Increase In mtDNA Content Research Articles

Mitochondrial DNA replication is essential for neurogenesis but not gliogenesis in fetal neural stem cells.

Mitochondria are unique organelles that have their own genome (mtDNA) and perform various pivotal functions within a cell. Recently, evidence has highlighted the role of mitochondria in the process of stem cell differentiation, including differentiation of neural stem cells (NSCs). Here we studied the importance of mtDNA function in the early differentiation process of NSCs in two cell culture models: the CGR8-NS cell line that was derived from embryonic stem cells by a lineage selection technique, and primary NSCs that were isolated from embryonic day 14 mouse fetal forebrain. We detected a dramatic increase in mtDNA content upon NSC differentiation to adapt their mtDNA levels to their differentiated state, which was not accompanied by changes in mitochondrial transcription factor A expression. As chemical mtDNA depletion by ethidium bromide failed to generate living ρ° cell lines from both NSC types, we used inhibition of mtDNA polymerase-γ by 2'-3'-dideoxycytidine to reduce mtDNA replication and subsequently cellular mtDNA content. Inhibition of mtDNA replication upon NSC differentiation reduced neurogenesis but not gliogenesis. The mtDNA depletion did not change energy production/consumption or cellular reactive oxygen species (ROS) content in the NSC model used. In conclusion, mtDNA replication is essential for neurogenesis but not gliogenesis in fetal NSCs through as yet unknown mechanisms, which, however, are largely independent of energy/ROS metabolism.

FNDC4 reduces hepatocyte inflammatory cell death via AMPKα in metabolic dysfunction-associated steatotic liver disease

The molecular mediators responsible for the progression of metabolic dysfunction-associated steatotic liver disease (MASLD) to steatohepatitis (MASH) have not yet been completely disentangled. We sought to analyze whether FNDC4, an hepatokine and adipokine with anti-inflammatory properties, is involved in TNF-α-induced inflammatory cell death in patients with MASLD. Plasma FNDC4 (n=168) and hepatic FNDC4 and inflammatory cell death (n=65) were measured in samples from patients with severe obesity with available liver biopsy-proven MASLD diagnosis. The effect of FNDC4 on TNF-α-induced pyroptosis, apoptosis and necroptosis (PANoptosis) and mitochondrial dysfunction was studied invitro using human HepG2 hepatocytes. Compared with individuals with normal liver, patients with type 2 diabetes and MASLD exhibited decreased hepatic FNDC4 mRNA and protein levels, which were related to liver inflammation. An overexpression of TNF-α, its receptor TNF-R1 and factors involved in inflammatory cell death was also found in the liver of these patients. FNDC4-knockdown in HepG2 hepatocytes increased apoptotic cell death, while FNDC4 treatment blunted NLRP3 inflammasome-induced pyroptosis, apoptosis and necroptosis in TNF-α-stimulated hepatocytes. Moreover, FNDC4 improved TNF-α-induced hepatocyte mitochondrial dysfunction by enhancing mitochondrial DNA (mtDNA) copy number and OXPHOS complex subunits I, II, III and V protein expression. Mechanistically, AMP-activated protein kinase α (AMPKα) was required for the FNDC4-mediated inhibition of cell death and increase in mtDNA content. FNDC4 acts as a hepatocyte survival factor favouring mitochondrial homeostasis and decreasing inflammatory cell death via AMPKα. Collectively, our study identifies FNDC4 as an attractive target to prevent hepatocellular damage in patients with MASLD.

Bioenergetic function is decreased in peripheral blood mononuclear cells of veterans with Gulf War Illness.

