Functional glutamate transporters are expressed in the carotid chemoreceptor.

BACKGROUND: The carotid body (CB) plays a critical role in cyclic intermittent hypoxia (CIH)-induced chemosensitivity; however, the underlying mechanism remains uncertain. We have demonstrated the presence of multiple inotropic glutamate receptors (iGluRs) in CB, and that CIH exposure alters the level of some iGluRs in CB. This result implicates glutamatergic signaling in the CB response to hypoxia. The glutamatergic neurotransmission is not only dependent on glutamate and glutamate receptors, but is also dependent on glutamate transporters, including vesicular glutamate transporters (VGluTs) and excitatory amino acid transporters (EAATs). Here, we have further assessed the expression and distribution of VGluTs and EAATs in human and rat CB and the effect of CIH exposure on glutamate transporters expression. METHODS: The mRNA of VGluTs and EAATs in the human CB were detected by RT-PCR. The protein expression of VGluTs and EAATs in the human and rat CB were detected by Western blot. The distribution of VGluT3, EAAT2 and EAAT3 were observed by immunohistochemistry staining and immunofluorescence staining. Male Sprague-Dawley (SD) rats were exposed to CIH (FIO2 10-21%, 3 min/3 min for 8 h per day) for 2 weeks. The unpaired Student's t-test was performed. RESULTS: Here, we report on the presence of mRNAs for VGluT1-3 and EAAT1-3 in human CB, which is consistent with our previous results in rat CB. The proteins of VGluT1 and 3, EAAT2 and 3, but not VGluT2 and EAAT1, were detected with diverse levels in human and rat CB. Immunostaining showed that VGluT3, the major type of VGluTs in CB, was co-localized with tyrosine hydroxylase (TH) in type I cells. EAAT2 and EAAT3 were distributed not only in type I cells, but also in glial fibrillary acidic protein (GFAP) positive type II cells. Moreover, we found that exposure of SD rats to CIH enhanced the protein level of EAAT3 as well as TH, but attenuated the levels of VGluT3 and EAAT2 in CB. CONCLUSIONS: Our study suggests that glutamate transporters are expressed in the CB, and that glutamate transporters may contribute to glutamatergic signaling-dependent carotid chemoreflex to CIH.

GLT-1 Upregulation as a Potential Therapeutic Target for Ischemic Brain Injury.

Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system, which plays an important role in many aspects of normal brain function such as neural development, motor functions, learning and memory etc. However, excessive accumulation of glutamate in the extracellular fluid will induce excitotoxicity which is considered to be a major mechanism of cell death in brain ischemia. There is no enzyme to decompose the glutamate in extracellular fluid, so extracellular glutamate homeostasis within the central nervous system is mainly regulated by the uptake activity of excitatory amino acid transporters. Among the five excitatory amino acid transporters, glial glutamate transporter-1 (GLT-1) is responsible for 90% of total glutamate uptake. Thus, GLT-1 is essential for maintaining the appropriate level of extracellular glutamate, and then limiting excitotoxicity of glutamate in central nervous system. Therefore, the regulation of GLT-1 might be a potential therapeutic target for ischemic brain injury. This review summarizes recent advances including our findings in the methods or medicine that could protect neurons against brain ischemic injury via upregulation of GLT-1 and discuss the possible application of these strategies.

Mesenchymal stem cells deliver exogenous miRNAs to neural cells and induce their differentiation and glutamate transporter expression.

