Nuclear factor erythroid 2-related factor 2 facilitates neuronal glutathione synthesis by upregulating neuronal excitatory amino acid transporter 3 expression.

Astrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression.

Regulation of the glutamate transporter EAAT3 by mammalian target of rapamycin mTOR.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is stimulated by insulin, growth factors and nutrients and confers survival of several cell types. The kinase has previously been shown to stimulate amino acid uptake. In neurons, the cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus confers protection against excitotoxicity. In epithelia, EAAT3 accomplishes transepithelial glutamate and aspartate transport. The present study explored, whether mTOR regulates EAAT3 (SLC1A1). To this end, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without cRNA encoding mTOR and the glutamate induced current (I(glu)), a measure of glutamate transport, determined by dual electrode voltage clamp. Moreover, EAAT3 protein abundance was determined utilizing chemiluminescence. As a result, I(glu) was observed in Xenopus oocytes expressing EAAT3 but not in water injected oocytes. Coexpression of mTOR significantly increased I(glu), an effect reversed by rapamycin (100 nM). mTOR coexpression increased EAAT3 protein abundance in the cell membrane. The decay of I(glu) following inhibition of carrier insertion with brefeldin A in oocytes coexpressing EAAT3 with mTOR was similar in the presence and absence of rapamycin (100 nM). In conclusion, mTOR is a novel powerful regulator of EAAT3 and may thus contribute to protection against neuroexcitotoxicity.

Effects of pregabalin on the activity of glutamate transporter type 3.

BACKGROUND: Pregabalin, (S)-3-aminomethyl-5-methyl hexanoic acid, is a ligand for the α2δ subunit (a component of voltage-gated calcium channels) and has analgesic and anticonvulsant properties. Glutamate uptake by glutamate transporters may be a mechanism for these properties. We investigated the effects of pregabalin on the activity of the neuronal glutamate transporter type 3 (EAAT3). METHODS: EAAT3 was expressed in Xenopus laevis oocytes. Two-electrode voltage clamping was used to record membrane currents before, during, and after applying l-glutamate (30 µM) in the presence or absence of pregabalin. Currents were also measured in oocytes pretreated with a protein kinase C (PKC) activator (phorbol-12-myristate-13-acetate, PMA), PKC inhibitors (chelerythrine or staurosporine), or a phosphatidylinositol-3-kinase (PI3K) inhibitor wortmannin. RESULTS: The exposure of the oocytes injected with EAAT3 mRNA to serial concentrations of pregabalin (0.06-60 µM) significantly increased their responses to 30 µM l-glutamate. A kinetic study showed that pregabalin significantly increased V(max) without changing K(m). Treatment of oocytes with PMA, pregabalin, or pregabalin plus PMA significantly increased transporter currents vs controls, but treatment with PMA plus pregabalin did not increase the responses further vs PMA or pregabalin alone. In addition, pretreatment of oocytes with two PKC inhibitors (chelerythrine or staurosporine), or inhibitor wortmannin, significantly reduced basal and pregabalin-enhanced EAAT3 activity. CONCLUSIONS: Pregabalin increased EAAT3 activity and PKC and PI3K were involved. This may explain the analgesic effect of pregabalin in neuropathic pain.

Regulation of glutamate transporter GLT-1 by MAGI-1.

The sodium-dependent glutamate transporter, glutamate transporter subtype 1 (GLT-1) is one of the main glutamate transporters in the brain. GLT-1 contains a COOH-terminal sequence similar to one in an isoform of Slo1 K(+) channel protein previously shown to bind MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1). MAGI-1 is a scaffold protein which allows the formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. The glutathione S-transferase pull-down assay demonstrated that MAGI-1 was a binding partner of GLT-1. The interaction between MAGI-1 and GLT-1 was confirmed by co-immunoprecipitation. Immunofluorescence of MAGI-1 and GLT-1 demonstrated that the distribution of MAGI-1 and GLT-1 overlapped in astrocytes. Co-expression of MAGI-1 with GLT-1 in C6 Glioma cells resulted in a significant reduction in the surface expression of GLT-1, as assessed by cell-surface biotinylation. On the other hand, partial knockdown of endogenous MAGI-1 expression by small interfering RNA in differentiated cultured astrocytes increased glutamate uptake and the surface expression of endogenous GLT-1. Knockdown of MAGI-1 increased dihydrokainate-sensitive, Na(+) -dependent glutamate uptake, indicating that MAGI-1 regulates GLT-1 mediated glutamate uptake. These data suggest that MAGI-1 regulates surface expression of GLT-1 and the level of glutamate in the hippocampus.

