Schwann cells in neuromuscular in vitro models.

Neuromuscular cell culture models are used to investigate synapse formation and function, as well as mechanisms of de-and regeneration in neuromuscular diseases. Recent developments including 3D culture technique and hiPSC technology have propelled their ability to complement insights from in vivo models. However, most cultures have not considered Schwann cells, the glial part of NMJs. In the following, a brief overview of different types of neuromuscular cocultures is provided alongside examples for studies that included Schwann cells. From these, findings concerning the effects of Schwann cells on those cultures are summarized and future lines of research are proposed.

Sympathetic innervation in skeletal muscle and its role at the neuromuscular junction.

Neuromuscular junctions are the synapses between motor neurons and skeletal muscle fibers, which mediate voluntary muscle movement. Since neuromuscular junctions are also tightly associated with the capping function of terminal Schwann cells, these synapses have been classically regarded as tripartite chemical synapses. Although evidences from sympathetic innervation of neuromuscular junctions was described approximately a century ago, the essential presence and functional relevance of sympathetic contribution to the maintenance and modulation of neuromuscular junctions was demonstrated only recently. These findings shed light on the pathophysiology of different clinical conditions and can optimize surgical and clinical treatment modalities for skeletal muscle disorders.

An alternative pathway for sweet sensation: possible mechanisms and physiological relevance.

Sweet substances are detected by taste-bud cells upon binding to the sweet-taste receptor, a T1R2/T1R3 heterodimeric G protein-coupled receptor. In addition, experiments with mouse models lacking the sweet-taste receptor or its downstream signaling components led to the proposal of a parallel "alternative pathway" that may serve as metabolic sensor and energy regulator. Indeed, these mice showed residual nerve responses and behavioral attraction to sugars and oligosaccharides but not to artificial sweeteners. In analogy to pancreatic ß cells, such alternative mechanism, to sense glucose in sweet-sensitive taste cells, might involve glucose transporters and KATP channels. Their activation may induce depolarization-dependent Ca2+ signals and release of GLP-1, which binds to its receptors on intragemmal nerve fibers. Via unknown neuronal and/or endocrine mechanisms, this pathway may contribute to both, behavioral attraction and/or induction of cephalic-phase insulin release upon oral sweet stimulation. Here, we critically review the evidence for a parallel sweet-sensitive pathway, involved signaling mechanisms, neural processing, interactions with endocrine hormonal mechanisms, and its sensitivity to different stimuli. Finally, we propose its physiological role in detecting the energy content of food and preparing for digestion.

GFPT1 deficiency in muscle leads to myasthenia and myopathy in mice.

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway which yields precursors required for protein and lipid glycosylation. Mutations in GFPT1 and other genes downstream of this pathway cause congenital myasthenic syndrome (CMS) characterized by fatigable muscle weakness owing to impaired neurotransmission. The precise pathomechanisms at the neuromuscular junction (NMJ) owing to a deficiency in GFPT1 is yet to be discovered. One of the challenges we face is the viability of Gfpt1-/- knockout mice. In this study, we use Cre/LoxP technology to generate a muscle-specific GFPT1 knockout mouse model, Gfpt1tm1d/tm1d, characteristic of the human CMS phenotype. Our data suggest a critical role for muscle derived GFPT1 in the development of the NMJ, neurotransmission, skeletal muscle integrity and highlight that a deficiency in skeletal muscle alone is sufficient to cause morphological postsynaptic NMJ changes that are accompanied by presynaptic alterations despite the conservation of neuronal GFPT1 expression. In addition to the conventional morphological NMJ changes and fatigable muscle weakness, Gfpt1tm1d/tm1d mice display a progressive myopathic phenotype with the presence of tubular aggregates in muscle, characteristic of the GFPT1-CMS phenotype. We further identify an upregulation of skeletal muscle proteins glypican-1, farnesyltransferase/geranylgeranyltransferase type-1 subunit α and muscle-specific kinase, which are known to be involved in the differentiation and maintenance of the NMJ. The Gfpt1tm1d/tm1d model allows for further investigation of pathophysiological consequences on genes and pathways downstream of GFPT1 likely to involve misglycosylation or hypoglycosylation of NMJs and muscle targets.

