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Abstract The recently discovered interaction between presenilin 1 (PS1), a subunit of γ-secretase involved in amyloid-β (Aβ) peptide production, and GLT-1, the major brain glutamate transporter (EAAT2 in the human), may link two pathological aspects of Alzheimer’s disease: abnormal Aβ occurrence and neuronal network hyperactivity. In the current study, we employed a FRET-based fluorescence lifetime imaging microscopy (FLIM) to characterize the PS1/GLT-1 interaction in brain tissue from sporadic AD (sAD) patients. sAD brains showed significantly less PS1/GLT-1 interaction than those with frontotemporal lobar degeneration or non-demented controls. Familial AD (fAD) PS1 mutations, inducing a “closed” PS1 conformation similar to that in sAD brain, and gamma-secretase modulators (GSMs), inducing a “relaxed” conformation, respectively reduced and increased the interaction. Furthermore, PS1 influences GLT-1 cell surface expression and homomultimer formation, acting as a chaperone but not affecting GLT-1 stability. The diminished PS1/GLT-1 interaction suggests that these functions may not work properly in AD.

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases. [ABSTRACT FROM AUTHOR]

Alterations in the activity of the regulator of cell metabolism AMP-activated protein kinase (AMPK) have been reported in motor neurons from patients and animal models of amyotrophic lateral sclerosis (ALS). Considering the key role played by astrocytes in modulating energy metabolism in the nervous system and their compromised support towards neurons in ALS, we examined whether a putative alteration in AMPK expression/activity impacted astrocytic functions such as their metabolic plasticity and glutamate handling capacity. We found a reduced expression of AMPK mRNA in primary cultures of astrocytes derived from transgenic rats carrying an ALS-associated mutated superoxide dismutase (hSOD1G93A). The activation of AMPK after glucose deprivation was reduced in hSOD1G93A astrocytes compared to non-transgenic. This was accompanied by a lower increase in ATP levels and increased vulnerability to this insult, although the ATP production rate did not differ between the two cell types. Furthermore, soliciting the activity of glutamate transporters was found to induce similar AMPK activity in these cells. However, manipulation of AMPK activity did not influence glutamate transport. Together, these results suggest that the altered AMPK responsiveness in ALS might be context dependent and may compromise the metabolic adaptation of astrocytes in response to specific cellular stress. [ABSTRACT FROM AUTHOR]

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.

Alterations in the activity of the regulator of cell metabolism AMP-activated protein kinase (AMPK) have been reported in motor neurons from patients and animal models of amyotrophic lateral sclerosis (ALS). Considering the key role played by astrocytes in modulating energy metabolism in the nervous system and their compromised support towards neurons in ALS, we examined whether a putative alteration in AMPK expression/activity impacted astrocytic functions such as their metabolic plasticity and glutamate handling capacity. We found a reduced expression of AMPK mRNA in primary cultures of astrocytes derived from transgenic rats carrying an ALS-associated mutated superoxide dismutase (hSOD1G93A). The activation of AMPK after glucose deprivation was reduced in hSOD1G93A astrocytes compared to non-transgenic. This was accompanied by a lower increase in ATP levels and increased vulnerability to this insult, although the ATP production rate did not differ between the two cell types. Furthermore, soliciting the activity of glutamate transporters was found to induce similar AMPK activity in these cells. However, manipulation of AMPK activity did not influence glutamate transport. Together, these results suggest that the altered AMPK responsiveness in ALS might be context dependent and may compromise the metabolic adaptation of astrocytes in response to specific cellular stress.

LRP1, the low-density lipoprotein receptor 1, would be a novel candidate gene of epilepsy according to our bioinformatic results and the animal study. In this study, we explored the role of LRP1 in epilepsy and whether beta-hydroxybutyrate, the principal ketone body of the ketogenic diet, can treat epilepsy caused by LRP1 deficiency in drosophila. UAS/GAL4 system was used to establish different genotype models. Flies were given standard, high-sucrose, and ketone body food randomly. The bang-sensitive test was performed on flies and seizure-like behavior was assessed. In morphologic experiments, we found that LRP1 deficiency caused partial loss of the ellipsoidal body and partial destruction of the fan-shaped body. Whole-body and glia LRP1 defect flies had a higher seizure rate compared to the control group. Ketone body decreased the seizure rate in behavior test in all LRP1 defect flies, compared to standard and high sucrose diet. Overexpression of glutamate transporter gene Eaat1 could mimic the ketone body effect on LRP1 deficiency flies. This study demonstrated that LRP1 defect globally or in glial cells or neurons could induce epilepsy in drosophila. The ketone body efficaciously rescued epilepsy caused by LRP1 knockdown. The results support screening for LRP1 mutations as discriminating conduct for individuals who require clinical attention and further clarify the mechanism of the ketogenic diet in epilepsy, which could help epilepsy patients make a precise treatment case by case. [ABSTRACT FROM AUTHOR]

The sodium-dependent glutamate transporter GLT-1 (EAAT2, SLC1A2) has been well-described as an important regulator of extracellular glutamate homeostasis in the central nervous system (CNS), a function that is performed mainly through its presence on astrocytes. There is, however, increasing evidence for the expression of GLT-1 in CNS cells other than astrocytes and in functional roles that are mediated by mechanisms downstream of glutamate uptake. In this context, GLT-1 expression has been reported for both neurons and oligodendrocytes (OLGs), and neuronal presynaptic presence of GLT-1 has been implicated in the regulation of glutamate uptake, gene expression, and mitochondrial function. Much less is currently known about the functional roles of GLT-1 expressed by OLGs. The data presented here provide first evidence that GLT-1 expressed by maturing OLGs contributes to the modulation of developmental myelination in the CNS. More specifically, using inducible and conditional knockout mice in which GLT-1 was deleted in maturing OLGs during a peak period of myelination (between 2 and 4 weeks of age) revealed hypomyelinated characteristics in the corpus callosum of preferentially male mice. These characteristics included reduced percentages of smaller diameter myelinated axons and reduced myelin thickness. Interestingly, this myelination phenotype was not found to be associated with major changes in myelin gene expression. Taken together, the data presented here demonstrate that GLT-1 expressed by maturing OLGs is involved in the modulation of the morphological aspects associated with CNS myelination in at least the corpus callosum and during a developmental window that appears of particular vulnerability in males compared to females. [ABSTRACT FROM AUTHOR]

The sodium-dependent glutamate transporter GLT-1 (EAAT2, SLC1A2) has been well-described as an important regulator of extracellular glutamate homeostasis in the central nervous system (CNS), a function that is performed mainly through its presence on astrocytes. There is, however, increasing evidence for the expression of GLT-1 in CNS cells other than astrocytes and in functional roles that are mediated by mechanisms downstream of glutamate uptake. In this context, GLT-1 expression has been reported for both neurons and oligodendrocytes (OLGs), and neuronal presynaptic presence of GLT-1 has been implicated in the regulation of glutamate uptake, gene expression, and mitochondrial function. Much less is currently known about the functional roles of GLT-1 expressed by OLGs. The data presented here provide first evidence that GLT-1 expressed by maturing OLGs contributes to the modulation of developmental myelination in the CNS. More specifically, using inducible and conditional knockout mice in which GLT-1 was deleted in maturing OLGs during a peak period of myelination (between 2 and 4 weeks of age) revealed hypomyelinated characteristics in the corpus callosum of preferentially male mice. These characteristics included reduced percentages of smaller diameter myelinated axons and reduced myelin thickness. Interestingly, this myelination phenotype was not found to be associated with major changes in myelin gene expression. Taken together, the data presented here demonstrate that GLT-1 expressed by maturing OLGs is involved in the modulation of the morphological aspects associated with CNS myelination in at least the corpus callosum and during a developmental window that appears of particular vulnerability in males compared to females.

