RESUMEN
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.
Asunto(s)
Antimaníacos/farmacología , Ginsenósidos/farmacología , Ácido Glutámico/metabolismo , Panax/química , Privación de Sueño/tratamiento farmacológico , Sueño REM/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Conducta Animal , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Privación de Sueño/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismoRESUMEN
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.
Asunto(s)
Lóbulo Frontal/patología , Lóbulo Frontal/fisiopatología , Ácido Glutámico/metabolismo , Quinurenina/metabolismo , Estrés Psicológico/metabolismo , Estrés Psicológico/fisiopatología , Transmisión Sináptica , Animales , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Enfermedad Crónica , Citocinas/metabolismo , Lóbulo Frontal/efectos de los fármacos , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Mediadores de Inflamación/metabolismo , Ácido Quinurénico/metabolismo , Masculino , Redes y Vías Metabólicas/efectos de los fármacos , Ácido Quinolínico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Estrés Psicológico/tratamiento farmacológico , Transmisión Sináptica/efectos de los fármacos , Triptófano/metabolismoRESUMEN
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.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Piridazinas/química , Piridinas/farmacología , Animales , Técnicas de Química Sintética , Transportador 2 de Aminoácidos Excitadores , Masculino , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Fármacos Neuroprotectores/síntesis química , Compuestos Organoplatinos/toxicidad , Oxaliplatino , Piridazinas/síntesis química , Piridazinas/farmacología , Piridinas/química , Ratas Sprague-DawleyRESUMEN
Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological regulator of multiple membrane glutamate transporters that impacts on overall cellular glutamate handling.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Transportador 3 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/metabolismo , Ácido Glutámico/farmacología , Glándulas Mamarias Humanas/citología , Vitamina D/análogos & derivados , Sistema de Transporte de Aminoácidos y+/genética , Línea Celular , Medios de Cultivo/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores , Transportador 4 de Aminoácidos Excitadores/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Humanos , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Isoformas de Proteínas/metabolismo , Vitamina D/farmacologíaRESUMEN
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.
Asunto(s)
Astrocitos/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ácido Glutámico/metabolismo , Anciano , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Proteínas Portadoras/genética , Femenino , Expresión Génica , Humanos , Masculino , Núcleo Talámico Mediodorsal/metabolismo , Núcleo Talámico Mediodorsal/fisiopatología , Ratones , Persona de Mediana Edad , Neuronas/metabolismo , ARN Mensajero/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Tálamo/fisiopatologíaRESUMEN
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.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Moléculas de Adhesión Celular/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , VIH-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/patología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Moléculas de Adhesión Celular/genética , Regulación hacia Abajo/genética , Transportador 2 de Aminoácidos Excitadores/genética , Femenino , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , VIH-1/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Proteínas de Unión al ARN , Factor de Transcripción ReIA/genética , Factor de Transcripción YY1/genética , Factor de Transcripción YY1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
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.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Receptores de Glutamato/genética , Glutamato de Sodio/metabolismo , Porcinos/metabolismo , Administración Oral , Animales , Transporte Biológico , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/crecimiento & desarrollo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ácido Glutámico/metabolismo , Receptores de Glutamato/metabolismo , Glutamato de Sodio/administración & dosificación , Porcinos/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Conexina 43/metabolismo , Glucosa/farmacología , Líquido Intracelular/metabolismo , Neuroglía/efectos de los fármacos , Animales , Animales Recién Nacidos , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Cationes/metabolismo , Células Cultivadas , Conexina 43/antagonistas & inhibidores , Citocalasina B/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Glucoquinasa/metabolismo , Glucosa/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Hipotálamo/citología , Antígeno Ki-67/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Técnicas de Placa-Clamp , Probenecid/farmacología , Ratas , Ratas Sprague-Dawley , Factor de von Willebrand/metabolismoRESUMEN
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.
Asunto(s)
Neoplasias Encefálicas/patología , Moléculas de Adhesión Celular/metabolismo , Glioma/patología , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ácido Glutámico/metabolismo , Degeneración Nerviosa/patología , Oncogenes , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Proteína de Unión a CREB/metabolismo , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Transportador 2 de Aminoácidos Excitadores , Glioma/metabolismo , Humanos , Proteínas de la Membrana , Degeneración Nerviosa/metabolismo , Regiones Promotoras Genéticas , Proteínas de Unión al ARN , Ratas , Factor de Transcripción YY1/metabolismoRESUMEN
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.
