TFAM Gene Research Articles

Expression of genes related to mitochondrial function in Nellore cattle divergently ranked on residual feed intake.

Several measures have been proposed to investigate and improve feed efficiency in cattle. One of the most commonly used measure of feed efficiency is residual feed intake (RFI), which is estimated as the difference between actual feed intake and expected feed intake based on the animal's average live weight. This measure permits to identify and select the most efficient animals without selecting for higher mature weight. Mitochondrial function has been indicated as a major factor that influences RFI. The analysis of genes involved in mitochondrial function is therefore an alternative to identify molecular markers associated with higher feed efficiency. This study analyzed the expression of PGC1α, TFAM, UCP2 and UCP3 genes by quantitative real-time PCR in liver and muscle tissues of two groups of Nellore cattle divergently ranked on RFI values in order to evaluate the relationship of these genes with RFI. In liver tissue, higher expression of TFAM and UCP2 genes was observed in the negative RFI group. Expression of PGC1α gene did not differ significantly between the two groups, whereas UCP3 gene was not expressed in liver tissue. In muscle tissue, higher expression of TFAM gene was observed in the positive RFI group. Expression of PGC1α, UCP2 and UCP3 genes did not differ significantly between the two groups. These results suggest the use of TFAM and UCP2 as possible candidate gene markers in breeding programs designed to increase the feed efficiency of Nellore cattle.

Upregulation of TFAM and mitochondria copy number in human lymphoblastoid cells

Mitochondria are central to several physiological and pathological conditions in humans. In the present study, we performed copy number analysis of nuclear encoded mitochondrial genes, in peripheral blood mononuclear cells (PBMCs) and its representative lymphoblastoid cells (LCLs). We have observed hyper diploid copies of mitochondrial transcription factor A (TFAM) gene in the LCLs along with increased mtDNA copy number, mitochondrial mass, intracellular ROS and mitochondrial membrane potential, suggesting elevated mitochondrial biogenesis in LCLs. Gene expression analysis confirmed TFAM over-expression in LCLs when compared to PBMC. Based on our observation, we suggest that increased copy number of TFAM gene upregulates its expression, increases mtDNA copy numbers and protects it from oxidative stress induced damage in the transformed LCLs.

Impaired transport of mitochondrial transcription factor A (TFAM) and the metabolic memory phenomenon associated with the progression of diabetic retinopathy

Diabetes damages retinal mitochondrial DNA (mtDNA) and compromises the mtDNA transcription. In the transcription and replication of mtDNA, nuclear-encoded mitochondrial transcription factor A (TFAM) is considered a key activator. We have shown that in diabetes, although retinal TFAM gene expression is increased, its mitochondrial levels are decreased. This study investigates the role of mitochondrial outer and inner membrane transport systems in the transfer of TFAM into the mitochondria in diabetes and how reversal of hyperglycaemia affects the ability of TFAM to reach the mitochondria. Components of the membrane transport system, Tom70, Tom40, Tim23, and Tim44, were analysed in the retina from streptozotocin-induced diabetic rats maintained in poor control or in good control for 8 months, or in poor control for 4 months followed by in good control for 4 months. The binding of TFAM with Tom70 and Tim44 was determined by co-immunoprecipitation and that with mtDNA by chromatin immunoprecipitation. Retinal expressions of Tom70, Tom40, and Tim44 were significantly decreased in diabetes, and the binding of TFAM with Tom70, Tim44, and mtDNA was impaired. Reversal of hyperglycaemia had no beneficial effect on the decreased binding of TFAM to Tom proteins and mtDNA. Thus, subnormal membrane transport to systems in diabetes impair the transfer of TFAM into the mitochondria, and decreased TFAM-mtDNA binding that results in subnormal mitochondria transcription. These processes continue to be dysfunctional even after the hyperglycaemic insult is terminated. Strategies targeting mitochondrial membrane transport proteins could have the potential of improving mitochondrial biogenesis and slowing or halting the progression of diabetic retinopathy.

