RESUMEN
Disruption of Interleukin-1ß (IL-1ß) signaling sensitized mice to convulsant stimuli, suggesting that this quintessential cytokine of the innate immune system contributes to maintenance of the innate seizure threshold (ST). However, much remains unknown about where and how IL-1ß secretion occurs in the normal brain. This study examined the possibility that neurons of the hippocampus are key sources of constitutive IL-1ß secretion and that the release from these cells is dependent on the purinoceptor, P2X7. It was posited that treatment with the P2X7 antagonist, JNJ-47965567 (JNJ), would cause IL-1ß to accumulate in cells that produce it, and consequently, lower the ST. No IL-1ß immunoreactivity was detected in any region of the hippocampal formation of mice treated with the JNJ vehicle, Sulfobutylether-ß-cyclodextrin. In contrast, prominent immunoreactivity was discovered in the pyramidal neurons of the CA3 region 60 min after treatment with the P2X7 antagonist. Lower levels were found in CA1 neurons, and no immunoreactivity was detected in granule cells of the dentate gyrus. JNJ also increased IL-1ß immunoreactivity in the cell bodies of hippocampal neurons in culture. Interestingly, JNJ potentiated bicuculline-induced Fos and COX-2 mRNA expression in the cultures and this was blocked by an NMDA receptor antagonist. Moreover, pentylenetetrazole-induced seizure severity and incidence of convulsions were increased in mice treated with JNJ and this resembled that observed with IL-1 signaling-deficient mice. Overall, the results from this study support the notion that constitutive P2X7-dependent IL-1ß release from hippocampal pyramidal neurons contributes to maintenance of the ST in the normal brain, perhaps by modulating neuronal excitability. These findings may have implications for epilepsy, a brain disorder in which the ST is compromised.
Asunto(s)
Células Piramidales , Convulsiones , Animales , Encéfalo/metabolismo , Hipocampo/metabolismo , Interleucina-1beta , Ratones , Células Piramidales/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Convulsiones/inducido químicamente , Convulsiones/metabolismoRESUMEN
OBJECTIVE: Although the cystine/glutamate antiporter System xc - (Sxc -) plays a permissive role in glioma-associated seizures, its contribution to other acquired epilepsies has not been determined. As such, the present study investigates whether and how Sxc - contributes to the pentylenetetrazole (PTZ) chemical kindling model of epileptogenesis. METHODS: Male Sxc - null (sut/sut) mice and their wild-type littermates were administered PTZ (i.p.) daily for up to 21 days (kindling paradigm). Seizure severity was scored on a 5-point behavioral scale. Mossy fiber sprouting, cellular degeneration, and Sxc - light chain (xCT) messenger RNA (mRNA) were explored using Timm staining, thionin staining, and real-time quantitative polymerase chain reaction (qPCR), respectively. Levels of reduced and oxidized glutathione and cysteine were determined via high-performance liquid chromatography (HPLC). Plasma membrane protein levels of glutamate and γ-aminobutyric acid (GABA) receptor subunits as well as the K+/Cl- co-transporter KCC2 were quantified via western blot analysis. RESULTS: Repeated administration of PTZ produced chemical kindling in only 50% of Sxc - null mice as compared to 82% of wild-type littermate control mice. Kindling did not result in any changes in xCT mRNA levels assessed in wild-type mice. No cellular degeneration or mossy fiber sprouting was discernible in either genotype. Except for a small, but significant, decrease in oxidized cysteine in the hippocampus, no other change in measured redox couples was determined in Sxc - null mice. Cortical levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 were decreased in Sxc - null mice as compared to wild-type littermates, whereas all other proteins tested showed no difference between genotypes. SIGNIFICANCE: This study provides the first evidence that Sxc - signaling contributes to epileptogenesis in the PTZ kindling model of acquired epilepsy. Further data indicate that a reduction in AMPA receptor signaling could underlie the resistance to PTZ kindling uncovered in Sxc - null mice.
