Milestone Review: Excitatory amino acid transporters - Beyond their expected function.

Glutamate is the major excitatory neurotransmitter in the vertebrate brain, it is critically involved in the function and dysfunction of the central nervous system. The molecular cloning of its ionotropic receptors in the last decade of the past century increased exponentially the interest in this neurotransmitter system. Since then, a plethora of knowledge of the structure, function, and regulation of its receptors and transporters has advanced our understanding of glutamate-mediated neurochemical transactions. Moreover, the characterization of glial glutamate receptors together with the compulsory participation of surrounding astrocytes in glutamate turnover and in the known metabolic coupling with neurons has supported what is now known as the tripartite synapses. The molecular characterization of the various glutamate transporters has also been fundamental for the involvement of glial cells in glutamatergic synapses. Using radial glial cultures, over the years, we have demonstrated an alternative glutamate-mediated signaling system triggered by sodium-dependent glutamate transporters. A detailed account of these findings and the signaling through other glutamate transporters are presented here. The role of this signaling system in the context of glutamatergic transmission is discussed as well as the future directions in the field.

Cooperative and competitive regulation of the astrocytic transcriptome by neurons and endothelial cells: Impact on astrocyte maturation.

Astrocytes have essential roles in central nervous system (CNS) health and disease. During development, immature astrocytes show complex interactions with neurons, endothelial cells, and other glial cell types. Our work and that of others have shown that these interactions are important for astrocytic maturation. However, whether and how these cells work together to control this process remains poorly understood. Here, we test the hypothesis that cooperative interactions of astrocytes with neurons and endothelial cells promote astrocytic maturation. Astrocytes were cultured alone, with neurons, endothelial cells, or a combination of both. This was followed by astrocyte sorting, RNA sequencing, and bioinformatic analysis to detect transcriptional changes. Across culture configurations, 7302 genes were differentially expressed by 4 or more fold and organized into 8 groups that demonstrate cooperative and antagonist effects of neurons and endothelia on astrocytes. We also discovered that neurons and endothelial cells caused splicing of 200 and 781 mRNAs, respectively. Changes in gene expression were validated using quantitative PCR, western blot (WB), and immunofluorescence analysis. We found that the transcriptomic data from the three-culture configurations correlated with protein expression of three representative targets (FAM107A, GAT3, and GLT1) in vivo. Alternative splicing results also correlated with cortical tissue isoform representation of a target (Fibronectin 1) at different developmental stages. By comparing our results to published transcriptomes of immature and mature astrocytes, we found that neurons or endothelia shift the astrocytic transcriptome toward a mature state and that the presence of both cell types has a greater effect on maturation than either cell alone. These results increase our understanding of cellular interactions/pathways that contribute to astrocytic maturation. They also provide insight into how alterations to neurons and/or endothelial cells may alter astrocytes with implications for astrocytic changes in CNS disorders and diseases.

Brain endothelial cells induce astrocytic expression of the glutamate transporter GLT-1 by a Notch-dependent mechanism.

Neuron-secreted factors induce astrocytic expression of the glutamate transporter, GLT-1 (excitatory amino acid transporter 2). In addition to their elaborate anatomic relationships with neurons, astrocytes also have processes that extend to and envelop the vasculature. Although previous studies have demonstrated that brain endothelia contribute to astrocyte differentiation and maturation, the effects of brain endothelia on astrocytic expression of GLT-1 have not been examined. In this study, we tested the hypothesis that endothelia induce expression of GLT-1 by co-culturing astrocytes from mice that utilize non-coding elements of the GLT-1 gene to control expression of reporter proteins with the mouse endothelial cell line, bEND.3. We found that endothelia increased steady state levels of reporter and GLT-1 mRNA/protein. Co-culturing with primary rat brain endothelia also increases reporter protein, GLT-1 protein, and GLT-1-mediated glutamate uptake. The Janus kinase/signal transducer and activator of transcription 3, bone morphogenic protein/transforming growth factor ß, and nitric oxide pathways have been implicated in endothelia-to-astrocyte signaling; we provide multiple lines of evidence that none of these pathways mediate the effects of endothelia on astrocytic GLT-1 expression. Using transwells with a semi-permeable membrane, we demonstrate that the effects of the bEND.3 cell line are dependent upon contact. Notch has also been implicated in endothelia-astrocyte signaling in vitro and in vivo. The first step of Notch signaling requires cleavage of Notch intracellular domain by γ-secretase. We demonstrate that the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester blocks endothelia-induced increases in GLT-1. We show that the levels of Notch intracellular domain are higher in nuclei of astrocytes co-cultured with endothelia, an effect also blocked by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Finally, infection of co-cultures with shRNA directed against recombination signal binding protein for immunoglobulin kappa J, a Notch effector, also reduces endothelia-dependent increases in enhanced green fluorescent protein and GLT-1. Together, these studies support a novel role for Notch in endothelia-dependent induction of GLT-1 expression. Cover Image for this issue: doi. 10.1111/jnc.13825.

Methylphenidate Increases Glutamate Uptake in Bergmann Glial Cells.

Glutamate, the main excitatory transmitter in the vertebrate brain, exerts its actions through the activation of specific membrane receptors present in neurons and glial cells. Over-stimulation of glutamate receptors results in neuronal death, phenomena known as excitotoxicity. A family of glutamate uptake systems, mainly expressed in glial cells, removes the amino acid from the synaptic cleft preventing an excessive glutamatergic stimulation and thus neuronal damage. Autism spectrum disorders comprise a group of syndromes characterized by impaired social interactions and anxiety. One or the most common drugs prescribed to treat these disorders is Methylphenidate, known to increase dopamine extracellular levels, although it is not clear if its sedative effects are related to a plausible regulation of the glutamatergic tone via the regulation of the glial glutamate uptake systems. To gain insight into this possibility, we used the well-established model system of cultured chick cerebellum Bergmann glia cells. A time and dose-dependent increase in the activity and protein levels of glutamate transporters was detected upon Methylphenidate exposure. Interestingly, this increase is the result of an augmentation of both the synthesis as well as the insertion of these protein complexes in the plasma membrane. These results favour the notion that glial cells are Methylphenidate targets, and that by these means could regulate dopamine turnover.

Glutamate-dependent translational control through ribosomal protein S6 phosphorylation in cultured bergmann glial cells.

Glutamate (Glu) the main excitatory neurotransmitter of the central nervous system regulates gene expression at different levels through the activation of specific membrane receptors and transporters expressed in neurons and glia cells. A membrane to nucleus signaling cascade triggered by this neurotransmitter has been described in cultured cerebellar Bergmann glia cells isolated from chick embryos. Furthermore, it has also been described that Glu receptors activation is linked to a modulation of [(35)S]-methionine incorporation into newly synthesized polypeptides. In order to gain insight into the signal transduction cascades that participate in this effect, in the present study we characterized the phosphorylation of a critical component of the translational machinery, namely the ribosomal protein S6. The phosphorylation sites in rpS6 have been mapped to five clustered residues, Ser235, Ser236, Ser240, Ser244 and Ser247. Nevertheless, Ser236 phosphorylation is the primary phosphorylation site. The kinases responsible of this modification are p70(S6K) and p90(RSK). rpS6 phosphorylation increases the affinity of 40s subunit for mRNAs and thus facilitates translational initiation. Glutamate exposure of cultured cerebellar Bergmann glia cells results in a time- and dose-dependent increase in rpS6 phosphorylation. This effect is mainly observed at cytoplasm, and involves the phosphoinositol-3 kinase/protein kinase B pathway. Our results favor the notion of a continuous neuronal signaling to glia cells that regulates the proteome of these cells not only at the transcriptional level but also at the level of protein synthesis.

Glutamatergic Transmission: A Matter of Three.

Glutamatergic transmission in the vertebrate brain requires the involvement of glia cells, in a continuous molecular dialogue. Glial glutamate receptors and transporters are key molecules that sense synaptic activity and by these means modify their physiology in the short and long term. Posttranslational modifications that regulate protein-protein interactions and modulate transmitter removal are triggered in glial cells by neuronal released glutamate. Moreover, glutamate signaling cascades in these cells are linked to transcriptional and translational control and are critically involved in the control of the so-called glutamate/glutamine shuttle and by these means in glutamatergic neurotransmission. In this contribution, we summarize our current understanding of the biochemical consequences of glutamate synaptic activity in their surrounding partners and dissect the molecular mechanisms that allow neurons to take control of glia physiology to ensure proper glutamate-mediated neuronal communication.

