Publications by authors named "Jorgina Satrustegui"

58 Publications

Mitochondria and calcium defects correlate with axonal dysfunction in GDAP1-related Charcot-Marie-Tooth mouse model.

Authors:
Azahara Civera-Tregón Laura Domínguez Paula Martínez-Valero Clàudia Serrano Alex Vallmitjana Raúl Benítez Janet Hoenicka Jorgina Satrústegui Francesc Palau

Neurobiol Dis 2021 Feb 11;152:105300. Epub 2021 Feb 11.

Laboratory of Neurogenetics and Molecular Medicine - IPER, Institut de Recerca Sant Joan de Déu, Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Madrid, Spain; Department of Genetic and Molecular Medicine - IPER, Hospital Sant Joan de Déu, Barcelona, Spain; Clinic Institute of Medicine and Dermatology, Hospital Clínic, and Division of Pediatrics, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain. Electronic address:

Ganglioside-induced differentiation associated protein 1 (GDAP1) gene encodes a protein of the mitochondrial outer membrane and of the mitochondrial membrane contacts with the endoplasmic reticulum (MAMs) and lysosomes. Since mutations in GDAP1 cause Charcot-Marie-Tooth, an inherited motor and sensory neuropathy, its function is essential for peripheral nerve physiology. Our previous studies showed structural and functional defects in mitochondria and their contacts when GDAP1 is depleted. Nevertheless, the underlying axonal pathophysiological events remain unclear. Here, we have used embryonic motor neurons (eMNs) cultures from Gdap1 knockout (Gdap1) mice to investigate in vivo mitochondria and calcium homeostasis in the axons. We imaged mitochondrial axonal transport and we found a defective pattern in the Gdap1 eMNs. We also detected pathological and functional mitochondria membrane abnormalities with a drop in ATP production and a deteriorated bioenergetic status. Another consequence of the loss of GDAP1 in the soma and axons of eMNs was the in vivo increase calcium levels in both basal conditions and during recovery after neuronal stimulation with glutamate. Further, we found that glutamate-stimulation of respiration was lower in Gdap1 eMNs showing that the basal bioenergetics failure jeopardizes a full respiratory response and prevents a rapid return of calcium to basal levels. Together, our results demonstrate that the loss of GDAP1 critically compromises the morphology and function of mitochondria and its relationship with calcium homeostasis in the soma and axons, offering important insight into the cellular mechanisms associated with axonal degeneration of GDAP1-related CMT neuropathies and the relevance that axon length may have.
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http://dx.doi.org/10.1016/j.nbd.2021.105300DOI Listing
February 2021

βOHB Protective Pathways in Aralar-KO Neurons and Brain: An Alternative to Ketogenic Diet.

Authors:
Irene Pérez-Liébana María José Casarejos Andrea Alcaide Eduardo Herrada-Soler Irene Llorente-Folch Laura Contreras Jorgina Satrústegui Beatriz Pardo

J Neurosci 2020 11 21;40(48):9293-9305. Epub 2020 Oct 21.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease characterized by hypomyelination, hypotonia, developmental arrest, and epilepsy. We have investigated whether β-hydroxybutyrate (βOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in -knock-out (KO) neurons and mice. We report that βOHB efficiently recovers -KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring βOHB entry into the neuron, and protects from glutamate excitotoxicity. -deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, -KO pups were treated without distinction of gender with daily intraperitoneal injections of βOHB during 5 d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of βOHB-treated -KO mice. Although brain Asp and NAA levels did not change by βOHB administration, a 4-d βOHB treatment to -KO, but not to control, neurons led to a substantial increase in Asp (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain Asp and NAA is possibly because of its active utilization by the -KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of βOHB as a therapeutic treatment in AGC1 deficiency deserves further investigation. deficiency induces a fatal phenotype in humans and mice and is associated with impaired neurodevelopment, epilepsy, and hypomyelination. In neurons, highly expressing , its deficiency causes a metabolic blockade hampering mitochondrial energetics and respiration. Here, we find that βOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in -deficient neurons. βOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate (Asp) and NAA, which is impeded by deficiency, presumably through citrate-malate shuttle. In -knock-out (KO) mice, βOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The βOHB-induced myelin synthesis occurring together with the marked increment in neuronal NAA synthesis supports the role of NAA as a lipid precursor during postnatal myelination.
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http://dx.doi.org/10.1523/JNEUROSCI.0711-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687055PMC
November 2020

Mitochondrial movement in Aralar/Slc25a12/AGC1 deficient cortical neurons.

Authors:
Guillermo Puertas-Frías Araceli Del Arco Beatriz Pardo Jorgina Satrústegui Laura Contreras

Neurochem Int 2019 12 28;131:104541. Epub 2019 Aug 28.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAM-CSIC, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049, Madrid, Spain; Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IISFJD), 28049, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28049, Madrid, Spain. Electronic address:

The elevated energy demands in the brain are fulfilled mainly by glucose catabolism. In highly polarized neurons, about 10-50% of mitochondria are transported along microtubules using mitochondrial-born ATP to locations with high energy requirements. In this report, we have investigated the impact of Aralar deficiency on mitochondrial transport in cultured cortical neurons. Aralar/slc25a12/AGC1 is the neuronal isoform of the aspartate-glutamate mitochondrial carrier, a component of the malate-aspartate shuttle (MAS) which plays an important role in redox balance, which is essential to maintain glycolytic pyruvate supply to neuronal mitochondria. Using live imaging microscopy we observed that the lack of Aralar does not affect the number of moving mitochondria nor the Ca-induced stop, the only difference being a 10% increase in mitochondrial velocity in Aralar deficient neurons. Therefore, we evaluated the possible fuels used in each case by studying the relative contribution of oxidative phosphorylation and glycolysis to mitochondrial movement using specific inhibitors. We found that the ATP synthase inhibitor oligomycin caused a smaller inhibition of mitochondrial movement in Aralar-KO than control neurons, whereas the glycolysis inhibitor iodoacetate had similar effects in neurons from both genotypes. In line with these findings, the decrease in cytosolic ATP/ADP ratio caused by oligomycin was more pronounced in control than in Aralar-KO neurons, but no differences were observed with iodoacetate. Oligomycin effect was reverted by aralar re-expression in knock out cultures. As mitochondrial movement is not reduced in Aralar-KO neurons, these results suggest that these neurons may use an additional pathway for mitochondria movement and ATP/ADP ratio maintenance.
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http://dx.doi.org/10.1016/j.neuint.2019.104541DOI Listing
December 2019

The Response to Stimulation in Neurons and Astrocytes.