Asunto(s)
Alcoholismo/fisiopatología , Astrocitos/fisiología , Trastorno Depresivo Mayor/fisiopatología , Transportador 1 de Aminoácidos Excitadores/metabolismo , Lóbulo Frontal/fisiopatología , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/fisiología , Corteza Prefrontal/fisiopatología , Transmisión Sináptica/fisiología , Adulto , Anciano , Alcoholismo/epidemiología , Alcoholismo/patología , Astrocitos/patología , Comorbilidad , Trastorno Depresivo Mayor/epidemiología , Trastorno Depresivo Mayor/patología , Transportador 2 de Aminoácidos Excitadores , Femenino , Lóbulo Frontal/patología , Humanos , Masculino , Persona de Mediana Edad , Corteza Prefrontal/patología , Valores de ReferenciaRESUMEN
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.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Deficiencia de Tiamina/fisiopatología , Encefalopatía de Wernicke/fisiopatología , Acetilcisteína/farmacología , Adulto , Anciano , Animales , Astrocitos/metabolismo , Lóbulo Frontal/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Neuronas/fisiología , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Tálamo/metabolismo , Deficiencia de Tiamina/tratamiento farmacológicoAsunto(s)
Líquido Extracelular/efectos de los fármacos , Lóbulo Frontal/citología , Ácido Glutámico/metabolismo , Alucinógenos/administración & dosificación , Fenciclidina/administración & dosificación , Sinaptofisina/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Animales , Lóbulo Frontal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ratas , Factores de TiempoRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Glutamato en la Membrana Plasmática/biosíntesis , Esquizofrenia/fisiopatología , Tálamo/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Antipsicóticos/farmacología , Complejo del Señalosoma COP9 , Clozapina/farmacología , Femenino , Proteínas de Transporte de Glutamato en la Membrana Plasmática/efectos de los fármacos , Ácido Glutámico/metabolismo , Factores de Intercambio de Guanina Nucleótido/biosíntesis , Haloperidol/farmacología , Proteínas de Choque Térmico/biosíntesis , Humanos , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de Transporte de Membrana , Persona de Mediana Edad , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Factores de Intercambio de Guanina Nucleótido Rho , Esquizofrenia/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Glutamato en la Membrana Plasmática/metabolismo , Ácido Glutámico/metabolismo , Aniones/metabolismo , Sitios de Unión , Transporte Biológico/fisiología , Encéfalo/metabolismo , Línea Celular , Electrofisiología , Transportador 4 de Aminoácidos Excitadores/fisiología , Líquido Extracelular/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática/genética , Humanos , Cinética , Modelos Biológicos , Potasio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sodio/metabolismoRESUMEN
At present, the ability of polyunsaturated fatty acids (PUFAs) to regulate individual glutamate transporter subtypes is poorly understood and very little information exists on the mechanism(s) by which PUFAs achieve their effects on the transport process. Here we investigate the effect of cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) on the activity of the mammalian glutamate transporter subtypes, GLT1, GLAST and EAAC1 individually expressed in human embryonic kidney (HEK) cells. Exposure of cells to 100 muM DHA increased the rate of d-[(3)H]aspartate uptake by over 72% of control in HEK(GLT1) cells, and by 45% of control in HEK(EAAC1) cells. In contrast, exposure of HEK(GLAST) cells to 200 muM DHA resulted in almost 40% inhibition of d-[(3)H]aspartate transport. Removal of extracellular calcium increased the inhibitory potential of DHA in HEK(GLAST) cells. In contrast, in the absence of extracellular calcium, the stimulatory effect of DHA on d-[(3)H]aspartate uptake in HEK(GLT1) and HEK(EAAC1) cells was abolished, and significant inhibition of the transport process by DHA was observed. Inhibition of CaM kinase II or PKC had no effect on the ability of DHA to inhibit transport into HEK(GLAST) cells but abolished the stimulatory effect of DHA on d-[(3)H]aspartate transport into HEK(GLT1) and HEK(EAAC1) cells. Inhibition of PKA had no effect on the modulation of d-[(3)H]aspartate transport by DHA in any of the cell lines. We conclude that DHA differentially modulates the GLT1, GLAST and EAAC1 glutamate transporter subtypes via different mechanisms. In the case of GLT1 and EAAC1, DHA appears to stimulate d-[(3)H]aspartate uptake via a mechanism requiring extracellular calcium and involving CaM kinase II and PKC, but not PKA. In contrast, the inhibitory effect of DHA on GLAST does not require extracellular calcium and does not involve CaM kinase II, PKC or PKA.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Egtácico/análogos & derivados , Transportador 2 de Aminoácidos Excitadores/metabolismo , Simportadores/metabolismo , Ácido Aspártico/metabolismo , Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Línea Celular , Quelantes/farmacología , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , ADN Complementario/biosíntesis , ADN Complementario/genética , Ácido Egtácico/farmacología , Transportador 1 de Aminoácidos Excitadores , Transportador 3 de Aminoácidos Excitadores , Espacio Extracelular/enzimología , Espacio Extracelular/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Humanos , Plásmidos/genética , Proteína Quinasa C/antagonistas & inhibidores , TransfecciónRESUMEN
Glutamate transporter proteins are essential for the control of interstitial glutamate levels, with an impairment of their function or levels being a major potential contributor to excitotoxicity. We have investigated the effects of lateral fluid percussion on the levels of the glutamate transporter proteins GLT-1alpha, its splice variant GLT-1v, GLAST, and EAAC1 in the rat in order to evaluate their pathogenetic role in this model of traumatic brain injury (TBI). Immunoblot analysis revealed neuronal loss in the cerebral cortex was accompanied by a 54% decrease in GLT-1v 6 h following the insult which progressed to an 83% loss of the transporter after 24 h. No changes in GLT-1alpha, GLAST, or EAAC1 were observed in this brain region at either time point. GLT-1v content was also decreased by 55% and 68% in the hippocampus and thalamus, respectively, at 6 h post-injury, but recovered fully after 24 h in both brain regions. In contrast, levels of GLT-1alpha were increased in the hippocampus at 6 h and 24 h post-TBI. These alterations in transporter protein content were also confirmed using immunohistochemical methods. Our results show for the first time a pattern of early, dynamic changes in the levels of GLT-1 transporter splice variants in different brain regions in this trauma model. In addition, correlation of GLT-1v levels with both neuronal cell loss and alpha-internexin content in the injured cortex suggests that loss of this novel glutamate transporter may be a key factor in determining cerebral vulnerability following this type of brain injury.
Asunto(s)
Lesiones Encefálicas/metabolismo , Corteza Cerebral/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Degeneración Nerviosa/metabolismo , Neuronas/metabolismo , Empalme Alternativo/genética , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Animales , Lesiones Encefálicas/patología , Lesiones Encefálicas/fisiopatología , Proteínas Portadoras/metabolismo , Corteza Cerebral/patología , Corteza Cerebral/fisiopatología , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Transportador 1 de Aminoácidos Excitadores , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Inmunohistoquímica , Proteínas de Filamentos Intermediarios , Masculino , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Simportadores/metabolismo , Tálamo/metabolismo , Tálamo/fisiopatología , Factores de Tiempo , Heridas no PenetrantesRESUMEN
PURPOSE: The serum- and glucocorticoid-inducible kinase SGK1 regulates several epithelial channels and transporters, the related protein kinase B (PKB) regulates glucose transport. SGK1 is expressed in the brain and could thus regulate glial and/or neuronal transport processes. The present study explores whether SGK1 is expressed in the retina and whether it regulates EAAT3, a Na(+)-coupled glutamate transporter. EAAT3 is expressed in retinal ganglion cells and accomplishes the clearance of glutamate from synaptic clefts. METHODS: Immunohistochemistry was performed to test for retinal SGK1 expression. For functional analysis, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without additional injection of wild-type SGK1, constitutively active (S422D)SGK1, inactive (K127N)SGK1, and/or constitutively active (T308D,S473D)PKB. Glutamate induced current (I(GLU)) was taken as a measure for transport. RESULTS: SGK1 is indeed expressed in several retinal cells including retinal ganglion cells where it is colocalized with EAAT3. In EAAT3-expressing Xenopus oocytes, glutamate-induced current was stimulated by coexpression of wild-type SGK1, constitutively active (S422D)SGK1, and constitutively active (T308D,S473D)PKB, but not by inactive (K127N)SGK1. CONCLUSIONS: SGK1 and EAAT3 are coexpressed in retinal neurons, and SGK1 serves to stimulate EAAT3. This function is shared by protein kinase B (PKB). The experiments reveal a novel mechanism regulating EAAT3, which may be essential for the function of the retinal ganglion cells.