Abstract
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.
Highlights
Endocytosis of signaling transmembrane proteins, such as EGFR (Caldieri et al, 2018), CD81 (Hosokawa et al, 2020), NMDA-R (Scott et al, 2004), AMPA-R (Ehlers, 2000), or ion channels in general (Estadella et al, 2020), is initiated by Clathrin-dependent or -independent invagination of plasma membrane, resulting in transmembrane receptor-containing early endosomes
Concentration on details demonstrated ample ramifications of sympathetic neurons that were often found initiating from major branches of blood vessels (Figure 1C; shown region is boxed in Figure 1A; exemplary sympathetic ramifications branching from large blood vessels, see arrowheads) and ran along muscle fibers and small blood vessels (Figure 1D; shown region is boxed in Figure 1C; exemplary sympathetic fibers running along small blood vessels, see arrowheads)
We investigated whether sympathetic innervation might act on endo/lysosomal trafficking at the neuromuscular junction (NMJ) by means of in vivo confocal microscopy, proteomic, Western blot, and immunofluorescence analyses of muscles treated with or without chemical sympathectomy
Summary
Endocytosis of signaling transmembrane proteins, such as EGFR (Caldieri et al, 2018), CD81 (Hosokawa et al, 2020), NMDA-R (Scott et al, 2004), AMPA-R (Ehlers, 2000), or ion channels in general (Estadella et al, 2020), is initiated by Clathrin-dependent or -independent invagination of plasma membrane, resulting in transmembrane receptor-containing early endosomes. Rab 5 is exchanged by Rab 7 to reach a state of late endosomes They are delivered to lysosomal degradation either directly (Chavrier et al, 1990; Soldati et al, 1995) or via a process known as macroautophagy (hereafter short autophagy) (Amaya et al, 2015). While Rab 11 is known to be associated with recycling endosomes and post-Golgi vesicular trafficking (Lock and Stow, 2005; Welz et al, 2014), some studies point toward an additional role of this Rab GTPase in autophagy regulation (Fader et al, 2008; Longatti et al, 2012; Szatmári et al, 2014; Puri et al, 2018)
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