RESUMO
The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis.
Assuntos
Agrina/fisiologia , Junção Neuromuscular/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Agrina/deficiência , Agrina/genética , Processamento Alternativo , Animais , Diafragma/embriologia , Diafragma/crescimento & desenvolvimento , Ativação Enzimática , Feminino , Proteínas Relacionadas a Receptor de LDL , Longevidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/deficiência , Proteínas Musculares/fisiologia , Junção Neuromuscular/embriologia , Junção Neuromuscular/crescimento & desenvolvimento , Doenças da Junção Neuromuscular/enzimologia , Doenças da Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/fisiopatologia , Fosforilação , Densidade Pós-Sináptica/fisiologia , Processamento de Proteína Pós-Traducional , Receptores Colinérgicos/fisiologia , Receptores de LDL/deficiência , Receptores de LDL/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Teste de Desempenho do Rota-RodRESUMO
In the adult forebrain, new interneurons are continuously generated and integrated into the existing circuitry of the olfactory bulb (OB). In an attempt to identify signals that regulate this synaptic integration process, we found strong expression of agrin in adult generated neuronal precursors that arrive in the olfactory bulb after their generation in the subventricular zone. While the agrin receptor components MuSK and Lrp4 were below detection level in neuron populations that represent synaptic targets for the new interneurons, the alternative receptor α3-Na(+)K(+)-ATPase was strongly expressed in mitral cells. Using a transplantation approach, we demonstrate that agrin-deficient interneuron precursors migrate correctly into the OB. However, in contrast to wild-type neurons, which form synapses and survive for prolonged periods, mutant neurons do not mature and are rapidly eliminated. Using in vivo brain electroporation of the olfactory system, we show that the transmembrane form of agrin alone is sufficient to mediate integration and demonstrate that excess transmembrane agrin increases the number of dendritic spines. Last, we provide in vivo evidence that an interaction between agrin and α3-Na(+)K(+)-ATPase is of functional importance in this system.
Assuntos
Agrina/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Transdução de Sinais/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Fatores Etários , Agrina/biossíntese , Agrina/deficiência , Animais , Células Cultivadas , Feminino , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/enzimologia , Bulbo Olfatório/enzimologia , Bulbo Olfatório/crescimento & desenvolvimento , Transdução de Sinais/genética , ATPase Trocadora de Sódio-Potássio/biossíntese , Sinapses/genéticaRESUMO
Nestin is expressed in many different progenitors during development including those of the CNS, heart, skeletal muscle, and kidney. The adult expression is mainly restricted to the subependymal zone and dentate gyrus of the brain, the neuromuscular junction, and renal podocytes. In addition, this intermediate filament protein has served as a marker of neural stem/progenitor cells for close to 20 years. Therefore it is surprising that its function in development and adult physiology is still poorly understood. Here we report that nestin deficiency is compatible with normal development of the CNS. The mutant mice, however, show impaired motor coordination. Furthermore, we found that the number of acetylcholine receptor clusters, the nerve length, and the endplate bandwidth are significantly increased in neuromuscular junction area of nestin-deficient mice. This is similar to the phenotype described for deficiency of cyclin-dependent kinase 5 (Cdk5), a candidate downstream affecter of nestin. Moreover, we demonstrate that nestin deficiency can rescue maintenance of acetylcholine receptor clusters in the absence of agrin, similar to Cdk5/agrin double knock-outs, suggesting that the observed nestin deficiency phenotype is the consequence of aberrant Cdk5 activity.
Assuntos
Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Quinase 5 Dependente de Ciclina/deficiência , Proteínas de Filamentos Intermediários/deficiência , Proteínas do Tecido Nervoso/deficiência , Junção Neuromuscular/metabolismo , Agregação de Receptores/fisiologia , Receptores Colinérgicos/metabolismo , Agrina/deficiência , Agrina/genética , Agrina/metabolismo , Animais , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/fisiologia , Feminino , Marcação de Genes/métodos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/fisiologia , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Nestina , Junção Neuromuscular/fisiologia , Agregação de Receptores/genética , Receptores Colinérgicos/genética , Receptores Colinérgicos/fisiologiaRESUMO
Motor axons approach muscles that are regionally prespecialized, as acetylcholine receptors are clustered in the central region of muscle before and independently of innervation. This muscle prepattern requires MuSK, a receptor tyrosine kinase that is essential for synapse formation. It is not known how muscle prepatterning is established, and whether motor axons recognize this prepattern. Here we show that expression of Musk is prepatterned in muscle and that early Musk expression in developing myotubes is sufficient to establish muscle prepatterning. We further show that ectopic Musk expression promotes ectopic synapse formation, indicating that muscle prepatterning normally has an instructive role in directing where synapses will form. In addition, ectopic Musk expression stimulates synapse formation in the absence of Agrin and rescues the lethality of Agrn mutant mice, demonstrating that the postsynaptic cell, and MuSK in particular, has a potent role in regulating the formation of synapses.
Assuntos
Axônios/fisiologia , Neurônios Motores/citologia , Receptores Proteína Tirosina Quinases/metabolismo , Sinapses/fisiologia , Agrina/deficiência , Animais , Animais Recém-Nascidos , Padronização Corporal/genética , Antígeno CD24/genética , Células Cultivadas , Diafragma/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Mutação , Receptores Proteína Tirosina Quinases/genética , Receptores Colinérgicos/metabolismoRESUMO
Drugs that inhibit Na,K-ATPases, such as digoxin and ouabain, alter cardiac myocyte contractility. We recently demonstrated that agrin, a protein first identified at the vertebrate neuromuscular junction, binds to and regulates the activity of alpha3 subunit-containing isoforms of the Na,K-ATPase in the mammalian brain. Both agrin and the alpha3 Na,K-ATPase are expressed in heart, but their potential for interaction and effect on cardiac myocyte function was unknown. Here we show that agrin binds to the alpha3 subunit of the Na,K-ATPase in cardiac myocyte membranes, inducing tyrosine phosphorylation and inhibiting activity of the pump. Agrin also triggers a rapid increase in cytoplasmic Na(+) in cardiac myocytes, suggesting a role in cardiac myocyte function. Consistent with this hypothesis, spontaneous contraction frequencies of cultured cardiac myocytes prepared from mice in which agrin expression is blocked by mutation of the Agrn gene are significantly higher than in the wild type. The Agrn mutant phenotype is rescued by acute treatment with recombinant agrin. Furthermore, exposure of wild type myocytes to an agrin antagonist phenocopies the Agrn mutation. These data demonstrate that the basal frequency of myocyte contraction depends on endogenous agrin-alpha3 Na,K-ATPase interaction and suggest that agrin modulation of the alpha3 Na,K-ATPase is important in regulating heart function.
Assuntos
Agrina/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Agrina/química , Agrina/deficiência , Agrina/genética , Animais , Sítios de Ligação , Células Cultivadas , Reagentes de Ligações Cruzadas , Coração Fetal/citologia , Coração Fetal/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Knockout , Complexos Multiproteicos , Mutação , Contração Miocárdica/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , ATPase Trocadora de Sódio-Potássio/química , Tirosina/químicaRESUMO
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
Neuromuscular junctions (NMJs) normally form in the central region of developing muscle. In this process, agrin released from motor neurons has been considered to initiate the formation of synaptic acetylcholine receptor (AChR) clusters (neurocentric model). However, in muscle developing in the absence of nerves and thus of agrin, AChR clusters still form in the muscle center. This raises the possibility that the region of NMJ formation is determined by muscle-derived cues that spatially restrict the nerve to form synapses from aneural AChR clusters, e.g., by patterned expression of the agrin receptor MuSK (muscle-specific kinase) (myocentric model). Here we examine at initial stages of synaptogenesis whether the responsiveness of myotubes to agrin is spatially restricted, whether the regions of NMJ formation in wild-type muscle and of aneural AChR cluster formation in agrin-deficient animals correlate, and whether AChR cluster growth depends on the presence of agrin. We show that primary myotubes form AChR clusters in response to exogenous agrin in their central region only, a pattern that can spatially restrict NMJ formation. However, the nerve also makes synapses in regions in which aneural AChR clusters do not form, and agrin promotes synaptic cluster growth from the first stages of neuromuscular contact formation. These data indicate that aneural AChR clusters per se are not required for NMJ formation. A model is proposed that explains either the neurocentric or the myocentric mode of NMJ formation depending on a balance between the levels of MuSK expression and the availability of nerve-released agrin.