Sensing Senses: Optical Biosensors to Study Gustation.

The five basic taste modalities, sweet, bitter, umami, salty and sour induce changes of Ca2+ levels, pH and/or membrane potential in taste cells of the tongue and/or in neurons that convey and decode gustatory signals to the brain. Optical biosensors, which can be either synthetic dyes or genetically encoded proteins whose fluorescence spectra depend on levels of Ca2+, pH or membrane potential, have been used in primary cells/tissues or in recombinant systems to study taste-related intra- and intercellular signaling mechanisms or to discover new ligands. Taste-evoked responses were measured by microscopy achieving high spatial and temporal resolution, while plate readers were employed for higher throughput screening. Here, these approaches making use of fluorescent optical biosensors to investigate specific taste-related questions or to screen new agonists/antagonists for the different taste modalities were reviewed systematically. Furthermore, in the context of recent developments in genetically encoded sensors, 3D cultures and imaging technologies, we propose new feasible approaches for studying taste physiology and for compound screening.

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.

A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells.

BACKGROUND: Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive. METHODS: This study used low-attachment based generation of spheroids composed of up to three cell types. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student's t-test. RESULTS: The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. Regularly, some melanoma cells were also found to invade the fibroblast core. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity. CONCLUSIONS: In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.

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.

Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction.

Inherited deficiency in ether lipids, a subgroup of phospholipids whose biosynthesis needs peroxisomes, causes the fatal human disorder rhizomelic chondrodysplasia punctata. The exact roles of ether lipids in the mammalian organism and, therefore, the molecular mechanisms underlying the disease are still largely enigmatic. Here, we used glyceronephosphate O-acyltransferase knockout (Gnpat KO) mice to study the consequences of complete inactivation of ether lipid biosynthesis and documented substantial deficits in motor performance and muscle strength of these mice. We hypothesized that, probably in addition to previously described cerebellar abnormalities and myelination defects in the peripheral nervous system, an impairment of neuromuscular transmission contributes to the compromised motor abilities. Structurally, a morphologic examination of the neuromuscular junction (NMJ) in diaphragm muscle at different developmental stages revealed aberrant axonal branching and a strongly increased area of nerve innervation in Gnpat KO mice. Post-synaptically, acetylcholine receptor (AChR) clusters colocalized with nerve terminals within a widened endplate zone. In addition, we detected atypical AChR clustering, as indicated by decreased size and number of clusters following stimulation with agrin, in vitro. The turnover of AChRs was unaffected in ether lipid-deficient mice. Electrophysiological evaluation of the adult diaphragm indicated that although evoked potentials were unaltered in Gnpat KO mice, ether lipid deficiency leads to fewer spontaneous synaptic vesicle fusion events but, conversely, an increased post-synaptic response to spontaneous vesicle exocytosis. We conclude from our findings that ether lipids are essential for proper development and function of the NMJ and may, therefore, contribute to motor performance. Read the Editorial Highlight for this article on page 463.

A compact unc45b-promoter drives muscle-specific expression in zebrafish and mouse.

Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off-target effects the therapeutic gene should be driven by a tissue-specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue-specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle-specific expression but presents heat-shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b-deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle-specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431-438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

Neuromuscular junction degeneration in muscle wasting.