In order to meet the energetic demands of cell-to-cell signaling, increases in local neuronal signaling are matched by a coordinated increase in local blood flow, termed neurovascular coupling. Multiple different signals from neurons, astrocytes, and pericytes contribute to this control of blood flow. Previously, several groups demonstrated that inhibition/ablation of glutamate transporters attenuates the neurovascular response. However, it was not determined if glutamate transporter activation was sufficient to increase blood flow. Here, we used multiphoton imaging to monitor the diameter of fluorescently labeled cortical arterioles in anesthetized C57/B6J mice. We delivered vehicle, glutamate transporter substrates, or a combination of a glutamate transporter substrate with various pharmacologic agents via a glass micropipette while simultaneously visualizing changes in arteriole diameter. We developed a novel image analysis method to automate the measurement of arteriole diameter in these time-lapse analyses. Using this workflow, we first conducted pilot experiments in which we focally applied L-glutamate, D-aspartate, or L- threo -hydroxyaspartate (L-THA) and measured arteriole responses as proof of concept. We subsequently applied the selective glutamate transport substrate L-THA (applied at concentrations that do not activate glutamate receptors). We found that L-THA evoked a significantly larger dilation than that observed with focal saline application. This response was blocked by co-application of the potent glutamate transport inhibitor, L-(2S,3S)-3-[3-[4-(trifluoromethyl)-benzoylamino]benzyloxy]-aspartate (TFB-TBOA). Conversely, we were unable to demonstrate a reduction of this effect through co-application of a cocktail of glutamate and GABA receptor antagonists. These studies provide the first direct evidence that activation of glutamate transport is sufficient to increase arteriole diameter. We explored potential downstream mechanisms mediating this transporter-mediated dilation by using a Ca2+ chelator or inhibitors of reversed-mode Na+/Ca2+ exchange, nitric oxide synthetase, or cyclo-oxygenase. The estimated effects and confidence intervals suggested some form of inhibition for a number of these inhibitors. Limitations to our study design prevented definitive conclusions with respect to these downstream inhibitors; these limitations are discussed along with possible next steps. Understanding the mechanisms that control blood flow are important because changes in blood flow/energy supply are implicated in several neurodegenerative disorders and are used as a surrogate measure of neuronal activity in widely used techniques such as functional magnetic resonance imaging (fMRI). [ABSTRACT FROM AUTHOR]

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). A family of five Na+-dependent transporters maintain low levels of extracellular glutamate and shape excitatory signaling. Shortly after the research group of the person being honored in this special issue (Dr. Baruch Kanner) cloned one of these transporters, his group and several others showed that their activity can be acutely (within minutes to hours) regulated. Since this time, several different signals and post-translational modifications have been implicated in the regulation of these transporters. In this review, we will provide a brief introduction to the distribution and function of this family of glutamate transporters. This will be followed by a discussion of the signals that rapidly control the activity and/or localization of these transporters, including protein kinase C, ubiquitination, glutamate transporter substrates, nitrosylation, and palmitoylation. We also include the results of our attempts to define the role of palmitoylation in the regulation of GLT-1 in crude synaptosomes. In some cases, the mechanisms have been fairly well-defined, but in others, the mechanisms are not understood. In several cases, contradictory phenomena have been observed by more than one group; we describe these studies with the goal of identifying the opportunities for advancing the field. Abnormal glutamatergic signaling has been implicated in a wide variety of psychiatric and neurologic disorders. Although recent studies have begun to link regulation of glutamate transporters to the pathogenesis of these disorders, it will be difficult to determine how regulation influences signaling or pathophysiology of glutamate without a better understanding of the mechanisms involved. [ABSTRACT FROM AUTHOR]

In order to meet the energetic demands of cell-to-cell signaling, increases in local neuronal signaling are matched by a coordinated increase in local blood flow, termed neurovascular coupling. Multiple different signals from neurons, astrocytes, and pericytes contribute to this control of blood flow. Previously, several groups demonstrated that inhibition/ablation of glutamate transporters attenuates the neurovascular response. However, it was not determined if glutamate transporter activation was sufficient to increase blood flow. Here, we used multiphoton imaging to monitor the diameter of fluorescently labeled cortical arterioles in anesthetized C57/B6J mice. We delivered vehicle, glutamate transporter substrates, or a combination of a glutamate transporter substrate with various pharmacologic agents via a glass micropipette while simultaneously visualizing changes in arteriole diameter. We developed a novel image analysis method to automate the measurement of arteriole diameter in these time-lapse analyses. Using this workflow, we first conducted pilot experiments in which we focally applied L-glutamate, D-aspartate, or L-threo-hydroxyaspartate (L-THA) and measured arteriole responses as proof of concept. We subsequently applied the selective glutamate transport substrate L-THA (applied at concentrations that do not activate glutamate receptors). We found that L-THA evoked a significantly larger dilation than that observed with focal saline application. This response was blocked by co-application of the potent glutamate transport inhibitor, L-(2S,3S)-3-[3-[4-(trifluoromethyl)-benzoylamino]benzyloxy]-aspartate (TFB-TBOA). Conversely, we were unable to demonstrate a reduction of this effect through co-application of a cocktail of glutamate and GABA receptor antagonists. These studies provide the first direct evidence that activation of glutamate transport is sufficient to increase arteriole diameter. We explored potential downstream mechanisms mediating this transporter-mediated dilation by using a Ca2+ chelator or inhibitors of reversed-mode Na+/Ca2+ exchange, nitric oxide synthetase, or cyclo-oxygenase. The estimated effects and confidence intervals suggested some form of inhibition for a number of these inhibitors. Limitations to our study design prevented definitive conclusions with respect to these downstream inhibitors; these limitations are discussed along with possible next steps. Understanding the mechanisms that control blood flow are important because changes in blood flow/energy supply are implicated in several neurodegenerative disorders and are used as a surrogate measure of neuronal activity in widely used techniques such as functional magnetic resonance imaging (fMRI).

