Irene Llorente-Folch

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation

The glutamate–glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate–glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content. In vivo 13C nuclear magnetic resonance labeling with 13C2acetate or (1-13C) glucose showed that the drop in brain glutamine is due to a failure in glial glutamate synthesis. Aralar deficiency induces a decrease in aspartate content, an increase in lactate production, and lactate-to-pyruvate ratio in cultured neurons but not in cultured astrocytes, indicating that Aralar is only functional in neurons. We find that aspartate, but not other amino acids, increases glutamate synthesis in both control and aralar-deficient astrocytes, mainly by serving as amino donor. These findings suggest the existence of a neuron-to-astrocyte aspartate transcellular pathway required for astrocyte glutamate synthesis and subsequent glutamine formation. This pathway may provide a mechanism to transfer neuronal-born redox equivalents to mitochondria in astrocytes.

Calcium-Regulation of Mitochondrial Respiration Maintains ATP Homeostasis and Requires ARALAR/AGC1-Malate Aspartate Shuttle in Intact Cortical Neurons

Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5–5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

The regulation of neuronal mitochondrial metabolism by calcium

Calcium signalling is fundamental to the function of the nervous system, in association with changes in ionic gradients across the membrane. Although restoring ionic gradients is energetically costly, a rise in intracellular Ca2+ acts through multiple pathways to increase ATP synthesis, matching energy supply to demand. Increasing cytosolic Ca2+ stimulates metabolite transfer across the inner mitochondrial membrane through activation of Ca2+‐regulated mitochondrial carriers, whereas an increase in matrix Ca2+ stimulates the citric acid cycle and ATP synthase. The aspartate–glutamate exchanger Aralar/AGC1 (Slc25a12), a component of the malate–aspartate shuttle (MAS), is stimulated by modest increases in cytosolic Ca2+ and upregulates respiration in cortical neurons by enhancing pyruvate supply into mitochondria. Failure to increase respiration in response to small (carbachol) and moderate (K+‐depolarization) workloads and blunted stimulation of respiration in response to high workloads (veratridine) in Aralar/AGC1 knockout neurons reflect impaired MAS activity and limited mitochondrial pyruvate supply. In response to large workloads (veratridine), acute stimulation of respiration occurs in the absence of MAS through Ca2+ influx through the mitochondrial calcium uniporter (MCU) and a rise in matrix [Ca2+]. Although the physiological importance of the MCU complex in work‐induced stimulation of respiration of CNS neurons is not yet clarified, abnormal mitochondrial Ca2+ signalling causes pathology. Indeed, loss of function mutations in MICU1, a regulator of MCU complex, are associated with neuromuscular disease. In patient‐derived MICU1 deficient fibroblasts, resting matrix Ca2+ is increased and mitochondria fragmented. Thus, the fine tuning of Ca2+ signals plays a key role in shaping mitochondrial bioenergetics.

Ca2 + regulation of mitochondrial function in neurons

Calcium is thought to regulate respiration but it is unclear whether this is dependent on the increase in ATP demand caused by any Ca(2+) signal or to Ca(2+) itself. [Na(+)]i, [Ca(2+)]i and [ATP]i dynamics in intact neurons exposed to different workloads in the absence and presence of Ca(2+) clearly showed that Ca(2+)-stimulation of coupled respiration is required to maintain [ATP]i levels. Ca(2+) may regulate respiration by activating metabolite transport in mitochondria from outer face of the inner mitochondrial membrane, or after Ca(2+) entry in mitochondria through the calcium uniporter (MCU). Two Ca(2+)-regulated mitochondrial metabolite transporters are expressed in neurons, the aspartate-glutamate exchanger ARALAR/AGC1/Slc25a12, a component of the malate-aspartate shuttle, and the ATP-Mg/Pi exchanger SCaMC-3/APC2/Slc25a23, with S0.5 for Ca(2+) of 300nM and 3.4μM, respectively. The lack of SCaMC-3 results in a smaller Ca(2+)-dependent stimulation of respiration only at high workloads, as caused by veratridine, whereas the lack of ARALAR reduced by 46% basal OCR in intact neurons using glucose as energy source and the Ca(2+)-dependent responses to all workloads: a reduction of about 65-70% in the response to the high workload imposed by veratridine, and completely suppression of the OCR responses to moderate (K(+)-depolarization) and small (carbachol) workloads, effects reverted by pyruvate supply. For K(+)-depolarization, this occurs in spite of the presence of large [Ca(2+)]mit signals and increased formation of mitochondrial NAD(P)H. These results show that ARALAR-MAS is a major contributor of Ca(2+)-stimulated respiration in neurons by providing increased pyruvate supply to mitochondria. In its absence and under moderate workloads, matrix Ca(2+) is unable to stimulate pyruvate metabolism and entry in mitochondria suggesting a limited role of MCU in these conditions. This article was invited for a Special Issue entitled: 18th European Bioenergetic Conference.

