Publications by authors named "Araceli del Arco"

35 Publications

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuint.2019.104541DOI Listing
December 2019

The Response to Stimulation in Neurons and Astrocytes.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11064-019-02803-7DOI Listing
October 2019

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.23574DOI Listing
April 2019

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2017.00363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735122PMC
December 2017

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2017.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673633PMC
November 2017

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep42993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5318958PMC
February 2017

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbabio.2016.04.003DOI Listing
August 2016

Calcium regulation of mitochondrial carriers.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2016.03.024DOI Listing
October 2016

PKA Phosphorylates the ATPase Inhibitory Factor 1 and Inactivates Its Capacity to Bind and Inhibit the Mitochondrial H(+)-ATP Synthase.

Cell Rep 2015 Sep 17;12(12):2143-55. Epub 2015 Sep 17.

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Centro de Investigación Biomédica en Red de Enfermedades Raras CIBERER-ISCIII, Instituto de Investigación Hospital 12 de Octubre, Universidad Autónoma de Madrid, 28049 Madrid, Spain. Electronic address:

The mitochondrial H(+)-ATP synthase synthesizes most of cellular ATP requirements by oxidative phosphorylation (OXPHOS). The ATPase Inhibitory Factor 1 (IF1) is known to inhibit the hydrolase activity of the H(+)-ATP synthase in situations that compromise OXPHOS. Herein, we demonstrate that phosphorylation of S39 in IF1 by mitochondrial protein kinase A abolishes its capacity to bind the H(+)-ATP synthase. Only dephosphorylated IF1 binds and inhibits both the hydrolase and synthase activities of the enzyme. The phosphorylation status of IF1 regulates the flux of aerobic glycolysis and ATP production through OXPHOS in hypoxia and during the cell cycle. Dephosphorylated IF1 is present in human carcinomas. Remarkably, mouse heart contains a large fraction of dephosphorylated IF1 that becomes phosphorylated and inactivated upon in vivo β-adrenergic stimulation. Overall, we demonstrate the essential function of the phosphorylation of IF1 in regulating energy metabolism and speculate that dephosho-IF1 might play a role in signaling mitohormesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.08.052DOI Listing
September 2015

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.2702-14.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605552PMC
February 2015

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbabio.2014.04.010DOI Listing
October 2014

Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

J Neurosci 2013 Aug;33(35):13957-71, 13971a

Departament of Molecular Biology, Centre for Molecular Biology Severo Ochoa UAM-CSIC, Autonomous University of Madrid, Madrid, Spain.

Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0929-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618512PMC
August 2013

Glucagon regulation of oxidative phosphorylation requires an increase in matrix adenine nucleotide content through Ca2+ activation of the mitochondrial ATP-Mg/Pi carrier SCaMC-3.

J Biol Chem 2013 Mar 23;288(11):7791-802. Epub 2013 Jan 23.

Departmento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-CSIC, Madrid, Spain.

It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca(2+)-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/Pi carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca(2+) signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca(2+)-dependent way with an S0.5 for Ca(2+) activation of 3.3 ± 0.9 μm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca(2+)-mobilizing agents, possibly by allowing a Ca(2+)-dependent accumulation of mitochondrial AdNs and matrix Ca(2+), events permissive for other glucagon actions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.409144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597818PMC
March 2013

Clonal human fetal ventral mesencephalic dopaminergic neuron precursors for cell therapy research.

PLoS One 2012 31;7(12):e52714. Epub 2012 Dec 31.

Department of Molecular Biology-U.A.M. and Center of Molecular Biology "Severo Ochoa"-U.A.M.-C.S.I.C. Nicolás Cabrera, 1 Universidad Autónoma de Madrid, Campus Cantoblanco, Madrid, Spain.

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH⁺ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052714PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534109PMC
June 2013

SCaMC-1Like a member of the mitochondrial carrier (MC) family preferentially expressed in testis and localized in mitochondria and chromatoid body.

PLoS One 2012 6;7(7):e40470. Epub 2012 Jul 6.

