Mitochondrial DNA Copy Research Articles

Phosphorylation of Human TFAM in Mitochondria Impairs DNA Binding and Promotes Degradation by the AAA+ Lon Protease

Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription, and inheritance. Little is known about the mechanisms underlying its posttranslational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anticancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However, in cells with severe mtDNA deficits, nonphosphorylated TFAM is also degraded, as it is DNA free. Depleting Lon in these cells increases levels of TFAM and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapid fine-tuning of TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.

Autophagy and skeletal muscles in sepsis.

BackgroundMitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles.Methodology/Principal FindingsAdult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca++ retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor.Conclusion/SignificanceWe conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor.

Mammalian NUMT insertion is non-random

It is well known that remnants of partial or whole copies of mitochondrial DNA, known as Nuclear MiTochondrial sequences (NUMTs), are found in nuclear genomes. Since whole genome sequences have become available, many bioinformatics studies have identified putative NUMTs and from those attempted to infer the factors involved in NUMT creation. These studies conclude that NUMTs represent randomly chosen regions of the mitochondrial genome. There is less consensus regarding the nuclear insertion sites of NUMTs — previous studies have discussed the possible role of retrotransposons, but some recent ones have reported no correlation or even anti-correlation between NUMT sites and retrotransposons. These studies have generally defined NUMT sites using BLAST with default parameters. We analyze a redefined set of human NUMTs, computed with a carefully considered protocol. We discover that the inferred insertion points of NUMTs have a strong tendency to have high-predicted DNA curvature, occur in experimentally defined open chromatin regions and often occur immediately adjacent to A + T oligomers. We also show clear evidence that their flanking regions are indeed rich in retrotransposons. Finally we show that parts of the mitochondrial genome D-loop are under-represented as a source of NUMTs in primate evolution.

Mitochondrial biogenesis: CO and NO too

The cell’s mitochondrial mass and respiratory capacity are protected by a number of structurally distinct events within the cell, including changes in cytoskeletal morphology, the mitochondrial fusion and fission cycle, mitophagy, and mitochondrial biogenesis. These events safeguard mitochondrial function at times of increased cell stress by protecting existing mitochondria, by disposing of damaged organelles, and by replacing them with new ones. This process of mitochondrial quality control involves intricate nuclear and bi-genomic programs over which the endogenous gaseous mediators, nitric oxide (NO) and carbon monoxide (CO), exert substantial regulatory control. This talk addresses novel molecular mechanisms by which NO and CO stimulate the nuclear transcriptional regulators, including energy-sensing and redox-regulated transcription factors and co-activators involved in the induction of mitochondrial biogenesis. These mechanisms include the regulation of NO and CO production by the NO synthases and heme oxygenase-1 (HO-1), biochemical interactions of NO and CO with a limited number of specific mitochondrial proteins, and activation of unique, hierarchical, and overlapping signal transduction pathways that participate in the nuclear-encoded gene expression required for the synthesis of mitochondrial proteins, their importation into mitochondria, transcription and replication of mitochondrial DNA, and mitochondrial proliferation. The production of one or both gases effects tailored adjustments in mitochondrial mass, number, distribution, and/or phenotype in response to increasing requirements for cellular work, cell repair, and proliferation, particularly in response to inflammation and oxidative stress in the heart, liver, kidney, and other major organs. Emphasis is given to the contribution of the innate immune system to oxidative/nitrosative cycles of mitochondrial damage to which the host cell, to protect its energy supply and survive, induces mitochondrial biogenesis under the control of NO and CO in concert with the expression of a complement of anti-oxidant and anti-inflammatory defense genes. This multifunctional network increases cellular resistance to metabolic failure and cell death and promotes immune tolerance while the inflammatory stress is brought under control.

