Rafael Garesse | Universidad Autónoma de Madrid (original) (raw)

Papers by Rafael Garesse

Schematic diagram of the sequence organization of mtDNA helicase; eight amino acid seque... more Schematic diagram of the sequence organization of mtDNA helicase; eight amino acid sequence motifs common to ring primases and helicases are indicated in gray. The bacteriophage T7 gp4 linker region is indicated in the middle of the diagram. Mutations in the human mtDNA helicase gene (Twinkle/C10orf2) found in adPEO are shown above the scheme (reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043954#pone.0043954-Wanrooij1" target="_blank">[46]</a>), the orthologous adPEO mutations W441C and A442P in the Drosophila mtDNA helicase gene (d-mtDNA helicase), and the “Walker A” mutation K388A are shown below the scheme. The underlined mutations indicate autosomal recessive PEO mutations. The positions of mutations in d-mtDNA helicase used in this study are shown in bold. Sequence alignment of the regions containing altered amino acids is shown in the lower panel. Dm, fly; Ag, mosquito; Mm, mouse; Hs, human; T7, bacteriophage T7.</p

Biophysical Journal, 2009

Mutations in OPAI, a dynamin-related GTPase involved in mitochondrial fusion, cristae organizatio... more Mutations in OPAI, a dynamin-related GTPase involved in mitochondrial fusion, cristae organization and control of apoptosis, have been linked to non-syndromic optic neuropathy transmitted as an autosomal-dominant trait (DOA).We here report on eight patients from six independent families showing that mutations in the OPAI gene can also be responsible for a syndromic form of DOA associated with sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia and mitochondrial myopathy with cytochrome c oxidase negative and Ragged Red Fibres. Most remarkably, we demonstrate that these patients all harboured multiple deletions of mitochondrial DNA (mtDNA) in their skeletal muscle, thus revealing an unrecognized role of the OPAI protein in mtDNA stability. The five OPAI mutations associated with these DOA'plus' phenotypes were all mis-sense point mutations affecting highly conserved amino acid positions and the nuclear genes previously known to induce mtDNA multiple deletions such as POLGI, PEOI (Twinkle) and SLC25A4 (ANTI) were ruled out. Our results show that certain OPAI mutations exert a dominant negative effect responsible for multi-systemic disease, closely related to classical mitochondrial cytopathies, by a mechanism involving mtDNA instability.

(A) Left panel: Steady-state levels of fully assembled complex I were evaluated in the t... more (A) Left panel: Steady-state levels of fully assembled complex I were evaluated in the triple mutant cybrid clones and in controls by BN-PAGE. Fully assembled complex I, and complex II, was determined by western blotting using antibodies recognizing NDUFA9 and CII 70 kDa, respectively. Right panel: quantification of 4 control clones and 2 triple clones from two independent gels and normalized to complex II, represented as percentage of the controls. (B) BN-SDS-PAGE of individual clones after 48 h of recovery from chloramphenicol treatment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146816#sec002" target="_blank">Materials and methods</a>). Experiments were performed at least by duplicate. SC indicates supercomplexes containing complex I.</p

Mitochondrial Diseases, 2021

Annals of the rheumatic diseases, Jan 5, 2016

To evaluate the influence of the mitochondrial DNA (mtDNA) haplogroups in the risk of incident kn... more To evaluate the influence of the mitochondrial DNA (mtDNA) haplogroups in the risk of incident knee osteoarthritis (OA) and to explain the functional consequences of this association to identify potential diagnostic biomarkers and therapeutic targets. Two prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 2579 subjects of the incidence subcohort, and the cohort hip and cohort knee (CHECK) included 635, both with 8-year follow-up. The analysis included the association of mtDNA haplogroups with the rate of incident knee OA in subjects from both cohorts followed by a subsequent meta-analysis. Transmitochondrial cybrids harbouring haplogroup J or H were constructed to detect differences between them in relation to physiological features including specific mitochondrial metabolic parameters, reactive oxygen species production, oxidative stress and apoptosis. Compared with H, the haplogroup J associates with decreased risk of incident knee OA in sub...

