Aspartate-glutamate Carrier Research Articles

The mitochondrial aspartate/glutamate carrier does not transport GABA

ɣ-aminobutyric acid (GABA) is a four‑carbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown.

A rare case report: multiple intrahepatic masses in a pediatric patient with citrin deficiency

Deficiency of citrin, the liver-type aspartate-glutamate carrier, arises from biallelic mutations of the gene SLC25A13. Although citrin deficiency (CD) is associated with higher risk of hepatocellular carcinoma (HCC) in adult patients, this association remains inconclusive in pediatric cases. The patient in this paper had been diagnosed to have CD by SLC25A13 analysis at the age 10 months, and then in response to dietary therapy, her prolonged jaundice and marked hepatosplenomegaly resolved gradually. However, she was referred to the hospital once again due to recurrent abdominal distention for 2 weeks at her age 4 years and 9 months, when prominently enlarged liver and spleen were palpated, along with a strikingly elevated serum alpha-fetoprotein (AFP) level of 27605 ng/mL as well as a large mass in the right liver lobe and a suspected tumor thrombus within the portal vein on enhanced computed tomography. After 4 rounds of adjuvant chemotherapy, right hepatic lobectomy and portal venous embolectomy were performed at her age 5 years and 3 months, and metastatic hepatoblastoma was confirmed by histopathological analysis. Afterwards, the patient underwent 5 additional cycles of chemotherapy and her condition remained stable for 7 months after surgery. Unfortunately, hepatoblastoma recurred in the left lobe at the age 5 years and 10 months, which progressed rapidly into liver failure, and led to death at the age 6 years and 1 month. As far as we know, this is the the first case of hepatoblastoma in a patient with CD, raising the possibility of an association between these two conditions.

Pathogenesis and Management of Citrin Deficiency.

Citrin deficiency (CD) is a hereditary disorder caused by SLC25A13 mutations that manifests as neonatal intrahepatic cholestasis caused by CD (NICCD), failure to thrive and dyslipidemia caused by CD (FTTDCD), and adult-onset type 2 citrullinemia (CTLN2). Citrin, an aspartate-glutamate carrier primarily expressed in the liver, is a component of the malate-aspartate shuttle, which is essential for glycolysis. Citrin-deficient hepatocytes have primary defects in glycolysis and de novo lipogenesis and exhibit secondarily downregulated PPARα, leading to impaired β-oxidation. They are unable to utilize glucose and free fatty acids as energy sources, resulting in energy deficiencies. Medium-chain triglyceride (MCT) supplements are effective for treating CD by providing energy to hepatocytes, increasing lipogenesis, and activating the malate-citrate shuttle. However, patients with CD often exhibit growth impairment and irreversible brain and/or liver damage. To improve the quality of life and prevent irreversible damage, MCT supplementation with a diet containing minimal carbohydrates is recommended promptly after the diagnosis.

Aspartic Acid in Health and Disease.

Aspartic acid exists in L- and D-isoforms (L-Asp and D-Asp). Most L-Asp is synthesized by mitochondrial aspartate aminotransferase from oxaloacetate and glutamate acquired by glutamine deamidation, particularly in the liver and tumor cells, and transamination of branched-chain amino acids (BCAAs), particularly in muscles. The main source of D-Asp is the racemization of L-Asp. L-Asp transported via aspartate-glutamate carrier to the cytosol is used in protein and nucleotide synthesis, gluconeogenesis, urea, and purine-nucleotide cycles, and neurotransmission and via the malate-aspartate shuttle maintains NADH delivery to mitochondria and redox balance. L-Asp released from neurons connects with the glutamate-glutamine cycle and ensures glycolysis and ammonia detoxification in astrocytes. D-Asp has a role in brain development and hypothalamus regulation. The hereditary disorders in L-Asp metabolism include citrullinemia, asparagine synthetase deficiency, Canavan disease, and dicarboxylic aminoaciduria. L-Asp plays a role in the pathogenesis of psychiatric and neurologic disorders and alterations in BCAA levels in diabetes and hyperammonemia. Further research is needed to examine the targeting of L-Asp metabolism as a strategy to fight cancer, the use of L-Asp as a dietary supplement, and the risks of increased L-Asp consumption. The role of D-Asp in the brain warrants studies on its therapeutic potential in psychiatric and neurologic disorders.

