Motor neuron-derived microRNAs cause astrocyte dysfunction in amyotrophic lateral sclerosis.

We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate transporter in astrocytes [excitatory amino acid transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.

Descending Noradrenergic Inhibition: An Important Mechanism of Gabapentin Analgesia in Neuropathic Pain.

Gabapentinoids are effective in a wide range of animal pain models and in patients with neuropathic pain and has become one of first-line treatments for neuropathic pain. Because spinal plasticity and sensitization have been intensely studied in neuropathic pain, most laboratory studies have focused on actions of gabapentinoids in the spinal cord, where they reduce primary afferent traffic and excitation of spinal nociceptive neurons, via interaction with α2δ subunits of voltage-gated Ca2+ channels. However, a recent clinical study questioned the relevance of this in vitro and in vivo rodent studies by demonstrating a complete lack of clinical efficacy of intrathecal gabapentin in patients with chronic pain. Curiously, preclinical studies continue to focus on spinal cord actions of gabapentinoids despite this lack of translation to humans.We and others demonstrated that gabapentin inhibits presynaptic GABA release and induces glutamate release from astrocytes in the locus coeruleus (LC), thereby increasing LC neuron activity and spinal noradrenaline release, and that gabapentin relies on this action in the LC for its analgesia. We also recently discovered that, with prolonged time after neuropathic injury, noradrenergic neurons in the LC become less responsive to gabapentin, leading to impaired gabapentin analgesia, and that astroglial glutamate dysregulation is critical to this impaired LC response. The clinically available drug valproate increases glutamate transporter-1 (GLT-1) expression in the LC to restore this impaired gabapentin analgesia.

Glial EAAT2 regulation of extracellular nTS glutamate critically controls neuronal activity and cardiorespiratory reflexes.

KEY POINTS: Excitatory amino acid transporter 2 (EAAT2) is present on astrocytes in the nucleus tractus solitarii (nTS), an important nucleus in cardiorespiratory control. Its specific role in influencing nTS neuronal activity and thereby basal and reflex cardiorespiratory function is unknown. The specific role of nTS EAAT2 was determined via whole animal and brainstem slice patch clamp experiments. Astrocytic EAAT2 buffers basal glutamate activation of AMPA-type glutamate receptors and therefore decreases baseline excitability of nTS neurons. EAAT2 modulates cardiorespiratory control and tempers excitatory cardiorespiratory responses to activation of the peripheral chemoreflex. This study supports the concept that nTS astrocyte transporters influence sympathetic nervous system activity and cardiorespiratory reflex function in health and disease. ABSTRACT: Glutamatergic signalling is critical in the nucleus tractus solitarii (nTS) for cardiorespiratory homeostasis and initiation of sensory reflexes, including the chemoreflex activated during hypoxia. Maintenance of nTS glutamate concentration occurs in part through astrocytic excitatory amino acid transporters (EAATs). We previously established the importance of EAATs in the nTS by demonstrating their inhibition produced neuronal excitation to alter basal cardiorespiratory function. Since EAAT2 is the most expressed EAAT in the nTS, this study specifically determined EAAT2's role in nTS astrocytes, its influence on neuronal and synaptic properties, and ultimately on basal and reflex cardiorespiratory function. The EAAT2-specific antagonist dihydrokainate (DHK) was microinjected into the anaesthetized rat nTS or applied to rat nTS slices. DHK produced depressor, bradycardic and sympathoinhibitory responses and reduced neural respiration in the intact rat, mimicking responses to glutamate excitation. DHK also enhanced responses to glutamate microinjection. DHK elevated extracellular nTS glutamate concentration, depolarized neurons and enhanced spontaneous EPSCs. EAAT2 block also augmented action potential discharge in chemosensitive nTS neurons. Glial recordings confirmed EAAT2 is functional on nTS astrocytes. Neuronal excitation and cardiorespiratory effects following EAAT2 inhibition were due to activation of putative extrasynaptic AMPA receptors as their antagonism blocked DHK responses in the intact rat nTS and the slice. The DHK-induced elevation of extracellular glutamate and neuronal excitation augmented chemoreflex-mediated pressor, sympathoexcitatory and minute neural ventilation responses in the rat. These data shed new light on the important role astrocytic EAAT2 plays on buffering nTS excitation and overall cardiorespiratory function.

