Antimania-Like Effect of Panax ginseng Regulating the Glutamatergic Neurotransmission in REM-Sleep Deprivation Rats.

Previous studies have shown the therapeutic properties of ginseng and ginsenosides on hyperactive and impulsive behaviors in several psychiatric diseases. Herein, we investigated the effect of Panax ginseng Meyer (PG) on hyperactive/impulsive behaviors in a manic-like animal model, sleep deprivation (SD) rats. Male rats were sleep-deprived for 48 h, and PG (200 mg/kg) was administered for 4 days, from 2 days prior to the start of SD to the end date of SD. The elevated plus maze (EPM) test showed that PG alleviated the increased frequency of entries into and spent time within open arms by SD. In order to investigate the molecular mechanism on this effect of PG, we assessed differentially expressed genes (DEGs) in the prefrontal cortex of PG-treated SD rats using RNA sequencing (RNA-seq) and performed gene-enrichment analysis for DEGs. The gene-enrichment analysis showed that PG most prominently affected the glutamatergic synapse pathway. Among the glutamatergic synapse pathway genes, particularly, PG enhanced the expressions of glutamate transporter Slc1a3 and Slc1a2 reduced in SD rats. Moreover, we found that PG could inhibit the SD-induced phosphorylation of the NR2A subunit of the NMDA receptor. These results suggested that PG might have a therapeutic effect against the manic-like behaviors, regulating the glutamatergic neurotransmission.

Chronic Mild Stress Alters Kynurenine Pathways Changing the Glutamate Neurotransmission in Frontal Cortex of Rats.

Immune stimulation might be involved in the pathophysiology of major depressive disorder (MDD). This stimulation induces indoleamine 2,3-dioxygenase (IDO), an enzyme that reduces the tryptophan bioavailability to synthesize serotonin. IDO products, kynurenine metabolites, exert neurotoxic/neuroprotective actions through glutamate receptors. Thus, we study elements of these pathways linked to kynurenine metabolite activity examining whether antidepressants (ADs) can modulate them. Male Wistar rats were exposed to chronic mild stress (CMS), and some of them were treated with ADs. The expression of elements of the IDO pathway, including kynurenine metabolites, and their possible modulation by ADs was studied in the frontal cortex (FC). CMS increased IDO expression in FC compared to control group, and ADs restored the IDO expression levels to control values. CMS-induced IDO expression led to increased levels of the excitotoxic quinolinic acid (QUINA) compared to control, and ADs prevented the rise in such levels. Neither CMS nor ADs changed significantly the antiexcitotoxic kynurenic acid (KYNA) levels. The QUINA/KYNA ratio, calculated as excitotoxicity risk indicator, increased after CMS and ADs prevented this increase. CMS lowered excitatory amino acid transporter (EAAT)-1 and EAAT-4 expression, and some ADs restored their expression levels. Furthermore, CMS decreased N-methyl-D-aspartate receptor (NMDAR)-2A and 2B protein expression, and ADs mitigated this decrease. Our research examines the link between CMS-induced pro-inflammatory cytokines and the kynurenine pathway; it shows that CMS alters the kynurenine pathway in rat FC. Importantly, it also reveals the ability of classic ADs to prevent potentially harmful situations related to the brain scenario caused by CMS.

Synthesis and Biological Evaluation of Novel Neuroprotective Pyridazine Derivatives as Excitatory Amino Acid Transporter 2 (EAAT2) Activators.

LDN-212320 (3) was found to be a potent EAAT2 activator at a translational level, restoring the normal clearance of glutamate and providing neuronal protection. Since the pharmacologic activation of EAAT2 represents a valuable strategy to relieve neuropathic pain, we synthesized novel activators (4a-f) of EAAT2. Among them 4f, analyzed in comparison with 3 by different paradigms in a rat model of oxaliplatin-induced neuropathic pain, showed the better antihypersensitive profile being able to fully counteract the oxaliplatin-induced neuropathy.

Cell-specific abnormalities of glutamate transporters in schizophrenia: sick astrocytes and compensating relay neurons?

