Reduced mtDNA Copy Number Research Articles

Aerobic Exercise Counteracts Mitochondrial Dysfunction In The Insulin Resistant Brain

Insulin resistance (IR) increases the risk for Alzheimer’s disease and other dementias; however, the underpinning mechanisms for this increased risk remain to be fully defined. Impaired mitochondrial function is one component contributing to cognitive impairment. PURPOSE: As insulin resistance impairs mitochondrial oxidative metabolism and increases reactive oxygen species (ROS) in skeletal muscle, we considered whether similar events occur in the brain, which like muscle is rich in insulin receptors and mitochondria. Further, we sought to determine whether aerobic exercise (AE) could prevent the hypothesized deficits in mitochondrial function accompanying diet-induced IR. METHODS: 12-week-old, male, C57BL6 mice were fed a standard (Chow) or high fat diet (HFD) (60% kcal from fat) for four-weeks and provided access to running wheels (EX) or sedentary (SED) conditions (n = 9-10 per group). Following treatment, mitochondria were freshly isolated from the cerebrum to assess mitochondrial respiration, ROS production, and ATP production. Insulin resistance was determined ex-vivo in the hippocampus by the ability of insulin to stimulate AKT-phosphorylation. mtDNA copy number, mRNA expression, and proteomic measurements were performed on isolated hippocampal tissue. RESULTS: HFD induced hippocampal insulin resistance (p < 0.001), which was corrected by AE. HFD decreased ATP production 12% (p = 0.01) and increased ROS emission 79% (p < 0.01) in isolated cerebral mitochondria, which were rescued with AE. Impairments in mitochondrial function with HFD were paralleled by reductions in mtDNA copy number (1.00 ± 0.06 vs 0.85 ± 0.06; p = 0.02) and mRNA expression of mitochondrial genes, such as PGC1a1 (1.00 ± 0.05 vs 0.78 ± 0.07; p = 0.03) and TFAM (1.00 ± 0.08 vs 0.62 ± 0.11; p = 0.03), which were corrected by AE. Proteomic analysis of the hippocampus showed that HFD led to oxidative post-translational modifications (PTMs) to 17 mitochondrial proteins (corrected p-value ≤ 0.05 and absolute log2 fold change ≥ 0.5); however, this increase in oxidative PTMs to mitochondrial proteins with HFD was almost completely reversed by AE. CONCLUSIONS: HFD induces IR in the cerebrum and hippocampus, which associates with mitochondrial dysfunction. Brain IR and mitochondrial dysfunction accompanying HFD are prevented with AE.

Tfam Knockdown Results in Reduction of mtDNA Copy Number, OXPHOS Deficiency and Abnormalities in Zebrafish Embryos.

High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (tfam), a regulator of mtDNA replication, during early zebrafish development. Reduction of tfam using a splice-modifying morpholino (MO) resulted in a 42 ± 17% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart, and muscle, as well as a 50 ± 22% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA, and down-regulation of genes involved in haem production and the metabolism of metabolites, which appear to trigger increased rRNA and tRNA synthesis in the nucleoli. However, this stress or compensatory response appears to fall short as pathology emerges and expression of genes related to eye development are severely down-regulated. Taken together, this study highlights the importance of sufficient mtDNA copies for early zebrafish development. Zebrafish is an excellent model to manipulate the mtDNA bottleneck and study its effect on embryogenesis rapidly and in large numbers of offspring.

N6-Deoxyadenosine Methylation in Mammalian Mitochondrial DNA

N6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA.

