GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.     

The ionic stoichiometry of the GLAST glutamate transporter in salamander retinal glia

Maintaining a low extracellular glutamate concentration in the central nervous system is important for terminating synaptic transmission and preventing excitotoxic cell death. The stoichiometry of the most abundant glutamate transporter, GLT-1, predicts that a very low glutamate concentration, ∼2 n m , should be reached in the absence of glutamate release, yet microdialysis measurements give a value of ∼1 μ m . If other glutamate transporters had a different stoichiometry, the predicted minimum glutamate concentration could be higher, for example if those transporters were driven by the cotransport of 2 Na⁺ (rather than of 3 Na⁺ as for GLT-1). Here we investigated the ionic stoichiometry of the glutamate transporter GLAST, which is the major glutamate transporter expressed in the retina and cerebellum, is expressed in other adult brain areas at a lower level than GLT-1, and is present throughout the brain early in development when expression of GLT-1 is low. Glutamate transport by GLAST was found to be driven, as for GLT-1, by the cotransport of 3 Na⁺ and 1 H⁺ and the counter-transport of 1 K⁺, suggesting that the minimum extracellular glutamate concentration should be similar during development and in the adult brain. A less powerful accumulation of glutamate by GLAST than by GLT-1 cannot be used to explain the high glutamate concentration measured by microdialysis.     

Contribution of glutamate transporter GLT-1 to removal of synaptically released glutamate at climbing fiber-Purkinje cell synapses

Rapid removal of synaptically released glutamate from the extracellular space ensures a high signal-to-noise ratio in excitatory neurotransmission. In the cerebellum, glial glutamate transporters, GLAST and GLT-1, are co-localized in the processes of Bergmann glia wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, the decay kinetics of climbing fiber-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(−/−) mice are not different from those in wild-type (WT) mice. This raises a possibility that GLT-1 plays a significant role in clearing glutamate at CF-PC synapses despite its smaller amount of expression. Here, we studied the functions of GLT-1 and GLAST in the clearance of glutamate using GLAST(−/−) mice and GLT-1(−/−) mice. In the presence of cyclothiazide (CTZ) that attenuates the desensitization of AMPA receptors, the decay time constant of CF-EPSCs (τ_w) in GLT-1(−/−) mice was slower than that in WT mice. However, the degree of this prolongation of τ_w was less prominent compared to that in GLAST(−/−) mice. The values of τ_w in GLT-1(−/−) mice and GLAST(−/−) mice were comparable to those estimated in WT mice in the presence of a potent blocker of glial glutamate transporters (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA) at 10 and 100 nM, which reduced the amplitudes of glutamate transporter currents elicited by CF stimulation in Bergmann glia to ∼81 and ∼28%, respectively. We conclude that GLT-1 plays a minor role compared to GLAST in clearing synaptically released glutamate at CF-PC synapses.     

Up-regulation of spinal glutamate transporters contributes to anti-hypersensitive effects of valproate in rats after peripheral nerve injury

Valproate produces analgesia in animals and humans, however, its mechanisms of action are yet unknown. The present study examined effects of repeated administration of valproate on behavioral hypersensitivity and expression of glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in the spinal dorsal horn in rats after L5-L6 spinal nerve ligation (SNL). SNL significantly reduced mechanical withdrawal threshold and expression of GLT-1 and GLAST in the spinal dorsal horn. Repeated oral administration of valproate reduced hypersensitivity, restored down-regulated expression of GLT-1 and GLAST in the spinal dorsal horn, and enhanced analgesia from the glutamate transporter activator riluzole. This analgesia from valproate was blocked by the selective GLT-1 blocker dihydrokainic acid (DHK). These data suggest that valproate restores down-regulated expression of glutamate transporters in the spinal cord to presumably reduce glutamate signaling and to reduce hypersensitivity after nerve injury, and that combination of valproate with riluzole produces enhanced analgesia which relies on the spinal glutamate transporters.     