Mitochondrial aspartate/glutamate carrier AGC1 regulates cardiac function via Drp1-mediated mitochondrial fission in doxorubicin-induced cardiomyopathy

Mitochondrial fission has been noted in the pathogenesis of dilated cardiomyopathy (DCM), but the underlying specific regulatory mechanism, especially in the development of doxorubicin (DOX)-induced cardiomyopathy remains unclear. In the present study, we explore whether the aspartate-glutamate carrier1 (AGC1) interacts with the fission protein dynamin-related protein 1 (Drp1) and reveal the functional and molecular mechanisms contributing to DOX-induced cardiomyopathy. Results of co-immunoprecipitation mass spectrometry (CO-IP MS) analysis based on heart tissue of DCM patients revealed that AGC1 expression was significantly upregulated in DCM-induced injury and AGC1 level was closely correlated with mitochondrial morphogenesis and function. We showed that AGC1 knockdown protected mice from DOX-induced cardiomyopathy by preventing mitochondrial fission, while the overexpression of AGC1 in the mouse heart led to impairment of cardiac function. Mechanistically, AGC1 overexpression could upregulate Drp1 expression and contribute to subsequent excessive mitochondrial fission. Specifically, AGC1 knockdown or the use of Drp1-specific inhibitor Mdivi-1 alleviated cardiomyocyte apoptosis and inhibited impairment of mitochondrial function induced by DOX exposure. In summary, our data illustrate that AGC1, as a novel contributor to DCM, regulates cardiac function via Drp1-mediated mitochondrial fission, indicating that targeting AGC1-Drp1 axis could be a potential therapeutic strategy for DOX-induced cardiomyopathy.

Aspartate-glutamate carrier 2 (citrin): a role in glucose and amino acid metabolism in the liver

Aspartate-glutamate carrier 2 (citrin): a role in glucose and amino acid metabolism in the liver

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 deficiency.

Role of Mitochondrial Transporters on Metabolic Rewiring of Pancreatic Adenocarcinoma: A Comprehensive Review.

Pancreatic cancer is among the deadliest cancers worldwide and commonly presents as pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming is a hallmark of PDAC. Glucose and glutamine metabolism are extensively rewired in order to fulfil both energetic and synthetic demands of this aggressive tumour and maintain favorable redox homeostasis. The mitochondrial pyruvate carrier (MPC), the glutamine carrier (SLC1A5_Var), the glutamate carrier (GC), the aspartate/glutamate carrier (AGC), and the uncoupling protein 2 (UCP2) have all been shown to influence PDAC cell growth and progression. The expression of MPC is downregulated in PDAC and its overexpression reduces cell growth rate, whereas the other four transporters are usually overexpressed and the loss of one or more of them renders PDAC cells unable to grow and proliferate by altering the levels of crucial metabolites such as aspartate. The aim of this review is to comprehensively evaluate the current experimental evidence about the function of these carriers in PDAC metabolic rewiring. Dissecting the precise role of these transporters in the context of the tumour microenvironment is necessary for targeted drug development.

Generation and characterization of the adult neuron-specific Aralar/AGC1 knock-out mice.