Heart failure after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous DES-p.R349P knock-in mice

BackgroundMutations in the human desmin gene (DES) cause autosomal-dominant and -recessive cardiomyopathies, leading to heart failure, arrhythmias, and AV blocks. We analyzed the effects of vascular pressure overload in a patient-mimicking p.R349P desmin knock-in mouse model that harbors the orthologue of the frequent human DES missense mutation p.R350P.Methods and resultsTransverse aortic constriction (TAC) was performed on heterozygous (HET) DES-p.R349P mice and wild-type (WT) littermates. Echocardiography demonstrated reduced left ventricular ejection fraction in HET-TAC (WT-sham: 69.5 ± 2.9%, HET-sham: 64.5 ± 4.7%, WT-TAC: 63.5 ± 4.9%, HET-TAC: 55.7 ± 5.4%; p<0.01). Cardiac output was significantly reduced in HET-TAC (WT sham: 13088 ± 2385 μl/min, HET sham: 10391 ± 1349μl/min, WT-TAC: 8097 ± 1903μl/min, HET-TAC: 5793 ± 2517μl/min; p<0.01). Incidence and duration of AV blocks as well as the probability to induce ventricular tachycardias was highest in HET-TAC. We observed reduced mtDNA copy numbers in HET-TAC (WT-sham: 12546 ± 406, HET-sham: 13526 ± 781, WT-TAC: 11155 ± 3315, HET-TAC: 8649 ± 1582; p = 0.025), but no mtDNA deletions. The activity of respiratory chain complexes I and IV showed the greatest reductions in HET-TAC.ConclusionPressure overload in HET mice aggravated the clinical phenotype of cardiomyopathy and resulted in mitochondrial dysfunction. Preventive avoidance of pressure overload/arterial hypertension in desminopathy patients might represent a crucial therapeutic measure.

Decrease in T-Lymphocyte Mitochondrial DNA Copy Number Is Associated with Acute Graft Versus Host Disease

Introduction Mitochondria play an important role in T-lymphocyte activation and initiation of the inflammatory response, two pathways that are important in the pathogenesis of acute graft-versus-host disease (aGvHD). We evaluated whether change in mitochondrial DNA (mtDNA) copy number in CD3+ T-lymphocytes measured at two time-points pre and post aGvHD could be used as a biomarker for aGvHD diagnosis that correlates with clinical and/or biopsy proven aGvHD. Methods 99 patients who underwent an allogeneic hematopoietic cell transplant (allo-HCT) between March 2012 and December 2016, were diagnosed with aGvHD and had samples available before and after aGvHD diagnosis were selected for this analysis. We measured mtDNA copy number in CD3+ T-lymphocytes at two time points using real time quantitative polymerase chain reaction (PCR); once prior to diagnosis of aGvHD (median (Interquartile range): 11 (5 – 22) days prior to aGvHD diagnosis) and once after aGvHD diagnosis (median: 23 (12-34) days after aGvHD diagnosis) in 99 patients. 16% (n=16) of the patients had grade I aGvHD and 84% (n=83) had grade II-IV aGvHD. We used linear mixed models with random subject and batch effects to evaluate the change in mtDNA copy number before to after aGvHD diagnosis within each patient. We also used Fine and Gray regression models to evaluate whether reduction in mtDNA copy number after aGvHD diagnosis was associated with one year non relapse mortality (NRM). Results The median age was 58 (interquartile range 38 to 65). 35% patients underwent a transplant from a HLA-identical sibling donor, 19% matched unrelated donor, 2% mismatched related donor, 14% single and 30% double umbilical cord blood. Conditioning was myeloablative in 36% and reduced intensity in 64%. aGvHD was diagnosed at a median of 42 (range 14-180) days from HCT. 16 (16%) of the patients were diagnosed with grade 1, 38% with grade II, 34% grade III and 11% grade IV aGvHD. Overall, geometric mean mtDNA copy number decreased 14% after aGvHD diagnosis (95% CI: 3% to 26%; p=0.01). MtDNA copy number decreased from pre-aGvHD to post-aGvHD in all grades of aGvHD though a decrease was seen in a larger proportion of patients with grade IV aGvHD (9/11; 82%) vs. patients with grade I aGvHD (8/16; 50%). There were 25 patients who experienced NRM within the first year of transplant. Exploratory analysis showed that mortality was lower among patients in whom mtDNA copy number increased post-aGvHD diagnosis (6/38; 16%) as compared to those in whom mtDNA copy number decreased post-aGvHD diagnosis (19/61; 31%) (p=0.12). Conclusion This study shows that mtDNA copy number was more likely to decrease after diagnosis of aGvHD. Thus, change in mtDNA copy number among CD3+ T lymphocytes may be a useful biomarker to identify individuals with aGvHD. The prognostic significance of reduced mtDNA copy number on NRM needs to be evaluated in larger studies.