Assuntos
Proteínas Musculares/metabolismo , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/fisiologia , Agrina/deficiência , Animais , Diafragma/citologia , Diafragma/embriologia , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Técnicas de Cultura de Órgãos , Agregação de Receptores/fisiologia , Receptores Proteína Tirosina Quinases/deficiência , Fatores de Transcrição/genéticaRESUMO
Adult neurogenesis in the hippocampus may represent a form of plasticity in brain functions including mood, learning and memory. However, mechanisms underlying neural stem/progenitor cells (NSPCs) proliferation are not well understood. We found that Agrin, a factor critical for neuromuscular junction formation, is elevated in the hippocampus of mice that are stimulated by enriched environment (EE). Genetic deletion of the Agrn gene in excitatory neurons decreases NSPCs proliferation and increases depressive-like behavior. Low-density lipoprotein receptor-related protein 4 (Lrp4), a receptor for Agrin, is expressed in hippocampal NSPCs and its mutation blocked basal as well as EE-induced NSPCs proliferation and maturation of newborn neurons. Finally, we show that Lrp4 interacts with and activates receptor tyrosine kinase-like orphan receptor 2 (Ror2); and Ror2 mutation impairs NSPCs proliferation. Together, these observations identify a role of Agrin-Lrp4-Ror2 signaling for adult neurogenesis, uncovering previously unexpected functions of Agrin and Lrp4 in the brain.
Assuntos
Agrina/metabolismo , Hipocampo/crescimento & desenvolvimento , Proteínas Relacionadas a Receptor de LDL/metabolismo , Células-Tronco Neurais/fisiologia , Neurogênese , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Transdução de Sinais , Agrina/deficiência , Animais , Proliferação de Células , Técnicas de Inativação de Genes , Proteínas Relacionadas a Receptor de LDL/deficiência , Camundongos , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/deficiênciaRESUMO
The patterning of skeletal muscle is thought to depend upon signals provided by motor neurons. We show that AChR gene expression and AChR clusters are concentrated in the central region of embryonic skeletal muscle in the absence of innervation. Neurally derived Agrin is dispensable for this early phase of AChR expression, but MuSK, a receptor tyrosine kinase activated by Agrin, is required to establish this AChR prepattern. The zone of AChR expression in muscle lacking motor axons is wider than normal, indicating that neural signals refine this muscle-autonomous prepattern. Neuronal Neuregulin-1, however, is not involved in this refinement process, nor indeed in synapse-specific AChR gene expression. Our results demonstrate that AChR expression is patterned in the absence of innervation, raising the possibility that similarly prepatterned muscle-derived cues restrict axon growth and initiate synapse formation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/inervação , Receptores Colinérgicos/genética , Receptores Acoplados a Proteínas G , Agrina/deficiência , Agrina/genética , Agrina/metabolismo , Animais , Axônios/fisiologia , Padronização Corporal/fisiologia , Desenvolvimento Embrionário e Fetal , Camundongos , Camundongos Knockout , Denervação Muscular , Neurregulinas/genética , Neurregulinas/fisiologia , Neurônios Aferentes/fisiologia , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores de Lisofosfolipídeos , Recombinação Genética , Sinapses/fisiologiaRESUMO
Agrin-deficient mice die at birth because of aberrant development of the neuromuscular junctions. Here, we examined the role of agrin at brain synapses. We show that agrin is associated with excitatory but not inhibitory synapses in the cerebral cortex. Most importantly, we examined the brains of agrin-deficient mice whose perinatal death was prevented by the selective expression of agrin in motor neurons. We find that the number of presynaptic and postsynaptic specializations is strongly reduced in the cortex of 5- to 7-week-old mice. Consistent with a reduction in the number of synapses, the frequency of miniature postsynaptic currents was greatly decreased. In accordance with the synaptic localization of agrin to excitatory synapses, changes in the frequency were only detected for excitatory but not inhibitory synapses. Moreover, we find that the muscle-specific receptor tyrosine kinase MuSK, which is known to be an essential component of agrin-induced signaling at the neuromuscular junction, is also localized to a subset of excitatory synapses. Finally, some components of the mitogen-activated protein (MAP) kinase pathway, which has been shown to be activated by agrin in cultured neurons, are deregulated in agrin-deficient mice. In summary, our results provide strong evidence that agrin plays an important role in the formation and/or the maintenance of excitatory synapses in the brain, and we provide evidence that this function involves MAP kinase signaling.