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 (OMIM #612949, also named early infantile epileptic encephalopathy 39) characterized by hypomyelination, hypotonia, developmental arrest and epilepsy. In mice, the global aralar knockout shows hyperreactivity, growth retardation, motor discoordination, seizures and hypomyelination; thus, recapitulating the pathology of the human disease. Aralar brain expression is mainly restricted to neurons, while glial expression of Aralar has been demonstrated to be scarce. However, Aralar deficiency manifests alteration in fundamental glial functions such as glutamine and myelination synthesis by astrocytes and oligodendrocytes respectively. In order to dissect the role of neuronal Aralar in the disease mechanism of AGC1 deficiency, we have ablated the expression of Aralar in mature (from PND30 onward) excitatory cortical and hippocampal neurons using the CaMKIIα-driven Cre recombinase expression in Aralarlox/lox mice. Here, we present the characterization of the mature neuron-specific aralar knock out mice. We demonstrated that the excision of exon 3 aralar starts from PND30 and continues to occur during mouse adulthood. Consequently, Aralar protein levels gradually decay across the 2nd to 6th month up to a 70- 75% decrease in the cortex and a 50% decrease in the hippocampus. The drop in Aralar levels drive an alteration in cortical metabolites causing a 25% and 50% decrease in aspartate and serine levels, the metabolites most affected in the brain of the global aralar-KO mice. This partial decreases in ARALAR and metabolites caused by cortical CaMKIIα-driven aralar ablation are not accompanied by growth or behavioral alterations in a battery of motor, memory and cognitive tests. The results suggest that the absence of Aralar in CaMKIIα expressing neurons (cortical and hippocampal mature excitatory neurons), does not mimic the alterations caused by global deficiency of Aralar/AGC1. However, since the levels of cortical ARALAR in this model resemble those of healthy aralar+/- mice, further experiments are being carried out to explore the origin (or location) of the ARALAR protein remnants and the possible metabolic compensation by other neurons and/or cell types in these mice. Without ruling out either that the ARALAR-MAS pathway could play a more critical role during neurodevelopment than adulthood in brain physiology and metabolism.

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.

Histone Acetylation Defects in Brain Precursor Cells: A Potential Pathogenic Mechanism Causing Proliferation and Differentiation Dysfunctions in Mitochondrial Aspartate-Glutamate Carrier Isoform 1 Deficiency.

Mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) deficiency is an ultra-rare genetic disease characterized by global hypomyelination and brain atrophy, caused by mutations in the SLC25A12 gene leading to a reduction in AGC1 activity. In both neuronal precursor cells and oligodendrocytes precursor cells (NPCs and OPCs), the AGC1 determines reduced proliferation with an accelerated differentiation of OPCs, both associated with gene expression dysregulation. Epigenetic regulation of gene expression through histone acetylation plays a crucial role in the proliferation/differentiation of both NPCs and OPCs and is modulated by mitochondrial metabolism. In AGC1 deficiency models, both OPCs and NPCs show an altered expression of transcription factors involved in the proliferation/differentiation of brain precursor cells (BPCs) as well as a reduction in histone acetylation with a parallel alteration in the expression and activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, histone acetylation dysfunctions have been dissected in in vitro models of AGC1 deficiency OPCs (Oli-Neu cells) and NPCs (neurospheres), in physiological conditions and following pharmacological treatments. The inhibition of HATs by curcumin arrests the proliferation of OPCs leading to their differentiation, while the inhibition of HDACs by suberanilohydroxamic acid (SAHA) has only a limited effect on proliferation, but it significantly stimulates the differentiation of OPCs. In NPCs, both treatments determine an alteration in the commitment toward glial cells. These data contribute to clarifying the molecular and epigenetic mechanisms regulating the proliferation/differentiation of OPCs and NPCs. This will help to identify potential targets for new therapeutic approaches that are able to increase the OPCs pool and to sustain their differentiation toward oligodendrocytes and to myelination/remyelination processes in AGC1 deficiency, as well as in other white matter neuropathologies.

Citrin deficiency: Does the reactivation of liver aralar-1 come into play and promote HCC development?

Hepatocellular carcinoma (HCC) is a longstanding issue in clinical practice and metabolic research. New clues in better understanding the pathogenesis of HCC might relate to the metabolic context in patients with citrin (aspartate-glutamate carrier 1) deficiency (CD). Because citrin-deficient liver (CDL) is subject to HCC, it represents a unique metabolic model to highlight the mechanisms of HCC promotion, offering different angles of study than the classical metabolic syndrome/obesity/non-alcoholic fatty liver disease (NAFLD)/HCC study axis. In turn, the metabolic features of HCC could shed light on the pathogenesis of CDL. Among these, HCC-induced re-activation of aralar-1 (aspartate-glutamate carrier 2), physiologically not expressed in the adult liver, might take place in CDL, so gene redundancy for mitochondrial aspartate-glutamate carriers would be exploited by the CDL. This proposed (aralar-1 re-activation) and known (citrate/malate cycle) adaptive mechanisms may substitute for the impaired function in CD and are consistent with the clinical remission stage of CD and CD improvement by medium-chain triglycerides (MCT). However, these metabolic adaptive benefits could also promote HCC development. In CD, as a result of PPARα down-regulation, liver mitochondrial fatty acid-derived acetyl-CoA would, like glucose-derived acetyl-CoA, be used for lipid anabolism and fuel nuclear acetylation events which might trigger aralar-1 re-activation as seen in non-CD HCC. A brief account of these metabolic events which might lead to aralar-1 re-activation in CDL is here given. Consistency of this account for CDL events further relies on the protective roles of PPARα and inhibition of mitochondrial and plasma membrane citrate transporters in non-CD HCC.

