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1.
Cell ; 180(6): 1178-1197.e20, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32200800

RESUMO

Social impairment is frequently associated with mitochondrial dysfunction and altered neurotransmission. Although mitochondrial function is crucial for brain homeostasis, it remains unknown whether mitochondrial disruption contributes to social behavioral deficits. Here, we show that Drosophila mutants in the homolog of the human CYFIP1, a gene linked to autism and schizophrenia, exhibit mitochondrial hyperactivity and altered group behavior. We identify the regulation of GABA availability by mitochondrial activity as a biologically relevant mechanism and demonstrate its contribution to social behavior. Specifically, increased mitochondrial activity causes gamma aminobutyric acid (GABA) sequestration in the mitochondria, reducing GABAergic signaling and resulting in social deficits. Pharmacological and genetic manipulation of mitochondrial activity or GABA signaling corrects the observed abnormalities. We identify Aralar as the mitochondrial transporter that sequesters GABA upon increased mitochondrial activity. This study increases our understanding of how mitochondria modulate neuronal homeostasis and social behavior under physiopathological conditions.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Ácido gama-Aminobutírico/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Animais Geneticamente Modificados , Ácido Aspártico/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Glucose/metabolismo , Homeostase , Humanos , Masculino , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Comportamento Social , Transmissão Sináptica , Ácido gama-Aminobutírico/genética
2.
Cell Mol Biol Lett ; 29(1): 44, 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38553684

RESUMO

Aspartate-glutamate carrier isoform 1 (AGC1) is a carrier responsible for the export of mitochondrial aspartate in exchange for cytosolic glutamate and is part of the malate-aspartate shuttle, essential for the balance of reducing equivalents in the cells. In the brain, mutations in SLC25A12 gene, encoding for AGC1, cause an ultra-rare genetic disease, reported as a neurodevelopmental encephalopathy, whose symptoms include global hypomyelination, arrested psychomotor development, hypotonia and seizures. Among the biological components most affected by AGC1 deficiency are oligodendrocytes, glial cells responsible for myelination processes, and their precursors [oligodendrocyte progenitor cells (OPCs)]. The AGC1 silencing in an in vitro model of OPCs was documented to cause defects of proliferation and differentiation, mediated by alterations of histone acetylation/deacetylation. Disrupting AGC1 activity could possibly reduce the availability of acetyl groups, leading to perturbation of many biological pathways, such as histone modifications and fatty acids formation for myelin production. Here, we explore the transcriptome of mouse OPCs partially silenced for AGC1, reporting results of canonical analyses (differential expression) and pathway enrichment analyses, which highlight a disruption in fatty acids synthesis from both a regulatory and enzymatic stand. We further investigate the cellular effects of AGC1 deficiency through the identification of most affected transcriptional networks and altered alternative splicing. Transcriptional data were integrated with differential metabolite abundance analysis, showing downregulation of several amino acids, including glutamine and aspartate. Taken together, our results provide a molecular foundation for the effects of AGC1 deficiency in OPCs, highlighting the molecular mechanisms affected and providing a list of actionable targets to mitigate the effects of this pathology.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , Doenças Mitocondriais , Células Precursoras de Oligodendrócitos , Transtornos Psicomotores , Camundongos , Animais , Regulação para Baixo/genética , Células Precursoras de Oligodendrócitos/metabolismo , Ácido Aspártico/metabolismo , Isoformas de Proteínas/metabolismo , Ácidos Graxos
3.
J Neurosci ; 42(19): 3879-3895, 2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35387872

RESUMO

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.


Assuntos
Ácido Aspártico , Fosforilação Oxidativa , Trifosfato de Adenosina/metabolismo , Animais , Ácido Aspártico/metabolismo , Cálcio/metabolismo , Glucose/metabolismo , Glicólise , Malatos/metabolismo , Camundongos , Neurônios/fisiologia , Piruvatos/metabolismo
4.
Biochem Biophys Res Commun ; 643: 1-7, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36584587

RESUMO

The study aimed to explore the role of age-associated elevated cytosolic Ca2+ in changes of brain mitochondria energetic processes. Two groups of rats, young adults (4 months) and advanced old (24 months), were evaluated for potential alterations of mitochondrial parameters, the oxidative phosphorylation (OxPhos), membrane potential, calcium retention capacity, activity of glutamate/aspartate carrier (aralar), and ROS formation. We demonstrated that the brain mitochondria of older animals have a lower resistance to Ca2+ stress with resulting consequences. The suppressed complex I OxPhos and decreased membrane potential were accompanied by reduction of the Ca2+ threshold required for induction of mPTP. The Ca2+ binding sites of mitochondrial aralar mediated a lower activity of old brain mitochondria. The altered interaction between aralar and mPTP may underlie mitochondrial dysregulation leading to energetic depression during aging. At the advanced stages of aging, the declined metabolism is accompanied by the diminished oxidative background.


