daily. Mice had critical velocity (CV) and anaerobic running capacity (ARC) evaluated at the beginning, middle course (4th week) and at the end of study (8th week). Despite non-significant differences in each week LC-mice were more active than SC-mice by considering all SPA values obtained in the entire period of 8 weeks. The difference in SPA over the whole day was mainly due to light phase activity, but also due to activity at dark period (from 6 pm to 9 pm and from 5 am to 6 am). LC-mice also exhibited higher food and water intake over the entire 8-wk period. LC-mice had lower content of fat mass (% of the eviscerated carcass) than SC-mice (SC: 8.4 ±â€¯0.4 vs LC: 6.3 ±â€¯0.3, p < 0.05). LC-mice also exhibited reduced epididymal fat pads (% of body mass) compared to SC-mice (SC: 1.3 ±â€¯0.1 vs LC: 0.9 ±â€¯0.1, p < 0.05) and retroperitoneal fat pads (SC: 0.4 ±â€¯0.05 vs LC: 0.2 ±â€¯0.02, p < 0.05). The LC-group showed significantly higher critical velocity than SC-group at the fourth week (SC: 14.9 ±â€¯0.6 m·min-1 vs LC: 18.0 ±â€¯0.3 m·min-1, p < 0.05) and eighth week (SC: 17.1 ±â€¯0.5 m·min-1 vs LC: 18.8 ±â€¯0.6 m·min-1, p < 0.05). Our findings demonstrate that eight weeks of LC housing increases SPA of C57BL/6J mice, and this may lead to reduced fat accumulation as well as higher aerobic fitness. Importantly, our study implies that SC limits SPA, possibly generating experimental artifacts in long-term rodent studies.

The effects of AICAR and rapamycin on mitochondrial function in immortalized mitochondrial DNA mutator murine embryonic fibroblasts.

Mitochondrial DNA mutations accumulate with age and may play a role in stem cell aging as suggested by the premature aging phenotype of mitochondrial DNA polymerase gamma (POLG) exonuclease-deficient mice. Therefore, E1A immortalized murine embryonic fibroblasts (MEFs) from POLG exonuclease-deficient and wild-type (WT) mice were constructed. Surprisingly, when some E1A immortalized MEF lines were cultured in pyruvate-containing media they slowly became addicted to the pyruvate. The POLG exonuclease-deficient MEFs were more sensitive to several mitochondrial inhibitors and showed increased reactive oxygen species (ROS) production under standard conditions. When cultured in pyruvate-containing media, POLG exonuclease-deficient MEFs showed decreased oxygen consumption compared to controls. Increased AMP-activated protein kinase (AMPK) signaling and decreased mammalian target of rapamycin (mTOR) signaling delayed aging and influenced mitochondrial function. Therefore, the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, or rapamycin, an mTOR inhibitor, on measures of mitochondrial function were determined. Rapamycin treatment transiently increased respiration only in WT MEFs and, under most conditions, increased ATP levels. Short term AICAR treatment transiently increased ROS production and, under most conditions, decreased ATP levels. Chronic AICAR treatment decreased respiration and ROS production in WT MEFs. These results demonstrate the context-dependent effects of AICAR and rapamycin on mitochondrial function.

Identification of tissue-specific transcriptional markers of caloric restriction in the mouse and their use to evaluate caloric restriction mimetics.

Caloric restriction (CR) without malnutrition has been shown to retard several aspects of the aging process and to extend lifespan in different species. There is strong interest in the identification of CR mimetics (CRMs), compounds that mimic the beneficial effects of CR on lifespan and healthspan without restriction of energy intake. Identification of CRMs in mammals is currently inefficient due to the lack of screening tools. We have performed whole-genome transcriptional profiling of CR in seven mouse strains (C3H/HeJ, CBA/J, DBA/2J, B6C3F1/J, 129S1/SvImJ, C57BL/6J, and BALB/cJ) in white adipose tissue (WAT), gastrocnemius muscle, heart, and brain neocortex. This analysis has identified tissue-specific panels of genes that change in expression in multiple mouse strains with CR. We validated a subset of genes with qPCR and used these to evaluate the potential CRMs bezafibrate, pioglitazone, metformin, resveratrol, quercetin, 2,4-dinitrophenol, and L-carnitine when fed to C57BL/6J 2-month-old mice for 3 months. Compounds were also evaluated for their ability to modulate previously characterized biomarkers of CR, including mitochondrial enzymes citrate synthase and SIRT3, plasma inflammatory cytokines TNF-α and IFN-γ, glycated hemoglobin (HbA1c) levels and adipocyte size. Pioglitazone, a PPAR-γ agonist, and L-carnitine, an amino acid involved in lipid metabolism, displayed the strongest effects on both the novel transcriptional markers of CR and the additional CR biomarkers tested. Our findings provide panels of tissue-specific transcriptional markers of CR that can be used to identify novel CRMs, and also represent the first comparative molecular analysis of several potential CRMs in multiple tissues in mammals.

