RESUMEN

Elevated levels of the inflammatory cytokine interleukin-6 (IL-6) occur in a number of CNS disorders. However, little is known about how this condition affects CNS neuronal function. Transgenic mice that express elevated levels of IL-6 in the CNS show cognitive changes, increased propensity for hippocampal seizures and reduced number of inhibitory interneurons, suggesting that elevated levels of IL-6 can cause neuroadaptive changes that alter hippocampal function. To identify these neuroadaptive changes, we measured the levels of protein expression using Western blot analysis and synaptic function using field potential recordings in hippocampus from IL-6 transgenic mice (IL-6 tg) and their non-transgenic (non-tg) littermates. Western blot analysis showed enhanced levels of the GFAP and STAT3 in the IL-6 tg hippocampus compared with the non-tg hippocampus, but no difference for several other proteins. Field potential recordings of synaptic transmission at the Schaffer collateral to CA1 synapse showed enhanced dendritic excitatory postsynaptic potentials and somatic population spikes in the CA1 region of hippocampal slices from IL-6 tg mice compared with slices from non-tg littermate controls. No differences were observed for several forms of short-term and long-term synaptic plasticity between hippocampal slices from IL-6 tg and non-tg mice. These results demonstrate that elevated levels of IL-6 can alter mechanisms involved in the excitability of hippocampal neurons and synapses, an effect consistent with recent evidence indicating that elevated production of IL-6 plays an important role in conditions associated with seizure activity and in other impairments observed in CNS disorders with a neuroinflammatory component.

Asunto(s)

Sistema Nervioso Central/metabolismo , Hipocampo/fisiología , Interleucina-6/biosíntesis , Interleucina-6/genética , Transmisión Sináptica/genética , Animales , Western Blotting , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/fisiología , Dendritas/efectos de los fármacos , Dendritas/fisiología , Fenómenos Electrofisiológicos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción STAT3/metabolismo

RESUMEN

We examined the effect of chronic CCL3 treatment on the properties of cultured rat hippocampal neurons to gain an understanding of the neuronal effects of CCL3 during neuroinflammatory disorders. Western blot assays showed that chronic exposure to CCL3 altered the level of specific neuronal and glial proteins and that CCL3 had no effect on neuronal survival. CCL3 treatment also altered intracellular Ca(2+) dynamics and increased Ca(2+) levels in hippocampal neurons, measured by fura-2 imaging techniques. Additionally, chronic CCL3 increased NMDA-evoked Ca(2+) signals in the hippocampal neurons and increased NMDA receptor levels. These CCL3-induced neuroadaptive changes could play an important role in the CNS dysfunction associated with CNS disorders with a neuroinflammatory component.

Asunto(s)

Quimiocina CCL3/farmacología , Hipocampo/citología , Red Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacos , 2-Amino-5-fosfonovalerato/farmacología , Animales , Animales Recién Nacidos , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Descarboxilasa/metabolismo , Hipocampo/efectos de los fármacos , Neuroglía/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fosfopiruvato Hidratasa/metabolismo , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/metabolismo , Potenciales Sinápticos/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacología

RESUMEN

Glutamine synthetase (GS) is a pivotal glial enzyme in the glutamate-glutamine cycle. GS is important in maintaining low extracellular glutamate concentrations and is downregulated in the hippocampus of temporal lobe epilepsy patients with mesial-temporal sclerosis, an epilepsy syndrome that is frequently associated with early life febrile seizures (FS). Human congenital loss of GS activity has been shown to result in brain malformations, seizures and death within days after birth. Recently, we showed that GS knockout mice die during embryonic development and that haploinsufficient GS mice have no obvious abnormalities or behavioral seizures. In the present study, we investigated whether reduced expression/activity of GS in haploinsufficient GS mice increased the susceptibility to experimentally induced FS. FS were elicited by warm-air-induced hyperthermia in 14-day-old mice and resulted in seizures in most animals. FS susceptibility was measured as latencies to four behavioral FS characteristics. Our phenotypic data show that haploinsufficient mice are more susceptible to experimentally induced FS (P < 0.005) than littermate controls. Haploinsufficient animals did not differ from controls in hippocampal amino acid content, structure (Nissl and calbindin), glial properties (glial fibrillary acidic protein and vimentin) or expression of other components of the glutamate-glutamine cycle (excitatory amino acid transporter-2 and vesicular glutamate transporter-1). Thus, we identified GS as a FS susceptibility gene. GS activity-disrupting mutations have been described in the human population, but heterozygote mutations were not clearly associated with seizures or epilepsy. Our results indicate that individuals with reduced GS activity may have reduced FS seizure thresholds. Genetic association studies will be required to test this hypothesis.