Biochemistry, 2016

The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mito... more The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mitochondrial respiratory chain, requires a large number of accessory proteins (the so-called assembly factors). Human COX assembly factor 3 (hCOA3), also known as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56), interacts with the first subunit protein of COX to form its catalytic core and promotes its assemblage with the other units. Therefore, hCOA3 is involved in COX biogenesis in humans and can be exploited as a drug target in patients with mitochondrial dysfunctions. However, to be considered a molecular target, its structure and conformational stability must first be elucidated. We have embarked on the description of such features by using spectroscopic and hydrodynamic techniques, in aqueous solution and in the presence of detergents, together with computational methods. Our results show that hCOA3 is an oligomeric protein, forming aggregates of different molecular masses in aqueous solution. Moreover, on the basis of fluorescence and circular dichroism results, the protein has (i) its unique tryptophan partially shielded from solvent and (ii) a relatively high percentage of secondary structure. However, this structure is highly flexible and does not involve hydrogen bonding. Experiments in the presence of detergents suggest a slightly higher content of nonrigid helical structure. Theoretical results, based on studies of the primary structure of the protein, further support the idea that hCOA3 is a disordered protein. We suggest that the flexibility of hCOA3 is crucial for its interaction with other proteins to favor mitochondrial protein translocation and assembly of proteins involved in the respiratory chain. M itochondria are the "powerhouses" of cells, generating the bulk of cellular ATP. Cellular respiration occurs within them, involving oxygen consumption and ATP release. This process is known as the "electron transport chain" and involves five protein complexes: four enzymatic respiratory ones and the ATP synthase. Electrons delivered from NADH and succinate go through the electron transport chain to O 2 , which is finally reduced to H 2 O. The cytochrome c oxidase (COX) or complex IV, the fourth enzyme of the electron transport chain, catalyzes the oxidation of cytochrome c, transferring its electrons to O 2. The mammalian complex IV is formed by 13 protein subunits: three of them (COX1 to

Human molecular genetics, Jan 19, 2015

The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease p... more The heart is a muscle with high energy demands. Hence, most patients with mitochondrial disease produced by defects in the Oxidative Phosphorylation System (OXPHOS) are susceptible to cardiac involvement. The presentation of mitochondrial cardiomyopathy includes hypertrophic, dilated, and left ventricular (LV) noncompaction, but the molecular mechanisms involved in cardiac impairment are unknown. One of the most frequent OXPHOS defect in humans frequently associated with cardiomyopathy is cytochrome c oxidase (COX) deficiency caused by mutations in COX assembly factors like Sco1 and Sco2. To investigate the molecular mechanisms that underlie the cardiomyopathy associated with Sco deficiency, we have heart specifically interfered scox, the single Drosophila Sco orthologue. Cardiac-specific knockdown of scox reduces fly lifespan, and it severely compromises heart function and structure, producing dilated cardiomyopathy. Cardiomyocytes with low levels of scox have a significant reducti...

Experimental Dermatology, 2005

Mitochondrial dysfunction might play a role in the pathogenesis of liver damage in erythropoietic... more Mitochondrial dysfunction might play a role in the pathogenesis of liver damage in erythropoietic protoporphyria (EPP). Changes in mitochondrial respiratory chain activities were evaluated in the Fech m1pas /Fech m1pas mouse model for EPP. Mice from different strains congenic for the same ferrochelatase germline mutation manifest variable degrees of hepatobiliary injury. Protoporphyric animals bred into the C57BL/6J background showed a higher degree of hepatomegaly and liver damage as well as higher protoporphyrin (PP) accumulation than those bred into the SJL/J and BALB/cJ backgrounds. Whereas mitochondrial respiratory chain activities remained unchanged in the liver of protoporphyric mice C57BL/ 6J, they were increased in protoporphyric mice from both SJL/J and BALB/cJ backgrounds, when compared to wild-type animals. Mitochondrial respiratory chain activities were increased in Hep G2 cell line after accumulation of PP following addition of aminolevulinic acid. As a direct effect of these elevated mitochondrial activities, in both hepatic cells from mutant mouse strains and Hep G2 cells, adenosine 5 0triphosphate (ATP) levels significantly increased as the intracellular PP concentration was reduced. These results indicate that PP modifies intracellular ATP requirements as well as hepatic mitochondrial respiratory chain enzymatic activities and further suggest that an increase of these activities may provide a certain degree of protection against liver damage in protoporphyric mice.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2006