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

Mitochondrial carriers (MC) form a highly conserved family involved in solute transport across the inner mitochondrial membrane in eukaryotes. In mammals, ATP-Mg/Pi carriers, SCaMCs, form the most complex subgroup with four paralogs, SCaMC-1, -2, -3 and -3L, and several splicing variants. Here, we report the tissue distribution and subcellular localization of a mammalian-specific SCaMC paralog, 4930443G12Rik/SCaMC-1Like (SCaMC-1L), which displays unanticipated new features. SCaMC-1L proteins show higher amino acid substitution rates than its closest paralog SCaMC-1. In mouse, SCaMC-1L expression is restricted to male germ cells and regulated during spermatogenesis but unexpectedly its localization is not limited to mitochondrial structures. In mature spermatids SCaMC-1L is detected in the mitochondrial sheath but in previous differentiation stages appears associated to cytosolic granules which colocalize with specific markers of the chromatoid body (CB) in post-meiotic round spermatids and inter-mitochondrial cement (IMC) in spermatocytes. The origin of this atypical distribution was further investigated by transient expression in cell lines. Similarly to male germ cells, in addition to mitochondrial and cytosolic distribution, a fraction of SCaMC-1L-expressing COS-7 cells display cytosolic SCaMC-1L-aggregates which exhibit aggresomal-like features as the CB. Our results indicate that different regions of SCaMC-1L hinder its import into mitochondria and this apparently favours the formation of cytosolic aggregates in COS-7 cells. This mechanism could be also operational in male germ cells and explain the incorporation of SCaMC-1L into germinal granules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040470PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391283PMC
April 2013

Deficiency of the mitochondrial transporter of aspartate/glutamate aralar/AGC1 causes hypomyelination and neuronal defects unrelated to myelin deficits in mouse brain.

J Neurosci Res 2011 Dec 23;89(12):2008-17. Epub 2011 May 23.

Departament of Molecular Biology, Centre for Molecular Biology Severo Ochoa UAM-CSIC, and CIBER of Rare Diseases (CIBERER), Autonomous University of Madrid, Madrid, Spain.

The aralar/AGC1 knockout (KO) mouse shows a drastic decrease in brain aspartate and N-acetylaspartate levels and global hypomyelination, which are attributed to the lack of neuron-produced NAA used by oligodendrocytes as precursor of myelin lipid synthesis. In addition, these mice have a gradual drop in brain glutamine synthesis. We show here that hypomyelination is more pronounced in gray than in white matter regions. We find a lack of neurofilament-labelled processes in hypomyelinated fiber tracks from cerebral cortex but not from those of the cerebellar granule cell layer, which correspond to Purkinje neurons. Therefore, the impaired development or degeneration of neuronal processes in cerebral cortex is independent of hypomyelination. An increase in O4-labelled, immature oligodendrocytes is observed in gray and white matter regions of the aralar KO brain, suggesting a block in maturation compatible with the lack of NAA supplied by neurons. However, no defects in oligodendrocyte maturation were observed in in-vitro-cultured mixed astroglial cultures. We conclude that the primary defect of pyramidal neurons in cerebral cortex is possibly associated with a progressive failure in glutamatergic neurotransmission and may be among the main causes of the pathology of aralar/AGC1 deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jnr.22639DOI Listing
December 2011

Adenine nucleotide transporters in organelles: novel genes and functions.

Cell Mol Life Sci 2011 Apr 5;68(7):1183-206. Epub 2011 Jan 5.

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

In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-010-0612-3DOI Listing
April 2011

S-resistin inhibits adipocyte differentiation and increases TNFalpha expression and secretion in 3T3-L1 cells.

Biochim Biophys Acta 2010 Oct 17;1803(10):1131-41. Epub 2010 Jul 17.

Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Toledo, Spain.