Authors:
Inés Juaristi Laura Contreras Paloma González-Sánchez Irene Pérez-Liébana Luis González-Moreno Beatriz Pardo Araceli Del Arco Jorgina Satrústegui

Neurochem Res 2019 Oct 23;44(10):2385-2391. Epub 2019 Apr 23.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Nicolás Cabrera, 1, 28049, Madrid, Spain.

The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this high oxidative index, contrasts with the metabolic traits of the major cell types in the brain studied in culture, neurons and astrocytes, including the selective use of the malate-aspartate shuttle (MAS) in neurons and the glycerol-phosphate shuttle in astrocytes. Metabolic interactions among these cell types may partly explain the high oxidative index of the brain. In vivo, neuronal activation results in a decrease in the oxygen glucose index, which has been attributed to a stimulation of glycolysis and lactate production in astrocytes in response to glutamate uptake (astrocyte-neuron lactate shuttle, ANLS). Recent findings indicate that this is accompanied with a stimulation of pyruvate formation and astrocyte respiration, indicating that lactate formation is not the only astrocytic response to neuronal activation. ANLS proposes that neurons utilize lactate produced by neighboring astrocytes. Indeed, neurons can use lactate to support an increase in respiration with different workloads, and this depends on the Ca activation of MAS. However, whether this activation operates in the brain, particularly at high stimulation conditions, remains to be established.
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http://dx.doi.org/10.1007/s11064-019-02803-7DOI Listing
October 2019

Calcium Deregulation and Mitochondrial Bioenergetics in GDAP1-Related CMT Disease.

Authors:
Paloma González-Sánchez Jorgina Satrústegui Francesc Palau Araceli Del Arco

Int J Mol Sci 2019 Jan 18;20(2). Epub 2019 Jan 18.

Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28029 Madrid, Spain.

The pathology of Charcot-Marie-Tooth (CMT), a disease arising from mutations in different genes, has been associated with an impairment of mitochondrial dynamics and axonal biology of mitochondria. Mutations in () cause several forms of CMT neuropathy, but the pathogenic mechanisms involved remain unclear. GDAP1 is an outer mitochondrial membrane protein highly expressed in neurons. It has been proposed to play a role in different aspects of mitochondrial physiology, including mitochondrial dynamics, oxidative stress processes, and mitochondrial transport along the axons. Disruption of the mitochondrial network in a neuroblastoma model of -related CMT has been shown to decrease Ca entry through the store-operated calcium entry (SOCE), which caused a failure in stimulation of mitochondrial respiration. In this review, we summarize the different functions proposed for GDAP1 and focus on the consequences for Ca homeostasis and mitochondrial energy production linked to CMT disease caused by different mutations.
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http://dx.doi.org/10.3390/ijms20020403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359725PMC
January 2019

Extracellular ATP and glutamate drive pyruvate production and energy demand to regulate mitochondrial respiration in astrocytes.

Authors:
Inés Juaristi Irene Llorente-Folch Jorgina Satrústegui Araceli Del Arco

Glia 2019 04 9;67(4):759-774. Epub 2019 Jan 9.

Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.

Astrocytes respond to energetic demands by upregulating glycolysis, lactate production, and respiration. This study addresses the role of respiration and calcium regulation of respiration as part of the astrocyte response to the workloads caused by extracellular ATP and glutamate. Extracellular ATP (100 μM to 1 mM) causes a Ca -dependent workload and fall of the cytosolic ATP/ADP ratio which acutely increases astrocytes respiration. Part of this increase is related to a Ca -dependent upregulation of cytosolic pyruvate production. Conversely, glutamate (200 μM) causes a Na , but not Ca , dependent workload even though glutamate-induced Ca signals readily reach mitochondria. The glutamate workload triggers a rapid fall in the cytosolic ATP/ADP ratio and stimulation of respiration. These effects are mimicked by D-aspartate a nonmetabolized agonist of the glutamate transporter, but not by a metabotropic glutamate receptor agonist, indicating a major role of Na -dependent workload in stimulated respiration. Glutamate-induced increase in respiration is linked to a rapid increase in glycolytic pyruvate production, suggesting that both glutamate and extracellular ATP cause an increase in astrocyte respiration fueled by workload-induced increase in pyruvate production. However, glutamate-induced pyruvate production is partly resistant to glycolysis blockers (iodoacetate), indicating that oxidative consumption of glutamate also contributes to stimulated respiration. As stimulation of respiration by ATP and glutamate are similar and pyruvate production smaller in the first case, the results suggest that the response to extracellular ATP is a Ca -dependent upregulation of respiration added to glycolysis upregulation. The global contribution of astrocyte respiratory responses to brain oxygen consumption is an open question.
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http://dx.doi.org/10.1002/glia.23574DOI Listing
April 2019

Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients.

Authors:
Bruna Calsina Maria Currás-Freixes Alexandre Buffet Tirso Pons Laura Contreras Rocío Letón Iñaki Comino-Méndez Laura Remacha María Calatayud Berta Obispo Antoine Martin Regis Cohen Susan Richter Judith Balmaña Esther Korpershoek Elena Rapizzi Timo Deutschbein Laurent Vroonen Judith Favier Ronald R de Krijger Martin Fassnacht Felix Beuschlein Henri J Timmers Graeme Eisenhofer Massimo Mannelli Karel Pacak Jorgina Satrústegui Cristina Rodríguez-Antona Laurence Amar Alberto Cascón Nicole Dölker Anne-Paule Gimenez-Roqueplo Mercedes Robledo

Genet Med 2018 12 16;20(12):1652-1662. Epub 2018 Jul 16.

Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Purpose: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype.

Methods: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach.

Results: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL.

Conclusion: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.
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http://dx.doi.org/10.1038/s41436-018-0068-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456538PMC
December 2018

Store-Operated Calcium Entry Is Required for mGluR-Dependent Long Term Depression in Cortical Neurons.

Authors:
Paloma González-Sánchez Araceli Del Arco José A Esteban Jorgina Satrústegui

Front Cell Neurosci 2017 14;11:363. Epub 2017 Dec 14.

Department of Molecular Biology, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain.

Store-operated calcium entry (SOCE) is a Calcium (Ca) influx pathway activated by depletion of intracellular stores that occurs in eukaryotic cells. In neurons, the presence and functions of SOCE are still in question. Here, we show evidences for the existence of SOCE in primary mouse cortical neurons. Endoplasmic reticulum (ER)-Ca depletion using thapsigargin (Tg) triggered a maintained cytosolic Ca increase, which rapidly returned to basal level in the presence of the SOCE blockers 2-Aminoethoxydiphenyl borate (2-APB) and YM-58483. Neural SOCE is also engaged by activation of metabotropic glutamate receptors (mGluRs) with (S)-3,5-dihydroxyphenylglycine (DHPG) (agonist of group I mGluRs), being an essential mechanism to maintain the mGluR-driven Ca signal. Activation of group I of mGluRs triggers long-term depression (LTD) in many brain regions, but the underlying mechanism and, specifically, the necessity of Ca increase in the postsynaptic neuron is controversial. In primary cortical neurons, we now show that the inhibition of Ca influx through SOCE impaired DHPG-LTD, pointing out a key function of calcium and SOCE in synaptic plasticity.
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http://dx.doi.org/10.3389/fncel.2017.00363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735122PMC
December 2017

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Authors:
Niamh M C Connolly Pierre Theurey Vera Adam-Vizi Nicolas G Bazan Paolo Bernardi Juan P Bolaños Carsten Culmsee Valina L Dawson Mohanish Deshmukh Michael R Duchen Heiko Düssmann Gary Fiskum Maria F Galindo Giles E Hardingham J Marie Hardwick Mika B Jekabsons Elizabeth A Jonas Joaquin Jordán Stuart A Lipton Giovanni Manfredi Mark P Mattson BethAnn McLaughlin Axel Methner Anne N Murphy Michael P Murphy David G Nicholls Brian M Polster Tullio Pozzan Rosario Rizzuto Jorgina Satrústegui Ruth S Slack Raymond A Swanson Russell H Swerdlow Yvonne Will Zheng Ying Alvin Joselin Anna Gioran Catarina Moreira Pinho Orla Watters Manuela Salvucci Irene Llorente-Folch David S Park Daniele Bano Maria Ankarcrona Paola Pizzo Jochen H M Prehn

Cell Death Differ 2018 03 11;25(3):542-572. Epub 2017 Dec 11.

Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.
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http://dx.doi.org/10.1038/s41418-017-0020-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864235PMC
March 2018

De Novo Mutations in SLC25A24 Cause a Disorder Characterized by Early Aging, Bone Dysplasia, Characteristic Face, and Early Demise.

Authors:
Karin Writzl Ales Maver Lidija Kovačič Paula Martinez-Valero Laura Contreras Jorgina Satrustegui Marco Castori Laurence Faivre Pablo Lapunzina André B P van Kuilenburg Slobodanka Radović Christel Thauvin-Robinet Borut Peterlin Araceli Del Arco Raoul C Hennekam

Am J Hum Genet 2017 Nov;101(5):844-855

Department of Pediatrics, Academic Medical Center, University of Amsterdam, 1105AZ Amsterdam, the Netherlands.

A series of simplex cases have been reported under various diagnoses sharing early aging, especially evident in congenitally decreased subcutaneous fat tissue and sparse hair, bone dysplasia of the skull and fingers, a distinctive facial gestalt, and prenatal and postnatal growth retardation. For historical reasons, we suggest naming the entity Fontaine syndrome. Exome sequencing of four unrelated affected individuals showed that all carried the de novo missense variant c.649C>T (p.Arg217Cys) or c.650G>A (p.Arg217His) in SLC25A24, a solute carrier 25 family member coding for calcium-binding mitochondrial carrier protein (SCaMC-1, also known as SLC25A24). SLC25A24 allows an electro-neutral and reversible exchange of ATP-Mg and phosphate between the cytosol and mitochondria, which is required for maintaining optimal adenine nucleotide levels in the mitochondrial matrix. Molecular dynamic simulation studies predict that p.Arg217Cys and p.Arg217His narrow the substrate cavity of the protein and disrupt transporter dynamics. SLC25A24-mutant fibroblasts and cells expressing p.Arg217Cys or p.Arg217His variants showed altered mitochondrial morphology, a decreased proliferation rate, increased mitochondrial membrane potential, and decreased ATP-linked mitochondrial oxygen consumption. The results suggest that the SLC25A24 mutations lead to impaired mitochondrial ATP synthesis and cause hyperpolarization and increased proton leak in association with an impaired energy metabolism. Our findings identify SLC25A24 mutations affecting codon 217 as the underlying genetic cause of human progeroid Fontaine syndrome.
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http://dx.doi.org/10.1016/j.ajhg.2017.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673633PMC
November 2017

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

Authors:
Laura Remacha Iñaki Comino-Méndez Susan Richter Laura Contreras María Currás-Freixes Guillermo Pita Rocío Letón Antonio Galarreta Rafael Torres-Pérez Emiliano Honrado Scherezade Jiménez Lorena Maestre Sebastian Moran Manel Esteller Jorgina Satrústegui Graeme Eisenhofer Mercedes Robledo Alberto Cascón

Clin Cancer Res 2017 Oct 18;23(20):6315-6324. Epub 2017 Jul 18.

Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes. We used downregulation and methylation of as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases. One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in A variant in , c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio. This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2250DOI Listing
October 2017

Micro-sampling method based on high-resolution continuum source graphite furnace atomic absorption spectrometry for calcium determination in blood and mitochondrial suspensions.

Authors:
Beatriz Gómez-Nieto Mª Jesús Gismera Mª Teresa Sevilla Jorgina Satrústegui Jesús R Procopio

Talanta 2017 Aug 30;170:15-21. Epub 2017 Mar 30.

Departamento de Química Analítica y Análisis Instrumental, Facultad de Ciencias, Avda. Francisco Tomás y Valiente, 7, Universidad Autónoma de Madrid, 28049 Madrid, Spain.

A micro-sampling and straightforward method based on high resolution continuum source atomic absorption spectrometry (HR-CS AAS) was developed to determine extracellular and intracellular Ca in samples of interest in clinical and biomedical analysis. Solid sampling platforms were used to introduce the micro-samples into the graphite furnace atomizer. The secondary absorption line for Ca, located at 239.856nm, was selected to carry out the measurements. Experimental parameters such as pyrolysis and atomization temperatures and the amount of sample introduced for the measurements were optimized. Calibration was performed using aqueous standards and the approach to measure at the wings of the absorption lines was employed for the expansion of the linear response range. The limit of detection was of 0.02mgL Ca (0.39ng Ca) and the upper limit of linear range was increased up to 8.0mgL Ca (160ng Ca). The proposed method was used to determine Ca in mitochondrial suspensions and whole blood samples with successful results. Adequate recoveries (within 91-107%) were obtained in the tests performed for validation purposes.
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http://dx.doi.org/10.1016/j.talanta.2017.03.086DOI Listing
August 2017

ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.

Authors:
Inés Juaristi María L García-Martín Tiago B Rodrigues Jorgina Satrústegui Irene Llorente-Folch Beatriz Pardo

J Neurochem 2017 07 22;142(1):132-139. Epub 2017 May 22.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid, Spain.

ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells.
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http://dx.doi.org/10.1111/jnc.14047DOI Listing
July 2017

CMT-linked loss-of-function mutations in GDAP1 impair store-operated Ca entry-stimulated respiration.

Authors:
Paloma González-Sánchez David Pla-Martín Paula Martínez-Valero Carlos B Rueda Eduardo Calpena Araceli Del Arco Francesc Palau Jorgina Satrústegui

Sci Rep 2017 02 21;7:42993. Epub 2017 Feb 21.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, 28049, Spain.

GDAP1 is an outer mitochondrial membrane protein involved in Charcot-Marie-Tooth (CMT) disease. Lack of GDAP1 gives rise to altered mitochondrial networks and endoplasmic reticulum (ER)-mitochondrial interactions resulting in a decreased ER-Ca levels along with a defect on store-operated calcium entry (SOCE) related to a misallocation of mitochondria to subplasmalemmal sites. The defect on SOCE is mimicked by MCU silencing or mitochondrial depolarization, which prevent mitochondrial calcium uptake. Ca release from de ER and Ca inflow through SOCE in neuroblastoma cells result in a Ca-dependent upregulation of respiration which is blunted in GDAP1 silenced cells. Reduced SOCE in cells with CMT recessive missense mutations in the α-loop of GDAP1, but not dominant mutations, was associated with smaller SOCE-stimulated respiration. These cases of GDAP1 deficiency also resulted in a decreased ER-Ca levels which may have pathological implications. The results suggest that CMT neurons may be under energetic constraints upon stimulation by Ca mobilization agonists and point to a potential role of perturbed mitochondria-ER interaction related to energy metabolism in forms of CMT caused by some of the recessive or null mutations of GDAP1.
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http://dx.doi.org/10.1038/srep42993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5318958PMC
February 2017

Deficient glucose and glutamine metabolism in knockout mice contributes to altered visual function.

Authors:
Laura Contreras Laura Ramirez Jianhai Du James B Hurley Jorgina Satrústegui Pedro de la Villa

Mol Vis 2016 12;22:1198-1212. Epub 2016 Oct 12.

Department of Systems Biology, University of Alcalá, Alcalá de Henares, Spain.

Purpose: To characterize the vision phenotype of mice lacking , the mitochondrial aspartate-glutamate carrier mutated in global cerebral hypomyelination (OMIM 612949).

Methods: We tested overnight dark-adapted control and aralar-deficient mice for the standard full electroretinogram (ERG) response. The metabolic stress of dark-adaptation was reduced by 5 min illumination after which the ERG response was monitored in darkness. We used the electrical response to two identical saturating light flashes (paired-flash stimulation) to isolate the inner retina and photoreceptor responses. Retinal morphology was examined with hematoxylin and eosin staining, immunohistochemistry of antibodies against retinal cells, and 4',6-diamidino-2-phenylindole (DAPI) labeling.

Results: Aralar plays a pivotal role in retina metabolism as aralar provides de novo synthesis pathway for glutamine, protects glutamate from oxidation, and is required for efficient glucose oxidative metabolism. Aralar-deficient mice are not blind as their retinas have light-evoked activity. However, we report an approximate 50% decrease in the ERG amplitude response in the light-evoked activity of dark-adapted retinas from aralar-deficient mice, in spite of normal retina histology. The defective response is partly reversed by exposure to a brief illumination period, which lowers the metabolic stress of dark-adaptation. The metabolic stress and ERG alteration takes place primarily in photoreceptors, but the response to two flashes applied in fast succession also revealed an alteration in synaptic transmission consistent with an imbalance of glutamate and an energy deficit in the inner retina neurons.

Conclusions: We propose that compromised glucose oxidation and altered glutamine and glutamate metabolism in the absence of aralar are responsible for the phenotype reported.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5063090PMC
January 2018

Heme-Oxygenase I and PCG-1α Regulate Mitochondrial Biogenesis via Microglial Activation of Alpha7 Nicotinic Acetylcholine Receptors Using PNU282987.