Excitatory amino-acid transporters (EAATs) bind and transport glutamate, limiting spillover from synapses due to their dense perisynaptic expression primarily on astroglia. Converging evidence suggests that abnormalities in the astroglial glutamate transporter localization and function may underlie a disease mechanism with pathological glutamate spillover as well as alterations in the kinetics of perisynaptic glutamate buffering and uptake contributing to dysfunction of thalamo-cortical circuits in schizophrenia. We explored this hypothesis by performing cell- and region-level studies of EAAT1 and EAAT2 expression in the mediodorsal nucleus of the thalamus in an elderly cohort of subjects with schizophrenia. We found decreased protein expression for the typically astroglial-localized glutamate transporters in the mediodorsal and ventral tier nuclei. We next used laser-capture microdissection and quantitative polymerase chain reaction to assess cell-level expression of the transporters and their splice variants. In the mediodorsal nucleus, we found lower expression of transporter transcripts in a population of cells enriched for astrocytes, and higher expression of transporter transcripts in a population of cells enriched for relay neurons. We confirmed expression of transporter protein in neurons in schizophrenia using dual-label immunofluorescence. Finally, the pattern of transporter mRNA and protein expression in rodents treated for 9 months with antipsychotic medication suggests that our findings are not due to the effects of antipsychotic treatment. We found a compensatory increase in transporter expression in neurons that might be secondary to a loss of transporter expression in astrocytes. These changes suggest a profound abnormality in astrocyte functions that support, nourish and maintain neuronal fidelity and synaptic activity.

Astrocyte elevated gene-1 is a novel modulator of HIV-1-associated neuroinflammation via regulation of nuclear factor-κB signaling and excitatory amino acid transporter-2 repression.

Astrocyte elevated gene-1 (AEG-1), a novel human immunodeficiency virus (HIV)-1 and tumor necrosis factor (TNF)-α-inducible oncogene, has generated significant interest in the field of cancer research as a therapeutic target for many metastatic aggressive tumors. However, little is known about its role in astrocyte responses during HIV-1 central nervous system (CNS) infection and whether it contributes toward the development of HIV-associated neurocognitive disorders (HAND). Therefore, in this study, we investigated changes in AEG-1 CNS expression in HIV-1-infected brain tissues and elucidated a potential mechanism of AEG-1-mediated regulation of HAND. Immunoblotting and immunohistochemical analyses of HIV-1 seropositive and HIV-1 encephalitic human brain tissues revealed significantly elevated levels of AEG-1 protein. Immunohistochemical analyses of HIV-1 Tat transgenic mouse brain tissues also showed a marked increase in AEG-1 staining. Similar to in vivo observations, cultured astrocytes expressing HIV-1 Tat also revealed AEG-1 and cytokine up-regulation. Astrocytes treated with HAND-relevant stimuli, TNF-α, interleukin (IL)-1ß, and HIV-1, also significantly induced AEG-1 expression and nuclear translocation via activation of the nuclear factor (NF)-κB pathway. Co-immunoprecipitation studies demonstrated IL-1ß- or TNF-α-induced AEG-1 interaction with NF-κB p65 subunit. AEG-1 knockdown decreased NF-κB activation, nuclear translocation, and transcriptional output in TNF-α-treated astrocytes. Moreover, IL-1ß treatment of AEG-1-overexpressing astrocytes significantly lowered expression of excitatory amino acid transporter 2, increased expression of excitatory amino acid transporter 2 repressor ying yang 1, and reduced glutamate clearance, a major transducer of excitotoxic neuronal damage. Findings from this study identify a novel transcriptional co-factor function of AEG-1 and further implicate AEG-1 in HAND-associated neuroinflammation.

Oral administration of MSG increases expression of glutamate receptors and transporters in the gastrointestinal tract of young piglets.

Glutamate receptors and transporters, including T1R1 and T1R3 (taste receptor 1, subtypes 1 and 3), mGluRs (metabotropic glutamate receptors), EAAC-1 (excitatory amino acid carrier-1), GLAST-1 (glutamate-aspartate transporter-1), and GLT-1 (glutamate transporter-1), are expressed in the gastrointestinal tract. This study determined effects of oral administration of monosodium glutamate [MSG; 0, 0.06, 0.5, or 1 g/kg body weight (BW)/day] for 21 days on expression of glutamate receptors and transporters in the stomach and jejunum of sow-reared piglets. Both mRNA and protein levels for gastric T1R1, T1R3, mGluR1, mGluR4, EAAT1, EAAT2, EAAT3, and EAAT4 and mRNA levels for jejunal T1R1, T1R3, EAAT1, EAAT2, EAAT3 and EAAT4 were increased (P < 0.05) by MSG supplementation. Among all groups, mRNA levels for gastric EAAT1, EAAT2, EAAT3, and EAAT4 were highest (P < 0.05) in piglets receiving 1 g MSG/kg BW/day. EAAT1 and EAAT2 mRNA levels in the stomach and jejunum of piglets receiving 0.5 g MSG/kg BW/day, as well as jejunal EAAT3 and EAAT4 mRNA levels in piglets receiving 1 g MSG/kg BW/day, were higher (P < 0.05) than those in the control and in piglets receiving 0.06 g MSG/kg BW/day. Furthermore, protein levels for jejunal T1R1 and EAAT3 were higher (P < 0.05) in piglets receiving 1 g MSG/kg BW/day than those in the control and in piglets receiving 0.06 g MSG/kg BW/day. Collectively, these findings indicate that dietary MSG may beneficially stimulate glutamate signaling and sensing in the stomach and jejunum of young pigs, as well as their gastrointestinal function.