Group I mGluR-regulated translation of the neuronal glutamate transporter, excitatory amino acid carrier 1.

Recently, we demonstrated that mRNA for the neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), is found in dendrites of hippocampal neurons in culture and in dendrites of hippocampal pyramidal cells after pilocarpine-induced status epilepticus (SE). We also showed that SE increased the levels of EAAC1 mRNA ~15-fold in synaptoneurosomes. In this study, the effects of SE on the distribution EAAC1 protein in hippocampus were examined. In addition, the effects of Group 1 mGluR receptor activation on the levels of EAAC1 protein were examined in synaptoneurosomes prepared from sham control animals and from animals that experience pilocarpine-induced SE. We find that EAAC1 immunoreactivity increases in pyramidal cells of the hippocampus after 3 h of SE. In addition, the group I mGluR agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), caused an increase in EAAC1 protein levels in hippocampal synaptoneurosomes; this effect of DHPG was much larger (~3- to 5-fold) after 3 h of SE. The DHPG-induced increases in EAAC1 protein were blocked by two different inhibitors of translation but not by inhibitors of transcription. mGluR1 or mGluR5 antagonists completely blocked the DHPG-induced increases in EAAC1 protein. DHPG also increased the levels of glutamate receptor 2/3 protein, but this effect was not altered by SE. The DHPG-induced increase in EAAC1 protein was blocked by an inhibitor of the mammalian target of rapamycin or an inhibitor of extracellular signal-regulated kinase. These studies provide the first evidence EAAC1 translation can be regulated, and they show that regulated translation of EAAC1 is up-regulated after SE.

Evidence for the interaction of COX-2 with mGluR5 in the regulation of EAAT1 and EAAT3 protein levels in the mouse hippocampus. The influence of oxidative stress mechanisms.

Since we found that inhibition of cyclooxygenase-2 (COX-2) with concomitant application of a metabotropic glutamate receptor subtype 5 (mGluR5) antagonist (MTEP) down-regulates mGluR7 in the hippocampus (HC) and changes behavior of mice, our team decided to investigate the mechanism responsible for the observed changes. The amino acid glutamate (Glu) is a major excitatory neurotransmitter in the brain. Glu uptake is regulated by excitatory amino acid transporters (EAAT). There are five transporters with documented expression in neurons and glia in the central nervous system (CNS). EAATs, maintain the correct transmission of the Glu signal and prevent its toxic accumulation by removing Glu from the synapse. It has been documented that the toxic level of Glu is one of the main causes of mental and cognitive abnormalities. Given the above mechanisms involved in the functioning of the Glu synapse, we hypothesized modification of Glu uptake, involving EAATs as the cause of the observed changes. This study investigated the level of selected EAATs in the HC after chronic treatment with mGluR5 antagonist MTEP, NS398, and their combination using Western blot. Concomitant MTEP treatment with NS398 or a single administration of the above causes changes in LTP and modulation of EAAT levels in mouse HC. As EAATs are cellular markers of oxidative stress mechanisms, the E. coli lipopolysaccharide (LPS) challenge was performed. The modified Barnes maze test (MBM) revealed alterations in the mouse spatial learning abilities. This study reports an interaction between the mGluR5 and COX-2 in the HC, with EAAT1 and EAAT3 involvement.

The ATRA-dependent overexpression of the glutamate transporter EAAC1 requires RARbeta induction.

