Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters

"This is the peer reviewed version of the following article: Journal of Neuroscience Research 88.5 (2010): 1009-1016, which has been published in final form at https://doi.org/10.1002/jnr.22283. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions"The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are components of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, b-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no β-galactosidase was found in brain glial cells. It was confined to neuronal cells within discrete neuronal clusters. Double-immunolabelling experiments showed that β-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where β-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver β-galactosidase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neuron

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

Ca2+ activation kinetics of the two aspartate-glutamate mitochondrial carriers, aralar and citrin: Role in the heart malate-aspartate NADH shuttle

Ca2+ regulation of the Ca2+ binding mitochondrial carriers for aspartate/glutamate (AGCs) is provided by their N-terminal extensions, which face the intermembrane space. The two mammalian AGCs, aralar and citrin, are members of the malate-aspartate NADH shuttle. We report that their N-terminal extensions contain up to four pairs of EF-hand motifs plus a single vestigial EF-hand, and have no known homolog. Aralar and citrin contain one fully canonical EF-hand pair and aralar two additional half-pairs, in which a single EF-hand is predicted to bind Ca2+. Shuttle activity in brain or skeletal muscle mitochondria, which contain aralar as the major AGC, is activated by Ca2+ with S0.5 values of 280-350 nM; higher than those obtained in liver mitochondria (100-150 nM) that contain citrin as the major AGC. We have used aralar- and citrin-deficient mice to study the role of the two isoforms in heart, which expresses both AGCs. The S0.5 for Ca 2+ activation of the shuttle in heart mitochondria is about 300 nM, and it remains essentially unchanged in citrin-deficient mice, although it undergoes a drastic reduction to about 100 nM in aralar-deficient mice. Therefore, aralar and citrin, when expressed as single isoforms in heart, confer differences in Ca2+ activation of shuttle activity, probably associated with their structural differences. In addition, the results reveal that the two AGCs fully account for shuttle activity in mouse heart mitochondria and that no other glutamate transporter can replace the AGCs in this pathwayThis work was supported in part by grants from the Ministerio de Educacio´ n y Ciencia (BFU2005-C02-01, GEN2003-20235-C05-03/NAC), Instituto de Salud Carlos III del Ministerio de Sanidad (PI042457), European Union (LSHM-CT-2006-518153) (to J. S.), by Grant SAF2004-06843-C03 from the Ministerio de Educacio´ n y Ciencia (to P. G.-P.), by an institutional grant from the Fundacio´ n Ramo´ n Areces to the CBMSO, and by Grants-in-Aid for Scientific Research (16390100) from the Japan Society for the Promotion of Science (to K. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fac

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

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

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

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 cell