Accelerated hippocampal biological aging in bipolar disorder.

Evidence suggests accelerated aging mechanisms in bipolar disorder (BD), including DNA methylation (DNAm) aging in blood. However, it is unknown whether such mechanisms are also evident in the brain, in particular in association with other biological clocks. To investigate this, we interrogated genome-wide DNAm in postmortem hippocampus from 32 BD-I patients and 32 non-psychiatric controls group-matched for age and sex from the NIMH Human Brain Collection Core. DNAm age and epigenetic aging acceleration were estimated using the Horvath method. Telomere length (TL) and mitochondrial DNA (mtDNA) copy number were quantified by real-time PCR. Between-group differences were assessed by linear regression and univariate general linear models with age, sex, race, postmortem interval, tissue pH, smoking, and body mass index included as co-variates. Groups did not differ for epigenetic aging acceleration when considering the entire sample. However, after splitting the sample by the median age, an epigenetic aging acceleration was detected in patients compared to controls among older subjects (P=.042). While TL did not differ between groups, a reduction in mtDNA copy number was observed in patients compared to controls (P=.047). In addition, significant correlations were observed between epigenetic aging acceleration and TL (r=-.337, P=.006), as well as between TL and mtDNA copy number (r=.274, P=.028). Hippocampal aging may underlie neurocognitive dysfunctions observed in BD patients. Moreover, our results suggest a complex cross-talk between biological clocks in hippocampus that may underlie clinical manifestations of premature aging in BD.

P.79Malignant cardiac phenotype after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous R349P desmin knock-in mice

Which etiological factors significantly aggravate the disease progression in genetically determined forms of cardiomyopathies? In the present study, we investigated the influence of pressure overload on cardiac function in our R349P desmin knock-in mouse model for human desminopathies. We performed transverse aortic constriction (TAC) operations in heterozygous R349P desmin knock-in mice and wild-type siblings. Left heart catheterization demonstrated effective TAC procedures by marked increases in systolic carotid pressure and end-systolic and end-diastolic left ventricular pressure values. Echocardiography demonstrated a significant reduction of the left ventricular ejection fraction only in TAC R349P desmin knock-in mice as compared to controls. Electrophysiological investigation revealed an increase in spontaneous AV blocks and the longest AV block duration in the mutant TAC mice. Furthermore, the probability of induction of ventricular tachycardias was also significantly increased in these mice. Mitochondrial analyses demonstrated marked reductions of the activities of respiratory chain complexes I and IV as well as significantly reduced mtDNA copy numbers in the TAC-operated desminopathy mice. While young, 3-month-old, non-operated heterozygous R349P desmin knock-in mice do not show obvious signs of cardiomyopathy, they develop marked signs of cardiomyopathy with accompanying arrhythmias and AV blocks when subjected to pressure overload by TAC operation. The cardiac disease manifestations are associated with reductions in respiratory chain complex activities and mtDNA copy numbers. Preventive avoidance of pressure overload, i.e. arterial hypertension, to the heart might represent a crucial therapeutic measure.

Association of Folate Metabolites and Mitochondrial Function in Peripheral Blood Cells in Alzheimer's Disease: A Matched Case-Control Study.