Role of the major glutamate transporter GLT1 in nucleus accumbens core versus shell in cue-induced cocaine-seeking behavior.

Relapse to cocaine-seeking behavior requires an increase in nucleus accumbens (NAc) core glutamate transmission. Decreased expression of glutamate type I transporter (GLT1), which is responsible for >90% of glutamate clearance, occurs in the core of rats withdrawn from cocaine self-administration, while treatment with ceftriaxone, a ß-lactam antibiotic previously shown to increase GLT1 expression and function in rodents, upregulates GLT1 and attenuates cue-induced cocaine reinstatement. Here, we tested the effects of increasing GLT1 expression on cue-induced cocaine seeking in rats exposed to either limited (2 h/d) or extended (6 h/d) cocaine access followed by short (2 d) or long (45 d) withdrawal periods. Treatment with ceftriaxone (200 mg/kg, i.p.) upregulated core GLT1 expression and attenuated cue-induced cocaine-seeking behavior only in rats exposed to long withdrawal periods, with a greater effect in the extended-access condition. Pearson's correlation revealed GLT1 expression in core to be inversely correlated with cue-induced cocaine-seeking behavior. To localize the effects of GLT1 upregulation within NAc, we tested the hypothesis that blockade of GLT1 in NAc core, but not shell, would reverse the ceftriaxone-mediated effect. Rats withdrawn from cocaine self-administration were treated with the same dose of ceftriaxone followed by intracore or intrashell infusions of one of two GLT1 blockers, dihydrokainic acid (500 µM) or DL-threo-ß-benzyloxyaspartate (250 µM), or saline. Our results reveal that the ceftriaxone-mediated attenuation of cue-induced cocaine reinstatement is reversed by GLT1 blockade in core, but not shell, and further implicate core GLT1 as a potential therapeutic target for cocaine relapse.

Astrocytes modulate a postsynaptic NMDA-GABAA-receptor crosstalk in hypothalamic neurosecretory neurons.

A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABA(A)ergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABA(A) receptors (GABA(A)Rs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABA(A)Rs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR-GABA(A)R crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate-GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate-GABA interactions in MNCs, which involves crosstalk between NMDA and GABA(A) postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABA(A)Rs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure.

Role of astrocytes in thiamine deficiency.

Thiamine deficiency (TD) is the underlying cause of Wernicke's encephalopathy (WE), an acute neurological disorder characterized by structural damage to key periventricular structures in the brain. Increasing evidence suggests these focal histological lesions may be representative of a gliopathy in which astrocyte-related changes are a major feature of the disorder. These changes include a loss of the glutamate transporters GLT-1 and GLAST concomitant with elevated interstitial glutamate levels, lowered brain pH associated with increased lactate production, decreased levels of GFAP, reduction in the levels of glutamine synthetase, swelling, alterations in levels of aquaporin-4, and disruption of the blood-brain barrier. This review focusses on how these manifestations contribute to the pathophysiology of TD and possibly WE.

DCPIB, the proposed selective blocker of volume-regulated anion channels, inhibits several glutamate transport pathways in glial cells.

4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB) was identified as the selective blocker of volume-regulated anion channels (VRAC). VRAC are permeable to small inorganic and organic anions, including the excitatory neurotransmitter glutamate. In recent years DCPIB has been increasingly used for probing the physiologic and pathologic roles of VRAC and was found to potently suppress pathologic glutamate release in cerebral ischemia. Because ischemic glutamate release can be mediated by a plethora of mechanisms, in this study we explored the selectivity of DCPIB toward the majority of previously identified glutamate transporters and permeability pathways. l-[(3)H]glutamate, d-[(3)H]aspartate, and l-[(14)C]cystine were used to trace amino acid release and uptake. We found that in addition to its well-characterized effect on VRAC, DCPIB potently inhibited glutamate release via connexin hemichannels and glutamate uptake via the glutamate transporter GLT-1 in rat glial cells. In contrast, DCPIB had no direct effect on vesicular glutamate release from rat brain synaptosomes or the cystine/glutamate exchange in astrocytes. The compound did not affect the astrocytic glutamate transporter GLAST, nor did it block glutamate release via the P2X(7)/pannexin permeability pathway. The ability of DCPIB to directly block connexin hemichannels was confirmed using a gene-specific siRNA knockdown approach. Overall, our data demonstrate that DCPIB influences several glutamate transport pathways and that its effects on VRAC in vivo should be verified using additional pharmacological controls.

Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes.

Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins L-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal DL-threo-ß-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes.

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death.

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal ß-cell lines and human islet ß-cells but not in α-cells. In human islets, glutamate-induced ß-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of ß-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced ß-cell death, whereas GLT1 up-regulation promoted ß-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that ß-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

The equivalent of a thallium binding residue from an archeal homolog controls cation interactions in brain glutamate transporters.

Glutamate transporters maintain low synaptic concentrations of neurotransmitter by coupling uptake to flux of other ions. Their transport cycle consists of two separate translocation steps, namely cotransport of glutamic acid with three Na(+) followed by countertransport of K(+). Two Tl(+) binding sites, presumed to serve as sodium sites, were observed in the crystal structure of a related archeal homolog and the side chain of a conserved aspartate residue contributed to one of these sites. We have mutated the corresponding residue of the eukaryotic glutamate transporters GLT-1 and EAAC1 to asparagine, serine, and cysteine. Remarkably, these mutants exhibited significant sodium-dependent radioactive acidic amino acid uptake when expressed in HeLa cells. Reconstitution experiments revealed that net uptake by the mutants in K(+)-loaded liposomes was impaired. However, with Na(+) and unlabeled L-aspartate inside the liposomes, exchange levels were around 50-90% of those by wild-type. In further contrast to wild-type, where either substrate or K(+) stimulated the anion conductance by the transporter, substrate but not K(+) modulated the anion conductance of the mutants expressed in oocytes. Both with wild-type EAAC1 and EAAC1-D455N, not only sodium but also lithium could support radioactive acidic amino acid uptake. In contrast, with D455S and D455C, radioactive uptake was only observed in the presence of sodium. Thus the conserved aspartate is required for transporter-cation interactions in each of the two separate translocation steps and likely participates in an overlapping sodium and potassium binding site.

Role of excitatory amino acid transporter-2 (EAAT2) and glutamate in neurodegeneration: opportunities for developing novel therapeutics.

Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Analysis of the 2.5 kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many ß-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a ß-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes through the NF-κB signaling pathway. The NF-κB binding site at -272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

Riluzole normalizes early-life stress-induced visceral hypersensitivity in rats: role of spinal glutamate reuptake mechanisms.

BACKGROUND & AIMS: The molecular basis underlying visceral hypersensitivity in functional irritable bowel syndrome remains elusive, resulting in poor treatment effectiveness. Because alterations in spinal non-neuronal (astrocytic) glutamate reuptake are suspected to participate in chronic pain, we asked whether such processes occur in visceral hypersensitivity. METHODS: Visceral hypersensitivity was induced in Sprague-Dawley rats by maternal separation. Separated adults were given a systemic administration of riluzole (5 mg/kg), an approved neuroprotective agent activating glutamate reuptake. Visceral hypersensitivity was assessed using colorectal distension (40 mm Hg). Somatic nociception was quantified using Hot Plate, Randall-Sellito, and Hargreaves tests. Spinal proteins were quantified using immunofluorescence and Western blot. The dependence of visceral sensory function upon spinal glutamate transport was evaluated by intrathecal injection of glutamate transport antagonist DL-threo-beta-benzyloxyaspartate (TBOA). For in vitro testing of riluzole and TBOA, primary cultures of astrocytes were used. RESULTS: We show that riluzole counteracts stress-induced visceral hypersensitivity without affecting visceral response in nonseparated rats or altering nociceptive responses to somatic pain stimulation. In addition, maternal separation produces a reduction in glial excitatory amino acid transporter (EAAT)-1 with no change in EAAT-2 or gamma-amino butyric acid transporters. Stress was not associated with changes in glial fibrillary acidic protein or astrocytic morphology per se. Furthermore, visceral normosensitivity relies on spinal EAAT, as intrathecal TBOA is sufficient to induce hypersensitivity in normal rats. CONCLUSIONS: We identify spinal EAAT as a therapeutic target, and establish riluzole as a candidate to counteract gastrointestinal hypersensitivity in disorders such as irritable bowel syndrome.