Authors:
Elisa Navarro Laura Gonzalez-Lafuente Irene Pérez-Liébana Izaskun Buendia Elia López-Bernardo Cristina Sánchez-Ramos Ignacio Prieto Antonio Cuadrado Jorgina Satrustegui Susana Cadenas Maria Monsalve Manuela G López

Antioxid Redox Signal 2017 07 30;27(2):93-105. Epub 2016 Sep 30.

1 Instituto Teófilo Hernando, Departamento Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid , Madrid, Spain .

Aims: A loss in brain acetylcholine and cholinergic markers, subchronic inflammation, and impaired mitochondrial function, which lead to low-energy production and high oxidative stress, are common pathological factors in several neurodegenerative diseases (NDDs). Glial cells are important for brain homeostasis, and microglia controls the central immune response, where α7 acetylcholine nicotinic receptors (nAChR) seem to play a pivotal role; however, little is known about the effects of this receptor in metabolism. Therefore, the aim of this study was to evaluate if glial mitochondrial energetics could be regulated through α7 nAChR.

Results: Primary glial cultures treated with the α7 nicotinic agonist PNU282987 increased their mitochondrial mass and their mitochondrial oxygen consumption without increasing oxidative stress; these changes were abolished when nuclear erythroid 2-related factor 2 (Nrf2) was absent, heme oxygenase-1 (HO-1) was inhibited, or peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) was silenced. More specifically, microglia of animals treated intraperitoneally with the α7 nAChR agonist PNU282987 (10 mg/kg) showed a significant increase in mitochondrial mass. Interestingly, LysM-Hmox1 and PGC-1α animals showed lower microglial mitochondrial levels and treatment with PNU282987 did not produce effects on mitochondrial levels.

Innovation: Increases in microglial mitochondrial mass and metabolism can be achieved via α7 nAChR by a mechanism that implicates Nrf2, HO-1, and PGC-1α. This signaling pathway could open a new strategy for the treatment of NDDs, such as Alzheimer's, characterized by a reduction of cholinergic markers.

Conclusion: α7 nAChR signaling increases glial mitochondrial mass, both in vitro and in vivo, via HO-1 and PCG-1α. These effects could be of potential benefit in the context of NDDs. Antioxid. Redox Signal. 27, 93-105.
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http://dx.doi.org/10.1089/ars.2016.6698DOI Listing
July 2017

Uncoupling Protein 2 (UCP2) Function in the Brain as Revealed by the Cerebral Metabolism of (1-C)-Glucose.

Authors:
Laura Contreras Eduardo Rial Sebastian Cerdan Jorgina Satrustegui

Neurochem Res 2017 Jan 12;42(1):108-114. Epub 2016 Jul 12.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, CSIC-UAM-Universidad Autónoma de Madrid, c/Nicolás Cabrera 1, 28049, Madrid, Spain.

The mitochondrial aspartate/glutamate transporter Aralar/AGC1/Slc25a12 is critically involved in brain aspartate synthesis, and AGC1 deficiency results in a drastic fall of brain aspartate levels in humans and mice. It has recently been described that the uncoupling protein UCP2 transports four carbon metabolites including aspartate. Since UCP2 is expressed in several brain cell types and AGC1 is mainly neuronal, we set to test whether UCP2 could be a mitochondrial aspartate carrier in the brain glial compartment. The study of the cerebral metabolism of (1-C)-glucose in vivo in wild type and UCP2-knockout mice showed no differences in C3 or C2 labeling of aspartate, suggesting that UCP2 does not function as a mitochondrial aspartate carrier in brain. However, surprisingly, a clear decrease (of about 30-35 %) in the fractional enrichment of glutamate, glutamine and GABA was observed in the brains of UCP2-KO mice which was not associated with differences in either glucose or lactate enrichments. The results suggest that the dilution in the labeling of glutamate and its downstream metabolites could originate from the uptake of an unlabeled substrate that could not leave the matrix via UCP2 becoming trapped in the matrix. Understanding the nature of the unlabeled substrate and its precursor(s) as alternative substrates to glucose is of interest in the context of neurological diseases associated with UCP2.
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http://dx.doi.org/10.1007/s11064-016-1999-5DOI Listing
January 2017

L-Lactate-Mediated Neuroprotection against Glutamate-Induced Excitotoxicity Requires ARALAR/AGC1.

Authors:
Irene Llorente-Folch Carlos B Rueda Irene Pérez-Liébana Jorgina Satrústegui Beatriz Pardo

J Neurosci 2016 Apr;36(16):4443-56

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid 28049, Spain, Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid 28029, Spain, and Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid 28006, Spain

Unlabelled: ARALAR/AGC1/Slc25a12, the aspartate-glutamate carrier from brain mitochondria, is the regulatory step in the malate-aspartate NADH shuttle, MAS. MAS is used to oxidize cytosolic NADH in mitochondria, a process required to maintain oxidative glucose utilization. The role of ARALAR was analyzed in two paradigms of glutamate-induced excitotoxicity in cortical neurons: glucose deprivation and acute glutamate stimulation. ARALAR deficiency did not aggravate glutamate-induced neuronal death in vitro, although glutamate-stimulated respiration was impaired. In contrast, the presence of L-lactate as an additional source protected against glutamate-induced neuronal death in control, but not ARALAR-deficient neurons.l-Lactate supplementation increased glutamate-stimulated respiration partially prevented the decrease in the cytosolic ATP/ADP ratio induced by glutamate and substantially diminished mitochondrial accumulation of 8-oxoguanosine, a marker of reactive oxygen species production, only in the presence, but not the absence, of ARALAR. In addition,l-lactate potentiated glutamate-induced increase in cytosolic Ca(2+), in a way independent of the presence of ARALAR. Interestingly,in vivo, the loss of half-a-dose of ARALAR in aralar(+/-)mice enhanced kainic acid-induced seizures and neuronal damage with respect to control animals, in a model of excitotoxicity in which increased L-lactate levels and L-lactate consumption have been previously proven. These results suggest that,in vivo, an inefficient operation of the shuttle in the aralar hemizygous mice prevents the protective role of L-lactate on glutamate excitotoxiciy and that the entry and oxidation of L-lactate through ARALAR-MAS pathway is required for its neuroprotective function.