Asunto(s)

Predisposición Genética a la Enfermedad/genética , Glutamato-Amoníaco Ligasa/genética , Ácido Glutámico/metabolismo , Haplotipos/genética , Convulsiones Febriles/genética , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Encéfalo/enzimología , Encéfalo/fisiopatología , Química Encefálica/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Transportador 2 de Aminoácidos Excitadores/análisis , Transportador 2 de Aminoácidos Excitadores/metabolismo , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones , Ratones Noqueados , Tiempo de Reacción/genética , Convulsiones Febriles/enzimología , Convulsiones Febriles/fisiopatología , Proteína 1 de Transporte Vesicular de Glutamato/análisis , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Vimentina/análisis , Vimentina/metabolismo

RESUMEN

Febrile seizures (FS) are the most common seizure type in children and recurrent FS are a risk factor for developing temporal lobe epilepsy. Although the mechanisms underlying FS are largely unknown, recent family, twin and animal studies indicate that genetics are important in FS susceptibility. Here, a forward genetic strategy was used employing mouse chromosome substitution strains (CSS) to identify novel FS susceptibility quantitative trait loci (QTLs). FS were induced by exposure to warm air at postnatal day 14. Video electroencephalogram monitoring identified tonic-clonic convulsion onset, defined as febrile seizure latency (FSL), as a reliable phenotypic parameter to determine FS susceptibility. FSL was determined in both sexes of the host strain (C57BL/6J), the donor strain (A/J) and CSS. C57BL/6J mice were more susceptible to FS than A/J mice. Phenotypic screening of the CSS panel identified six strains(CSS1, -2, -6 -10, -13 and -X) carrying QTLs for FS susceptibility. CSS1, -10 and -13 were less susceptible (protective QTLs), whereas CSS2, -6 and -X were more susceptible (susceptibility QTLs) to FS than the C57BL/6J strain. Our data show that mouse FS susceptibility is determined by complex genetics, which is distinct from that for chemically induced seizures. This is the first dataset using CSS to screen for a seizure trait in mouse pups. It provides evidence for common FS susceptibility QTLs that serve as starting points to fine map FS susceptibility QTLs and to identify FS susceptibility genes. This will increase our understanding of human FS, working toward the identification of new therapeutic targets.

Asunto(s)

Cromosomas de los Mamíferos/genética , Sitios de Carácter Cuantitativo/genética , Convulsiones Febriles/genética , Animales , Conducta Animal/fisiología , Temperatura Corporal/genética , Temperatura Corporal/fisiología , Interpretación Estadística de Datos , Electroencefalografía , Femenino , Ligamiento Genético/genética , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Fenotipo , Convulsiones Febriles/psicología

RESUMEN

The generation of motor activity levels is under tight neural control to execute essential behaviors, such as movement toward food or for social interaction. To identify novel neurobiological mechanisms underlying motor activity levels, we studied a panel of chromosome substitution (CS) strains derived from mice with high (C57BL/6J strain) or low motor activity levels (A/J strain) using automated home cage behavioral registration. In this study, we genetically mapped the expression of baseline motor activity levels (horizontal distance moved) to mouse chromosome 1. Further genetic mapping of this trait revealed an 8.3-Mb quantitative trait locus (QTL) interval. This locus is distinct from the QTL interval for open-field anxiety-related motor behavior on this chromosome. By data mining, an existing phenotypic and genotypic data set of 2445 genetically heterogeneous mice (http://gscan.well.ox.ac.uk/), we confirmed linkage to the peak marker at 79 970 253 bp and refined the QTL to a 312-kb interval containing a single gene (A830043J08Rik). Sequence analysis showed a nucleotide deletion in the 3' untranslated region of the Riken gene. Genome-wide microarray gene expression profiling in brains of discordant F(2) individuals from CS strain 1 showed a significant upregulation of Epha4 in low-active F(2) individuals. Inclusion of a genetic marker for Epha4 confirmed that this gene is located outside of the QTL interval. Both Epha4 and A830043J08Rik are expressed in brain motor circuits, and similar to Epha4 mutants, we found linkage between reduced motor neurons number and A/J chromosome 1. Our findings provide a novel QTL and a potential downstream target underlying motor circuitry development and the expression of physical activity levels.