Expression, Purification of Recombinant Human Mitochondrial Transcription Termination Factor 3 (hMTERF3) and Preparation of Polyclonal Antibody Against hMTERF3

In mammalian cells, a family of mitochondrial transcription termination factors (MTERFs) regulates mitochondrial gene expression. Mitochondrial transcription termination factor 3 (MTERF3) is the most conserved member of the MTERF family and a negative regulator of mammalian mitochondrial DNA transcription. To create a specific polyclonal antibody against human MTERF3 (hMTERF3), we first cloned hMTERF3 into prokaryotic expression vector pGEX-4T-1, and GST-hMTERF3 was efficiently expressed in Escherichia coli after induction by IPTG. The expressed GST-tagged hMTERF3 fusion protein was purified by passive electro-elution process and then used to immunize BALB/c mice, we obtained anti-GST-hMTERF3 polyclonal antibody purified by protein A column and determined the sensitivity and specificity of the antibody against human MTERF3 by enzyme-linked immunosorbent assay and Western blot assay. Furthermore, the full-length hMTERF3 protein expressed in human embryonic kidney 293T cells was detected by anti-GST-hMTERF3 in western blot analysis and immunofluorescence staining. Taken together, our results demonstrate the functionality of the mouse anti-GST-hMTERF3 polyclonal antibody which will provide a useful tool for further characterization of hMTERF3.

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.

Next‐generation Sequencing Facilitates the Diagnosis in a Child With Twinkle Mutations Causing Cholestatic Liver Failure

Next‐generation Sequencing Facilitates the Diagnosis in a Child With Twinkle Mutations Causing Cholestatic Liver Failure

Clinical significance of mitochondrial transcription factor A expression in patients with colorectal cancer

Mitochondrial transcription factorA (mtTFA) is necessary for both the transcription and maintenance of mitochondrial DNA (mtDNA). The present study investigated the relationship between clinicopathological factors, prognosis and the immunohistochemical expression of mtTFA in the tumors of patients diagnosed with primary colorectal cancer (CRC). Surgical specimens from 105 colorectal patients were immunohistochemically stained using a polyclonal anti-mtTFA antibody. The relationships among the mtTFA expression, clinicopathological factors and prognosis were evaluated. A total of 47 (44.8%) of the 105 patients with CRC were determined to have positive mtTFA expression. The positive expression of mtTFA significantly correlated with lymph node metastasis, distant metastasis and advanced TNM staging. On the other hand, negative mtTFA expression showed a tendency to correlate with high Ki-67 index. The survival of patients with positive mtTFA expression was significantly worse than that of patients with negative mtTFA expression. The positive mtTFA expression appears to be a useful marker for tumor progression and poor prognosis in patients with CRC.

High-dose clevudine impairs mitochondrial function and glucose-stimulated insulin secretion in INS-1E cells

BackgroundClevudine is a nucleoside analog reverse transcriptase inhibitor that exhibits potent antiviral activity against hepatitis B virus (HBV) without serious side effects. However, mitochondrial myopathy has been observed in patients with chronic HBV infection taking clevudine. Moreover, the development of diabetes was recently reported in patients receiving long-term treatment with clevudine. In this study, we investigated the effects of clevudine on mitochondrial function and insulin release in a rat clonal β-cell line, INS-1E.MethodsThe mitochondrial DNA (mtDNA) copy number and the mRNA levels were measured by using quantitative PCR. MTT analysis, ATP/lactate measurements, and insulin assay were performed.ResultsBoth INS-1E cells and HepG2 cells, which originated from human hepatoma, showed dose-dependent decreases in mtDNA copy number and cytochrome c oxidase-1 (Cox-1) mRNA level following culture with clevudine (10 μM-1 mM) for 4 weeks. INS-1E cells treated with clevudine had reduced total mitochondrial activities, lower cytosolic ATP contents, enhanced lactate production, and more lipid accumulation. Insulin release in response to glucose application was markedly decreased in clevudine-treated INS-1E cells, which might be a consequence of mitochondrial dysfunction.ConclusionsOur data suggest that high-dose treatment with clevudine induces mitochondrial defects associated with mtDNA depletion and impairs glucose-stimulated insulin secretion in insulin-releasing cells. These findings partly explain the development of diabetes in patients receiving clevudine who might have a high susceptibility to mitochondrial toxicity.