Association of Thr12Ser polymorphism in mitochondrial transcription factor A TFAM gene with coronary artery disease

In this study we genotyped polymorphism Thr12Ser (rs1937) in TFAM gene in two groups: patients with coronary artery disease and control group (people from Tomsk population). For genotyping was used restriction fragment length polymorphism. We have found significant higher genotype CC frequency in men with coronary artery disease — 4.52% compared with control men — 0.45% (χ 2 = 6.315; р = 0.043; OR = 10.45). Thus genotype CC is associated with coronary artery disease in men fromTomsk population.

Intracellular and extracellular ATP coordinately regulate the inverse correlation between osteoclast survival and bone resorption.

Osteoclasts, highly differentiated bone-resorbing cells of hematopoietic origin, have two conflicting tendencies: a lower capacity to survive and a higher capacity to execute energy-consuming activities such as bone resorption. Here, we report that when compared with their precursors, mature mitochondria-rich osteoclasts have lower levels of intracellular ATP, which is associated with receptor activator of nuclear factor κ-B ligand (RANKL)-induced Bcl-x(L) down-regulation. Severe ATP depletion, caused by disrupting mitochondrial transcription factor A (Tfam) gene, leads to increased bone-resorbing activity despite accelerated apoptosis. Although AMP-activated protein kinase (AMPK) activation by ATP depletion is not involved in the regulation of osteoclast function, the release of ATP from intracellular stores negatively regulates bone-resorbing activity through an autocrine/paracrine feedback loop by altering cytoskeletal structures. Furthermore, osteoclasts derived from aged mice exhibit reduced mitochondrial DNA (mtDNA) and intracellular ATP levels with increased bone-resorbing activity, implicating the possible involvement of age-related mitochondrial dysfunction in osteoporosis. Thus, our study provides evidence for a mechanism underlying the control of cellular functions by reciprocal changes in intracellular and extracellular ATP, which regulate the negative correlation between osteoclast survival and bone resorption.

Positive effect of curcumin on inflammation and mitochondrial dysfunction in obese mice with liver steatosis

Obesity contributes to the increased risk of liver- related morbidity and mortality. The accumulation of macrophages in adipose tissue in an inflammatory state is a hallmark of obesity-induced hepatic steatosis. In this study, we reveal the role of curcumin in the molecular mechanisms underlying inflammatory events in a model consisting of obese mice with hepatic steatosis. Obese mice fed with curcumin experienced significant weight loss and significantly reduced serum triglyceride (TG) levels. The adipose tumor necrocis factor-α, interleukin-6 (IL-6) and monocyte chemotactic protein-1 levels were significantly higher in obese mice compared to mice fed with curcumin. Compared to the obese mice, curcumin decreased the number of F4/80-positive macrophages in epididymal adipose and liver tissue. The induction of signal transducer and activator of transcription 3 phosphorylation by curcumin resulted in the downregulation of the suppressor of cytokine signaling 3 in the liver of obese mice. Curcumin decreased hepatic TG in obese mice by downregulating the gene expression of sterol regulatory element-binding protein-1c in the liver. The hepatic expression of several mitochondrial biogenesis genes decreased in the obese mice, including mitochondrial DNA (mtDNA), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam), which are responsible for the lower mitochondrial respiratory chain (MRC) complex I activity and adenosine triphosphate production. By contrast, obese mice treated with curcumin showed normalized mtDNA, NRF1 and Tfam gene expression, reduced hepatic nuclear factor-κB activities and levels of thiobarbituric acid reactive substances (TBARS) and restored mitochondrial oxidative metabolism and biogenesis. The results from the present sudy show that curcumin prevents fatty liver disease through multiple mechanisms, and suggest that curcumin may be used to prevent the development and progression of non-alcoholic fatty liver disease (NAFLD).