The nutrition state plays an important role in the progress of aging. Folate may play a role in protecting mitochondrial (mt) DNA by reducing oxidative stress. The primary aim of this study was to examine the association of mitochondrial oxidative damage with risk of Alzheimer's disease (AD), and to explore the possible role of folate metabolites in this association in a matched case-control study. Serum folate metabolites and mitochondrial function in peripheral blood cells were determined in 82 AD cases and 82 healthy controls, individually matched by age, gender, and education. AD patients had lower serum levels of folate and higher homocysteine (Hcy) concentration. AD patients had a reduced mtDNA copy number, higher mtDNA deletions, and increased 8-OHdG content in mtDNA indicative of reduced mitochondrial function. The highest level of mtDNA copy number would decrease the risk of AD (OR = 0.157, 95% CI: 0.058-0.422) compared to the lowest level, independently of serum folate, and Hcy levels. Serum folate levels correlated with low 8-OHdG content in mtDNA both in AD patients and controls, independently of serum Hcy level. Moreover, serum Hcy levels correlated with low copy number in mtDNA both in AD patients and controls, independently of serum folate levels. In conclusion, mitochondrial function in peripheral blood cells could be associated with risk of AD independent of multiple covariates. AD patients with a folate deficiency or hyperhomocysteinemia had low mitochondrial function in peripheral blood cells. However, further randomized controlled trials are need to determine a causal effect.

Ginkgolic Acids Impair Mitochondrial Function by Decreasing Mitochondrial Biogenesis and Promoting FUNDC1-Dependent Mitophagy.

Ginkgolic acids (GAs) are found in the leaves, nuts, and testa of Ginkgo biloba and have been reported to exhibit antitumor, antibacterial, and pro-apoptotic activities. However, their role in mitochondrial function is still unclear. Our previous study showed that genes related to the mitochondria present significant changes in GA-treated mouse bone marrow stromal cells. We hypothesize that GAs may regulate mitochondrial function. Here, we found that GA treatment induced mitochondrial fragmentation, reduced mtDNA copy numbers and mitochondrial protein levels, and impaired mitochondrial adenosine 5'-triphosphate production and oxygen consumption. The GA-induced mitochondrial mass loss may be due to decreased mitochondrial biogenesis. In addition, abolishing autophagy by Atg7 knockout or the administration of an autophagy inhibitor can restore the GA-induced decrease in mitochondrial mass. Furthermore, FUNDC1 knockdown restored the GA-induced changes in mitochondrial mass reduction and mitochondrial membrane potential loss. Together, our studies demonstrated that GAs impaired mitochondrial function by decreasing mitochondrial biogenesis and promoting FUNDC1-dependent mitophagy.

289-OR: Aerobic Exercise Counteracts Mitochondrial Dysfunction in the Insulin Resistant Brain

Insulin resistance increases the risk for Alzheimer’s disease and other dementias; however, the underpinning mechanisms for this increased risk remain to be fully defined. Insulin resistance impairs mitochondrial oxidative metabolism and increases reactive oxygen species (ROS) in skeletal muscle, both of which are counteracted by aerobic exercise (AE). Therefore, we considered whether similar events occur in the brain, specifically in regions such as cerebrum and hippocampus which are rich in insulin receptors and mitochondria. We determined whether AE would counteract mitochondrial deficits in hippocampal and cerebral mitochondrial function in high fat diet (HFD) induced insulin resistance. Four weeks of HFD in mice induced hippocampal insulin resistance which was corrected by AE. HFD decreased ATP production and increased ROS emission in isolated cerebral mitochondria, which were rescued with AE. Impairments in mitochondrial function with HFD were paralleled by reductions in mtDNA copy number, mRNA expression of mitochondrial genes, and oxidative enzyme activities (e.g., citrate synthase, COX) in the hippocampus, which were corrected by AE. We also observed excessive mitochondrial fission in the hippocampus following HFD, which was prevented by AE. Proteomic analysis of the hippocampus showed that HFD led to oxidative post-translational modifications (PTMs) of mitochondrial proteins; however, this increase in oxidative PTMs to mitochondrial proteins with HFD was almost completely reversed by AE. Further, intranasal administration of the insulin receptor antagonist S961 prevented AE-induced improvements in ATP production and ROS emission in isolated cerebral mitochondrial during HFD, demonstrating that AE enhances cerebral mitochondrial function during HFD by resensitizing brain insulin signaling. Disclosure G. Ruegsegger: None. P.M. Vanderboom: None. S. Dasari: Consultant; Self; The Binding Site. K. Klaus: None. K. Nair: None.