Impaired mitochondrial function in murine oocytes is associated with controlled ovarian hyperstimulationand in vitro maturation

The present study was designed to determine whether controlled ovarian hyperstimulation (COH) and in vitro maturation (IVM), two common clinical procedures in human IVF treatment, have an impact on mitochondrial DNA (mtDNA) copy number and mitochondrial function in oocytes. Matured mouse oocytes recovered following COH, IVM and natural cycles (NC), which simulated those treatments in human clinic IVF treatment. The copies of mtDNA, the activity of mitochondria as determined by inner mitochondrial membrane potential and oocyte adenosine trisphosphate (ATP) content, pattern of mitochondrial distribution, reactive oxygen species (ROS) levels and the integrity of the cytoskeleton were evaluated in oocytes. Significant differences were detected between COH and NC groups in all measures, except the pattern of mitochondrial distribution and ROS levels. There were also significant differences detected between IVM and NC treatment groups in the copies of mitochondrial DNA, the level of ROS and the integrity of the cytoskeleton in oocytes. In conclusion, the results of this investigation indicate that non-physiological COH and IVM treatments inhibit mtDNA replication, alter mitochondrial function and increase the percentage of abnormal cytoskeleton and ROS production. Damage related to the mitochondria may partly explain the low efficiency of IVF and high rate of embryonic loss associated with these clinical procedures.

Large heterogeneity of mitochondrial DNA transcription and initiation of replication exposed by single-cell imaging

Mitochondrial DNA (mtDNA) replication and transcription are crucial for cell function, but these processes are poorly understood at the single-cell level. We describe a novel fluorescence in situ hybridization protocol, called mTRIP (mitochondrial transcription and replication imaging protocol), that reveals simultaneously mtDNA and RNA, and that can also be coupled to immunofluorescence for in situ protein examination. mTRIP reveals mitochondrial structures engaged in initiation of DNA replication by identification of a specific sequence in the regulatory D-loop, as well as unique transcription profiles in single human cells. We observe and quantify at least three classes of mitochondrial structures: (i) replication initiation active and transcript-positive (Ia-Tp); (ii) replication initiation silent and transcript-positive (Is-Tp); and (iii) replication initiation silent and transcript-negative (Is-Tn). Thus, individual mitochondria are dramatically heterogeneous within the same cell. Moreover, mTRIP exposes a mosaic of distinct nucleic acid patterns in the D-loop, including H-strand versus L-strand transcripts, and uncoupled rRNA transcription and mtDNA initiation of replication, which might have functional consequences in the regulation of the mtDNA. Finally, mTRIP identifies altered mtDNA processing in cells with unbalanced mtDNA content and function, including in human mitochondrial disorders. Thus, mTRIP reveals qualitative and quantitative alterations that provide additional tools for elucidating the dynamics of mtDNA processing in single cells and mitochondrial dysfunction in diseases.

New Roles of Mitochondrial Transcription Factor A in Cancer

Mitochondria are the main sites of energy production in almost all eukaryotic cells. Mitochondria use oxidative phosphorylation to convert redox energy of substrates into adenosine triphosphate (ATP). Mitochondrial transcription factor A (mtTFA; also known as TFAM) is necessary for both transcription and maintenance of mitochondrial DNA (mtDNA), and it is one of the high mobility group (HMG) proteins that preferentially recognizes cisplatin-damaged DNA and oxidized DNA. Loss of mtTFA causes depletion of mtDNA, loss of mitochondrial transcripts, loss of mtDNA-encoded polypeptides, and severe respiratory chain deficiency. Mitochondria play a critical role in cancer cell metabolism and are also essential for cell proliferation. It is well known that mitochondrial uncoupling mediates the metabolic shift to aerobic glycolysis in cancer cells. Thus, mitochondria control cell survival and growth. In addition, the number of mitochondria correlates with the growth rate of cancer cells. The information gleaned from this review may provide critical clues to novel therapeutic interventions aimed at overcoming cancer. More detailed functional analyses of mtTFA should further elucidate its role in mitochondrial genome instability and apoptosis.c

Evaluation of Hepatic Mitochondria and Hematological Parameters in Zidovudine-TreatedB6C3F1Mice