Activation of sodium-dependent glutamate transporters regulates the morphological aspects of oligodendrocyte maturation via signaling through calcium/calmodulin-dependent kinase IIß's actin-binding/-stabilizing domain.

Signaling via the major excitatory amino acid glutamate has been implicated in the regulation of various aspects of the biology of oligodendrocytes, the myelinating cells of the central nervous system (CNS). In this respect, cells of the oligodendrocyte lineage have been described to express a variety of glutamate-responsive transmembrane proteins including sodium-dependent glutamate transporters. The latter have been well characterized to mediate glutamate clearance from the extracellular space. However, there is increasing evidence that they also mediate glutamate-induced intracellular signaling events. Our data presented here show that the activation of oligodendrocyte expressed sodium-dependent glutamate transporters, in particular GLT-1 and GLAST, promotes the morphological aspects of oligodendrocyte maturation. This effect was found to be associated with a transient increase in intracellular calcium levels and a transient phosphorylation event at the serine (S)(371) site of the calcium sensor calcium/calmodulin-dependent kinase type IIß (CaMKIIß). The potential regulatory S(371) site is located within CaMKIIß's previously defined actin-binding/-stabilizing domain, and phosphorylation events within this domain were identified in our studies as a requirement for sodium-dependent glutamate transporter-mediated promotion of oligodendrocyte maturation. Furthermore, our data provide good evidence for a role of these phosphorylation events in mediating detachment of CaMKIIß from filamentous (F)-actin, and hence allowing a remodeling of the oligodendrocyte's actin cytoskeleton. Taken together with our recent findings, which demonstrated a crucial role of CaMKIIß in regulating CNS myelination in vivo, our data strongly suggest that a sodium-dependent glutamate transporter-CaMKIIß-actin cytoskeleton axis plays an important role in the regulation of oligodendrocyte maturation and CNS myelination.

GLAST/EAAT1-induced glutamine release via SNAT3 in Bergmann glial cells: evidence of a functional and physical coupling.

Glutamate, the major excitatory transmitter in the vertebrate brain, is removed from the synaptic cleft by a family of sodium-dependent glutamate transporters profusely expressed in glial cells. Once internalized, it is metabolized by glutamine synthetase to glutamine and released to the synaptic space through sodium-dependent neutral amino acid carriers of the N System (SNAT3/slc38a3/SN1, SNAT5/slc38a5/SN2). Glutamine is then taken up by neurons completing the so-called glutamate/glutamine shuttle. Despite of the fact that this coupling was described decades ago, it is only recently that the biochemical framework of this shuttle has begun to be elucidated. Using the established model of cultured cerebellar Bergmann glia cells, we sought to characterize the functional and physical coupling of glutamate uptake and glutamine release. A time-dependent Na⁺-dependent glutamate/aspartate transporter/EAAT1-induced System N-mediated glutamine release could be demonstrated. Furthermore, D-aspartate, a specific glutamate transporter ligand, was capable of enhancing the co-immunoprecipitation of Na⁺-dependent glutamate/aspartate transporter and Na⁺-dependent neutral amino acid transporter 3, whereas glutamine tended to reduce this association. Our results suggest that glial cells surrounding glutamatergic synapses may act as sensors of neuron-derived glutamate through their contribution to the neurotransmitter turnover.

Glutamate-dependent translational control in cultured Bergmann glia cells: eIF2α phosphorylation.

Glutamate (Glu), the major excitatory amino acid, activates a wide variety of signal transduction cascades. Synaptic plasticity relies on activity-dependent differential protein expression. Glu receptors have been critically involved in long-term synaptic changes, although recent findings suggest that Na(+)-dependent Glu transporters participate in Glu-induced signalling. Within the cerebellum, Bergmann glia cells are in close proximity to glutamatergic synapses and through their receptors and transporters, sense and respond to neuronal glutamatergic activity. Translational control represents the fine-tuning stage of protein expression regulation and Glu modulates this event in glial cells. In this context, we decided to explore the involvement of Glu receptors and transporters in the regulation of the initiation phase of protein synthesis. To this end, Bergmann glia cells were exposed to glutamatergic ligands and the serine 51-phosphorylation pattern of the main regulator of the initiation phase of translation, namely the α subunit of eukaryotic initiation factor 2 (eIF2α), determined. A time and dose-dependent increase in eIF2α phosphorylation was detected. The signalling cascade included Ca(2+) influx, activation of the Ca(2+)/calmodulin-dependent protein kinase II and protein kinase C. These results provide an insight into the molecular targets of the Glu effects at the translational level and strengthen the notion of the critical involvement of glia cells in glutamatergic synaptic function.

