Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Department of Pathology and Anatomy, Eastern Virginia Medical School, Norfolk, VA 23501, USA.
Developmental Biology (Impact Factor: 3.55). 10/2004; 273(2):276-84. DOI: 10.1016/j.ydbio.2004.06.003
Source: PubMed


Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.

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Available from: Earl W Godfrey, Nov 11, 2014
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    • "The NOS1 isozyme is expressed in neuronal tissue, often during periods of growth cone extension and synapse formation [1], [12]–[23] and can influence both synapse assembly [24]–[37] and maintenance [6], [8], [38], [39]. Recent evidence also suggests NO has developmental effects on the developing neuromuscular junction (NMJ): in Xenopus and chick embryos chronic NO treatment promotes acetylcholine (ACh) receptor clustering [2]–[5]. In addition, acute exposure to NO donors depresses spontaneous and evoked synaptic transmission at the NMJ of developing amphibians [40], an effect which may contribute to activity-dependent maturation of neuromuscular synapses. "
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    ABSTRACT: Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.
    PLoS ONE 01/2014; 9(1):e86930. DOI:10.1371/journal.pone.0086930 · 3.23 Impact Factor
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    • "Prolonged NOS inhibition could impair functional recovery, as we reported after hypoglossal nerve injury (Sunico et al., 2008). This hypothesis is supported by data on embryonic neuromuscular junctions, wherein the application of NO donors to muscle cells induced acetylcholine receptor clustering and NOS activity was necessary for postsynaptic differentiation (Schwarte and Godfrey, 2004). Since the 3 major isoforms of NOS are dysregulated in activated satellite cells or myotubes in denervated skeletal muscle after sciatic nerve crushing (Chen et al., 2008), NOS inhibition after the second week could be detrimental for muscle re-innervation. "
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    ABSTRACT: Physical injury to a nerve is the most frequent cause of acquired peripheral neuropathy, which is responsible for loss of motor, sensory and/or autonomic functions. Injured axons in the peripheral nervous system maintain the capacity to regenerate in adult mammals. However, after nerve transection, stumps of damaged nerves must be surgically joined to guide regenerating axons into the distal nerve stump. Even so, severe functional limitations persist after restorative surgery. Therefore, the identification of molecules that regulate degenerative and regenerative processes is indispensable in developing therapeutic tools to accelerate and improve functional recovery. Here, I consider the role of nitric oxide (NO) synthesized by the three major isoforms of NO synthases (NOS) in motor neuropathy. Neuronal NOS (nNOS) seems to be the primary source of NO that is detrimental to the survival of injured motoneurons. Endothelial NOS (eNOS) appears to be the major source of NO that interferes with axonal regrowth, at least soon after injury. Finally, NO derived from inducible NOS (iNOS) or nNOS is critical to the process of lipid breakdown for Wallerian degeneration and thereby benefits axonal regrowth. Specific inhibitors of these isoforms can be used to protect injured neurons from degeneration and promote axonal regeneration. A cautious proposal for the treatment of acquired motor neuropathy using therapeutic tools that locally interfere with eNOS/nNOS activities seems to merit consideration.
    Journal of Neuroscience Research 07/2010; 88(9):1846-57. DOI:10.1002/jnr.22353 · 2.59 Impact Factor
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    • "At later stages (more than 2 weeks post-injury) NO could be necessary for re-connection and maturation of the neuromuscular junction and as a result prolonged NOS inhibition impaired the recovery of muscle function. This hypothesis is supported by data obtained in embryonic neuromuscular junctions since the application of NO donors to muscle cells induced acetylcholine receptors clustering and NOS activity was necessary for postsynaptic differentiation (Schwarte and Godfrey, 2004). The three major isoforms of NOS are up-regulated in activated satellite cells or myotubes in the denervated skeletal muscle after sciatic nerve crushing, which support the involvement of NO in muscle regeneration after nerve injury (Chen et al., 2008). "
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    ABSTRACT: Physical injury to a nerve is the most common cause of acquired peripheral neuropathy. Identification of molecules involved in degenerative and regenerative processes is a key step toward development of therapeutic tools in order to accelerate motor, sensory and/or autonomic function recovery. We have studied the role of nitric oxide (NO) using as a model the severe crushing of a motor nerve in adult rats. This type of injury up-regulates the three isoforms of nitric oxide synthase (NOS) in the affected nerve. Chronic systemic inhibition of NOS accelerated the onset of functional muscle reinnervation evaluated by the recording of compound muscle action potential evoked by electrical stimulation of the injured nerve. Besides, it increased the number of back-labeled motoneurons by application, 2 days after injury, of a retrograde marker 10 mm distal to the crushing site. These effects were mimicked by chronic specific inhibition of the endothelial isoform of nitric oxide synthase (eNOS), but not by specific inhibitors of the neuronal or inducible isoform. Next, we intraneurally injected a replication-deficient adenoviral vector directing the expression of a dominant negative mutant of eNOS (Ad-TeNOS). A single injection of Ad-TeNOS on the day of crushing significantly accelerated functional recovery of neuromuscular junction and increased axonal regeneration. Moreover, Ad-TeNOS did not compromise motoneuron viability or stability of reestablished neuromuscular junctions. Taken together, these results suggest that NO of endothelial origin slows down muscle reinnervation by means of detrimental actions on axonal regeneration after peripheral nerve injury. These experiments identify eNOS as a potential therapeutic target for treatment of traumatic nerve injuries and highlight the potential of gene therapy in treating injuries of this type using viral vectors to suppress the activity of eNOS.
    Neuroscience 10/2008; 157(1):40-51. DOI:10.1016/j.neuroscience.2008.09.001 · 3.36 Impact Factor
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