scholarly journals Subtle Neuromuscular Defects in Utrophin-deficient Mice

1997 ◽  
Vol 136 (4) ◽  
pp. 871-882 ◽  
Author(s):  
R. Mark Grady ◽  
John P. Merlie ◽  
Joshua R. Sanes
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Cultured Cells ◽  
Neuromuscular Junctions ◽  
Becker Muscular Dystrophy ◽  
Postsynaptic Membrane ◽  
Mutant Mice ◽  
Nonmuscle Cells ◽  
And Behavior

Utrophin is a large cytoskeletal protein that is homologous to dystrophin, the protein mutated in Duchenne and Becker muscular dystrophy. In skeletal muscle, dystrophin is broadly distributed along the sarcolemma whereas utrophin is concentrated at the neuromuscular junction. This differential localization, along with studies on cultured cells, led to the suggestion that utrophin is required for synaptic differentiation. In addition, utrophin is present in numerous nonmuscle cells, suggesting that it may have a more generalized role in the maintenance of cellular integrity. To test these hypotheses we generated and characterized utrophin-deficient mutant mice. These mutant mice were normal in appearance and behavior and showed no obvious defects in muscle or nonmuscle tissue. Detailed analysis, however, revealed that the density of acetylcholine receptors and the number of junctional folds were reduced at the neuromuscular junctions in utrophin-deficient skeletal muscle. Despite these subtle derangements, the overall structure of the mutant synapse was qualitatively normal, and the specialized characteristics of the dystrophin-associated protein complex were preserved at the mutant neuromuscular junction. These results point to a predominant role for other molecules in the differentiation and maintenance of the postsynaptic membrane.

Distribution of acetylcholine receptors in the vicinity of nerve terminals on skeletal muscle of the frog

1972 ◽  
Vol 181 (1065) ◽  
pp. 431-440 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Connective Tissue ◽  
Neuromuscular Junction ◽  
Muscle Fibre ◽  
Nerve Terminal ◽  
Acetylcholine Receptors ◽  
Postsynaptic Membrane ◽  
Muscle Membrane ◽  
Muscle Fibres ◽  
Nerve Terminals

1. The acetylcholine (ACh) sensitivity of muscle fibres at the neuromuscular junction of the frog was investigated in preparations in which the nerve terminals could be clearly seen. 2. ACh released iontophoretically from a micropipette that was precisely positioned at various points along the muscle fibre in the vicinity of the synapse showed that the peak chemosensitivity (up to 1900 mV/nC) is confined to an area of postsynaptic membrane within a few micra of the nerve terminal; a tenfold decline in sensitivity was obtained when the ACh was released only 5 to 10 μm from the terminal’s edge. It is estimated that most of the response obtained when ACh is released within 40 μm from the terminal (the area covered in this study) is due to diffusion to the immediate postsynaptic area. The extrasynaptic chemosensitivity of the muscle membrane was too low to be measured with the present methods. 3. The accuracy with which micropipettes could be positioned in synaptic areas and the clarity of viewing nerve terminals were improved by bathing the tissue in collagenase, which reduced the amount of connective tissue. The distribution of chemosensitivity remained unchanged by such treatment. The ACh response was not detectably altered when nerve terminals were lifted off the muscle, exposing the subsynaptic muscle surface.

scholarly journals Characterisation of alpha-dystrobrevin in muscle

1998 ◽  
Vol 111 (17) ◽  
pp. 2595-2605 ◽  
Author(s):  
R. Nawrotzki ◽  
N.Y. Loh ◽  
M.A. Ruegg ◽  
K.E. Davies ◽  
D.J. Blake
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Synapse Formation ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Related Protein ◽  
Receptor Clustering

Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of alpha-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce alpha-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct alpha-dystrobrevin isoforms. In myotubes, alpha-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, alpha-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of alpha-dystrobrevin-1 by agrin. Furthermore, we show that alpha-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus alpha-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that alpha-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.

scholarly journals HDAC6 regulates microtubule stability and clustering of AChRs at neuromuscular junctions