The mechanisms underlying trafficking and membrane targeting of EAAC1, the rodent counterpart of the human EAAT3 carrier for anionic amino acids, are well characterized. In contrast, much less is known on the regulation of Slc1a1, the gene that encodes for the transporter. We have recently found that all-trans retinoic acid (ATRA) stimulates EAAC1 expression and anionic amino acid transport in C6 rat glioma cells. We report here that the ATRA effect on EAAC1 activity was inhibited by the specific RAR antagonist LE540 and mimicked by Am80, a RAR agonist, but not by the RXR agonist HX630. Moreover, the ATRA-dependent induction of Slc1a1 mRNA required the synthesis of a protein intermediate and was not associated with changes in the messenger half-life. ATRA treatment induced the expression of both Rarb mRNA and RARbeta protein several hours before the induction of Slc1a1, while the mRNA for RFX1, a transcription factor recently involved in Slc1a1 transcription, was unchanged. In addition, Rarb silencing markedly inhibited the ATRA-dependent increase of both Rarb and Slc1a1 mRNAs. We conclude that in C6 glioma cells the induction of Slc1a1 by ATRA requires the synthesis of RARbeta, suggesting that the receptor is involved in the regulation of the transporter gene.

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology.

VPS10P domain receptors emerge as central regulators of intracellular protein sorting in neurons with relevance for various brain pathologies. Here, we identified a role for the family member SorCS2 in protection of neurons from oxidative stress and epilepsy-induced cell death. We show that SorCS2 acts as sorting receptor that sustains cell surface expression of the neuronal amino acid transporter EAAT3 to facilitate import of cysteine, required for synthesis of the reactive oxygen species scavenger glutathione. Lack of SorCS2 causes depletion of EAAT3 from the plasma membrane and impairs neuronal cysteine uptake. As a consequence, SorCS2-deficient mice exhibit oxidative brain damage that coincides with enhanced neuronal cell death and increased mortality during epilepsy. Our findings highlight a protective role for SorCS2 in neuronal stress response and provide a possible explanation for upregulation of this receptor seen in surviving neurons of the human epileptic brain.

Plasma membrane expression of the neuronal glutamate transporter EAAC1 is regulated by glial factors: evidence for different regulatory pathways associated with neuronal maturation.

At the glutamatergic synapse the neurotransmitter is removed from the synaptic cleft by high affinity amino acid transporters located on neurons (EAAC1) and astrocytes (GLAST and GLT1), and a coordinated action of these cells is necessary in order to regulate glutamate extracellular concentration. We show here that treatment of neuronal cultures with glial soluble factors (GCM) is associated with a redistribution of EAAC1 and GLAST to the cell membrane and we analysed the effect of membrane cholesterol depletion on this regulation. In enriched neuronal culture (90% neurons and 10% astrocytes), GCM treatment for 10 days increases EAAC1 and GLAST cell surface expression with no change in total expression. In opposite, GLT1 surface expression is not modified by GCM but total expression is increased. When cholesterol is acutely depleted from the membrane by 10 mM methyl-beta-cyclodextrin (beta5-MCD, 30 min), glutamate transport activity and cell surface expressions of EAAC1 and GLAST are decreased in the enriched neuronal culture treated by GCM. In pure neuronal culture addition of GCM also increases EAAC1 cell membrane expression but surprisingly acute treatment with beta5-MCD decreases glutamate uptake activity but not EAAC1 cell membrane expression. By immunocytochemistry a modification in the distribution of EAAC1 within neurons was undetectable whatever the treatment but we show that EAAC1 was no more co localized with Thy-1 in the enriched neuronal culture treated by GCM suggesting that GCM have stimulated polarity formation in neurons, an index of maturation. In conclusion we suggest that different regulatory mechanisms are involved after GCM treatment, glutamate transporter trafficking to and from the plasma membrane in enriched neuronal culture and modulation of EAAC1 intrinsic activity and/or association with regulatory proteins at the cell membrane in the pure neuronal culture. These different regulatory pathways of EAAC1 are associated with different neuronal maturation stages.

Altered hippocampal expression of glutamate receptors and transporters in GRM2 and GRM3 knockout mice.