Human mitochondrial diseases are associated with a wide range of clinical symptoms, and those tha... more Human mitochondrial diseases are associated with a wide range of clinical symptoms, and those that result from mutations in mitochondrial DNA affect at least 1 in 8500 individuals. The development of animal models that reproduce the variety of symptoms associated with this group of complex human disorders is a major focus of current research. Drosophila represents an attractive model, in large part because of its short life cycle, the availability of a number of powerful techniques to alter gene structure and regulation, and the presence of orthologs of many human disease genes. We describe here Drosophila models of mitochondrial DNA depletion, deafness, encephalopathy, Freidreich's ataxia, and diseases due to mitochondrial DNA mutations. We also describe several genetic approaches for gene manipulation in flies, including the recently developed method of targeted mutagenesis by recombinational knock-in.

Archives of Neurology, 2006

Background: Both dominant and recessive mutations were reported in the gene encoding the mitochon... more Background: Both dominant and recessive mutations were reported in the gene encoding the mitochondrial (mt) DNA polymerase ␥ (POLG) in patients with progressive external ophthalmoplegia (PEO). Phenotypes other than PEO were recently documented in patients with mutations in the POLG gene. Objective: To screen patients with mitochondrial disease and multiple mtDNA deletions in muscle for mutations in the coding regions of the POLG, PEO1, and SLC25A4 genes. Design: To identify the underlying molecular defect in a group of patients with multiple mtDNA deletions comparing their molecular genetic findings with those of healthy controls. Patients: Twenty-four patients (16 men and 8 women) diagnosed with mitochondrial disease and having multiple mtDNA deletions in muscle by Southern blot analysis. Thirteen patients had PEO; 2 had PEO alone, 4 had PEO and myopathy, and 5 had PEO and multisystem involvement. Four patients had multisystem disease without PEO. The remaining 9 patients had isolated myopathy. DNA from 100 healthy individuals was also studied. Results: No mutation was identified in the PEO1 or SLC25A4 genes. Nine POLG mutations were observed in 6 of 24 patients. Four novel mutations were detected and mapped in the linker region (M603L) and in the pol domain of the enzyme (R853W; D1184N; R1146C). Five patients with PEO had mutations: 2 were compound heterozygotes, 1 was homozygous, and another showed a mutation in a single allele. The remaining patient also showed a sole mutation and had an unusual phenotype lacking ocular involvement. Conclusions: POLG molecular defects were found in 25% of our patients with multiple mtDNA deletions and mitochondrial disease. The uncommon phenotype found in 1 of these patients stresses the clinical variability of patients harboring POLG mutations. Molecular studies in the POLG gene should be addressed in patients with mitochondrial disease, particularly in those with PEO, and multiple mtDNA deletions.

Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico, Jan 24, 2016

The potential role of the mitochondrial genome has recently attracted interest because of its hig... more The potential role of the mitochondrial genome has recently attracted interest because of its high mutation frequency in tumors. Different aspects of mtDNA make it relevant for cancer's biology, such as it encodes a limited but essential number of genes for OXPHOS biogenesis, it is particularly susceptible to mutations, and its copy number can vary. Moreover, most ROS in mitochondria are produced by the electron transport chain. These characteristics place the mtDNA in the center of multiple signaling pathways, known as mitochondrial retrograde signaling, which modifies numerous key processes in cancer. Cybrid studies support that mtDNA mutations are relevant and exert their effect through a modification of OXPHOS function and ROS production. However, there is still much controversy regarding the clinical relevance of mtDNA mutations. New studies should focus more on OXPHOS dysfunction associated with a specific mutational signature rather than the presence of mutations in the m...