Effects of calorie restriction on the lifespan and healthspan of POLG mitochondrial mutator mice.

Mitochondrial DNA (mtDNA) mutations are thought to have a causative role in age-related pathologies. We have shown previously that mitochondrial mutator mice (PolgD257A/D257A), harboring a proofreading-deficient version of the mtDNA polymerase gamma (POLG), accumulate mtDNA mutations in multiple tissues and display several features of accelerated aging. Calorie restriction (CR) is known to delay the onset of age-related diseases and to extend the lifespan of a variety of species, including rodents. In the current study we investigated the effects of CR on the lifespan and healthspan of mitochondrial mutator mice. Long-term CR did not increase the median or maximum lifespan of PolgD257A/D257A mice. Furthermore, CR did not reduce mtDNA deletions in the heart and muscle, accelerated sarcopenia, testicular atrophy, nor improve the alterations in cardiac parameters that are present in aged mitochondrial mutator mice. Therefore, our findings suggest that accumulation of mtDNA mutations may interfere with the beneficial action of CR in aging retardation.

Sirt1 deficiency protects cochlear cells and delays the early onset of age-related hearing loss in C57BL/6 mice.

Hearing gradually declines with age in both animals and humans, and this condition is known as age-related hearing loss (AHL). Here, we investigated the effects of deficiency of Sirt1, a member of the mammalian sirtuin family, on age-related cochlear pathology and associated hearing loss in C57BL/6 mice, a mouse model of early-onset AHL. Sirt1 deficiency reduced age-related oxidative damage of cochlear hair cells and spiral ganglion neurons and delayed the early onset of AHL. In cultured mouse inner ear cell lines, Sirt1 knockdown increased cell viability under oxidative stress conditions, induced nuclear translocation of Foxo3a, and increased acetylation status of Foxo3a. This resulted in increased activity of the antioxidant enzyme catalase. In young wild-type mice, both Sirt1 and Foxo3a proteins resided in the cytoplasm of the supporting cells within the organ of Corti of the cochlea. Therefore, our findings suggest that SIRT1 promotes early-onset AHL through suppressing FOXO3a-mediated oxidative stress resistance in the cochlea of C57BL/6 mice.

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice.

BACKGROUND: Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. RESULTS: Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. CONCLUSIONS: Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.

Accumulation of Mitochondrial DNA Mutations Disrupts Cardiac Progenitor Cell Function and Reduces Survival.

Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.

SIRT3 mediates multi-tissue coupling for metabolic fuel switching.

SIRT3 is a member of the Sirtuin family of NAD(+)-dependent deacylases and plays a critical role in metabolic regulation. Organism-wide SIRT3 loss manifests in metabolic alterations; however, the coordinating role of SIRT3 among metabolically distinct tissues is unknown. Using multi-tissue quantitative proteomics comparing fasted wild-type mice to mice lacking SIRT3, innovative bioinformatic analysis, and biochemical validation, we provide a comprehensive view of mitochondrial acetylation and SIRT3 function. We find SIRT3 regulates the acetyl-proteome in core mitochondrial processes common to brain, heart, kidney, liver, and skeletal muscle, but differentially regulates metabolic pathways in fuel-producing and fuel-utilizing tissues. We propose an additional maintenance function for SIRT3 in liver and kidney where SIRT3 expression is elevated to reduce the acetate load on mitochondrial proteins. We provide evidence that SIRT3 impacts ketone body utilization in the brain and reveal a pivotal role for SIRT3 in the coordination between tissues required for metabolic homeostasis.

A conserved transcriptional signature of delayed aging and reduced disease vulnerability is partially mediated by SIRT3.