Assuntos
Agrina/deficiência , Córtex Cerebral/metabolismo , Sinapses/metabolismo , Fatores Etários , Agrina/genética , Agrina/fisiologia , Animais , Animais Recém-Nascidos , Córtex Cerebral/patologia , Galinhas , Feminino , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Taxa de Sobrevida , Sinapses/patologiaRESUMO
Establishment of cell-cell adhesion is crucial in embryonic development as well as within the stem cell niches of an adult. Adhesion between macrophages and erythroblasts is required for the formation of erythroblastic islands, specialized niches where erythroblasts proliferate and differentiate to produce red blood cells throughout life. The Eph family is the largest known family of receptor tyrosine kinases (RTKs) and controls cell adhesion, migration, invasion and morphology by modulating integrin and adhesion molecule activity and by modifying the actin cytoskeleton. Here, we identify the proteoglycan agrin as a novel regulator of Eph receptor signaling and characterize a novel mechanism controlling cell-cell adhesion and red cell development within the erythroid niche. We demonstrate that agrin induces clustering and activation of EphB1 receptors on developing erythroblasts, leading to the activation of α5ß1 integrins. In agreement, agrin knockout mice display severe anemia owing to defective adhesion to macrophages and impaired maturation of erythroid cells. These results position agrin-EphB1 as a novel key signaling couple regulating cell adhesion and erythropoiesis.
Assuntos
Agrina/metabolismo , Adesão Celular/fisiologia , Transdução de Sinais/fisiologia , Agrina/deficiência , Agrina/genética , Animais , Sobrevivência Celular , Eritrócitos/citologia , Eritrócitos/metabolismo , Receptores de Hialuronatos/metabolismo , Integrina alfa5beta1/metabolismo , Proteínas Relacionadas a Receptor de LDL , Células Progenitoras de Megacariócitos/citologia , Células Progenitoras de Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Fosforilação , Receptor EphB1/metabolismo , Receptores de LDL/metabolismo , Baço/patologiaRESUMO
The dystrophin glycoprotein complex (DGC) is a multimolecular complex that links the extracellular matrix to the cytoskeleton. The DGC is present at the skeletal neuromuscular junction and required for its maturation and maintenance. Members of the DGC are also expressed in brain. We used cultured hippocampal neurons to analyze the distribution, regulation, and role in synaptogenesis of the major transmembrane component of the DGC, dystroglycan; one of its extracellular ligands, agrin; and one of its cytoskeletal binding partners, dystrophin. alpha-Dystroglycan, beta-dystroglycan, and dystrophin clustered at a subset of inhibitory synapses containing GABA(A)R subunits alpha1, alpha2, and gamma2, and the inhibitory receptor anchoring protein gephyrin. DGC components were not detected at excitatory glutamatergic synapses. Dystroglycan is the first identified adhesive macromolecule at mature GABA synapses. Developmentally, dystroglycan clustered at synaptic loci after synaptic vesicles, GABA(A)R, and gephyrin, the latter being closely associated with GABA(A)R at all stages of synaptogenesis analyzed. Analysis of gephyrin -/-, agrin -/-, and mdx mouse hippocampal neurons in culture indicated that synaptic clustering of dystroglycan occurs independently of gephyrin, agrin, and dystrophin. In dystroglycan-deficient neurons, cultured from a conditional mutant strain, GABAergic synapses differentiated with clusters of gephyrin and GABA(A)R apposed to synaptic terminals, but these synapses did not contain detectable dystrophin. Thus the DGC is not essential for GABAergic synaptogenesis but is likely to function in modulating inhibitory synapses or conferring specialized properties on a subset of them.