Evaluation of gliovascular functions of AQP4 readthrough isoforms.

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Reciprocal communication between astrocytes and endothelial cells is required for astrocytic glutamate transporter 1 (GLT-1) expression.

Astrocytes have diverse functions that are supported by their anatomic localization between neurons and blood vessels. One of these functions is the clearance of extracellular glutamate. Astrocytes clear glutamate using two Na+-dependent glutamate transporters, GLT-1 (also called EAAT2) and GLAST (also called EAAT1). GLT-1 expression increases during synaptogenesis and is a marker of astrocyte maturation. Over 20 years ago, several groups demonstrated that astrocytes in culture express little or no GLT-1 and that neurons induce expression. We recently demonstrated that co-culturing endothelia with mouse astrocytes also induced expression of GLT-1 and GLAST. These increases were blocked by an inhibitor of γ-secretase. This and other observations are consistent with the hypothesis that Notch signaling is required, but the ligands involved were not identified. In the present study, we used rat astrocyte cultures to further define the mechanisms by which endothelia induce expression of GLT-1 and GLAST. We found that co-cultures of astrocytes and endothelia express higher levels of GLT-1 and GLAST protein and mRNA. That endothelia activate Hes5, a transcription factor target of Notch, in astrocytes. Using recombinant Notch ligands, anti-Notch ligand neutralizing antibodies, and shRNAs, we provide evidence that both Dll1 and Dll4 contribute to endothelia-dependent regulation of GLT-1. We also provide evidence that astrocytes secrete a factor(s) that induces expression of Dll4 in endothelia and that this effect is required for Notch-dependent induction of GLT-1. Together these studies indicate that reciprocal communication between astrocytes and endothelia is required for appropriate astrocyte maturation and that endothelia likely deploy additional non-Notch signals to induce GLT-1.

Characterization of the cystine/glutamate antiporter in cultured Bergmann glia cells.

Glutamate, the major excitatory transmitter in the vertebrate brain is a potent neurotoxin through the over-stimulation of its specific membrane receptors. In accordance, a tight regulation of its extracellular levels by plasma membrane transporters is present. A family of excitatory amino acid transporters is expressed in neurons and glia cells and is responsible of the removal of the neurotransmitter from the synaptic cleft. Glial transporters account for more than 80% of the brain uptake activity. The cystine/glutamate antiporter is another plasma membrane-bound protein critically involved in glutamatergic transmission. Upon oxidative stress, it begins to pump out glutamate in exchange for cystine, mostly needed for glutathione production. Taking into consideration that all of these glutamate transporter proteins are present in glia cells that surround glutamatergic synapses, we reasoned that a functional coupling of them should exist to prevent an excitotoxic insult to the neighboring neuronal cells. To this end, we used the established model of chick cerebellar Bergmann glia cultures. Once we could establish the expression of the cystine/glutamate antiporter in our system, we characterized its kinetic properties and started to gain insight into its regulation and plausible coupling to other transporters. Exposure to glutamate reduces the uptake activity and favors a physical interaction with the excitatory amino acid transporter 1 and the Na+-dependent neutral amino acids transporter 3. In contrast, treatment of the cultured cells with a nitric oxide donor such as sodium nitroprussiate augments the exchanger activity. Longer sodium nitroprussiate exposure periods down-regulates the cystine/glutamate protein levels. These results suggest that a coordinated interplay between glutamate transporters and exchangers takes place in glia cells to prevent excitotoxic insults.

GLAST/EAAT1 regulation in cultured Bergmann glia cells: role of the NO/cGMP signaling pathway.