Effects of Short and Prolonged Mild Intracellular Nitric Oxide Manipulations on Various Aspects of Insulin Secretion in INS-1E β-Cells

We aimed at evaluating the impact of short and prolonged mild manipulations of intracellular nitric oxide (NO) bioavailability on the main features of insulin secretion and whether NO promotes mitochondrial biogenesis in isolated β-cells. INS-1E β-cells were exposed to either the intracellular NO donor, hydroxylamine (HA), or the NO synthase inhibitor, L-nitro-arginine-methyl-ester (L-NAME), at concentrations lower than 2.0 mM. Glucose and arginine-induced insulin secretion (GIIS and AIIS) were measured after short (1 h) or prolonged (48 h) exposure to L-NAME 1.0 and 2.0 mM or HA 0.4 and 0.8 mM, lower concentrations were also evaluated for the 1 h effects. Basal insulin secretion (BIS), with either HA or L-NAME added to culture media, and peroxisome proliferators-activated receptor γ coactivator 1α (PGC-1α), nuclear respiratory factor-1 (NRF-1), and mitochondrial DNA transcription factor-A (Tfam) gene expression during chronic HA supplementation were also measured. Neither L-NAME nor HA affected insulin release at glucose 3.3 mM or in cell culture (BIS). Both short and prolonged cell exposure to L-NAME potentiated GIIS though with a flat dose-response curve while HA inhibited GIIS only at the highest concentration. AIIS was prevented by short exposure to L-NAME and potentiated by HA, while it did not respond to prolonged incubations. Prolonged cell exposure to HA had no effect on PGC-1α, NRF-1 or Tfam gene expression. In INS1E cells an intact NO synthesis is necessary to limit insulin release in response to acute glucose gradients and to fully respond to arginine while intracellular NO enrichment above the physiologic levels further inhibits GIIS and potentiate AIIS only when excessive. Prolonged NO manipulations do not affect AIIS, BIS or mitochondrial biogenesis.

Mitochondrial transcription factor A ( TFAM) polymorphisms and risk of late-onset Alzheimer's disease in Han Chinese

Chronic mitochondria DNA (mtDNA) damage and mitochondrial dysfunction induced by increased reactive oxygen species (ROS) have been proved to contribute to the development of Alzheimer's disease (AD). Mitochondrial transcription factor A (TFAM) plays an important role in the maintenance of mtDNA integrity. Recently, some studies suggested two single nucleotide polymorphisms (SNPs) (rs1937 and rs2306604) in the TFAM gene are associated with sporadic late-onset AD (LOAD) in Caucasians. To explore the correlation between TFAM gene and LOAD, we performed a case-control study in a large Chinese cohort consisting of 394 patients and 390 healthy controls. The results showed that there were significant differences in genotype ( P = 0.03) and allele ( P = 0.04) frequencies of the SNP rs1937 between LOAD patients and controls. The minor C allele of rs1937 acted as a moderate protective factor of LOAD ( P = 0.04, odds ratios/OR = 0.76). The logistic regression analysis also suggested an association of LOAD with SNP rs1937 (dominant model: P = 0.03, OR = 0.71; recessive model: P = 0.02, OR = 0.25; additive model: P = 0.01, OR = 0.68). No significant association was observed between rs2306604 and LOAD. Haplotype analysis identified the haplotype CC as a protective factor of LOAD ( P = 0.038, OR = 0.76). This study provides the evidence that variations in TFAM are involved in the pathogenesis of sporadic LOAD in the Han Chinese population.

Mitochondrial DNA and TFAM gene variation in early-onset myocardial infarction: Evidence for an association to haplogroup H

The main objective of this research was to define the association between common mitochondrial DNA (mtDNA) polymorphisms and mitochondrial transcription A gene ( TFAM) variants and myocardial infarction (MI) in patients with atherosclerotic diseased vessels. Ten mitochondrial polymorphisms that defined the nine common European haplogroups were genotyped in 500 male patients with early onset MI (< 55 years) and at least one atherosclerotic coronary vessel (angiographically confirmed), and 500 healthy controls. In addition, we searched for DNA variants in the coding region of the TFAM gene and compared patients and controls for the allele and genotype frequencies. Early onset MI was strongly associated with male gender and tobacco smoking in our population. MtDNA haplogroup H (defined by allele 7028 °C) was significantly more frequent in a first group of patients ( n = 250) compared to controls ( n = 300), and the association was confirmed in a second group of only smokers (250 patients and 200 controls). For total patients and controls, we obtained a p = 0.002 (OR = 1.50; 95% CI = 1.17–1.92) for H vs. the other haplogroups. We found four common TFAM polymorphisms, with allele/genotype frequencies that did not differ between patients and controls. In conclusion, mitochondrial haplogroup H was associated with early onset MI in male smokers. Our work supported a role for the mtDNA variation in the risk for atherosclerosis and ischemic associated events, likely due to differences in mitochondrial function and reactive oxygen production between the different haplogroups.