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Alexis Osseni ◽  
Aymeric Ravel-Chapuis ◽  
Jean-Luc Thomas ◽  
Vincent Gache ◽  
Laurent Schaeffer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Relative Stability ◽  
Neuromuscular Junctions ◽  
Postsynaptic Membrane ◽  
Post Translational Modification ◽  
Acetylation Status ◽  
Microtubule Stability ◽  
Hdac6 Inhibitor ◽  
The Stability

Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NMJ), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.

scholarly journals Processing of ARIA and release from isolated nerve terminals

1999 ◽  
Vol 354 (1381) ◽  
pp. 411-416 ◽  
Author(s):  
Bomie Han ◽  
Gerald D. Fischbach
Keyword(s):  
Neuromuscular Junction ◽  
Action Potential ◽  
Acetylcholine Receptors ◽  
Molecular Layer ◽  
Postsynaptic Membrane ◽  
High Density ◽  
Small Contribution ◽  
Presynaptic Nerve Terminal ◽  
Voltage Gated

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage–gated Na + channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage–gated Na + channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo . ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.

scholarly journals The mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1833-1840 ◽  
Author(s):  
L S Musil ◽  
D E Frail ◽  
J P Merlie
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptor ◽  
Cell Lines ◽  
Neuromuscular Junctions ◽  
Cell Types ◽  
Cardiac Cells ◽  
Mrna Levels ◽  
Receptor Associated Protein ◽  
Mouse Tissues ◽  
Nonmuscle Cells

Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes (Frail, D.E., L.S. Musil, a. Bonanno, and J.P. Merlie, 1989. Neuron. 2:1077-1086) led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with [35S]methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.

scholarly journals The gut microbiota influences skeletal muscle mass and function in mice

2019 ◽  
Vol 11 (502) ◽  
pp. eaan5662 ◽  
Author(s):  
Shawon Lahiri ◽  
Hyejin Kim ◽  
Isabel Garcia-Perez ◽  
Musarrat Maisha Reza ◽  
Katherine A. Martin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Gut Microbiota ◽  
Muscle Mass ◽  
Skeletal Muscle Mass ◽  
Neuromuscular Junctions ◽  
Short Chain Fatty Acids ◽  
Germ Free ◽  
Mouse Skeletal Muscle ◽  
And Function

The functional interactions between the gut microbiota and the host are important for host physiology, homeostasis, and sustained health. We compared the skeletal muscle of germ-free mice that lacked a gut microbiota to the skeletal muscle of pathogen-free mice that had a gut microbiota. Compared to pathogen-free mouse skeletal muscle, germ-free mouse skeletal muscle showed atrophy, decreased expression of insulin-like growth factor 1, and reduced transcription of genes associated with skeletal muscle growth and mitochondrial function. Nuclear magnetic resonance spectrometry analysis of skeletal muscle, liver, and serum from germ-free mice revealed multiple changes in the amounts of amino acids, including glycine and alanine, compared to pathogen-free mice. Germ-free mice also showed reduced serum choline, the precursor of acetylcholine, the key neurotransmitter that signals between muscle and nerve at neuromuscular junctions. Reduced expression of genes encoding Rapsyn and Lrp4, two proteins important for neuromuscular junction assembly and function, was also observed in skeletal muscle from germ-free mice compared to pathogen-free mice. Transplanting the gut microbiota from pathogen-free mice into germ-free mice resulted in an increase in skeletal muscle mass, a reduction in muscle atrophy markers, improved oxidative metabolic capacity of the muscle, and elevated expression of the neuromuscular junction assembly genes Rapsyn and Lrp4. Treating germ-free mice with short-chain fatty acids (microbial metabolites) partly reversed skeletal muscle impairments. Our results suggest a role for the gut microbiota in regulating skeletal muscle mass and function in mice.

scholarly journals Basal lamina directs acetylcholinesterase accumulation at synaptic sites in regenerating muscle.