Deficiency of the mitochondrial transporter of aspartate/glutamate aralar/AGC1 causes hypomyelination and neuronal defects unrelated to myelin deficits in mouse brain.

The aralar/AGC1 knockout (KO) mouse shows a drastic decrease in brain aspartate and N‐acetylaspartate levels and global hypomyelination, which are attributed to the lack of neuron‐produced NAA used by oligodendrocytes as precursor of myelin lipid synthesis. In addition, these mice have a gradual drop in brain glutamine synthesis. We show here that hypomyelination is more pronounced in gray than in white matter regions. We find a lack of neurofilament‐labelled processes in hypomyelinated fiber tracks from cerebral cortex but not from those of the cerebellar granule cell layer, which correspond to Purkinje neurons. Therefore, the impaired development or degeneration of neuronal processes in cerebral cortex is independent of hypomyelination. An increase in O4‐labelled, immature oligodendrocytes is observed in gray and white matter regions of the aralar KO brain, suggesting a block in maturation compatible with the lack of NAA supplied by neurons. However, no defects in oligodendrocyte maturation were observed in in‐vitro‐cultured mixed astroglial cultures. We conclude that the primary defect of pyramidal neurons in cerebral cortex is possibly associated with a progressive failure in glutamatergic neurotransmission and may be among the main causes of the pathology of aralar/AGC1 deficiency.

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.

L-Lactate-Mediated Neuroprotection against Glutamate-Induced Excitotoxicity Requires ARALAR/AGC1.

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 Ca2+, 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.

AGC1‐malate aspartate shuttle activity is critical for dopamine handling in the nigrostriatal pathway

The mitochondrial transporter of aspartate‐glutamate Aralar/AGC1 is a regulatory component of the malate‐aspartate shuttle. Aralar deficiency in mouse and human causes a shutdown of brain shuttle activity and global cerebral hypomyelination. A lack of neurofilament‐labeled processes is detected in the cerebral cortex, but whether different types of neurons are differentially affected by Aralar deficiency is still unknown. We have now found that Aralar‐knockout (Aralar‐KO) post‐natal mice show hyperactivity, anxiety‐like behavior, and hyperreactivity with a decrease of dopamine (DA) in terminal‐rich regions. The striatum is the brain region most affected in terms of size, amino acid and monoamine content. We find a decline in vesicular monoamine transporter‐2 (VMAT2) levels associated with increased DA metabolism through MAO activity (DOPAC/DA ratio) in Aralar‐KO striatum. However, no decrease in DA or in the number of nigral tyrosine hydroxylase‐positive cells was detected in Aralar‐KO brainstem. Adult Aralar‐hemizygous mice presented also increased DOPAC/DA ratio in striatum and enhanced sensitivity to amphetamine. Our results suggest that Aralar deficiency causes a fall in GSH/GSSG ratio and VMAT2 in striatum that might be related to a failure to produce mitochondrial NADH and to an increase of reactive oxygen species (ROS) in the cytosol. The results indicate that the nigrostriatal dopaminergic system is a target of Aralar deficiency.

Altered postnatal development of cortico- hippocampal neuronal electric activity in mice deficient for the mitochondrial aspartate-glutamate transporter

The deficiency in the mitochondrial aspartate/glutamate transporter Aralar/AGC1 results in a loss of the malate-aspartate NADH shuttle in the brain neurons, hypomyelination, and additional defects in the brain metabolism. We studied the development of cortico/hippocampal local field potential (LFP) in Aralar/AGC1 knockout (KO) mice. Laminar profiles of LFP, evoked potentials, and unit activity were recorded under anesthesia in young (P15 to P22) Aralar-KO and control mice as well as control adults. While LFP power increased 3 to 7 times in both cortex and hippocampus of control animals during P15 to P22, the Aralar-KO specimens hardly progressed. The divergence was more pronounced in the CA3/hilus region. In parallel, spontaneous multiunit activity declined severely in KO mice. Postnatal growth of hippocampal-evoked potentials was delayed in KO mice, and indicated abnormal synaptic and spike electrogenesis and reduced output at P20 to P22. The lack of LFP development in KO mice was accompanied by the gradual appearance of epileptic activity in the CA3/hilus region that evolved to status epilepticus. Strikingly, CA3 bursts were poorly conducted to the CA1 field. We conclude that disturbed substrate supply to neuronal mitochondria impairs development of cortico—hippocampal LFPs. Aberrant neuronal electrogenesis and reduced neuron output may explain circuit dysfunction and phenotype deficiencies.

ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate

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.