In the rodent forebrain, the majority of astrocytes are generated during the early postnatal phase. Following differentiation, astrocytes undergo maturation which accompanies the development of the neuronal network. Neonate astrocytes exhibit a distinct morphology and domain size which differs to their mature counterparts. Moreover, many of the plasma membrane proteins prototypical for fully developed astrocytes are only expressed at low levels at neonatal stages. These include connexins and Kir4.1, which define the low membrane resistance and highly negative membrane potential of mature astrocytes. Newborn astrocytes moreover express only low amounts of GLT‐1, a glutamate transporter critical later in development. Furthermore, they show specific differences in the properties and spatio‐temporal pattern of intracellular calcium signals, resulting from differences in their repertoire of receptors and signalling pathways. Therefore, roles fulfilled by mature astrocytes, including ion and transmitter homeostasis, are underdeveloped in the young brain. Similarly, astrocytic ion signalling in response to neuronal activity, a process central to neuron–glia interaction, differs between the neonate and mature brain. This review describes the unique functional properties of astrocytes in the first weeks after birth and compares them to later stages of development. We conclude that with an immature neuronal network and wider extracellular space, astrocytic support might not be as demanding and critical compared to the mature brain. The delayed differentiation and maturation of astrocytes in the first postnatal weeks might thus reflect a reduced need for active, energy‐consuming regulation of the extracellular space and a less tight control of glial feedback onto synaptic transmission. [ABSTRACT FROM AUTHOR]

High-affinity, Na+-dependent glutamate transporters are the primary means by which synaptically released glutamate is removed from the extracellular space. They restrict the spread of glutamate from the synaptic cleft into the perisynaptic space and reduce its spillover to neighboring synapses. Thereby, glutamate uptake increases the spatial precision of synaptic communication. Its dysfunction and the entailing rise of the extracellular glutamate concentration accompanied by an increased spread of glutamate result in a loss of precision and in enhanced excitation, which can eventually lead to neuronal death via excitotoxicity. Efficient glutamate uptake depends on a negative resting membrane potential as well as on the transmembrane gradients of the co-transported ions (Na+, K+, and H+) and thus on the proper functioning of the Na+/K+-ATPase. Consequently, numerous studies have documented the impact of an energy shortage, as occurring for instance during an ischemic stroke, on glutamate clearance and homeostasis. The observations range from rapid changes in the transport activity to altered expression of glutamate transporters. Notably, while astrocytes account for the majority of glutamate uptake under physiological conditions, they may also become a source of extracellular glutamate elevation during metabolic stress. However, the mechanisms of the latter phenomenon are still under debate. Here, we review the recent literature addressing changes of glutamate uptake and homeostasis triggered by acute metabolic stress, i.e., on a timescale of seconds to minutes.

The recently discovered interaction between Presenilin 1 (PS1), a catalytic subunit of γ-secretase responsible for generating amyloid-β peptides, and GLT-1, a major glutamate transporter in the brain (EAAT2), provides a mechanistic link between these two key factors involved in Alzheimer's disease (AD) pathology. Modulating this interaction can be crucial to understand the consequence of such crosstalk in AD context and beyond. However, the interaction sites between these two proteins are unknown. Herein, we utilized an alanine scanning approach coupled with FRET-based fluorescence lifetime imaging microscopy to identify the interaction sites between PS1 and GLT-1 in their native environment within intact cells. We found that GLT-1 residues at position 276 to 279 (TM5) and PS1 residues at position 249 to 252 (TM6) are crucial for GLT-1-PS1 interaction. These results have been cross validated using AlphaFold Multimer prediction. To further investigate whether this interaction of endogenously expressed GLT-1 and PS1 can be prevented in primary neurons, we designed PS1/GLT-1 cell-permeable peptides (CPPs) targeting the PS1 or GLT-1 binding site. We used HIV TAT domain to allow for cell penetration which was assayed in neurons. First, we assessed the toxicity and penetration of CPPs by confocal microscopy. Next, to ensure the efficiency of CPPs, we monitored the modulation of GLT-1-PS1 interaction in intact neurons by fluorescence lifetime imaging microscopy. We saw significantly less interaction between PS1 and GLT-1 with both CPPs. Our study establishes a new tool to study the functional aspect of GLT-1-PS1 interaction and its relevance in normal physiology and AD models., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetically encoded fluorescent glutamate indicators (iGluSnFRs) enable neurotransmitter release and diffusion to be visualized in intact tissue. Synaptic iGluSnFR signal time courses vary widely depending on experimental conditions, often lasting 10–100 times longer than the extracellular lifetime of synaptically released glutamate estimated with uptake measurements. iGluSnFR signals typically also decay much more slowly than the unbinding kinetics of the indicator. To resolve these discrepancies, here we have modeled synaptic glutamate diffusion, uptake and iGluSnFR activation to identify factors influencing iGluSnFR signal waveforms. Simulations suggested that iGluSnFR competes with transporters to bind synaptically released glutamate, delaying glutamate uptake. Accordingly, synaptic transporter currents recorded from iGluSnFR-expressing astrocytes in mouse cortex were slower than those in control astrocytes. Simulations also suggested that iGluSnFR reduces free glutamate levels in extrasynaptic spaces, likely limiting extrasynaptic receptor activation. iGluSnFR and lower affinity variants, nonetheless, provide linear indications of vesicle release, underscoring their value for optical quantal analysis.

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems—the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues GltPh and GltTk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors. [ABSTRACT FROM AUTHOR]

In addition to being an amino acid that is incorporated into proteins, glutamate is the most abundant neurotransmitter in the mammalian CNS, the precursor for the inhibitory neurotransmitter γ-aminobutyric acid, and one metabolic step from the tricarboxylic acid cycle intermediate α-ketoglutarate. Extracellular glutamate is cleared by a family of Na+-dependent transporters. These transporters are variably expressed by all cell types in the nervous system, but the bulk of clearance is into astrocytes. GLT-1 and GLAST (also called EAAT2 and EAAT1) mediate this activity and are extremely abundant proteins with their expression enriched in fine astrocyte processes. In this review, we will focus on three topics related to these astrocytic glutamate transporters. First, these transporters co-transport three Na+ ions and a H+ with each molecule of glutamate and counter-transport one K+; they are also coupled to a Cl− conductance. The movement of Na+ is sufficient to cause profound astrocytic depolarization, and the movement of H+ is linked to astrocytic acidification. In addition, the movement of Na+ can trigger the activation of Na+ co-transporters (e.g. Na+–Ca2+ exchangers). We will describe the ways in which these ionic movements have been linked as signals to brain function and/or metabolism. Second, these transporters co-compartmentalize with mitochondria, potentially providing a mechanism to supply glutamate to mitochondria as a source of fuel for the brain. We will provide an overview of the proteins involved, discuss the evidence that glutamate is oxidized, and then highlight some of the un-resolved issues related to glutamate oxidation. Finally, we will review evidence that ischemic insults (stroke or oxygen/glucose deprivation) cause changes in these astrocytic mitochondria and discuss the ways in which these changes have been linked to glutamate transport, glutamate transport-dependent signaling, and altered glutamate metabolism. We conclude with a broader summary of some of the unresolved issues. [ABSTRACT FROM AUTHOR]