Spinal glial glutamate transporters downregulate in rats with taxol-induced hyperalgesia

Changes in the expression of glial glutamate transporters (GLAST and GLT-1) were examined in the spinal cord of rats with chemotherapy (taxol)-induced mechanical hyperalgesia. Immunohistochemical studies show that the expression of both GLAST and GLT-1 in the L4-L5 spinal dorsal horn is decreased by 24% (P<0.001) and 23% (P<0.001), respectively, in rats with taxol-induced hyperalgesia as compared with those in control rats. These changes were further confirmed using an enzyme-linked immunosorbent assay that confirmed downregulation of GLAST by 36% (P<0.05) and GLT-1 by 18% (P<0.05) in the L4-L5 spinal cord of taxol-treated rats. These data indicate that downregulation of glutamate transporters may contribute to the development of hyperalgesia induced by taxol and suggest that glutamate transporters may be a new target for treatment of pain.     

急性眼高压大鼠视网膜谷氨酸/天冬氨酸转运体和谷氨酰胺合成酶的表达

目的：通过检测急性眼高压后大鼠视网膜谷氨酸/天冬氨酸转运体(GLAST)和谷氨酰胺合成酶(GS)的表达变化,探讨急性青光眼视网膜节细胞(RGCs)损伤的可能机制。方法：成年大鼠,根据不同存活时间分组。左眼眼压升高至闪光视网膜电图b波消失的临界眼压且维持缺血60min。实验动物分别存活1、3、7、14d后通过免疫组织化学检测大鼠视网膜GLAST和GS的表达变化。结果：GLAST和GS在存活1d和3d时表达上调然后下调,GS还存在重新分布。结论：GLAST和GS的表达与存活时间密切相关,两者表达的相似变化是急性青光眼RGCs损伤的重要原因。     

Distribution of the glutamate transporters GLT-1 (SLC1A2) and GLAST (SLC1A3) in peripheral organs

The glutamate transporters GLT-1 and GLAST are widely expressed in astrocytes in the brain where they fulfill important functions during glutamatergic neurotransmission. The present study examines their distribution in peripheral organs using in situ hybridization (ISH) and immunocytochemistry. GLAST was found to be more widely distributed than GLT-1. GLAST was expressed primarily in epithelial cells, cells of the macrophage-lineage, lymphocytes, fat cells, interstitial cells, and salivary gland acini. GLT-1 was primarily expressed in glandular tissue, including mammary gland, lacrimal gland, and ducts and acini in salivary glands, but also by perivenous hepatocytes and follicular dendritic cells in spleen and lymph nodes. The findings demonstrate that, although expressed by the same cells in the brain, these two glutamate transporters have different distribution patterns in peripheral tissues and that they fulfill glutamate transport functions apart from glutamatergic neurotransmission in these areas.     

Neuronal stimulation leading to upregulation of glutamate transporter-1 (GLT-1) in rat microglia in vitro

As previously reported, activated microglia facilitate the expression of a glial cell-type glutamate transporter, glutamate transporter-1 (GLT-1; EAAT2), around injured motoneurons in axotomized rat facial nucleus. This phenomenon suggests that the motoneurons stimulate microglia to enhance the levels of GLT-1. In the present study, we investigated the effects of neuronal stimulus on the uptake of glutamate (Glu) by microglia and on the expression of GLT-1 protein in microglia in vitro. A 14C-Glu uptake experiment revealed that microglia enhance uptake of Glu by stimulation with neuronal conditioned medium (NCM). The NCM-stimulated uptake was significantly suppressed in the presence of dihydrokinate (a specific GLT-1 inhibitor), suggesting that GLT-1 is a major glutamate transporter for the uptake. Furthermore, immunoblotting analysis revealed that the amounts of GLT-1, but not another glial cell-type glutamate transporter glutamate-aspartate transporter (GLAST: EAAT1), increased significantly in microglia by treatment with NCM. Altogether, neuronal stimulus was found to promote the uptake of Glu in microglia, probably due to the increased levels of GLT-1.     