Assuntos
Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial , Ratos , Animais , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Encéfalo/metabolismo , Fosforilação Oxidativa , Cálcio/metabolismo
5.
Int J Mol Sci ; 23(1)2022 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-35008954

RESUMO

AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named "early infantile epileptic encephalopathy 39" (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.


Assuntos
Agrecanas/genética , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Predisposição Genética para Doença , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/etiologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Doenças Mitocondriais/etiologia , Doenças Mitocondriais/metabolismo , Transtornos Psicomotores/etiologia , Transtornos Psicomotores/metabolismo , Agrecanas/deficiência , Agrecanas/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/metabolismo , Biomarcadores , Encéfalo/metabolismo , Terapia Combinada , Gerenciamento Clínico , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Metabolismo Energético , Estudos de Associação Genética , Ácido Glutâmico/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/terapia , Humanos , Malatos/metabolismo , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/terapia , Bainha de Mielina/metabolismo , Oxirredução , Fenótipo , Transtornos Psicomotores/diagnóstico , Transtornos Psicomotores/terapia
6.
J Neurosci ; 40(48): 9293-9305, 2020 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-33087477

RESUMO

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 ß-hydroxybutyrate (ßOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that ßOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring ßOHB 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 ßOHB 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 ßOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by ßOHB administration, a 4-d ßOHB 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 ßOHB 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 ßOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in aralar-deficient neurons. ßOHB 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, ßOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The ßOHB-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.


Assuntos
Ácido 3-Hidroxibutírico/fisiologia , Agrecanas/fisiologia , Encéfalo/fisiologia , Dieta Cetogênica , Vias Neurais/fisiologia , Neurônios/fisiologia , Ácido 3-Hidroxibutírico/administração & dosagem , Ácido 3-Hidroxibutírico/farmacologia , Agrecanas/genética , Aminoácidos/metabolismo , Animais , Dopamina/fisiologia , Feminino , Ácido Glutâmico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/genética , Bainha de Mielina/fisiologia , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/fisiologia , Consumo de Oxigênio/fisiologia , Respiração/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Monoamina/fisiologia
7.
J Neurosci ; 36(16): 4443-56, 2016 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-27098689

RESUMO

ARALAR/AGC1/Slc25a12, the aspartate-glutamate carrier from brain mitochondria, is the regulatory step in the malate-aspartate NADH shuttle, MAS. MAS is used to oxidize cytosolic NADH in mitochondria, a process required to maintain oxidative glucose utilization. The role of ARALAR was analyzed in two paradigms of glutamate-induced excitotoxicity in cortical neurons: glucose deprivation and acute glutamate stimulation. ARALAR deficiency did not aggravate glutamate-induced neuronal death in vitro, although glutamate-stimulated respiration was impaired. In contrast, the presence of L-lactate as an additional source protected against glutamate-induced neuronal death in control, but not ARALAR-deficient neurons.l-Lactate supplementation increased glutamate-stimulated respiration partially prevented the decrease in the cytosolic ATP/ADP ratio induced by glutamate and substantially diminished mitochondrial accumulation of 8-oxoguanosine, a marker of reactive oxygen species production, only in the presence, but not the absence, of ARALAR. In addition,l-lactate potentiated glutamate-induced increase in cytosolic Ca(2+), in a way independent of the presence of ARALAR. Interestingly,in vivo, the loss of half-a-dose of ARALAR in aralar(+/-)mice enhanced kainic acid-induced seizures and neuronal damage with respect to control animals, in a model of excitotoxicity in which increased L-lactate levels and L-lactate consumption have been previously proven. These results suggest that,in vivo, an inefficient operation of the shuttle in the aralar hemizygous mice prevents the protective role of L-lactate on glutamate excitotoxiciy and that the entry and oxidation of L-lactate through ARALAR-MAS pathway is required for its neuroprotective function. SIGNIFICANCE STATEMENT: Lactate now stands as a metabolite necessary for multiple functions in the brain and is an alternative energy source during excitotoxic brain injury. Here we find that the absence of a functional malate-aspartate NADH shuttle caused by aralar/AGC1 disruption causes a block in lactate utilization by neurons, which prevents the protective role of lactate on excitotoxicity, but not glutamate excitotoxicity itself. Thus, failure to use lactate is detrimental and is possibly responsible for the exacerbated in vivo excitotoxicity in aralar(+/-)mice.