It has recently been proposed using a multi-compartmental mathematical model that negatively fixed charged membrane-associated sites constrain the flow of cations in perisynaptic astroglial processes. This restricted movement of ions between the perisynaptic cradle (PsC), principal astroglial processes and the astrocyte soma gives rise to potassium (K+) and sodium (Na+) microdomains at the PsC. The present paper extends the above model to demonstrate that the formation of an Na+ microdomain can reverse the Na+/Ca2+ exchanger (NCX) thus providing an additional source of calcium (Ca2+) at the PsC. Results presented clearly show that reversal of the Na+/Ca2+ exchanger is instigated by a glutamate transporter coupled increase in concentration of cytoplasmic [Na+]i at the PsC, which and instigates Ca2+ influx through the NCX. As the flow of Ca2+ along the astrocyte process and away from the PsC is also constrained by Ca2+ binding proteins, then a Ca2+ microdomain forms at the PsC. The paper also serves to demonstrate that the EAAT, NKA, and NCX represent the minimal requirement necessary and sufficient for the development of a Ca2+ microdomain and that these mechanisms directly link neuronal activity and glutamate release to the formation of localized Na+ and Ca2+ microdomains signals at the PsC. This local source of Ca2+ can provide a previously underexplored form of astroglial Ca2+ signaling.

It has recently been proposed using a multi-compartmental mathematical model that negatively fixed charged membrane-associated sites constrain the flow of cations in perisynaptic astroglial processes. This restricted movement of ions between the perisynaptic cradle (PsC), principal astroglial processes and the astrocyte soma gives rise to potassium (K+) and sodium (Na+) microdomains at the PsC. The present paper extends the above model to demonstrate that the formation of an Na+ microdomain can reverse the Na+/Ca2+ exchanger (NCX) thus providing an additional source of calcium (Ca2+) at the PsC. Results presented clearly show that reversal of the Na+/Ca2+ exchanger is instigated by a glutamate transporter coupled increase in concentration of cytoplasmic [Na+]i at the PsC, which and instigates Ca2+ influx through the NCX. As the flow of Ca2+ along the astrocyte process and away from the PsC is also constrained by Ca2+ binding proteins, then a Ca2+ microdomain forms at the PsC. The paper also serves to demonstrate that the EAAT, NKA, and NCX represent the minimal requirement necessary and sufficient for the development of a Ca2+ microdomain and that these mechanisms directly link neuronal activity and glutamate release to the formation of localized Na+ and Ca2+ microdomains signals at the PsC. This local source of Ca2+ can provide a previously underexplored form of astroglial Ca2+ signaling. [ABSTRACT FROM AUTHOR]

Abstract We have analysed post-mortem samples of prefrontal cortex from control and alcoholic human brains by the technique of Western blotting to estimate and compare the expressions of glutamate transporter GLAST (Excitatory Amino Acid Transporter One; EAAT1). Furthermore, using the non-alcoholic prefrontal cortex and custom-made GLAST (EAAT1) antibody we determined GLAST (EAAT1) "interactome" i.e. the set of proteins selectively bound by GLAST (EAAT1). We found that GLAST (EAAT1) was significantly more abundant (about 1.6-fold) in the cortical tissue from alcoholic brains compared to that from non-alcoholic controls. The greatest increase in the level of GLAST (EAAT1) was found in plasma membrane fraction (2.2-fold). Additionally, using the prefrontal cortical tissue from control brains, we identified 38 proteins specifically interacting with GLAST (EAAT1). These can be classified as contributing to the cell structure (6 proteins; 16%), energy and general metabolism (18 proteins; 47%), neurotransmitter metabolism (three proteins; 8%), signalling (6 proteins: 16%), neurotransmitter storage/release at synapses (three proteins; 8%) and calcium buffering (two proteins; 5%). We discuss possible consequences of the increased expression of GLAST (EAAT1) in alcoholic brain tissue and whether or how this could disturb the function of the proteins potentially interacting with GLAST (EAAT1) in vivo. The data represent an extension of our previous proteomic and metabolomic studies of human alcoholism revealing another aspect of the complexity of changes imposed on brain by chronic long-term consumption of ethanol. Highlights • We studied glutamate transporter GLAST in human prefrontal cortex. • GLAST expression was significantly increased in alcoholic brains. • The increase in GLAST appeared to be most pronounced in plasma membranes. • In healthy brains, we used proteomics to determine GLAST "interactome". • GLAST interacted with 38 proteins; 18 of those were involved in energy metabolism. [ABSTRACT FROM AUTHOR]

Developmental cortical malformations (DCMs) are linked with severe epilepsy and are caused by both genetic and environmental insults. DCMs include several neurological diseases, such as focal cortical dysplasia, polymicrogyria, schizencephaly, and others. Human studies have implicated astrocyte reactivity and dysfunction in the pathophysiology of DCMs, but their specific role is unknown. As astrocytes powerfully regulate glutamate neurotransmission, and glutamate levels are known to be increased in human epileptic foci, understanding the role of astrocytes in the pathological sequelae of DCMs is extremely important. Additionally, recent studies examining astrocyte glutamate uptake in DCMs have reported conflicting results, adding confusion to the field. In this study we utilized the freeze lesion (FL) model of DCM, which is known to induce reactive astrocytosis and cause significant changes in astrocyte morphology, proliferation, and distribution. Using whole-cell patch clamp recording from astrocytes, we recorded both UV-uncaging and synaptically evoked glutamate transporter currents (TCs), widely accepted assays of functional glutamate transport by astrocytes. With this approach, we set out to test the hypothesis that astrocyte membrane properties and glutamate transport were disrupted in this model of DCM. Though we found that the developmental maturation of astrocyte membrane resistance was disrupted by FL, glutamate uptake by individual astrocytes was robust throughout FL development. Interestingly, using an immunolabeling approach, we observed spatial and developmental differences in excitatory amino acid transporter (EAAT) expression in FL cortex. Spatially specific differences in EAAT2 (GLT-1) and EAAT1 (GLAST) expression suggest that the relative contribution of each EAAT to astrocytic glutamate uptake may be altered in FL cortex. Lastly, we carefully analyzed the amplitudes and onset times of both synaptically- and UV uncaging-evoked TCs. We found that in the FL cortex, synaptically-evoked, but not UV uncaging-evoked TCs, were larger in amplitude. Additionally, we found that the amount of electrical stimulation required to evoke a synaptic TC was significantly reduced in the FL cortex. Both of these findings are consistent with increased excitatory input to the FL cortex, but not with changes in how individual astrocytes remove glutamate. Taken together, our results demonstrate that the maturation of astrocyte membrane resistance, local distribution of glutamate transporters, and glutamatergic input to the cortex are altered in the FL model, but that single-cell astrocytic glutamate uptake is robust.