Regulation of glutamate transport in developing rat oligodendrocytes.

Glutamate released from synaptic vesicles mediates excitatory neurotransmission by stimulating glutamate receptors. Glutamate transporters maintain low synaptic glutamate levels critical for this process, a role primarily attributed to astrocytes. Recently, vesicular release of glutamate from unmyelinated axons in the rat corpus callosum has been shown to elicit AMPA receptor-mediated currents in glial progenitor cells. Glutamate transporters are the only mechanism of glutamate clearance, yet very little is known about the role of glutamate transporters in normal development of oligodendrocytes (OLs) or in excitotoxic injury to OLs. We found that OLs in culture are capable of sodium-dependent glutamate uptake with a K(m) of 10 +/- 2 microm and a V(max) of 2.6, 5.0, and 3.8 nmol x min(-1) x mg(-1) for preoligodendrocytes, immature, and mature OLs, respectively. Surprisingly, EAAC1, thought to be exclusively a neuronal transporter, contributes more to [(3)H]l-glutamate uptake in OLs than GLT1 or GLAST. These data suggest that glutamate transporters on oligodendrocytes may serve a critical role in maintaining glutamate homeostasis at a time when unmyelinated callosal axons are engaging in glutamatergic signaling with glial progenitors. Furthermore, GLT1 was significantly increased in cultured mature OLs contrary to in vivo data in which we have shown that, although GLT1 is present on developing OLs when unmyelinated axons are prevalent in the developing rat corpus callosum, after myelination, GLT1 is not expressed on mature OLs. The absence of GLT1 in mature OLs in the rat corpus callosum and its presence in mature rat cultured OLs may indicate that a signaling process in vivo is not activated in vitro.

Palmitoylation and function of glial glutamate transporter-1 is reduced in the YAC128 mouse model of Huntington disease.

Excitotoxicity plays a key role in the selective vulnerability of striatal neurons in Huntington disease (HD). Decreased glutamate uptake by glial cells could account for the excess glutamate at the synapse in patients as well as animal models of HD. The major molecule responsible for clearing glutamate at the synapses is glial glutamate transporter GLT-1. In this study, we show that GLT-1 is palmitoylated at cysteine38 (C38) and further, that this palmitoylation is drastically reduced in HD models both in vitro and in vivo. Palmitoylation is required for normal GLT-1 function. Blocking palmitoylation either with the general palmitoylation inhibitor, 2-bromopalmitate, or with a GLT-1 C38S mutation, severely impairs glutamate uptake activity. In addition, GLT-1-mediated glutamate uptake is indeed impaired in the YAC128 HD mouse brain, with the defect in the striatum evident as early as 3 months prior to obvious neuropathological findings, and in both striatum and cortex at 12 months. These phenotypes are not a result of changes in GLT1 protein expression, suggesting a crucial role of palmitoylation in GLT-1 function. Thus, it appears that impaired GLT-1 palmitoylation is present early in the pathogenesis of HD, and may influence decreased glutamate uptake, excitotoxicity, and ultimately, neuronal cell death in HD.

Amyloid Fibril-Induced Astrocytic Glutamate Transporter Disruption Contributes to Complement C1q-Mediated Microglial Pruning of Glutamatergic Synapses.