MicroRNAs (miRNAs) are potential therapeutic targets in a variety of pathological conditions in the brain; however, their clinical application is hampered by lack of efficient delivery modes. Mesenchymal stromal stem cells (MSCs) migrate to sites of injury and inflammation and exert therapeutic effects in various neurological disorders. Here, we examined the ability of MSCs to deliver exogenous miRNA mimics and pre-miRNAs to human neural progenitor cells (NPCs) and astrocytes and characterized the functional impact of this delivery. We found that MSCs efficiently delivered fluorescent-labeled miR-124 and miR-145 mimics to cocultured NPCs and astrocytes. We further demonstrated the delivery of the miRNAs using novel reporter plasmids that contain a sequence complementary to miR-124 or miR-145 downstream of luciferase or mCherry. Binding of the specific miRNAs to these sequences results in decreased luciferase activity or mCherry fluorescence and therefore enable analysis of miRNA delivery in living cells. The delivered exogenous miR-124 significantly decreased the expression of the target gene Sox9 by targeting its 3'-UTR, and increased the neuronal differentiation of the NPCs. In addition, the delivered miR-124 increased the expression of the glutamate transporters, EAAT1 in NPCs and EAAT2 in both NPCs and astrocytes. Similar results were obtained with MSCs transfected with pre-miR-124. The miRNA delivery was mediated by MSC-derived exosomes and was cell contact independent. These results suggest that MSCs can functionally deliver exogenous miRNAs to neural cells and provide an efficient route of therapeutic miRNA delivery to the brain in pathological conditions with clinical implications for regenerative medicine.

Exploring effects of EAAT polymorphisms on cognitive functions in schizophrenia.

AIM: To evaluate the effect of functional polymorphisms (rs4354668 and rs2731880) of the excitatory amino acid transporters (EAAT1 and 2) on the cognitive dysfunction that characterizes schizophrenia. MATERIALS & METHODS: One hundred and ninety two subjects diagnosed with schizophrenia were assessed with Brief Assessment of Cognition in Schizophrenia, Wisconsin Card Sorting Test, Continuous Performance Test and N-back test and genotyped for rs4354668 and rs2731880. RESULTS: ANOVA showed a significant difference among both EAAT1 and EAAT2 genotype groups on different cognitive measures. Worse performances were observed among carriers of the genotypes associated with lower EAAT expression. CONCLUSION: RESULTS suggest that impaired activity and EAAT expression could influence cognitive performances in schizophrenia, thus representing a target of interest for development of pharmacological strategies aimed to improve cognition.

Expression of astrocytic genes coding for proteins implicated in neural excitation and brain edema is altered after acute liver failure.

In vitro and in vivo studies have suggested that reduced astrocytic uptake of neuronally released glutamate, alterations in expression of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP-4) contribute to brain edema in acute liver failure (ALF). However, there is no evidence to date to suggest that these alterations occur in patients with ALF. We analyzed the mRNA expression of excitatory amino acid transporters (EAAT-1, EAAT-2), GFAP, and AQP-4 in the cerebral cortex obtained at autopsy from eight patients with ALF and from seven patients with no evidence of hepatic or neurological disorders by real-time PCR, and protein expression was assessed using immunoblotting and immunohistochemistry. We demonstrated a significant decrease in GFAP mRNA and protein levels in ALF patients compared to controls. While the loss of EAAT-2 protein in ALF samples was post-translational in nature, EAAT-1 protein remained within normal limits. Immunohistochemistry confirmed that, in all cases, the losses of EAAT-2 and GFAP were uniquely astrocytic in their localization. AQP-4 mRNA expression was significantly increased and its immunohistochemistry demonstrated increased AQP-4 immunoreactivity in the glial end-feet process surrounding the microvessels. These findings provide evidence of selective alterations in the expression of genes coding for key astrocytic proteins implicated in central nervous system (CNS) excitability and brain edema in human ALF. We investigated the gene expression of astrocytic proteins involved in astrocyte swelling causing brain edema in autopsied brain tissues of patients with acute liver failure. This study demonstrated loss of GFAP expression and up-regulation of AQP-4 protein expression leading to cerebral edema, and loss of EAAT-2 expression implicated in excitatory neurotransmission. These findings may provide new drug targets against CNS complications of acute liver failure.

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 deficits in pontine locus coeruleus astrocytes in men with major depressive disorder.