Gulf War Illness (GWI) is a major health problem for approximately 250,000 Gulf War (GW) veterans, but the etiology of GWI is unclear. We hypothesized that mitochondrial dysfunction is an important contributor to GWI, based on the similarity of some GWI symptoms to those occurring in some mitochondrial diseases; the plausibility that certain pollutants to which GW veterans were exposed affect mitochondria; mitochondrial effects observed in studies in laboratory models of GWI; and previous evidence of mitochondrial outcomes in studies in GW veterans. A primary role of mitochondria is generation of energy via oxidative phosphorylation. However, direct assessment of mitochondrial respiration, reflecting oxidative phosphorylation, has not been carried out in veterans with GWI. In this case-control observational study, we tested multiple measures of mitochondrial function and integrity in a cohort of 114 GW veterans, 80 with and 34 without GWI as assessed by the Kansas definition. In circulating white blood cells, we analyzed multiple measures of mitochondrial respiration and extracellular acidification, a proxy for non-aerobic energy generation; mitochondrial DNA (mtDNA) copy number; mtDNA damage; and nuclear DNA damage. We also collected detailed survey data on demographics; deployment; self-reported exposure to pesticides, pyridostigmine bromide, and chemical and biological warfare agents; and current biometrics, health and activity levels. We observed a 9% increase in mtDNA content in blood in veterans with GWI, but did not detect differences in DNA damage. Basal and ATP-linked oxygen consumption were respectively 42% and 47% higher in veterans without GWI, after adjustment for mtDNA amount. We did not find evidence for a compensatory increase in anaerobic energy generation: extracellular acidification was also lower in GWI (12% lower at baseline). A subset of 27 and 26 veterans returned for second and third visits, allowing us to measure stability of mitochondrial parameters over time. mtDNA CN, mtDNA damage, ATP-linked OCR, and spare respiratory capacity were moderately replicable over time, with intraclass correlation coefficients of 0.43, 0.44, 0.50, and 0.57, respectively. Other measures showed higher visit-to-visit variability. Many measurements showed lower replicability over time among veterans with GWI compared to veterans without GWI. Finally, we found a strong association between recalled exposure to pesticides, pyridostigmine bromide, and chemical and biological warfare agents and GWI (p < 0.01, p < 0.01, and p < 0.0001, respectively). Our results demonstrate decreased mitochondrial respiratory function as well as decreased glycolytic activity, both of which are consistent with decreased energy availability, in peripheral blood mononuclear cells in veterans with GWI.

The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0h after warming, and 10 aneuploid blastocysts cultured for 4-5h after warming. A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Mitochondrial augmentation of hematopoietic stem cells in children with single large-scale mitochondrial DNA deletion syndromes

Patients with single large-scale mitochondrial DNA (mtDNA) deletion syndromes (SLSMDs) usually present with multisystemic disease, either as Pearson syndrome in early childhood or as Kearns-Sayre syndrome later in life. No disease-modifying therapies exist for SLSMDs. We have developed a method to enrich hematopoietic cells with exogenous mitochondria, and we treated six patients with SLSMDs through a compassionate use program. Autologous CD34+ hematopoietic cells were augmented with maternally derived healthy mitochondria, a technology termed mitochondrial augmentation therapy (MAT). All patients had substantial multisystemic disease involvement at baseline, including neurologic, endocrine, or renal impairment. We first assessed safety, finding that the procedure was well tolerated and that all study-related severe adverse events were either leukapheresis-related or related to the baseline disorder. After MAT, heteroplasmy decreased in the peripheral blood in four of the six patients. An increase in mtDNA content of peripheral blood cells was measured in all six patients 6 to 12 months after MAT as compared baseline. We noted some clinical improvement in aerobic function, measured in patients 2 and 3 by sit-to-stand or 6-min walk testing, and an increase in the body weight of five of the six patients suffering from very low body weight before treatment. Quality-of-life measurements as per caregiver assessment and physical examination showed improvement in some parameters. Together, this work lays the ground for clinical trials of MAT for the treatment of patients with mtDNA disorders.

Age-Related Decline in Nrf2/ARE Signaling Is Associated with the Mitochondrial DNA Damage and Cognitive Impairments

In this research, we compared the cognitive parameters of 2-, 7-, and 15-month-old mice, changes in mitochondrial DNA (mtDNA) integrity and expression of genes involved in the nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signaling pathway. We showed an age-related decrease in the Nfe2l2 expression in the cerebral cortex, not in the hippocampus. At the same time, we find an increase in the mtDNA copy number in the cerebral cortex, despite the lack of an increase in gene expression, which is involved in the mitochondrial biogenesis regulation. We suppose that increase in mtDNA content is associated with mitophagy downregulation. We supposed that mitophagy downregulation may be associated with an age-related increase in the mtDNA damage. In the hippocampus, we found a decrease in the Bdnf expression, which is involved in the pathways, which play an essential role in regulating long-term memory formation. We showed a deficit of working and reference memory in 15-month-old-mice in the water Morris maze, and a decrease in the exploratory behavior in the open field test. Cognitive impairments in 15-month-old mice correlated with a decrease in Bdnf expression in the hippocampus, Nfe2l2 expression, and an increase in the number of mtDNA damage in the cerebral cortex. Thus, these signaling pathways may be perspective targets for pharmacological intervention to maintain mitochondrial quality control, neuronal plasticity, and prevent the development of age-related cognitive impairment.

Mitochondrial genome recovery by ATFS-1 is essential for development after starvation

Nutrient availability regulates the C.elegans life cycle as well as mitochondrial physiology. Food deprivation significantly reduces mitochondrial genome (mtDNA) numbers and leads to aging-related phenotypes. Here we show that the bZIP (basic leucine zipper) protein ATFS-1, a mediator of the mitochondrial unfolded protein response (UPRmt), is required to promote growth and establish a functional germline after prolonged starvation. We find that recovery of mtDNA copy numbers and development after starvation requires mitochondrion-localized ATFS-1 but not its nuclear transcription activity. We also find that the insulin-like receptor DAF-2 functions upstream of ATFS-1 to modulate mtDNA content. We show that reducing DAF-2 activity represses ATFS-1 nuclear function while causing an increase in mtDNA content, partly mediated by mitochondrion-localized ATFS-1. Our data indicate the importance of the UPRmt in recovering mitochondrial mass and suggest that atfs-1-dependent mtDNA replication precedes mitochondrial network expansion after starvation.

MITOCHONDRIAL DNA CONTENT BEFORE VITRIFICATION (Mitoscore®) IS NOT A RELIABLE MARKER OF MITOCHONDRIAL MASS AND ACTIVITY IN THE HUMAN BLASTOCYST: A CONFOCAL AND ELECTRON MICROSCOPY APPROACH

To evaluate whether there is a correlation between the increase in mitochondrial DNA (mtDNA) content observed in post-warming human blastocysts, their redox state and their mitochondrial mass measured by confocal and electron microscopy, respectively. A prospective cohort study was carried out with a total of 17 aneuploid blastocysts. To calculate the redox ratio, NADH and FAD autofluorescence quantification was performed with 10 of them, 5 blastocysts cultured 5 hours after warming and other 5 blastocysts cultured a maximum of 1 hour after warming. Moreover, an electronic microscopy analysis of 7 blastocysts was performed, 3 of them cultured 5 hours after warming and another 4 cultured a maximum of 1 hour after warming by visual count of mitochondria. The statistical study for both experiments was conducted by correlation analysis and T-test. No significant differences (P>0.05) were observed regarding the redox ratio between just-warmed blastocysts (average redox ratio= 1.49) and blastocysts recovering after warming (average redox ratio= 1.26). Regarding the mitochondrial mass, no significant differences (P> 0.05) in the number of mitochondria were observed (just-warmed average mitochondria per cell= 30.77 vs recovering-after-warming average mitochondria per cell= 30.94). Furthermore, we do not observe differences in the mitochondrial matrix configuration that could indicate differences at the level of mitochondrial activity between the two groups. Despite the increase in mtDNA content observed after the stressful vitrification-warming process in human blastocysts (Pérez-Sánchez, ESHRE 2019), no consequential increase in redox activity or mitochondrial mass is observed.