Downregulation of glutamate transporters is associated with elevation in extracellular glutamate concentration following rat microsphere embolism

Sodium-dependent glutamate transporters expressed in astroglial cells and neurons are essential for clearance of extracellular glutamate. In the present study, we found elevation of extracellular glutamate concentration associated with concomitant downregulation of glutamate transporters following rat microsphere embolism (ME). A marked increase in extracellular glutamate in the rat striatum was observed by microdialysis immediately after ME induction, and glutamate remained elevated at least 12 h after ischemia. Concomitantly, impairment of high KCl (146 mM)-induced glutamate release was observed in the striatum 12 h after ME. Consistent with the persistent increase in extracellular glutamate, expression of the glutamate transporters EAAC1 and GLT-1 significantly decreased 6 h after insult without a change in GLAST levels. GLT-1 expression was restored to basal levels within 48 h, whereas EAAC1 expression remained decreased up to at least 72 h after ME. Restoration of GLT-1 was associated with increased expression of the astroglial marker GFAP, whereas markedly reduced EACC1 levels were correlated with reduced levels of the neuronal marker MAP2, likely due to loss of vulnerable neurons. Taken together, downregulation of glutamate transporters after ME is associated with dysregulation of basal glutamate concentrations and KCl-induced glutamate release in the brain.     

Propentofylline-induced astrocyte modulation leads to alterations in glial glutamate promoter activation following spinal nerve transection

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.     

Interleukin-1 attenuates normal tension glaucoma-like retinal degeneration in EAAC1-deficient mice

Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of retinal ganglion cells (RGCs) and optic nerves. Although glaucoma is often associated with elevated intraocular pressure, recent studies have shown a relatively high prevalence of normal tension glaucoma (NTG) in glaucoma patient populations. In the mammalian retina, glutamate/aspartate transporter (GLAST) is localized to Müller glial cells, whereas excitatory amino acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs. Since the loss of GLAST or EAAC1 leads to retinal degeneration similar to that seen in NTG, we examined the effects of interleukin-1 (IL-1) on RGC death in GLAST- and EAAC1-deficient mice. IL-1 promoted increased glutamate uptake in Müller cells by suppressing intracellular Na⁺ accumulation, which is necessary to counteract Na⁺-glutamate cotransport. The observed trends for the glutamate uptake increase in the wild-type (WT), GLAST- and EAAC1-deficient mice were similar; however, the baseline glutamate uptake and intracellular Na⁺ concentration in the GLAST-deficient mice were significantly lower than those in the wild-type mice. Consistently, pretreatment with IL-1 exhibited no beneficial effects on glutamate-induced RGC degeneration in the GLAST-deficient mice. In contrast, IL-1 significantly increased glutamate uptake by Müller cells and the number of surviving RGCs in the wild-type and EAAC1-deficient mice. Our findings suggest that the use of IL-1 for enhancing the function of glutamate transporters may be useful for neuroprotection in retinal degenerative disorders including NTG.     

Roles of glial glutamate transporters in shaping EPSCs at the climbing fiber-Purkinje cell synapses

Glial glutamate transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia (BG) wrapping excitatory synapses on Purkinje cells (PCs). Although GLAST is expressed six-fold more abundantly than GLT-1, no change is detected in the kinetics of climbing fiber (CF)-mediated excitatory postsynaptic currents (CF-EPSCs) in PCs in GLAST(-/-) mice compared to the wild-type mice (WT). Here we aimed to clarify the mechanism(s) underlying this unexpected finding using a selective GLT-1 blocker, dihydrokainate (DHK), and a novel antagonist of glial glutamate transporter, (2S,3S)-3-[3-(4-methoxybenzoylamino)benzyloxy]aspartate (PMB-TBOA). In the presence of cyclothiazide (CTZ), which attenuates the desensitization of AMPA receptors, DHK prolonged the decay time constant (tau(w)) of CF-EPSCs in WT, indicating that GLT-1 plays a partial role in the removal of glutamate. The application of 100 nM PMB-TBOA, which inhibited CF-mediated transporter currents in BG by approximately 80%, caused no change in tau(w) in WT in the absence of CTZ, whereas it prolonged tau(w) in the presence of CTZ. This prolonged value of tau(w) was similar to that in GLAST(-/-) mice in the presence of CTZ. These results indicate that glial glutamate transporters can apparently retain the fast decay kinetics of CF-EPSCs if a small proportion ( approximately 20%) of functional transporters is preserved.     

