scholarly journals The mechanism of agrin-induced acetylcholine receptor aggregation

1991 ◽  
Vol 331 (1261) ◽  
pp. 273-280 ◽  
Keyword(s):  
Neuromuscular Junction ◽  
Tyrosine Phosphorylation ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Electric Organ ◽  
Motor Axon ◽  
Β Subunit ◽  
Axon Terminals ◽  
Receptor Aggregation

Agrin, a protein isolated from the synapse-rich electric organ of Torpedo californica , induces the formation of specializations on myotubes in culture which resemble the post-synaptic apparatus at the vertebrate skeletal neuromuscular junction. For example, the specializations contain aggregates of acetylcholine receptors and acetylcholinesterase. This report summarizes the evidence that the formation of the postsynaptic apparatus at developing and regenerating neuromuscular junctions is triggered by the release of agrin from motor axon terminals and describes results of recent experiments which suggest that agrininduced tyrosine phosphorylation of the β subunit of the acetylcholine receptor may play a role in receptor aggregation.

scholarly journals Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester.

1988 ◽  
Vol 107 (1) ◽  
pp. 267-278 ◽  
Author(s):  
B G Wallace
Keyword(s):  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Electric Organ ◽  
Lateral Migration ◽  
Axon Terminals ◽  
Kinase C ◽  
Receptor Aggregation ◽  
Mean Size ◽  
Cultured Myotubes ◽  
The Mean

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.

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 Identification of agrin in electric organ extracts and localization of agrin-like molecules in muscle and central nervous system

1987 ◽  
Vol 132 (1) ◽  
pp. 223-230 ◽  
Author(s):  
M. A. Smith ◽  
Y. M. Yao ◽  
N. E. Reist ◽  
C. Magill ◽  
B. G. Wallace ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Electric Organ ◽  
Synaptic Cleft ◽  
Muscle Fibres ◽  
Motor Neurones ◽  
Cultured Myotubes ◽  
Torpedo Electric Organ

The portion of the muscle fibre's basal lamina that occupies the synaptic cleft at the neuromuscular junction contains molecules that cause the aggregation of acetylcholine receptors and acetylcholinesterase on regenerating muscle fibres. Agrin, which is extracted from basal lamina-containing fractions of the Torpedo electric organ and causes the formation of acetylcholine receptor and acetylcholinesterase aggregates on cultured myotubes, may be similar, if not identical, to the acetylcholine receptor- and acetylcholinesterase-aggregating molecules at the neuro-muscular junction. Here we summarize experiments which led to the identification of agrin and established that the basal lamina at the neuromuscular junction contains molecules antigenically similar to agrin. We also discuss results which raise the possibility that agrin-like molecules at the neuromuscular junction are produced by motor neurones.

scholarly journals Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

1984 ◽  
Vol 32 (9) ◽  
pp. 973-981 ◽  
Author(s):  
B W Lubit
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Morphological Changes ◽  
Muscle Cells ◽  
High Concentration ◽  
Rat Muscle ◽  
Cytoplasmic Actin ◽  
High Concentrations

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

scholarly journals Regulatory Function of Sympathetic Innervation on the Endo/Lysosomal Trafficking of Acetylcholine Receptor

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatjana Straka ◽  
Charlotte Schröder ◽  
Andreas Roos ◽  
Laxmikanth Kollipara ◽  
Albert Sickmann ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Ribosomal Proteins ◽  
Neuromuscular Junctions ◽  
Sympathetic Neurons ◽  
Sympathetic Innervation ◽  
Regulatory Function ◽  
Hindlimb Muscles ◽  
Nerve Muscle