Significance Statement: Lactate now stands as a metabolite necessary for multiple functions in the brain and is an alternative energy source during excitotoxic brain injury. Here we find that the absence of a functional malate-aspartate NADH shuttle caused by aralar/AGC1 disruption causes a block in lactate utilization by neurons, which prevents the protective role of lactate on excitotoxicity, but not glutamate excitotoxicity itself. Thus, failure to use lactate is detrimental and is possibly responsible for the exacerbated in vivo excitotoxicity in aralar(+/-)mice.
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http://dx.doi.org/10.1523/JNEUROSCI.3691-15.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601833PMC
April 2016

Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs).

Authors:
Carlos B Rueda Irene Llorente-Folch Javier Traba Ignacio Amigo Paloma Gonzalez-Sanchez Laura Contreras Inés Juaristi Paula Martinez-Valero Beatriz Pardo Araceli Del Arco Jorgina Satrustegui

Biochim Biophys Acta 2016 Aug 7;1857(8):1158-1166. Epub 2016 Apr 7.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain; CIBER de Enfermedades Raras (CIBERER), Spain; Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD), Spain.

Glutamate elicits Ca(2+) signals and workloads that regulate neuronal fate both in physiological and pathological circumstances. Oxidative phosphorylation is required in order to respond to the metabolic challenge caused by glutamate. In response to physiological glutamate signals, cytosolic Ca(2+) activates respiration by stimulation of the NADH malate-aspartate shuttle through Ca(2+)-binding to the mitochondrial aspartate/glutamate carrier (Aralar/AGC1/Slc25a12), and by stimulation of adenine nucleotide uptake through Ca(2+) binding to the mitochondrial ATP-Mg/Pi carrier (SCaMC-3/Slc25a23). In addition, after Ca(2+) entry into the matrix through the mitochondrial Ca(2+) uniporter (MCU), it activates mitochondrial dehydrogenases. In response to pathological glutamate stimulation during excitotoxicity, Ca(2+) overload, reactive oxygen species (ROS), mitochondrial dysfunction and delayed Ca(2+) deregulation (DCD) lead to neuronal death. Glutamate-induced respiratory stimulation is rapidly inactivated through a mechanism involving Poly (ADP-ribose) Polymerase-1 (PARP-1) activation, consumption of cytosolic NAD(+), a decrease in matrix ATP and restricted substrate supply. Glutamate-induced Ca(2+)-activation of SCaMC-3 imports adenine nucleotides into mitochondria, counteracting the depletion of matrix ATP and the impaired respiration, while Aralar-dependent lactate metabolism prevents substrate exhaustion. A second mechanism induced by excitotoxic glutamate is permeability transition pore (PTP) opening, which critically depends on ROS production and matrix Ca(2+) entry through the MCU. By increasing matrix content of adenine nucleotides, SCaMC-3 activity protects against glutamate-induced PTP opening and lowers matrix free Ca(2+), resulting in protracted appearance of DCD and protection against excitotoxicity in vitro and in vivo, while the lack of lactate protection during in vivo excitotoxicity explains increased vulnerability to kainite-induced toxicity in Aralar +/- mice. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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http://dx.doi.org/10.1016/j.bbabio.2016.04.003DOI Listing
August 2016

Calcium regulation of mitochondrial carriers.

Authors:
Araceli Del Arco Laura Contreras Beatriz Pardo Jorgina Satrustegui

Biochim Biophys Acta 2016 10 28;1863(10):2413-21. Epub 2016 Mar 28.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain; CIBER de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain; Instituto de Investigaciones Sanitarias Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain. Electronic address:

Mitochondrial function is regulated by calcium. In addition to the long known effects of matrix Ca(2+), regulation of metabolite transport by extramitochondrial Ca(2+) represents an alternative Ca(2+)-dependent mechanism to regulate mitochondrial function. The Ca(2+) regulated mitochondrial transporters (CaMCs) are well suited for that role, as they contain long N-terminal extensions harboring EF-hand Ca(2+) binding domains facing the intermembrane space. They fall in two groups, the aspartate/glutamate exchangers, AGCs, major components of the NADH malate aspartate shuttle (MAS) and urea cycle, and the ATP-Mg(2+)/Pi exchangers or short CaMCs (APCs or SCaMCs). The AGCs are activated by relatively low Ca(2+) levels only slightly higher than resting Ca(2+), whereas all SCaMCs studied so far require strong Ca(2+) signals, above micromolar, for activation. In addition, AGCs are not strictly Ca(2+) dependent, being active even in Ca(2+)-free conditions. Thus, AGCs are well suited to respond to small Ca(2+) signals and that do not reach mitochondria. In contrast, ATP-Mg(2+)/Pi carriers are inactive in Ca(2+) free conditions and activation requires Ca(2+) signals that will also activate the calcium uniporter (MCU). By changing the net content of adenine nucleotides of the matrix upon activation, SCaMCs regulate the activity of the permeability transition pore, and the Ca(2+) retention capacity of mitochondria (CRC), two functions synergizing with those of the MCU. The different Ca(2+) activation properties of the two CaMCs are discussed in relation to their newly obtained structures. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.
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http://dx.doi.org/10.1016/j.bbamcr.2016.03.024DOI Listing
October 2016

Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.

Authors:
Jianhai Du Austin Rountree Whitney M Cleghorn Laura Contreras Ken J Lindsay Martin Sadilek Haiwei Gu Danijel Djukovic Dan Raftery Jorgina Satrústegui Mark Kanow Lawrence Chan Stephen H Tsang Ian R Sweet James B Hurley

J Biol Chem 2016 Feb 16;291(9):4698-710. Epub 2015 Dec 16.

From the Department of Biochemistry, Department of Ophthalmology, University of Washington, Seattle, Washington 98109,

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5'-GMP, ribose-5-phosphate, ketone bodies, and purines.
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http://dx.doi.org/10.1074/jbc.M115.698985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813492PMC
February 2016

Down-regulation of oxidative phosphorylation in the liver by expression of the ATPase inhibitory factor 1 induces a tumor-promoter metabolic state.