BACKGROUND: Norepinephrine and glutamate are among several neurotransmitters implicated in the neuropathology of major depressive disorder (MDD). Glia deficits have also been demonstrated in people with MDD, and glia are critical modulators of central glutamatergic transmission. We studied glia in men with MDD in the region of the brain (locus coeruleus; LC) where noradrenergic neuronal cell bodies reside and receive glutamatergic input. METHODS: The expression of 3 glutamate-related genes (SLC1A3, SLC1A2, GLUL) concentrated in glia and a glia gene (GFAP) were measured in postmortem tissues from men with MDD and from paired psychiatrically healthy controls. Initial gene expression analysis of RNA isolated from homogenized tissue (n = 9-10 pairs) containing the LC were followed by detailed analysis of gene expressions in astrocytes and oligodendrocytes (n = 6-7 pairs) laser captured from the LC region. We assessed protein changes in GFAP using immunohistochemistry and immunoblotting (n = 7-14 pairs). RESULTS: Astrocytes, but not oligodendrocytes, demonstrated robust reductions in the expression of SLC1A3 and SLC1A2, whereas GLUL expression was unchanged. GFAP expression was lower in astrocytes, and we confirmed reduced GFAP protein in the LC using immunostaining methods. LIMITATIONS: Reduced expression of protein products of SLC1A3 and SLC1A2 could not be confirmed because of insufficient amounts of LC tissue for these assays. Whether gene expression abnormalities were associated with only MDD and not with suicide could not be confirmed because most of the decedents who had MDD died by suicide. CONCLUSION: Major depressive disorder is associated with unhealthy astrocytes in the noradrenergic LC, characterized here by a reduction in astrocyte glutamate transporter expression. These findings suggest that increased glutamatergic activity in the LC occurs in men with MDD.

Abnormal expression of glutamate transporters in temporal lobe areas in elderly patients with schizophrenia.

Glutamate transporters facilitate the buffering, clearance and cycling of glutamate and play an important role in maintaining synaptic and extrasynaptic glutamate levels. Alterations in glutamate transporter expression may lead to abnormal glutamate neurotransmission contributing to the pathophysiology of schizophrenia. In addition, alterations in the architecture of the superior temporal gyrus and hippocampus have been implicated in this illness, suggesting that synapses in these regions may be remodeled from a lifetime of severe mental illness and antipsychotic treatment. Thus, we hypothesize that glutamate neurotransmission may be abnormal in the superior temporal gyrus and hippocampus in schizophrenia. To test this hypothesis, we examined protein expression of excitatory amino acid transporter 1-3 and vesicular glutamate transporter 1 and 2 in subjects with schizophrenia (n=23) and a comparison group (n=27). We found decreased expression of EAAT1 and EAAT2 protein in the superior temporal gyrus, and decreased EAAT2 protein in the hippocampus in schizophrenia. We didn't find any changes in expression of the neuronal transporter EAAT3 or the presynaptic vesicular glutamate transporters VGLUT1-2. In addition, we did not detect an effect of antipsychotic medication on expression of EAAT1 and EAAT2 proteins in the temporal association cortex or hippocampus in rats treated with haloperidol for 9 months. Our findings suggest that buffering and reuptake, but not presynaptic release, of glutamate is altered in glutamate synapses in the temporal lobe in schizophrenia.

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.

Plasma membrane and vesicular glutamate transporter expression in chromaffin cells of bovine adrenal medulla.

The study of the functional expression of glutamate signaling molecules in peripheral tissues has received relatively little attention. However, evidence is increasing for a role of glutamate as an extracellular signal mediator in endocrine systems, in addition to having an excitatory amino acid neurotransmitter role in the CNS. Chromaffin cells are good models of catecholaminergic neurons, in which previous work from our group demonstrated the existence of both functional glutamate receptors and specific exocytotic and nonexocytotic glutamate release. In this work, the presence of specific plasma membrane (EAATs) and vesicular glutamate (VGLUTs) transporters has been investigated by using confocal microscopy, flow cytometric analysis, Western blot, and qRT-PCR techniques. We found specific expression of EAAT3, EAAT2, VGLUT1, and VGLUT3 in about 95%, 65%, 55%, and 25%, respectively, of the whole chromaffin cell population. However, chromaffin cells do not express VGLUT2 and have a very low expression of EAAT1. VGLUTs are localized mainly in the membrane fraction, and EAATs share their subcellular location between membrane and cytosolic fractions. Their estimated molecular weights were about 70 kDa for EAAT2, about 65 kDa for EAAT3, about 50 kDa for VGLUT1, and about 60 kDa for VGLUT3. RT-qPCR techniques confirm the expression of these glutamate transporters at the mRNA level and show a different regulation by cytokines and glucocorticoids between VGLUT1 and -3 and EAAT2 and -3 subfamilies. These interesting results support the participation of these glutamate transporters in the process of glutamate release in chromaffin cells and in the regulation of their neurosecretory function in adrenal medulla.