Association of mitochondrial transcription factor (TFAM) gene polymorphism with physical performance in athletes

The aim of the study was to investigate allelic distribution of the mitochondrial transcription factor gene (TFAM) Ser12Thr polymorphism in athletes (n = 1537) and controls (n = 1113), and to find interrelation between genotypes and aerobic capacity in rowers (n = 90). Genotyping was performed by restriction fragment length polymorphism analysis. Aerobic capacity (maximal oxygen consumption (VO2max) and maximal power production capacity (Wmax)) was determined using an incremental test to exhaustion by rower ergometer. The frequency of TFAM 12Thr allele was significantly higher in endurance-oriented athletes (n = 588) than in controls (14.0% vs. 9.1%; p <0.0001), and increased with the growth of skills. Furthermore, TFAM 12Thr allele was associated with high values of aerobic performance (when Wmax and VO2max were measured). Thus, TFAM gene Ser12Thr polymorphism is associated with physical performance of athletes.

Methylation of TFAM gene promoter in peripheral white blood cells is associated with insulin resistance in adolescents

PurposeTo explore whether DNA methylation of the mitochondrial transcription factor A (TFAM) promoter is associated with insulin resistance in a sample of adolescents with features of metabolic syndrome. MethodsThe data and blood samples were collected from 122 adolescents out of a cross-sectional study of 934 high-school students. The population was divided into two groups: noninsulin resistance (NIR) and insulin resistance (IR). After bisulfite treatment of genomic DNA from peripheral leukocytes, we used methylation-specific polymerase chain reaction (PCR) to assess DNA methylation of three putative methylation target sites (CpG) in the TFAM promoter. ResultsThe ratio of the promoter methylated DNA/unmethylated DNA was 0.012±0.0009 (1.2% of alleles), and inversely correlated with the biochemical features of insulin resistance (plasma fasting insulin R: −0.26, p<0.004 and homeostasis model assessment (HOMA) index R: −0.27, p<0.002), and obesity (R: −0.27, p<0.002). Multiple regression analysis showed that the log-transformed HOMA index correlated with the status of promoter methylation of TFAM, independently of body mass index (BMI) Z score (β: −0.33±0.094, p=0.00094). Finally, the TFAM promoter methylated DNA/unmethylated DNA ratio was found to be significantly associated with insulin resistance as dichotomous variable (NIR n=45, 0.014±0.002 and IR n=77, 0.011±0.001, respectively, p<0.016). ConclusionOur findings suggest a potential role of promoter TFAM methylation in the pathogenesis of insulin resistance in adolescents.

Evaluation of TFAM and FABP4 gene polymorphisms in three lines of Nellore cattle selected for growth

We analyzed the polymorphisms TFAM HaeIII, TFAM MboI and FABP4 MspA1I in three Nellore lines selected for growth in order to evaluate how selection affects the frequencies of these polymorphisms and evaluate their association with growth and carcass traits in Zebu cattle. Birth, weaning and yearling weights, rump height, longissimus muscle area, backfat thickness, and rump fat thickness were analyzed. The sample was constituted of animals from two lines selected for yearling weight (NeS and NeT), and a control line (NeC), established in 1980, at the São Paulo Instituto de Zootecnia. Two hundred and seventy-two heifers were genotyped for TFAM gene SNPs, and 325 heifers were genotyped for the FABP4 SNP. High frequencies were observed for the alleles A (TFAM HaeIII), C (TFAM MboI) and C (FABP4 MspA1I). Significant differences in allele frequencies between NeS and NeT were observed for the TFAM HaeIII, and between the line NeT and lines NeC and NeS for the FABP4 MspA1I SNP. Five haplotypes were observed for the two polymorphisms in the TFAM gene, haplotype AACC being the most frequent. None of the markers separately or according to haplotype was significantly associated with the growth and carcass traits. The low frequencies of alleles that are associated with high marbling scores and thick subcutaneous fat in taurine breeds might explain the low means for these traits in Nellore cattle.