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/metabolismo , Retina/metabolismo , Simportadores/metabolismo , Sistema de Transporte de Aminoácidos X-AG/genética , Animales , Transporte Biológico , Transportador 3 de Aminoácidos Excitadores , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Ácido Glutámico/metabolismo , Proteínas Inmediatas-Precoces , Técnicas de Placa-Clamp , ARN Complementario/metabolismo , Ratas , Ratas Endogámicas BN , Células Ganglionares de la Retina/metabolismo , Sodio/metabolismo , Simportadores/genética , Xenopus laevisRESUMEN
This study's rationale was that the expression and activity of aspartate transporters in hypertrophied hearts might be different from normal hearts, which could affect the use of aspartate in myocardial protection of hypertrophied hearts. mRNA expression of system X(ag)(-) transporters in hearts from normal (Wistar Kyoto) and hypertrophied (spontaneously hypertensive rat) rats was investigated by RT-PCR. EAAT3 protein expression in isolated cells and vesicles from normal and hypertrophied hearts was investigated by Western blotting. The same vesicles were also used to measure aspartate uptake. The effects of 0.5 mmol l(-1) aspartate supplementation on cardiac performance during ischaemia-reperfusion were investigated in isolated and perfused hearts. Both normal and hypertrophied hearts expressed EAAT1 and EAAT3 mRNA. EAAT3 protein expression was significantly greater in cells and vesicles from hypertrophied hearts compared to normal hearts. The velocity (V(max)) of aspartate uptake was faster at 24.4 +/- 2.2 pmol mg(-1) s(-1) in vesicles from hypertrophied hearts compared to 8.2 +/- 0.8 pmol mg(-1) s(-1) (P < 0.001, t test, n= 6, means +/-s.e.m.) in normal heart vesicles. The affinity (K(m)) was similar for both preparations. When recoveries were matched, 0.5 mmol l(-1) aspartate addition reduced reperfusion injury and increased functional recovery of hypertrophied hearts but not normal hearts. This was associated with a greater preservation of ATP, glutamate and glutamine and less lactate production during ischaemia in aspartate-treated hypertrophied hearts compared to all other experimental groups. These results suggest that increased aspartate transporter expression and activity in hypertrophy helps facilitate aspartate entry into hypertrophied cardiomyocytes, which in turn leads to improved myocardial protection.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Cardiomegalia/metabolismo , Daño por Reperfusión/fisiopatología , Simportadores/metabolismo , Adenosina Trifosfato/metabolismo , Alanina/sangre , Alanina/metabolismo , Sistema de Transporte de Aminoácidos X-AG/genética , Animales , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacocinética , Western Blotting , Cardiomegalia/genética , Cardiomegalia/fisiopatología , Transportador 1 de Aminoácidos Excitadores , Transportador 3 de Aminoácidos Excitadores , Glutamato Deshidrogenasa/metabolismo , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Ácido Glutámico/sangre , Ácido Glutámico/metabolismo , Glutamina/sangre , Glutamina/metabolismo , Corazón/efectos de los fármacos , Corazón/fisiopatología , Frecuencia Cardíaca/fisiología , Isquemia/fisiopatología , Cinética , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Masculino , Miocardio/química , Miocardio/metabolismo , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Reperfusión , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcolema/química , Sarcolema/metabolismo , Simportadores/genética , Presión Ventricular/fisiologíaRESUMEN