Fragile X-associated tremor/ataxia syndrome: Regional decrease of mitochondrial DNA copy number relates to clinical manifestations.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in at least one-third of adult carriers of a premutation (55-200 CGG repeats) in the fragile X mental retardation 1 (FMR1) gene. Several studies have shown that mitochondrial dysfunction may play a central role in aging and also in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease as well as in FXTAS. It has been recently proposed that mtDNA copy number, measured by the number of mitochondrial genomes per nuclear genome (diploid), could be a useful biomarker of mitochondrial dysfunction. In order to elucidate the role of mtDNA variation in the pathogenesis of FXTAS, mtDNA copy number was quantified by digital droplet Polymerase chain reaction. In human brain samples, mtDNA levels were measured in the cerebellar vermis, dentate nucleus, parietal and temporal cortex, thalamus, caudate nucleus and hippocampus from a female FXTAS patient, a FMR1 premutation male carrier without FXTAS and from three male controls. The mtDNA copy number was further analyzed using this technology in dermal fibroblasts primary cultures derived from three FXTAS patients and three controls as well as in cortex and cerebellum of a CGG knock in FXTAS mice model. Finally, qPCR was carried out in human blood samples. Results indicate reduced mtDNA copy number in the specific brain region associated with disease progression in FXTAS patients, providing new insights into the role of mitochondrial dysfunction in the pathogenesis of FXTAS.

Chronic fetal hypoxia disrupts the peri-conceptual environment in next-generation adult female rats.

Key points Exposure to chronic hypoxia during gestation influences long‐term health and development, including reproductive capacity, across generations.If the peri‐conceptual environment in the developing oviduct is affected by gestational hypoxia, then this could have implications for later fertility and the health of future generations.In the present study, we show that the oviducts of female rats exposed to chronic hypoxia in utero have reduced telomere length, decreased mitochondrial DNA biogenesis and increased oxidative stressThe results of the present study show that exposure to chronic gestational hypoxia leads to accelerated ageing of the oviduct in early adulthood and they help us understand how exposure to hypoxia during development could influence reproductive health across generations. Exposure to chronic hypoxia during fetal development has important effects on immediate and long‐term outcomes in offspring. Adverse impacts in adult offspring include impairment of cardiovascular function, metabolic derangement and accelerated ovarian ageing. However, it is not known whether other aspects of the female reproductive system may be similarly affected. In the present study, we examined the impact of chronic gestational hypoxia on the developing oviduct. Wistar rat dams were randomized to either normoxia (21%) or hypoxia (13%) from day 6 post‐mating until delivery. Post‐delivery female offspring were maintained in normoxia until 4 months of age. Oviductal gene expression was assayed at the RNA (quantitative RT‐PCR) and protein (western blotting) levels. Oviductal telomere length was assayed using Southern blotting. Oviductal telomere length was reduced in the gestational hypoxia‐exposed animals compared to normoxic controls (P < 0.01). This was associated with a specific post‐transcriptional reduction in the KU70 subunit of DNA‐pk in the gestational hypoxia‐exposed group (P < 0.05). Gestational hypoxia‐exposed oviducts also showed evidence of decreased mitochondrial DNA biogenesis, reduced mtDNA copy number (P < 0.05) and reduced gene expression of Tfam (P < 0.05) and Pgc1α (P < 0.05). In the hypoxia‐exposed oviducts, there was upregulation of mitochondrial‐specific anti‐oxidant defence enzymes (MnSOD; P < 0.01). Exposure to chronic gestational hypoxia leads to accelerated ageing of the oviduct in adulthood. The oviduct plays a central role in early development as the site of gamete transport, syngamy, and early development; hence, accelerated ageing of the oviductal environment could have important implications for fertility and the health of future generations.

Differential impacts of gonadotrophins, IVF and embryo culture on mouse blastocyst development.