Asunto(s)

Mapeo Cromosómico , Actividad Motora/genética , Animales , Cromosomas/genética , Cartilla de ADN , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Receptor EphA4/genética

RESUMEN

Febrile seizures (FS) are the most prevalent seizures in children. Although FS are largely benign, complex FS increase the risk to develop temporal lobe epilepsy (TLE). Studies in rat models for FS have provided information about functional changes in the hippocampus after complex FS. However, our knowledge about the genes and pathways involved in the causes and consequences of FS is still limited. To enable molecular, genetic and knockout studies, we developed and characterized an FS model in mice and used it as a phenotypic screen to analyze FS susceptibility. Hyperthermia was induced by warm air in 10- to 14-day-old mice and induced FS in all animals. Under the conditions used, seizure-induced behavior in mice and rats was similar. In adulthood, treated mice showed increased hippocampal Ih current and seizure susceptibility, characteristics also seen after FS in rats. Of the seven genetically diverse mouse strains screened for FS susceptibility, C57BL/6J mice were among the most susceptible, whereas A/J mice were among the most resistant. Strains genetically similar to C57BL/6J also showed a susceptible phenotype. Our phenotypic data suggest that complex genetics underlie FS susceptibility and show that the C57BL/6J strain is highly susceptible to FS. As this strain has been described as resistant to convulsants, our data indicate that susceptibility genes for FS and convulsants are distinct. Insight into the mechanisms underlying seizure susceptibility and FS may help to identify markers for the early diagnosis of children at risk for complex FS and TLE and may provide new leads for treatment.

Asunto(s)

Predisposición Genética a la Enfermedad/genética , Ratones Endogámicos C57BL/genética , Convulsiones Febriles/genética , Convulsiones Febriles/fisiopatología , Animales , Conducta Animal , Convulsivantes/farmacología , Electrofisiología , Fiebre/genética , Fiebre/fisiopatología , Hipocampo/fisiopatología , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Pentilenotetrazol/farmacología , Fenotipo , Ratas , Ratas Sprague-Dawley , Convulsiones Febriles/inducido químicamente , Especificidad de la Especie

RESUMEN

Synthetic corticosteroids, such as dexamethasone, are frequently administered to pregnant women at risk for preterm delivery. Endogenous corticosteroids are essential for normal development, but exposure to therapeutic doses at critical developmental stages may have adverse effects on the central nervous system. Major concern has arisen about long-term effects of corticosteroid treatment on brain plasticity, particularly in the hippocampus. Therefore, we analyzed the molecular, cellular, and behavioral effects of prenatal dexamethasone treatment on the adult hippocampus. Pregnant mice were treated at embryonic day 15.5 with a single dose of dexamethasone or saline. Adult offspring was analyzed for hippocampal neuron loss, cell proliferation, and NMDA receptor subunit expression. Hippocampal function was assessed in the Morris water maze and synaptic plasticity in the CA1 field by determining frequency dependence of LTP and LTD in hippocampal slices. Prenatal dexamethasone treatment decreased hippocampal cell proliferation in the dentate gyrus. Treated mice showed reduced LTD, impaired spatial learning, and a marked reduction in lifespan. Our data show long-term adverse effects of prenatal dexamethasone treatment on hippocampal function in mice and suggest accelerated aging. These findings indicate that it is important to be restrictive with corticosteroid administration during fetal development because of the lifelong consequences.

Asunto(s)

Corticoesteroides/efectos adversos , Hipocampo/efectos de los fármacos , Longevidad/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Animales , Proliferación Celular/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Giro Dentado/fisiopatología , Femenino , Hipocampo/metabolismo , Hipocampo/fisiopatología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/fisiología , Longevidad/fisiología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Plasticidad Neuronal/fisiología , Técnicas de Cultivo de Órganos , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Factores de Tiempo

RESUMEN

Women at risk for preterm delivery are treated with synthetic glucocorticoids (GCs) to enhance fetal lung maturation. GCs can bind to two intracellular receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), which function as transcription factors. Both are highly expressed in the hippocampus. Several studies have focused on adverse side effects of antenatal GC treatment. However, relatively little is known about the ontogeny of GR and MR, especially in human. Therefore, we studied the ontogeny of both receptors in the human and mouse hippocampus and investigated the effects of antenatal dexamethasone (dex) treatment, a synthetic glucocorticoid, on MR and GR mRNA levels during hippocampal development. The results demonstrate that MR mRNA was first expressed in mouse hippocampus at embryonic day (E)15.5, at the timepoint when dex was administered. In contrast, GR mRNA expression was first observed after birth at postnatal day (P)5. However, in the human hippocampus both receptors are expressed at 24 weeks of gestation, when antenatal GCs are administered in clinical practice. Quantitative in situ hybridization demonstrated that MR mRNA levels were reduced only shortly after dex treatment at E16, but were unaffected from E18 onwards. These findings indicate that a single antenatal dex administration at E15.5 transiently affects MR mRNA levels in the mouse hippocampus. No effect of antenatal dex treatment was found on the human hippocampus at the third trimester of pregnancy. These data on the prenatal ontogeny of both corticosteroid receptors in the human hippocampus is important for understanding the significance of fetal glucocorticoid or stress exposure and its potential effects on health and disease.

Asunto(s)

Glucocorticoides/efectos adversos , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Femenino , Glucocorticoides/uso terapéutico , Hipocampo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Especificidad de la Especie , Factores de Tiempo

RESUMEN

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and glutamate excitotoxicity has been implicated in its pathogenesis. Platelets contain a glutamate uptake system and express components of the glutamate-glutamine cycle, such as the predominant glial excitatory amino acid transporter 2 (EAAT2). In several neurological diseases platelets have proven to be systemic markers for the disease. We compared properties of key components of the glutamate-glutamine cycle in blood platelets of ALS patients and healthy controls. Platelets were analyzed for (3)H-glutamate uptake in the presence or absence of thrombin and for EAAT2 and glutamine synthetase protein expression by Western blotting. Platelets of ALS patients showed a 37% increase in expression of glutamine synthetase, but normal expression of glutamate transporter EAAT2. Glutamate uptake in resting or thrombin-stimulated platelets did not differ significantly between platelets from ALS patients and controls. Thrombin-stimulation resulted in about a seven-fold increase in glutamate uptake. Our data suggest that glutamine synthetase may be a peripheral marker of ALS and encourage further investigation into the role of this enzyme in ALS.

Asunto(s)

Esclerosis Amiotrófica Lateral/metabolismo , Plaquetas/metabolismo , Transportador 2 de Aminoácidos Excitadores/sangre , Glutamato-Amoníaco Ligasa/sangre , Adulto , Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/enzimología , Plaquetas/enzimología , Western Blotting , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

Glutamate toxicity has been implicated in the pathogenesis of various neurological diseases. Glial glutamate transporters play a key role in the regulation of extracellular glutamate levels in the brain by removing glutamate from the extracellular fluid. Since human blood platelets possess an active glutamate uptake system, they have been used as a peripheral model of glutamate transport in the central nervous system (CNS). The present study is aimed at identifying the glutamate transporter on blood platelets, and to asses the influence of platelet activation on glutamate uptake. Platelets from healthy donors showed Na+-dependent glutamate uptake (Km, 3.5+/-0.9 microM; Vmax, 2.8+/-0.2 pmol glutamate/75 x 10(6)platelets/30 min), which could be blocked dose-dependently by the EAAT specific inhibitors DL-threo-E-benzyloxyaspartate (TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and high concentrations of the EAAT2 inhibitor dihydrokainate (DHK). Analysis of platelet homogenates on Western blots showed EAAT2 as the predominant glutamate transporter. Platelet activation by thrombin caused an increase in glutamate uptake, which could be inhibited by TBOA and the EAAT2 inhibitor DHK. Kinetic analysis showed recruitment of new transporters to the membrane. Indeed, Western blot analysis of subcellular fractions revealed that alpha-granules, which fuse with the membrane upon thrombin stimulation, contained significant EAAT2 immunoreactivity. Inhibition of the second messengers involved in alpha-granule secretion (protein kinase C, phosphatidylinositol-3-kinase) inhibited thrombin-stimulated uptake, but not basal uptake. These data show that the glial EAAT2 is the predominant glutamate transporter on blood platelets and suggest, that thrombin increases glutamate uptake capacity by recruiting new transporters (EAAT2) from alpha-granules.

Asunto(s)

Plaquetas/metabolismo , Transportador 2 de Aminoácidos Excitadores/fisiología , Ácido Glutámico/sangre , Neuroglía/metabolismo , Trombina/farmacología , Adenosina Difosfato/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Western Blotting , Separación Celular , Forma de la Célula/efectos de los fármacos , Cromatografía en Gel , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Humanos , Técnicas In Vitro , Cinética , Neuroglía/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Sodio/fisiología

