Hippocampal astrocytic neogenin regulating glutamate uptake, a critical pathway for preventing epileptic response.

Epilepsy, a common neurological disorder, is featured with recurrent seizures. Its underlying pathological mechanisms remain elusive. Here, we provide evidence for loss of neogenin (NEO1), a coreceptor for multiple ligands, including netrins and bone morphological proteins, in the development of epilepsy. NEO1 is reduced in hippocampi from patients with epilepsy based on transcriptome and proteomic analyses. Neo1 knocking out (KO) in mouse brains displays elevated epileptiform spikes and seizure susceptibility. These phenotypes were undetectable in mice, with selectively depleted NEO1 in excitatory (NeuroD6-Cre+) or inhibitory (parvalbumin+) neurons, but present in mice with specific hippocampal astrocytic Neo1 KO. Additionally, neurons in hippocampal dentate gyrus, a vulnerable region in epilepsy, in mice with astrocyte-specific Neo1 KO show reductions in inhibitory synaptic vesicles and the frequency of miniature inhibitory postsynaptic current(mIPSC), but increase of the duration of miniature excitatory postsynaptic current and tonic NMDA receptor currents, suggesting impairments in both GABAergic transmission and extracellular glutamate clearance. Further proteomic and cell biological analyses of cell-surface proteins identified GLAST, a glutamate-aspartate transporter that is marked reduced in Neo1 KO astrocytes and the hippocampus. NEO1 interacts with GLAST and promotes GLAST surface distribution in astrocytes. Expressing NEO1 or GLAST in Neo1 KO astrocytes in the hippocampus abolishes the epileptic phenotype. Taken together, these results uncover an unrecognized pathway of NEO1-GLAST in hippocampal GFAP+ astrocytes, which is critical for GLAST surface distribution and function, and GABAergic transmission, unveiling NEO1 as a valuable therapeutic target to protect the brain from epilepsy.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Genetic inhibition of collapsin response mediator protein-2 phosphorylation ameliorates retinal ganglion cell death in normal-tension glaucoma models.

Glaucoma is a neurodegenerative disorder caused by the death of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) is a cause of glaucoma. However, glaucoma often develops with normal IOP and is known as normal-tension glaucoma (NTG). Glutamate neurotoxicity is considered as one of the significant causes of NTG, resulting in excessive stimulation of retinal neurons via the N-methyl-D-aspartate (NMDA) receptors. The present study examined the phosphorylation of collapsin response mediator protein-2 (CRMP2), a protein that is abundantly expressed in neurons and involved in their development. In two mouse models, NMDA-injection and glutamate/aspartate transporter (GLAST) mutant, CRMP2 phosphorylation at the cyclin-dependent kinase-5 (Cdk5) site was elevated in RGCs. We confirmed that the decrease in the number of RGCs and thickness of the inner retinal layer (IRL) could be suppressed after NMDA administration in CRMP2KI/KI mice with genetically inhibited CRMP2 phosphorylation. Next, we investigated GLAST heterozygotes (GLAST+/-) with CRMP2KI/KI (GLAST+/-;CRMP2KI/KI) and GLAST knockout (GLAST-/-) mice with CRMP2KI/KI (GLAST-/-;CRMP2KI/KI) mice and compared them with GLAST+/- and GLAST-/- mice. pCRMP2 (S522) inhibition significantly reduced RGC loss and IRL thinning. These results suggest that the inhibition of CRMP2 phosphorylation could be a novel strategy for treating NTG.

Astrocyte Responses Influence Local Effects of Whole-Brain Magnetic Stimulation in Parkinsonian Rats.