Recent studies have demonstrated that neuromuscular junctions are co-innervated by sympathetic neurons. This co-innervation has been shown to be crucial for neuromuscular junction morphology and functional maintenance. To improve our understanding of how sympathetic innervation affects nerve–muscle synapse homeostasis, we here used in vivo imaging, proteomic, biochemical, and microscopic approaches to compare normal and sympathectomized mouse hindlimb muscles. Live confocal microscopy revealed reduced fiber diameters, enhanced acetylcholine receptor turnover, and increased amounts of endo/lysosomal acetylcholine-receptor-bearing vesicles. Proteomics analysis of sympathectomized skeletal muscles showed that besides massive changes in mitochondrial, sarcomeric, and ribosomal proteins, the relative abundance of vesicular trafficking markers was affected by sympathectomy. Immunofluorescence and Western blot approaches corroborated these findings and, in addition, suggested local upregulation and enrichment of endo/lysosomal progression and autophagy markers, Rab 7 and p62, at the sarcomeric regions of muscle fibers and neuromuscular junctions. In summary, these data give novel insights into the relevance of sympathetic innervation for the homeostasis of muscle and neuromuscular junctions. They are consistent with an upregulation of endocytic and autophagic trafficking at the whole muscle level and at the neuromuscular junction.

scholarly journals Identification of agrin, a synaptic organizing protein from Torpedo electric organ.

1987 ◽  
Vol 105 (6) ◽  
pp. 2471-2478 ◽  
Author(s):  
R M Nitkin ◽  
M A Smith ◽  
C Magill ◽  
J R Fallon ◽  
Y M Yao ◽  
...  
Keyword(s):  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Gel Filtration ◽  
Electric Organ ◽  
Molecular Characteristics ◽  
Active Components ◽  
Cultured Myotubes ◽  
Aggregation Activity ◽  
Two Peaks

Extracts of the electric organ of Torpedo californica contain a proteinaceous factor that causes the formation of patches on cultured myotubes at which acetylcholine receptors (AChR), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) are concentrated. Results of previous experiments indicate that this factor is similar to the molecules in the synaptic basal lamina that direct the aggregation of AChR and AChE at regenerating neuromuscular junctions in vivo. We have purified the active components in the extracts 9,000-fold. mAbs against four different epitopes on the AChR/AChE/BuChE-aggregating molecules each immunoprecipitated four polypeptides from electric organ extracts, with molecular masses of 150, 135, 95, and 70 kD. Gel filtration chromatography of electric organ extracts revealed two peaks of AChR/AChE/BuChE-aggregation activity; one comigrated with the 150-kD polypeptide, the other with the 95-kD polypeptide. The 135- and 70-kD polypeptides did not cause AChR/AChE/BuChE aggregation. Based on these molecular characteristics and on the pattern of staining seen in sections of muscle labeled with the mAbs, we conclude that the electric organ-aggregating factor is distinct from previously identified molecules, and we have named it "agrin."

scholarly journals An Agonist of the CXCR4 Receptor Strongly Promotes Regeneration of Degenerated Motor Axon Terminals

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1183 ◽  
Author(s):  
Negro ◽  
Zanetti ◽  
Mattarei ◽  
Valentini ◽  
Megighian ◽  
...  
Keyword(s):  
Functional Recovery ◽  
Neuromuscular Junctions ◽  
Recovery Of Function ◽  
Motor Axon ◽  
Cxcr4 Antagonist ◽  
Axon Terminals ◽  
Stem Cell Migration ◽  
Natural Ligand ◽  
Nerve Recovery ◽  
Strong Candidate

The activation of the G-protein coupled receptor CXCR4 by its ligand CXCL12α is involved in a large variety of physiological and pathological processes, including the growth of B cells precursors and of motor axons, autoimmune diseases, stem cell migration, inflammation, and several neurodegenerative conditions. Recently, we demonstrated that CXCL12α potently stimulates the functional recovery of damaged neuromuscular junctions via interaction with CXCR4. This result prompted us to test the neuroregeneration activity of small molecules acting as CXCR4 agonists, endowed with better pharmacokinetics with respect to the natural ligand. We focused on NUCC-390, recently shown to activate CXCR4 in a cellular system. We designed a novel and convenient chemical synthesis of NUCC-390, which is reported here. NUCC-390 was tested for its capability to induce the regeneration of motor axon terminals completely degenerated by the presynaptic neurotoxin α-Latrotoxin. NUCC-390 was found to strongly promote the functional recovery of the neuromuscular junction, as assayed by electrophysiology and imaging. This action is CXCR4 dependent, as it is completely prevented by AMD3100, a well-characterized CXCR4 antagonist. These data make NUCC-390 a strong candidate to be tested in human therapy to promote nerve recovery of function after different forms of neurodegeneration.

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.

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