Assuntos
Agrecanas/deficiência , Ácido Glutâmico/toxicidade , Ácido Láctico/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Neuroproteção/efeitos dos fármacos , Animais , Células Cultivadas , Ácido Láctico/farmacologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroproteção/fisiologia
8.
Biochim Biophys Acta ; 1857(8): 1158-1166, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27060251

RESUMO

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.


Assuntos
Antiporters/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antiporters/genética , Canais de Cálcio/genética , Respiração Celular/efeitos dos fármacos , Expressão Gênica , Ácido Glutâmico/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade Mitocondrial , Proteínas Mitocondriais/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
9.
Biochim Biophys Acta ; 1863(10): 2394-412, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27132995

RESUMO

In this review we discuss the structure and functions of the aspartate/glutamate carriers (AGC1-aralar and AGC2-citrin). Those proteins supply the aspartate synthesized within mitochondrial matrix to the cytosol in exchange for glutamate and a proton. A structure of an AGC carrier is not available yet but comparative 3D models were proposed. Moreover, transport assays performed by using the recombinant AGC1 and AGC2, reconstituted into liposome vesicles, allowed to explore the kinetics of those carriers and to reveal their specific transport properties. AGCs participate to a wide range of cellular functions, as the control of mitochondrial respiration, calcium signaling and antioxydant defenses. AGC1 might also play peculiar tissue-specific functions, as it was found to participate to cell-to-cell metabolic symbiosis in the retina. On the other hand, AGC1 is involved in the glutamate-mediated excitotoxicity in neurons and AGC gene or protein alterations were discovered in rare human diseases. Accordingly, a mice model of AGC1 gene knock-out presented with growth delay and generalized tremor, with myelinisation defects. More recently, AGC was proposed to play a crucial role in tumor metabolism as observed from metabolomic studies showing that the asparate exported from the mitochondrion by AGC1 is employed in the regeneration of cytosolic glutathione. Therefore, given the central role of AGCs in cell metabolism and human pathology, drug screening are now being developed to identify pharmacological modulators of those carriers. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.


Assuntos
Ácido Aspártico/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Transportadores de Ânions Orgânicos/fisiologia , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Bovinos , Sequência Consenso , Humanos , Malatos/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Proteínas de Transporte da Membrana Mitocondrial/genética , Modelos Moleculares , NAD/metabolismo , Proteínas de Neoplasias/fisiologia , Especificidade de Órgãos , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/genética , Oxirredução , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
10.
J Neurochem ; 142(1): 132-139, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28429368

RESUMO

ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells.


Assuntos
Agrecanas/genética , Agrecanas/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Ácido Láctico/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Transtornos Psicomotores/genética , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Antiporters/genética , Astrócitos/metabolismo , Química Encefálica/genética , Glucose/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Consumo de Oxigênio/genética
11.
Biochim Biophys Acta Bioenerg ; 1865(4): 149487, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38945283

RESUMO

É£-aminobutyric acid (GABA) is a four­carbon 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.

12.
Genes (Basel) ; 13(7)2022 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-35886006

RESUMO

We investigated two litters of distantly related Nova Scotia Duck Tolling Retrievers (NSDTR), of which four puppies were affected by cerebellar signs with or without neuromuscular weakness. The phenotype was termed cerebellar degeneration­myositis complex (CDMC). We suspected a heritable condition and initiated a genetic analysis. The genome of one affected dog was sequenced and compared to 565 control genomes. This search yielded a private protein-changing SLC25A12 variant in the affected dog, XM_038584842.1:c.1337C>T, predicted to result in the amino acid change XP_038440770.1:(p.Pro446Leu). The genotypes at the variant co-segregated with the phenotype as expected for a monogenic autosomal recessive mode of inheritance in both litters. Genotyping of 533 additional NSDTR revealed variant allele frequencies of 3.6% and 1.3% in a European and a North American cohort, respectively. The available clinical and biochemical data, together with current knowledge about SLC25A12 variants and their functional impact in humans, mice, and dogs, suggest the p.Pro446Leu variant is a candidate causative defect for the observed phenotype in the affected dogs.