BACKGROUND: Excessive glutamatergic transmission in the striatum is implicated in Parkinson's disease (PD) progression. Astrocytes maintain glutamate homeostasis, protecting from excitotoxicity through the glutamate-aspartate transporter (GLAST), whose alterations have been reported in PD. Noninvasive brain stimulation using intermittent theta-burst stimulation (iTBS) acts on striatal neurons and glia, inducing neuromodulatory effects and functional recovery in experimental parkinsonism. OBJECTIVE: Because PD is associated with altered astrocyte function, we hypothesized that acute iTBS, known to rescue striatal glutamatergic transmission, exerts regional- and cell-specific effects through modulation of glial functions. METHODS: 6-Hydroxydopamine-lesioned rats were exposed to acute iTBS, and the areas predicted to be more responsive by a biophysical, hyper-realistic computational model that faithfully reconstructs the experimental setting were analyzed. The effects of iTBS on glial cells and motor behavior were evaluated by molecular and morphological analyses, and CatWalk and Stepping test, respectively. RESULTS: As predicted by the model, the hippocampus, cerebellum, and striatum displayed a marked c-FOS activation after iTBS, with the striatum showing specific morphological and molecular changes in the astrocytes, decreased phospho-CREB levels, and recovery of GLAST. Striatal-dependent motor performances were also significantly improved. CONCLUSION: These data uncover an unknown iTBS effect on astrocytes, advancing the understanding of the complex mechanisms involved in TMS-mediated functional recovery. Data on numerical dosimetry, obtained with a degree of anatomical details never before considered and validated by the biological findings, provide a framework to predict the electric-field induced in different specific brain areas and associate it with functional and molecular changes. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

In Vitro Functional Characterization of Type-I Taste Bud Cells as Monocytes/Macrophages-like Which Secrete Proinflammatory Cytokines.

The sense of taste determines the choice of nutrients and food intake and, consequently, influences feeding behaviors. The taste papillae are primarily composed of three types of taste bud cells (TBC), i.e., type I, type II, and type III. The type I TBC, expressing GLAST (glutamate--aspartate transporter), have been termed as glial-like cells. We hypothesized that these cells could play a role in taste bud immunity as glial cells do in the brain. We purified type I TBC, expressing F4/80, a specific marker of macrophages, from mouse fungiform taste papillae. The purified cells also express CD11b, CD11c, and CD64, generally expressed by glial cells and macrophages. We further assessed whether mouse type I TBC can be polarized toward M1 or M2 macrophages in inflammatory states like lipopolysaccharide (LPS)-triggered inflammation or obesity, known to be associated with low-grade inflammation. Indeed, LPS-treatment and obesity state increased TNFα, IL-1ß, and IL-6 expression, both at mRNA and protein levels, in type I TBC. Conversely, purified type I TBC treated with IL-4 showed a significant increase in arginase 1 and IL-4. These findings provide evidence that type I gustatory cells share many features with macrophages and may be involved in oral inflammation.

Diverse cellular origins of adult blood vascular endothelial cells.

During embryonic stages, vascular endothelial cells (ECs) originate from the mesoderm, at specific extraembryonic and embryonic regions, through a process called vasculogenesis. In the adult, EC renewal/replacement mostly depend on local resident ECs or endothelial progenitor cells (EPCs). Nevertheless, contribution from circulating ECs/EPCs was also reported. In addition, cells lacking from EC/EPC markers with in vitro extended plasticity were shown to originate endothelial-like cells (ELCs). Most of these cells consist of mesenchymal stromal progenitors, which would eventually get mobilized from the bone marrow after injury. Based on that, current knowledge on different mouse and human bone marrow stromal cell (BM-SC) subpopulations, able to contribute with mesenchymal stromal/stem cells (MSCs), is herein reviewed. Such analyses underline an unexpected heterogeneity among sinusoidal LepR+ stromal/CAR cells. For instance, in a recent report a subgroup of LepR+ stromal/CAR progenitors, which express GLAST and is traced in Wnt1Cre;R26RTom mice, was found to contribute with ELCs in vivo. These GLAST â€‹+ â€‹Wnt1+ BM-SCs were shown to get mobilized to the peripheral blood and to contribute with liver regeneration. Other sources of ELCs, such as adipose, neural and dental pulp tissues, were also published. Finally, mechanisms likely involved in the enhanced cellular plasticity properties of bone marrow/adipose tissue stromal cells, able to originate ELCs, are assessed. In the future, strategies to analyze the in vivo expression profile of stromal cells, with MSC properties, in combination with screening of active genomic regions at the single cell-level, during early postnatal development and/or after injury, will likely help understanding properties of these ELC sources.

The expression and function of glutamate aspartate transporters in Bergmann glia are decreased in neuronal nitric oxide synthase-knockout mice during postnatal development.