Conception via assisted reproductive technology (ART) increases the risk of type 2 diabetes and cardiovascular disease in adulthood. Underlying differences between ART-conceived and in-vivo-conceived embryos that contribute to this increased risk are, however, not known. This study examined the developmental characteristics of mouse blastocysts derived from ART- compared with in-vivo-conceived embryos. To determine the effect of ovarian stimulation versus IVF versus in-vitro embryo culture on phenotype, six distinct groups of blastocysts were generated. Female mice were naturally cycling or treated with high or mild doses of gonadotrophin, followed by natural mating or IVF under clinical conditions. Embryo morphokinetics were assessed by continuous time-lapse monitoring. Cell lineage allocation to the inner cell mass (Oct4+) or trophectoderm (Cdx2+) was determined by immunohistochemistry, and mitochondrial DNA (mtDNA) copy number was measured by quantitative PCR. Ovarian stimulation increased embryo number but reduced the percentage of blastocysts. Morphokinetic analysis showed that gonadotrophin treatment led to advanced development (P<0.05) due to earlier post-pronuclear breakdown. The blastocyst rate was reduced in IVF embryos compared with those fertilized in vivo before culture (P<0.001). Morphokinetics showed that embryo development was slower in all the IVF groups (P<<0.05), due to a delay from the 3-cell stage. A reduced total and trophectoderm cell number was observed in all groups of cultured blastocysts compared with naturally conceived blastocysts (P<0.01). Gonadotrophin treatment did not affect the blastocyst mtDNA copy number; however, IVF embryos exhibited reduced mtDNA copy number compared with naturally conceived embryos. Ovarian stimulation, IVF and in-vitro culture differentially impair blastocyst developmental kinetics, differentiation and mtDNA copy number.

Molecular Characterization of Peroxisome Proliferator-Activated Receptor-Gamma Coactivator-1α (PGC1α) and Its Role in Mitochondrial Biogenesis in Blunt Snout Bream (Megalobrama amblycephala).

PGC1α is a transcriptional coactivator that plays key roles in mitochondrial biogenesis, so exploring its molecular characterization contributes to the understanding of mitochondrial function in cultured fish. In the present study, a full-length cDNA coding PGC1α was cloned from the liver of blunt snout bream (Megalobrama amblycephala) which covered 3741 bp with an open reading frame of 2646 bp encoding 881 amino acids. Sequence alignment and phylogenetic analysis revealed high conservation with other fish species, as well as other higher vertebrates. Comparison of the derived amino acid sequences indicates that, as with other fish, there is a proline at position 176 (RIRP) compared to a Thr in the mammalian sequences (RIRT). To investigate PGC1α function, three in vitro tests were carried out using primary hepatocytes of blunt snout bream. The effect of AMPK activity on the expression of PGC1α was determined by the culture of the hepatocytes with an activator (Metformin) or inhibitor (Compound C) of AMPK. Neither AMPK activation nor inhibition altered PGC1α expression. Knockdown of PGC1α expression in hepatocytes using small interfering RNA (si-RNA) was used to determine the role of PGC1α in mitochondrial biogenesis. No significant differences in the expression of NRF1 and TFAM, and mtDNA copy number were found between control and si-RNA groups. Also, hepatocytes were cultured with oleic acid, and the findings showed the significant reduction of mtDNA copy number in oleic acid group compared to control. Moreover, oleic acid down-regulated the expression of NRF1 and TFAM genes, while PGC1α expression remained unchanged. Our findings support the proposal that PGC1α may not play a role in mitochondrial biogenesis in blunt snout bream hepatocytes.

Homocysteine impairs porcine oocyte quality via deregulation of one-carbon metabolism and hypermethylation of mitochondrial DNA†.

Homocysteine (Hcy) is an intermediate in the one-carbon metabolism that donates methyl groups for methylation processes involved in epigenetic gene regulation. Although poor oocyte quality in polycystic ovarian syndrome (PCOS) patients is associated with elevated Hcy concentration in serum and follicular fluid, whether Hcy directly affects oocyte quality and its mechanisms are poorly understood. Here we show that Hcy treatment impaired oocyte quality and developmental competence, indicated by significantly reduced survival rate, polar body extrusion rate, and cleavage rate. Hcy treatment resulted in mitochondrial dysfunction, with increased production of mitochondrial ROS, reduced mtDNA copy number, and the expression of 7 out of 13 mtDNA-encoded genes and 2 ribosome RNA genes, 12S rRNA and 16S rRNA. Upon Hcy treatment, the expression of one-carbon metabolic enzymes and DNMT1 was enhanced. Interestingly, DNA methyltransferase inhibitor 5'AZA rescued Hcy-induced mitochondrial dysfunction, impaired oocyte quality and developmental competence. Concurrently, expression of one-carbon metabolic enzymes and methylation status of mtDNA coding sequences were also normalized, at least partially, by 5'AZA treatment. Our findings not only extend the understanding about how Hcy induces poor oocyte quality, but also contribute to a novel angle of identifying targets for enhancing the quality of oocyte from PCOS patients.

