Degeneration of neuromuscular junction in age and dystrophy

Functional denervation is a hallmark of aging sarcopenia as well as of muscular dystrophy. It is thought to be a major factor reducing skeletal muscle mass, particularly in the case of sarcopenia. Neuromuscular junctions (NMJs) serve as the interface between the nervous and skeletal muscular systems, and thus they may receive pathophysiological input of both pre- and post-synaptic origin. Consequently, NMJs are good indicators of motor health on a systemic level. Indeed, upon sarcopenia and dystrophy, NMJs morphologically deteriorate and exhibit altered characteristics of primary signaling molecules, such as nicotinic acetylcholine receptor and agrin. Since a remarkable reversibility of these changes can be observed by exercise, there is significant interest in understanding the molecular mechanisms underlying synaptic deterioration upon aging and dystrophy and how synapses are reset by the aforementioned treatments. Here, we review the literature that describes the phenomena observed at the NMJ in sarcopenic and dystrophic muscle as well as to how these alterations can be reversed and to what extent. In a second part, the current information about molecular machineries underlying these processes is reported

Motor Endplate—Anatomical, Functional, and Molecular Concepts in the Historical Perspective

By mediating voluntary muscle movement, vertebrate neuromuscular junctions (NMJ) play an extraordinarily important role in physiology. While the significance of the nerve-muscle connectivity was already conceived almost 2000 years back, the precise cell and molecular biology of the NMJ have been revealed in a series of fascinating research activities that started around 180 years ago and that continues. In all this time, NMJ research has led to fundamentally new concepts of cell biology, and has triggered groundbreaking advancements in technologies. This review tries to sketch major lines of thought and concepts on NMJ in their historical perspective, in particular with respect to anatomy, function, and molecular components. Furthermore, along these lines, it emphasizes the mutual benefit between science and technology, where one drives the other. Finally, we speculate on potential major future directions for studies on NMJ in these fields

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

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

Autophagy Impairment in Muscle Induces Neuromuscular Junction Degeneration and Precocious Aging

The cellular basis of age-related tissue deterioration remains largely obscure. The ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions. Autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins. We report that specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and, consequently, on muscle strength, ultimately affecting the lifespan of animals. Inhibition of autophagy also exacerbates aging phenotypes in muscle, such as mitochondrial dysfunction, oxidative stress, and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses. These results demonstrate that age-related deterioration of synaptic structure and function is exacerbated by defective autophagy

Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo

<p>Endocytosed nicotinic acetylcholine receptors (CHRN) are degraded via macroautophagy/autophagy during atrophic conditions and are accompanied by the autophagic regulator protein SH3GLB1. The present study addressed the functional role of SH3GLB1 on CHRN trafficking and its implementation. We found an augmented ratio of total SH3GLB1 to threonine-145 phosphorylated SH3GLB1 (SH3GLB1:p-SH3GLB1) under conditions of increased CHRN vesicle numbers. Overexpression of T145 phosphomimetic (T145E) and phosphodeficient (T145A) mutants of SH3GLB1, was found to either slow down or augment the processing of endocytic CHRN vesicles, respectively. Co-expression of the early endosomal orchestrator RAB5 largely rescued the slow processing of endocytic CHRN vesicles induced by T145E. SH3GLB1 phosphomutants did not modulate the expression or colocalization of RAB5 with CHRN vesicles, but instead altered the expression of RAB5 activity regulators. In summary, these findings suggest that SH3GLB1 controls CHRN endocytic trafficking in a phosphorylation- and RAB5-dependent manner at steps upstream of autophagosome formation.</p

Regulation of the COPII secretory machinery via focal adhesions and extracellular matrix signaling

Proteins that enter the secretory pathway are transported from their place of synthesis in the endoplasmic reticulum to the Golgi complex by COPII-coated carriers. The networks of proteins that regulate these components in response to extracellular cues have remained largely elusive. Using high-throughput microscopy, we comprehensively screened 378 cytoskeleton-associated and related proteins for their functional interaction with the coat protein complex II (COPII) components SEC23A and SEC23B. Among these, we identified a group of proteins associated with focal adhesions (FERMT2, MACF1, MAPK8IP2, NGEF, PIK3CA, and ROCK1) that led to the downregulation of SEC23A when depleted by siRNA. Changes in focal adhesions induced by plating cells on ECM also led to the downregulation of SEC23A and decreases in VSVG transport from ER to Golgi. Both the expression of SEC23A and the transport defect could be rescued by treatment with a focal adhesion kinase inhibitor. Altogether, our results identify a network of cytoskeleton-associated proteins connecting focal adhesions and ECM-related signaling with the gene expression of the COPII secretory machinery and trafficking

Response to cAMP agonists differs between wildtype and mdx synapses. Tibialis anterior muscles of wildtype and mdx mice were transfected with RIα-EPAC.

<p>Ten days later, muscles were injected with BGT-AF647 to stain NMJs and monitored with in vivo confocal (A–D, G–J) or two-photon (E–F, K–M) microscopy. Scale bars depict 50 µm. <b>A and G:</b> Shown are single mdx NMJs with normal (A1) or fragmented morphology (A2, G1, G2). <b>B and H:</b> BGT-AF647 fluorescence signals. Boxed regions are shown enlarged in A and G. <b>C and I:</b> RIα-EPAC fluorescence signals in the same field as in B and H. <b>D and J:</b> Overlays of B and C (D) and H and I (J). BGT-AF647 and RIα-EPAC signals are in red and green, respectively. <b>E and K:</b> Same field as in B and H showing FRET-ratios in pseudo-colors before application of CGRP or NE (indicated). <b>F and L:</b> Same field as in E and K showing FRET-ratio in pseudo-colors after application of CGRP or NE (indicated). <b>M:</b> Quantification of several experiments. Shown is the percentage of increase in CFP/YFP ratio values (F(485 nm)/F(535 nm)) compared to basal upon application of 50 µl of either 10 µM CGRP or 10 µM NE as indicated. Data represent mean ± SEM (n = 10 and n = 14 wildtype NMJs for CGRP and NE, respectively; n = 13 and n = 9 mdx NMJs for CGRP and NE, respectively).</p

Notexin treatment transiently reduces the subsynaptic accumulation of PKA-RI and myosin Va.

<p>EDL muscles were injected with Notexin to induce muscle degeneration. 6, 10, and 30 days after treatment muscles were resected and sliced transversally. <b>A:</b> Slices were stained with wheat germ agglutinin-AlexaFluor488 for plasma membranes (green), and with DRAQ5 for nuclei (red). Images show confocal sections through muscles harvested 6, 10, or 30 days after Notexin, as indicated. Scale bar, 100 µm. <b>B–D:</b> Slices were stained with BGT-AF647 (NMJs) and with antibodies against PKA-RI (B), myosin Va (C), and utrophin (D). Confocal images were taken and then analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040860#s4" target="_blank">Methods</a> section. Scatter plots (3 left columns) show diameters of all analyzed fibers as a function of their subsynaptic enrichment of immunofluorescence. Vertical dotted lines indicate the separation between immuno-negative (left halves of plots) and immuno-positive NMJs (right halves of plots). Horizontal dotted lines indicate the separation between fibers smaller and larger than 40 µm in diameter. Column graphs (right) summarize data in scatter plots and depict the fractions of immuno-positive NMJs obtained 6, 10, and 30 days after Notexin treatment. Data are mean ± SEM (n = 6, 4, and 4 muscles for 6, 10, and 30 day time points). White and grey columns represent values for fibers smaller and larger than 40 µm in diameter, respectively.</p