PURPOSE OF REVIEW: Denervation is a hallmark of age-related and other types of muscle wasting. This review focuses on recent insights and current viewpoints regarding the mechanisms and clinical relevance of maintaining the neuromuscular junction to counteract muscle wasting resulting from aging or neural disease/damage. RECENT FINDINGS: Activity-dependent regulation of autophagy, the agrin-muscle specific kinase-Lrp4 signaling axis, and sympathetic modulation are principal mechanisms involved in stabilizing the neuromuscular junction. These findings are derived from several animal models and were largely confirmed by human gene expression analysis as well as insights from rare neuromuscular diseases such as amyotrophic lateral sclerosis and congenital myasthenic syndromes. Based on these insights, agrin-derived fragments are currently being evaluated as biomarkers for age-related muscle wasting. Tuning of autophagy, of the agrin pathway, and of sympathetic input are being studied as clinical treatment of muscle wasting disorders. SUMMARY: Basic research has revealed that maintenance of neuromuscular junctions and a few signaling pathways are important in the context of age-dependent and other forms of muscle wasting. These findings have recently started to enter clinical practice, but further research needs to substantiate and refine our knowledge.

Turnover of acetylcholine receptors at the endplate revisited: novel insights into nerve-dependent behavior.

The turnover of nicotinic acetylcholine receptors (AChR) is a critical factor that determines function and safety of neuromuscular transmission at the nerve-muscle synapses, i.e. neuromuscular junctions (NMJs). Previously, three different populations of AChRs exhibiting distinct stereotypic and activity-dependent half-life values were observed in mouse muscles. To address AChR turnover in more detail, we here employed a recently developed longitudinal radioiodine assay that is based on repetitive measurements of radio emission from the same animals over long periods of time in combination with systematic variation of the time elapsed between AChR pulse-labeling and muscle denervation. Modeling of the data revealed profiles of AChR de novo synthesis and receptor incorporation into the postsynaptic membrane. Furthermore, decay of pre-existing AChRs upon denervation showed a peculiar pattern corroborating earlier findings of a two-step stabilization of AChRs.

Mitochondrial fission and remodelling contributes to muscle atrophy.

Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin-proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.

Rapsyn mediates subsynaptic anchoring of PKA type I and stabilisation of acetylcholine receptor in vivo.

The stabilisation of acetylcholine receptors (AChRs) at the neuromuscular junction depends on muscle activity and the cooperative action of myosin Va and protein kinase A (PKA) type I. To execute its function, PKA has to be present in a subsynaptic microdomain where it is enriched by anchoring proteins. Here, we show that the AChR-associated protein, rapsyn, interacts with PKA type I in C2C12 and T-REx293 cells as well as in live mouse muscle beneath the neuromuscular junction. Molecular modelling, immunoprecipitation and bimolecular fluorescence complementation approaches identify an α-helical stretch of rapsyn to be crucial for binding to the dimerisation and docking domain of PKA type I. When expressed in live mouse muscle, a peptide encompassing the rapsyn α-helical sequence efficiently delocalises PKA type I from the neuromuscular junction. The same peptide, as well as a rapsyn construct lacking the α-helical domain, induces severe alteration of acetylcholine receptor turnover as well as fragmentation of synapses. This shows that rapsyn anchors PKA type I in close proximity to the postsynaptic membrane and suggests that this function is essential for synapse maintenance.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Aberrant evoked calcium signaling and nAChR cluster morphology in a SOD1 D90A hiPSC-derived neuromuscular model.

Familial amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder that is due to mutations in one of several target genes, including SOD1. So far, clinical records, rodent studies, and in vitro models have yielded arguments for either a primary motor neuron disease, or a pleiotropic pathogenesis of ALS. While mouse models lack the human origin, in vitro models using human induced pluripotent stem cells (hiPSC) have been recently developed for addressing ALS pathogenesis. In spite of improvements regarding the generation of muscle cells from hiPSC, the degree of maturation of muscle cells resulting from these protocols has remained limited. To fill these shortcomings, we here present a new protocol for an enhanced myotube differentiation from hiPSC with the option of further maturation upon coculture with hiPSC-derived motor neurons. The described model is the first to yield a combination of key myogenic maturation features that are consistent sarcomeric organization in association with complex nAChR clusters in myotubes derived from control hiPSC. In this model, myotubes derived from hiPSC carrying the SOD1 D90A mutation had reduced expression of myogenic markers, lack of sarcomeres, morphologically different nAChR clusters, and an altered nAChR-dependent Ca2+ response compared to control myotubes. Notably, trophic support provided by control hiPSC-derived motor neurons reduced nAChR cluster differences between control and SOD1 D90A myotubes. In summary, a novel hiPSC-derived neuromuscular model yields evidence for both muscle-intrinsic and nerve-dependent aspects of neuromuscular dysfunction in SOD1-based ALS.

