The Drosophila nuclear factor DREF positively regulates the expression of the mitochondrial transcription termination factor DmTTF

et al.The DREF [DRE (DNA replication-related element)-binding factor], which regulates the transcription of a group of cell proliferation-related genes in Drosophila, also controls the expression of three genes involved in mtDNA (mitochondrial DNA) replication and maintenance. In the present study, by in silico analysis, we have identified DREs in the promoter region of a gene participating in mtDNA transcription, the DmTTF (Drosophila mitochondrial transcription termination factor). Transient transfection assays in Drosophila S2 cells, with mutated versions of DmTTF promoter region, showed that DREs control DmTTF transcription; moreover, gel-shift and Chip (chromatin immunoprecipitation) assays demonstrated that the analysed DRE sites interact with DREF in vitro and in vivo. Accordingly, DREF knock-down in S2 cells by RNAi (RNA interference) induced it considerable decrease in DmTTF mRNA level. These results clearly demonstrate that DREF positively controls DmTTF expression. On the other hand, mtRNApol (mitochondrial RNA polymerase) lacks DREs in its promoter and is not regulated in vivo by DREF. In situ RNA hybridization Studies showed that DmTTF was transcribed almost ubiquitously throughout all stages of Drosophila embryogenesis, whereas mRNApol was efficiently transcribed from stages 11-12. Territories where transcription occurred mostly were the gut and Malpighi tubes for DmTTF, and the gut, mesoderm, pharyngeal muscle and Malpighi tubes for mtRNApol. The partial overlapping in the temporal and spatial mRNA expression patterns confirms that transcription of the two genes is differentially regulated during embryogenesis and suggests that DmTTF might play multiple roles in the mtDNA transcription process, for which different levels of the protein with respect to mtRNApol are required.Peer reviewe

Evaluation of mitochondrial function and metabolic reprogramming during tumor progression in a cell model of skin carcinogenesis

Metabolic reprogramming from mitochondrial aerobic respiration to aerobic glycolysis is a hallmark of cancer. However, whether it is caused by a dysfunction in the oxidative phosphorylation pathway is still under debate. In this work, we have analyzed the bioenergetic cellular (BEC) index and the relative cell ability to grow in the presence of either galactose or glucose as sources of sugar (Gal/Glu index) of a system formed by four epidermal cell lines with increasing tumorigenic potentials, ranging from nontumorigenic to highly malignant. We find that the BEC index gradually decreases whereas the Gal/Glu index increases with tumorigenicity, indicating that a progressive metabolic adaptation to aerobic glycolysis occurs in tumor cells associated with malignancy. Interestingly, this metabolic adaptation does not appear to be caused by damaged respiration, since the expression and activity of components of the respiratory chain complexes were unchanged in the cell lines. Moreover, the corresponding mitochondrial ATP synthetic abilities of the cell lines were found similar. The production of reactive oxygen species was also measured. A shift in ROS generation was found when compared nontumorigenic with tumorigenic cell lines, the latter exhibiting about threefold higher ROS levels than nontumorigenic cells. This result indicates that oxidative stress is an early event during tumor progression. © 2013 Elsevier Masson SAS. All rights reserved.This work was supported by grants of the Center for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III (grants PI 07/0167 and PI 10/0703 to RG), Comunidad Autónoma de Madrid (grant number GEN-0269 to RG and grant S2010/BMD-2359, SkinModel-CM, to MQ) and the Spanish Ministry of Science and Innovation (grant SAF2010-19152 to MQ).Peer Reviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Functional characterization of three concomitant MtDNA LHON mutations shows no synergistic effect on mitochondrial activity

This is an open access article distributed under the terms of the Creative Commons Attribution License.-- et al.The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Leber's hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.This work was supported by Instituto de Salud Carlos III, http://www.isciii.es/, PI 04/1001 to M.A.F.M.; Instituto de Salud Carlos III, http://www.isciii.es/, PI12/01683 to M.A.M.; Instituto de Salud Carlos III, http://www.isciii.es/, PI13/00556 to R.G.; Comunidad Autónoma de Madrid, http://www.madrid.org/cs/Satellite?pagename=ComunidadMadrid/Home, S2010/BMD-2402 to R.G.; Fundación Mutua Madrileña, http://www.fundacionmutua.es/, 10.04.02.0064 to M.A.F.M.; The Spanish Ministry of Economy and Competitiveness, http://www.mineco.gob.es/, BFU2011-25763 to J.A.; FEDER funds from the E.U. to R.G., http://ec.europa.eu/regional_policy/es/funding/erdf/.Peer Reviewe

