RESUMO
Understanding the origins and evolution of synapses may provide insight into species diversity and the organization of the brain. Using comparative proteomics and genomics, we examined the evolution of the postsynaptic density (PSD) and membrane-associated guanylate kinase (MAGUK)-associated signaling complexes (MASCs) that underlie learning and memory. PSD and MASC orthologs found in yeast carry out basic cellular functions to regulate protein synthesis and structural plasticity. We observed marked changes in signaling complexity at the yeast-metazoan and invertebrate-vertebrate boundaries, with an expansion of key synaptic components, notably receptors, adhesion/cytoskeletal proteins and scaffold proteins. A proteomic comparison of Drosophila and mouse MASCs revealed species-specific adaptation with greater signaling complexity in mouse. Although synaptic components were conserved amongst diverse vertebrate species, mapping mRNA and protein expression in the mouse brain showed that vertebrate-specific components preferentially contributed to differences between brain regions. We propose that the evolution of synapse complexity around a core proto-synapse has contributed to invertebrate-vertebrate differences and to brain specialization.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Estudos de Avaliação como Assunto , Proteínas do Tecido Nervoso/metabolismo , Proteoma , Sinapses/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Comportamento Animal , Encéfalo/citologia , Encéfalo/metabolismo , Mapeamento Encefálico , Proteínas Adaptadoras de Sinalização CARD , Proteínas do Citoesqueleto/genética , Drosophila , Expressão Gênica , Camundongos , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteômica/métodos , Transdução de Sinais/fisiologiaRESUMO
Phosphorylation-dependent changes in AMPA receptor function have a crucial role in activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although three previously identified phosphorylation sites in AMPA receptor glutamate receptor 1 (GluR1) subunits (S818, S831, and S845) appear to have important roles in LTP and LTD, little is known about the role of other putative phosphorylation sites in GluR1. Here, we describe the characterization of a recently identified phosphorylation site in GluR1 at threonine 840. The results of in vivo and in vitro phosphorylation assays suggest that T840 is not a substrate for protein kinases known to phosphorylate GluR1 at previously identified phosphorylation sites, such as protein kinase A, protein kinase C, and calcium/calmodulin-dependent kinase II. Instead, in vitro phosphorylation assays suggest that T840 is a substrate for p70S6 kinase. Although LTP-inducing patterns of synaptic stimulation had no effect on GluR1 phosphorylation at T840 in the hippocampal CA1 region, bath application of NMDA induced a strong, protein phosphatase 1- and/or 2A-mediated decrease in T840 phosphorylation. Moreover, GluR1 phosphorylation at T840 was transiently decreased by a chemical LTD induction protocol that induced a short-term depression of synaptic strength and persistently decreased by a chemical LTD induction protocol that induced a lasting depression of synaptic transmission. Together, our results show that GluR1 phosphorylation at T840 is regulated by NMDA receptor activation and suggest that decreases in GluR1 phosphorylation at T840 may have a role in LTD.
Assuntos
Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Treonina/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Análise de Variância , Animais , Células Cultivadas , Colforsina/farmacologia , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/citologia , Humanos , Técnicas In Vitro , Isoproterenol/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/efeitos da radiação , Análise Serial de Proteínas/métodos , Transfecção/métodosRESUMO
During development of the amniote peripheral nervous system, the initial trajectory of primary sensory axons is determined largely by the action of axon repellents. We have shown previously that tissues flanking dorsal root ganglia, the notochord lying medially and the dermamyotomes lying laterally, are sources of secreted molecules that prevent axons from entering inappropriate territories. Although there is evidence suggesting that SEMA3A contributes to the repellent activity of the dermamyotome, the nature of the activity secreted by the notochord remains undetermined. We have employed an expression cloning strategy to search for axon repellents secreted by the notochord, and have identified SEMA3A as a candidate repellent. Moreover, using a spectrum of different axon populations to assay the notochord activity, together with neuropilin/Fc receptor reagents to block semaphorin activity in collagen gel assays, we show that SEMA3A probably contributes to notochord-mediated repulsion. Sympathetic axons that normally avoid the midline in vivo are also repelled, in part, by a semaphorin-based notochord activity. Although our results implicate semaphorin signalling in mediating repulsion by the notochord, repulsion of early dorsal root ganglion axons is only partially blocked when using neuropilin/Fc reagents. Moreover, retinal axons, which are insensitive to SEMA3A, are also repelled by the notochord. We conclude that multiple factors act in concert to guide axons in this system, and that further notochord repellents remain to be identified.