Distribution of Glutamate Transporter Subtypes During Human Brain Development

Abstract: In the mature brain, removal of glutamate from the synaptic cleft plays an important role in the maintenance of subtoxic levels of glutamate. This requirement is handled by a family of glutamate transporters, EAAT1, EAAT2, EAAT3, and EAAT4. Due to the involvement of glutamate also in neuronal development, it is believed that glutamate transport plays a role in developmental processes as well. Therefore, we have used immunohistochemical and immunoblot analysis to determine the distribution of the four glutamate transporters during human brain development using human pre- and postnatal brain tissue. Regional analysis showed that each transporter subtype has a unique distribution during development. EAAT2 was the most prominent glutamate transporter subtype and was highly enriched in cortex, basal ganglia, cerebellum, and thalamus in all ages examined. EAAT1 immunoreactivity was lower than that of EAAT2, with predominant localization in cortex, basal ganglia, hippocampus, and periventricular region. EAAT3 was located mainly in cortex, basal ganglia, and hippocampus, and EAAT4 was found only in cortex, hippocampus, and cerebellar cortex. The distinct regional distribution of various EAAT subtypes and also the transient expression of specific EAAT subtypes during development suggest multiple functional roles for glutamate transporters in the developing brain.     

Impairment of glutamate transport and increased vulnerability to oxidative stress in neuroblastoma SH-SY5Y cells expressing a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis

Human neuroblastoma SH-SY5Y cells transfected with either familial amyotrophic lateral sclerosis-typical G93A mutant or wild-type copper/zinc superoxide dismutase were compared to untransfected cells in term of glutamate transport. Vmax of glutamate uptake was reduced in mutant cells, with no change in Km. No difference in EAAT1, EAAT2 and EAAT3 glutamate transporter mRNAs and immunoreactive proteins was found, suggesting that one or more transporters are functionally inactivated, possibly due to increased oxidative stress induced by the G93A mutation. Mutant cells showed a marked sensitivity to oxidants, resulting in a more pronounced reduction of glutamate uptake. Short-term antioxidant treatment did not reverse the impairment of glutamate uptake in G93A cells. Interestlingly, N-acetylcysteine was partially effective in preventing glutamate uptake reduction due to exogenous oxidative insults. Since the inhibition of the EAAT2 transporter subtype had no effect on glutamate re-uptake in this model, our study suggests an impaired function of the EAAT1/3 transporter subtypes, possibly due to oxidative inactivation, in the presence of mutant copper/zinc superoxide dismutase. Therefore, this model might prove to be a valuable tool to study the effects of mutant copper/zinc superoxide dismutase associated with amyotrophic lateral sclerosis on glutamate transport in neuronal cells, without the specific contribution of glial cells. These findings might lead to the identification of new therapeutic strategies aimed at preventing the damage associated with ALS.     

Association of excitatory amino acid transporters, especially EAAT2, with cholesterol-rich lipid raft microdomains: importance for excitatory amino acid transporter localization and function

In the present study, we investigated the role of membrane cholesterol in the function of glutamate transporters. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced Na(+)-dependent glutamate uptake in primary cortical cultures. Glial glutamate transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-beta-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na(+)-dependent glutamate uptake. Our data suggest that the glutamate transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for excitatory amino acid transporter localization and function.     

Benzodiazepines differently modulate EAAT1/GLAST and EAAT2/GLT1 glutamate transporters expressed in CHO cells.

It has been described recently that low concentrations of benzodiazepines stimulate the transport activity of the neuronal glutamate transporter EAAT3, whereas high concentrations inhibit it. The present study is aimed to investigate whether benzodiazepines have similar effects on the two glial glutamate transporter, EAAT1 and EAAT2. To this end, the transporters were transiently expressed in CHO cells and transport activity was determined by isotope fluxes using D-aspartate as non-metabolizable homologue of L-glutamate. At low D-aspartate concentrations (1 micromol/l) EAAT1-mediated uptake was reduced significantly by low concentrations of oxazepam (1 micromol/l) and diazepam (1 and 10 micromol/l). At 100 micromol/l D-aspartate oxazepam stimulated EAAT1-mediated uptake up to 150% in a dose dependent manner, whereas the inhibition by low concentrations of diazepam was attenuated. In contrast, a significant effect of diazepam on EAAT2-mediated uptake was only observed at 1000 micromol/l where uptake was inhibited by 60%. A similar inhibition was observed for EAAT1. These studies demonstrate a different modulation of EAAT1 and EAAT2 by benzodiazepines. Furthermore the glial transporters differ from the neuronal glutamate transporter. Thus, a complex in vivo response of the various transporters to benzodiazepines can be expected.     

