Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2010; 107(23):10702-7. DOI: 10.1073/pnas.1004956107
Source: PubMed


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.

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    • "Before innervation, a pre-pattern of AChR clusters forms on the muscle surface to guide the subsequent positioning of motor axons for NMJ assembly (Lin et al., 2001; Vock et al., 2008; Jing et al., 2009). Upon nerve-muscle contact, most pre-patterned AChR clusters are disassembled by the inhibitory effect of acetylcholine (ACh) (Lin et al., 2005; Misgeld et al., 2005; An et al., 2010), except the ones located in close apposition with the motor axon which are stabilized by the motor neuron-derived proteoglycan agrin (Lin et al., 2005; Misgeld et al., 2005). Agrin signals through the muscle-specific tyrosine kinase receptor MuSK (Valenzuela et al., 1995; Dechiara et al., 1996; Glass et al., 1996), by forming a membrane complex with the low density lipoprotein receptor-related protein 4 (Lrp4) (Weatherbee et al., 2006; Kim et al., 2008; Zhang et al., 2008). "
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    ABSTRACT: Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain-and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, β-catenin-dependent signaling on the assembly of the vertebrate NMJ.
    Full-text · Article · Apr 2014 · Frontiers in Cellular Neuroscience
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    • "Alternatively MuSK/dok-7 may function in the processing only after AChR/rapsyn reaches the plasma membrane. Several elegant in vivo studies examined the dynamics of rapsyn/AChRs on the plasma membrane (Bruneau and Akaaboune, 2007; 2010). However, it will be very useful to examine their interactions before they reach the plasma membrane. "
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    ABSTRACT: The accumulation of acetylcholine receptors (AChRs) at nerve terminals is critical for signal transmission at the neuromuscular junction, and rapsyn is essential for this process. Previous studies suggest that AChRs might direct rapsyn self-clusters to the synapse. In vivo experiments with fluorescently tagged AChR or rapsyn in zebrafish larvae revealed that rapsyn self-clusters separate from AChRs did not exist before synapse formation. Examination of rapsyn in the AChR-less mutant sofa potato revealed that rapsyn in the absence of AChR was localized in the Golgi complex. Expression of muscle-type AChR in sofa potato restored synaptic clustering of rapsyn, while neuronal type AChR had no effect. To determine whether this requirement of protein interaction is reciprocal, we examined the mutant twitch once, which has a missense mutation in rapsyn. While the AChRs distributed nonsynaptically on the plasma membrane in twitch once, mutant rapsyn was retained in the Golgi complex. We conclude that AChRs enable the transport of rapsyn from the Golgi complex to the plasma membrane through a molecule-specific interaction.
    Full-text · Article · May 2012 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    • "In addition, ChAT/agrin double mutants rescued the inhibition of AChR clustering observed in agrin single mutants at late stages of NMJ assembly [33,34]. Interestingly, recent findings in animals rendered null only for muscle AChRs, reveal that the negative effects that ACh plays on AChR clustering seem not to rely on the activation of muscle receptors [35]. In addition, the “anti-synaptogenic” effect of acetylcholine is antagonized in synaptic domains by the “pro-synaptogenic” role of motor neuron-derived agrin [33–35]. "
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    ABSTRACT: The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction.
    Full-text · Article · Dec 2011 · International Journal of Molecular Sciences
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