RESUMO
Activation of PKCÉ, an abundant and developmentally regulated PKC isoform in the brain, has been implicated in memory throughout life and across species. Yet, direct evidence for a mechanistic role for PKCÉ in memory is still lacking. Hence, we sought to evaluate this in rats, using short-term treatments with two PKCÉ-selective peptides, the inhibitory ÉV1-2 and the activating ψÉRACK, and the novel object recognition task (NORT). Our results show that the PKCÉ-selective activator ψÉRACK, did not have a significant effect on recognition memory. In the short time frames used, however, inhibition of PKCÉ activation with the peptide inhibitor ÉV1-2 significantly impaired recognition memory. Moreover, when we addressed at the molecular level the immediate proximal signalling events of PKCÉ activation in acutely dissected rat hippocampi, we found that ψÉRACK increased in a time-dependent manner phosphorylation of MARCKS and activation of Src, Raf, and finally ERK1/2, whereas ÉV1-2 inhibited all basal activity of this pathway. Taken together, these findings present the first direct evidence that PKCÉ activation is an essential molecular component of recognition memory and point toward the use of systemically administered PKCÉ-regulating peptides as memory study tools and putative therapeutic agents.
Assuntos
Memória/fisiologia , Proteína Quinase C-épsilon/metabolismo , Reconhecimento Psicológico/fisiologia , Animais , Western Blotting , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Produtos do Gene tat/farmacologia , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Neurônios/enzimologia , Fosforilação , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Ratos Wistar , Reconhecimento Psicológico/efeitos dos fármacos , Quinases raf/fisiologia , Quinases da Família src/fisiologiaRESUMO
G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C ε-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a pre-requisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation.
Assuntos
Córtex Cerebral/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/citologia , Receptor CB1 de Canabinoide/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Imunoprecipitação/métodos , Microdomínios da Membrana/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Cannabinoid receptors 1 (CB1Rs) play important roles in the regulation of dendritic branching, synapse density, and synaptic transmission through multiple G-protein-coupled signaling systems, including the activation of the extracellular signal-regulated kinases ERK1/2. The proximal signaling interactions leading to ERK1/2 activation by CB1R in CNS remain, however, unclear. Here, we present evidence that the CB1R agonist methanandamide induced a biphasic and sustained activation of ERK1/2 in primary neurons derived from E7 telencephalon. We show that E7 neurons natively express high levels of CB1R message and protein, the majority of which associates with PKCÉ at basal conditions. We now demonstrate that the first peak of ERK activation by CB1R was mediated by the sequential activation of G(q), PLC, and PKCÉ, selectively, and that the CB1R-activated PKCÉ acutely formed transient signaling modules containing activated Src and Fyn. A second pool of CB1Rs, coupled to PTX-sensitive activation of G(i/o), utilized as effectors additional Src and Fyn molecules to generate a second, additional wave of ERK activation at 15 min. Concurrently to these intermolecular signaling interactions, cytoskeleton-associated proteins MARCKS and p120catenin were drastically modified by phosphorylation of PKC and Src, respectively. These receptor-proximal signaling events correlated well with induction of neuritic outgrowth in the long term. Our data provide evidence for multiprotein signaling complex formation in the coupling of CB1R to activation of ERK in CNS neurons, and may elucidate several of the less understood acute effects of cannabinoid drugs.