S-resistin is a non-secretable resistin spliced variant described in white adipose tissue from Wistar rats. Since resistin has been implicated in adipogenesis regulation, here we have investigated the possible role of this new isoform in this process. For that, we have studied the adipocyte development in 3T3-L1 pre-adipocyte cell line stably expressing s-resistin and resistin. Both isoforms are able to restrain 3T3-L1 pre-adipocyte differentiation though affecting differently the expression pattern of pro-adipogenic transcription factors such CCAAT/enhancer binding proteins alpha and beta (C/EBPalpha and C/EBPbeta) and peroxisome proliferator-activated receptor gamma (PPARgamma), as well of proteins implicated in lipid metabolism such perilipin, fatty acid synthase (FAS), adipocyte lipid binding protein (ALBP/aP2) and carnitine palmitoyltransferase1 (CPT1). Likewise, both resistin isoforms impair insulin-stimulated glucose transport by decreasing glucose transport 4 (GLUT4) expression but to a different degree. In addition, s-resistin expressing 3T3-L1 cells display other remarkable differences. Thus, in these cells, endogenous resistin expression falls down while tumor necrosis factor alpha (TNFalpha) and interleukine 6 (IL-6) productions are increased along differentiation. These findings indicate that s-resistin isoform also impairs adipocyte differentiation affecting the expression pattern of key pro-adipogenic transcription factors and insulin sensitivity. Additionally, s-resistin may play a role in inflammatory processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2010.06.012DOI Listing
October 2010

Transport of adenine nucleotides in the mitochondria of Saccharomyces cerevisiae: interactions between the ADP/ATP carriers and the ATP-Mg/Pi carrier.

Mitochondrion 2009 Apr 17;9(2):79-85. Epub 2009 Jan 17.

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

The ADP/ATP and ATP-Mg/Pi carriers are widespread among eukaryotes and constitute two systems to transport adenine nucleotides in mitochondria. ADP/ATP carriers carry out an electrogenic exchange of ADP for ATP essential for oxidative phosphorylation, whereas ATP-Mg/Pi carriers perform an electroneutral exchange of ATP-Mg for phosphate and are able to modulate the net content of adenine nucleotides in mitochondria. The functional interplay between both carriers has been shown to modulate viability in Saccharomyces cerevisiae. The simultaneous absence of both carriers is lethal. In the light of the new evidence we suggest that, in addition to exchange of cytosolic ADP for mitochondrial ATP, the specific function of the ADP/ATP carriers required for respiration, both transporters have a second function, which is the import of cytosolic ATP in mitochondria. The participation of these carriers in the generation of mitochondrial membrane potential is discussed. Both are necessary for the function of the mitochondrial protein import and assembly systems, which are the only essential mitochondrial functions in S. cerevisiae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mito.2009.01.001DOI Listing
April 2009

The expression of rat resistin isoforms is differentially regulated in visceral adipose tissues: effects of aging and food restriction.

Metabolism 2009 Feb;58(2):204-11

Area de Bioquímica, Centro Regional de Investigaciones Biomédicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, Av Carlos III s/n 45071 Toledo, Spain.

Two variants of the adipose hormone resistin are generated by alternative splicing in Wistar rats. Here we analyzed the expression of these resistin variants in 2 main visceral adipose depots, epididymal and retroperitoneal, as well as the resistin serum concentration during aging and food restriction. Total protein levels of resistin were also analyzed in extracts from both visceral adipose depots. Resistin variants show similar patterns of relative expression in visceral adipose tissues in 3-month-old rats, representing the short variant, s-resistin, which is 15% of the full-length transcript. However, only epididymal, but not retroperitoneal, fat pad shows a decrease in both messenger RNA and protein levels of resistin isoforms with aging. Food restriction decreases adiposity index in 8- and 24-month-old animals to values even lower than those of 3-month-old animals. Food restriction decreases resistin expression in both adipose tissues in 8-month-old but not in 24-month-old rats. Interestingly, concomitant with the improvement of insulin sensitivity asserted by homeostasis model assessment, resistin serum levels decrease only in food-restricted 8-month-old animals. In contrast, food restriction up-regulates s-resistin messenger RNA in epididymal adipose tissue, whereas no significant changes are appreciated in retroperitoneal adipose tissue. These data indicate that both forms of resistin are differentially regulated by fat depot location, aging, and even nutritional status, suggesting that alternative splicing plays a key role in this differential regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.metabol.2008.09.014DOI Listing
February 2009

Requirement for aralar and its Ca2+-binding sites in Ca2+ signal transduction in mitochondria from INS-1 clonal beta-cells.

J Biol Chem 2009 Jan 7;284(1):515-24. Epub 2008 Nov 7.

Departamento de Biología Molecular, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain.

Aralar, the mitochondrial aspartate-glutamate carrier present in beta-cells, is a component of the malate-aspartate NADH shuttle (MAS). MAS is activated by Ca2+ in mitochondria from tissues with aralar as the only AGC isoform with an S0.5 of approximately 300 nm. We have studied the role of aralar and its Ca2+-binding EF-hand motifs in glucose-induced mitochondrial NAD(P)H generation by two-photon microscopy imaging in INS-1 beta-cells lacking aralar or expressing aralar mutants blocked for Ca2+ binding. Aralar knock-down in INS-1 beta-cell lines resulted in undetectable levels of aralar protein and complete loss of MAS activity in isolated mitochondria and in a 25% decrease in glucose-stimulated insulin secretion. MAS activity in mitochondria from INS-1 cells was activated 2-3-fold by extramitochondrial Ca2+, whereas aralar mutants were Ca2+ insensitive. In Ca2+-free medium, glucose-induced increases in mitochondrial NAD(P)H were small (1.3-fold) and unchanged regardless of the lack of aralar. In the presence of 1.5 mm Ca2+, glucose induced robust increases in mitochondrial NAD(P)H (approximately 2-fold) in cell lines with wild-type or mutant aralar. There was a approximately 20% reduction in NAD(P)H response in cells lacking aralar, illustrating the importance of MAS in glucose action. When small Ca2+ signals that resulted in extremely small mitochondrial Ca2+ transients were induced in the presence of glucose, the rise in mitochondrial NAD(P)H was maintained in cells with wild-type aralar but was reduced by approximately 50% in cells lacking or expressing mutant aralar. These results indicate that 1) glucose-induced activation of MAS requires Ca2+ potentiation; 2) Ca2+ activation of MAS represents a larger fraction of glucose-induced mitochondrial NAD(P)H production under conditions where suboptimal Ca2+ signals are associated with a glucose challenge (50 versus 20%, respectively); 3) inactivation of EF-hand motifs in aralar prevents activation of MAS by small Ca2+ signals. The results suggest a possible role for aralar and MAS in priming the beta-cell by Ca2+-mobilizing neurotransmitter or hormones.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M806729200DOI Listing
January 2009

Characterization of SCaMC-3-like/slc25a41, a novel calcium-independent mitochondrial ATP-Mg/Pi carrier.

Biochem J 2009 Feb;418(1):125-33

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

The SCaMCs (small calcium-binding mitochondrial carriers) constitute a subfamily of mitochondrial carriers responsible for the ATP-Mg/P(i) exchange with at least three paralogues in vertebrates. SCaMC members are proteins with two functional domains, the C-terminal transporter domain and the N-terminal domain which harbours calcium-binding EF-hands and faces the intermembrane space. In the present study, we have characterized a shortened fourth paralogue, SCaMC-3L (SCaMC-3-like; also named slc25a41), which lacks the calcium-binding N-terminal extension. SCaMC-3L orthologues are found exclusively in mammals, showing approx. 60% identity to the C-terminal half of SCaMC-3, its closest paralogue. In mammalian genomes, SCaMC-3 and SCaMC-3L genes are adjacent on the same chromosome, forming a head-to-tail tandem array, and show identical exon-intron boundaries, indicating that SCaMC-3L could have arisen from an SCaMC-3 ancestor by a partial duplication event which occurred prior to mammalian radiation. Expression and functional data suggest that, following the duplication event, SCaMC-3L has acquired more restrictive functions. Unlike the broadly expressed longer SCaMCs, mouse SCaMC-3L shows a limited expression pattern; it is preferentially expressed in testis and, at lower levels, in brain. SCaMC-3L transport activity was studied in yeast deficient in Sal1p, the calcium-dependent mitochondrial ATP-Mg/P(i) carrier, co-expressing SCaMC-3L and mitochondrial-targeted luciferase, and it was found to perform ATP-Mg/P(i) exchange, in a similar manner to Sal1p or other ATP-Mg/P(i) carriers. However, metabolite transport through SCaMC-3L is calcium-independent, representing a novel mechanism involved in adenine nucleotide transport across the inner mitochondrial membrane, different to ADP/ATP translocases or long SCaMC paralogues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20081262DOI Listing
February 2009

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose.