Authors:
Fulvio Santacatterina Laura Sánchez-Cenizo Laura Formentini Maysa A Mobasher Estela Casas Carlos B Rueda Inmaculada Martínez-Reyes Cristina Núñez de Arenas Javier García-Bermúdez Juan M Zapata María Sánchez-Aragó Jorgina Satrústegui Ángela M Valverde José M Cuezva

Oncotarget 2016 Jan;7(1):490-508

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.

The ATPase Inhibitory Factor 1 (IF1) is an inhibitor of the mitochondrial H+-ATP synthase that regulates the activity of both oxidative phosphorylation (OXPHOS) and cell death. Here, we have developed transgenic Tet-On and Tet-Off mice that express a mutant active form of hIF1 in the hepatocytes to restrain OXPHOS in the liver to investigate the relevance of mitochondrial activity in hepatocarcinogenesis. The expression of hIF1 promotes the inhibition of OXPHOS in both Tet-On and Tet-Off mouse models and induces a state of metabolic preconditioning guided by the activation of the stress kinases AMPK and p38 MAPK. Expression of the transgene significantly augmented proliferation and apoptotic resistance of carcinoma cells, which contributed to an enhanced diethylnitrosamine-induced liver carcinogenesis. Moreover, the expression of hIF1 also diminished acetaminophen-induced apoptosis, which is unrelated to differences in permeability transition pore opening. Mechanistically, cell survival in hIF1-preconditioned hepatocytes results from a nuclear factor-erythroid 2-related factor (Nrf2)-guided antioxidant response. The results emphasize in vivo that a metabolic phenotype with a restrained OXPHOS in the liver is prone to the development of cancer.
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http://dx.doi.org/10.18632/oncotarget.6357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808013PMC
January 2016

Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle: Implications for Neuronal Energy Metabolism.

Authors:
Jorgina Satrústegui Lasse K Bak

Neurochem Res 2015 Dec 3;40(12):2425-30. Epub 2015 Jul 3.

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, 2100, Copenhagen, Denmark.

The malate-aspartate NADH shuttle (MAS) operates in neurons and other cells to translocate reducing equivalents from the cytosol to the mitochondrial matrix, thus allowing a continued flux through the glycolytic pathway and metabolism of extracellular lactate. Recent discoveries have taught us that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium uniporter, there is a positive correlation between Ca(2+) and MAS activity; however, if cytosolic Ca(2+) increases above the threshold, MAS activity is thought to be reduced by an intricate mechanism. The latter forces the neurons to partly rely on anaerobic glycolysis producing lactate that may be metabolized subsequently, by neurons or other cells. In this review, we will discuss the evidence for Ca(2+)-mediated regulation of MAS that have been uncovered over the last decade or so, together with the need for further verification, and examine the metabolic ramifications for neurons.
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http://dx.doi.org/10.1007/s11064-015-1652-8DOI Listing
December 2015

Whole-exome sequencing identifies MDH2 as a new familial paraganglioma gene.

Authors:
Alberto Cascón Iñaki Comino-Méndez María Currás-Freixes Aguirre A de Cubas Laura Contreras Susan Richter Mirko Peitzsch Veronika Mancikova Lucía Inglada-Pérez Andrés Pérez-Barrios María Calatayud Sharona Azriel Rosa Villar-Vicente Javier Aller Fernando Setién Sebastian Moran Juan F Garcia Ana Río-Machín Rocío Letón Álvaro Gómez-Graña María Apellániz-Ruiz Giovanna Roncador Manel Esteller Cristina Rodríguez-Antona Jorgina Satrústegui Graeme Eisenhofer Miguel Urioste Mercedes Robledo

J Natl Cancer Inst 2015 Mar 11;107(5). Epub 2015 Mar 11.

: Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre, Madrid, Spain (AC, ICM, MCF, AAdC, VM, LIP, RL, AGG, MAR, CRA, MR); Centro de Investigación Biomédica en Red de Enfermedades Raras, Madrid, Spain (AC, LC, LIP, CRA, JS, MU, MR); Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAM-CSIC, Universidad Autónoma de Madrid and Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Madrid, Spain (LC, JS); Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany (SR, MP, GE); Departments of Pathology (APB) and Endocrinology and Nutrition Service (MC), Hospital 12 de Octubre, Madrid, Spain; Endocrinology Service, Hospital Infanta Sofía, San Sebastián de los Reyes, Spain (SA); Department of Endocrinology and Nutrition Service, Hospital de Fuenlabrada, Madrid, Spain (RVV); Endocrinology Service, Hospital Puerta de Hierro, Majadahonda, Madrid, Spain (JA); Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, L'Hospitalet, Barcelona, Spain (FS, SM, ME); Department of Pathology, MD Anderson Cancer Center Madrid, Madrid, Spain (JFG); Molecular Cytogenetics Group (ARM), Monoclonal Antibodies Unit, Biotechnology Programme (GR), and Familial Cancer Clinical Unit (MU), Spanish National Cancer Research Centre, Madrid, Spain.

Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Student's t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene.
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http://dx.doi.org/10.1093/jnci/djv053DOI Listing
March 2015

Mitochondrial ATP-Mg/Pi carrier SCaMC-3/Slc25a23 counteracts PARP-1-dependent fall in mitochondrial ATP caused by excitotoxic insults in neurons.

