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

Lifetime of AChRs is reduced in dystrophic muscles and correlates with synaptic integrity and subsynaptic enrichment of myosin Va and PKA-RI.

<p>A: Tibialis anterior muscles of wildtype (wt) and mdx mice were pulse-labeled with ¹²⁵I-BGT on day 0. Subsequently, residual ¹²⁵I-emission was measured repetitively in the live animals at indicated time points from these muscles in situ. For the duration of measurements (10 min), mice were anaesthetized with isofluorane. Data represent mean ± SEM (n = 5 mice). Welch-test revealed significant differences between wildtype and mdx values, ** p<0.01. B–F: Tibialis anterior muscles were injected with BGT-AF647 (old receptors). Ten days later, muscles were exposed, injected with BGT-AF555 (new receptors), and then monitored with in vivo confocal microscopy (B–D). Subsequently, muscles were sliced, immunostained, and analyzed with confocal microscopy (E–F). B: Representative maximum z-projections of wildtype and mdx NMJs as indicated. Old and new receptor signals are shown in green and red, respectively. Pixels with similar intensities of both dyes appear in yellow. Scale bar, 50 µm. C: Graph showing the fraction of pixels with new receptor signals dominating over old receptor signals in individual NMJs as a function of the number of fragments per NMJ. Data represent mean ± SEM (n = 4 wildtype muscles, n = 8 mdx muscles. 109 and 191 NMJs were analyzed for wildtype and mdx, respectively). Significance was tested with Welch test, ** p<0.01. D: Graph depicts all individual values of the fractions of pixels with new receptor signals dominating over old receptor signals in NMJs. Values were grouped in NMJs with less than 3 fragments and NMJs with 3 or more fragments. Red lines indicate medians. Note the large variance in mdx. Same data set as in C. E–F: Correlations of subsynaptic myosin Va (E) and PKA-RI enrichment (F) with the apparent turnover of AChRs. Tibialis anterior muscles used for in vivo imaging (B–D) were sliced transversally and immunostained for myosin Va or PKA-RI. Ratio of old and new receptors and the accumulation of myosin Va or PKA-RI were determined for each synapse (n = 5 muscles; 651 and 340 NMJs were quantified for myosin Va and PKA-RI, respectively). Significance was tested with Welch test, ** p<0.01.</p

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

Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

<p>Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in <i>trim63^−/−</i> mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.</p