RESUMEN
OBJECTIVE: Studies have addressed the potential involvement of L-12/15-lipoxygenases (LOs), a polyunsaturated fatty acid metabolizing enzyme, in experimental models of acute stroke and chronic neurodegeneration; however, none to our knowledge has explored its role in epilepsy development. Thus, this study characterizes the cell-specific expression of L-12/15 -LO in the brain and examines its contribution to epileptogenesis. METHODS: L-12/15-LO messenger RNA (mRNA) and protein expression and activity were characterized via polymerase chain reaction (PCR), immunocytochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. To assess its role in epileptogenesis, L-12/15 -LO-deficient mice and their wild-type littermates were treated with pentylenetetrazole (PTZ, ip) every other day for up to 43 days (kindling paradigm). The innate seizure threshold was assessed by the acute PTZ-induced seizure response of naive mice. RESULTS: L-12/15 -LO mRNA is expressed in hippocampal and cortical tissue from wild-type C57BL/6 mice. In addition, it is physically and functionally expressed by microglia, neurons, and brain microvessel endothelial cells, but not by astrocytes. Mice deficient in L-12/15 -LO were resistant to PTZ-induced kindling and demonstrated an elevated innate seizure threshold. Despite this, a significant increase in seizure-related mortality was observed during the kindling paradigm in L-12/15 -LO nulls relative to their wild-type littermates. SIGNIFICANCE: The present study is the first to detail the role of L-12/15-LO in the epileptogenic process. The results suggest that constitutive L-12/15-LO expression contributes to a lower innate set point for PTZ acute seizure generation, translating to higher rates of kindling acquisition. Nevertheless, increased seizure-related deaths in mice lacking activity of L-12/15-LO suggests that its products may influence endogenous mechanisms involved in termination of seizure activity.
RESUMEN
Activity of neuronal cyclooxygenase-2 (COX-2), a primary source of PG synthesis in the normal brain, is enhanced by excitatory neurotransmission and this is thought to be involved in seizure suppression. Results herein showing that the incidence of pentylenetetrazole (PTZ)-induced convulsions is suppressed in transgenic mice overexpressing COX-2 in neurons support this notion. T-cell intracellular antigen-1 (TIA-1) is an mRNA binding protein that is known to bind to COX-2 mRNA and repress its translation in non-neuronal cell types. An examination of the expression profile of TIA-1 protein in the normal brain indicated that it is expressed broadly by neurons, including those that express COX-2. However, whether TIA-1 regulates COX-2 protein levels in neurons is not known. The purpose of this study was to test the possibility that deletion of TIA-1 increases basal COX-2 expression in neurons and consequently raises the seizure threshold. Results demonstrate that neither the basal nor seizure-induced expression profiles of COX-2 were altered in mice lacking a functional TIA-1 gene suggesting that TIA-1 does not contribute to regulation of COX-2 protein expression in neurons. The acute PTZ-induced seizure threshold was also unchanged in mice lacking TIA-1 protein, indicating that this RNA binding protein does not influence the innate seizure threshold. Nevertheless, the results raise the possibility that the level of neuronal COX-2 expression may be a determinant of the innate seizure threshold and suggest that a better understanding of the regulation of COX-2 expression in the brain could provide new insight into the molecular mechanisms that suppress seizure induction.
Asunto(s)
Encéfalo/metabolismo , Ciclooxigenasa 2/metabolismo , Neuronas/metabolismo , Convulsiones/metabolismo , Animales , Encéfalo/patología , Ciclooxigenasa 2/genética , Modelos Animales de Enfermedad , Expresión Génica , Masculino , Ratones Endogámicos C57BL , Neuronas/patología , Pentilenotetrazol , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Distribución Aleatoria , Convulsiones/patologíaRESUMEN
Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , Ácido Glutámico/metabolismo , Plasticidad Neuronal , Neuronas/metabolismo , Factor de Transcripción Sp1/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Ciclooxigenasa 2/genética , Regulación de la Expresión Génica , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuronas/citología , Regiones Promotoras Genéticas/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/genética , Transcripción Genética/genéticaRESUMEN
System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1ß, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1ß increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1ß induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1ß-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1ß-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1ß-induced stabilization of astrocyte xCT mRNA.