Assuntos
Doenças do Cão , Miosite , Animais , Doenças do Cão/diagnóstico , Cães , Testes Genéticos , Genótipo , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Miosite/genética , Nova Escócia
13.
Biochim Biophys Acta Gen Subj ; 1865(5): 129854, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33497735

RESUMO

BACKGROUND: In 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The 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 SIGNIFICANCE: The 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.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Antiporters/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Sistemas de Transporte de Aminoácidos Acídicos/química , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/química , Antiporters/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Evolução Molecular , Humanos , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
14.
Front Cell Neurosci ; 15: 773709, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35095421

RESUMO

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.

15.
Biochimie ; 190: 20-23, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34228977

RESUMO

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.


Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ânions Orgânicos/deficiência , Acetilcoenzima A/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/metabolismo , Humanos , NAD/metabolismo , Triglicerídeos/metabolismo
16.
Cancer Metab ; 8(1): 26, 2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33292758

RESUMO

BACKGROUND: Aspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of intracellular aspartate levels on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis. RESULTS: Low AGC1 expression correlates with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells. CONCLUSION: This study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation, do not always reflect metastatic capacity in vivo.

17.
Neurochem Int ; 131: 104541, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31472174

RESUMO

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 maintenance.


Assuntos
Agrecanas/metabolismo , Córtex Cerebral/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Agrecanas/genética , Animais , Ácido Aspártico/metabolismo , Transporte Biológico Ativo , Cálcio/metabolismo , Cálcio/farmacologia , Córtex Cerebral/citologia , Glicólise/efeitos dos fármacos , Imuno-Histoquímica , Malatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/genética , Fosforilação Oxidativa , Ácido Pirúvico/metabolismo
18.
Mol Cell Oncol ; 6(5): e1536843, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31528687

RESUMO

Cancer cells rely on glutamine to fuel mitochondria, however it remains unclear whether this is needed for bioenergetic or biosynthetic pathways. Our study suggests that an essential function of mitochondrial glutamine metabolism is to provide aspartate to the cytosol where it can be used for nucleotide and protein synthesis.

19.
Cell Metab ; 28(5): 706-720.e6, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30122555

RESUMO

Mitochondrial function is important for aspartate biosynthesis in proliferating cells. Here, we show that mitochondrial aspartate export via the aspartate-glutamate carrier 1 (AGC1) supports cell proliferation and cellular redox homeostasis. Insufficient cytosolic aspartate delivery leads to cell death when TCA cycle carbon is reduced following glutamine withdrawal and/or glutaminase inhibition. Moreover, loss of AGC1 reduces allograft tumor growth that is further compromised by treatment with the glutaminase inhibitor CB-839. Together, these findings argue that mitochondrial aspartate export sustains cell survival in low-glutamine environments and AGC1 inhibition can synergize with glutaminase inhibition to limit tumor growth.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiporters/metabolismo , Ácido Aspártico/metabolismo , Sobrevivência Celular , Citosol/metabolismo , Glutamina/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Ciclo do Ácido Cítrico , Feminino , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Neoplasias/metabolismo
20.
Neurochem Int ; 88: 38-46, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25889949

RESUMO

Brain energetic requirements are elevated due to the high cost of impulse transmission and information storage, and are met mainly by glucose oxidation. The energy needs are closely matched by metabolic regulation, which requires the close cooperation of neurons and astrocytes and involves highly regulated fluxes of metabolites between cells. The metabolism in each type of cell is determined in part by its proteomic profile, which has been regarded as complementary. This review will consider the cellular distribution of the mitochondrial aspartate-glutamate carrier, aralar/AGC1/SLC25A12, and its role in the synergic metabolism between neurons and astrocytes.


Assuntos
Agrecanas/fisiologia , Metabolismo Energético/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Humanos
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