Bergmann glia (BG) predominantly use glutamate/aspartate transporters (GLAST) for glutamate uptake in the cerebellum. Recently, nitric oxide (NO) treatment has been shown to upregulate GLAST function and increase glutamate uptake in vitro. We previously discovered that neuronal nitric oxide synthase knockout (nNOS-/- ) mice displayed structural and functional neuronal abnormalities in the cerebellum during development, in addition to previously reported motor deficits. Although these developmental deficits have been identified in the nNOS-/- cerebellum, it is unknown whether BG morphology and GLAST expression are also affected in the absence of nNOS in vivo. This study is the first to characterize BG morphology and GLAST expression during development in nNOS-/- mice using immunohistochemistry and western blotting across postnatal development. Results showed that BG in nNOS-/- mice exhibited abnormal morphology and decreased GLAST expression compared with wildtype (WT) mice across postnatal development. Treating ex vivo WT cerebellar slices with the NOS inhibitor L-NAME decreased GLAST expression while treating nNOS-/- slices with the slow-release NO-donor NOC-18 increased GLAST expression when compared with their respective controls. In addition, treating primary BG isolated from WT mice with the selective nNOS inhibitor 7N decreased the membrane expression of GLAST and influx of Ca2+ /Na+ , while treating nNOS-/- BG with SNAP increased the membrane expression of GLAST and Ca2+ /Na+ influx. Moreover, the effects of SNAP on GLAST expression and Ca2+ /Na+ influx in nNOS-/- BG were significantly reduced by a PKG inhibitor. Together, these results reveal a novel role for nNOS/NO signaling in BG development, regulated by a PKG-mediated mechanism.

Rapid Regulation of Glutamate Transport: Where Do We Go from Here?

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.

Overlapping distributions of mammalian types I, II, and III taste cell markers in chicken taste buds.

Mammalian taste buds comprise types I, II, and III taste cells, with each type having specific characteristics: glia-like supporting cells (type I), taste receptor cells (type II), and presynaptic cells (type III). In this study, to characterize the peripheral taste-sensing systems in chickens, we analyzed the distributions of the mammalian types I, II, and III taste cell markers in chicken taste buds: glutamate-aspartate transporter (GLAST) for type I; taste receptor type 1 members 1 and 3 (T1R1 and T1R3), taste receptor type 2 member 7 (T2R7), and α-gustducin for type II; and synaptosomal protein 25 (SNAP25) and neural cell adhesion molecule (NCAM) for type III. We found that most GLAST+ taste cells expressed α-gustducin and SNAP25 and that high percentages of T1R3+ or α-gustducin+ taste cells expressed SNAP25 and NCAM. These results demonstrated a unique subset of chicken taste cells expressing multiple taste cell type marker proteins. Taken together, these results provide new insights into the taste-sensing mechanisms in vertebrate taste buds.

Reduction of glutamate neurotoxicity: A novel therapeutic approach for Niemann-Pick disease, type C1.

Niemann-Pick disease, type C1 is a progressive, lethal, neurodegenerative disorder due to endolysosomal storage of unesterified cholesterol. Cerebellar ataxia, as a result of progressive loss of cerebellar Purkinje neurons, is a major symptom of Nieman-Pick disease, type C1. Comparing single cell RNAseq data from control (Npc1+/+) and mutant (Npc1-/-) mice, we observed significantly decreased expression of Slc1a3 in Npc1-/- astrocytes. Slc1a3 encodes a glutamate transporter (GLAST, EAAT1) which functions to decrease glutamate concentrations in the post synaptic space after neuronal firing. Glutamate is an excitatory neurotransmitter and elevated extracellular levels of glutamate can be neurotoxic. Impaired EAAT1 function underlies type-6 episodic ataxia, a rare disorder with progressive cerebellar dysfunction, thus suggesting that impaired glutamate uptake in Niemann-Pick disease, type C1 could contribute to disease progression. We now show that decreased expression of Slc1a3 in Npc1-/- mice has functional consequences that include decreased surface protein expression and decreased glutamate uptake by Npc1-/- astrocytes. To test whether glutamate neurotoxicity plays a role in Niemann-Pick disease, type C1 progression, we treated NPC1 deficient mice with ceftriaxone and riluzole. Ceftriaxone is a ß-lactam antibiotic that is known to upregulate the expression of Slc1a2, an alternative glial glutamate transporter. Although ceftriaxone increased Slc1a2 expression, we did not observe a treatment effect in NPC1 mutant mice. Riluzole is a glutamate receptor antagonist that inhibits postsynaptic glutamate receptor signaling and reduces the release of glutamate. We found that treatment with riluzole increased median survival in Npc1-/- by 12%. Given that riluzole is an approved drug for the treatment of amyotrophic lateral sclerosis, repurposing of this drug may provide a novel therapeutic approach to decrease disease progression in Niemann-Pick disease type, C1 patients.