The complement C1q plays a critical role in microglial phagocytosis of glutamatergic synapses and in the pathogenesis of neuroinflammation in Alzheimer's disease (AD). We recently reported that upregulation of metabotropic glutamate receptor signaling is associated with increased synaptic C1q production and subsequent microglial phagocytosis of synapses in the rodent models of AD. Here, we explored the role of astrocytic glutamate transporter in the synaptic C1q production and microglial phagocytosis of hippocampal glutamatergic synapses in a rat model of AD. Activation of astrocyte and reduction glutamate transporter 1 (GLT1) were noted after bilateral microinjection of amyloid-beta (Aß1-40) fibrils into the hippocampal CA1 area of rats. Ceftriaxone is a ß-lactam antibiotic that upregulates GLT1 expression. Bilateral microinjection of ceftriaxone recovered GLT1 expression, decreased synaptic C1q production, suppressed microglial phagocytosis of glutamatergic synapses in the hippocampal CA1, and attenuated synaptic and cognitive deficits in rats microinjected with Aß1-40. In contrast, artificial suppression of GLT1 activity by DL-threo-beta-benzyloxyaspartate (DL-TBOA) in naïve rats induced synaptic C1q expression and microglial phagocytosis of glutamatergic synapses in the hippocampal CA1 area, resulting in synaptic and cognitive dysfunction. These findings demonstrated that impairment of astrocytic glutamate transporter plays a role in the pathogenesis of AD.

The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies.

The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Förster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

Up-regulation of GLT-1 severely impairs LTD at mossy fibre--CA3 synapses.

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. By this action, they maintain low levels of ambient glutamate, thus preventing excitotoxic damage, and contribute to shaping synaptic currents. We show that up-regulation of the glutamate transporter GLT-1 by ceftriaxone severely impaired mGluR-dependent long-term depression (LTD), induced at rat mossy fibre (MF)-CA3 synapses by repetitive stimulation of afferent fibres. This effect involved GLT-1, since LTD was rescued by the selective GLT-1 antagonist dihydrokainate (DHK). DHK per se produced a modest decrease in fEPSP amplitude that rapidly regained control levels after DHK wash out. Moreover, the degree of fEPSP inhibition induced by the low-affinity glutamate receptor antagonist gamma-DGG was similar during basal synaptic transmission but not during LTD, indicating that in ceftriaxone-treated rats LTD induction did not alter synaptic glutamate transient concentration. Furthermore, ceftriaxone-induced GLT-1 up-regulation significantly reduced the magnitude of LTP at MF-CA3 synapses but not at Schaffer collateral-CA1 synapses. Postembedding immunogold studies in rats showed an increased density of gold particles coding for GLT-1a in astrocytic processes and in mossy fibre terminals; in the latter, gold particles were located near and within the active zones. In both CEF-treated and untreated GLT-1 KO mice used for verifying the specificity of immunostaining, the density of gold particles in MF terminals was comparable to background levels. The enhanced expression of GLT-1 at release sites may prevent activation of presynaptic receptors, thus revealing a novel mechanism by which GLT-1 regulates synaptic plasticity in the hippocampus.

Complete but not partial inhibition of glutamate transporters exacerbates cortical excitability in the R6/2 mouse model of Huntington's disease.

AIM: Deficient glutamate reuptake occurs in the cerebral cortex of Huntington's disease (HD) patients and murine models. Here, we examine the effects of partial or complete blockade of glutamate transporters on excitatory postsynaptic currents (EPSCs) of cortical pyramidal neurons (CPNs). METHODS: Whole-cell patch clamp recordings of CPNs in slices from symptomatic R6/2 mice and wild-type (WT) littermates were used to examine the effects of selective or concurrent inhibition of glutamate reuptake transporters. RESULTS: Selective inhibition of the glial glutamate transporter 1 (GLT-1) or the glutamate aspartate transporter (GLAST) produced slight decreases in decay time of evoked EPSCs in CPNs from WT and R6/2 mice with no significant differences between genotypes. In contrast, concurrent inhibition of both transporters with DL-TBOA induced a significant increase in area and decay time and this effect was significantly greater in R6/2 CPNs. Furthermore, full blockade also reduced spontaneous EPSC frequency and exacerbated epileptiform activity in CPNs from symptomatic R6/2 mice. CONCLUSIONS: R6/2 CPNs are more sensitive to glutamate accumulation during full inhibition of both glutamate transporters, and these neurons have homeostatic mechanisms to cope with inhibition of GLT-1 or GLAST by a mechanism that involves upregulation of either transporter when the other is deficient.