P–189 The transcriptional profile of arrested cleavage stage human embryos

Abstract Study question What are the molecular pathways overactivated in arrested cleavage stage human embryos? Summary answer There is an upregulation of mitochondrial activity and cellular stress-related pathways in arrested cleavage embryos, which is in agreement with the “quiet hypothesis”. What is known already mtDNA content decreases during embryo development, however there is a high increase in mtDNA content in arrested cleavage embryos that may correspond to a response to intrinsic or extrinsic factor creating stress. This reasoning would be in agreement with stablished hypothesis showing that a basal metabolism with a moderate-low energy consumption is actually a sign of embryo health, so a viable embryo does not need to use an extra energy to accommodate to the environment (Leese, 2012). The study of the transcriptional profile during human embryo development will give further information about key molecular process involved in in-vitro embryonic competence. Study design, size, duration A prospective cohort study was performed with 11 MII oocytes (average age= 22,9 years), 10 non-arrested cleavage embryos (average age= 29,9 years), 5 arrested cleavage embryos (average age= 38,8 years) and 8 blastocysts (average age= 39,1 years). All specimens were warmed and sampled in PCR tubes with 2 μl of suitable reaction buffer for the RNA sequencing protocol. Participants/materials, setting, methods Specimens were analyzed by single-cell RNA sequencing (scRNA-seq). Correlation studies, principal component and differential expression analysis were performed with DESeq2 package. Differential gene expression analyses were done using the parametric Wald test, with Benjamini-Hochberg multiple test correction (padj). Finally, Fgsea algorithm was used for enrichment analysis on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene ontology (GO) terms. Main results and the role of chance We do not observe mitochondria-related activity pathways significantly (P&amp;gt;.05) deregulated between MII oocytes and non-arrested cleavage embryos considering GO and KEGG categories. When comparing non-arrested cleavage embryos versus blastocysts, we observe several ATP production/consumption and cristae formation-related pathways significantly (P&amp;lt;.05) upregulated in blastocysts compared to non-arrested cleavage embryos considering GO and KEGG categories. This change in activity coincides with the metabolic activation event that occurs in the blastocyst stage. However, when we analyze arrested cleavage embryos versus non-arrested cleavage embryos, we observe several ATP production related-pathways and mitochondria-related apoptosis pathways significantly (P&amp;lt;.05) upregulated in arrested cleavage embryos compared to non-arrested cleavage embryos considering GO categories. With KEGG categories, we notice a significant (P&amp;lt;.05) upregulation of oxidative phosphorylation in arrested cleavage embryos. On the other hand, when we analyze the differences between arrested cleavage embryos and blastocysts, taking into account the differences related to the change of embryo stage, we do not observe ATP production or consumption-related pathways significantly (P&amp;gt;.05) deregulated considering GO and KEGG categories. Then, human arrested embryos in parallel with the increase in mtDNA content, display an upregulation of mitochondrial activity and cellular stress which is in line with the expected overactive metabolism of non-viable embryos. Limitations, reasons for caution All analyzed blastocyst were aneuploid, so we are unable to determine what the results would be with euploid blastocysts. Also, although age can it be ruled out, no differences were observed between mean age from cleavage stage embryos (arrested and non-arrested ones) and blastocyst. Wider implications of the findings: This study evidences the relation between extreme mtDNA content increase and the identification of the pathways involved in active metabolism and apoptosis in arrested cleavage stage human embryos. Trial registration number Not applicable

Daily Oral Supplementation with 60 mg of Elemental Iron for 12 Weeks Alters Blood Mitochondrial DNA Content, but Not Leukocyte Telomere Length in Cambodian Women.

There is limited evidence regarding the potential risk of untargeted iron supplementation, especially among individuals who are iron-replete or have genetic hemoglobinopathies. Excess iron exposure can increase the production of reactive oxygen species, which can lead to cellular damage. We evaluated the effect of daily oral supplementation on relative leukocyte telomere length (rLTL) and blood mitochondrial DNA (mtDNA) content in non-pregnant Cambodian women (18–45 years) who received 60 mg of elemental iron as ferrous sulfate (n = 190) or a placebo (n = 186) for 12 weeks. Buffy coat rLTL and mtDNA content were quantified by monochrome multiplex quantitative polymerase chain reaction. Generalized linear mixed-effects models were used to predict the absolute and percent change in rLTL and mtDNA content after 12 weeks. Iron supplementation was not associated with an absolute or percent change in rLTL after 12 weeks compared with placebo (ß-coefficient: −0.04 [95% CI: −0.16, 0.08]; p = 0.50 and ß-coefficient: −0.96 [95% CI: −2.69, 0.77]; p = 0.28, respectively). However, iron supplementation was associated with a smaller absolute and percent increase in mtDNA content after 12 weeks compared with placebo (ß-coefficient: −11 [95% CI: −20, −2]; p = 0.02 and ß-coefficient: −11 [95% CI: −20, −1]; p= 0.02, respectively). Thus, daily oral iron supplementation for 12 weeks was associated with altered mitochondrial homeostasis in our study sample. More research is needed to understand the risk of iron exposure and the biological consequences of altered mitochondrial homeostasis in order to inform the safety of the current global supplementation policy.

Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research

To assess the mitochondrial DNA (mtDNA) load and variation in human oocytes and during preimplantation embryo development using specimens donated for research. Prospective cohort study. Not applicable. A total of 50 invitro fertilization patients and 11 oocyte donors whose specimens were obtained between July 2017 and July2018. None. All specimens were separately collected. Quantitative polymerase chain reaction was performed with SurePlex DNA Amplification System (Illumina). Primers for the adenosine triphosphate 8 mitochondrial gene and the β-actin were used. Data were statistically analyzed by analysis of variance with the Scheffé multiple pairwise comparison for categorical variables and by linear regression for numerical variables. Human metaphase II (MII) oocytes had significantly more total mtDNA copy number than day 3 embryos, and day 3 embryos had more total and per-cell mtDNA copy number than aneuploid blastocysts. There was a significant decrease in mtDNA content associated with failed-fertilized oocytes compared to noninseminated metaphase II oocytes. During preimplantation development, before implantation, human embryos undergo a significant decrease in total mtDNA content and no increase in mtDNA content at the blastocyst stage. Oocytes need to carry a correct threshold of mitochondrial load in the oocyte in order to successfully fertilize. An active degradation of mtDNA before implantation occurs after fertilization takes place. These findings could be used to improve knowledge about the best embryo culture conditions and would serve as a basis for further studies addressing again the use of mtDNA content as an embryo viability marker.

Deletion of OGG1 Results in a Differential Signature of Oxidized Purine Base Damage in mtDNA Regions.

Mitochondrial oxidative stress accumulates with aging and age-related diseases and induces alterations in mitochondrial DNA (mtDNA) content. Since mtDNA qualitative alterations are also associated with aging, repair of mtDNA damage is of great importance. The most relevant form of DNA repair in this context is base excision repair (BER), which removes oxidized bases such as 8-oxoguanine (8-oxoG) and thymine glycol through the action of the mitochondrial isoform of the specific 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase (OGG1) or the endonuclease III homolog (NTH1). Mouse strains lacking OGG1 (OGG1−/−) or NTH1 (NTH1−/−) were analyzed for mtDNA alterations. Interestingly, both knockout strains presented a significant increase in mtDNA content, suggestive of a compensatory mtDNA replication. The mtDNA “common deletion” was not detected in either knockout mouse strain, likely because of the young age of the mice. Formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites accumulated in mtDNA from OGG1−/− but not from NTH1−/− mice. Interestingly, the D-loop region was most severely affected by the absence of OGG1, suggesting that this region may be a hotspot for oxidative damage. Thus, we speculate that mtDNA alterations may send a stress message to evoke cell changes through a retrograde mitochondrial–nucleus communication.

Altered cord blood mitochondrial DNA content and pregnancy lead exposure in the PROGRESS cohort