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, also called mGlu2 and mGlu3, encoded by GRM2 and GRM3, respectively) are therapeutic targets for several psychiatric disorders. GRM3 may also be a schizophrenia susceptibility gene. mGluR2-/- and mGluR3-/- mice provide the only unequivocal means to differentiate between these receptors, yet interpretation of in vivo findings may be complicated by secondary effects on expression of other genes. To address this issue, we examined the expression of NMDA receptor subunits (NR1, NR2A, NR2B) and glutamate transporters (EAAT1-3), as well as the remaining group II mGluR, in the hippocampus of mGluR2-/- and mGluR3-/- mice, compared with wild-type controls. mGluR2 mRNA was increased in mGluR3-/- mice, and vice versa. NR2A mRNA was increased in both knockout mice. EAAT1 (GLAST) mRNA and protein, and EAAT2 (GLT-1) protein, were reduced in mGluR3-/- mice, whereas EAAT3 (EAAC1) mRNA was decreased in mGluR2-/- mice. Transcripts for NR1 and NR2B were unchanged. The findings show a compensatory upregulation of the remaining group II metabotropic glutamate receptor in the knockout mice. Upregulation of NR2A expression suggests modified NMDA receptor signaling in mGluR2-/- and mGluR3-/- mice, and downregulation of glutamate transporter expression suggests a response to altered synaptic glutamate levels. The results show a mutual interplay between mGluR2 and mGluR3, and also provide a context in which to interpret behavioral and electrophysiological results in these mice.

The expression of glutamate transporters in chest compression-induced audiogenic epilepsy: a comparative study.

BACKGROUND: This study was conducted to compare the expression of three glutamate transporter subtypes (GLAST, GLT-1 and EAAC1) in rats undergoing chest compression-induced global cerebral ischemia in the presence and absence of cerebral ischemia-related epilepsy. MATERIAL AND METHODS: A reliable rat model of global cerebral ischemia-related epilepsy was established. The rats were divided into the following groups: sham surgery group (Group S), global cerebral ischemia without epilepsy (Group I) and global cerebral ischemia with epilepsy (Group E). The latter two groups were further divided into four subgroups based on time (24 hours, 72 hours, 5 days and 7 days) after 8 minutes of chest compression. Electroencephalographic recordings were obtained in all rats. Hippocampal tissue samples were prepared, and the expression of GLAST, GLT-1 and EAAC1 in the hippocampal CA1 region and the motor cortex area was studied using immunohistochemical methods. RESULTS: Seizure developed in 32 (64%) of 50 rats. Compared with that in group I, the expression of GLT-1 in the hippocampal CA1 region and the motor cortex area in group E was down-regulated, and EAAC1 was up-regulated in those regions. CONCLUSION: Altering the expression of GLT-1 and EAAC1 through some means might lead them to be potential targets for therapy in cerebral ischemia-related epilepsy.

EAAC1 glutamate transporter expression in the rat lithium-pilocarpine model of temporal lobe epilepsy.

Glutamate excitotoxicity has been involved in the pathophysiology of epilepsy. Normal functioning of glutamate transporters clears the synaptically released glutamate to prevent excitotoxic neuronal death. Using densitometric immunohistochemical analysis, we examined the temporal expression of the neuronal glutamate transporter (EAAC1) in the lithium-pilocarpine rat model of temporal lobe epilepsy. During the acute period of lithium-pilocarpine-induced status epilepticus, EAAC1 transporter expression increased in the pyramidal neurons of cornus ammonis (CA)1, CA2 and CA3 (fields of the hippocampus), in dentate gyrus (DG) granule cells and in olfactory tubercle (Tu). During the latent period, EAAC1 expression was strongly expressed in the DG granular and molecular layers, Tu, cerebral cortex and septum, and went back to control levels in CA1, CA2 and CA3 layers. The overexpression of EAAC1 occurred mainly in structures prone to develop Fluoro-Jade-B-positive degenerating neurons. It is, however, not clear to what extent the overexpression of EAAC1 contributes to epileptogenesis and in which area it may represent a preventive or compensatory or response to injury.

A dominant-negative variant of SNAP-23 decreases the cell surface expression of the neuronal glutamate transporter EAAC1 by slowing constitutive delivery.

A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1.

EAAC1 is expressed in rat and human prostate epithelial cells; functions as a high-affinity L-aspartate transporter; and is regulated by prolactin and testosterone.

