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

Ca2+-regulated mitochondrial carriers of ATP-Mg2+/Pi: evolutionary insights in protozoans

In addition to its uptake across the Ca2+ uniporter, intracellular calcium signals can stimulate mitochondrial metabolism activating metabolite exchangers of the inner mitochondrial membrane belonging to the mitochondrial carrier family (SLC25). One of these Ca2+-regulated mitochondrial carriers (CaMCs) are the reversible ATP-Mg2+/Pi transporters, or SCaMCs, required for maintaining optimal adenine nucleotide (AdN) levels in the mitochondrial matrix representing an alternative transporter to the ADP/ATP translocases (AAC). This CaMC has a distinctive Calmodulin-like (CaM-like) domain fused to the carrier domain that makes its transport activity strictly dependent on cytosolic Ca2+ signals. Here we investigate about its origin analysing its distribution and features in unicellular eukaryotes. Unexpectedly, we find two types of ATP-Mg2+/Pi carriers, the canonical ones and shortened variants lacking the CaM-like domain. Phylogenetic analysis shows that both SCaMC variants have a common origin, unrelated to AACs, suggesting in turn that recurrent losses of the regulatory module have occurred in the different phyla. They are excluding variants that show a more limited distribution and less conservation than AACs. Interestingly, these truncated variants of SCaMC are found almost exclusively in parasitic protists, such as apicomplexans, kinetoplastides or animal-patogenic oomycetes, and in green algae, suggesting that its lost could be related to certain life-styles. In addition, we find an intricate structural diversity in these variants that may be associated with their pathogenicity. The consequences on SCaMC functions of these new SCaMC-b variants are discussedThis work has been funded by a grant from the Spanish Ministry of Science SAF2017-82560-R (to AdelA), and by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa (CBMSO

Mitochondrial movement in Aralar/Slc25a12/AGC1 deficient cortical neurons

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 Ca2+-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 maintenanceThis work was supported by grants S2010/BMD-2402, and a grant from Fundación Ramón Areces to JS; SAF2014-56929-R to JS and BP; SAF2017-82560-R to BP and AdA and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa. LC has been the recipient of a Junta de Ampliación de Estudios Consejo Superior de Investigaciones Científicas and CIBERER postdoctoral contracts. The authors declare no competing financial interest

Proteína antagonista del receptor de la interleuquina-1 (IRAP) para el tratamiento de enfermedades articulares en los caballos de carreras

En la actualidad existen numerosas técnicas para el tratamiento de las enfermedades articulares en los caballos de carreras. Sin embargo todas ellas presentan ciertas limitaciones. La interleuquina-1 es una citoquina que juega un papel importante en la degeneración de las estructuras músculo-esqueléticas debido a su potente acción inflamatoria. Por ello, el uso de la proteína antagonista del receptor de la interleuquina-1 (IRAP) consigue disminuir drásticamente el dolor y la inflamación de la articulación afectada. El objetivo de este trabajo es describir y dar a conocer esta técnica que creemos que es de gran utilidad. Además tratamos de mostrar su eficacia en un caso clínico. Esta técnica se aplicó en un Pura Sangre Inglés de cinco años que presentaba signos radiológicos compatibles con osteoartritis. Se le aplicaron cuatro inyecciones intraarticulares separadas por un intervalo de 8-10 días. Actualmente, el caballo ya presenta una notable mejoría y ha regresado al entrenamiento. El tratamiento con IRAP ha demostrado gran eficacia frente a diversas enfermedades articulares y supone una buena alternativa frente al controvertido uso de los corticoesteroides intraarticulares.Currently, there are numerous techniques described for the treatment of joint diseases in racehorses. Nevertheless, all of these show certain limitations. Interleukin-1 is a citoquin that plays an important role in the deterioration of the skeletal muscle structures due to its powerful inflammatory action. Due to this, the use of the protein antagonist of the interleukin-1 receptor (IRAP) manages to drastically decrease the pain and the inflammation of the affected joint. The objective of this issue is to describe and to inform of this technique that we believe is of great use. In addition to this, we have tried to show its efficiency in a clinical case. This technique was applied on a five-year-old thoroughbred that had radiological compatible signs with osteoarthritis. Four intra-articular injections were applied with an 8 to 10 day interval. At present, the horse shows a great improvement and has returned to its training. The treatment with IRAP has demonstrated great efficiency towards diverse joint diseases and is a good alternative to the use of the controversial treatment of intra-articular corticosteroids

Role of aralar, the mitochondrial transporter of aspartate-glutamate, in brain N-acetylaspartate formation and Ca2+ signaling in neuronal mitochondria

"This is the peer reviewed version of the following article: Journal of Neuroscience Research 85.15 (2007): 3359-3366, which has been published in final form at https://doi.org/10.1002/jnr.21299. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions"Aralar, the Ca2+-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 Ca2+ signals to mitochondria via an increase in mitochondrial NADHMinisterio de Educación y Ciencia; Contract grant numbers: BFU2005-C02-01 and GEN2003-20235-C05-03/NAC; Contract grant sponsor: Instituto de Salud Carlos III del Ministerio de Sanidad; Contract grant numbers: PI042457; Contract grant sponsor: European Union; Contract grant numbers: LSHM-CT-2006-518153 (to J.S.); Contract grant sponsor: Fundación Ramón Areces (institutional grant to Centro de Biología Molecular Severo Ochoa

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

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

Regulation of neuronal energy metabolism by calcium: role of MCU and aralar/malate-aspartate shuttle

Calcium is a major regulator of cellular metabolism. Calcium controls mitochondrial respiration, and calcium signaling is used to meet cellular energetic demands through energy production in the organelle. Although it has been widely assumed that Ca2+-actions require its uptake by mitochondrial calcium uniporter (MCU), alternative pathways modulated by cytosolic Ca2+ have been recently proposed. Recent findings have indicated a role for cytosolic Ca2+ signals acting on mitochondrial NADH shuttles in the control of cellular metabolism in neurons using glucose as fuel. It has been demonstrated that AGC1/Aralar, the component of the malate/aspartate shuttle (MAS) regulated by cytosolic Ca2+, participates in the maintenance of basal respiration exerted through Ca2+-fluxes between ER and mitochondria, whereas mitochondrial Ca2+-uptake by MCU does not contribute. Aralar/MAS pathway, activated by small cytosolic Ca2+ signals, provides in fact substrates, redox equivalents and pyruvate, fueling respiration. Upon activation and increases in workload, neurons upregulate OxPhos, cytosolic pyruvate production and glycolysis, together with glucose uptake, in a Ca2+-dependent way, and part of this upregulation is via Ca2+ signaling. Both MCU and Aralar/MAS contribute to OxPhos upregulation, Aralar/MAS playing a major role, especially at small and submaximal workloads. Ca2+ activation of Aralar/MAS, by increasing cytosolic NAD+/NADH provides Ca2+-dependent increases in glycolysis and cytosolic pyruvate production priming respiration as a feed-forward mechanism in response to workload. Thus, except for glucose uptake, these processes are dependent on Aralar/MAS, whereas MCU is the relevant target for Ca2+ signaling when MAS is bypassed, by using pyruvate or β-hydroxybutyrate as substrate

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver

The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficienc