SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology.

Sympathetic innervation controls homeostasis of neuromuscular junctions in health and disease.

The distribution and function of sympathetic innervation in skeletal muscle have largely remained elusive. Here we demonstrate that sympathetic neurons make close contact with neuromuscular junctions and form a network in skeletal muscle that may functionally couple different targets including blood vessels, motor neurons, and muscle fibers. Direct stimulation of sympathetic neurons led to activation of muscle postsynaptic ß2-adrenoreceptor (ADRB2), cAMP production, and import of the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PPARGC1A) into myonuclei. Electrophysiological and morphological deficits of neuromuscular junctions upon sympathectomy and in myasthenic mice were rescued by sympathicomimetic treatment. In conclusion, this study identifies the neuromuscular junction as a target of the sympathetic nervous system and shows that sympathetic input is crucial for synapse maintenance and function.

Postnatal Development and Distribution of Sympathetic Innervation in Mouse Skeletal Muscle.

Vertebrate neuromuscular junctions (NMJs) have been conceived as tripartite synapses composed of motor neuron, Schwann cell, and muscle fiber. Recent work has shown the presence of sympathetic neurons in the immediate vicinity of NMJs and experimental and clinical findings suggest that this plays an eminent role in adult NMJ biology. The present study examined the postnatal development and distribution of sympathetic innervation in different muscles using immunofluorescence, confocal microscopy, and Western blot. This demonstrates the proximity of sympathetic neurons in diaphragm, extensor digitorum longus, tibialis anterior, soleus, and levator auris longus muscles. In extensor digitorum longus muscle, sympathetic innervation of NMJs was quantified from perinatal to adult stage and found to increase up to two months of age. In diaphragm muscle, an extensive network of sympathetic neurons was prominent along the characteristic central synapse band. In summary, these data demonstrate that an elaborate sympathetic innervation is present in several mouse skeletal muscles and that this is often next to NMJs. Although the presence of sympathetic neurons at the perisynaptic region of NMJs increased during postnatal development, many synapses were already close to sympathetic neurons at birth. Potential implications of these findings for treatment of neuromuscular diseases are discussed.

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.

Effects of ASC Application on Endplate Regeneration Upon Glycerol-Induced Muscle Damage.

Amongst other approaches, adipose-derived stromal cells (ASCs) have recently been tested with respect to their regenerative capacity for treatment of neuromuscular disorders. While beneficial effects of ASCs on muscle recovery were observed previously, their impact on regeneration of neuromuscular junctions (NMJs) is unclear. Here, we used a murine glycerol damage model to study disruption and regeneration of NMJs and to evaluate the effects of systemic application of ASCs on muscle and NMJ recovery. In mice that were not treated with ASCs, a differential response of NMJ pre- and post-synapses to glycerol-induced damage was observed. While post-synapses were still present in regions that were necrotic and lacking actin and dystrophin, pre-synapses disappeared soon in those affected areas. Partial regeneration of NMJs occurred within 11 days after damage. ASC treatment slightly enhanced NMJ recovery and reduced the loss of presynaptic sites, but also led to a late phase of muscle necrosis and fibrosis. In summary, the results suggest a differential sensitivity of NMJ pre- and post-synapses to glycerol-induced muscle damage and that the use of ASC for the treatment of neuromuscular disorders needs further careful evaluation.

Nicotinic acetylcholine receptor at vertebrate motor endplates: Endocytosis, recycling, and degradation.

At vertebrate motor endplates, the conversion of nerve impulses into muscle contraction is initiated by binding of acetylcholine to its nicotinic receptor (nAChR) at the postsynapse. Efficiency and safety of this process are dependent on proper localization, density, and molecular composition of the receptors. To warrant this, intricate machineries regulating the turnover of nAChR are in place. They control and execute the processes of i) expression, ii) delivery to the postsynaptic membrane, iii) clustering at the plasma membrane, iv) endocytic retrieval, v) activity-dependent recycling, and vi) degradation of nAChR. Concentrating on aspects iv-vi, this review addresses the current status of techniques, concepts, and open questions on endocytosis, recycling, and degradation of nAChR. A picture is emerging, that shows connections between executing machineries and their regulators. The first group includes the actin cytoskeleton, myosin motor proteins, Rab G-proteins, and the autophagic cascade. The second group features protein kinases A and C, Cdk5, and CaMKII as well as other components like the E3-ligase MuRF1 and the membrane shaping regulator, SH3GLB1. Recent studies have started to shed light onto nerve inputs that appear to master the tuning of the postsynaptic protein trafficking apparatus and the expression of critical components for nAChR turnover.

A Novel Optical Tissue Clearing Protocol for Mouse Skeletal Muscle to Visualize Endplates in Their Tissue Context.

Neuromuscular junctions (NMJs) mediate skeletal muscle contractions and play an important role in several neuromuscular disorders when their morphology and function are compromised. However, due to their small size and sparse distribution throughout the comparatively large, inherently opaque muscle tissue the analysis of NMJ morphology has been limited to teased fiber preparations, longitudinal muscle sections, and flat muscles. Consequently, whole mount analyses of NMJ morphology, numbers, their distribution, and assignment to a given muscle fiber have also been impossible to determine in muscle types that are frequently used in experimental paradigms. This impossibility is exacerbated by the lack of optical tissue clearing techniques that are compatible with clear and persistent NMJ stains. Here, we present MYOCLEAR, a novel and highly reproducible muscle tissue clearing protocol. Based on hydrogel-based tissue clearing methods, this protocol permits the labeling and detection of all NMJs in adult hindleg extensor digitorum longus muscles from wildtype and diseased mice. The method is also applicable to adult mouse diaphragm muscles and can be used for different staining agents, including toxins, lectins, antibodies, and nuclear dyes. It will be useful in understanding the distribution, morphological features, and muscle tissue context of NMJs in hindleg muscle whole mounts for biomedical and basic research.

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

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.

Modulation of amyloid-ß protein precursor expression by HspB1.

Upregulation of heat shock proteins, such as Hsp70 and HspB1/Hsp27, have been associated with an amelioration of the deficits in animal models of Alzheimer's disease (AD). HspB1 is reported to be increased in AD brains and to accumulate in plaques, but whether this localization is an attempt by HspB1 to ameliorate the detrimental effects of amyloid-ß (Aß) on cells or part of the disease process is unknown. Here we explore the potential effects of the HspB1 on amyloid-ß protein precursor (AßPP) processing and distribution within HEK293 stable cell lines expressing either AßPPwt or AßPPsw. We compare AßPP production, distribution, and release of proteolytic products (including Aß40 and Aß42) to determine possible modifications in the presence of HspB1. We also investigate whether HspB1 interacts with Aß or its precursor, AßPP, and whether, through this interaction, it is able to alter AßPP processing or release of Aß peptide. Coexpression of HspB1 resulted in increased cellular holoAßPP as well as C-terminal fragments. Further, expression of HspB1 attenuated the release of Aß42 from the AßPPsw cells. In summary, we have shown that expression of HspB1 alters AßPP expression and processing in cell lines expressing AßPPwt and AßPPsw. Furthermore, the presence of HspB1 decreased the amount of Aß42 released by the cell lines. Thus in addition to its effects on protecting cells from the potentially toxic effects of Aß, HspB1 also appears to be involved in modulating cellular levels of AßPP, although an understanding of the underlying mechanisms requires further investigation.