Journal of Biological Chemistry, Aug 4, 2000

The developmental pattern of expression of the genes encoding the catalytic (␣) and accessory (␤)... more The developmental pattern of expression of the genes encoding the catalytic (␣) and accessory (␤) subunits of mitochondrial DNA polymerase (pol ␥) has been examined in Drosophila melanogaster. The steady-state level of pol ␥-␤ mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. In contrast, the steady-state level of pol ␥-␣ mRNA decreases as development proceeds and is low in stages of active mtDNA replication. This difference in mRNA abundance results at least in part from differences in the rates of mRNA synthesis. The pol ␥ genes are located in a compact cluster of five genes that contains three promoter regions (P1-P3). The P1 region directs divergent transcription of the pol ␥-␤ gene and the adjacent rpII33 gene. P1 contains a DNA replication-related element (DRE) that is essential for pol ␥-␤ promoter activity, but not for rpII33 promoter activity in Schneider's cells. A second divergent promoter region (P2) controls the expression of the orc5 and sop2 genes. The P2 region contains two DREs that are essential for orc5 promoter activity, but not for sop2 promoter activity. The expression of the pol ␥-␣ gene is directed by P3, a weak promoter that does not contain DREs. Electrophoretic mobility shift experiments demonstrate that the DREbinding factor (DREF) regulatory protein binds to the DREs in P1 and P2. DREF regulates the expression of several genes encoding key factors involved in nuclear DNA replication. Its role in controlling the expression of the pol ␥-␤ and orc5 genes establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication. Overall, our results suggest that the accessory subunit of mtDNA polymerase plays an important role in the control of mtDNA replication in Drosophila. Animal mitochondrial DNAs, with few exceptions, encode 13 subunits of the respiratory complexes I and III-V located in the inner mitochondrial membrane. The remainder of the ϳ1000

Thyroid hormone (T3) plays a critical role in the develop-ment of the central nervous system and ... more Thyroid hormone (T3) plays a critical role in the develop-ment of the central nervous system and its deficiency during the early neonatal period results in severe brain damage. However the mechanisms involved and the genes specifically regulated by T3 during brain development are largely un-known. By using a subtractive hybridization technique we have isolated a number of cDNAs that represented mito-chondrial genes (12S and 16S rRNAs and cytochrome c oxidase subunit I). The steady state level of all three RNAs was reduced in hypothyroid animals during the postnatal period and T3 administration restored control levels. During fetal life the level of 16S rRNA was decreased in the brain of hypothyroid animals, suggesting a prenatal effect of thyroid hormone on brain development. Since T3 does not affect the amount of mitochondrial DNA, the results suggest that the effect of T3 is at transcriptional and/or postranscriptional level. In addition, the transcript levels for two nuclear-en-c...

1Departamento de Bioquímica, Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC, and... more 1Departamento de Bioquímica, Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC, and Centro de Investigación Biomédica en Red en Enfermedades Raras (CIBERER), Facultad de Medicina, Universidad Autónoma de Madrid, Spain 2Instituto de Investigación Santitaria Hospital 12 de Octubre (i+12) 3Centro de Biología Molecular “Severo Ochoa” CSIC-UAM, Madrid, Spain. 4Department of Biochemistry and Molecular Biology and Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824-1319, USA. 5Department of Biomedical Sciences, MRC Center for Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, UK. 6Present address: Department of Biomedical Sciences, MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, UK. 7Present address: University of Miami Miller School of Medicine. Department of Neurology and Cell Biology and Anatomy. 1420 NW 9 Avenue, Miami, F1 33136. 8Present address:...

Resumen del poster presentado a la VIII Reunion Cientifica Anual del Centro de Investigacion Biom... more Resumen del poster presentado a la VIII Reunion Cientifica Anual del Centro de Investigacion Biomedica En Red de Enfermedades Raras, celebrada en San Lorenzo del Escorial (Madrid) los dias 12 y 13 de marzo de 2015.

International Journal of Molecular Sciences

Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characteriz... more Leigh syndrome (LS) is the most frequent infantile mitochondrial disorder (MD) and is characterized by neurodegeneration and astrogliosis in the basal ganglia or the brain stem. At present, there is no cure or treatment for this disease, partly due to scarcity of LS models. Current models generally fail to recapitulate important traits of the disease. Therefore, there is an urgent need to develop new human in vitro models. Establishment of induced pluripotent stem cells (iPSCs) followed by differentiation into neurons is a powerful tool to obtain an in vitro model for LS. Here, we describe the generation and characterization of iPSCs, neural stem cells (NSCs) and iPSC-derived neurons harboring the mtDNA mutation m.13513G>A in heteroplasmy. We have performed mitochondrial characterization, analysis of electrophysiological properties and calcium imaging of LS neurons. Here, we show a clearly compromised oxidative phosphorylation (OXPHOS) function in LS patient neurons. This is also...