RESUMEN

The chemokine CCL2 is produced at high levels in the central nervous system (CNS) during infection, injury, neuroinflammation and other pathological conditions. Cells of the CNS including neurons and glia express receptors for CCL2 and these receptors may contribute to a signaling system through which pathologic conditions in the CNS are communicated. However, our understanding of the consequences of activation of chemokine signaling in the CNS is limited, especially for neurons. In many cell types, chemokine signaling alters intracellular Ca(2+) dynamics. Therefore, we investigated the potential involvement of this mechanism in neuronal signaling activated by CCL2. In addition, we examined the effects of CCL2 on neuronal excitability. The studies focused on the rat cerebellar Purkinje neuron, an identified CNS neuronal type reported to express both CCL2 and its receptor, CCR2. Immunohistochemical studies of Purkinje neurons in situ confirmed that they express CCR2 and CCL2. The effect of exogenous application on Purkinje neurons was studied in a cerebellar culture preparation. CCL2 was tested by micropressure or bath application, at high concentrations (13-100 nm) to simulate conditions during a pathologic state. Results show that Purkinje neurons express receptors for CCL2 and that activation of these receptors alters several neuronal properties. CCL2 increased resting Ca(2+) levels, enhanced the Ca(2+) response evoked by activation of metabotropic glutamate receptor 1 and depressed action potential generation in the cultured Purkinje neurons. Passive membrane properties were unaltered. These modulatory effects of CCL2 on neuronal properties are likely to contribute to the altered CNS function associated with CNS disease and injury.

Asunto(s)

Potenciales de Acción/inmunología , Señalización del Calcio/inmunología , Membrana Celular/metabolismo , Quimiocina CCL2/metabolismo , Neuroinmunomodulación/fisiología , Células de Purkinje/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/inmunología , Quimiocina CCL2/inmunología , Quimiocina CCL2/farmacología , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Inhibición Neural/efectos de los fármacos , Inhibición Neural/inmunología , Neuroinmunomodulación/efectos de los fármacos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Células de Purkinje/efectos de los fármacos , Células de Purkinje/inmunología , Ratas , Ratas Sprague-Dawley , Receptores CCR2 , Receptores de Quimiocina/efectos de los fármacos , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Receptores de Glutamato Metabotrópico/metabolismo

RESUMEN

BACKGROUND: Increased levels of glutamate have been reported in the epileptogenic hippocampus of patients with temporal lobe epilepsy (TLE). This sustained increase, which may contribute to the initiation and propagation of seizure activity, indicates impaired clearance of glutamate released by neurons. Glutamate is predominantly cleared by glial cells through the excitatory amino acid transporter 2 (EAAT2) and its subsequent conversion to glutamine by the glial enzyme glutamine synthetase (GS). METHODS: The authors examined the hippocampal distribution of GS, EAAT2, and glial fibrillary acidic protein (GFAP) by immunohistochemistry in TLE patients with (HS group) and without hippocampal sclerosis (non-HS group), and in autopsy controls. In hippocampal homogenates the authors measured relative protein amounts by immunoblotting and GS enzyme activity. RESULTS: In the autopsy control and non-HS group GS immunoreactivity (IR) was predominantly found in glia in the neuropil of the subiculum, of the pyramidal cell layer of all CA fields, and in the supragranular layer of the dentate gyrus. In the HS group, GS and EAAT2 IR were markedly reduced in subfields showing neuron loss (CA1 and CA4), whereas GFAP IR was increased. The reduction in GS IR in the HS group was confirmed by immunoblotting and paralleled by decreased GS enzyme activity. CONCLUSIONS: Glial glutamine synthetase is downregulated in the hippocampal sclerosis (HS) hippocampus of temporal lobe epilepsy (TLE) patients in areas with severe neuron loss. This downregulation appears to be pathology-related, rather than seizure-related, and may be part of the mechanism underlying impaired glutamate clearance found in the hippocampus of TLE patients with HS.

Asunto(s)

Epilepsia del Lóbulo Temporal/enzimología , Glutamato-Amoníaco Ligasa/deficiencia , Hipocampo/enzimología , Neuronas/patología , Adulto , Anciano , Lobectomía Temporal Anterior , Anticonvulsivantes/uso terapéutico , Biomarcadores , Neoplasias Encefálicas/enzimología , Muerte Celular , Terapia Combinada , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/cirugía , Transportador 2 de Aminoácidos Excitadores/análisis , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Glutamato-Amoníaco Ligasa/análisis , Ácido Glutámico/metabolismo , Hipocampo/patología , Humanos , Masculino , Persona de Mediana Edad , Neuroglía/enzimología , Esclerosis

RESUMEN

In a tuberous sclerosis patient with a mutation in the TSC1 tumor suppressor gene, no second-hit mutation was found in a resected cortical tuber. Tuber giant cells showed predominantly nuclear hamartin, cytosolic tuberin, and hyperphosphorylation of S6. Differential accumulation of hamartin and tuberin in separate cellular compartments of giant cells may prevent formation of the hamartin-tuberin complex, resulting in increased S6 phosphorylation. These data provide an alternative mechanism for tuberogenesis.

