RESUMO
We have previously reported the characterization of a novel immunoglobulin supergene family member, designated class-I MHC-restricted T cell associated molecule (CRTAM). Here we further characterize human CRTAM and find that it is highly expressed in the cerebellum, notably in Purkinje neurons. We identify CRTAM as a new member of the nectin-like (Necls) family and find significant expression of Necl-2 (IGSF4), a protein known to bind CRTAM and another member of the nectin superfamily, in the cerebellum. These findings suggest that CRTAM/Necl-2 binding may contribute to neuronal interactions. We also show that, in the immune system, CRTAM expression is restricted to activated class-I MHC-restricted T cells, including NKT and CD8 T cells. CRTAM represents one of the most highly expressed surface markers of activated human CD8 T cells and NKT cells, suggesting it may have diagnostic uses in various human viral and autoimmune diseases.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Cerebelo/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Imunoglobulinas/metabolismo , Ativação Linfocitária/fisiologia , Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting/métodos , Molécula 1 de Adesão Celular , Moléculas de Adesão Celular , Cerebelo/citologia , Relação Dose-Resposta a Droga , Ativação Enzimática/fisiologia , Feminino , Citometria de Fluxo/métodos , Humanos , Imuno-Histoquímica/métodos , Ionomicina/farmacologia , Leucócitos Mononucleares/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Filogenia , Células de Purkinje/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alinhamento de Sequência/métodos , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The role of brain insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in neuroprotection was further investigated using in vitro and in vivo models of cerebral ischemia by assessing the effects of IGF-I, IGF-II, and high affinity IGFBP ligand inhibitors (the peptide [Leu24, 59, 60, Ala31]hIGF-I (IGFBP-LI) and the small molecule NBI-31772 (1-(3,4-dihydroxybenzoyl)-3-hydroxycarbonyl-6, 7-dihydroxyisoquinoline), which pharmacologically displace and elevate endogenous, bioactive IGFs from IGFBPs. Treatment with IGF-I, IGF-II, or IGFBP-LI (2 microg/mL) significantly (P < 0.05) reduced CA1 damage in organotypic hippocampal cultures resulting from 35 minutes of oxygen and glucose deprivation by 71%, 60%, and 40%, respectively. In the subtemporal middle cerebral artery occlusion (MCAO) model of focal ischemia, intracerebroventricular (icv) administration of IGF-I and IGF-II at the time of artery occlusion reduced ischemic brain damage in a dose-dependent manner, with maximum reductions in total infarct size of 37% (P < 0.01) and 38% (P < 0.01), respectively. In this model of MCAO, i.c.v. administration of NBI-31772 at the time of ischemia onset also dose-dependently reduced infarct size, and the highest dose (100 microg) significantly reduced both total (by 40%, P < 0.01) and cortical (by 43%, P < 0.05) infarct volume. In the intraluminal suture MCAO model, administration of NBI-31772 (50 microg i.c.v.) at the time of artery occlusion reduced both cortical infarct volume (by 40%, P < 0.01) and brain swelling (by 24%, P < 0.05), and it was still effective when treatment was delayed up to 3 hours after the induction of ischemia. These results further define the neuroprotective properties of IGFs and IGFBP ligand inhibitors in experimental models of cerebral ischemia.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Catecóis/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Isoquinolinas/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Isquemia Encefálica/metabolismo , Catecóis/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/metabolismo , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like II/farmacologia , Isoquinolinas/metabolismo , Fármacos Neuroprotetores/metabolismo , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-DawleyRESUMO
We have used a rat model of focal cerebral ischemia to investigate changes in gene expression that occur during stroke. To monitor these changes, we employed representational difference analysis-polymerase chain reaction (PCR). A total of 128 unique gene fragments were isolated, and we selected 13 of these for quantitative reverse transcriptase-PCR analysis. Of these 13 genes, we found seven that were differentially expressed. Four of these genes have not previously been implicated in stroke, and include neuronal activity regulated pentraxin (Narp), cysteine rich protein 61 (Cyr61), Bcl-2 binding protein BIS (Bcl-2-interacting death suppressor), and lectin-like ox-LDL receptor (LOX-1). We demonstrated differential expression of each gene by quantitative PCR analysis, and in the case of LOX-1, we further confirmed differential expression by in situ hybridization. LOX-1 expression is induced greater than ten fold at the core lesion site, and is essentially localized to the ipsilateral half of the brain. LOX-1 appears to be expressed in a non-neuronal cell type, and it does not appear to be expressed in vascular endothelial cells within the brain. This suggests that LOX-1 may serve a novel function in the brain.