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses.

Cocaine use disorder is a chronically relapsing disease without FDA-approved treatments. Using a rodent model of cocaine relapse, we and others have previously demonstrated that the beta-lactam antibiotic ceftriaxone attenuates cue- and cocaine-primed reinstatement of cocaine-seeking. Ceftriaxone restores cocaine-induced deficits in both system xc- and GLT-1 expression and function in the nucleus accumbens core (NAc). We recently demonstrated that restoration of GLT-1 expression in the NAc is necessary for ceftriaxone to attenuate reinstatement of cocaine-seeking. Here we used an adeno-associated virus (AAV) to overexpress GLT-1a in the NAc to investigate whether such restoration is sufficient to attenuate cue- and cocaine-primed reinstatement. Rats self-administered cocaine for two weeks and received injections of either AAV-GFAP-GLT-1a or AAV-GFAP-eGFP in the NAc following the last day of self-administration. Rats then underwent three weeks of extinction training (during which time transduction and expression occurred) before undergoing a cue- or cocaine-primed reinstatement test. Microdialysis for the quantification of glutamate efflux in the NAc was conducted during the cocaine-primed test. Rats that received AAV-GFAP-GLT-1a reinstated cue-primed cocaine-seeking in a similar manner as rats that received the control AAV-GFAP-eGFP. Upregulation of GLT-1a attenuated glutamate efflux during a cocaine-primed reinstatement test, but was not sufficient to attenuate reinstatement. We confirmed that GLT-1a upregulation resulted in functional upregulation of glutamate transport and expression, without affecting sodium-independent glutamate uptake, indicating system xc-was not altered. These results indicate that upregulation of NAc GLT-1 transporters alone is not sufficient to prevent the reinstatement of cocaine-seeking and implicate additional mechanisms in regulating glutamate efflux. [ABSTRACT FROM AUTHOR]

Hepatic glutamate uptake and conversion to glutamine is critical for whole-body N metabolism, but how this process is regulated during growth is poorly described. The hepatic glutamate uptake activities, protein content of system XAG- transporters (EAAC1, GLT-1) and regulatory proteins (GTRAP3-18, ARL6IP1), glutamine synthetase (GS) activity and content, and glutathione (GSH) content, were compared in liver tissue of weaned Angus steers randomly assigned (n = 8) to predominantly lean (growing) or predominantly lipid (finished) growth regimens. Steers were fed a cotton seed hull-based diet to achieve final body weights of 301 or 576 kg, respectively, at a constant rate of growth. Liver tissue was collected at slaughter and hepatic membranes fractionated. Total (75%), Na+-dependent (90%), system XAG--dependent (abolished) glutamate uptake activity, and EAAC1 content (36%) in canalicular membrane-enriched vesicles decreased as steers developed from growing (n = 6) to finished (n = 4) stages, whereas Na+-independent uptake did not change. In basolateral membrane-enriched vesicles, total (60%), Na+-dependent (60%), and Na+-independent (56%) activities decreased, whereas neither system XAG--dependent uptake nor protein content changed. EAAC1 protein content in liver homogenates (n = 8) decreased in finished vs. growing steers, whereas GTRAP3-18 and ARL6IP1 content increased and GLT-1 content did not change. Concomitantly, hepatic GS activity decreased (32%) as steers fattened, whereas GS and GSH contents did not differ. We conclude that hepatic glutamate uptake and GS synthesis capacities are reduced in livers of finished versus growing beef steers, and that hepatic system XAG- transporter activity/EAAC1 content is inversely proportional to GTRAP3-18 content. [ABSTRACT FROM AUTHOR]

Summary: Aims: This review summarizes evidence for dysfunctional connectivity between cortical and striatal neurons in Huntington's disease (HD), a fatal neurodegenerative condition caused by a single gene mutation. The focus is on data derived from recording of electrophysiological signals in behaving transgenic mouse models. Discussions: Firing patterns of individual neurons and the frequency oscillations of local field potentials indicate a disruption in corticostriatal processing driven, in large part, by interactions between cells that contain the mutant gene rather than the mutant gene alone. Dysregulation of glutamate, an excitatory amino acid released by cortical afferents, plays a key role in the breakdown of corticostriatal communication, a process modulated by ascorbate, an antioxidant vitamin found in high concentration in striatum. Up‐regulation of glutamate transport by drug administration or viral‐vector delivery improves ascorbate homeostasis and neurobehavioral processing in HD mice. Further analysis of electrophysiological data, including the use of sophisticated computational strategies, is required to discern how behavioral demands modulate the flow of corticostriatal information and its disruption by HD. Conclusions: Long before massive cell loss occurs, HD impairs the mechanisms by which cortical and striatal neurons communicate. A key problem identified in transgenic animal models is dysregulation of the dynamic changes in extracellular glutamate and ascorbate. Improved understanding of how these neurochemical systems impact corticostriatal communication is necessary before an effective therapeutic strategy can emerge. [ABSTRACT FROM AUTHOR]

Astrocytes have long been regarded as essentially unexcitable cells that do not contribute to active signaling and information processing in the brain. Contrary to this classical view, it is now firmly established that astrocytes can specifically respond to glutamate released from neurons. Astrocyte glutamate signaling is initiated upon binding of glutamate to ionotropic and/or metabotropic receptors, which can result in calcium signaling, a major form of glial excitability. Release of so-called gliotransmitters like glutamate, ATP and D-serine from astrocytes in response to activation of glutamate receptors has been demonstrated to modulate various aspects of neuronal function in the hippocampus. In addition to receptors, glutamate binds to high-affinity, sodium-dependent transporters, which results in rapid buffering of synaptically-released glutamate, followed by its removal from the synaptic cleft through uptake into astrocytes. The degree to which astrocytes modulate and control extracellular glutamate levels through glutamate transporters depends on their expression levels and on the ionic driving forces that decrease with ongoing activity. Another major determinant of astrocytic control of glutamate levels could be the precise morphological arrangement of fine perisynaptic processes close to synapses, defining the diffusional distance for glutamate, and the spatial proximity of transporters in relation to the synaptic cleft. In this review, we will present an overview of the mechanisms and physiological role of glutamate-induced ion signaling in astrocytes in the hippocampus as mediated by receptors and transporters. Moreover, we will discuss the relevance of astroglial glutamate uptake for extracellular glutamate homeostasis, focusing on how activity-induced dynamic changes of perisynaptic processes could shape synaptic transmission at glutamatergic synapses. [ABSTRACT FROM AUTHOR]