IntroductionLead (Pb) crosses the placenta and can cause oxidative stress, reduced fetal growth and neurological problems. The principal source of oxidative stress in human cells is mitochondria. Therefore, disruption of normal mitochondrial function during pregnancy may represent a primary mechanism behind the adverse effects of lead. We sought to assess the association of Pb exposure during pregnancy with mitochondrial DNA (mtDNA) content, a sensitive marker of mitochondrial function, in cord blood. Materials and methodsThis study comprised mother-infant pairs from the Programming Research in Obesity, Growth, Environment and Social Stressors (PROGRESS) study, a prospective birth-cohort that enrolled 1050 pregnant women from Mexico City who were receiving prenatal care between December 2007 and July 2011. Quantitative PCR was used to calculate relative MtDNA content (mitochondrial-to-nuclear DNA ratio (mtDNA/nDNA)) in cord blood. Lead concentrations in both maternal blood (2nd and 3rd trimester and at delivery day) and in cord blood were measured by ICP-MS. Multivariable regression models adjusting for multiple confounders were fitted with 410 mother-infant pairs for whom complete data for mtDNA content, lead levels, and covariates were available. ResultsMaternal blood Pb measured in the second (mean 3.79 μg/dL, SD 2.63; β = 0.059, 95% CI 0.008, 0.111) and third trimester (mean 3.90 μg/dL; SD 2.84; β = 0.054, 95% CI 0.002, 0.107) during pregnancy and PB in cord blood (mean 3.50 μg/dL, SD 2.59; β = 0.050, 95% CI 0.004; 0.096) were associated with increased cord blood mtDNA content (mean 1.46, SD 0.44). In two-way interaction analyses, cord blood Pb marginally interacted with gestational age leading to an increase in mtDNA content for pre-term births (Benjamini-Hochberg False Discovery Rate correction; BH-FDR = 0.08). ConclusionThis study shows that lead exposure in pregnancy alters mtDNA content in cord blood; therefore, alteration of mtDNA content might be a mechanism underlying the toxicity of lead.

Leber\u2019s hereditary optic neuropathy, intellectual disability and epilepsy presenting with variable penetrance associated to the m.3460G\u2009>A mutation and a heteroplasmic expansion of the microsatellite in MTRNR1 gene \u2013 case report

BackgroundLeber’s hereditary optic neuropathy (LHON) associated with mutations in mitochondrial DNA (mtDNA) typically manifests only optic nerve involvement but in some patients may develop additional neurological complications. The cause of this association is not clear.Case presentationWe present a case of a 24-year-old male with a history of subacute, painless, and rapidly progressive bilateral vision loss. We performed ophthalmological, neurological and neuropsychological investigations in the proband and his LHON family. The proband showed optic neuropathy, epilepsy, migraine, and intellectual disability; all the maternal relatives did not manifest optic neuropathy but a moderate to severe intellectual disability. Genetic screening revealed a novel association of the LHON m.3460G > A primary mutation with the m.T961delT + C(n)ins within the mitochondrial encoded 12S RNA (MTRNR1) gene which segregates with the intellectual disability through the maternal branch of the family. We also found a significant increase of mtDNA content in all the unaffected homo/heteroplasmic mutation carriers with respect to either affected or control subjects.ConclusionThis is the first case reporting the co-segregation of a mutation in MTRNR1 gene with a LHON primary mutation, which may be a risk factor of the extraocular signs complicating LHON phenotype. In addition, the data herein reported, confirmed that the key factor modulating the penetrance of optic atrophy in the family is the amount of mtDNA.

Mutant eIF2B leads to impaired mitochondrial oxidative phosphorylation in vanishing white matter disease.

Eukaryotic translation initiation factor 2B (eIF2B) is a master regulator of protein synthesis under normal and stress conditions. Mutations in any of the five genes encoding its subunits lead to vanishing white matter (VWM) disease, a recessive genetic deadly illness caused by progressive loss of white matter in the brain. In this study we used fibroblasts, which are not involved in the disease, to demonstrate the involvement of eIF2B in mitochondrial function and abundance. Mass spectrometry of total proteome of mouse embryonic fibroblasts (MEFs) isolated from Eif2b5R132H/R132H mice revealed unbalanced stoichiometry of proteins involved in oxidative phosphorylation and of mitochondrial translation machinery components, among others. Mutant MEFs exhibit 55% decrease in oxygen consumption rate per mtDNA content and 47% increase in mitochondrial abundance (p<0.005), reflecting adaptation to energy requirements. A more robust eIF2B-associated oxidative respiration deficiency was found in mutant primary astrocytes, which exhibit >3-fold lower ATP-linked respiration per cell despite a 2-fold increase in mtDNA content (p<0.03). The 2-fold increase in basal and stimulated glycolysis in mutant astrocytes (p≤0.03), but not in MEFs, demonstrates their higher energetic needs and further explicates their involvement in the disease. The data demonstrate the critical role of eIF2B in tight coordination of expression from nuclear and mitochondrial genomes and illuminates the importance of mitochondrial function in VWM pathology. Further dissection of the signaling network associated with eIF2B function will help generating therapeutic strategies for VWM disease and possibly other neurodegenerative disorders.