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Sistema de Transporte de Aminoácidos y+/metabolismo , Astrocitos/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Regulación de la Expresión Génica , Interleucina-1beta/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Sistema de Transporte de Aminoácidos y+/genética , Animales , Células Cultivadas , Proteína 1 Similar a ELAV/genética , Ácido Glutámico/metabolismo , Interleucina-1beta/genética , Ratones , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Regulación hacia ArribaRESUMEN
The astrocyte cystine/glutamate antiporter (system xc(-)) contributes substantially to the excitotoxic neuronal cell death facilitated by glucose deprivation. The purpose of this study was to determine the mechanism by which this occurred. Using pure astrocyte cultures, as well as, mixed cortical cell cultures containing both neurons and astrocytes, we found that neither an enhancement in system xc(-) expression nor activity underlies the excitotoxic effects of aglycemia. In addition, using three separate bioassays, we demonstrate no change in the ability of glucose-deprived astrocytes--either cultured alone or with neurons--to remove glutamate from the extracellular space. Instead, we demonstrate that glucose-deprived cultures are 2 to 3 times more sensitive to the killing effects of glutamate or N-methyl-D-aspartate when compared with their glucose-containing controls. Hence, our results are consistent with the weak excitotoxic hypothesis such that a bioenergetic deficiency, which is measureable in our mixed but not astrocyte cultures, allows normally innocuous concentrations of glutamate to become excitotoxic. Adding to the burgeoning literature detailing the contribution of astrocytes to neuronal injury, we conclude that under our experimental paradigm, a cytotoxic, co-operative interaction between energy deprivation and glutamate release from astrocyte system xc(-) mediates aglycemic neuronal cell death.
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Sistema de Transporte de Aminoácidos y+/metabolismo , Astrocitos/fisiología , Muerte Celular/fisiología , Ácido Glutámico/metabolismo , Neuronas/fisiología , Adenosina Trifosfato/metabolismo , Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Animales , Ácido Aspártico/metabolismo , Astrocitos/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Técnicas de Cocultivo , Cistina/metabolismo , Espacio Extracelular/metabolismo , Ácido Glutámico/toxicidad , Ratones , N-Metilaspartato/metabolismo , N-Metilaspartato/toxicidad , Neuronas/efectos de los fármacos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Factores de TiempoRESUMEN
Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system xc---an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents--whereas addition of L-cystine restores--GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc-. Indeed, drugs known to inhibit system xc- ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system xc- (xCT). Finally, enhancement of astrocytic system xc- expression and function via IL-1ß (interleukin-1ß) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc- inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc-, have a direct, non-cell autonomous effect on cortical neuron survival.
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Sistema de Transporte de Aminoácidos y+/metabolismo , Astrocitos/metabolismo , Comunicación Celular/fisiología , Hipoglucemia/metabolismo , Neuronas/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Muerte Celular/fisiología , Técnicas de Cocultivo , Glucosa/deficiencia , Ácido Glutámico/metabolismo , Ratones , Ratones Mutantes , Neuronas/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The function of endogenous interleukin-1ß (IL-1ß) signaling in acute seizure activity was examined using transgenic mice harboring targeted deletions in the genes for either IL-1ß (Il1b) or its signaling receptor (Il1r1). Acute epileptic seizure activity was modeled using two mechanistically distinct chemoconvulsants, kainic acid (KA) and pentylenetetrazole (PTZ). KA-induced seizure activity was more severe in homozygous null (-/-) Il1b mice compared to their wild-type (+/+) littermate controls, as indicated by an increase in the incidence of sustained generalized convulsive seizure activity. In the PTZ seizure model, the incidence of acute convulsive seizures was increased in both Il1b and Il1r1-/- mice compared to their respective +/+ littermate controls. Interestingly, the selective cyclooxygenase (COX)-2 inhibitor, rofecoxib, mimicked the effect of IL-1ß deficiency on PTZ-induced convulsions in Il1r1+/+ but not -/- mice. Together, these results suggest that endogenous IL-1ß possesses anticonvulsive properties that may be mediated by arachidonic acid metabolites derived from the catalytic action of COX-2.
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Ciclooxigenasa 2/metabolismo , Interleucina-1beta/metabolismo , Convulsiones/metabolismo , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Interleucina-1beta/genética , Ácido Kaínico , Lactonas/farmacología , Ratones , Ratones Noqueados , Pentilenotetrazol , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Convulsiones/inducido químicamente , Convulsiones/genética , Sulfonas/farmacologíaRESUMEN
Microglia, resident phagocytic cells of the central nervous system, are frequent contaminants of astrocyte cultures. Unfortunately and not always fully appreciated, contamination by microglia can confound results of studies designed to elucidate the molecular mechanisms underlying astrocyte-specific responses. The paradigm described herein employs the mitotic inhibitor, cytosine ß-D: -arabinofuranoside, followed by the lysosomotropic agent, leucine methylester, to maximally deplete microglia, thereby generating highly enriched astrocyte monolayers that remain viable and functional. Successful removal of microglia from confluent monolayers of primary astrocyte cultures is achieved without the need for cell passage and successful reduction is confirmed by depletion of microglial-specific markers.