Glucose increases intracellular free Ca(2+) in tanycytes via ATP released through connexin 43 hemichannels.

The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal-glial cells, termed tanycytes. The latter express glucose-sensing proteins, including glucose transporter 2, glucokinase, and ATP-sensitive K(+) (K(ATP) ) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose-induced rise of intracellular free Ca(2+) concentration ([Ca(2+) ](i) ) in cultured ß-tanycytes was examined. Fura-2AM time-lapse fluorescence images revealed that glucose increases the intracellular Ca(2+) signal in a concentration-dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a K(ATP) channel-dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y(1) receptors followed by inositol trisphosphate receptor activation and Ca(2+) release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca(2+) ](i) in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes.

Oncogene AEG-1 promotes glioma-induced neurodegeneration by increasing glutamate excitotoxicity.

Aggressive tumor growth, diffuse tissue invasion, and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogene that is overexpressed in several types of human cancers, including more than 90% of brain tumors. In addition, AEG-1 promotes gliomagenesis, particularly in the context of tumor growth and invasion, 2 primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from glioma patients indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.

Glial and glutamatergic markers in depression, alcoholism, and their comorbidity.

BACKGROUND: Alteration of glutamatergic neurotransmission in the prefrontal cortex (PFC) may contribute to the pathophysiology of alcoholism and major depressive disorder (MDD). Among glial cells, astrocytes are mostly responsible for recycling synaptic glutamate by uptake through excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2), and conversion to glutamine with glutamine synthetase (GS). Low density of astrocytes in the PFC of "uncomplicated' alcoholics and MDD subjects may parallel altered glutamate transporters and GS in the PFC. METHODS: Immunohistochemistry and Western blotting for glutamate transporters, GS and glial fibrillary acidic protein (GFAP) were applied to postmortem tissue of the left orbitofrontal cortex from 13 subjects with MDD, 13 with alcoholism, 10 with comorbid alcoholism plus MDD (MDA), and 13 non-psychiatric controls. Area fraction of immunoreactivity was measured in sections, and protein levels in Western blots. RESULTS: EAAT2 immunoreactivity was significantly lower in MDD and MDA subjects than in controls. EAAT1 levels were lower in MDA and MDD subjects as compared to controls, while GS levels in MDA were significantly lower than in alcoholics and controls, and lower in MDD subjects than in alcoholics. Area fraction of GFAP was lower in MDD, but not in MDA subjects as compared to controls or alcoholics. LIMITATIONS: High variability of protein levels in some groups and effects of antidepressant treatment, although appearing to be limited, cannot be fully evaluated. CONCLUSIONS: There are differential changes in the expression of glial glutamatergic markers in depression and alcoholism, suggesting a depletion of certain aspects of glutamatergic processing in depression.

Loss of astrocytic glutamate transporters in Wernicke encephalopathy.

Wernicke encephalopathy (WE), a neurological disorder caused by thiamine deficiency (TD), is characterized by structural damage in brain regions that include the thalamus and cerebral cortex. The basis for these lesions is unclear, but may involve a disturbance of glutamatergic neurotransmission. We have therefore investigated levels of the astrocytic glutamate transporters EAAT1 and EAAT2 in order to evaluate their role in the pathophysiology of this disorder. Histological assessment of the frontal cortex revealed a significant loss of neurons in neuropathologically confirmed cases of WE compared with age-matched controls, concomitant with decreases in alpha-internexin and synaptophysin protein content of 67 and 52% by immunoblotting. EAAT2 levels were diminished by 71% in WE, with levels of EAAT1 also reduced by 62%. Loss of both transporter sites was confirmed by immunohistochemical methods. Development of TD in rats caused a profound loss of EAAT1 and EAAT2 in the thalamus accompanied by decreases in other astrocyte-specific proteins. Treatment of TD rats with N-acetylcysteine prevented the downregulation of EAAT2 in the medial thalamus, and ameliorated the loss of several other astrocyte proteins, concomitant with increased neuronal survival. Our results suggest that (1) loss of EAAT1 and EAAT2 glutamate transporters is associated with structural damage to the frontal cortex in patients with WE, (2) oxidative stress plays an important role in this process, and (3) TD has a profound effect on the functional integrity of astrocytes. Based on these findings, we recommend that early treatment using a combination of thiamine AND antioxidant approaches should be an important consideration in cases of WE.

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism.

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.