Oxidative DNA damage and alteration of glutamate transporter expressions in the hippocampal Ca1 area immediately after ischemic insult

Although oxidative stress and excitotoxicity may be interdependent mechanisms that are involved in delayed neuronal death, the temporal participation of these events in the early stage after ischemia-reperfusion insult is unclear. Therefore, in the present study, using the gerbil global ischemic model we investigated whether oxidative stress could be correlated with the expression of the glutamate transporters in the hippocampus, and whether these events are related and cooperate in the events that link ischemia to neuronal death in vivo. Thirty minutes after ischemia, the intensities of glutamate transporter-1 (GLT-1), glutamate/aspar-tate transporter (GLAST), and 8-hydroxy2'-deoxy-guanosine (8-OHdG) immunoreactivities were markedly increased in the hippocampal CA1 area. In contrast, excitatory amino acid carrier-1 (EAAC-1) immunoreactivity was 30% lower in the CA1 area than in the sham level. At 3 h post-reperfusion, the EAAC-1 expression began to increase in the CA1 area. Twelve hours after reperfusion, the reduction of both GLT-1 and GLAST immunoreactivity was salient, while the EAAC-1 immunoreactivity level intensified significantly. The 8-OHdG immunoreactivity peaked at this time point. These findings suggest that oxidative stress and alterations in the glutamate transporter expression in the CA1 area may simultaneously trigger neuronal damages very early after ischemia.     

Neuronal Expression of Splice Variants of “Glial” Glutamate Transporters in Brains Afflicted by Alzheimer’s Disease: Unmasking an Intrinsic Neuronal Property

Anomalies in glutamate homeostasis may contribute to the pathological processes involved in Alzheimer’s disease (AD). Glutamate released from neurons or glial cells is normally rapidly cleared by glutamate transporters, most of which are expressed at the protein level by glial cells. However, in some patho-physiological situations, expression of glutamate transporters that are normally considered to be glial types, appears to be evoked in populations of distressed neurons. This study analysed the expression of exon-skipping forms of the three predominant excitatory amino acid (glutamate) transporters (EAATs1-3) in brains afflicted with AD. We demonstrate by immunocytochemistry in temporal cortex, the expression of these proteins particularly in limited subsets of neurons, some of which appeared to be dys-morphic. Whilst the neuronal expression of the “glial” glutamate transporters EAAT1 and EAAT2 is frequently considered to represent the abnormal and ectopic expression of such transporters, we suggest this may be a misinterpretation, since neurons such as cortical pyramidal cells normally express abundant mRNA for these EAATs (but little if any EAAT protein expression). We hypothesize instead that distressed neurons in the AD brain can turn on the translation of pre-existent mRNA pools, or suppress the degradation of alternately spliced glutamate transporter protein, leading to the “unmasking” of, rather than evoked expression of “glial” glutamate transporters in stressed neurons. Special issue article in honor of Dr. Graham Johnston.     

Water and urea permeation pathways of the human excitatory amino acid transporter EAAT1

Glutamate transport is coupled to the co-transport of 3 Na(+) and 1 H(+) followed by the counter-transport of 1 K(+). In addition, glutamate and Na(+) binding to glutamate transporters generates an uncoupled anion conductance. The human glial glutamate transporter EAAT1 (excitatory amino acid transporter 1) also allows significant passive and active water transport, which suggests that water permeation through glutamate transporters may play an important role in glial cell homoeostasis. Urea also permeates EAAT1 and has been used to characterize the permeation properties of the transporter. We have previously identified a series of mutations that differentially affect either the glutamate transport process or the substrate-activated channel function of EAAT1. The water and urea permeation properties of wild-type EAAT1 and two mutant transporters were measured to identify which permeation pathway facilitates the movement of these molecules. We demonstrate that there is a significant rate of L-glutamate-stimulated passive and active water transport. Both the passive and active L-glutamate-stimulated water transport is most closely associated with the glutamate transport process. In contrast, L-glutamate-stimulated [(14)C]urea permeation is associated with the anion channel of the transporter. However, there is also likely to be a transporter-specific, but glutamate independent, flux of water via the anion channel.     

Localization and expression of the glutamate transporter,excitatory amino acid transporter 4, within astrocytes of the rat retina

Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.This work was supported by grants from the National Health and Medical Research Council (Grant #208950) and Retina Australia.     

Regional Deafferentiation Down-Regulates Subtypes of Glutamate Transporter Proteins

Abstract: Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by d -[³H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. d -[³H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and d -[³H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.     

