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 deathin vitro, although glutamate-stimulated respiration was impaired. In contrast, the presence ofl-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 inaralar^+/−mice enhanced kainic acid-induced seizures and neuronal damage with respect to control animals, in a model of excitotoxicity in which increasedl-lactate levels andl-lactate consumption have been previously proven. These results suggest that,in vivo, an inefficient operation of the shuttle in thearalarhemizygous mice prevents the protective role ofl-lactate on glutamate excitotoxiciy and that the entry and oxidation ofl-lactate through ARALAR-MAS pathway is required for its neuroprotective function.SIGNIFICANCE STATEMENTLactate 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 byaralar/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 exacerbatedin vivoexcitotoxicity inaralar^+/−mice.

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.

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.

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 inaralar-knock-out (KO) neurons and mice. We report that βOHB efficiently recoversaralar-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-treatedaralar-KO mice. Although brain Asp and NAA levels did not change by βOHB administration, a 4-d βOHB treatment toaralar-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 thearalar-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 STATEMENTAralardeficiency induces a fatal phenotype in humans and mice and is associated with impaired neurodevelopment, epilepsy, and hypomyelination. In neurons, highly expressingaralar, 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 inaralar-deficient neurons. βOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate (Asp) and NAA, which is impeded byaralardeficiency, presumably through citrate-malate shuttle. Inaralar-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.

Charcot–Marie–Tooth (CMT) disease is a neuropathy that lacks effective therapy. CMT patients show degeneration of peripheral nerves, leading to muscle weakness and loss of proprioception. Loss of mitochondrial oxidative phosphorylation proteins and enzymes of the antioxidant response accompany degeneration of nerves in skin biopsies of CMT patients. Herein, we followed a drug-repurposing approach to find drugs in a Food and Drug Administration-approved library that could prevent development of CMT disease in the Gdap1-null mouse model. We found that the antibiotic florfenicol is a mitochondrial uncoupler that prevents the production of reactive oxygen species and activates respiration in human GDAP1-knockdown neuroblastoma cells and in dorsal root ganglion neurons of Gdap1-null mice. Treatment of CMT-affected Gdap1-null mice with florfenicol has no beneficial effect in the course of the disease. However, administration of florfenicol, or the antioxidant MitoQ, to pre-symptomatic GDAP1-null mice prevented weight gain and ameliorated the motor coordination deficiencies that developed in the Gdap1-null mice. Interestingly, both florfenicol and MitoQ halted the decay in mitochondrial and redox proteins in sciatic nerves of Gdap1-null mice, supporting that oxidative damage is implicated in the etiology of the neuropathy. These findings support the development of clinical trials for translation of these drugs for treatment of CMT patients.