Assuntos
Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Inibição Neural/fisiologia , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Agrina/deficiência , Agrina/genética , Agrina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Distroglicanas , Distrofina/deficiência , Distrofina/genética , Distrofina/metabolismo , Glutamato Descarboxilase/metabolismo , Hipocampo , Substâncias Macromoleculares , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Endogâmicos mdx , Camundongos Knockout , Distrofia Muscular Animal , Neurônios/citologia , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/ultraestruturaRESUMO
Approximately half of the motoneurons produced during development die before birth or shortly after birth. Although it is believed that survival depends on a restricted supply of a trophic sustenance produced by the synaptic target tissue (i.e., muscle), it is unclear whether synapse formation per se is involved in motoneuron survival. To address this issue, we counted cranial motoneurons in a set of mutant mice in which formation of neuromuscular junctions is dramatically impaired (i.e., null mutants for agrin, nerve-derived agrin, rapsyn, and MuSK). We demonstrate that in the absence of synaptogenesis, there is an 18-34% increase in motoneuron survival in the facial, trochlear, trigeminal motor, and hypoglossal nuclei; the highest survival occurred in the MuSK-deficient animals in which synapse formation is most severely compromised. There was no change in the size of the mutant motoneurons as compared with control animals, and the morphology of the mutant motoneurons appeared normal. We postulate that the increased axonal branching observed in these mutants leads to a facilitated "access" of the motoneurons to muscle-derived trophic factors at sites other than synapses or that inactivity increases the production of such factors. Finally, we examined motoneurons in double mutants of CNTFRalpha(-/-) (in which there is a partial loss of motoneurons) and MuSK(-/-) (in which there is an increased survival of motoneurons). The motoneuron numbers in the double mutants parallel those of the single MuSK-deficient mice, indicating that synapse disruption can even overcome the deleterious effect of CNTFRalpha ablation.
Assuntos
Neurônios Motores/metabolismo , Doenças da Junção Neuromuscular/metabolismo , Doenças da Junção Neuromuscular/patologia , Junção Neuromuscular/patologia , Receptores Colinérgicos , Agrina/deficiência , Agrina/genética , Animais , Animais Recém-Nascidos , Axônios/patologia , Contagem de Células , Sobrevivência Celular/genética , Nervos Cranianos/citologia , Nervos Cranianos/embriologia , Camundongos , Camundongos Mutantes , Neurônios Motores/citologia , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Junção Neuromuscular/embriologia , Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/embriologia , Doenças da Junção Neuromuscular/genética , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Receptor do Fator Neutrófico Ciliar/deficiência , Receptor do Fator Neutrófico Ciliar/genéticaRESUMO
Nerve-derived agrin is a specific isoform of agrin that promotes clustering of nicotinic acetylcholine receptors (AChR) and other components of the neuromuscular junction (NMJ). We investigated the effects of agrin on functional maturation of NMJs at the early stages of synaptogenesis in human muscle. Specifically, we assessed the importance of agrin for the differentiation of developing NMJs to the stage where they are able to transmit signals that result in contractions of myotubes. We utilized an in vitro model in which human myotubes are innervated by neurons extending from spinal cord explants of fetal rat. This model is suitable for functional studies because all muscle contractions are the result of neuromuscular transmission and can be quantitated. An anti-agrin antibody, Agr 33, was applied to co-cultures during de novo NMJ formation. Quantitative analyses demonstrated that Agr 33 reduced the number of AChR clusters to 20% and their long axes to 50% of control, yet still permitted early, NMJ-mediated muscle contractions that are normally observed in 7-10-day-old co-cultures. However, at later times of development, the same treatment completely prevented the increase in the number of contracting units as compared with untreated co-cultures. It is concluded that there are two modes of functional maturation of NMJs with regard to agrin effects: one that is insensitive and the other that is sensitive to agrin blockade. Agrin-insensitive mode is limited to the small population of NMJs that become functional at the earlier stages of functional innervation. However, most of the NMJs become contraction-competent at the later stages of the innervation process. These NMJs become functional only if agrin action is uncompromised. This is the first characterization of the contribution of agrin to NMJ development on human muscle.
