Glutamate is the major excitatory neurotransmitter in the central nervous system. Beyond this function, glutamate also plays a key role in intermediary metabolism in all organs and tissues, linking carbohydrate and amino acid metabolism via the tricarboxylic acid cycle. Under both physiological and pathological conditions, we have recently found that the ability of glutamate to fuel cell metabolism selectively relies on the activity of two main transporters: the sodium-calcium exchanger (NCX) and the sodium-dependent excitatory amino-acid transporters (EAATs). In ischemic settings, when glutamate is administered at the onset of the reoxygenation phase, the coordinate activity of EAAT and NCX allows glutamate to improve cell viability by stimulating ATP production. So far, this phenomenon has been observed in both cardiac and neuronal models. In this review, we focus on the most recent findings exploring the unusual activity of glutamate as a potential survival factor in different settings. KeywordsAmino-acid transporters • ATP • Cell viability • Ischemia/reperfusion • Sodium-calcium exchanger Abbreviations AD Alzheimer's disease ALS Amyotrophic lateral sclerosis AGCs Aspartate/glutamate carriers AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid DL-TBOA DL-Threo-β-benzyloxyaspartic acid EAAC1 Excitatory amino acid carrier 1 EAATs Excitatory amino-acid transporters KA Kainate GLAST Glutamate-aspartate transporter GLT1 Glutamate transporter 1 mGluR Metabotropic glutamate receptors NCX Sodium-calcium exchanger NMDA N-Methyl-d-aspartate SN-6 2-[[4-[(4-Nitrophenyl)methoxy]phenyl] methyl]-4-thiazolidinecarboxylic acid ethyl ester Glutamate as neurotransmitterThe role of the glutamatergic neurotransmission into the human central nervous system (CNS) has been consolidated through a long research history over more than 70 years [1].The presence of high concentrations of glutamate in the brain was first identified in the 1930s, but at that time it was mainly considered an energy source for the CNS [2], as it has been ubiquitously found, with special regard to cytosolic and mitochondrial compartments. Only in 1984 was glutamate fully recognized as the major excitatory neurotransmitter in the mammalian brain [3] and now its functions within the CNS are well established. Glutamate is involved in many aspects of normal brain function, including cognition, memory and learning, cell migration, differentiation and death during the development of the CNS [4]. Furthermore, glutamate seems to have a role also in the peripheral nervous system and in endocrine cells as well [4,5]. Despite its ubiquitous nature, glutamate concentrations are maintained at homeostatic levels through the tightly Cellular and Molecular Life Sciences * Simona Magi

Na+-dependent excitatory amino acid transporters (EAATs) are the major transport mechanisms for extracellular glutamate removal in the central nervous system (CNS). The primary function assigned to EAATs is the maintenance of low extracellular glutamate levels, thus allowing glutamate to be used as a signaling molecule in the brain and to avoid excitotoxicity. However, glutamate has other recognized functions. For instance, it is a key anaplerotic substrate for the tricarboxylic acid (TCA) cycle, as it can be converted to α-ketoglutarate by transaminases or glutamate dehydrogenase. Furthermore, glutamate is a precursor of the main antioxidant glutathione, which plays a pivotal role in preventing oxidative cell death. Therefore, glutamate signaling/use is at the crossroad of multiple metabolic pathways and accordingly, it can influence a plethora of cell functions, both in health and disease. Here, we provide an overview of the main functions of glutamate and its transport systems, analyzing its role as a neurotransmitter and at the same time, the possible metabolic fates it can undergo in the intracellular milieu. Specifically, the metabolic role of glutamate and the molecular machinery proposed to metabolically support its transport will be further analyzed.