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Astrocitos/citología , Técnicas de Cultivo de Célula/métodos , Citarabina/farmacología , Leucina/análogos & derivados , Leucina/farmacología , Microglía/citología , Microglía/efectos de los fármacos , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismoRESUMEN
Astrocytes produce numerous mediators under conditions of inflammation in the central nervous system. One such mediator is nitric oxide (NO) derived from nitric oxide synthase-2 (NOS-2), the high output, inducible NOS isoform. Expression of NOS-2 and production of NO can be stimulated in astrocyte cultures by combinations of cytokines and lipopolysaccharide, a gram-negative bacterial endotoxin. This chapter details methods to induce and analyze NOS-2 expression and NO production in astrocyte cultures.
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Astrocitos/metabolismo , Técnicas de Cultivo de Célula/métodos , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico/biosíntesis , Animales , Astrocitos/fisiología , Citocinas , Inducción Enzimática/fisiología , Inmunohistoquímica , Interferón gamma , Interleucina-1beta , Lipopolisacáridos , Ratones , Factor de Crecimiento Transformador beta1RESUMEN
PURPOSE: The goal of this study was to determine whether prophylactic prandial administration of rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, could alter seizure generation, kindling acquisition, and/or kindling maintenance in the mouse pentylenetetrazole (PTZ) epilepsy model. METHODS: Male CD-1 mice were fed ad libitum with control chow or chow formulated to deliver 30 mg/kg/day rofecoxib. After 5 days, mice were treated with a single dose of 40 or 55 mg/kg PTZ (acute paradigm) or 40 mg/kg PTZ delivered daily (kindling paradigm). Seizure severity was scored on a four-point behavioral scale and COX-2 expression was assessed in brain slices from a subset of mice 3 h or 72 h after acute PTZ or following establishment of kindling. KEY FINDINGS: Hippocampal COX-2 expression was transiently upregulated 3 h after an acute PTZ-induced convulsion and returned to baseline levels within 72 h, whereas it remained elevated for at least 72 h after the final seizure in the kindling paradigm. Despite this increase, chronic rofecoxib treatment did not attenuate the severity of acute PTZ-induced seizures and failed to alter kindling development or maintenance. SIGNIFICANCE: The present study demonstrates that prophylactic, prandial rofecoxib treatment lacks efficacy against acute PTZ-induced seizure generation and kindling acquisition, and does not reverse the kindled state once established.
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Anticonvulsivantes , Convulsivantes , Inhibidores de la Ciclooxigenasa 2/farmacología , Excitación Neurológica/efectos de los fármacos , Lactonas/farmacología , Pentilenotetrazol , Convulsiones/prevención & control , Sulfonas/farmacología , Animales , Conducta Animal/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Ciclooxigenasa 2/metabolismo , Dieta , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos , Extinción Psicológica/efectos de los fármacos , Interacciones Alimento-Droga , Inmunohistoquímica , Ácido Kaínico/farmacología , Masculino , Ratones , Convulsiones/inducido químicamenteRESUMEN
We previously demonstrated that transforming growth factor-beta1 (TGF-beta1), while having no effect alone, enhances nitric oxide (NO) production in primary, purified mouse astrocytes induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma), by recruiting a latent population of astrocytes to respond, thereby enhancing the total number of cells that express Nos2. In this investigation, we evaluated the molecular signaling pathway by which this occurs. We found that purified murine primary astrocytes express mRNA for TGFbetaRII as well as the TGFbetaRI subunit activin-like kinase 5 (ALK5), but not ALK1. Immunofluorescence microscopy confirmed the expression of TGFbetaRII and ALK5 protein in astrocytes. Consistent with ALK5 signaling, Smad3 accumulated in the nucleus of astrocytes as early as 30 min after TGF-beta1 (3 ng/mL) treatment and persisted upto 32 hr after TGF-beta1 administration. Addition of ALK5 inhibitors prevented TGF-beta1-mediated Smad3 nuclear accumulation and NO production when given prior to the Nos2 induction stimuli, but not after. Finally, astrocyte cultures derived from Smad3 null mutant mice did not exhibit a TGF-beta1-mediated increase in iNOS expression. Overall, this data suggests that ALK5 signaling and Smad3 nuclear accumulation is required for optimal enhancement of LPS plus IFNgamma-induced NO production in astrocytes by TGF-beta1.