Circadian vulnerability of cisplatin-induced ototoxicity in the cochlea.

The chemotherapeutic agent cisplatin is renowned for its ototoxic effects. While hair cells in the cochlea are established targets of cisplatin, less is known regarding the afferent synapse, which is an essential component in the faithful temporal transmission of sound. The glutamate aspartate transporter (GLAST) shields the auditory synapse from excessive glutamate release, and its loss of function increases the vulnerability to noise, salicylate, and aminoglycosides. Until now, the involvement of GLAST in cisplatin-mediated ototoxicity remains unknown. Here, we test in mice lacking GLAST the effects of a low-dose cisplatin known not to cause any detectable change in hearing thresholds. When administered at nighttime, a mild hearing loss in GLAST KO mice was found but not at daytime, revealing a potential circadian regulation of the vulnerability to cisplatin-mediated ototoxicity. We show that the auditory synapse of GLAST KO mice is more vulnerable to cisplatin administration during the active phase (nighttime) when compared to WT mice and treatment during the inactive phase (daytime). This effect was not related to the abundance of platinum compounds in the cochlea, rather cisplatin had a dose-dependent impact on cochlear clock rhythms only after treatment at nighttime suggesting that cisplatin can modulate the molecular clock. Our findings suggest that the current protocols of cisplatin administration in humans during daytime may cause a yet undetectable damage to the auditory synapse, more so in already damaged ears, and severely impact auditory sensitivity in cancer survivors.

LncRNA-UCA1 inhibits the astrocyte activation in the temporal lobe epilepsy via regulating the JAK/STAT signaling pathway.

This article aimed to reveal the mechanism of long noncoding RNA (lncRNA) urothelial cancer-associated 1 (UCA1) regulated astrocyte activation in temporal lobe epilepsy (TLE) rats via mediating the activation of the JAK/STAT signaling pathway. A model of TLE was established based on rats via kainic acid (KA) injection. All rats were divided into the Sham group (without any treatments), KA group, normal control (NC; injection with empty vector) + KA group, and UCA1 + KA group. The Morris water maze was used to test the learning and memory ability of rats, and the expression of UCA1 in the hippocampus was determined by quantitative real time polymerase chain reaction (qRT-PCR). Surviving neurons were counted by Nissl staining, and expression levels of glial cells glial fibrillary acidic protein (GFAP), p-JAK1, and p-STAT3 and glutamate/aspartate transporter (GLAST) were analyzed by immunofluorescence and Western blot analysis. A rat model of TLE was established by intraperitoneal injection of KA. qRT-PCR and fluorescence analyses showed that UCA1 inhibited astrocyte activation in the hippocampus of epileptic rats. Meanwhile, the Morris water maze analysis indicated that UCA1 improved the learning and memory in epilepsy rats. Moreover, the Nissl staining showed that UCA1 might have a protective effect on neuronal injury induced by KA injection. Furthermore, the immunofluorescence and Western blot analysis revealed that the overexpression of UCA1 inhibited KA-induced abnormal elevation of GLAST, astrocyte activation of the JAK/STAT signaling pathway, as well as hippocampus of epilepsy rats. UCA1 inhibited hippocampal astrocyte activation and JAK/STAT/GLAST expression in TLE rats and improved the adverse reactions caused by epilepsy.

GLAST Activity is Modified by Acute Manganese Exposure in Bergmann Glial Cells.

Glutamate is the major excitatory amino acid neurotransmitter in the vertebrate brain. It exerts its actions through the activation of specific plasma membrane receptors expressed in neurons and glial cells. Overactivation of glutamate receptors results in neuronal death, known as excitotoxicity. A family of sodium-dependent glutamate transporters enriched in glial cells are responsible of the vast majority of the removal of this amino acid form the synaptic cleft. Therefore, a precise and exquisite regulation of these proteins is required not only for a proper glutamatergic transmission but also for the prevention of an excitotoxic insult. Manganese is a trace element essential as a cofactor for several enzymatic systems, although in high concentrations is involved in the disruption of brain glutamate homeostasis. The molecular mechanisms associated to manganese neurotoxicity have been focused on mitochondrial function, although energy depletion severely compromises the glutamate uptake process. In this context, in this contribution we analyze the effect of manganese exposure in glial glutamate transporters function. To this end, we used the well-established model of chick cerebellar Bergmann glia cultures. A time and dose dependent modulation of [3H]-D-aspartate uptake was found. An increase in the transporter catalytic efficiency, most probably linked to a discrete increase in the affinity of the transporter was detected upon manganese exposure. Interestingly, glucose uptake was reduced by this metal. These results favor the notion of a direct effect of manganese on glial cells, this in turn alters their coupling with neurons and might lead to changes in glutamatergic transmission.

