MAGI-1c: a synaptic MAGUK interacting with muSK at the vertebrate neuromuscular junction.
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The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ. (+info)
Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation.
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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. (+info)
Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.
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Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo. (+info)
Muscle activity and muscle agrin regulate the organization of cytoskeletal proteins and attached acetylcholine receptor (AchR) aggregates in skeletal muscle fibers.
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In innervated skeletal muscle fibers, dystrophin and beta-dystroglycan form rib-like structures (costameres) that appear as predominantly transverse stripes over Z and M lines. Here, we show that the orientation of these stripes becomes longitudinal in denervated muscles and transverse again in denervated electrically stimulated muscles. Skeletal muscle fibers express nonneural (muscle) agrin whose function is not well understood. In this work, a single application of > or = 10 nM purified recombinant muscle agrin into denervated muscles preserved the transverse orientation of costameric proteins that is typical for innervated muscles, as did a single application of > or = 1 microM neural agrin. At lower concentration, neural agrin induced acetylcholine receptor aggregates, which colocalized with longitudinally oriented beta-dystroglycan, dystrophin, utrophin, syntrophin, rapsyn, and beta 2-laminin in denervated unstimulated fibers and with the same but transversely oriented proteins in innervated or denervated stimulated fibers. The results indicate that costameres are plastic structures whose organization depends on electrical muscle activity and/or muscle agrin. (+info)
At mammalian neuromuscular junctions (NMJs), innervation induces and maintains the metabolic stability of acetylcholine receptors (AChRs). To explore whether neural agrin may cause similar receptor stabilization, we injected neural agrin cDNA of increasing transfection efficiencies into denervated adult rat soleus (SOL) muscles. As the efficiency increased, the amount of recombinant neural agrin expressed in the muscles also increased. This agrin aggregated AChRs on muscle fibers, whose half-life increased in a dose-dependent way from 1 to 10 days. Electrical muscle stimulation enhanced the stability of AChRs with short half-lives. Therefore, neural agrin can stabilize aggregated AChRs in a concentration- and activity-dependent way. However, there was no effect of stimulation on AChRs with a long half-life (10 days). Thus, at sufficiently high concentrations, neural agrin alone can stabilize AChRs to levels characteristic of innervated NMJs. (+info)
Agrin differentially regulates the rates of axonal and dendritic elongation in cultured hippocampal neurons.
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In the present study, we examined the role of agrin in axonal and dendritic elongation in central neurons. Dissociated hippocampal neurons were grown in the presence of either recombinant agrin or antisense oligonucleotides designed to block agrin expression. Our results indicate that agrin differentially regulates axonal and dendritic growth. Recombinant agrin decreased the rate of elongation of main axons but induced the formation of axonal branches. On the other hand, agrin induced both dendritic elongation and dendritic branching. Conversely, cultured hippocampal neurons depleted of agrin extended longer, nonbranched axons and shorter dendrites when compared with controls. These changes in the rates of neurite elongation and branching were paralleled by changes in the composition of the cytoskeleton. In the presence of agrin, there was an upregulation of the expression of microtubule-associated proteins MAP1B, MAP2, and tau. In contrast, a downregulation of the expression of these MAPs was detected in agrin-depleted cells. Taken collectively, these results suggest an important role for agrin as a trigger of the transcription of neuro-specific genes involved in neurite elongation and branching in central neurons. (+info)
Induction of multiple signaling loops by MuSK during neuromuscular synapse formation.
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At the neuromuscular junction, two motor neuron-derived signals have been implicated in the regulation of synaptogenesis. Neuregulin-1 is thought to induce transcription of acetylcholine receptor (AChR) genes in subsynaptic muscle nuclei by activating ErbB receptors. Neural agrin aggregates AChRs by activating the receptor tyrosine kinase MuSK. Here, we show that these two signals act sequentially. Agrin, by activating MuSK, induces the synthesis and aggregation of both MuSK and ErbB receptors. ErbB acts downstream of MuSK in synapse formation. In this way, MuSK activation leads to the establishment of a neuregulin-1-dependent signaling complex that maintains MuSK, ErbB, and AChR expression at the synapse of electrically active muscle fibers. (+info)
Schwann cells express active agrin and enhance aggregation of acetylcholine receptors on muscle fibers.
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To explore novel roles of glial cells in synaptic function and formation, we examined the expression of agrin in frog Schwann cells and tested their role in the aggregation of acetylcholine receptors (AChRs). Using reverse transcription-PCR, we found that Schwann cells along nerve fibers in tadpoles expressed only the inactive agrin isoform B0 but began to also express active agrin isoforms B11 and B19 at approximately metamorphosis. During nerve regeneration in the adult, the expression of these active agrin isoforms in Schwann cells was upregulated, including the appearance of the most potent isoform, B8. This upregulation was induced by regenerating axons but not by nerve injury per se. In muscle cultures, the presence of adult Schwann cells enhanced the number and the total area of AChR aggregates 2.2- and 4.5-fold, respectively, and this enhancement was eliminated by heparin treatment. Furthermore, adult Schwann cells in culture expressed active agrin isoforms and produced agrin protein. Using a novel technique to selectively ablate perisynaptic Schwann cells (PSCs) at the neuromuscular junction, we found that PSCs also expressed active agrin isoforms B11 and B19, and these active isoforms were upregulated, including the appearance of B8, during reinnervation. Observation in vivo showed that extrajunctional AChR aggregates were associated with PSC sprouts after nerve injury and subsequent reinnervation. These results suggest that, contrary to the prevailing view that only neurons express active agrin, glial cells also express active agrin and play a role in the aggregation of AChRs both in vitro and in vivo. (+info)