Asunto(s)

Proteínas del Tejido Nervioso/metabolismo , Proteínas Represoras/metabolismo , Proteína S6 Ribosómica/metabolismo , Esclerosis Tuberosa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Corteza Cerebral/metabolismo , Niño , Epilepsia/etiología , Epilepsia/metabolismo , Femenino , Mutación de Línea Germinal , Humanos , Técnicas para Inmunoenzimas , Fosforilación , Mutación Puntual , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética

RESUMEN

RATIONALE: Altered expression of glutamate transporter EAAT2 protein has been reported in the hippocampus of patients with temporal lobe epilepsy (TLE). Two alternative EAAT2 mRNA splice forms, one resulting from a partial retention of intron 7 (I7R), the other from a deletion of exon 9 (E9S), were previously implicated in the loss of EAAT2 protein in patients with amyotrophic lateral sclerosis. METHODS: By RT-PCR we studied the occurrence of I7R and E9S in neocortical and hippocampal specimens from TLE patients and non-neurological controls. RESULTS: Both splice forms were found in all neocortical specimens from TLE patients (100% I7R, 100% E9S). This was significantly more than in controls (67% I7R, 60% E9S; P < 0.05). We also detected I7R and E9S in all seven motor cortex post-mortem samples from patients with amyotrophic lateral sclerosis. Within the TLE patient group, both splice variants appeared significantly more in non-sclerotic (100%), than in sclerotic hippocampi (69%, P < 0.05). CONCLUSION: These data indicate that the epileptic brain, especially that of TLE patients without hippocampal sclerosis, is highly prone to alternative EAAT2 mRNA splicing. Our data confirm that the presence of alternative EAAT2 splice forms is not disease specific.

Asunto(s)

Empalme Alternativo/genética , Epilepsia del Lóbulo Temporal/genética , Transportador 2 de Aminoácidos Excitadores/genética , Hipocampo/metabolismo , Neocórtex/metabolismo , ARN Mensajero/metabolismo , Adulto , Anciano , Empalme Alternativo/fisiología , Distribución de Chi-Cuadrado , Epilepsia del Lóbulo Temporal/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Humanos , Persona de Mediana Edad , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética

RESUMEN

Glutamate metabolism is known to be important for growth and development of the human fetus. The glutamate transporters EAAT1, EAAT2 and EAAT3 are key components of the glutamate-glutamine cycle and responsible for active transport of glutamate over the cell membrane. The placenta is thought to regulate glutamate transport during fetal development. Glutamate transporters have been found in placentae of rats, but their distribution in the human placenta is unknown. Therefore, the distribution of glutamate transporters EAAT1, EAAT2 and EAAT3 were analysed in the human placenta during normal pregnancies ending between 8 and 40 weeks of gestation and in placentae of intrauterine growth restricted infants with gestational ages between 28 and 35 weeks of pregnancy. Using immunohistochemistry, EAAT1 expression was found in the syncytiotrophoblast layer, while EAAT2 was detected in the syncytiotrophoblast layer and in endothelial cells of about 5 per cent of all fetal blood vessels. EAAT3 was observed in the endothelium of the fetal blood vessels in all placentae examined. However, expression was also found in the syncytio- and the cytotrophoblast layer of the fetal villi at 8 weeks of gestational age. The expression patterns of EAAT1, EAAT2 and EAAT3 suggest involvement in active transport of glutamate between the fetal and maternal blood circulation. No differences were found in the distribution of the glutamate transporters between control and IUGR placentae. Our data show specific localization of EAAT1, EAAT2 and EAAT3 in the human placenta during development.