Early Postnatal Exposure to a Low Dose of Nanoparticulate Silver Induces Alterations in Glutamate Transporters in Brain of Immature Rats.

Due to strong antimicrobial properties, silver nanoparticles (AgNPs) are used in a wide range of medical and consumer products, including those dedicated for infants and children. While AgNPs are known to exert neurotoxic effects, current knowledge concerning their impact on the developing brain is scarce. During investigations of mechanisms of neurotoxicity in immature rats, we studied the influence of AgNPs on glutamate transporter systems which are involved in regulation of extracellular concentration of glutamate, an excitotoxic amino acid, and compared it with positive control-Ag citrate. We identified significant deposition of AgNPs in brain tissue of exposed rats over the post-exposure time. Ultrastructural alterations in endoplasmic reticulum (ER) and Golgi complexes were observed in neurons of AgNP-exposed rats, which are characteristics of ER stress. These changes presumably underlie substantial long-lasting downregulation of neuronal glutamate transporter EAAC1, which was noted in AgNP-exposed rats. Conversely, the expression of astroglial glutamate transporters GLT-1 and GLAST was not affected by exposure to AgNPs, but the activity of the transporters was diminished. These results indicate that even low doses of AgNPs administered during an early stage of life create a substantial risk for health of immature organisms. Hence, the safety of AgNP-containing products for infants and children should be carefully considered.

Calcineurin Controls Expression of EAAT1/GLAST in Mouse and Human Cultured Astrocytes through Dynamic Regulation of Protein Synthesis and Degradation.

Alterations in the expression of glutamate/aspartate transporter (GLAST) have been associated with several neuropathological conditions including Alzheimer's disease and epilepsy. However, the mechanisms by which GLAST expression is altered are poorly understood. Here we used a combination of pharmacological and genetic approaches coupled with quantitative PCR and Western blot to investigate the mechanism of the regulation of GLAST expression by a Ca2+/calmodulin-activated phosphatase calcineurin (CaN). We show that treatment of cultured hippocampal mouse and fetal human astrocytes with a CaN inhibitor FK506 resulted in a dynamic modulation of GLAST protein expression, being downregulated after 24-48 h, but upregulated after 7 days of continuous FK506 (200 nM) treatment. Protein synthesis, as assessed by puromycin incorporation in neo-synthesized polypeptides, was inhibited already after 1 h of FK506 treatment, while the use of a proteasome inhibitor MG132 (1 µM) shows that GLAST protein degradation was only suppressed after 7 days of FK506 treatment. In astrocytes with constitutive genetic ablation of CaN both protein synthesis and degradation were significantly inhibited. Taken together, our data suggest that, in cultured astrocytes, CaN controls GLAST expression at a posttranscriptional level through regulation of GLAST protein synthesis and degradation.

Serial exposure to ethanol drinking and methamphetamine enhances glutamate excitotoxicity.

A significant comorbidity exists between alcohol and methamphetamine (Meth) abuse but the neurochemical consequences of this co-abuse are unknown. Alcohol and Meth independently and differentially affect glutamatergic transmission but the unique effects of their serial exposure on glutamate signaling in mediating damage to dopamine neurons are unknown. Sprague-Dawley rats had intermittent voluntary access to 10% ethanol (EtOH) every other day and water over 28 days and were then administered a binge injection regimen of Meth or saline. EtOH drinking decreased the glutamate aspartate transporter and increased basal extracellular concentrations of glutamate within the striatum when measured after the last day of drinking. Ceftriaxone is known to increase the expression and/or activity of glutamate transporters in the brain and prevented both the decreases in glutamate aspartate transporter and the increases in basal extracellular glutamate when administered during EtOH drinking. EtOH drinking also exacerbated the acute increases in extracellular glutamate observed upon Meth exposure, the subsequent increases in spectrin proteolysis, and the long-term decreases in dopamine content in the striatum, all of which were attenuated by ceftriaxone administration during EtOH drinking only. These results implicate EtOH-induced increases in extracellular glutamate and corresponding decreases in glutamate uptake as mechanisms that contribute to the vulnerability produced by EtOH drinking and the unique neurotoxicity observed after serial exposure to Meth that is not observed with either drug alone. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Altered function of the glutamate-aspartate transporter GLAST, a potential therapeutic target in glioblastoma.