Urea Memory: Transient Cell Exposure to Urea Causes Persistent Mitochondrial ROS Production and Endothelial Dysfunction.

Urea at post-dialysis levels induces increased ROS in a number of cell types. The aim of this study was to determine whether urea-induced production of ROS remains elevated after urea is no longer present, and, if it does, to characterize its origin and effects. Human arterial endothelial cells were incubated with 20 mM urea for two days, and then cells were incubated for an additional two days in medium alone. Maximal ROS levels induced by initial urea continued at the same level despite urea being absent. These effects were prevented by either MnSOD expression or by Nox1/4 inhibition with GKT13781. Sustained urea-induced ROS caused a persistent reduction in mtDNA copy number and electron transport chain transcripts, a reduction in transcription of mitochondrial fusion proteins, an increase in mitochondrial fission proteins, and persistent expression of endothelial inflammatory markers. The SOD-catalase mimetic MnTBAP reversed each of these. These results suggest that persistent increases in ROS after cells are no long exposed to urea may play a major role in continued kidney damage and functional decline despite reduction of urea levels after dialysis.

Characterization of the human homozygous R182W POLG2 mutation in mitochondrial DNA depletion syndrome.

Mutations in mitochondrial DNA (mtDNA) have been linked to a variety of metabolic, neurological and muscular diseases which can present at any time throughout life. MtDNA is replicated by DNA polymerase gamma (Pol γ), twinkle helicase and mitochondrial single-stranded binding protein (mtSSB). The Pol γ holoenzyme is a heterotrimer consisting of the p140 catalytic subunit and a p55 homodimeric accessory subunit encoded by the nuclear genes POLG and POLG2, respectively. The accessory subunits enhance DNA binding and promote processive DNA synthesis of the holoenzyme. Mutations in either POLG or POLG2 are linked to disease and adversely affect maintenance of the mitochondrial genome, resulting in depletion, deletions and/or point mutations in mtDNA. A homozygous mutation located at Chr17: 62492543G>A in POLG2, resulting in R182W substitution in p55, was previously identified to cause mtDNA depletion and fatal hepatic liver failure. Here we characterize this homozygous R182W p55 mutation using in vivo cultured cell models and in vitro biochemical assessments. Compared to control fibroblasts, homozygous R182W p55 primary dermal fibroblasts exhibit a two-fold slower doubling time, reduced mtDNA copy number and reduced levels of POLG and POLG2 transcripts correlating with the reported disease state. Expression of R182W p55 in HEK293 cells impairs oxidative-phosphorylation. Biochemically, R182W p55 displays DNA binding and association with p140 similar to WT p55. R182W p55 mimics the ability of WT p55 to stimulate primer extension, support steady-state nucleotide incorporation, and suppress the exonuclease function of Pol γ in vitro. However, R182W p55 has severe defects in protein stability as determined by differential scanning fluorimetry and in stimulating function as determined by thermal inactivation. These data demonstrate that the Chr17: 62492543G>A mutation in POLG2, R182W p55, severely impairs stability of the accessory subunit and is the likely cause of the disease phenotype.