The effects of 12-week exposure to zidovudine (AZT) at 400, 500, and 600 mg/kg/d were examined on expression of 542 mitochondria-related genes and mitochondrial DNA (mtDNA) copy number in the liver of male and female B6C3F1 mice to understand mitochondrial role in sex-related differences in development of lactic acidosis. Plasma lactate levels and hematologic parameters were also examined. Results indicated increased red blood cell (RBC) count in vehicle-treated controls, whereas a dose-related decline in the RBC count was noted in AZT-treated mice compared to the basal levels before treatments began. These decreases were associated with significant dose-related increases in mean corpuscular volume and mean corpuscular hemoglobin levels. This effect was greater in AZT-treated females compared to males. In both sexes, 12-week AZT or vehicle exposure significantly reduced plasma lactate levels compared to the basal levels. Results also showed modest, but significant, changes in the expression of genes associated with apoptosis and lipid metabolism at 600 mg/kg/d AZT. Neither drug nor sex influenced hepatic mtDNA copy number. Altogether, 12-week AZT exposure as high as 600 mg/kg/d did not impair hepatic mitochondria or induce lactic acidosis in B6C3F1 mice. However, AZT-mediated hematologic toxicity appeared to be greater in females compared to males.

HIV Metabolic Disease: The Mitochondria–Adipocyte Connection

HIV metabolic disease in lipodystropic patients includes changes in fat tissue that are associated with insulin resistance, hypertriglyceridemia, and increased cardiovascular risk. Nucleoside reverse transcriptase inhibitors (NRTIs) have been shown to contribute to lipodystrophy by inhibiting mitochondrial DNA replication and transcription in fat tissue and adipocytes. Novel data from two recent studies show that immune activation and protease inhibitors (PIs) can also affect mitochondrial function. In a cross-sectional study aimed at characterizing the effects of HIV infection and antiretroviral therapy (ART) in adipose tissue, muscle, and peripheral blood mononuclear cells (PBMCs), Morse and colleagues compared mitochondrial DNA (mtDNA) …

Mitochondrial transcription factor A regulates BCL2L1 gene expression and is a prognostic factor in serous ovarian cancer

Mitochondrial transcription factor A (mtTFA) is necessary for both transcription and maintenance of mitochondrial DNA (mtDNA). Recently, we reported that mtTFA is expressed not only in mitochondria, but also in nuclei. However, the function of mtTFA in the nucleus has not been clearly elucidated. In the present study, we examined nuclear mtTFA expression in 60 tissue samples of serous ovarian cancer using immunohistochemical analysis and found that 56.7% of serous ovarian cancer patients were positive for mtTFA, whereas 43.3% were negative. Univariate survival analysis showed that the overall 5-year survival rate was significantly worse for patients with mtTFA-positive cancer compared with mtTFA-negative cancer (32%vs 42%, respectively; P = 0.021). To elucidate the function of mtTFA in the nucleus, we investigated BCL2L1, a target gene of mtTFA. There was a significant correlation between nuclear mtTFA expression and BCL2L1 expression in seven ovarian cancer cell lines and in specimens of clinical ovarian cancer. Cellular BCL2L1 was downregulated following transfection of siRNA against mtTFA. BCL2L1 promoter activity was increased after transfection of mtTFA expression plasmid, but decreased after siRNA knockdown of mtTFA. Chromatin immunoprecipitation assays showed that mtTFA was bound to the BCL2L1 promoter region. These results suggest that mtTFA is a prognostic factor for a poor outcome of ovarian cancer and may function as an antiapoptotic factor, regulating genes such as BCL2L1. Furthermore, mtTFA may be a promising molecular target for novel therapeutic strategies for the treatment of ovarian cancer.

191 SUPPLEMENTATION OF MATURATION MEDIUM WITH FOLLICULAR FLUID, EPIDERMAL GROWTH FACTOR AND NEUREGULIN AFFECTS MITOCHONDRIAL DNA REPLICATION, OOCYTE MATURATION AND EMBRYO DEVELOPMENT IN PIGS