Effect of High Folic Acid Diet on Mitochondrial DNA Content in Young Adult and Aged Mice

Mitochondrial DNA (mtDNA) codes for proteins of oxidative phosphorylation system and maintaining adequate mtDNA copy number is necessary for cell survival. Studies in animal models have shown that folate deficiency can affect mtDNA content. Current data suggests excess folic acid intake can result in conditions that resemble folate deficiency. We tested the hypothesis that excess folic acid in diet can influence mtDNA gene content in old and young mice. Young (3 months) and old mice (16 months) mice were fed a control (1xRDA) or high folic acid (20 x RDA) diet for 3 months (n=8–11/ group). The colonic mucosal mtDNA content was determined by real‐time PCR using primers specific for mtDNA and normalized to an endogenous reference gene, mapK. The data were analyzed using student's t‐test. Among mice on control diet, mtDNA content of old mice was 0.4 fold that of the young mice (p=0.004). There was no difference in mtDNA content of young mice on control vs high folic acid diet. MtDNA content of the old mice fed a high folic acid diet was 7‐fold higher than those fed a control diet (p=0.001). While higher mtDNA copy number is considered to have beneficial effects on survival, very high mtDNA copy number has been shown to be accompanied by nucleoid enlargement, defective transcription and mtDNA deletions in old mice. Thus the dramatic increase in mtDNA content due to excess folic acid diet may be detrimental. Further research is required to establish if the high mtDNA content due a high folic acid diet can result in mitochondrial dysfunction in aged mice.Support or Funding InformationSupport from USDA cooperative agreement 51520‐008‐04S and pilot grant from JM USDA HNRCA on Aging at Tufts University

Role of mtDNA Haplogroups in the Prevalence of Knee Osteoarthritis in a Southern Chinese Population

Mitochondrial DNA (mtDNA) has been implicated in various human degenerative diseases. However, the role of mtDNA in Osteoarthritis (OA) is less known. To investigate whether mtDNA haplogroups contribute to the prevalence of knee OA, we have carried out a comprehensive case-control study on 187 knee OA patients and 420 geographically matched controls in southern China. OA patients were classified on the Kellgren/Lawrence scale from two to four for the disease severity study and the data were analyzed by adjusting for age and sex. We found that patients with haplogroup G (OR = 3.834; 95% CI 1.139, 12.908; p = 0.03) and T16362C (OR = 1.715; 95% CI 1.174, 2.506; p = 0.005) exhibited an increased risk of OA occurrence. Furthermore, patients carrying haplogroup G had a higher severity progression of knee OA (OR = 10.870; 95% CI 1.307, 90.909; p = 0.007). On the other hand, people with haplogroup B/B4 (OR = 0.503; 95% CI 0.283, 0.893; p = 0.019)/(OR = 0.483; 95% CI 0.245, 0.954; p = 0.036) were less susceptible for OA occurrence. Interestingly, we found OA patients also exhibited a general increase in mtDNA content. Our study indicates that the mtDNA haplogroup plays a role in modulating OA development.

Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane is the most effective medical therapy for adrenocortical carcinoma, but its molecular mechanism of action remains poorly understood. Although mitotane is known to have mitochondrial (mt) effects, a direct link to mt dysfunction has never been established. We examined the functional consequences of mitotane exposure on proliferation, steroidogenesis, and mt respiratory chain, biogenesis and morphology, in two human adrenocortical cell lines, the steroid-secreting H295R line and the non-secreting SW13 line. Mitotane inhibited cell proliferation in a dose- and a time-dependent manner. At the concentration of 50 μM (14 mg/l), which corresponds to the threshold for therapeutic efficacy, mitotane drastically reduced cortisol and 17-hydroxyprogesterone secretions by 70%. This was accompanied by significant decreases in the expression of genes encoding mt proteins involved in steroidogenesis (STAR, CYP11B1, and CYP11B2). In both H295R and SW13 cells, 50 μM mitotane significantly inhibited (50%) the maximum velocity of the activity of the respiratory chain complex IV (cytochrome c oxidase (COX)). This effect was associated with a drastic reduction in steady-state levels of the whole COX complex as revealed by blue native PAGE and reduced mRNA expression of both mtDNA-encoded COX2 (MT-CO2) and nuclear DNA-encoded COX4 (COX4I1) subunits. In contrast, the activity and expression of respiratory chain complexes II and III were unaffected by mitotane treatment. Lastly, mitotane exposure enhanced mt biogenesis (increase in mtDNA content and PGC1α (PPARGC1A) expression) and triggered fragmentation of the mt network. Altogether, our results provide first evidence that mitotane induced a mt respiratory chain defect in human adrenocortical cells.