Mol Microbiol 2008 Aug 28;69(3):570-85. Epub 2008 Jun 28.

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

Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.03 mM and 0.28 +/- 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V(max) with a S(0.5) of 15 muM, and no changes in the K(m) for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2958.2008.06300.xDOI Listing
August 2008

Role of aralar, the mitochondrial transporter of aspartate-glutamate, in brain N-acetylaspartate formation and Ca(2+) signaling in neuronal mitochondria.

J Neurosci Res 2007 Nov;85(15):3359-66

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma, 28049, Cantoblanco, Madrid, Spain

Aralar, the Ca(2+)-dependent mitochondrial aspartate-glutamate carrier expressed in brain and skeletal muscle, is a member of the malate-aspartate NADH shuttle. Disrupting the gene for aralar, SLC25a12, in mice has enabled the discovery of two new roles of this carrier. On the one hand, it is required for synthesis of brain aspartate and N-acetylaspartate, a neuron-born metabolite that supplies acetate for myelin lipid synthesis; and on the other, it is essential for the transmission of small Ca(2+) signals to mitochondria via an increase in mitochondrial NADH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jnr.21299DOI Listing
November 2007

Mitochondrial transporters as novel targets for intracellular calcium signaling.

Physiol Rev 2007 Jan;87(1):29-67

Departamento de Biología Molecular Centro de Biología Molecular "Severo Ochoa" UAM-CSIC, Facultad de Ciencias, Universidad Autónoma, Madrid, Spain.

Ca(2+) signaling in mitochondria is important to tune mitochondrial function to a variety of extracellular stimuli. The main mechanism is Ca(2+) entry in mitochondria via the Ca(2+) uniporter followed by Ca(2+) activation of three dehydrogenases in the mitochondrial matrix. This results in increases in mitochondrial NADH/NAD ratios and ATP levels and increased substrate uptake by mitochondria. We review evidence gathered more than 20 years ago and recent work indicating that substrate uptake, mitochondrial NADH/NAD ratios, and ATP levels may be also activated in response to cytosolic Ca(2+) signals via a mechanism that does not require the entry of Ca(2+) in mitochondria, a mechanism depending on the activity of Ca(2+)-dependent mitochondrial carriers (CaMC). CaMCs fall into two groups, the aspartate-glutamate carriers (AGC) and the ATP-Mg/P(i) carriers, also named SCaMC (for short CaMC). The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle, and citrin, the liver AGC, is also a member of the urea cycle. Both types of CaMCs are activated by Ca(2+) in the intermembrane space and function together with the Ca(2+) uniporter in decoding the Ca(2+) signal into a mitochondrial response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/physrev.00005.2006DOI Listing
January 2007

Cloning, tissue expression and metal inducibility of an ubiquitous metallothionein from Panulirus argus.

Gene 2005 Nov 26;361:140-8. Epub 2005 Sep 26.

Area de Bioquímica, Facultad de Químicas, Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Camilo José Cela, 10, 13071 Ciudad Real, Spain.