Aggressive triple negative breast cancers have unique molecular signature on the basis of mitochondrial genetic and functional defects

Metastatic breast cancer is a leading cause of cancer-related deaths in women worldwide. Patients with triple negative breast cancer (TNBCs), a highly aggressive tumor subtype, have a particularly poor prognosis. Multiple reports demonstrate that altered content of the multicopy mitochondrial genome (mtDNA) in primary breast tumors correlates with poor prognosis. We earlier reported that mtDNA copy number reduction in breast cancer cell lines induces an epithelial-mesenchymal transition associated with metastasis. However, it is unknown whether the breast tumor subtypes (TNBC, Luminal and HER2+) differ in the nature and amount of mitochondrial defects and if mitochondrial defects can be used as a marker to identify tumors at risk for metastasis. By analyzing human primary tumors, cell lines and the TCGA dataset, we demonstrate a high degree of variability in mitochondrial defects among the tumor subtypes and TNBCs, in particular, exhibit higher frequency of mitochondrial defects, including reduced mtDNA content, mtDNA sequence imbalance (mtRNR1:ND4), impaired mitochondrial respiration and metabolic switch to glycolysis which is associated with tumorigenicity. We identified that genes involved in maintenance of mitochondrial structural and functional integrity are differentially expressed in TNBCs compared to non-TNBC tumors. Furthermore, we identified a subset of TNBC tumors that contain lower expression of epithelial splicing regulatory protein (ESRP)-1, typical of metastasizing cells. The overall impact of our findings reported here is that mitochondrial heterogeneity among TNBCs can be used to identify TNBC patients at risk of metastasis and the altered metabolism and metabolic genes can be targeted to improve chemotherapeutic response.

Histopathological liver findings in patients with hepatocerebral mitochondrial depletion syndrome with defined molecular basis.

Mitochondrial DNA depletion consisting of the systemic reduction of mtDNA copy number in cells may have a heterogenous genetic basis, resulting from a pathogenic change in the nuclear genes involved in mtDNA synthesis. The mode of inheritance is autosomal recessive. Severe hepatocerebral disease represents one of many different clinical forms of so-called mitochondrial depletion syndrome (MDS). We present the liver histopathology of 13 children who eventually died in the course of hepatocerebral MDS confirmed molecularly, harbouring mutations of DGUOK, MPV17, and POLG genes. Material comprising eight autopsy and five liver biopsy specimens showed a moderately reproducible pattern of parenchymal damage, which we consider potentially helpful in the differential diagnosis and planning of the diagnostic investigation in families of children who died due to early-onset acute liver failure and encephalopathy.

Independent impacts of aging on mitochondrial DNA quantity and quality in humans

BackgroundThe accumulation of mitochondrial DNA (mtDNA) mutations, and the reduction of mtDNA copy number, both disrupt mitochondrial energetics, and may contribute to aging and age-associated phenotypes. However, there are few genetic and epidemiological studies on the spectra of blood mtDNA heteroplasmies, and the distribution of mtDNA copy numbers in different age groups and their impact on age-related phenotypes. In this work, we used whole-genome sequencing data of isolated peripheral blood mononuclear cells (PBMCs) from the UK10K project to investigate in parallel mtDNA heteroplasmy and copy number in 1511 women, between 17 and 85 years old, recruited in the TwinsUK cohorts.ResultsWe report a high prevalence of pathogenic mtDNA heteroplasmies in this population. We also find an increase in mtDNA heteroplasmies with age (β = 0.011, P = 5.77e-6), and showed that, on average, individuals aged 70-years or older had 58.5% more mtDNA heteroplasmies than those under 40-years old. Conversely, mtDNA copy number decreased by an average of 0.4 copies per year (β = −0.395, P = 0.0097). Multiple regression analyses also showed that age had independent effects on mtDNA copy number decrease and heteroplasmy accumulation. Finally, mtDNA copy number was positively associated with serum bicarbonate level (P = 4.46e-5), and inversely correlated with white blood cell count (P = 0.0006). Moreover, the aggregated heteroplasmy load was associated with blood apolipoprotein B level (P = 1.33e-5), linking the accumulation of mtDNA mutations to age-related physiological markers.ConclusionsOur population-based study indicates that both mtDNA quality and quantity are influenced by age. An open question for the future is whether interventions that would contribute to maintain optimal mtDNA copy number and prevent the expansion of heteroplasmy could promote healthy aging.