Assuntos
Agrina/deficiência , Células do Corno Anterior/embriologia , Diferenciação Celular/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/inervação , Junção Neuromuscular/embriologia , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Agrina/antagonistas & inibidores , Animais , Células do Corno Anterior/citologia , Células do Corno Anterior/metabolismo , Anticorpos , Técnicas de Cocultura , Regulação para Baixo/fisiologia , Feminino , Feto , Cones de Crescimento/metabolismo , Cones de Crescimento/ultraestrutura , Humanos , Modelos Biológicos , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Ratos , Transmissão Sináptica/fisiologiaRESUMO
Differentiation of the postsynaptic membrane at the neuromuscular junction requires agrin, a nerve-derived signal; MuSK, a critical component of the agrin receptor in muscle; and rapsyn, a protein that interacts with acetylcholine receptors (AChRs). We showed previously that nerve-induced AChR aggregation is dramatically impaired in knockout mice lacking agrin, MuSK, or rapsyn. However, the phenotypes of these mutants differed in several respects, suggesting that the pathway from agrin to MuSK to rapsyn is complex. Here, we compared the effects of these mutations on two aspects of synaptic differentiation: AChR clustering and transcriptional specialization of synapse-associated myonuclei. First, we show that a plant lectin, VVA-B4, previously shown to act downstream of agrin, can induce AChR clusters on MuSK-deficient but not rapsyn-deficient myotubes in culture. Thus, although both MuSK and rapsyn are required for AChR clustering in vivo, only rapsyn is essential for cluster formation per se. Second, we show that neuregulin, a nerve-derived inducer of AChR gene expression, activates AChR gene expression in cultured agrin- and MuSK-deficient myotubes, even though synapse-specific transcriptional specialization is disrupted in agrin and MuSK mutants in vivo. We propose that agrin works through MuSK to determine a synaptogenic region within which synaptic differentiation occurs.
Assuntos
Agrina/fisiologia , Proteínas Musculares/fisiologia , Receptores de Fatores de Crescimento/fisiologia , Receptores Nicotínicos/fisiologia , Agrina/deficiência , Agrina/genética , Animais , Células Cultivadas , Cruzamentos Genéticos , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Mutagênese , Fenótipo , Receptores Colinérgicos/genética , Receptores Colinérgicos/fisiologia , Receptores de Fatores de Crescimento/deficiência , Receptores de Fatores de Crescimento/genética , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/genética , beta-Galactosidase/genéticaRESUMO
Astrocytic endfeet membranes are studded with aquaporin-4 (AQP4) containing orthogonal arrays of particles (OAP) which can be visualized exclusively by the freeze-fracturing method. They are predominantly expressed where the astroglial membrane is in contact with the superficial and perivascular basal lamina. This polarity seems to be essential for the integrity of the blood-brain barrier (BBB). The basal lamina containing many extracellular matrix (ECM) components such as collagen, laminin and heparansulfate proteoglycans like agrin is thought to influence this OAP-related polarity of astrocytes. Recently, we have shown that agrin, in particular the neuronal isoform A4B8, is capable of influencing the formation of OAPs in astrocytes when cultured in the presence of agrin-conditioned media. In this paper we wanted to investigate whether coating with exogenous agrin compared to coating with other ECM components would induce OAP formation in astrocytes of the agrin-null mouse. For this purpose, we cultured astrocytes from agrin-null and wild-type mice on agrin- or ECM-coated surfaces. Immunofluorescent cytochemical staining of AQP4 indicated a higher AQP4 expression level in cultures with agrin- or ECM-coated than in cultures with uncoated surfaces, whereas western blot analyses and PCR showed no differences. α-Dystroglycan is thought to be a potential receptor of agrin and was immunostained in wild-type as well as in agrin-null astrocytes. In freeze-fracture replicas, we observed an increase in OAP density in astrocytes when growing on agrin- and ECM-coatings. These results concurred with other experiments in which changes in volume were measured following hypotonic stress, which supported the positive influence of exogenous agrin on AQP4 insertion into the membrane, on OAP formation and on water transport.
Assuntos
Agrina/deficiência , Agrina/metabolismo , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Animais , Aquaporina 4/genética , Aquaporina 4/metabolismo , Encéfalo/citologia , Contagem de Células/métodos , Células Cultivadas , Distroglicanas/metabolismo , Matriz Extracelular/metabolismo , Técnica de Fratura por Congelamento/métodos , Humanos , Camundongos , Camundongos Knockout , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transfecção/métodosRESUMO
Synaptic organizing molecules and neurotransmission regulate synapse development. Here, we use the skeletal neuromuscular junction to assess the interdependence of effects evoked by an essential synaptic organizing protein, agrin, and the neuromuscular transmitter, acetylcholine (ACh). Mice lacking agrin fail to maintain neuromuscular junctions, whereas neuromuscular synapses differentiate extensively in the absence of ACh. We now demonstrate that agrin's action in vivo depends critically on cholinergic neurotransmission. Using double-mutant mice, we show that synapses do form in the absence of agrin provided that ACh is also absent. We provide evidence that ACh destabilizes nascent postsynaptic sites, and that one major physiological role of agrin is to counteract this "antisynaptogenic" influence. Similar interactions between neurotransmitters and synaptic organizing molecules may operate at synapses in the central nervous system.