Assuntos
Isquemia Encefálica/genética , Infarto Cerebral/genética , Regulação da Expressão Gênica/fisiologia , Traumatismo por Reperfusão/genética , Acidente Vascular Cerebral/genética , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Infarto Cerebral/metabolismo , Infarto Cerebral/fisiopatologia , DNA Complementar/análise , Endotélio Vascular/metabolismo , Masculino , Neostriado/metabolismo , Neostriado/patologia , Neostriado/fisiopatologia , Neuroglia/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de LDL/genética , Receptores de LDL Oxidado , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Receptores Depuradores Classe E , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Regulação para Cima/fisiologiaRESUMO
Glutamate is the major excitatory neurotransmitter in the central nervous system and is tightly regulated by cell surface transporters to avoid increases in concentration and associated neurotoxicity. Selective blockers of glutamate transporter subtypes are sparse and so knock-out animals and antisense techniques have been used to study their specific roles. Here we used WAY-855, a GLT-1-preferring blocker, to assess the role of GLT-1 in rat hippocampus. GLT-1 was the most abundant transporter in the hippocampus at the mRNA level. According to [(3)H]-l-glutamate uptake data, GLT-1 was responsible for approximately 80% of the GLAST-, GLT-1-, and EAAC1-mediated uptake that occurs within dissociated hippocampal tissue, yet when this transporter was preferentially blocked for 120 h with WAY-855 (100 microm), no significant neurotoxicity was observed in hippocampal slices. This is in stark contrast to results obtained with TBOA, a broad-spectrum transport blocker, which, at concentrations that caused a similar inhibition of glutamate uptake (10 and 30 microm), caused substantial neuronal death when exposed to the slices for 24 h or longer. Likewise, WAY-855, did not significantly exacerbate neurotoxicity associated with simulated ischemia, whereas TBOA did. Finally, intrahippocampal microinjection of WAY-855 (200 and 300 nmol) in vivo resulted in marginal damage compared with TBOA (20 and 200 nmol), which killed the majority of both CA1-4 pyramidal cells and dentate gyrus granule cells. These results indicate that selective inhibition of GLT-1 is insufficient to provoke glutamate build-up, leading to NMDA receptor-mediated neurotoxic effects, and suggest a prominent role of GLAST and/or EAAC1 in extracellular glutamate maintenance.
Assuntos
Inibidores Enzimáticos/toxicidade , Transportador 2 de Aminoácido Excitatório/fisiologia , Ácido Glutâmico/metabolismo , Heptanos/toxicidade , Compostos Heterocíclicos com 3 Anéis/toxicidade , Hipocampo/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Sistema X-AG de Transporte de Aminoácidos/fisiologia , Animais , Animais Recém-Nascidos , Ácido Aspártico/farmacologia , Western Blotting/métodos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Transportador 2 de Aminoácido Excitatório/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/citologia , Humanos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Fatores de Tempo , Trítio/metabolismoRESUMO
Fractalkine is a unique chemokine reported to be constitutively expressed by neurons. Its only receptor, CX3CR1, is expressed by microglia. Little is known about the expression of fractalkine and CX3CR1 in spinal cord. Given that peripheral nerve inflammation and/or injury gives rise to neuropathic pain, and neuropathic pain may be partially mediated by spinal cord glial activation and consequent glial proinflammatory cytokine release, there must be a signal released by affected neurons that triggers the activation of glia. We sought to determine whether there is anatomical evidence implicating spinal fractalkine as such a neuron-to-glia signal. We mapped the regional and cellular localization of fractalkine and CX3CR1 in the rat spinal cord and dorsal root ganglion, under basal conditions and following induction of neuropathic pain, employing both an inflammatory (sciatic inflammatory neuropathy; SIN) as well as a traumatic (chronic constriction injury; CCI) model. Fractalkine immunoreactivity and mRNA were observed in neurons, but not glia, in the rat spinal cord and dorsal root ganglia, and levels did not change following either CCI or SIN. By contrast, CX3CR1 was expressed by microglia in the basal state, and the microglial cellular concentration was up-regulated in a regionally specific manner in response to neuropathy. CX3CR1-expressing cells were identified as microglia by their cellular morphology and positive OX-42 and CD4 immunostaining. The cellular distribution of fractalkine and CX3CR1 in the spinal circuit associated with nociceptive transmission supports a potential role in the mechanisms that contribute to the exaggerated pain state in these models of neuropathy.
Assuntos
Quimiocinas CX3C/metabolismo , Gânglios Espinais/metabolismo , Proteínas de Membrana/metabolismo , Dor/metabolismo , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Medula Espinal/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Quimiocina CX3CL1 , Quimiocinas CX3C/análise , Gânglios Espinais/química , Masculino , Proteínas de Membrana/análise , Medição da Dor/métodos , Doenças do Sistema Nervoso Periférico/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Citocinas/análise , Receptores de HIV/análise , Medula Espinal/químicaRESUMO
Clinically used benzodiazepine and nonbenzodiazepine sedative-hypnotic agents for the treatment of insomnia produce their therapeutic effects through allosteric enhancement of the effects of the inhibitory neurotransmitter GABA at the GABA(A) receptor. Indiplon is a novel pyrazolopyrimidine sedative-hypnotic agent, currently in development for insomnia. Using radioligand binding studies, indiplon inhibited the binding of [(3)H]Ro 15-1788 (flumazenil) to rat cerebellar and cerebral cortex membranes with high affinity (K(i) values of 0.55 and 0.45 nM, respectively). [(3)H]Indiplon binding to rat cerebellar and cerebral cortex membranes was reversible and of high affinity, with K(D) values of 1.01 and 0.45 nM, respectively, with a pharmacological specificity consistent with preferential labeling of GABA(A) receptors containing alpha1 subunits. In "GABA shift" experiments and in measurements of GABA-induced chloride conductance in rat cortical neurons in culture, indiplon behaved as an efficacious potentiator of GABA(A) receptor function. In both the radioligand binding and electrophysiological experiments, indiplon had a higher affinity than zolpidem or zaleplon. These in vitro properties are consistent with the in vivo properties of indiplon as an effective sedative-hypnotic acting through allosteric potentiation of the GABA(A) receptor.