Integrated analysis of transcriptomic and proteomic alterations in mouse models of ALS/FTD identify early metabolic adaptions with similarities to mitochondrial dysfunction disorders.

OBJECTIVE: Sporadic and familial amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease that results in loss of motor neurons and, in some patients, associates with frontotemporal dementia (FTD). Apart from the accumulation of proteinaceous deposits, emerging literature indicates that aberrant mitochondrial bioenergetics may contribute to the onset and progression of ALS/FTD. Here we sought to investigate the pathophysiological signatures of mitochondrial dysfunction associated with ALS/FTD. METHODS: By means of label-free mass spectrometry (MS) and mRNA sequencing (mRNA-seq), we report pre-symptomatic changes in the cortices of TDP-43 and FUS mutant mouse models. Using tissues from transgenic mouse models of mitochondrial diseases as a reference, we performed comparative analyses and extracted unique and common mitochondrial signatures that revealed neuroprotective compensatory mechanisms in response to early damage. RESULTS: In this regard, upregulation of both Acyl-CoA Synthetase Long-Chain Family Member 3 (ACSL3) and mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) were the most representative change in pre-symptomatic ALS/FTD tissues, suggesting that fatty acid beta-oxidation and mitochondrial protein translation are mechanisms of adaptation in response to ALS/FTD pathology. CONCLUSIONS: Together, our unbiased integrative analyses unveil novel molecular components that may influence mitochondrial homeostasis in the earliest phase of ALS.

Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study.

Metabolic reprogramming is a hallmark of cancer. Somatic mutations in genes involved in oncogenic signaling pathways, including KRAS and TP53, rewire the metabolic machinery in cancer cells. We here set out to determine, at the single cell level, metabolic signatures in human colon cancer cells engineered to express combinations of activating KRAS gene mutations and TP53 gene deletions. Specifically, we explored how somatic mutations in these genes and substrate availability (lactate, glucose, substrate deprivation) from the extracellular microenvironment affect bioenergetic parameters, including cellular ATP, NADH and mitochondrial membrane potential dynamics. Employing cytosolic and mitochondrial FRET-based ATP probes, fluorescent NADH sensors, and the membrane-permeant cationic fluorescent probe TMRM in HCT-116 cells as a model system, we observed that TP53 deletion and KRAS mutations drive a shift in metabolic signatures enabling lactate to become an efficient metabolite to replenish both ATP and NADH following nutrient deprivation. Intriguingly, cytosolic, mitochondrial and overall cellular ATP measurements revealed that, in WT KRAS cells, TP53 deficiency leads to an enhanced ATP production in the presence of extracellular lactate and glucose, and to the greatest increase in ATP following a starvation period. On the other hand, oncogenic KRAS in TP53-deficient cells reversed the alterations in cellular ATP levels. Moreover, cell population measurements of mitochondrial and glycolytic metabolism using a Seahorse analyzer demonstrated that WT KRAS TP53-silenced cells display an increase of the basal respiration and tightly-coupled mitochondria, in the presence of glucose as substrate, compared to TP53 competent cells. Furthermore, cells possessing oncogenic KRAS, independently of TP53 status, showed less pronounced mitochondrial membrane potential changes in response to metabolic nutrients. Furthermore, analysis of cytosolic and mitochondrial NADH levels revealed that the simultaneous presence of TP53 deletion and oncogenic KRAS showed the most pronounced alteration in cytosolic and mitochondrial NADH during metabolic stress. In conclusion, our findings demonstrate how activating KRAS mutation and loss of TP53 remodel cancer metabolism and lead to alterations in bioenergetics under metabolic stress conditions by modulating cellular ATP production, NADH oxidation, mitochondrial respiration and function.

ßOHB 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 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.

Extracellular ATP and glutamate drive pyruvate production and energy demand to regulate mitochondrial respiration in astrocytes.

Astrocytes respond to energetic demands by upregulating glycolysis, lactate production, and respiration. This study addresses the role of respiration and calcium regulation of respiration as part of the astrocyte response to the workloads caused by extracellular ATP and glutamate. Extracellular ATP (100 µM to 1 mM) causes a Ca2+ -dependent workload and fall of the cytosolic ATP/ADP ratio which acutely increases astrocytes respiration. Part of this increase is related to a Ca2+ -dependent upregulation of cytosolic pyruvate production. Conversely, glutamate (200 µM) causes a Na+ , but not Ca2+ , dependent workload even though glutamate-induced Ca2+ signals readily reach mitochondria. The glutamate workload triggers a rapid fall in the cytosolic ATP/ADP ratio and stimulation of respiration. These effects are mimicked by D-aspartate a nonmetabolized agonist of the glutamate transporter, but not by a metabotropic glutamate receptor agonist, indicating a major role of Na+ -dependent workload in stimulated respiration. Glutamate-induced increase in respiration is linked to a rapid increase in glycolytic pyruvate production, suggesting that both glutamate and extracellular ATP cause an increase in astrocyte respiration fueled by workload-induced increase in pyruvate production. However, glutamate-induced pyruvate production is partly resistant to glycolysis blockers (iodoacetate), indicating that oxidative consumption of glutamate also contributes to stimulated respiration. As stimulation of respiration by ATP and glutamate are similar and pyruvate production smaller in the first case, the results suggest that the response to extracellular ATP is a Ca2+ -dependent upregulation of respiration added to glycolysis upregulation. The global contribution of astrocyte respiratory responses to brain oxygen consumption is an open question.

BCL2 and BCL(X)L selective inhibitors decrease mitochondrial ATP production in breast cancer cells and are synthetically lethal when combined with 2-deoxy-D-glucose.

Cancer cells display differences regarding their engagement of glycolytic vs. mitochondrial oxidative phosphorylation (OXPHOS) pathway. Triple negative breast cancer, an aggressive form of breast cancer, is characterized by elevated glycolysis, while estrogen receptor positive breast cancer cells rely predominantly on OXPHOS. BCL2 proteins control the process of mitochondrial outer membrane permeabilization during apoptosis, but also regulate cellular bioenergetics. Because BCL2 proteins are overexpressed in breast cancer and targetable by selective antagonists, we here analysed the effect of BCL2 and BCL(X)L selective inhibitors, Venetoclax and WEHI-539, on mitochondrial bioenergetics and cell death. Employing single cell imaging using a FRET-based mitochondrial ATP sensor, we found that MCF7 breast cancer cells supplied with mitochondrial substrates reduced their mitochondrial ATP production when treated with Venetoclax or WEHI-539 at concentrations that per se did not induce cell death. Treatments with lower concentrations of both inhibitors also reduced the length of the mitochondrial network and the dynamics, as evaluated by quantitative confocal microscopy. We next tested the hypothesis that mitochondrial ATP production inhibition with BCL2 or BCL(X)L antagonists was synthetically lethal when combined with glycolysis inhibition. Treatment with 2-deoxy-D-glucose in combination with Venetoclax or WEHI-539 synergistically reduced the cellular bioenergetics of ER+ and TNBC breast cancer cells and abolished their clonogenic potential. Synthetic lethality was also observed when cultures were grown in 3D spheres. Our findings demonstrate that BCL2 antagonists exert potent effects on cancer metabolism independent of cell death-inducing effects, and demonstrate a synthetic lethality when these are applied in combination with glycolysis inhibitors.

Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases.

Neurodegenerative diseases are a spectrum of chronic, debilitating disorders characterised by the progressive degeneration and death of neurons. Mitochondrial dysfunction has been implicated in most neurodegenerative diseases, but in many instances it is unclear whether such dysfunction is a cause or an effect of the underlying pathology, and whether it represents a viable therapeutic target. It is therefore imperative to utilise and optimise cellular models and experimental techniques appropriate to determine the contribution of mitochondrial dysfunction to neurodegenerative disease phenotypes. In this consensus article, we collate details on and discuss pitfalls of existing experimental approaches to assess mitochondrial function in in vitro cellular models of neurodegenerative diseases, including specific protocols for the measurement of oxygen consumption rate in primary neuron cultures, and single-neuron, time-lapse fluorescence imaging of the mitochondrial membrane potential and mitochondrial NAD(P)H. As part of the Cellular Bioenergetics of Neurodegenerative Diseases (CeBioND) consortium ( www.cebiond.org ), we are performing cross-disease analyses to identify common and distinct molecular mechanisms involved in mitochondrial bioenergetic dysfunction in cellular models of Alzheimer's, Parkinson's, and Huntington's diseases. Here we provide detailed guidelines and protocols as standardised across the five collaborating laboratories of the CeBioND consortium, with additional contributions from other experts in the field.

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.

Glutamate excitotoxicity and Ca2+-regulation of respiration: Role of the Ca2+ activated mitochondrial transporters (CaMCs).

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.

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 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.

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 Ca(2+) and Na(+) influx, activation of poly(ADP ribose) polymerase-1 (PARP-1), and delayed Ca(2+) 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 Ca(2+)-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 Ca(2+) deregulation, and SCaMC-3-deficient mitochondria incubated with ADP or ATP-Mg had reduced Ca(2+) retention capacity, suggesting a failure to maintain matrix adenine nucleotides as a cause for premature delayed Ca(2+) 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.

Ca(2+) 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.

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.

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.

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.

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 (13)C nuclear magnetic resonance labeling with (13)C(2)acetate or (1-(13)C) 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.