Glutamate, the major excitatory amino acid, activates a wide variety of signal transduction cascades. Ionotropic and metabotropic glutamate receptors are critically involved in long-term synaptic changes, although recent findings suggest that the electrogenic Na(+)-dependent glutamate transporters, responsible for its removal from the synaptic cleft participate in the signaling transactions triggered by this amino acid. Glutamate transporters are profusely expressed in glia therefore most of its uptake occurs in this cellular compartment. In the cerebellar cortex, Bergmann glial cells enwrap glutamatergic synapses and participate in the recycling of its neurotransmitter through the glutamate/glutamine shuttle. It has long been acknowledged that glutamatergic transmission in the cerebellar molecular layer results in cGMP accumulation within Bergmann glia cells. In this context, we decided to investigate a plausible role of the nitric oxide/cGMP-signaling pathway in the regulation of Bergmann glia glutamate transporters. To this end, the well-established model of primary cultures of chick cerebellar Bergmann glial cells was used. Confluent monolayers were exposed to the nitric oxide donor, sodium nitroprusside, or to the non-hydrolysable cGMP analog dbcGMP and the [(3)H] D-aspartate uptake activity measured. An increase in uptake activity, related to an augmentation in VMax, was detected with both treatments. The signaling cascade includes NO/cGMP/PKG and Ca(2+) influx through the Na(+)/Ca(2+) exchanger and might be related to the plasma membrane glutamate transporters turnover. Interestingly enough, an inhibitor of the cGMP dependent protein kinase was capable to abolish the sodium nitroprusside induced Ca(2+) influx. These results provide an insight into the physiological role of cGMP in the cerebellum.

Glutamate transporter-dependent mTOR phosphorylation in Müller glia cells.

Glu (glutamate), the excitatory transmitter at the main signalling pathway in the retina, is critically involved in changes in the protein repertoire through the activation of signalling cascades, which regulate protein synthesis at transcriptional and translational levels. Activity-dependent differential gene expression by Glu is related to the activation of ionotropic and metabotropic Glu receptors; however, recent findings suggest the involvement of Na+-dependent Glu transporters in this process. Within the retina, Glu uptake is aimed at the replenishment of the releasable pool, and for the prevention of excitotoxicity and is carried mainly by the GLAST/EAAT-1 (Na+-dependent glutamate/aspartate transporter/excitatory amino acids transporter-1) located in Müller radial glia. Based on the previous work showing the alteration of GLAST expression induced by Glu, the present work investigates the involvement of GLAST signalling in the regulation of protein synthesis in Müller cells. To this end, we explored the effect of D-Asp (D-aspartate) on Ser-2448 mTOR (mammalian target of rapamycin) phosphorylation in primary cultures of chick Müller glia. The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-ß-hydroxyaspartate). Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K. Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses. These results add a novel receptor-independent pathway for Glu signalling in Müller glia, and further strengthen the critical involvement of these cells in the regulation of glutamatergic transmission in the retina.

Signaling through EAAT-1/GLAST in cultured Bergmann glia cells.

Glutamate, the major excitatory amino acid, activates a wide variety of signal transduction cascades. Synaptic plasticity relies on activity-dependent differential protein expression. Ionotropic and metabotropic glutamate receptors have been critically involved in long-term synaptic changes, although recent findings suggest that the electrogenic Na(+)-dependent glutamate transporters, responsible of its removal from the synaptic cleft, participate in glutamate-induced signaling. Transporter proteins are expressed in neurons and glia cells albeit most of the glutamate uptake occurs in the glial compartment. Within the cerebellum, Bergmann glial cells are close to glutamatergic synapses and participate actively in the recycling of glutamate through the glutamate/glutamine shuttle. In this context, we decided to investigate a plausible role of Bergmann glia glutamate transporters as signaling entities. To this end, primary cultures of chick cerebellar Bergmann glial cells were exposed to d-aspartate (D-Asp) and other transporter ligands and the serine 2448 phosphorylation pattern of the master regulator of protein synthesis, namely the mammalian target of rapamycin (mTOR), determined. An increase in mTOR phosphorylation and activity was detected. The signaling cascade included Ca(2+) influx, activation of the phosphatidylinositol 3-kinase and protein kinase B. Furthermore, transporter signaling resulted also in an increase in activator protein-1 (AP-1) binding to DNA and the up-regulation of the transcription of an AP-1 driven gene construct. These results add a novel mediator of the glutamate effects at the translational and transcriptional levels and further strengthen the notion of the critical involvement of glia cells in synaptic function.