The Role of Glial Glutamate Transporters in Maintaining the Independent Operation of Juvenile Mouse Cerebellar Parallel Fibre Synapses

There is controversy over the extent to which glutamate released at one synapse can escape from the synaptic cleft and affect receptors at other synapses nearby, thereby compromising the synapse-specificity of information transmission. Here we show that the glial glutamate transporters GLAST and GLT-1 limit the activation of Purkinje cell AMPA receptors produced by glutamate diffusion between parallel fibre synapses in the cerebellar cortex of juvenile mice. For a single stimulus to the cerebellar molecular layer of wild-type mice, increasing the number of activated parallel fibres prolonged the parallel fibre EPSC, demonstrating an interaction between different synapses. Knocking out GLAST, or blocking GLT-1 in the absence of GLAST, prolonged the EPSC when many parallel fibres were stimulated but not when few were stimulated. When spatially separated parallel fibres were activated by granular layer stimulation, the EPSC prolongation produced by stimulating more fibres or reducing glutamate transport was greatly reduced. Thus, GLAST and GLT-1 curtail the EPSC produced by a single stimulus only when many nearby fibres are simultaneously activated. However when trains of stimuli were applied, even to a small number of parallel fibres, knocking out GLAST or blocking GLT-1 in the absence of GLAST greatly prolonged and enhanced the AMPA receptor-mediated current. These results show that glial cell glutamate transporters allow neighbouring synapses to operate more independently, and control the postsynaptic response to high frequency bursts of action potentials.     

Endothelins negatively regulate glial glutamate transporter expression

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system which at high extracellular levels leads to neuronal over-stimulation and subsequent excitotoxic neuronal cell death. Both the termination of glutamatergic neurotransmission and the prevention of neurotoxic extracellular glutamate concentrations are predominantly achieved by the uptake of extracellular glutamate into astroglia through the high-affinity glutamate transporters, excitatory amino acid transporter-2/glutamate transporter-1 (EAAT-2/GLT-1) and EAAT-1/glutamate aspartate transporter (GLAST). Although several injury-induced growth factors such as epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) potently stimulate the expression of glutamate transporters in cultured astroglia, GLT-1 and/or GLAST expression temporarily decreases during acute brain injuries eventually contributing to secondary neuronal cell death. We now demonstrate that the stimulatory influences of these injury-regulated growth factors are overridden by endothelins (ETs), a family of peptides also upregulated in the injured brain. Exposure of cultured cortical astroglia to ET-1, ET-2, and ET-3 resulted in a major loss of basal glutamate transporter expression after 72 hours and the complete prevention of the known stimulatory influences of dibutyryl cyclic (dbc)AMP, pituitary adenylate cyclase-activating polypeptide (PACAP), EGF, and TGFalpha on both GLT-1 and GLAST expression. With all ET isoforms, the inhibitory effects were detectable with similar low nanomolar concentrations and persisted in endothelin B-receptor deficient astroglia, suggesting that the inhibitory action is equally induced by endothelin A and B receptors. In astroglial cultures maintained with endothelins alone or in combination with PACAP, the inhibitory action was remarkably long-lasting and was still detectable after 7 days. In apparent contrast, glutamate transporter expression partially recovered between days 5 and 7 in cultures maintained with a combination of ETs and the injury-regulated growth factors EGF or TGFalpha. These findings point to ETs as major mediators of injury-dependent down-regulation of glial glutamate transporters and subsequent glutamate-induced brain damage.     

In vitro differentiated neural stem cells express functional glial glutamate transporters

The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35–40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na⁺-dependent [³H]-D-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters.