RESUMO
Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.
Assuntos
Acetilcolina/metabolismo , Junção Neuromuscular/metabolismo , Acetilação , Acetilcolina/agonistas , Animais , Carbacol/farmacologia , Diferenciação Celular , Agonistas Colinérgicos/farmacologia , Camundongos , Junção Neuromuscular/citologia , Junção Neuromuscular/efeitos dos fármacos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMO
Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.
Assuntos
Acetilcolina/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Inibição Neural/fisiologia , Junção Neuromuscular/fisiologia , Agregação de Receptores/fisiologia , Receptores Colinérgicos/fisiologia , Agrina/deficiência , Agrina/farmacologia , Animais , Western Blotting/métodos , Bungarotoxinas/farmacocinética , Carbacol/farmacologia , Carbocianinas/farmacocinética , Linhagem Celular , Colina O-Acetiltransferase/deficiência , Agonistas Colinérgicos/farmacologia , Quinase 5 Dependente de Ciclina , Diafragma/citologia , Interações Medicamentosas , Embrião de Mamíferos , Feminino , Proteínas de Homeodomínio , Imuno-Histoquímica/métodos , Imunoprecipitação , Hibridização In Situ/métodos , Camundongos , Camundongos Knockout , Muscarina/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Liso/efeitos dos fármacos , Músculo Liso/embriologia , Inibição Neural/efeitos dos fármacos , Gravidez , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Agregação de Receptores/efeitos dos fármacos , Roscovitina , Sinaptofisina/metabolismo , Fatores de Tempo , Fatores de Transcrição/deficiênciaRESUMO
Recent studies have shown that the survival of mammalian motoneurons in vitro is promoted by neurotrophins (NTs) and cAMP. There is also evidence that neurotrophins enhance transmitter release. We thus investigated whether these agents also promote synaptogenesis. Cultured Xenopus spinal cord neurons were treated with a mixture of BDNF, glia-derived neurotrophic factor, NT-3, and NT-4, in addition to forskolin and IBMX or the cell-permeant form of cAMP, to elevate the cAMP level. The outgrowth and survival of neurons were dramatically increased by this trophic stimulation. However, when these neurons were cocultured with muscle cells, the trophic agents resulted in a failure of synaptogenesis. Specifically, the induction of ACh receptor (AChR) clustering in cultured muscle cells was inhibited at nerve-muscle contacts, in sharp contrast to control, untreated cocultures. Because AChR clustering induced by agrin or growth factor-coated beads in muscle cells was unaffected by trophic stimulation, its effect on synaptogenesis is presynaptic in origin. In the control, agrin was deposited along the neurite and at nerve-muscle contacts. This was significantly downregulated in cultures treated with trophic stimuli. Reverse transcriptase-PCR analyses showed that this decrease in agrin deposition was caused by an inhibition of agrin synthesis by trophic stimuli. Both agrin synthesis and induction of AChR clustering were restored under trophic stimulation when Schwann cell-conditioned medium was introduced. These results suggest that trophic stimulation maintains spinal neurons in the growth state, and Schwann cell-derived factors allow them to switch to the synaptogenic state.
Assuntos
Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Células de Schwann/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Agrina/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , AMP Cíclico/metabolismo , Fibroblastos/citologia , Células Musculares/citologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Agregação de Receptores , Receptores Colinérgicos/metabolismo , Células de Schwann/citologia , Nervo Isquiático/citologia , Transdução de Sinais/fisiologia , Sinapses/fisiologia , XenopusRESUMO
Positive and negative regulation of neurotransmitter receptor aggregation on the postsynaptic membrane is a critical event during synapse formation. Acetylcholine (ACh) and agrin are two opposing signals that regulate ACh receptor (AChR) clustering during neuromuscular junction (NMJ) development. ACh induces dispersion of AChR clusters that are not stabilized by agrin via a cyclin-dependent kinase 5 (Cdk5)-mediated mechanism, but regulation of Cdk5 activation is poorly understood. We found that the intermediate filament protein nestin physically interacts with Cdk5 and is required for ACh-induced association of p35, the co-activator of Cdk5, with the muscle membrane. Blockade of nestin-dependent signaling inhibited ACh-induced Cdk5 activation and the dispersion of AChR clusters in cultured myotubes. Similar to the effects of Cdk5 gene inactivation, knockdown of nestin in agrin-deficient mouse embryos substantially restored AChR clusters. These results suggest that nestin is required for ACh-induced, Cdk5-dependent dispersion of AChR clusters during NMJ development.