Assuntos
Agrina/fisiologia , Neurotransmissores/fisiologia , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Acetilcolina/deficiência , Acetilcolina/fisiologia , Agrina/deficiência , Agrina/genética , Animais , Carbacol/farmacologia , Diferenciação Celular , Colina O-Acetiltransferase/deficiência , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/fisiologia , Feminino , Técnicas In Vitro , Camundongos , Camundongos Knockout , Modelos Neurológicos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/embriologia , Junção Neuromuscular/fisiologia , Neurotransmissores/deficiência , Neurotransmissores/farmacologia , GravidezRESUMO
The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.
Assuntos
Apoptose/fisiologia , Axônios/fisiologia , Embrião de Mamíferos/fisiologia , Neurônios Motores/fisiologia , Junção Neuromuscular/fisiologia , Agrina/deficiência , Agrina/metabolismo , Animais , Sobrevivência Celular/fisiologia , Camundongos , Contração Muscular/fisiologia , Proteínas Musculares/deficiência , Proteínas Musculares/metabolismo , Receptores Colinérgicos/deficiência , Receptores Colinérgicos/metabolismoRESUMO
Agrin is a synapse-organizing molecule that mediates the nerve-induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron-derived agrin is the AChR-aggregating factor, but the possible roles of muscle-derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin-deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin-deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin-deficient myotubes could also respond to neuron-induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin-deficient myotubes retain their ability to exhibit the agrin- or neuron-induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron-derived agrin but not the agrin from muscle.
Assuntos
Agrina/deficiência , Agrina/farmacologia , Músculo Esquelético/metabolismo , Agregação de Receptores/fisiologia , Receptores Colinérgicos/metabolismo , Acetilcolinesterase/metabolismo , Agrina/metabolismo , Animais , Linhagem Celular , Galinhas , Técnicas de Cocultura , DNA Complementar/genética , Fibroblastos/metabolismo , Fibroblastos/fisiologia , Músculo Esquelético/citologia , Neurônios/fisiologia , Distribuição Tecidual , TransfecçãoRESUMO
Agrin, a proteoglycan secreted by motoneurons, is a critical organizer of synaptic differentiation at skeletal neuromuscular junctions. Agrin is widely expressed in the nervous system so other functions seem likely, but none have been demonstrated. To test roles for agrin in interneuronal synapse formation, we studied hippocampi from mutant mice that completely lack the z+ splice form of agrin essential for neuromuscular differentiation and also exhibit severely ( approximately 90%) reduced levels of all agrin isoforms (M. Gautam et al., 1996, Cell 85, 525-535). The brains of neonatal homozygous agrin mutants were often smaller than those of heterozygous and wild-type littermates, but were morphologically and histologically indistinguishable. In particular, antibodies to pre- and postsynaptic components of glutamatergic synapses were similarly coaggregated at synaptic sites in both mutants and controls. Because mutants die at birth due to neuromuscular defects, we cultured neurons to assess later stages of synaptic maturation. In primary cultures, the agrin-deficient neurons formed MAP2-positive dendrites and tau-1-positive axons. Synaptic vesicle proteins, AMPA- and NMDA-type glutamate receptors, GABAA receptors, and the putative synapse-organizing proteins PSD-95, GKAP, and gephyrin formed numerous clusters at synaptic sites. Quantitatively, the number of SV2-labeled contacts per neuron at day 5 and the number of PSD-95 clusters per dendrite length at day 18 in culture showed no significant differences between genotypes. Furthermore, exogenous z+ agrin was unable to induce ectopic accumulation of components of central glutamatergic or GABAergic synapses as it does for neuromuscular cholinergic synapses. These results indicate that the z+ forms of agrin are dispensable for glutamatergic and GABAergic synaptic differentiation in the central nervous system.