Asunto(s)

Sistema de Transporte de Aminoácidos X-AG/análisis , Transportador 2 de Aminoácidos Excitadores/análisis , Placenta/química , Simportadores/análisis , Endotelio Vascular/química , Transportador 1 de Aminoácidos Excitadores , Transportador 3 de Aminoácidos Excitadores , Femenino , Retardo del Crecimiento Fetal/metabolismo , Feto/irrigación sanguínea , Edad Gestacional , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Humanos , Inmunohistoquímica , Placenta/irrigación sanguínea , Embarazo , Distribución Tisular , Trofoblastos/química

RESUMEN

High-affinity glutamate and GABA transporters found in the plasma membrane of neurons and glial cells terminate neurotransmission by rapidly removing extracellular transmitter. Impairment of transporter function has been implicated in the pathophysiologic mechanisms underlying epileptogenesis. We characterized glutamate and gamma-aminobutyric acid (GABA) transport in synaptosomes, isolated from neocortical and hippocampal biopsies of patients with temporal lobe epilepsy (TLE). We analyzed K(+)-evoked release in the presence and absence of Ca(2+) to determine vesicular and transporter-mediated release, respectively. We also analyzed (3)H-glutamate and (3)H-GABA uptake, the effect of glutamate uptake inhibitors L-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC) and DL-threo-beta-benzyloxyaspartate (TBOA), and GABA uptake inhibitor N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (SK&F 89976-A). Neocortical synaptosomes from TLE patients did not show vesicular glutamate release, strongly reduced transporter-mediated release, and an increased basal release compared to that in rat synaptosomes. Furthermore, basal release was less sensitive to tPDC, and (3)H-glutamate uptake was reduced compared to that in rat synaptosomes. Vesicular GABA release from neocortical synaptosomes of TLE patients was reduced compared to that in rat synaptosomes, whereas transporter-mediated release was hardly affected. Furthermore, basal GABA release was more than doubled, but neither basal nor stimulated release were increased by SK&F 89976-A, which did significantly increase both types of GABA release in rat synaptosomes. Finally, (3)H-GABA uptake by synaptosomes from TLE patients was reduced significantly in hippocampus (0.19 +/- 0.04%), compared to that in neocortex (0.32 +/- 0.04%). Control experiments with human peritumoral cortical tissue suggest that impaired uptake of glutamate, but not of GABA, was caused in part by the hypoxic state of the biopsy. Our findings provide evidence for impaired function of glutamate and GABA transporters in human TLE.

Asunto(s)

Sistema de Transporte de Aminoácidos X-AG/metabolismo , Proteínas Portadoras/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Ácido Glutámico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Sinaptosomas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Transporte Biológico/fisiología , Calcio/metabolismo , Epilepsia del Lóbulo Temporal/fisiopatología , Proteínas Transportadoras de GABA en la Membrana Plasmática , Humanos , Técnicas In Vitro , Potasio/metabolismo , Ratas

RESUMEN

Glial fibrillary acidic protein (GFAP) is considered to be a highly specific marker for glia. Here, we report on the expression of GFAP in neurons in the human hippocampus. Intriguingly, this neuronal GFAP is coded by out-of-frame splice variants and its expression is associated with Alzheimer pathology. We identified three novel GFAP splice forms: Delta 135 nt, Delta exon 6 and Delta 164 nt. Neuronal GFAP is mainly observed in the pyramidal neurons of the hippocampus of Alzheimer and Down syndrome patients and aged controls, but not in neurons of patients suffering from hippocampal sclerosis. Apparently, the hippocampal neurons in patients with Alzheimer's disease pathology are capable of expressing glia-specific genes.

Asunto(s)

Empalme Alternativo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Sistema de Lectura Ribosómico , Proteína Ácida Fibrilar de la Glía/metabolismo , Neuronas/metabolismo , Transcripción Genética , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Secuencia de Bases , Síndrome de Down/genética , Síndrome de Down/metabolismo , Síndrome de Down/patología , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/fisiopatología , Exones , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Masculino , Datos de Secuencia Molecular , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/patología , ARN Mensajero/análisis , Sistemas de Lectura/genética , Esclerosis