1985 ◽  
Vol 101 (3) ◽  
pp. 735-743 ◽  
Author(s):  
L Anglister ◽  
U J McMahan
Keyword(s):  
Plasma Membrane ◽  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Skeletal Muscles ◽  
Ache Activity ◽  
Neuromuscular Junctions ◽  
Muscle Fibers ◽  
Synaptic Cleft

In skeletal muscles that have been damaged in ways which spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fibers are characterized by junctional folds and accumulations of acetylcholine receptors and acetylcholinesterase (AChE). The formation of junctional folds and the accumulation of acetylcholine receptors is known to be directed by components of the synaptic portion of the myofiber basal lamina. The aim of this study was to determine whether or not the synaptic basal lamina contains molecules that direct the accumulation of AChE. We crushed frog muscles in a way that caused disintegration and phagocytosis of all cells at the neuromuscular junction, and at the same time, we irreversibly blocked AChE activity. New muscle fibers were allowed to regenerate within the basal lamina sheaths of the original muscle fibers but reinnervation of the muscles was deliberately prevented. We then stained for AChE activity and searched the surface of the new muscle fibers for deposits of enzyme they had produced. Despite the absence of innervation, AChE preferentially accumulated at points where the plasma membrane of the new muscle fibers was apposed to the regions of the basal lamina that had occupied the synaptic cleft at the neuromuscular junctions. We therefore conclude that molecules stably attached to the synaptic portion of myofiber basal lamina direct the accumulation of AChE at the original synaptic sites in regenerating muscle. Additional studies revealed that the AChE was solubilized by collagenase and that it remained adherent to basal lamina sheaths after degeneration of the new myofibers, indicating that it had become incorporated into the basal lamina, as at normal neuromuscular junctions.

scholarly journals Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions.

1981 ◽  
Vol 88 (2) ◽  
pp. 453-458 ◽  
Author(s):  
Y Nakajima ◽  
P C Bridgman
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Postsynaptic Membrane ◽  
Large Particles ◽  
Glutaraldehyde Solution ◽  
Active Zones ◽  
Low Cholesterol

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze-fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.

scholarly journals Salbutamol modifies the neuromuscular junction in a mouse model of ColQ myasthenic syndrome

2019 ◽  
Vol 28 (14) ◽  
pp. 2339-2351 ◽  
Author(s):  
Grace M McMacken ◽  
Sally Spendiff ◽  
Roger G Whittaker ◽  
Emily O’Connor ◽  
Rachel M Howarth ◽  
...  
Keyword(s):  
Muscle Strength ◽  
Neuromuscular Junction ◽  
Structural Changes ◽  
Neuromuscular Junctions ◽  
Skeletal Muscle Fibre ◽  
Postsynaptic Membrane ◽  
Adrenergic Agonists ◽  
Muscle Fibre Size ◽  
Gradual Improvement ◽  
Functional Benefit

Abstract The β-adrenergic agonists salbutamol and ephedrine have proven to be effective as therapies for human disorders of the neuromuscular junction, in particular many subsets of congenital myasthenic syndromes. However, the mechanisms underlying this clinical benefit are unknown and improved understanding of the effect of adrenergic signalling on the neuromuscular junction is essential to facilitate the development of more targeted therapies. Here, we investigated the effect of salbutamol treatment on the neuromuscular junction in the ColQ deficient mouse, a model of end-plate acetylcholinesterase deficiency. ColQ−/− mice received 7 weeks of daily salbutamol injection, and the effect on muscle strength and neuromuscular junction morphology was analysed. We show that salbutamol leads to a gradual improvement in muscle strength in ColQ−/− mice. In addition, the neuromuscular junctions of salbutamol treated mice showed significant improvements in several postsynaptic morphological defects, including increased synaptic area, acetylcholine receptor area and density, and extent of postjunctional folds. These changes occurred without alterations in skeletal muscle fibre size or type. These findings suggest that β-adrenergic agonists lead to functional benefit in the ColQ−/− mouse and to long-term structural changes at the neuromuscular junction. These effects are primarily at the postsynaptic membrane and may lead to enhanced neuromuscular transmission.

scholarly journals Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture.

1991 ◽  
Vol 115 (3) ◽  
pp. 765-778 ◽  
Author(s):  
M T Lupa ◽  
J H Caldwell
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptors ◽  
Muscle Fibers ◽  
Postsynaptic Membrane ◽  
Na Channel ◽  
High Concentration ◽  
Achr Cluster ◽  
Adult Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Alpha Bungarotoxin

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.

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