Identificación y caracterización de nuevos genes implicados en la función OXPHOS

Resumen del póster presentado a la VIII Reunión Científica Anual del Centro de Investigación Biomédica En Red de Enfermedades Raras, celebrada en San Lorenzo del Escorial (Madrid) los días 12 y 13 de marzo de 2015.La mitocondria es un orgánulo celular que juega un papel central en el metabolismo, destacando en: (i) el aporte de la energía en forma de ATP (ii) la producción de especies reactivas de oxígeno (ROS), (iii) la homeostasis del Ca2 intracelular y (iv) la muerte programada o apoptosis. La síntesis de ATP se realiza mediante el sistema cadena respiratoria (CR)/OXPHOS y su disfunción provoca las denominadas enfermedades mitocondriales. El CR/OXPHOS está formado por 13 subunidades estructurales codificadas en el genoma mitocondrial (mtDNA) y más de 80 en el DNA nuclear, necesitando además los elementos que las ensamblan y los pertenecientes a las maquinarias de replicación y decodificación del genoma mitocondrial. En más de la mitad de las patologías mitocondriales de base genética se desconoce el gen o genes responsables por lo que su identificación es actualmente un reto. En este sentido, la utilización de Drosophila puede suponer una herramienta adicional. El mtDNA de Drosophila y los elementos responsables de su mantenimiento y decodificación son similares a los de mamíferos. En el laboratorio hemos identificado genes esenciales para la función OXPHOS situados sobre mRNAs que codifican dos proteínas diferentes (bicistrones) y creemos que ello puede ser una tendencia. Tras un primer análisis in silico y la valoración de distintos parámetros (conservación evolutiva, probabilidad de destino mitocondrial, función desconocida, etc.) hemos comenzado un análisis funcional preliminar de un grupo de 8 genes seleccionados valorando la capacidad de crecimiento en galactosa como única fuente de carbono de knock-outs generados mediante el sistema CRISPR/Cas.Peer reviewe

Muscle-specific overexpression of the catalytic subunit of dna polymerase γ induces pupal lethality in Drosophila melanogaster

We show the physiological effects and molecular characterization of overexpression of the catalytic core of mitochondrial DNA (mtDNA) polymerase (pol γ-α) in muscle of Drosophila melanogaster. Muscle-specific overexpression of pol γ-α using the UAS/GAL4 (where UAS is upstream activation sequence) system produced more than 90% of lethality at the end of pupal stage at 25°C, and the survivor adult flies showed a significant reduction in life span. The survivor flies displayed a decreased mtDNA level that is accompanied by a corresponding decrease in the levels of the nucleoid-binding protein mitochondrial transcription factor A (mtTFA). Furthermore, an increase in apoptosis is detected in larvae and adults overexpressing pol γ-α. We suggest that the pupal lethality and reduced life span of survivor adult flies are both caused mainly by massive apoptosis of muscle cells induced by mtDNA depletion. © 2013 Wiley Periodicals, Inc.European Union Project (QLG1-CT-2001-00966); National Institutes of Health (GM45295); Spanish Ministerio de Ciencia e Innovación (PI10/00063)Peer Reviewe

Intergenomic nuclear-mitochondrial cross-talk in Drosophila melanogaster

Mitochondrial biogenesis is a complex process that depends on the coordinated expression of genes encoded in two different genomes, the nuclear and the mitochondrial genome. In response to extracellular signals and environmental cues and based on the communication between the mitochondria and nucleus, this process is strictly controlled during cell differentiation to supply different tissues with the correct number of functional mitochondria. To understand the physiopathology of mitochondrial biogenesis, it is critical to unravel the biochemical and molecular basis of this intergenomic cross‑talk. In mammals, cofactors of the PGC‑1 family are fundamental to integrate the response of specific transcription factors. In particular, Nuclear Respiratory Factors 1 and 2 activate the expression of a wide variety of respiratory genes. This regulatory transcriptional circuit is poorly documented in Drosophila, although a well conserved element, the Nuclear Respiratory Gene element, could play an important role in orchestrating energetic metabolism. In addition, the Drosophila DNA replication‑related element binding factor (DREF) regulates the expression of genes encoding critical elements of mtDNA metabolism, establishing a molecular link between mtDNA and nDNA replication. The use of Drosophila mutants with impaired oxidative phosphorylation has facilitated the study of several pathways involved in nuclear‑mitochondrial communication.Peer Reviewe

Glutamyl-tRNAGln amidotransferase is essential for mammalian mitochondrial translation in vivo