The solute carrier family 25 (SLC25) drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier SLC25A22 (also named GC1) have been identified in early epileptic encephalopathy (EEE) and migrating partial seizures in infancy (MPSI) but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an in vivo model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA) constructs using the Neon® Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing GC1 in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate) (NAD(P)H) formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC) was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP) in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes.Main Points:The mitochondrial respiratory chain is functional in absence of GC1Lack of glutamate oxidation results in a lower global ATP levelLack of mitochondrial glutamate transport results in intracellular glutamate accumulation

Amyloid-beta (Aβ) peptides are the major neuropathological hallmarks related with Alzheimer's disease (AD). Aβ peptides trigger several biochemical mechanisms of neurotoxicity, including neuroinflammation and glutamatergic neurotransmission impairment. Guanosine is the endogenous guanine-derived nucleoside that modulates the glutamatergic system and the cellular redox status, thus acting as a neuroprotective agent. Here, we investigated the putative neuroprotective effect of guanosine in an AD-like mouse model. Adult mice received a single intracerebroventricular injection of Aβ (400 pmol/site) or vehicle and then were treated immediately, 3 h later, and once a day during the subsequent 14 days with guanosine (8 mg/kg, intraperitoneally). Aβ or guanosine did not alter mouse locomotor activity and anxiety-related behaviors. Aβ-treated mice displayed short-term memory deficit in the object location task that was prevented by guanosine. Guanosine prevented the Aβ-induced increase in latency to grooming in the splash test, an indicative of anhedonia. Aβ increased Na-independent glutamate uptake in ex vivo hippocampal slices, and guanosine reversed it to control levels. The repeated administration of guanosine increased hippocampal GDP levels, which was not observed in the group treated with Aβ plus guanosine. Aβ induced an increase in hippocampal ADP levels. Aβ decreased GFAP expression in the hippocampal CA1 region, an effect not modified by guanosine. No differences were observed concerning synaptophysin and NeuN immunolabeling. Together, these results show that guanosine prevents memory deficit and anhedonic-like behavior induced by Aβ that seem to be linked to glutamate transport unbalance and alterations on purine and metabolite levels in mouse hippocampus. [ABSTRACT FROM AUTHOR]

The solute carrier family 25 (SLC25) drives the import of a large diversity of metabolites into mitochondria, a key cellular structure involved in many metabolic functions. Mutations of the mitochondrial glutamate carrier SLC25A22 (also named GC1) have been identified in early epileptic encephalopathy (EEE) and migrating partial seizures in infancy (MPSI) but the pathophysiological mechanism of GC1 deficiency is still unknown, hampered by the absence of an in vivo model. This carrier is mainly expressed in astrocytes and is the principal gate for glutamate entry into mitochondria. A sufficient supply of energy is essential for the proper function of the brain and mitochondria have a pivotal role in maintaining energy homeostasis. In this work, we wanted to study the consequences of GC1 absence in an in vitro model in order to understand if glutamate catabolism and/or mitochondrial function could be affected. First, short hairpin RNA (shRNA) designed to specifically silence GC1 were validated in rat C6 glioma cells. Silencing GC1 in C6 resulted in a reduction of the GC1 mRNA combined with a decrease of the mitochondrial glutamate carrier activity. Then, primary astrocyte cultures were prepared and transfected with shRNA-GC1 or mismatch-RNA (mmRNA) constructs using the Neonr Transfection System in order to target a high number of primary astrocytes, more than 64%. Silencing GC1 in primary astrocytes resulted in a reduced nicotinamide adenine dinucleotide (Phosphate) (NAD(P)H) formation upon glutamate stimulation. We also observed that the mitochondrial respiratory chain (MRC) was functional after glucose stimulation but not activated by glutamate, resulting in a lower level of cellular adenosine triphosphate (ATP) in silenced astrocytes compared to control cells. Moreover, GC1 inactivation resulted in an intracellular glutamate accumulation. Our results show that mitochondrial glutamate transport via GC1 is important in sustaining glutamate homeostasis in astrocytes. Main Points: • The mitochondrial respiratory chain is functional in absence of GC1 • Lack of glutamate oxidation results in a lower global ATP level • Lack of mitochondrial glutamate transport results in intracellular glutamate accumulation [ABSTRACT FROM AUTHOR]

Escherichia coli glutamate/aspartate-proton symporter GltP is a member of the Dicarboxylate/Amino Acid:Cation Symporter family of secondary active transport proteins. A range of computational, chemical, biochemical and biophysical methods characterised evolutionary relationships, structural features, substrate binding affinities and transport kinetics of wild-type and mutant forms of GltP. Sequence alignments and phylogenetic analysis revealed close homologies of GltP with human glutamate transporters involved in neurotransmission, neutral amino acid transporters and with the archaeal aspartate transporter GltPh. Topology predictions and comparisons with the crystal structure of GltPh were consistent with eight transmembrane-spanning α-helices and two hairpin re-entrant loops in GltP. Amplified expression of recombinant GltP with C-terminal affinity tags was achieved at 10% of total membrane protein in E. coli and purification to homogeneity with a yield of 0.8 mg/litre. Binding of substrates to GltP in native inner membranes and to purified protein solubilised in detergent was observed and quantified using solid-state NMR and fluorescence spectroscopy, respectively. A homology model of GltP docked with L-glutamate identified a putative binding site and residues predicted to interact with substrate. Sequence alignments identified further highly conserved residues predicted to have essential roles in GltP function. Residues were investigated by measuring transport activities, kinetics and response to thiol-specific reagents in 42 site-specific mutants compared with cysteine-less GltP (C256A) having an apparent affinity of initial rate transport (K m) for 3H-L-glutamate of 22.6 ± 5.5 μM in energised E. coli cells. This confirmed GltP residues involved in substrate binding and transport, especially in transmembrane helices VII and VIII. [ABSTRACT FROM AUTHOR]

• Glutamate uptake by cochlear glial cells is mediated mainly by a sodium- independent mechanism. • The cystine-glutamate exchanger (X CG −) is expressed in primary culture of cochlear glial cells • The activity of sodium-independent glutamate transporters is prominent in cochlear glial cells. Since glutamate is the primary excitatory neurotransmitter in the mammalian cochlea, the mechanisms for the removal of glutamate from the synaptic and extrasynaptic spaces are critical for maintaining normal function of this region. Glial cells of inner ear are crucial for regulation of synaptic transmission throughout since it closely interacts with neurons along the entire auditory pathway, however little is known about the activity and expression of glutamate transporters in the cochlea. In this study, using primary cochlear glial cells cultures obtained from newborn Balb/C mice, we determined the activity of a sodium-dependent and sodium-independent glutamate uptake mechanisms by means of High Performance Liquid Chromatography. The sodium-independent glutamate transport has a prominent contribution in cochlear glial cells which is similar to what has been demonstrated in other sensory organs, but it is not found in tissues less susceptible to continuous glutamate-mediated injuries. Our results showed that x CG − system is expressed in CGCs and is the main responsible for sodium-independent glutamate uptake. The identification and characterization of the x CG − transporter in the cochlea suggests a possible role of this transporter in the control of extracellular glutamate concentrations and regulation of redox state, that may aid in the preservation of auditory function. [ABSTRACT FROM AUTHOR]