Mitochondria supply the majority of ATP in a cell. Mitochondrial DNA (mtDNA) copy number in oocytes might be used as a marker of viability and might be a key determinant of pre-implantation embryo development. However, little is known about mtDNA copy number changes during porcine oocyte maturation and its regulation by extracellular growth factors. The objectives of the current study were to determine the effects of supplementation of in vitro maturation medium with porcine follicular fluid (pFF; 0, 10, 20 and 30%), epidermal growth factor (EGF; 10 ng mL–1), neuregulin 1 (NRG; 20 ng mL–1) and NRG + IGF1 (insulin-like growth factor-1; 100 ng mL–1 + NRG, 20 ng mL–1) during in vitro maturation on mtDNA copy number, oocyte meiotic maturation and subsequent embryo development after parthenogenic activation. Follicular fluid used for the pFF supplementation experiment was prepared from medium-sized (3–6 mm in diameter) healthy follicles. Cumulus–oocyte complexes (COCs) were collected from antral follicles (3–6 mm in diameter), cultured in LH- and FSH-containing maturation medium for 22 h at 38.5°C, transferred into basic maturation medium without FSH and LH and cultured for another 22 h. The basic maturation medium was TCM-199 supplemented with 0.1% polyvinylalcohol (w/v), 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 10 μg mL–1 of gentamicin, 0.57 mM cysteine and without or with different growth factors depending on the experimental design. In total, 177 germinal vesicle (GV) oocytes and 3837 MII oocytes were used for this study. All data were analyzed by the general linear model (GLM) procedure of SAS software (V9.2). The mtDNA copy number in oocytes increased (P < 0.05) from GV to MII stage oocytes (MII oocytes from all treatment groups pooled). Supplementation of IVM media with 10% pFF decreased mtDNA copy number (P < 0.05), whereas 20 and 30% pFF had no major effect on mtDNA copy number, resulting in a quadratic correlation between percentage of pFF and mtDNA copy number. There was a negative linear correlation between percentage of pFF and oocyte meiotic maturation, with a higher percentage of pFF inhibiting meiotic maturation (73.2 ± 5.2, 71.9 ± 4.8, 64.1 ± 8.5 and 65.8 ± 6.4% for 0, 10, 20 and 30% pFF groups, respectively). The mtDNA copy numbers in EGF and NRG-treated MII oocytes were significantly higher than those in GV oocytes, whereas the control was not different (EGF, 237 042.6 ± 22 198.2; NRG, 281 293.4 ± 22 893.5; and control, 231 856.8 ± 21 883.5 in MII oocytes vs 192 288.7 ± 21 675.4 in GV oocytes). The EGF, NRG and NRG+IGF1 treatments enhanced oocyte maturation as well. There was no difference in Day-7 blastocyst formation between EGF, NRG+IGF1 and the control, whereas the NRG treatment enhanced blastocyst formation as compared to the control (23.8 ± 2.4 vs 15.1 ± 2.1%; P < 0.05). This study demonstrated that there was an increase in mtDNA copy number during in vitro maturation. The EGF and NRG treatments stimulated mitochondria biogenesis, which may provide new means to increase oocyte quality and enhance embryonic development.

Biomarker for Benzopyrene Exposure Is Mitochondrial Mass and Mitochondrial DNA Copy in Blood Cells and Hematopoietic Tissues

Abstract 4889Benzopyrenes are well known pollutants and carcinogens. They can intercalate into DNA and interfere transcriptions, resulting in causing various human diseases. However, biomarkers of benzopyrene toxicity have not been comprehensively studied in blood and leukemia cells. The current study was investigated to discover biomarkers for benzopyrene exposure in blood cells and leukemia cell lines. Peripheral blood, peripheral blood hematopoietic stem cell and leukemia cells (THP-1, K562, Molt-4 and HL-60) were cultured in RPMI 1640 media with adding 0, 50, 100 and 200μM of benzopyrene. Viability and apoptosis were assessed by tryptophan blue dye exclusion test and flowcytometry using annexin V. Hydrogen peroxide was measured using enzyme immunoassay. Mitochondrial mass, membrane potential and mitochondrial DNA (mtDNA) copy number were measured using MitoTracker Green, Red probes and real time PCR, respectively. The number of cell remained constant for three weeks culture. Viability of four cell lines disclosed significant decrease after two weeks of benzopyrene treatment. Apoptosis was increased in time- and dose-dependent manner after two weeks of benzopyrene treatment. Mitochondrial contents and membrane potentials were dramatically increased in three-week culture at dose dependent manner. Hydrogen peroxide level was significantly elevated after two weeks treatment of benzopyrene compared to non-benzopyrene treatment group. The number of mtDNA copy increased gradually after exposure to benzopyrene. These results indicated that increased mitochondrial mass and mtDNA copy number were biomarkers for direct exposure of benzopyrene in blood cells and hematopoietic tissues. Disclosures:No relevant conflicts of interest to declare.

Where does this tiger come from?—A robust molecular technique for simultaneous identification of endangered species and subspecies

The Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES) monitors the international trade in endangered animal and plant species; a high profile example is the tiger, Panthera tigris. We report on the application of a SNaPshot multiplex technique to simultaneously identify tiger species and subspecies; this test is based on identification of SNPs within the tiger mitochondrial genome. Mitochondrial DNA sequences from four of the five extant putative tiger subspecies were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species. A total of 11 SNP loci were identified: five specific for tiger; three specific to Panthera tigris sumatrae and; three specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies, indicating that it will work on trace amounts of tissue, bone or hair.

Effect of bezafibrate treatment on late-onset mitochondrial myopathy in mice

Mitochondrial dysfunction is an important cause of metabolic disorders of children and adults, with no effective therapy options. Recently, induction of mitochondrial biogenesis, by transgenic overexpression of PGC1-alpha [peroxisome proliferator-activated receptor (PPAR)-gamma coactivator 1-alpha], was reported to delay progression of early-onset cytochrome-c-oxidase (COX) deficiency in skeletal muscle of two mouse models: a muscle-specific knock-out of COX10 (COX10-mKO) and a constitutive knock-out of Surf1 (Surf1-KO). A pan-PPAR agonist, bezafibrate, could similarly delay myopathy progression in COX10-mKOs, but not in SURF1-KOs. We asked whether bezafibrate affected disease progression in late-onset adult-type mitochondrial myopathy mice. These 'Deletor mice' express a dominant patient mutation in Twinkle-helicase, leading to accumulation of multiple mtDNA deletions and subsequent progressive respiratory chain (RC) deficiency with COX-negative muscle fibers at 12 months of age. The primary and secondary molecular findings in Deletor mice mimic closely those in patients with Twinkle myopathy. We applied 0.5% bezafibrate diet to Deletors for 22 weeks, starting at disease manifestation, mimicking patient treatment after diagnosis. Bezafibrate delayed significantly the accumulation of COX-negative fibers and multiple mtDNA deletions. However, mitochondrial biogenesis was not induced: mitochondrial DNA copy number, transcript and RC protein amounts decreased in both Deletors and wild-type mice. Furthermore, bezafibrate induced severe lipid oxidation effects, with hepatomegaly and loss of adipose tissue, the mechanism involving lipid mobilization by high hepatic expression of FGF21 cytokine. However, as bezafibrate has been tolerated well by humans, the beneficial muscle findings in Deletor mice support consideration of bezafibrate trials on adult patients with mitochondrial myopathy.

Mitochondrial Ribosomal Protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription

Basal transcription of human mitochondrial DNA (mtDNA) in vitro requires the single-subunit, bacteriophage-related RNA polymerase, POLRMT, and transcription factor h-mtTFB2. This two-component system is activated differentially at mtDNA promoters by human mitochondrial transcription factor A (h-mtTFA). Mitochondrial ribosomal protein L7/L12 (MRPL12) binds directly to POLRMT, but whether it does so in the context of the ribosome or as a "free" protein in the matrix is unknown. Furthermore, existing evidence that MRPL12 activates mitochondrial transcription derives from overexpression studies in cultured cells and transcription experiments using crude mitochondrial lysates, precluding direct effects of MRPL12 on transcription to be assigned. Here, we report that depletion of MRPL12 from HeLa cells by shRNA results in decreased steady-state levels of mitochondrial transcripts, which are not accounted for by changes in RNA stability. We also show that a significant "free" pool of MRPL12 exists in human mitochondria not associated with ribosomes. "Free" MRPL12 binds selectively to POLRMT in vivo in a complex distinct from those containing h-mtTFB2. Finally, using a fully recombinant mitochondrial transcription system, we demonstrate that MRPL12 stimulates promoter-dependent and promoter-independent transcription directly in vitro. Based on these results, we propose that, when not associated with ribosomes, MRPL12 has a second function in transcription, perhaps acting to facilitate the transition from initiation to elongation. We speculate that this is one mechanism to coordinate mitochondrial ribosome biogenesis and transcription in human mitochondria, where transcription of rRNAs from the mtDNA presumably needs to be adjusted in accordance with the rate of import and assembly of the nucleus-encoded MRPs into ribosomes.