Translational accuracy depends on the correct formation of aminoacyl-tRNAs, which, in the majority of cases, are produced by specific aminoacyl-tRNA synthetases that ligate each amino acid to its cognate isoaceptor tRNA. Aminoacylation of tRNAGln, however, is performed by various mechanisms in different systems. Since no mitochondrial glutaminyl-tRNA synthetase has been identified to date in mammalian mitochondria, GlntRNAGln has to be formed by an indirect mechanism in the organelle. It has been demonstrated that human mitochondria contain a non-discriminating glutamyl-tRNA synthetase and the heterotrimeric enzyme GatCAB (where Gat is glutamyltRNAGln amidotransferase), which are able to catalyse the formation of Gln-tRNAGln in vitro. In the present paper we demonstrate that mgatA (mouse GatA) interference inmouse cells produces a strong defect in mitochondrial translation without affecting the stability of the newly synthesized proteins. As a result, interfered cells present an impairment of the oxidative phosphorylation system and a significant increase in ROS (reactive oxygen species) levels. MS analysis of mitochondrial proteins revealed no glutamic acid found in the position of glutamines, strongly suggesting that misaminoacylated GlutRNAGln is rejected from the translational apparatus to maintain the fidelity of mitochondrial protein synthesis in mammals.This work was supported by the Center for Biomedical Research on Rare Diseases (CIBERER), the Instituto de Salud Carlos III [grant numbers PI 07/0167 and PI 10/0703 (to R.G.)] and the Comunidad de Madrid [grant number S2010/BMD-2402 (to R.G.)]. L.E. was funded by the Ministerio de Ciencia e Innovación (Spain) and P.C. by the Comunidad de Madrid.Peer Reviewe

Over-expression of the catalytic core of mitochondrial DNA (mtDNA) polymerase in the nervous system of Drosophila melanogaster reduces median life span by inducing mtDNA depletion

DNA polymerase γ (pol γ) is the sole DNA polymerase devoted to mitochondrial DNA (mtDNA) replication. We have characterized the molecular and physiological effects of over-expression of the catalytic subunit of pol γ, pol γ-α, in the nervous system of Drosophila melanogaster using the upstream activation sequence (UAS)/yeast transcriptional activator by binding to UAS (GAL4) system. Tissue-specific over-expression of pol γ-α was confirmed by immunoblot analysis, whereas the very low levels of endogenous protein are undetectable in UAS or GAL4 control lines. The transgenic flies over-expressing pol γ-α in the nervous system showed a moderate increase in pupal lethality, and a significant decrease in the median life span of adult flies. Moreover, these flies displayed a decrease in the rate of synthesis of mtDNA, which is accompanied by a significant mtDNA depletion, and a corresponding decrease in the levels of mitochondrial transcription factor A (mtTFA). Biochemical analysis showed an oxidative phosphorylation (OXPHOS) defect in transgenic flies, which were more susceptible to oxidative stress. Although we did not detect apoptosis in the nervous system of adult transgenic flies, brains of larvae over-expressing pol γ-α showed evidence of increased cell death that correlates with the observed phenotypes. Our data establish an animal model that mimics some of the features of human mtDNA depletion syndromes. © 2007 The Authors.This research was supported by European Union Project QLG1-CT-2001-00966, Ministerio de Ciencia y Tecnologia, Spain (Grants BMC01-1525 and BFU2004-04591) and Instituto de Salud Carlos III, Redes de centros RCMN (C03/08) and Tema´ticas (G03/ 011) (to RG) and National Institutes of Health Grant GM45295 (to LSK).Peer Reviewe

Drosophila nuclear factor DREF regulates the expression of the mitochondrial DNA helicase and mitochondrial transcription factor B2 but not the mitochondrial translation factor B1

et al.DREF [DRE (DNA replication-related element)-binding factor] controls the transcription of numerous genes in Drosophila, many involved in nuclear DNA (nDNA) replication and cell proliferation, three in mitochondrial DNA (mtDNA) replication and two in mtDNA transcription termination. In this work, we have analysed the involvement of DREF in the expression of the known remaining genes engaged in the minimal mtDNA replication (d-mtDNA helicase) and transcription (the activator d-mtTFB2) machineries and of a gene involved in mitochondrial mRNA translation (d-mtTFB1). We have identified their transcriptional initiation sites and DRE sequences in their promoter regions. Gel-shift and chromatin immunoprecipitation assays demonstrate that DREF interacts in vitro and in vivo with the d-mtDNA helicase and d-mtTFB2, but not with the d-mtTFB1 promoters. Transient transfection assays in Drosophila S2 cells with mutated DRE motifs and truncated promoter regions show that DREF controls the transcription of d-mtDNA helicase and d-mtTFB2, but not that of d-mtTFB1. RNA interference of DREF in S2 cells reinforces these results showing a decrease in the mRNA levels of d-mtDNA helicase and d-mtTFB2 and no changes in those of the d-mtTFB1. These results link the genetic regulation of nuclear DNA replication with the genetic control of mtDNA replication and transcriptional activation in Drosophila. © 2013 Elsevier B.V.This work was supported by grants from the Center for Biomedical Research on Rare Diseases (CIBERER) [INTRA/08/743.2] to R.G., the Instituto de Salud Carlos III (grants [PI 07/0167] and [PI 10/0703] to R.G.); the Comunidad de Madrid (grants [GEN-0269/2006] and [MITOLAB S2010/BMD-2402] to R.G); the Università di Bari, Progetto di Ricerca di Ateneo (to M.R.); and the National Institutes of Health (grant [GM45295] to L.S.K.).Peer Reviewe