The mitochondrial aspartate/glutamate carrier (AGC or Aralar1) isoforms in D. melanogaster: biochemical characterization, gene structure, and evolutionary analysis

BackgroundIn man two mitochondrial aspartate/glutamate carrier (AGC) isoforms, known as aralar and citrin, are required to accomplish several metabolic pathways. In order to fill the existing gap of knowledge in Drosophila melanogaster, we have studied aralar1 gene, orthologue of human AGC-encoding genes in this organism. MethodsThe blastp algorithm and the “reciprocal best hit” approach have been used to identify the human orthologue of AGCs in Drosophilidae and non-Drosophilidae. Aralar1 proteins have been overexpressed in Escherichia coli and functionally reconstituted into liposomes for transport assays. ResultsThe transcriptional organization of aralar1 comprises six isoforms, three constitutively expressed (aralar1-RA, RD and RF), and the remaining three distributed during the development or in different tissues (aralar1-RB, RC and RE). Aralar1-PA and Aralar1-PE, representative of all isoforms, have been biochemically characterized. Recombinant Aralar1-PA and Aralar1-PE proteins share similar efficiency to exchange glutamate against aspartate, and same substrate affinities than the human isoforms. Interestingly, although Aralar1-PA and Aralar1-PE diverge only in their EF-hand 8, they greatly differ in their specific activities and substrate specificity. ConclusionsThe tight regulation of aralar1 transcripts expression and the high request of aspartate and glutamate during early embryogenesis suggest a crucial role of Aralar1 in this Drosophila developmental stage. Furthermore, biochemical characterization and calcium sensitivity have identified Aralar1-PA and Aralar1-PE as the human aralar and citrin counterparts, respectively. General significanceThe functional characterization of the fruit fly mitochondrial AGC transporter represents a crucial step toward a complete understanding of the metabolic events acting during early embryogenesis.

Citrin deficiency mimicking mitochondrial depletion syndrome

BackgroundNeonatal intrahepatic cholestasis caused by citrin deficiency (CD) is a rare inborn error of metabolism due to variants in the SLC25A13 gene encoding the calcium-binding protein citrin. Citrin is an aspartate-glutamate carrier located within the inner mitochondrial membrane.Case presentationWe report on two siblings of Romanian-Vietnamese ancestry with citrin deficiency. Patient 1 is a female who presented at age 8 weeks with cholestasis, elevated lactate levels and recurrent severe hypoglycemia. Diagnosis was made by whole exome sequencing and revealed compound heterozygosity for the frameshift variant c.852_855del, p.Met285Profs*2 and a novel deletion c.(69 + 1_70–1)_(212 + 1_231–1)del in SLC25A13. The girl responded well to dietary treatment with a lactose-free, MCT-enriched formula. Her younger brother (Patient 2) was born 1 year later and also found to be carrying the same gene variants. Dietary treatment from birth was able to completely prevent clinical manifestation until his current age of 4.5 months.ConclusionsAs CD is a well-treatable disorder it should be ruled out early in the differential diagnosis of neonatal cholestasis. Due to the combination of hepatopathy, lactic acidosis and recurrent hypoglycemia the clinical presentation of CD may resemble hepatic mitochondrial depletion syndrome.