兴奋性氨基酸转运体2在神经退行性变中的作用

谷氨酸是脑内必需的兴奋性神经递质之一,兴奋性氨基酸转运体（Excitatory amino acid transporterEAAT）2是最主要的谷氨酸转运体,负责脑内90%以上的谷氨酸再摄取,调节突触间隙的谷氨酸浓度。EAAT2功能紊乱导致胞外谷氨酸过量积聚,在多种神经退行性疾病的发病过程中起重要作用,如阿尔茨海默病、亨廷顿舞蹈病、肌萎缩侧索硬化等。对于人EAAT2启动子的研究发现,NF-kB在星形胶质细胞中对EAAT2表达起关键作用。通过筛选1 040种FDA批准的化合物,发现多种β-内酰胺类抗生素如头孢曲松钠等是EAAT2的转录激活剂,可以增加EAAT2的蛋白表达水平,产生神经保护作用。     

Functional Properties of the Retinal Glutamate Transporters GLT-1c and EAAT5

In the mammalian retina, glutamate uptake is mediated by members of a family of glutamate transporters known as “excitatory amino acid transporters (EAATs).” Here we cloned and functionally characterized two retinal EAATs from mouse, the GLT-1/EAAT2 splice variant GLT-1c, and EAAT5. EAATs are glutamate transporters and anion-selective ion channels, and we used heterologous expression in mammalian cells, patch-clamp recordings and noise analysis to study and compare glutamate transport and anion channel properties of both EAAT isoforms. We found GLT-1c to be an effective glutamate transporter with high affinity for Na⁺ and glutamate that resembles original GLT-1/EAAT2 in all tested functional aspects. EAAT5 exhibits glutamate transport rates too low to be accurately measured in our experimental system, with significantly lower affinities for Na⁺ and glutamate than GLT-1c. Non-stationary noise analysis demonstrated that GLT-1c and EAAT5 also differ in single-channel current amplitudes of associated anion channels. Unitary current amplitudes of EAAT5 anion channels turned out to be approximately twice as high as single-channel amplitudes of GLT-1c. Moreover, at negative potentials open probabilities of EAAT5 anion channels were much larger than for GLT-1c. Our data illustrate unique functional properties of EAAT5, being a low-affinity and low-capacity glutamate transport system, with an anion channel optimized for anion conduction in the negative voltage range.     

Regulation of glial glutamate transporters by C-terminal domains

Excitatory amino acid transporter 2 (EAAT2) is a high affinity glutamate transporter predominantly expressed in astroglia. Human EAAT2 encompasses eight transmembrane domains and a 74-amino acid C-terminal domain that resides in the cytoplasm. We examined the role of this region by studying various C-terminal truncations and mutations using heterologous expression in mammalian cells, whole-cell patch clamp recording and confocal imaging. Removal of the complete C terminus (K498X EAAT2) results in loss of function because of intracellular retention of truncated proteins in the cytoplasm. However, a short stretch of amino acids (E500X EAAT2) within the C terminus results in correctly processed transporters. E500X reduced glutamate transport currents by 90%. Moreover, the voltage and substrate dependence of E500X EAAT2 anion currents was significantly altered. WT and mutant EAAT2 anion channels are modified by external Na(+) in the presence as well as in the absence of L-glutamate. Whereas Na(+) stimulates EAAT2 anion currents in the presence of L-glutamate, increased [Na(+)] reduces such currents without glutamate. In cells internally dialyzed with Na(+), WT, and truncated EAAT2 display comparable Na(+) dependence. With K(+) as main internal cation, E500X drastically increased the apparent dissociation constant for external Na(+). The effects of E500X can be represented by a kinetic model that allows translocation of the empty transporter from the outward- to the inward-facing conformation and stabilization of the inward-facing conformation by internal K(+). Our results demonstrate that the C terminus modifies the glutamate uptake cycle, possibly affecting the movements of the translocation domain of EAAT2 glutamate transporter.     

Preconditioning with cortical spreading depression decreases intraischemic cerebral glutamate levels and down-regulates excitatory amino acid transporters EAAT1 and EAAT2 from rat cerebal cortex plasma membranes

We previously reported a 50% reduction in cortical infarct volume following transient focal cerebral ischemia in rats preconditioned 3 days earlier with cortical spreading depression (CSD). The mechanism of the protective effect of prior CSD remains unknown. Recent studies demonstrate reversal of excitatory amino acid transporters (EAATs) to be a principal cause for elevated extracellular glutamate levels during cerebral ischemia. The present study measured the effect of CSD preconditioning on (a) intraischemic glutamate levels and (b) regulation of glutamate transporters within the ischemic cortex of the rat. Three days following either CSD or sham preconditioning, rats were subjected to 200 min of focal cerebral ischemia, and extracellular glutamate concentration was measured by in vivo microdialysis. Cortical glutamate exposure decreased 70% from 1,772.4 +/- 1,469.2 microM-min in sham-treated (n = 8) to 569.0 +/- 707.8 microM-min in CSD-treated (n = 13) rats (p <0.05). The effect of CSD preconditioning on glutamate transporter levels in plasma membranes (PMs) prepared from rat cerebral cortex was assessed by western blot analysis. Down-regulation of the glial glutamate transporter isoforms EAAT2 and EAAT1 from the PM fraction was observed at 1, 3, and 7 days but not at 0 or 21 days after CSD. Semiquantitative lane analysis showed a maximal decrease of 90% for EAAT2 and 50% for EAAT1 at 3 days post-CSD. The neuronal isoform EAAT3 was unaffected by CSD. This period of down-regulation coincides with the time frame reported for induced ischemic tolerance. These data are consistent with reversal of glutamate transporter function contributing to glutamate release during ischemia and suggest that down-regulation of these transporters may contribute to ischemic tolerance induced by CSD.     

Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down-regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron-astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron-glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.     

EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase

EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.     

Interactions between glutamate transporters and metabotropic glutamate receptors at excitatory synapses in the cerebellar cortex

Five glutamate transporter genes have been identified; two of these (EAAT3 and EAAT4) are expressed in neurons and are predominantly confined to the membranes of cell bodies and dendrites. At an ultrastructural level, glutamate transporters have been shown to surround excitatory synapses in hippocampus and cerebellum [J. Neurosci. 18 (1998) 3606; J. Comp. Neurol. 418 (2000) 255]. This pattern of localization overlaps the well-described perisynaptic distribution of Group I metabotropic glutamate receptors or mGluRs [Neuron 11 (1993) 771; J. Chem. Neuroanat. 13 (1997) 77]. Both of the principal excitatory synaptic inputs to cerebellar Purkinje neurons, the parallel fiber (PF) and climbing fiber (CF) synapses, express mGluR-dependent forms of synaptic plasticity [Nat. Neurosci. 4 (2001) 467]. Prompted by the colocalization of postsynaptic glutamate transporters and mGluRs, we have examined whether glutamate uptake limits mGluR-mediated signals and mGluR-dependent forms of plasticity at PF and CF synapses in cerebellar slices. We find that, at PF and, surprisingly also at CF synapses, mGluR activation generates a slow synaptic current and triggers intracellular calcium release. At both PF and CF synapses, mGluR responses are strongly limited by glutamate transporters under resting conditions and are facilitated by short trains of stimuli. Nearly every Purkinje neuron expresses an mGluR-mediated synaptic current upon inhibition of glutamate transport. Global applications of glutamate achieved by photolysis of chemically caged glutamate yield similar results and argue that the colocalized transporters can effectively limit glutamate access to the mGluRs even in the face of such a large amount of transmitter. We hypothesize that neuronal glutamate transporters and Group I mGluRs located in the perisynaptic space interact to sense and then regulate the amount of glutamate escaping excitatory synapses. This hypothesis is currently being tested using electrophysiological methods and the introduction of optically tagged glutamate transporter proteins. In the brain, synaptic signals are terminated mainly by neurotransmitter transporters. Families of genes encoding transporters for the major neurotransmitters (dopamine, GABA, glutamate, glycine, norepinephrine and 5-HT) have been identified. Although transporters serve as targets for important classes of therapeutic drugs (e.g. selective serotonin reuptake inhibitors) and drugs of abuse (amphetamine, cocaine), little is known about how they operate at a molecular level or contribute to synaptic transmission.     

Up-regulation of serum- and glucocorticoid-induced protein kinase 1 in the brain tissue of human and experimental epilepsy

Several studies have shown that serum- and glucocorticoid-induced protein kinase 1(SGK1) can regulate both glutamate receptors and glutamate transporters and may participate in the regulation of neuroexcitability in neuronal diseases. In our previous study, we analyzed differential gene expression in the anterior temporal neocortex of drug-refractory epilepsy patients relative to control patients using a complementary DNA microarray and found that the SGK1 gene was up-regulated more than twofold in the brain tissues of epileptic patients. In the current study, we measured SGK1 expression in the brain tissues of humans and in an experimental model of rat epilepsy in order to explore the relationship between SGK1 expression and epilepsy. The SGK1 expression was detected in thirty human brain tissues derived from patients undergoing operation for drug-refractory epilepsy and was also detected in eight samples from autopsies. Meanwhile, we investigated SGK1 expression during the epileptic process in rats using immunofluorescence, RT-PCR and western blot analysis. SGK1 expression was enhanced in the temporal neocortex of patients with drug-refractory epilepsy and was also highly expressed in the rat brain during different phases of the epileptic process. SGK1 expression was also related with the elevation of EAAT3, which expression reduced after knockdown SGK1. These results provide new insight into the potential role of SGK1 in the pathophysiology of epilepsy.