Aging is the most significant risk factor for a range of diseases, including many cancers, neurodegeneration, cardiovascular disease, and diabetes. Caloric restriction (CR) without malnutrition delays aging in diverse species, and therefore offers unique insights into age-related disease vulnerability. Previous studies suggest that there are shared mechanisms of disease resistance associated with delayed aging, however quantitative support is lacking. We therefore sought to identify a common response to CR in diverse tissues and species and determine whether this signature would reflect health status independent of aging. We analyzed gene expression datasets from eight tissues of mice subjected to CR and identified a common transcriptional signature that includes functional categories of mitochondrial energy metabolism, inflammation and ribosomal structure. This signature is detected in flies, rats, and rhesus monkeys on CR, indicating aspects of CR that are evolutionarily conserved. Detection of the signature in mouse genetic models of slowed aging indicates that it is not unique to CR but rather a common aspect of extended longevity. Mice lacking the NAD-dependent deacetylase SIRT3 fail to induce mitochondrial and anti-inflammatory elements of the signature in response to CR, suggesting a potential mechanism involving SIRT3. The inverse of this transcriptional signature is detected with consumption of a high fat diet, obesity and metabolic disease, and is reversed in response to interventions that decrease disease risk. We propose that this evolutionarily conserved, tissue-independent, transcriptional signature of delayed aging and reduced disease vulnerability is a promising target for developing therapies for age-related diseases.

Mito-protective autophagy is impaired in erythroid cells of aged mtDNA-mutator mice.

Somatic mitochondrial DNA (mtDNA) mutations contribute to the pathogenesis of age-related disorders, including myelodysplastic syndromes (MDS). The accumulation of mitochondria harboring mtDNA mutations in patients with these disorders suggests a failure of normal mitochondrial quality-control systems. The mtDNA-mutator mice acquire somatic mtDNA mutations via a targeted defect in the proofreading function of the mtDNA polymerase, PolgA, and develop macrocytic anemia similar to that of patients with MDS. We observed an unexpected defect in clearance of dysfunctional mitochondria at specific stages during erythroid maturation in hematopoietic cells from aged mtDNA-mutator mice. Mechanistically, aberrant activation of mechanistic target of rapamycin signaling and phosphorylation of uncoordinated 51-like kinase (ULK) 1 in mtDNA-mutator mice resulted in proteasome-mediated degradation of ULK1 and inhibition of autophagy in erythroid cells. To directly evaluate the consequence of inhibiting autophagy on mitochondrial function in erythroid cells harboring mtDNA mutations in vivo, we deleted Atg7 from erythroid progenitors of wild-type and mtDNA-mutator mice. Genetic disruption of autophagy did not cause anemia in wild-type mice but accelerated the decline in mitochondrial respiration and development of macrocytic anemia in mtDNA-mutator mice. These findings highlight a pathological feedback loop that explains how dysfunctional mitochondria can escape autophagy-mediated degradation and propagate in cells predisposed to somatic mtDNA mutations, leading to disease.

Heterozygous Polg mutation causes motor dysfunction due to mtDNA deletions.

OBJECTIVE: Mutations in nuclear-encoded mitochondrial DNA (mtDNA) polymerase (POLG) are known to cause autosomal dominant chronic progressive external ophthalmoplegia (adCPEO) with accumulation of multiple mtDNA deletions in muscles. However, no animal model with a heterozygous Polg mutation representing mtDNA impairment and symptoms of CPEO has been established. To understand the pathogenic mechanism of CPEO, it is important to determine the age dependency and tissue specificity of mtDNA impairment resulting from a heterozygous mutation in the Polg gene in an animal model. METHODS: We assessed behavioral phenotypes, tissue-specific accumulation of mtDNA deletions, and its age dependency in heterozygous Polg (D257A) knock-in mice carrying a proofreading-deficient mutation in the Polg. RESULTS: Heterozygous Polg (D257A) knock-in mice exhibited motor dysfunction in a rotarod test. Polg (+/D257A) mice had significant accumulation of multiple mtDNA deletions, but did not show significant accumulation of point mutations or mtDNA depletion in the brain. While mtDNA deletions increased in an age-dependent manner regardless of the tissue even in Polg (+/+) mice, the age-dependent accumulation of mtDNA deletions was enhanced in muscles and in the brain of Polg (+/D257A) mice. INTERPRETATION: Heterozygous Polg (D257A) knock-in mice showed tissue-specific, age-dependent accumulation of multiple mtDNA deletions in muscles and the brain which was likely to result in neuromuscular symptoms. Polg (+/D257A) mice may be used as an animal model of adCPEO associated with impaired mtDNA maintenance.