The mechanism of GLT-1 mediating cerebral ischemic injury depends on the activation of p38 MAPK.

The previous studies have shown that glial glutamate transporter-1 (GLT-1) participates in cerebral ischemic injury in rats. However, the mechanism involved remains to be elucidated. This study was undertaken to investigate whether p38 MAPK was involved in regulating GLT-1 in the process. At first, it was observed that global brain ischemia for 8 min led to obvious delayed neuronal death, GLT-1 down-regulation and p-p38 MAPK up-regulation in CA1 hippocampus in rats. Then, whether p-p38 MAPK was involved in regulating GLT-1 during cerebral ischemic injury was studied in vitro. Astrocyte-neuron co-cultures exposed to oxygen and glucose deprivation (OGD) were used to mimic brain ischemia. It was observed that lethal OGD (4-h OGD) decreased GLT-1 expression and increased p-p38 MAPK expression in astrocytes. The p-p38 MAPK protein rised from 0 min to 48 h that is the end time of the observation, and the peak value was at 12 h, which was 12.45 times of the control group. Moreover, pre-administration of p38 MAPK inhibitor SB203580 or its siRNA dose-dependently increased GLT-1 expression, and meanwhile alleviated the neuronal death induced by lethal OGD. The above results indicated that p38 MAPK signaling pathway participated in regulating GLT-1 during OGD injury in vitro. Finally, back to in vivo experiment, it was found that pre-administration of SB203580 by intracerebroventricular injection dose-dependently reversed the down-regulation of GLT-1 expression and attenuated the delayed neuronal death normally induced by global brain ischemia in CA1 hippocampus in rats. Taken together, it can be concluded that the mechanism of GLT-1 mediating cerebral ischemic injury depends on the activation of p38 MAPK.

In vivo knockdown of astroglial glutamate transporters GLT-1 and GLAST increases excitatory neurotransmission in mouse infralimbic cortex: Relevance for depressive-like phenotypes.

Alterations of energy metabolism and of astrocyte number/function in ventral anterior cingulate cortex (vACC) have been reported in major depressive disorder (MDD) patients and may contribute to MDD pathophysiology. We recently developed a mouse model of MDD mimicking these alterations. We knocked down the astroglial glutamate transporters GLAST and GLT-1 in infralimbic cortex (IL, rodent equivalent of vACC) using small interfering RNA (siRNA). GLAST and GLT-1 siRNA microinfusion in IL evoked a depressive-like phenotype, associated with a reduced serotonergic function and reduced forebrain BDNF expression. Neither effect occurred after siRNA application in the adjacent prelimbic cortex (PrL), thus emphasizing the critical role of vACC/IL in MDD pathogenesis. Here we examined the cellular/network basis of the changes induced in IL using intracellular recordings of layer V pyramidal neurons from mice microinjected with siRNA 24 h before. We analyzed (i) the electrophysiological characteristics of neurons; (ii) the synaptic transmission properties, by monitoring miniature, spontaneous and evoked EPSCs, and (iii) the gliotransmission, by monitoring slow inward currents (SICs), mediated by astrocytic glutamate release and activation of extra-synaptic NMDA receptors. GLT-1 and GLAST knockdown led to a more depolarized membrane potential and increased action potential firing rate of layer V pyramidal neurons, and enhanced excitatory synaptic transmission, as shown by the enhanced amplitude/frequency of spontaneous EPSCs. Gliotransmission was also increased, as indicated by the enhanced SIC amplitude/frequency. Hence, the depressive-like phenotype is associated with IL hyperactivity, likely leading to an excessive top-down inhibitory control of serotonergic activity through IL-midbrain descending pathways.