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Astrocitos/metabolismo , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico/biosíntesis , Transducción de Señal/fisiología , Proteína smad3/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Regulación hacia Arriba/fisiología , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/fisiología , Animales , Astrocitos/enzimología , Astrocitos/patología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Núcleo Celular/enzimología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Femenino , Regulación Enzimológica de la Expresión Génica/fisiología , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico/genética , Óxido Nítrico Sintasa de Tipo II/genética , Proteínas Serina-Treonina Quinasas/fisiología , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Proteínas Recombinantes/farmacología , Transducción de Señal/genética , Proteína smad3/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
We recently demonstrated that interleukin-1ß (IL-1ß) increases system x(c)(-) (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes, and microglia all express system x(c)(-) subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL-1ß stimulation increases mRNA for xCT--the light chain that confers substrate specificity--in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL-1ß exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1ß is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild-type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL-1ß does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL-1ß treatment in our mixed culture system was not observed in chimeric cultures consisting of wild-type neurons plated on top of sut astrocytes. Nor was it observed in wild-type cultures treated with a system x(c)(-) inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL-1ß selectively regulates system x(c)(-) activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1ß found under hypoxic conditions.
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Sistema de Transporte de Aminoácidos y+/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1beta/farmacología , Neuronas/fisiología , Sistema de Transporte de Aminoácidos y+/genética , Análisis de Varianza , Animales , Animales Recién Nacidos , Ácido Aspártico/metabolismo , Benzoatos/farmacología , Isótopos de Carbono/metabolismo , Muerte Celular/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Células Cultivadas , Corteza Cerebral/citología , Cistina/metabolismo , Dactinomicina/farmacología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Antagonistas de Aminoácidos Excitadores/farmacología , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/genética , Transportador 2 de Aminoácidos Excitadores/metabolismo , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/genética , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Regulación de la Expresión Génica/fisiología , Glicina/análogos & derivados , Glicina/farmacología , L-Lactato Deshidrogenasa/metabolismo , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Astrocytes are a major component of the resident non-neuronal glial cell population of the CNS. They are ubiquitously distributed throughout the brain and spinal cord, where they were initially thought to function in both structural and homeostatic capacities, providing the framework and environment in which neurons performed their parenchymal duties. However, this stroma-like view of astrocytes is no longer satisfactory. Mounting evidence particularly within the last decade indicates that astrocytes do not simply support neuronal activity but directly contribute to it. Congruent with this evolving view of astrocyte function in information processing is the emergent notion that these glial cells are not a homogeneous population of cells. Thus, astrocytes in various anatomically distinct regions of the normal CNS possess unique phenotypic characteristics that may directly influence the particular neuronal activities that define these regions. Remarkably, regional populations of astrocytes appear to exhibit local heterogeneity as well. Many phenotypic traits of the astrocyte lineage are responsive to local environmental cues (i.e., are adaptable), suggesting that plasticity contributes to this diversity. However, compelling evidence suggests that astrocytes arise from multiple distinct progenitor pools in the developing CNS, raising the intriguing possibility that some astrocyte heterogeneity may result from intrinsic differences between these progenitors. The purpose of this review is to explore the evidence for and mechanistic determinants of regional and local astrocyte diversity.
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Astrocitos/fisiología , Sistema Nervioso Central/citología , Sistema Nervioso Central/fisiología , Animales , Astrocitos/citología , Modelos BiológicosRESUMEN
Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.
Asunto(s)
Ácido Araquidónico/antagonistas & inhibidores , Citosol/enzimología , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Antagonistas de Prostaglandina/fisiología , Prostaglandinas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Ácido Araquidónico/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Citosol/efectos de los fármacos , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fosfolipasas A2 Grupo IV/metabolismo , Ratones , Prostaglandinas/biosíntesis , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
Transforming growth factor-beta1 (TGF-beta1) is upregulated by inflammatory mediators in several neurological diseases/disorders where it either participates in the pathology or provides protection. Often, the biological outcome of TGF-beta1 is dependent upon changes in gene expression. Recently, we demonstrated that TGF-beta1 enhances astrocytic nitric oxide production induced by lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) by increasing the number of astrocytes in a population that express NOS-2. The purpose of this study was twofold: (1) to determine whether this effect occurs more generally by assessing the effect of TGF-beta1 on another pro-inflammatory gene, cyclooxygenase-2 (COX-2); and (2) to assess stimulus specificity. We found that TGF-beta1 augmented LPS plus IFNgamma-induced COX-2 mRNA and protein expression, by nearly tripling the number of astrocytes that express COX-2. The effect was not stimulus-specific as TGF-beta1 enhanced the number of astrocytes that expressed both COX-2 and NOS-2 protein when either IL-1beta or TNFalpha was used in lieu of LPS. Collectively, these results suggest that TGF-beta1 augments overall protein expression levels of select pro-inflammatory genes in astrocytes in a promiscuous manner by reducing the magnitude of noise in the cellular population.