Folate deficiency increases mtDNA and D-1 mtDNA deletion in aged brain of mice lacking uracil-DNA glycosylase

Strong epidemiological and experimental evidence links folate deficiency and resultant hyperhomocysteinemia with cognitive decline and neurodegeneration. Here, we tested the hypothesis that uracil misincorporation contributes to mitochondrial pathology in aged brain following folate deprivation. In a 2 × 2 design, 14-month-old mice lacking uracil DNA glycosylase ( Ung−/−) versus wild-type controls were subjected to a folate-deficient versus a regular diet for six weeks. Folate-deficient feeding significantly enhanced mtDNA content and overall abundance of the D-1 mtDNA deletion in brain of Ung−/−, but not of wild-type mice. Independent of folate status, the frequency of the D-1 mtDNA deletion in mtDNA was significantly increased in Ung−/− mice. The rate of mitochondrial biogenesis as assessed at six weeks of the experimental diet by mRNA expression levels of transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and of mitochondrial transcription factor A (Tfam) was not affected by either Ung−/− genotype or short-term folate deficiency. Similarly, citrate synthase (CS) activity in the brain did not differ across experimental groups. By contrast, independent of genotype, lactate dehydrogenase (LDH) activity was significantly reduced in folate-deficient animals. Our results suggest that impaired uracil excision repair causes an increase in mitochondrial mutagenesis in aged brain along with a compensatory increase in mtDNA content in response to low folate status. Folate deficiency may contribute to neurodegeneration via mtDNA damage.

Improvement of Mitochondrial Toxicity in Patients Receiving a Nucleoside Reverse‐Transcriptase Inhibitor–Sparing Strategy: Results from the Multicenter Study with Nevirapine and Kaletra (MULTINEKA)

Nucleoside reverse-transcriptase inhibitor (NRTI)-related mitochondrial toxicity has been suggested as a key factor in the induction of antiretroviral-related lipoatrophy. This study aimed to evaluate in vivo the effects of NRTI withdrawal on mitochondrial parameters and body fat distribution. A multicenter, prospective, randomized trial assessed the efficacy and tolerability of switching to lopinavir-ritonavir plus nevirapine (nevirapine group; n = 34), compared with lopinavir-ritonavir plus 2 NRTIs (control group; n = 33) in a group of human immunodeficiency virus-infected adults with virological suppression. A subset of 35 individuals (20 from the nevirapine group and 15 from the control group) were evaluated for changes in the mitochondrial DNA (mtDNA) to nuclear DNA ratio and cytochrome c oxidase (COX) activity after NRTI withdrawal. Dual-energy X-ray absorptiometry (DEXA) scans were used to objectively quantify fat redistribution over time. The nevirapine group experienced a progressive increase in mtDNA content (a 40% increase at week 48; P = .039 for comparison between groups) and in the COX activity (26% and 32% at weeks 24 and 48, respectively; P = .01 and P = .09 for comparison between groups, respectively). There were no statistically significant between-group differences in DEXA scans at week 48, although a higher fat increase in extremities was observed in the nevirapine group. No virologic failures occurred in either treatment arm. Switching to a nucleoside-sparing regimen of nevirapine and lopinavir-ritonavir maintained full antiviral efficacy and led to an improvement in mitochondrial parameters, which suggests a reversion of nucleoside-associated mitochondrial toxicity. Although DEXA scans performed during the study only revealed slight changes in fat redistribution, a longer follow-up period may show a positive correlation between reduced mitochondrial toxicity and a clinical improvement of lipodystrophy.