The role of excitatory amino acid transporters in cerebral ischemia.

Glutamate is an excitatory neurotransmitter that plays a major role in the pathogenesis of ischemia brain injury. The regulation of glutamate neurotransmission is carried out by excitatory amino acid transporters (EAATs) that act through reuptake of glutamate into cells. EAATs may also release glutamate into the extracellular space in a calcium-independent manner during ischemia and dysfunction of EAATs is specifically implicated in the pathology of cerebral ischemia. Recent studies show that up-regulation of EAAT2 provides neuroprotection during ischemic insult. This review summarizes current knowledge regarding the role of EAATs in cerebral ischemia.

The effects of alcoholism on the human basolateral amygdala.

Alcohol affects gene expression in several brain regions. The amygdala is a key structure in the brain's emotional system and in recent years the crucial importance of the amygdala in drug-seeking and relapse has been increasingly recognized. In this study gene expression screening was used to identify genes involved in alcoholism in the human basolateral amygdala of male patients. The results show that alcoholism affects a broad range of genes and many systems including genes involved in synaptic transmission, neurotransmitter transport, structural plasticity, metabolism, energy production, transcription and RNA processing and the circadian cycle. In particular, genes involved in the glutamate system were affected in the alcoholic patients. In the amygdala the glutamate system is involved in the acquisition, consolidation, expression and extinction of associative learning, which is a vital part of addiction, and in alcohol abusers it is associated with withdrawal anxiety and neurodegeneration. Downregulation of the excitatory amino acid transporters GLAST, GLT-1 and the AMPA glutamate receptor 2 (GluR2) revealed by the microarray were confirmed by Western blots. The decreased expression of GLAST, GLT-1 and GluR2 in the alcoholic patients may increase glutamate tone and activity in the basolateral amygdala and this may contribute to neurodegeneration as well as the expression of associative memories and anxiety which underlie continued drug-seeking and chronic relapse.

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.

Human herpesvirus 6 (HHV-6) induces dysregulation of glutamate uptake and transporter expression in astrocytes.

Human herpesvirus 6 (HHV-6) infects and establishes latency in the central nervous system (CNS). Reactivation of latent HHV-6 has been associated with neurologic diseases including epilepsy and multiple sclerosis (MS). In vivo, HHV-6 has been localized to astrocytes and can infect human astrocytes in vitro, suggesting that this virus may have a tropism for glial cells and may affect glial cell function. An essential role of astrocytes in the CNS is active maintenance of the excitatory neurotransmitter glutamate. Dysregulation of glutamate has been implicated as a potential mechanism of disease in both epilepsy and MS. Both disorders have demonstrated elevated glutamate in CSF and may be associated with dysregulation of glutamate signaling, uptake, and metabolism. This study demonstrates dysregulation of glutamate uptake in human astrocytes infected with both variants of HHV-6, A and B, with differential effects of HHV-6 in acute and persistently infected cells. Whereas astrocytes acutely infected with HHV-6 demonstrated increased glutamate uptake, cells persistently infected with HHV-6A and HHV-6B demonstrated impaired glutamate uptake. Functional dysregulation of glutamate uptake was associated with early increases in mRNA and protein expression of the glial glutamate transporter EAAT-2 followed by a sustained decrease in mRNA expression in astrocytes infected with both HHV-6A and HHV-6B. Dysregulated glutamate uptake and transporter expression suggests a mechanism for dysregulation of glutamate levels in vivo and a potential mechanism for virus-associated neurologic disease.

A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke.