Asunto(s)
Astrocitos/enzimología , Ciclooxigenasa 2/biosíntesis , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Factor de Crecimiento Transformador beta1/fisiología , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , RatonesRESUMEN
Nitric oxide (NO) synthase-2 (NOS-2), a key source of NO at sites of neuroinflammation, is induced in astrocyte cultures treated with lipopolysaccharide (LPS) plus interferon-gamma (IFN gamma). A recent study examining the regulation of astrocytic NOS-2 expression demonstrated that transforming growth factor-beta1 (TGF beta 1) potentiated LPS plus IFN gamma-induced NOS-2 expression via expansion of the pool of astrocytes that express NOS-2. Results in the current report indicate that this population-based mechanism of increasing NOS-2 expression is not restricted to TGF beta 1, since it also accounts for the potentiation of NO production in astrocyte cultures by tumor necrosis factor-alpha (TNFalpha). In contrast to TGF beta 1, which required 24h preincubation for optimal potentiation of NO production, TNF alpha was maximally effective when added concurrently with LPS plus IFN gamma. Nevertheless, under conditions that optimally potentiated NO production, both cytokines recruited similar numbers of astrocytes to express NOS-2 (% NOS-2-positive cells after LPS plus IFN gamma alone or with TNFalpha or TGF beta 1 was 9.5+/-1.2, 25.3+/-2.9, and 32.4+/-3.0, respectively). Interestingly, stimulation of astrocytes in the presence of both TGF beta 1 and TNFalpha additively increased the number of astrocytes that expressed NOS-2 protein (% NOS-2-positive cells was 61.0+/-4.2) relative to each cytokine alone. Potentiation of NO production by either TNF alpha or TGF beta 1 was not ablated by neutralizing antibodies to TGF beta 1 or TNFalpha, respectively. Thus, the two cytokines act independently to recruit separate pools of astrocytes to express NOS-2. These results are consistent with the notion that astrocytes possess an innate heterogeneity with respect to responsiveness to these cytokines.
Asunto(s)
Astrocitos/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Anticuerpos/farmacología , Astrocitos/clasificación , Astrocitos/efectos de los fármacos , Células Cultivadas , Encefalitis/metabolismo , Encefalitis/fisiopatología , Gliosis/metabolismo , Gliosis/fisiopatología , Mediadores de Inflamación/farmacología , Ratones , Factor de Crecimiento Transformador beta1/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The purpose of this study was to determine whether the selective cyclooxygenase-2 (COX-2) inhibitor rofecoxib [4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone] could effectively prevent hippocampal neuronal injury in an animal model of excitotoxic neurodegeneration. COX-2 protein levels increased between 3 and 6 h, peaked at 12 h, and declined to near baseline levels 24 h after injection of N-methyl-d-aspartate (NMDA; 18 nmol) into the CA1 region of the left hippocampus. Mice that were fed ad libitum a control rodent diet for 4 days before and 3 days after injection of NMDA demonstrated marked neuronal loss in the primary cell layers of the ipsilateral CA1, CA3, and dentate gyrus (50, 30, and 20% cell loss, respectively). This injury was potently and dose-dependently reduced by feeding animals a diet standardized to deliver 15 or 30 mg/kg rofecoxib per day. Neurodegeneration in the CA1 region was reduced by 30.1 +/- 5.6 and 51.5 +/- 9.0%, respectively; in the CA3 by 64.6 +/- 12.4 and 69.0 +/- 14.1%, respectively; and in the dentate gyrus by 47.8 +/- 15.2 and 58.0 +/- 18.2%, respectively. Moreover, rofecoxib chow slightly but significantly reduced injury-induced brain edema. These findings demonstrate that rofecoxib can ameliorate excitotoxic neuronal injury in vivo and, as such, may be a particularly promising pharmaceutical for the treatment of neurological diseases associated with overactivation of NMDA receptors.