BACKGROUND: Prostate epithelial cells accumulate a high level of aspartate that is utilized as a substrate for their unique function of production and secretion of enormously high levels of citrate. In most mammalian cells aspartate is synthesized; and, therefore is a non-essential amino acid. In contrast, in citrate-producing prostate cells, aspartate is an essential amino acid that must be derived from circulation. The prostate intracellular/extracellular conditions present a 40:1 concentration gradient. Therefore, these cells must possess a plasma membrane-associated aspartate uptake transport process to achieve their functional activity. In earlier kinetic studies we identified the existence of a unique Na+-dependent high-affinity L-aspartate transport process in rat prostate secretory epithelial cells. The present report is concerned with the identification of this putative L-aspartate transporter in rat and human prostate cells. RESULTS: The studies show for the first time that EAAC1 is expressed in normal rat prostate epithelial cells, in normal and hyperplastic human prostate glands, and in human malignant prostate cell lines. EAAC1 expression and high-affinity L-aspartate transport are correspondingly down-regulated by EAAC1 siRNA knock down. Exposure of prostate cells to physiological levels of prolactin or testosterone results in an up-regulation of EAAC1 expression and a corresponding increase in the high-affinity transport of L-aspartate into the cells. CONCLUSION: This study shows that EAAC1 functions as the high-affinity L-aspartate transporter that is responsible for the uptake and accumulation of aspartate in prostate cells. In other cells (predominantly excitable tissue cells), EAAC1 has been reported to function as a glutamate transporter rather than as an aspartate transporter. The regulation of EAAC1 expression and L-aspartate transport by testosterone and prolactin is consistent with their regulation of citrate production in prostate cells. The identification of EAAC1 as the high-affinity L-aspartate transporter now permits studies to elucidate the mechanism of hormonal regulation of EAAC1 gene expression, and to investigate the mechanism by which the cellular environment effects the functioning of EAAC1 as an aspartate transporter or as a glutamate transporter.

EAAT3 promotes amino acid transport and proliferation of porcine intestinal epithelial cells.

Excitatory amino acid transporter 3 (EAAT3, encoded by SLC1A1) is an epithelial type high-affinity anionic amino acid transporter, and glutamate is the major oxidative fuel for intestinal epithelial cells. This study investigated the effects of EAAT3 on amino acid transport and cell proliferation through activation of the mammalian target of the rapamycin (mTOR) pathway in porcine jejunal epithelial cells (IPEC-J2). Anionic amino acid and cystine (Cys) transport were increased (P<0.05) by EAAT3 overexpression and decreased (P<0.05) by EAAT3 knockdown rather than other amino acids. MTT and cell counting assays suggested that IPEC-J2 cell proliferation increased (P<0.05) with EAAT3 overexpression. Phosphorylation of mTOR (Ser2448), ribosomal protein S6 kinase-1 (S6K1, Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1, Thr70) was increased by EAAT3 overexpression and decreased by EAAT3 knockdown (P<0.05), as were levels of activating transcription factor 4 (ATF4) and cystine/glutamate antiporter (xCT) (P<0.05). Our results demonstrate for the first time that EAAT3 facilitates anionic amino acid transport and activates the mTOR pathway, promoting Cys transport and IPEC-J2 cell proliferation.

GABA and glutamate transporters are expressed in human platelets.

GABA and glutamate are the major neurotransmitters in the human central nervous system. Disturbances in these transmitter systems have been suggested to influence a variety of neurological and psychiatric diseases. Human platelets have been used as a model for neural amino acid transport, although it has not been known exactly which transporters participate in the transport process. In this study, we identify with reverse transcription-polymerase chain reaction (RT-PCR) BGT-1 and EAAT3 as transporters for GABA and glutamate, respectively. We also show that platelets contain transporters for dopamine, taurine and creatine. The cloning of these transporters confirms that blood platelets can be used as a model for neurotransmitter transport in the CNS.

Hypoxia-inducible factors enhance glutamate signaling in cancer cells.

Signaling through glutamate receptors has been reported in human cancers, but the molecular mechanisms are not fully delineated. We report that in hepatocellular carcinoma and clear cell renal carcinoma cells, increased activity of hypoxia-inducible factors (HIFs) due to hypoxia or VHL loss-of-function, respectively, augmented release of glutamate, which was mediated by HIF-dependent expression of the SLC1A1 and SLC1A3 genes encoding glutamate transporters. In addition, HIFs coordinately regulated expression of the GRIA2 and GRIA3 genes, which encode glutamate receptors. Binding of glutamate to its receptors activated SRC family kinases and downstream pathways, which stimulated cancer cell proliferation, apoptosis resistance, migration and invasion in different cancer cell lines. Thus, coordinate regulation of glutamate transporters and receptors by HIFs was sufficient to activate key signal transduction pathways that promote cancer progression.