Transcript expression of mitochondria related genes is correlated with bovine oocyte selection by BCB test

This study was conducted in order to determine whether the level of G6PDH activity in immature bovine oocytes is correlated with the transcript expression of the mtDNA replication related genes, POLG, TFAM, NRF1 and mtDNA, encoded COX1 in immature and mature oocytes. G6PDH activity was assessed by the BCB test. Transcript level was assessed by real-time PCR. In immature oocytes, significant differences were noted in mRNA expression of three out of four of the genes analysed: TFAM mRNA expression differed ( P < 0.01) between BCB−, BCB+, and the control group; COX1 expression differed ( P < 0.05) between all analysed groups, and NRF1 transcript levels differed ( P < 0.01) between BCB− and BCB+, and between BCB− and the control group ( P < 0.05). No significant difference in transcript level of POLG gene between the analysed groups was noted. In mature oocytes, a significant difference ( P < 0.05) was noted only for mRNA level of TFAM gene between BCB− and BCB+, and between BCB− and the control group. The results suggest that immature BCB− oocytes do have significantly lower transcript level of genes involved in mitochondrial biogenesis, suggesting that this may be one of the reasons for their low developmental competence compared to BCB+ and control oocytes. Interestingly, we did not find significant differences in blastocyst rate between BCB+ and control oocytes. However, excluding BCB− oocytes from procedures relying on single oocyte can help in increasing the efficacy of the experiment. Our results showed a correlation between transcript level of mtDNA replication factors and G6PDH activity assessed by BCB staining in bovine oocytes.

The Porcine Mitochondrial Transcription Factor a Gene: Molecular Characterization, Radiation Hybrid Mapping and Genetic Diversity among 12 Pig Breeds

Problem statement: Mitochondrial transcription factor A (TFAM) is a nucleus-encoded protein that is a key activator of mitochondrial transcription as well as a major participant in mitochondrial genome replication. Genomic characterization of the porcine TFAM gene is, therefore, necessary to determine its involvement in regulation of fat depots and meat quality traits in pigs. Approach: Genomic DNA sequence was determined using a comparative in silico annotation approach. RT-PCR was used for analysis of alternative splicing. Genome location was determined using Radiation Hybrid (RH) mapping. Genetic marker was identified by sequencing and genotyped by the PCR-RFLP method with SacI. GENEPOP version 3.3 software was used for statistic analysis. Results: We determined both full-length cDNA and genomic DNA sequences of the porcine TFAM gene. Gene expression analysis revealed an alternative 5’ splice site, which excludes exon 4 of the pig gene. We assigned this gene to porcine chromosome 14 (SSC14). A G/A substitution was detected in intron 1 of porcine TFAM gene and genotyped on a total of 252 animals, including 165 from seven Chinese and 87 from five Western pig breeds. The Bayesian analysis via MCMC (Markov chain Monte Carlo) revealed that these two groups of pigs were well separated at this locus during the breed history; 95% of the posterior difference of TFAM allelic frequency between these two pig groups was greater than zero. Conclusion/Recommendations: All these data provided basic genomic information needed for further functional characterization of the porcine TFAM gene. Because marked differences in fat and lean tissue deposition exist between Western and Chinese pig breeds, the G557A mutation in the TFAM gene deserves further evaluation to determine its phenotypic effect on fattening and carcass traits in commercial pig populations.

Mitochondrial oxidative stress and dysfunction in myocardial remodelling

Recent experimental and clinical studies have suggested that oxidative stress is enhanced in myocardial remodelling and failure. The production of oxygen radicals is increased in the failing heart, whereas normal antioxidant enzyme activities are preserved. Mitochondrial electron transport is an enzymatic source of oxygen radical generation and can be a therapeutic target against oxidant-induced damage in the failing myocardium. Chronic increases in oxygen radical production in the mitochondria can lead to a catastrophic cycle of mitochondrial DNA (mtDNA) damage as well as functional decline, further oxygen radical generation, and cellular injury. Reactive oxygen species induce myocyte hypertrophy, apoptosis, and interstitial fibrosis by activating matrix metalloproteinases. These cellular events play an important role in the development and progression of maladaptive myocardial remodelling and failure. Therefore, oxidative stress and mtDNA damage are good therapeutic targets. Overexpression of the genes for peroxiredoxin-3 (Prx-3), a mitochondrial antioxidant, or mitochondrial transcription factor A (TFAM), could ameliorate the decline in mtDNA copy number in failing hearts. Consistent with alterations in mtDNA, the decrease in mitochondrial function was also prevented. Therefore, the activation of Prx-3 or TFAM gene expression could ameliorate the pathophysiological processes seen in mitochondrial dysfunction and myocardial remodelling. Inhibition of oxidative stress and mtDNA damage could be novel and effective treatment strategies for heart failure.

Expression of mitochondrial transcription factor A (TFAM) during porcine gametogenesis and preimplantation embryo development

Mitochondrial transcription factor A (TFAM) is responsible for stability, maintenance, and transcriptional control of mitochondrial DNA (mtDNA). We have studied the expression and distribution of TFAM in the gametes and preimplantation embryos of the domestic pig (Sus scrofa). We hypothesized that TFAM is not present in the boar sperm mitochondria to reduce the possibility of paternal mtDNA propagation in the progeny. In contrast, we anticipated that Tfam gene is expressed in a developmental stage-dependent manner in porcine oocytes and embryos. The appropriate TFAM band of 25 kDa was detected by Western blotting in ejaculated boar spermatozoa, as well as in porcine oocytes and zygotes. Boar sperm extracts also displayed several bands >25 kDa suggestive of post-translational modification by ubiquitination, confirmed by affinity purification of ubiquitinated proteins. TFAM immunoreactivity was relegated to the sperm tail principal piece and sperm head in fully differentiated spermatozoa. The content of Tfam mRNA increased considerably from the germinal vesicle to blastocyst stage and also between in vitro fertilized and cultured blastocysts compared to in vivo-derived blastocysts. TFAM protein accumulated in the oocytes during maturation and was reduced by proteolysis after fertilization. This pattern was not mirrored in parthenogenetically activated oocytes and zygotes reconstructed by SCNT, suggesting deviant processing of TFAM protein and transcript after oocyte/embryo manipulation. Thus, TFAM may exert a critical role in porcine gametogenesis and preimplantation embryo development. Altogether, our data on the role of TFAM in mitochondrial function and inheritance have broad implications for cell physiology and evolutionary biology.

Mitochondrial Transcription Factor A (TFAM) Gene Variation and Risk of Late-Onset Alzheimer's Disease

Impaired mitochondrial function and an increased number of mutations in mitochondrial DNA (mtDNA) has been found in brains of patients with late-onset Alzheimer's disease (LOAD). The TFAM-gene encodes the mitochondrial transcription factor A, a protein that controls the transcription, replication, damage sensing, and repair of mtDNA. TFAM is on human chromosome region 10q21.1, where a locus for LOAD has been mapped. Our objective was to determine the role of TFAM-gene variation in the risk of LOAD. The seven TFAM coding exons were analysed through single strand conformation analysis and direct sequencing in a cohort of Spanish LOAD-patients and healthy controls. We found four common polymorphisms, two in the flanquing intronic and two in the coding sequences. Polymorphism rs1937 (+35 G/C) was the only missense change (S12T). Genotyping of this polymorphism in 300 LOAD-patients and 183 healthy controls showed a significantly higher frequency of GG-homozygotes in the patients (92% vs. 86%; p=0.04; OR=1.91, 95%CI=1.02-3.50). This suggests that S12 is a risk factor for LOAD in our population. In conclusion, rare variants (mutations) in the TFAM gene were not found in LOAD-patients, but the S12T polymorphism was a moderate risk factor for LOAD in our population.