After synaptic release, glutamate is taken up by the nerve terminal via a plasma membrane-associated protein termed excitatory amino acid transporter 3 (EAAT3). Following entry into the nerve terminal, glutamate is pumped into synaptic vesicles by a vesicular transport system. Three different vesicular glutamate transporter proteins (VGLUT1-3) representing unique markers for glutamatergic neurons were recently characterized. The presence of EAAT3, glutaminase and VGLUT1-3 was examined in mouse, rat and rabbit species at mRNA and protein levels in hypothalamic neurons which are involved in the regulation of body weight using in situ hybridization and immunohistochemistry. EAAT3 and glutaminase mRNAs were demonstrated in all parts of the arcuate nucleus in the dorsomedial and ventromedial hypothalamic nuclei and lateral hypothalamic area. VGLUT1 mRNA was present in the magnocellular lateral hypothalamic nucleus. VGLUT2 mRNA was demonstrated in a subpopulation of neurons in the arcuate nucleus and in the ventromedial and dorsomedial hypothalamic nuclei and lateral hypothalamic area. Few VGLUT3 mRNA expressing neurons were scattered throughout the medial and lateral hypothalamus. EAAT3-like immunoreactivity (-li) was demonstrated in glutamate, neuropeptide Y (NPY), agouti-related peptide (AGRP), pro-opiomelanocortin (POMC), cocaine and amphetamine-regulated transcript (CART), melanin-concentrating hormone and orexin-immunoreactive (-ir) neurons. VGLUT2-li could only be demonstrated in POMC- and CART-ir neurons of the ventrolateral arcuate nucleus. The results show that key neurons involved in regulation of energy balance are glutamatergic and/or densely innervated by glutamatergic nerve terminals. Whereas orexigenic NPY/AGRP neurons situated in the ventromedial part of the arcuate nucleus are mainly GABAergic, it is shown that several anorexigenic POMC/CART neurons of the ventromedial arcuate nucleus are most likely glutamatergic [corrected].
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Membrana Celular/metabolismo , Hipotálamo/citología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de Transporte de Membrana , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Proteína Relacionada con Agouti , Sistema de Transporte de Aminoácidos X-AG/clasificación , Sistema de Transporte de Aminoácidos X-AG/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Peso Corporal/fisiología , Proteínas Portadoras/metabolismo , Ciclohexanos/metabolismo , Proteínas de Unión al ADN/metabolismo , Transportador 3 de Aminoácidos Excitadores , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Glutaminasa/metabolismo , Inmunohistoquímica/instrumentación , Inmunohistoquímica/métodos , Hibridación in Situ/mortalidad , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuropéptido Y/metabolismo , Neuropéptidos/metabolismo , Orexinas , Proopiomelanocortina/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3 , Especificidad de la Especie , Simportadores/metabolismo , Transactivadores/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato , Proteína 2 de Transporte Vesicular de Glutamato , Proteínas de Transporte Vesicular de GlutamatoRESUMEN
l-Glutamate is the major excitatory transmitter in the vertebrate retina and plays a central role in the transmission of the various retinal neurons. Glutamate is removed from the extracellular space by at least five different glutamate transporters. The cellular distribution of these has been studied so far mainly using immunocytochemistry. In the present study non-radioactive in situ hybridisation using complementary RNA probes was applied in order to identify the cell types of rat retina and optic nerve expressing generic GLT1, GLT1 variant (GLT1v or GLT1B), GLAST and EAAC1. The results were compared with immunocytochemical data achieved using affinity-purified antibodies against transporter peptides. In the immunohistochemical studies the human retina was included. The study showed that in the rat retina GLT1v and EAAC1 were coexpressed in various cell types, i.e. photoreceptor, bipolar, horizontal, amacrine, ganglion and Müller cells, whereas GLAST was only detected in Müller cells and astrocytes. In the rat optic nerve GLT1v and EAAC1 were preferentially expressed in oligodendrocytes, whereas GLAST was revealed to be present mainly in astrocytes. Generic GLT1 could not be detected in the retina or optic nerve. The cellular distribution of glutamate transporters (only immunocytochemistry) in the human retina was very similar to that of the rat retina. Remarkable results of our studies were that generic GLT1 was not detectable in the rat (and human) retina and that GLT1v and EAAC1 were demonstrable in most cell types of the retina (including photoreceptor cells and their terminals).