Authors:
Carlos B Rueda Javier Traba Ignacio Amigo Irene Llorente-Folch Paloma González-Sánchez Beatriz Pardo José A Esteban Araceli del Arco Jorgina Satrústegui

J Neurosci 2015 Feb;35(8):3566-81

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28049 Madrid, Spain, Centro de Investigación Biomédica en Red de Enfermedades Raras, 28029 Madrid, Spain, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, 28006 Madrid, Spain, and

Glutamate excitotoxicity is caused by sustained activation of neuronal NMDA receptors causing a large Ca(2+) and Na(+) influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca(2+) deregulation. Mitochondria undergo early changes in membrane potential during excitotoxicity, but their precise role in these events is still controversial. Using primary cortical neurons derived from mice, we show that NMDA exposure results in a rapid fall in mitochondrial ATP in neurons deficient in SCaMC-3/Slc25a23, a Ca(2+)-regulated mitochondrial ATP-Mg/Pi carrier. This fall is associated with blunted increases in respiration and a delayed decrease in cytosolic ATP levels, which are prevented by PARP-1 inhibitors or by SCaMC-3 activity promoting adenine nucleotide uptake into mitochondria. SCaMC-3 KO neurons show an earlier delayed Ca(2+) deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca(2+) retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca(2+) deregulation. SCaMC-3 KO neurons have higher vulnerability to in vitro excitotoxicity, and SCaMC-3 KO mice are more susceptible to kainate-induced seizures, showing that early PARP-1-dependent fall in mitochondrial ATP levels, counteracted by SCaMC-3, is an early step in the excitotoxic cascade.
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http://dx.doi.org/10.1523/JNEUROSCI.2702-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605552PMC
February 2015

Pyruvate kinase and aspartate-glutamate carrier distributions reveal key metabolic links between neurons and glia in retina.

Authors:
Ken J Lindsay Jianhai Du Stephanie R Sloat Laura Contreras Jonathan D Linton Sally J Turner Martin Sadilek Jorgina Satrústegui James B Hurley

Proc Natl Acad Sci U S A 2014 Oct 13;111(43):15579-84. Epub 2014 Oct 13.

Departments of Biochemistry, Ophthalmology, University of Washington, Seattle, WA 98195; and

Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell's ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1.
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http://dx.doi.org/10.1073/pnas.1412441111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4217417PMC
October 2014

Ca(2+) regulation of mitochondrial function in neurons.

Authors:
Carlos B Rueda Irene Llorente-Folch Ignacio Amigo Laura Contreras Paloma González-Sánchez Paula Martínez-Valero Inés Juaristi Beatriz Pardo Araceli del Arco Jorgina Satrústegui

Biochim Biophys Acta 2014 Oct 10;1837(10):1617-24. Epub 2014 May 10.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Departamento de Investigaciones-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD), ISCIII, Madrid, Spain. Electronic address:

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca(2+) signal or to Ca(2+) itself. [Na(+)]i, [Ca(2+)]i and [ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca(2+) clearly showed that Ca(2+)-stimulation of coupled respiration is required to maintain [ATP]i levels. Ca(2+) may regulate respiration by activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or after Ca(2+) entry in mitochondria through the calcium uniporter (MCU). Two Ca(2+)-regulated mitochondrial metabolite transporters are expressed in neurons, the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, and the ATP-Mg/Pi exchanger SCaMC-3/APC2/Slc25a23, with S0.5 for Ca(2+) of 300nM and 3.4μM, respectively. The lack of SCaMC-3 results in a smaller Ca(2+)-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca(2+)-dependent responses to all workloads: a reduction of about 65-70% in the response to the high workload imposed by veratridine, and completely suppression of the OCR responses to moderate (K(+)-depolarization) and small (carbachol) workloads, effects reverted by pyruvate supply. For K(+)-depolarization, this occurs in spite of the presence of large [Ca(2+)]mit signals and increased formation of mitochondrial NAD(P)H. These results show that ARALAR-MAS is a major contributor of Ca(2+)-stimulated respiration in neurons by providing increased pyruvate supply to mitochondria. In its absence and under moderate workloads, matrix Ca(2+) is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.
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http://dx.doi.org/10.1016/j.bbabio.2014.04.010DOI Listing
October 2014

Inhibition of mitochondrial pyruvate transport by zaprinast causes massive accumulation of aspartate at the expense of glutamate in the retina.

Authors:
Jianhai Du Whitney M Cleghorn Laura Contreras Ken Lindsay Austin M Rountree Andrei O Chertov Sally J Turner Ayse Sahaboglu Jonathan Linton Martin Sadilek Jorgina Satrústegui Ian R Sweet François Paquet-Durand James B Hurley

J Biol Chem 2013 Dec 1;288(50):36129-40. Epub 2013 Nov 1.

From the Department of Biochemistry.

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.
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http://dx.doi.org/10.1074/jbc.M113.507285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861660PMC
December 2013

De novo Synthesis of Glial Glutamate and Glutamine in Young Mice Requires Aspartate Provided by the Neuronal Mitochondrial Aspartate-Glutamate Carrier Aralar/AGC1.

Authors:
Beatriz Pardo Laura Contreras Jorgina Satrústegui

Front Endocrinol (Lausanne) 2013 Oct 15;4:149. Epub 2013 Oct 15.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAM-CSIC, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma de Madrid , Madrid, Spain.

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http://dx.doi.org/10.3389/fendo.2013.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796713PMC
October 2013

Cytosolic reducing power preserves glutamate in retina.

Authors:
Jianhai Du Whitney Cleghorn Laura Contreras Jonathan D Linton Guy C-K Chan Andrei O Chertov Takeyori Saheki Viren Govindaraju Martin Sadilek Jorgina Satrústegui James B Hurley

Proc Natl Acad Sci U S A 2013 Nov 14;110(46):18501-6. Epub 2013 Oct 14.

Departments of Biochemistry, Pharmacology, Chemistry, and Ophthalmology, University of Washington, Seattle, WA 98195.

Glutamate in neurons is an important excitatory neurotransmitter, but it also is a key metabolite. We investigated how glutamate in a neural tissue is protected from catabolism. Flux analysis using (13)C-labeled fuels revealed that retinas use activities of the malate aspartate shuttle to protect >98% of their glutamate from oxidation in mitochondria. Isolation of glutamate from the oxidative pathway relies on cytosolic NADH/NAD(+), which is influenced by extracellular glucose, lactate, and pyruvate.
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http://dx.doi.org/10.1073/pnas.1311193110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831988PMC
November 2013