βOHB 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 β-hydroxybutyrate (βOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that βOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring βOHB 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 βOHB during 5 d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of βOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by βOHB administration, a 4-d βOHB 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 βOHB as a therapeutic treatment in AGC1 deficiency deserves further investigation.SIGNIFICANCE STATEMENTAralar deficiency induces a fatal phenotype in humans and mice and is associated with impaired neurodevelopment, epilepsy, and hypomyelination. In neurons, highly expressing aralar, its deficiency causes a metabolic blockade hampering mitochondrial energetics and respiration. Here, we find that βOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in aralar-deficient neurons. βOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate (Asp) and NAA, which is impeded by aralar deficiency, presumably through citrate-malate shuttle. In aralar-knock-out (KO) mice, βOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The βOHB-induced myelin synthesis occurring together with the marked increment in neuronal NAA synthesis supports the role of NAA as a lipid precursor during postnatal myelination.

Metabolic Features of Mouse and Human Retinas: Rods versus Cones, Macula versus Periphery, Retina versus RPE.

SummaryPhotoreceptors, especially cones, which are enriched in the human macula, have high energy demands, making them vulnerable to metabolic stress. Metabolic dysfunction of photoreceptors and their supporting retinal pigment epithelium (RPE) is an important underlying cause of degenerative retinal diseases. However, how cones and the macula support their exorbitant metabolic demand and communicate with RPE is unclear. By profiling metabolite uptake and release and analyzing metabolic genes, we have found cone-rich retinas and human macula share specific metabolic features with upregulated pathways in pyruvate metabolism, mitochondrial TCA cycle, and lipid synthesis. Human neural retina and RPE have distinct but complementary metabolic features. Retinal metabolism centers on NADH production and neurotransmitter biosynthesis. The retina needs aspartate to sustain its aerobic glycolysis and mitochondrial metabolism. RPE metabolism is directed toward NADPH production and biosynthesis of acetyl-rich metabolites, serine, and others. RPE consumes multiple nutrients, including proline, to produce metabolites for the retina.

AGC2 (Citrin) Deficiency—From Recognition of the Disease till Construction of Therapeutic Procedures

Can you imagine a disease in which intake of an excess amount of sugars or carbohydrates causes hyperammonemia? It is hard to imagine the intake causing hyperammonemia. AGC2 or citrin deficiency shows their symptoms following sugar/carbohydrates intake excess and this disease is now known as a pan-ethnic disease. AGC2 (aspartate glutamate carrier 2) or citrin is a mitochondrial transporter which transports aspartate (Asp) from mitochondria to cytosol in exchange with glutamate (Glu) and H+. Asp is originally supplied from mitochondria to cytosol where it is necessary for synthesis of proteins, nucleotides, and urea. In cytosol, Asp can be synthesized from oxaloacetate and Glu by cytosolic Asp aminotransferase, but oxaloacetate formation is limited by the amount of NAD+. This means an increase in NADH causes suppression of Asp formation in the cytosol. Metabolism of carbohydrates and other substances which produce cytosolic NADH such as alcohol and glycerol suppress oxaloacetate formation. It is forced under citrin deficiency since citrin is a member of malate/Asp shuttle. In this review, we will describe history of identification of the SLC25A13 gene as the causative gene for adult-onset type II citrullinemia (CTLN2), a type of citrin deficiency, pathophysiology of citrin deficiency together with animal models and possible treatments for citrin deficiency newly developing.

Genetic Analysis of Peroxisomal Genes Required for Longevity in a Yeast Model of Citrin Deficiency.