Rhythmic oscillations of the microRNA miR-96-5p play a neuroprotective role by indirectly regulating glutathione levels.

Glutathione (GSH) is a key antioxidant that plays an important neuroprotective role in the brain. Decreased GSH levels are associated with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Here we show that a diurnal fluctuation of GSH levels is correlated with neuroprotective activity against oxidative stress in dopaminergic cells. In addition, we found that the cysteine transporter excitatory amino acid carrier 1 (EAAC1), which is involved in neuronal GSH synthesis, is negatively regulated by the microRNA miR-96-5p, which exhibits a diurnal rhythm. Blocking miR-96-5p by intracerebroventricular administration of an inhibitor increased the level of EAAC1 as well as that of GSH and had a neuroprotective effect against oxidative stress in the mouse substantia nigra. Our results suggest that the diurnal rhythm of miR-96-5p may play a role in neuroprotection by regulating neuronal GSH levels via EAAC1.

Chronic brain ischemia induces the expression of glial glutamate transporter EAAT2 in subcortical white matter.

Glutamate plays a central role in brain physiology and pathology. The involvement of excitatory amino acid transporters (EAATs) in neurodegenerative disorders including acute stroke has been widely studied, but little is known about the role of glial glutamate transporters in white matter injury after chronic cerebral hypoperfusion. The present study evaluated the expression of glial (EAAT1 and EAAT2) and neuronal (EAAT3) glutamate transporters in subcortical white matter and cortex, before and 3-28 days after the ligation of bilateral common carotid arteries (LBCCA) in rat brain. K-B staining showed a gradual increase of demyelination in white matter after ischemia, while there was no cortical involvement. Between 3 and 7 days after LBCCA, a significant increase in EAAT2 protein levels was observed in the ischemic brain and the number of EAAT2-positive cells also significantly increased both in the cortical and white matter lesions. EAAT2 was detected in glial-fibrillary acidic protein (GFAP)-positive astrocytes in both the cortex and white matter, but not in neuronal and oligodendroglial cells. EAAT1 was slightly elevated after ischemia only in the white matter, but EAAT3 was at almost similar levels both in the cortex and white matter after ischemia. A significant increase in EAAT2 expression level was also noted in the deep white matter of chronic human ischemic brain tissue compared to the control group. Our findings suggest important roles for up-regulated EAAT2 in chronic brain ischemia especially in the regulation of high-affinity of extracellular glutamate and minimization of white matter damage.

Gene expression of glutamate transporters SLC1A1, SLC1A3 and SLC1A6 in the cerebellar subregions of elderly schizophrenia patients and effects of antipsychotic treatment.

OBJECTIVES: The glutamatergic hypothesis of schizophrenia proposes alterations of excitatory amino acid transporters (solute carrier family, SLCs) expression and cerebellar dysfunctions. The influence of the neuregulin-1 (NRG1) risk genotype or effects of antipsychotics on expression of EAATs are unknown. METHODS: We compared post-mortem samples from the cerebellar hemispheres and vermis of 10 schizophrenia patients with nine normal subjects by investigating gene expression of SLC1A3, SLC1A1 and SLC1A6 by in-situ hybridization. We further assessed the allelic composition regarding the polymorphism rs35753505 (SNP8NRG221533) near the NRG1 gene. To control for effects due to antipsychotic treatment, we chronically treated rats with the antipsychotics haloperidol or clozapine and assessed gene expression of SLCs. RESULTS: Schizophrenia patients showed increased expression of SLC1A3 in the molecular layer of the vermis. Individuals carrying at least one C allele of rs35753505 (SNP8NRG221533) showed decreased expression of SLC1A6 in the molecular layer of both hemispheres, compared to individuals homozygous for the T allele. The animal model revealed suppression of SLC1A6 by clozapine. CONCLUSIONS: Increased SLC1A3 expression indicates facilitated transport and may result in reduced glutamate neurotransmission. Decreased SLC1A6 expression in NRG1 risk variant may be an adaptive effect to restore glutamate signalling, but treatment effects cannot be excluded.