In brain ischemia, reduction in oxygen and substrates affects mitochondrial respiratory chain and aerobic metabolism, culminating in ATP production impairment, ionic imbalance, and cell death. The restoration of blood flow and reoxygenation are frequently associated with exacerbation of tissue injury, giving rise to ischemia/reperfusion (I/R) injury. In this setting, the imbalance of brain bioenergetics induces important metabolic adaptations, including utilization of alternative energy sources, such as glutamate. Although glutamate has long been considered as a neurotoxin, it can also be used as intermediary metabolite for ATP synthesis, and both the Na+/Ca2+ exchanger (NCX) and the Na+-dependent excitatory amino-acid transporters (EAATs) are essential in this pathway. Here we analyzed the role of NCX in the potential of glutamate to improve metabolism and survival of neuronal cells subjected to hypoxia/reoxygenation (H/R). In SH-SY5Y neuroblastoma cells differentiated into a neuron-like state, H/R produced a significant cell damage, a decrease in ATP cellular content, and intracellular Ca2+ alterations. Exposure to glutamate at the onset of the reoxygenation phase attenuated H/R-induced cell damage and evoked a significant raise in intracellular ATP levels. Furthermore, we found that in H/R cells NCX reverse-mode activity was reduced, and that glutamate limited such reduction. All the effects induced by glutamate supplementation were lost when cells were transfected with small interfering RNA against NCX1 and EAAT3, suggesting the need of a specific functional interplay between these proteins for glutamate-induced protection. Collectively, our results revealed the potential beneficial effect of glutamate in an in vitro model of H/R injury and focused on the essential role exerted by NCX1. Although preliminary, these findings could be a starting point to further investigate in in vivo systems such protective effect in ischemic settings, shedding a new light on the classical view of glutamate as detrimental factor.

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Myocardial ischemia culminates in ATP production impairment, ionic derangement and cell death. The provision of metabolic substrates during reperfusion significantly increases heart tolerance to ischemia by improving mitochondrial performance. Under normoxia, glutamate contributes to myocardial energy balance as substrate for anaplerotic reactions, and we demonstrated that the Na+/Ca2+ exchanger1 (NCX1) provides functional support for both glutamate uptake and use for ATP synthesis. Here we investigated the role of NCX1 in the potential of glutamate to improve energy metabolism and survival of cardiac cells subjected to hypoxia/reoxygenation (H/R). Specifically, in H9c2-NCX1 myoblasts, ATP levels, mitochondrial activities and cell survival were significantly compromised after H/R challenge. Glutamate supplementation at the onset of the reoxygenation phase significantly promoted viability, improved mitochondrial functions and normalized the H/R-induced increase of NCX1 reverse-mode activity. The benefits of glutamate were strikingly lost in H9c2-WT (lacking NCX1 expression), or in H9c2-NCX1 and rat cardiomyocytes treated with either NCX or Excitatory Amino Acid Transporters (EAATs) blockers, suggesting that a functional interplay between these transporters is critically required for glutamate-induced protection. Collectively, these results revealed for the first time the key role of NCX1 for the beneficial effects of glutamate against H/R-induced cell injury.

Progressive accumulation of α-synuclein (α-syn) and exposure to environmental toxins are risk factors that may both concur to Parkinson’s disease (PD) pathogenesis. Electrophysiological recordings of field postsynaptic potentials (fEPSPs) and Ca2+ measures in striatal brain slices and differentiated SH-SY5Y cells showed that co-application of α-syn and the neurotoxic pesticide rotenone (Rot) induced Ca2+ dysregulation and alteration of both synaptic transmission and cell function. Interestingly, the presence of the mitochondrial NCX inhibitor CGP-37157 prevented these alterations. The specific involvement of the mitochondrial NCX was confirmed by the inability of the plasma membrane inhibitor SN-6 to counteract such phenomenon. Of note, using a siRNA approach, we found that NCX1 was the isoform specifically involved. These findings suggested that NCX1, operating on the mitochondrial membrane, may have a critical role in the maintenance of ionic Ca2+ homeostasis in PD and that its inhibition most likely exerts a protective effect in the toxicity induced by α-syn and Rot.

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson’s disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

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