Myosin Va cooperates with PKA RIalpha to mediate maintenance of the endplate in vivo.

Myosin V motor proteins facilitate recycling of synaptic receptors, including AMPA and acetylcholine receptors, in central and peripheral synapses, respectively. To shed light on the regulation of receptor recycling, we employed in vivo imaging of mouse neuromuscular synapses. We found that myosin Va cooperates with PKA on the postsynapse to maintain size and integrity of the synapse; this cooperation also regulated the lifetime of acetylcholine receptors. Myosin Va and PKA colocalized in subsynaptic enrichments. These accumulations were crucial for synaptic integrity and proper cAMP signaling, and were dependent on AKAP function, myosin Va, and an intact actin cytoskeleton. The neuropeptide and cAMP agonist, calcitonin-gene related peptide, rescued fragmentation of synapses upon denervation. We hypothesize that neuronal ligands trigger local activation of PKA, which in turn controls synaptic integrity and turnover of receptors. To this end, myosin Va mediates correct positioning of PKA in a postsynaptic microdomain, presumably by tethering PKA to the actin cytoskeleton.

Myosin Va: Capturing cAMP for synaptic plasticity.

The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo vesicles within the actin cortex. The latter is a preferred site of cAMP production, and the specificity of cAMP signaling is largely mediated through the formation of microdomains that spatially couple localized metabotropic receptor activity and cAMP production to selected effectors and downstream targets. This review summarizes the core literature on the role of myosin Va for the creation of such a cAMP microdomain at the mammalian nerve-muscle synapse that serves the activity-dependent recycling of nicotinic acetylcholine receptors (nAChRs)-a principal ligand-gated ion channel which is imperative for voluntary muscle contraction. It is discussed that i) the nerve-muscle synapse is a site with a unique actin-dependent microstructure, ii) myosin Va and protein kinase A regulatory subunit Iα as well as nAChR and its constitutive binding partner, rapsyn, colocalize in endocytic/recycling vesicles near the postsynaptic membrane, and iii) impairment of myosin Va or displacement of protein kinase A regulatory subunit Iα leads to the loss of nAChR stability. Regulation of this signaling process and underlying basic pieces of machinery were covered in previous articles, to which the present review refers.

Synthesis of large scale 3D microscopic images of 3D cell cultures for training and benchmarking.

The analysis of 3D microscopic cell culture images plays a vital role in the development of new therapeutics. While 3D cell cultures offer a greater similarity to the human organism than adherent cell cultures, they introduce new challenges for automatic evaluation, like increased heterogeneity. Deep learning algorithms are able to outperform conventional analysis methods in such conditions but require a large amount of training data. Due to data size and complexity, the manual annotation of 3D images to generate large datasets is a nearly impossible task. We therefore propose a pipeline that combines conventional simulation methods with deep-learning-based optimization to generate large 3D synthetic images of 3D cell cultures where the labels are known by design. The hybrid procedure helps to keep the generated image structures consistent with the underlying labels. A new approach and an additional measure are introduced to model and evaluate the reduced brightness and quality in deeper image regions. Our analyses show that the deep learning optimization step consistently improves the quality of the generated images. We could also demonstrate that a deep learning segmentation model trained with our synthetic data outperforms a classical segmentation method on real image data. The presented synthesis method allows selecting a segmentation model most suitable for the user's data, providing an ideal basis for further data analysis.