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST-depleted glioma. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.

Excitatory Amino Acid Transporters in Physiology and Disorders of the Central Nervous System.

Excitatory amino acid transporters (EAATs) encompass a class of five transporters with distinct expression in neurons and glia of the central nervous system (CNS). EAATs are mainly recognized for their role in uptake of the amino acid glutamate, the major excitatory neurotransmitter. EAATs-mediated clearance of glutamate released by neurons is vital to maintain proper glutamatergic signalling and to prevent toxic accumulation of this amino acid in the extracellular space. In addition, some EAATs also act as chloride channels or mediate the uptake of cysteine, required to produce the reactive oxygen speciesscavenger glutathione. Given their central role in glutamate homeostasis in the brain, as well as their additional activities, it comes as no surprise that EAAT dysfunctions have been implicated in numerous acute or chronic diseases of the CNS, including ischemic stroke and epilepsy, cerebellar ataxias, amyotrophic lateral sclerosis, Alzheimer's disease and Huntington's disease. Here we review the studies in cellular and animal models, as well as in humans that highlight the roles of EAATs in the pathogenesis of these devastating disorders. We also discuss the mechanisms regulating EAATs expression and intracellular trafficking and new exciting possibilities to modulate EAATs and to provide neuroprotection in course of pathologies affecting the CNS.

Na+, K+-ATPase inhibition induces neuronal cell death in rat hippocampal slice cultures: Association with GLAST and glial cell abnormalities.

Na+, K+-ATPase is a highly expressed membrane protein. Dysfunction of Na+, K+-ATPase has been implicated in the pathophysiology of several neurodegenerative and psychiatric disorders, however, the underlying mechanism of neuronal cell death resulting from Na+, K+-ATPase dysfunction is poorly understood. Here, we investigated the mechanism of neurotoxicity due to Na+, K+-ATPase inhibition using rat organotypic hippocampal slice cultures. Treatment with ouabain, a Na+, K+-ATPase inhibitor, increased the ratio of propidium iodide-positive cells among NeuN-positive cells in the hippocampal CA1 region, which was prevented by MK-801 and d-AP5, specific blockers of the N-methyl-d-aspartate (NMDA) receptor. EGTA, a Ca2+-chelating agent, also protected neurons from ouabain-induced injury. We observed that astrocytes expressed the glutamate aspartate transporter (GLAST), and ouabain changed the immunoreactive area of GFAP-positive astrocytes as well as GLAST. We also observed that ouabain increased the number of Iba1-positive microglial cells in a time-dependent manner. Furthermore, lithium carbonate, a mood-stabilizing drug, protected hippocampal neurons and reduced disturbances of astrocytes and microglia after ouabain treatment. Notably, lithium carbonate improved ouabain-induced decreases in GLAST intensity in astrocytes. These results suggest that glial cell abnormalities resulting in excessive extracellular concentrations of glutamate contribute to neurotoxicity due to Na+, K+-ATPase dysfunction in the hippocampal CA1 region.

Neural Progenitor Cells in Cerebral Cortex of Epilepsy Patients do not Originate from Astrocytes Expressing GLAST.

Adult neurogenesis in human brain is known to occur in the hippocampus, the subventricular zone, and the striatum. Neural progenitor cells (NPCs) were reported in the cortex of epilepsy patients; however, their identity is not known. Since astrocytes were proposed as the source of neural progenitors in both healthy and diseased brain, we tested the hypothesis that NPCs in the epileptic cortex originate from reactive, alternatively, de-differentiated astrocytes that express glutamate aspartate transporter (GLAST). We assessed the capacity to form neurospheres and the differentiation potential of cells dissociated from fresh cortical tissue from patients who underwent surgical treatment for pharmacologically intractable epilepsy. Neurospheres were generated from 57% of cases (8/14). Upon differentiation, the neurosphere cells gave rise to neurons, oligodendrocytes, and astrocytes. Sorting of dissociated cells showed that only cells negative for GLAST formed neurospheres. In conclusion, we show that cells with neural stem cell properties are present in brain cortex of epilepsy patients, and that these cells are not GLAST-positive astrocytes.