RESUMO
The Intracellular Fibroblast Growth Factor (iFGF) subfamily includes four members (FGFs 11-14) of the structurally related FGF superfamily. Previous studies showed that the iFGFs interact directly with the pore-forming (alpha) subunits of voltage-gated sodium (Nav) channels and regulate the functional properties of sodium channel currents. Sequence heterogeneity among the iFGFs is thought to confer specificity to this regulation. Here, we demonstrate that the two N-terminal alternatively spliced FGF14 variants, FGF14-1a and FGF14-1b, differentially regulate currents produced by Nav1.2 and Nav1.6 channels. FGF14-1b, but not FGF14-1a, attenuates both Nav1.2 and Nav1.6 current densities. In contrast, co-expression of an FGF14 mutant, lacking the N-terminus, increased Nav1.6 current densities. In neurons, both FGF14-1a and FGF14-1b localized at the axonal initial segment, and deletion of the N-terminus abolished this localization. Thus, the FGF14 N-terminus is required for targeting and functional regulation of Nav channels, suggesting an important function for FGF14 alternative splicing in regulating neuronal excitability.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/metabolismo , Canais de Sódio/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Células Cultivadas , Fatores de Crescimento de Fibroblastos/genética , Hipocampo/citologia , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.2 , Canal de Sódio Disparado por Voltagem NAV1.6 , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Canais de Sódio/genéticaRESUMO
Fibroblast growth factor 14 (FGF14) belongs to the intracellular FGF homologous factor subfamily of FGF proteins (iFGFs) that are not secreted and do not activate tyrosine kinase receptors. The iFGFs, however, have been shown to interact with the pore-forming (alpha) subunits of voltage-gated Na+ (Na(v)) channels. The neurological phenotypes seen in Fgf14-/- mice and the identification of an FGF14 missense mutation (FGF14(F145S)) in a Dutch family presenting with cognitive impairment and spinocerebellar ataxia suggest links between FGF14 and neuronal functioning. Here, we demonstrate that the expression of FGF14(F145S) reduces Na(v) alpha subunit expression at the axon initial segment, attenuates Na(v) channel currents, and reduces the excitability of hippocampal neurons. In addition, and in contrast with wild-type FGF14, FGF14(F145S) does not interact directly with Na(v) channel alpha subunits. Rather, FGF14(F145S) associates with wild-type FGF14 and disrupts the interaction between wild-type FGF14 and Na(v) alpha subunits, suggesting that the mutant FGF14(F145S) protein acts as a dominant negative, interfering with the interaction between wild-type FGF14 and Na(v) channel alpha subunits and altering neuronal excitability.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Mutação/fisiologia , Neurônios/fisiologia , Fenilalanina/genética , Serina/genética , Canais de Sódio/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Embrião de Mamíferos , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/biossíntese , Hipocampo/citologia , Humanos , Imunoprecipitação/métodos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/efeitos da radiação , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Ratos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/química , Canais de Sódio/genética , Tetrodotoxina/farmacologiaRESUMO
This study investigates proliferation and apoptosis of olfactory ensheathing cells in cocultures with spinal cord tissue. Proliferation of ensheathing cells was significantly increased when cocultured with explants from uninjured spinal cord, and spinal cord that had been subjected to chronic contusion or chronic needle stab injury, but not to acute needle stab injury. Proliferation rate was highest in cocultures with chronically stabbed cord tissue. Contaminating (p75NGFR-negative) cells in the cultures showed a significantly higher proliferation rate than ensheathing cells. Apoptosis of ensheathing cells was significantly increased in cocultures with acutely stabbed spinal cord explants compared with chronically contused spinal cord explants. These results suggest that delaying transplantation after spinal cord injury may be beneficial to ensheathing cell survival.
Assuntos
Regeneração Nervosa/fisiologia , Neuroglia/transplante , Mucosa Olfatória/citologia , Traumatismos da Medula Espinal/terapia , Animais , Apoptose , Divisão Celular , Células Cultivadas , Doença Crônica , Masculino , Neuroglia/citologia , Ratos , Ratos Wistar , Receptor de Fator de Crescimento Neural/metabolismo , Traumatismos da Medula Espinal/patologia , Ferimentos Perfurantes/terapiaRESUMO
Evidence suggests that CART (cocaine-amphetamine regulated transcript) peptides are mediators or modulators of the actions of psychostimulant drugs. In this study, the effects of intra-accumbal injections of rat long form (rl) CART 55-102 were examined. Injection of the peptide alone had no effect, but pretreatment with the peptide blunted or reduced the locomotor-inducing effects of cocaine after an i.p. injection. This effect was dose related and time limited, as expected. rlCART 1-27, a CART peptide fragment not active in other studies, was without effect on cocaine-induced locomotor activity. Because the actions of cocaine involve dopamine, the effect of rlCART 55-102 on dopamine-induced locomotor activity was examined. Intraaccumbal injection of dopamine produced a dose-related and time-limited increase in locomotor activity, as expected. Coinjection of rlCART 55-102 with dopamine blunted the effect. In summary, these data suggest that CART peptides in the nucleus accumbens would tend to oppose the actions of cocaine.
