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

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

In Vivo insulin-dependent glucose uptake of specific tissues is decreased during aging of mature wistar rats

Copyright (1997) The Endocrine Society .The paper can be found at the following URL on the website http://endo.endojournals.org

Glucagon regulation of oxidative phosphorylation requires an increase in matrixadenine nucleotide content through Ca2+-activation of the mitochondrial ATPMg/Pi carrier SCaMC-3

13 p.-6 fig.-1 tab.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.This work was supported in part by Ministerio de Educación y Ciencia Grants BFU2008-04084/BMC and BFU2011-30456, European Union Grant LSHMCT- 2006-518153, and CIBERER Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (an initiative of the ISCIII Instituto de SaludCarlos III) (to J. S.), Comunidad de Madrid Grants S-GEN-0269-2006 and S2010/BMD-2402 MITOLAB-CM (to J. S., E. R., and A. S.), by ISCIII Grant PI080610 (to A. delA), and an institutional grant from the Fundación Ramon Areces to the Centro de Biología Molecular Severo Ochoa.Peer reviewe

bOHB protective pathways in Aralar-Ko neurons and brain: An alternative to ketogenic diet

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 b-hydroxybutyrate (bOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that bOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring bOHB entry into the neuron, and protects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, aralar-KO pups were treated without distinction of gender with daily intraperitoneal injections of bOHB during 5d. 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 bOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by bOHB administration, a 4-d bOHB treatment to aralar-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 aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of bOHB as a therapeutic treatment in AGC1 deficiency deserves further investigationThis work was supported by Ministerio de Economía Grants SAF2014-56929R (to J.S. and B.P.) and SAF2017-82560R (AEI/FEDER, UE; to B.P.); the Centro de Investigación Biomédica en Red de Enfermedades Raras, an initiative of the Instituto de Salud Carlos III (ISCIII); a grant from the Fundación Ramon Areces (J.S.); the Irycis Chromatographic Services and Nervous System Markers Unit, UCS (2018/0135; to M.J.C.); and an institutional grant from the Fundación Ramon Areces to the Centro de Biología Molecular Severo Ochoa. I.P.-L. is the recipient of Contrato Predoctoral de Formación de Personal Investigador (FPI MINECO). We thank Dr. Antonio S. Herranz for his inputs as an expert in amino acid analysis by HPLC-UV, Dr. Araceli del Arco for critical reading of the manuscript, and Isabel Manso and Barbara Sesé for technical support. All experiments were conducted in compliance with the ARRIVE guideline

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

Glutamate excitotoxicity is caused by sustained activation of neuronal NMDA receptors causing a large Ca2+and Na+ influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca2+ 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 Ca2+-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 Ca2+ deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca2+retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca2+ 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.This work was supported by Ministerio de Economía Grant BFU2011-30456, by Centro de Investigación Biomédica en Red de Enfermedades Raras [an initiative of the Instituto de Salud Carlos III (ISCIII)], by Comunidad de Madrid Grant S2010/BMD-2402 MITOLAB-CM (to J.S.), by ISCIII Grant PI080610 (to A.d.A.), and by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. C.B.R. is the recipient of an Formacion Personal Universitario fellowship from the Ministerio de Educación y Ciencia. P.G.-S. is a recipient of a Formacion Personal Investigador-UAM fellowship from Universidad Autónoma de Madrid.Peer Reviewe

AGC1-malate aspartate shuttle activity is critical for dopaminehandling in the nigrostriatal pathway

This is the pre-peer reviewed version of the following article: Llorente-Folch, I. et al. "AGC1-malate aspartate shuttle activity is critical for dopaminehandling in the nigrostriatal pathway". Journal of Neurochemistry 124.3 (2013): 347-362, which has been published in final form at http://dx.doi.org/10.1111/jnc.12096This study was supported by grants from the Ministerio deEducacion y Ciencia BFU2008-04084/BMC (to JS), and Ciencia eInnovacion (SAF2010-16427 to MD), Comunidad de Madrid S-GEN-0269-2006 MITOLAB-CM (to JS), European Union GrantLSHM-CT-2006-518153 (to J.S.), and CureFXS E-Rare. EU/FISPS09102673, Spanish Ministry of Health (PI 082038 to MD),Marato TV3, Jerome Lejeune (JMLM/AC /08-044) to MD, Fundac-ion Medica Mutua Madrile~na (to BP), and by an institutional grantfrom the Fundacion Ramon Areces to the CBMSO. CIBERER is aninitiative of the ISCI

A Ca2+-Dependent Mechanism Boosting Glycolysis and OXPHOS by Activating Aralar-Malate-Aspartate Shuttle, upon Neuronal Stimulation

Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na+ and Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca2+-dependent way, independently of effects of Ca2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.This work was supported by Spanish Ministry of Science, Innovation and Universities SAF2014-56929R to J.S. and B.P.; SAF2017-82560-R to A.d.A. and B.P.; Fundación Ramón Areces to J.S.; and Fundación Ramón Areces institutional grant to Centro de Biología Molecular Severo Ochoa (CBMSO). I.P.-L. and L.G.-M. received predoctoral fellowships from MINECO. P.G.-S. received a postdoctoral research contract from Comunidad de Madri

Targeted exome sequencing of Krebs cycle genes reveals candidate cancer–predisposing mutations in pheochromocytomas and paragangliomas

Purpose: 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. Experimental Design: We used downregulation and methylation of RBP1, 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. Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1. A variant in GOT2, 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 SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered a-ketoglutarate/ isocitrate ratio. Conclusions: 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 cytosolThis work was supported by the Fondo de Investigaciones Sanitarias project PI15/00783, FEDER 2014-2020 (to A. Cascon) and the Deutsche Forschungs- gemeinschaft (grant RI 2684/1-1; to S. Richter). CEGEN-PRB2-ISCIII is supported by grant PT13/0001, ISCIII-SGEFI/FEDE

Inducción de diversas enzimas por aireación de células de Saccharomyces Cerevisiae cultivada en anaerobiosis: estudio de la influencia de los sistemas de síntesis de proteínas mitocondrial y citoplasmático

Tesis doctoral inédita leída e la Universidad Autónoma de Madrid. Facultad de Ciencias. Departamento de Bioquímica y Biología Molecular. Fecha de lectura:28-09-197

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

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 levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca fluctuations below the threshold of the mitochondrial calcium uniporter, there is a positive correlation between Ca and MAS activity; however, if cytosolic Ca 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-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.Peer Reviewe