Profound astrogliosis coincident with neuronal cell loss is universally described in human and animal models of temporal lobe epilepsy (TLE). In the kainic acid-induced status epilepticus (SE) model of TLE, astrocytes in the hippocampus become reactive soon after SE and before the onset of spontaneous seizures. To determine if astrocytes in the hippocampus exhibit changes in function soon after SE, we recorded from SR101-labeled astrocytes using the whole-cell patch technique in hippocampal brain slices prepared from control and kainic-acid-treated rats. Glutamate transporter-dependent currents were found to have significantly faster decay time kinetics and in addition, dye coupling between astrocytes was substantially increased. Consistent with an increase in dye coupling in reactive astrocytes, immunoblot experiments demonstrated a significant increase in both glial fibrillary acidic protein (GFAP) and connexin 43, a major gap junction protein expressed by astrocytes. In contrast to what has been observed in resected tissue from patients with refractory epilepsy, changes in potassium currents were not observed shortly after KA-induced SE. While many changes in neuronal function have been identified during the initial period of low seizure probability in this model of TLE, the present study contributes to the growing body of literature suggesting a role for astrocytes in the process of epileptogenesis.

l -Glutamate and l -aspartate are the main excitatory amino acids (EAAs) in the Central Nervous System (CNS) and their uptake regulation is critical for the maintenance of the excitatory balance. Excitatory amino acid transporters (EAATs) are widely distributed among central neurons and glial cells. GLAST and GLT1 are expressed in glial cells, whereas excitatory amino acid transporter 3/excitatory amino acid carrier 1 (EAAT3/EAAC1) is neuronal. Different signaling pathways regulate glutamate uptake by modifying the activity and expression of EAATs. In the present work we show that immature postnatal day 3 (PN3) rat retinas challenged by l -glutamate release [ 3 H]- d -Aspartate linked to the reverse transport, with participation of NMDA, but not of non-NMDA receptors. The amount of [ 3 H]- d -Aspartate released by l -glutamate is reduced during retinal development. Moreover, immature retinae at PN3 and PN7, but not PN14, exposed to a single dose of 200 or 500 μM caffeine or the selective A2A receptor (A2AR) antagonist 100 nM ZM241385 decreased [ 3 H]- d -Aspartate uptake. Caffeine also selectively increased total expression of EAAT3 at PN7 and its expression in membrane fractions. However, both EAAT1 and EAAT2 were reduced after caffeine treatment in P2 fraction. Addition of 100 nM DPCPX, an A1 receptor (A1R) antagonist, had no effect on the [ 3 H]- d -Aspartate uptake. [ 3 H]- d -Aspartate release was dependent on both extracellular sodium and D l -TBOA, but not calcium, implying a transporter-mediated mechanism. Our results suggest that in the developing rat retina caffeine modulates [ 3 H]- d -Aspartate uptake by blocking adenosine A2AR. [ABSTRACT FROM AUTHOR]

In the mammalian brain, a family of sodium-dependent transporters maintains low extracellular glutamate and shapes excitatory signaling. The bulk of this activity is mediated by the astroglial glutamate transporters GLT-1 and GLAST (also called EAAT2 and EAAT1). In this review, we will discuss evidence that these transporters co-localize with, form physical (co-immunoprecipitable) interactions with, and functionally couple to various ‘energy-generating’ systems, including the Na + /K + -ATPase, the Na + /Ca 2+ exchanger, glycogen metabolizing enzymes, glycolytic enzymes, and mitochondria/mitochondrial proteins. This functional coupling is bi-directional with many of these systems both being regulated by glutamate transport and providing the ‘fuel’ to support glutamate uptake. Given the importance of glutamate uptake to maintaining synaptic signaling and preventing excitotoxicity, it should not be surprising that some of these systems appear to ‘redundantly’ support the energetic costs of glutamate uptake. Although the glutamate–glutamine cycle contributes to recycling of neurotransmitter pools of glutamate, this is an over-simplification. The ramifications of co-compartmentalization of glutamate transporters with mitochondria for glutamate metabolism are discussed. Energy consumption in the brain accounts for ∼20% of the basal metabolic rate and relies almost exclusively on glucose for the production of ATP. However, the brain does not possess substantial reserves of glucose or other fuels. To ensure adequate energetic supply, increases in neuronal activity are matched by increases in cerebral blood flow via a process known as ‘neurovascular coupling’. While the mechanisms for this coupling are not completely resolved, it is generally agreed that astrocytes, with processes that extend to synapses and endfeet that surround blood vessels, mediate at least some of the signal that causes vasodilation. Several studies have shown that either genetic deletion or pharmacologic inhibition of glutamate transport impairs neurovascular coupling. Together these studies strongly suggest that glutamate transport not only coordinates excitatory signaling, but also plays a pivotal role in regulating brain energetics. [ABSTRACT FROM AUTHOR]

Recently, mitochondria have been localized to astrocytic processes where they shape Ca2+ signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca2+ channel blocker), two inhibitors of reversed Na+/Ca2+ exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-L-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA 1. Using the genetic Ca2+ indicator Lck-GCaMP-6S, we observed two types of Ca2+ signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ~2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca2+ through reversed Na+/Ca2+ exchange triggers mitochondrial loss and dramatic increases in Ca2+ signaling in astrocytic processes. [ABSTRACT FROM AUTHOR]

Lafora disease (LD, OMIM 254780 ) is a fatal rare disorder characterized by epilepsy and neurodegeneration. Although in recent years a lot of information has been gained on the molecular basis of the neurodegeneration that accompanies LD, the molecular basis of epilepsy is poorly understood. Here, we present evidence indicating that the homeostasis of glutamate transporter GLT-1 (EAAT2) is compromised in mouse models of LD. Our results indicate that primary astrocytes from LD mice have reduced capacity of glutamate transport, probably because they present a reduction in the levels of the glutamate transporter at the plasma membrane. On the other hand, the overexpression in cellular models of laforin and malin, the two proteins related to LD, results in an accumulation of GLT-1 (EAAT2) at the plasma membrane and in a severe reduction of the ubiquitination of the transporter. All these results suggest that the laforin/malin complex slows down the endocytic recycling of the GLT-1 (EAAT2) transporter. Since, defects in the function of this transporter lead to excitotoxicity and epilepsy, we suggest that the epilepsy that accompanies LD could be due, at least in part, to deficiencies in the function of the GLT-1 (EAAT2) transporter. [ABSTRACT FROM AUTHOR]