RESUMEN

In patients suffering from temporal lobe epilepsy (TLE), increased extracellular glutamate levels in the epileptogenic hippocampus both during and after clinical seizures have been reported. These increased glutamate levels could be the result of malfunctioning and/or downregulation of glutamate transporters (also known as EAATs; excitatory amino acid transporters). In this study, the distribution of protein and mRNA of EAAT subtypes was examined in the hippocampus of TLE patients with hippocampal sclerosis (HS group) and without hippocampal sclerosis (non-HS group), and in autopsy controls without neurological disorders. EAAT protein localization was studied by immunohistochemistry on paraffin sections using specific poly- and monoclonal antibodies against the glial glutamate transporters EAAT1 and EAAT2 and the neuronal glutamate transporter EAAT3. Antibody specificity was shown by immunoblotting. In the HS group, a small decrease in EAAT1-immunoreactivity (IR) was observed in CA4 and in the polymorphic and supragranular layer of the dentate gyrus, compared with the control group. The strongest changes were found for EAAT2 levels. In the non-HS group, increased EAAT2-IR was detected in the CA1 and CA2 field, compared with non-epileptic controls. EAAT2-IR was decreased in the HS compared with the non-HS group. Fewer EAAT3-positive cells were found in the HS group than in the non-HS and control group. In both TLE groups, increased EAAT3 levels were observed in individual neurones. In the HS group, the percentage of EAAT3-IR neurones was increased in CA2 and in the granule cell layer of the dentate gyrus. Radioactive in situ hybridization for EAAT1-3 confirmed our immunohistochemical results. Non-radioactive in situ hybridization showed that not only astrocytes, but also neurones express EAAT2 mRNA. Taken together, differences in both mRNA and protein levels of glutamate transporter subtypes were found in specific regions in the TLE hippocampus, with most severe changes found for EAAT2 and EAAT3 levels. The results indicate an upregulation of EAAT2 protein expression in CA1 and CA2 in neurones in the non-HS group. This is in line with decreased EAAT2 protein levels in the HS group, since these hippocampi are characterized by severe neuronal cell loss. The functional consequences (glutamate transport capacity) of the reported changes in EAAT2 and EAAT3 remain to be determined.

Asunto(s)

Sistema de Transporte de Aminoácidos X-AG/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Adulto , Sistema de Transporte de Aminoácidos X-AG/genética , Análisis de Varianza , Animales , Anticonvulsivantes/uso terapéutico , Resistencia a Medicamentos , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/fisiopatología , Femenino , Hipocampo/patología , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Masculino , Persona de Mediana Edad , Esclerosis

RESUMEN

In patients suffering from temporal lobe epilepsy (TLE) a highly variable degree of hippocampal sclerosis (HS) can be observed. For standard neuropathological evaluation after hippocampal resection, neuronal cell loss in the hippocampal subareas is assessed (Wyler score 0-4) [Wyler et al. (1992) J Epilepsy 5: 220-225]. Other marked morphological changes in the sclerotic hippocampus are gliosis and loss of mossy fibers in the hilus and mossy fiber sprouting in the supragranular layer. In this study we quantified changes in mossy fiber density using Timm's stain in resected hippocampal tissue from patients with various degrees of sclerosis. We found that tissue specimens from patients without sclerosis (W0) show almost no mossy fiber sprouting. Patients with moderate sclerosis show sprouting without fiber loss in the hilus, whereas specimens from patients with severe sclerosis show sprouting as well as fiber loss in the hilus. Thus, analysis of mossy fiber abundance in hilus and supragranular layer by the rapid and simple Timm's stain is a sensitive measure for hippocampal sclerosis. It provides a reliable rapid tool for neuropathological evaluation, even if the tissue only contains dentate gyrus due to the sectioning procedure.

Asunto(s)

Epilepsia del Lóbulo Temporal/patología , Hipocampo/patología , Fibras Musgosas del Hipocampo/ultraestructura , Índice de Severidad de la Enfermedad , Axones/ultraestructura , Biopsia , Gliosis/patología , Humanos , Esclerosis

RESUMEN

Induction of homosynaptic long term depression (LTD) in the CA1 field of the hippocampus is thought to require activation of N-methyl-d-aspartate receptors, an elevation of postsynaptic Ca(2+) levels, and a subsequent increase in phosphatase activity. To investigate the spatial and temporal changes in protein phosphatase activity following LTD induction, we determined the in situ phosphorylation state of a pre- (GAP-43/B-50) and postsynaptic (RC3) protein kinase C substrate during N-methyl-d-aspartate receptor-dependent LTD in the CA1 field of rat hippocampal slices. We show that LTD is associated with a transient (<30 min) and D-AP5-sensitive reduction in GAP-43/B-50 and RC3 phosphorylation and that LTD is prevented by the phosphatase inhibitors okadaic acid and cyclosporin A. Our data provide strong evidence for a transient increase in pre- and postsynaptic phosphatase activity during LTD. Since the in situ phosphorylation of the calmodulin-binding proteins GAP-43/B-50 and RC3 changes during both LTD and long term potentiation, these proteins may form part of the link between the Ca(2+) signal and Ca(2+)/calmodulin-dependent processes implicated in long term potentiation and LTD.

Asunto(s)

Proteínas de Unión a Calmodulina/metabolismo , Proteína GAP-43/metabolismo , Hipocampo/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteína Quinasa C/metabolismo , Animales , Técnicas In Vitro , Neurogranina , Fosforilación , Ratas