It remains unclear why some individuals are susceptible to excitotoxicity after stroke. A possible explanation is impaired glutamate uptake. We have found a highly prevalent polymorphism in the promoter of the glutamate transporter EAAT2 gene that abolishes a putative regulatory site for activator protein-2 (AP-2) and creates a new consensus binding site for the repressor transcription factor GC-binding factor 2 (GCF2). The mutant genotype is associated with increased plasma glutamate concentrations and with a higher frequency of early neurological worsening in human stroke. After transfection into astrocytes, the mutant promoter was not activated by AP-2 and was effectively repressed by GCF2, and its activity in the presence of GCF2 was reduced when compared with the AP-2-cotransfected wild-type promoter. We also show that GCF2 is expressed in ischemic rat brain, suggesting that decreased glutamate uptake occurs in individuals carrying the mutation after stroke. These findings may explain individual susceptibility to excitotoxic damage after stroke as well as the failure of glutamate antagonists in those patients without this polymorphism.

Expression of excitatory amino acid transporter interacting protein transcripts in the thalamus in schizophrenia.

The excitatory amino acid transporters (EAATs) are a family of plasma membrane proteins that maintain synaptic glutamate concentration by removing glutamate from the synaptic cleft. EAATs are expressed by glia (EAAT1 and EAAT2) and neurons (EAAT3 and EAAT4) throughout the brain. Glutamate reuptake is regulated, in part, by EAAT-interacting proteins that modulate subcellular localization and glutamate transport activity of the EAATs. Several lines of investigation support the hypothesis of glutamatergic abnormalities in schizophrenia. Previous work in our laboratory demonstrated increased expression of EAAT1 and EAAT2 transcripts in the thalamus, suggesting that alterations in synaptic glutamate levels may contribute to the pathophysiology of schizophrenia. Since EAAT-interacting proteins regulate EAAT function, directly impacting glutamatergic neurotransmission, we hypothesized that expression of EAAT-interacting proteins may also be altered in schizophrenia. Using in situ hybridization in subjects with schizophrenia and a comparison group, we detected increased expression of JWA and KIAA0302, molecules that regulate EAAT3 and EAAT4, respectively, in the thalamus in schizophrenia. In contrast, we did not find changes in the expression of transcripts for the EAAT2 and EAAT4 regulatory proteins GPS-1 and ARHGEF11. To address prior antipsychotic treatment in our schizophrenic subjects, we treated rats with haloperidol and clozapine for 4 weeks, and found changes in transcript expression of the EAAT-interacting proteins in clozapine-, but not haloperidol-, treated rats. These findings suggest that proteins associated with the regulation of glutamate reuptake may be abnormal in this illness, supporting the hypothesis of altered thalamic glutamatergic neurotransmission in schizophrenia.

Cellular distribution of glutamate transporters in the gastrointestinal tract of mice: an immunohistochemical and in situ hybridization approach.

L-Glutamate transport by intestinal epithelial cells is an initial step of the entire glutamate metabolism pathway in the gut mucosa. The present study examined the cellular distribution of glutamate transporters in the digestive tract of adult mice using immunohistochemistry and in situ hybridization technique. Expression of EAAC1 mRNA was more intense in the ileum, where the epithelium in crypts and the basal half of intestinal villi showed high levels of transcripts, suggesting an essential role of EAAC1 in differentiating or premature epithelial cells. Electron-microscopically, EAAC1 immunoreactivity was predominantly localized in the striated border of enterocytes. Immunoreactivity for GLT-1 was found in the lateral membrane of epithelial cells at the bottom of gastric glands and at the intestinal crypts, and also in the lateral membrane of secretory cells at the duodenal gland. GLAST immunoreactivity was restricted to the fundic and pyloric glands, and was especially intense in the neck portion of both glands. However, in situ hybridization analysis failed to confirm the expression of GLT-1 and GLAST at the mRNA level, possibly due to limited sensitivity. The strong and specific luminal localization of EAAC1 in the intestinal epithelium suggests that EAAC1 is a predominant transporter of glutamate, at least in the lower part of the small intestine.