Citrin is a liver-specific mitochondrial aspartate–glutamate carrier encoded by SLC25A13. Citrin deficiency caused by SLC25A13 mutation results in carbohydrate toxicity, citrullinemia type II, and fatty liver diseases, the mechanisms of some of which remain unknown. Citrin shows a functional homolog in yeast aspartate-glutamate carrier (Agc1p) and agc1Δ yeasts are used as a model organism of citrin deficiency. Here, we found that agc1Δ yeasts decreased fat utilization, impaired NADH balance in peroxisomes, and decreased chronological lifespan. The activation of GPD1-mediated NAD+ regeneration in peroxisomes by GPD1 over-expression or activation of the malate–oxaloacetate NADH peroxisomal shuttle, by increasing flux in this NADH shuttle and over-expression of MDH3, resulted in lifespan extension of agc1Δ yeasts. In addition, over-expression of PEX34 restored longevity of agc1Δ yeasts as well as wild-type cells. The effect of PEX34-mediated longevity required the presence of the GPD1-mediated NADH peroxisomal shuttle, which was independent of the presence of the peroxisomal malate–oxaloacetate NADH shuttle and PEX34-induced peroxisome proliferation. These data confirm that impaired NAD+ regeneration in peroxisomes is a key defect in the yeast model of citrin deficiency, and enhancing peroxisome function or inducing NAD+ regeneration in peroxisomes is suggested for further study in patients’ hepatocytes.

A Mutation in the Mitochondrial Aspartate/Glutamate Carrier Leads to a More Oxidizing Intramitochondrial Environment and an Inflammatory Myopathy in Dutch Shepherd Dogs.

Background:Inflammatory myopathies are characterized by infiltration of inflammatory cells into muscle. Typically, immune-mediated disorders such as polymyositis, dermatomyositis and inclusion body myositis are diagnosed.Objective:A small family of dogs with early onset muscle weakness and inflammatory muscle biopsies were investigated for an underlying genetic cause.Methods:Following the histopathological diagnosis of inflammatory myopathy, mutational analysis including whole genome sequencing, functional transport studies of the mutated and wild-type proteins, and metabolomic analysis were performed.Results:Whole genome resequencing identified a pathological variant in the SLC25A12 gene, resulting in a leucine to proline substitution at amino acid 349 in the mitochondrial aspartate-glutamate transporter known as the neuron and muscle specific aspartate glutamate carrier 1 (AGC1). Functionally reconstituting recombinant wild-type and mutant AGC1 into liposomes demonstrated a dramatic decrease in AGC1 transport activity and inability to transfer reducing equivalents from the cytosol into mitochondria. Targeted, broad-spectrum metabolomic analysis from affected and control muscles demonstrated a proinflammatory milieu and strong support for oxidative stress.Conclusions:This study provides the first description of a metabolic mechanism in which ablated mitochondrial glutamate transport markedly reduced the import of reducing equivalents into mitochondria and produced a highly oxidizing and proinflammatory muscle environment and an inflammatory myopathy.

Deficiency of Mitochondrial Aspartate-Glutamate Carrier 1 Leads to Oligodendrocyte Precursor Cell Proliferation Defects Both In Vitro and In Vivo.

Aspartate-Glutamate Carrier 1 (AGC1) deficiency is a rare neurological disease caused by mutations in the solute carrier family 25, member 12 (SLC25A12) gene, encoding for the mitochondrial aspartate-glutamate carrier isoform 1 (AGC1), a component of the malate–aspartate NADH shuttle (MAS), expressed in excitable tissues only. AGC1 deficiency patients are children showing severe hypotonia, arrested psychomotor development, seizures and global hypomyelination. While the effect of AGC1 deficiency in neurons and neuronal function has been deeply studied, little is known about oligodendrocytes and their precursors, the brain cells involved in myelination. Here we studied the effect of AGC1 down-regulation on oligodendrocyte precursor cells (OPCs), using both in vitro and in vivo mouse disease models. In the cell model, we showed that a reduced expression of AGC1 induces a deficit of OPC proliferation leading to their spontaneous and precocious differentiation into oligodendrocytes. Interestingly, this effect seems to be related to a dysregulation in the expression of trophic factors and receptors involved in OPC proliferation/differentiation, such as Platelet-Derived Growth Factor α (PDGFα) and Transforming Growth Factor βs (TGFβs). We also confirmed the OPC reduction in vivo in AGC1-deficent mice, as well as a proliferation deficit in neurospheres from the Subventricular Zone (SVZ) of these animals, thus indicating that AGC1 reduction could affect the proliferation of different brain precursor cells. These data clearly show that AGC1 impairment alters myelination not only by acting on N-acetyl-aspartate production in neurons but also on OPC proliferation and suggest new potential therapeutic targets for the treatment of AGC1 deficiency.