Invertebrate metallothioneins (MT) have mainly been reported in digestive tissues, but data about the existence of a ubiquitous isoform expressed also in nervous tissue, are not available. Here we report the identification of a new metallothionein gene (MTPA) from the lobster Panulirus argus, putatively encoding a 59 residue polypeptide including 19 Cys. Tissue specific analysis indicated that MTPA is ubiquitously expressed in the hepatopancreas, intestine, nervous tissue and muscle, with the highest levels in the hepatopancreas and the lowest in muscle and nervous tissue. In addition, our data showed that MTPA is differentially regulated by metals: tissue explants exposed to Cd exhibited increased MTPA mRNA levels in all cases, except in muscle, with the highest effects in the nervous tissue, while Zn was effective only in the hepatopancreas. Interestingly, Cu showed no effects in any of the analyzed tissues. Taken together, these results suggest that MTPA in the hepatopancreas likely plays an important role in Cd detoxification and Zn homeostasis. The potent Cd-inducibility of MTPA in the nervous tissue might suggest a key function of this protein in protecting this highly sensitive tissue from cadmium-induced neurotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gene.2005.07.026DOI Listing
November 2005

The calcium-dependent ATP-Mg/Pi mitochondrial carrier is a target of glucose-induced calcium signalling in Saccharomyces cerevisiae.

Biochem J 2005 Dec;392(Pt 3):537-44

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAM-CSIC, Facultad de Ciencias, Universidad Autónoma, 28049 Madrid, Spain.

Sal1p is a mitochondrial protein that belongs to the SCaMC (short calcium-binding mitochondrial carrier) subfamily of mitochondrial carriers. The presence of calcium-binding motifs facing the extramitochondrial space allows the regulation of the transport activity of these carriers by cytosolic calcium and provides a new mechanism to transduce calcium signals in mitochondria without the requirement of calcium entry in the organelle. We have studied its transport activity, finding that it is a carboxyatractyloside-resistant ATP-Mg carrier. Mitochondria from a disruption mutant of SAL1 have a 50% reduction in the uptake of ATP. We have also found a clear stimulation of ATP-transport activity by calcium, with an S(0.5) of approx. 30 microM. Our results also suggest that Sal1p is a target of the glucose-induced calcium signal which is non-essential in wild-type cells, but becomes essential for transport of ATP into mitochondria in yeast lacking ADP/ATP translocases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20050806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1316293PMC
December 2005

Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier.

J Biol Chem 2005 Sep 29;280(35):31333-9. Epub 2005 Jun 29.

Department of Molecular Metabolism and Biochemical Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan.

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M505286200DOI Listing
September 2005

Novel variants of human SCaMC-3, an isoform of the ATP-Mg/P(i) mitochondrial carrier, generated by alternative splicing from 3'-flanking transposable elements.

Authors:
Araceli Del Arco

Biochem J 2005 Aug;389(Pt 3):647-55

Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAM-CSIC, Facultad de Ciencias, Universidad Autónoma, 28049 Madrid, Spain.

CaMCs (calcium-dependent mitochondrial carriers) represent a novel subfamily of metabolite carriers of mitochondria. The ATP-Mg/P(i) co-transporter, functionally characterized more than 20 years ago, has been identified to be a CaMC member. There are three isoforms of the ATP-Mg/P(i) carrier in mammals, SCaMC-1 (short CaMC-1), -2 and -3 (or APC-1, -3 and -2 respectively), corresponding to the genes SLC25A24, SLC25A25 and SLC25A23 respectively, as well as six N-terminal variants generated by alternative splicing for SCaMC-1 and -2 isoforms. In the present study, we describe four new variants of human SCaMC-3 generated by alternative splicing. The new mRNAs use the exon 9 3'-donor site and distinct 5'-acceptor sites from repetitive elements, in regions downstream of exon 10, the last exon in all SCaMCs. Transcripts lacking exon 10 (SCaMC-3b, -3b', -3c and -3d) code for shortened proteins lacking the last transmembrane domain of 422, 456 and 435 amino acids, and were found in human tissues and HEK-293T cells. Mitochondrial targeting of overexpressed SCaMC-3 variants is incomplete. Surprisingly, the import impairment is overcome by removing the N-terminal extension of these proteins, suggesting that the hydrophilic N-terminal domain also participates in the mitochondrial import process, as shown for the CaMC members aralar and citrin [Roesch, Hynds, Varga, Tranebjaerg and Koehler (2004) Hum. Mol. Genet. 13, 2101-2111].
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20050283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1180714PMC
August 2005