Mitochondrial transcription factor A (TFAM) gene variation in Parkinson's disease

Mitochondrial function is necessary to supply the energy required for cell metabolism. Mutations/polymorphisms in mitochondrial DNA (mtDNA) have been implicated in Parkinson's disease (PD). The mitochondrial transcription factor A (TFAM) controls the transcription of mtDNA and regulates the mtDNA-copy number, thus being important for maintaining ATP production. TFAM dysfunction may also be involved in PD, and TFAM gene mutations/polymorphisms could contribute to the risk of developing PD. We searched for gene variants in the seven TFAM-exons in a total of 250 PD-patients. We found five common polymorphisms, and only one was a missense change (S12T in exon 1). Genotype and allele frequencies did not differ between patients and healthy controls ( n = 225) for the five polymorphisms. Our work suggests that TFAM-variants did not contribute to the risk of developing PD.

Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growth

To examine whether a reduction in the mtDNA level will compromise mitochondrial biogenesis and mitochondrial function, we created a cell model with depleted mtDNA. Stable transfection of small interfering (si)RNA of mitochondrial transcription factor A (Tfam) was used to interfere with Tfam gene expression. Selected stable clones showed 60-95% reduction in Tfam gene expression and 50-90% reduction in cytochrome b (Cyt b) gene expression. Tfam gene knockdown clones also showed decreased mtDNA-encoded cytochrome c oxidase subunit I (COX I) protein expression. However, no significant differences in protein expression were observed in nuclear DNA (nDNA)-encoded mitochondrial respiratory enzyme subunits. The cell morphology changed from a rhombus-like to a spindle-like form as determined in clones with decreased expressions of Tfam, mtRNA, and mitochondrial proteins. The mitochondrial respiratory enzyme activities and ATP production in such clones were significantly lower. The proportions of mtDNA mutations including 8-hydroxy-2'-deoxyguanosine (8-OHdG), a 4,977-bp deletion, and a 3,243-point mutation were also examined in these clones. No obvious increase in mtDNA mutations was observed in mitochondrial dysfunctional cell clones. The mitochondrial respiratory activity and ATP production ability recovered in cells with increased mtDNA levels after removal of the specific siRNA treatment. These experimental results provide direct evidence to substantiate that downregulation of mtDNA copy number and expression may compromise mitochondrial function and subsequent cell growth and morphology.

96 A novel heteroplasmic mutation in COXI leads to developmental delay, seizures and stroke-like episodes

Control over the number of mtDNA molecules per cell appears to be tightly regulated, but the mechanisms involved are largely unknown. Reversible alterations in the amount of mtDNA occur in response to stress suggesting that control over the amount of mtDNA is involved in stress-related diseases including major depressive disorder (MDD). Using low-coverage sequence data from 10,442 Chinese women to compute the normalized numbers of reads mapping to the mitochondrial genome as a proxy for the amount of mtDNA, we identified two loci that contribute to mtDNA levels: one within the TFAM gene on chromosome 10 (rs11006126, p value = 8.73 × 10−28, variance explained = 1.90%) and one over the CDK6 gene on chromosome 7 (rs445, p value = 6.03 × 10−16, variance explained = 0.50%). Both loci replicated in an independent cohort. CDK6 is thus a new molecule involved in the control of mtDNA. We identify increased rates of heteroplasmy in women with MDD, and show from an experimental paradigm using mice that the increase is likely due to stress. Furthermore, at least one heteroplasmic variant is significantly associated with changes in the amount of mtDNA (position 513, p value = 3.27 × 10−9, variance explained = 0.48%) suggesting site-specific heteroplasmy as a possible link between stress and increase in amount of mtDNA. These findings indicate the involvement of mitochondrial genome copy number and sequence in an organism’s response to stress.