At tripartite synapses, astrocytes undergo calcium signaling in response to release of neurotransmitters and this calcium signaling has been proposed to play a critical role in neuron–glia interaction. Recent work has now firmly established that, in addition, neuronal activity also evokes sodium transients in astrocytes, which can be local or global depending on the number of activated synapses and the duration of activity. Furthermore, astrocyte sodium signals can be transmitted to adjacent cells through gap junctions and following release of gliotransmitters. A main pathway for activity-related sodium influx into astrocytes is via high-affinity sodium-dependent glutamate transporters. Astrocyte sodium signals differ in many respects from the well-described glial calcium signals both in terms of their temporal as well as spatial distribution. There are no known buffering systems for sodium ions, nor is there store-mediated release of sodium. Sodium signals thus seem to represent rather direct and unbiased indicators of the site and strength of neuronal inputs. As such they have an immediate influence on the activity of sodium-dependent transporters which may even reverse in response to sodium signaling, as has been shown for GABA transporters for example. Furthermore, recovery from sodium transients through Na + /K + -ATPase requires a measurable amount of ATP, resulting in an activation of glial metabolism. In this review, we present basic principles of sodium regulation and the current state of knowledge concerning the occurrence and properties of activity-related sodium transients in astrocytes. We then discuss different aspects of the relationship between sodium changes in astrocytes and neuro-metabolic coupling, putting forward the idea that indeed sodium might serve as a new type of intracellular ion signal playing an important role in neuron–glia interaction and neuro-metabolic coupling in the healthy and diseased brain. [ABSTRACT FROM AUTHOR]

Developmental cortical malformations (DCMs) are linked with severe epilepsy and are caused by both genetic and environmental insults. DCMs include several neurological diseases, such as focal cortical dysplasia, polymicrogyria, schizencephaly, and others. Human studies have implicated astrocyte reactivity and dysfunction in the pathophysiology of DCMs, but their specific role is unknown. As astrocytes powerfully regulate glutamate neurotransmission, and glutamate levels are known to be increased in human epileptic foci, understanding the role of astrocytes in the pathological sequelae of DCMs is extremely important. Additionally, recent studies examining astrocyte glutamate uptake in DCMs have reported conflicting results, adding confusion to the field. In this study we utilized the freeze lesion (FL) model of DCM, which is known to induce reactive astrocytosis and cause significant changes in astrocyte morphology, proliferation, and distribution. Using whole-cell patch clamp recording from astrocytes, we recorded both UV-uncaging and synaptically evoked glutamate transporter currents (TCs), widely accepted assays of functional glutamate transport by astrocytes. With this approach, we set out to test the hypothesis that astrocyte membrane properties and glutamate transport were disrupted in this model of DCM. Though we found that the developmental maturation of astrocyte membrane resistance was disrupted by FL, glutamate uptake by individual astrocytes was robust throughout FL development. Interestingly, using an immunolabeling approach, we observed spatial and developmental differences in excitatory amino acid transporter (EAAT) expression in FL cortex. Spatially specific differences in EAAT2 (GLT-1) and EAAT1 (GLAST) expression suggest that the relative contribution of each EAAT to astrocytic glutamate uptake may be altered in FL cortex. Lastly, we carefully analyzed the amplitudes and onset times of both synaptically- and UV uncaging-evoked TCs. We found that in the FL cortex, synaptically-evoked, but not UV uncaging-evoked TCs, were larger in amplitude. Additionally, we found that the amount of electrical stimulation required to evoke a synaptic TC was significantly reduced in the FL cortex. Both of these findings are consistent with increased excitatory input to the FL cortex, but not with changes in how individual astrocytes remove glutamate. Taken together, our results demonstrate that the maturation of astrocyte membrane resistance, local distribution of glutamate transporters, and glutamatergic input to the cortex are altered in the FL model, but that single-cell astrocytic glutamate uptake is robust. [ABSTRACT FROM AUTHOR]

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After release from presynaptic nerve terminals, glutamate is quickly removed from the synaptic cleft by a family of five glutamate transporters, the so-called excitatory amino acid transporters (EAAT1-5). EAATs are prototypic members of the growing number of dual-function transport proteins: they are not only glutamate transporters, but also anion channels. Whereas the mechanisms underlying secondary active glutamate transport are well understood at the functional and at the structural level, mechanisms and cellular roles of EAAT anion conduction have remained elusive for many years. Recently, molecular dynamics simulations combined with simulation-guided mutagenesis and experimental analysis identified a novel anion-conducting conformation, which accounts for all experimental data on EAAT anion currents reported so far. We here review recent findings on how EAATs accommodate a transporter and a channel in one single protein. [ABSTRACT FROM AUTHOR]

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl) channels. The EAATs couple the transport of glutamate to the co-transport of three Na ions and one H ion into the cell, and the counter-transport of one K ion out of the cell. The EAAT Cl channel is activated by the binding of glutamate and Na, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, Glt, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl permeation and the mechanisms that underlie their split personality. [ABSTRACT FROM AUTHOR]

The Na+-dependent glutamate transporter GLT-1 (EAAT2) shows selective expression in astrocytes, and neurons induce the expression of GLT-1 in astrocytes. In an unpublished analysis of GLT-1 promoter reporter mice, we identified an evolutionarily conserved domain of 467 nucleotides ~ 8 kb upstream of the GLT-1 translation start site that is required for astrocytic expression. Using in silico approaches, we identified Pax6 as a transcription factor that could contribute to the control of GLT-1 expression by binding within this region. We demonstrated the expression of Pax6 protein in astrocytes in vivo. Lentiviral transduction of astrocytes with exogenous Pax6 increased the expression of enhanced green fluorescent protein ( eGFP) in astrocytes prepared from transgenic mice that use a bacterial artificial chromosome containing a large genomic region surrounding the GLT-1 gene to control expression of eGFP. It also increased GLT-1 protein and GLT-1-mediated uptake, whereas there was no effect on the levels of the other astroglial glutamate transporter, glutamate aspartate transporter (GLAST). Transduction of astrocytes with an sh RNA directed against Pax6 reduced neuron-dependent induction of GLT-1 or eGFP. Finally, we confirmed Pax6 interaction with the predicted DNA-binding site in electrophoretic mobility assays and chromatin immunoprecipitation (Ch IP). Together, these studies show that Pax6 contributes to the regulation of GLT-1 through an interaction with these distal elements and identify a novel role of Pax6 in astrocyte biology. [ABSTRACT FROM AUTHOR]