Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4⁻/⁻ NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.
... Interestingly, MuSK contains in its extracellular region a Frizzled-like domain (cysteinerich domain [CRD]) mediating its interaction with several WNTs, including WNT4, WNT11, and WNT9a in vitro [35][36][37]. Activation of the MuSK-Lrp4 complex regulates the prepatterning step, before muscle innervation, during which AChRs begin to aggregate in a central synaptic region of the muscle [38][39][40]. Moreover, in vivo knockdown of WNT4 and WNT11 affects muscle prepatterning and axon guidance, indicating a role for Wnt signaling [35,36,41]. ...
... Activation of the MuSK-Lrp4 complex regulates the prepatterning step, before muscle innervation, during which AChRs begin to aggregate in a central synaptic region of the muscle [38][39][40]. Moreover, in vivo knockdown of WNT4 and WNT11 affects muscle prepatterning and axon guidance, indicating a role for Wnt signaling [35,36,41]. Upon innervation, the MuSK-Lrp4 complex is further stimulated by neural AGRIN, which induces multiple signaling pathways leading to clustering and remodeling of aneural AChR clusters [42,43]. ...
... Previously, a different WNT3A response of Lrp5 or Lrp6 was found in osteoblasts arguing for a more important role of Lrp6 in comparison with Lrp5 [62]. Regarding the role of Lrp5 and/or Lrp6 at NMJs, this is even more exciting because an important regulator of NMJ formation and maintenance, the receptor tyrosine kinase MuSK, was reported to bind canonical Wnt ligands by its CRD and thereby act by itself like a canonical Wnt signaling receptor [35,63]. In patients, evidence accumulated for the pathophysiological importance of the MuSK CRD domain either by being linked to mutations in congenital myasthenic syndromes or by detection of autoantibodies in myasthenia gravis [64,65]. ...
Canonical Wnt signaling is involved in skeletal muscle cell biology. The exact way in which this pathway exerts its contribution to myogenesis or neuromuscular junctions (NMJ) is a matter of debate. Next to the common co-receptors of canonical Wnt signaling, Lrp5 and Lrp6, the receptor tyrosine kinase MuSK was reported to bind at NMJs WNT glycoproteins by its extracellular cysteine-rich domain. Previously, we reported canonical Wnt signaling being active in fast muscle fiber types. Here, we used conditional Lrp5 or Lrp6 knockout mice to investigate the role of these receptors in muscle cells. Conditional double knockout mice died around E13 likely due to ectopic expression of the Cre recombinase. Phenotypes of single conditional knockout mice point to a very divergent role for the two receptors. First, muscle fiber type distribution and size were changed. Second, canonical Wnt signaling reporter mice suggested less signaling activity in the absence of Lrps. Third, expression of several myogenic marker genes was changed. Fourth, NMJs were of fragmented phenotype. Fifth, recordings revealed impaired neuromuscular transmission. In sum, our data show fundamental differences in absence of each of the Lrp co-receptors and suggest a differentiated view of canonical Wnt signaling pathway involvement in adult skeletal muscle cells.
... Fragala et al. found that an increase in circulating Agrin fragments in response to short-term resistance exercise training in older adults [35]. Previous studies have reported that Wnts could bind to the extracellular carbohydrate recognition domain of MuSK, thereby increasing the phosphorylation levels of MuSK [63,64]. Additionally, there is substantial evidence suggesting that physical exercise could enhance the expression of Wnts [65,66]. ...
... A ce jour il a été identifié que parmi la famille des Wnt, certains régulent positivement ou négativement l'agrégation des AChR. Wnt4, 9a, 11 et 16 seuls ou en association auraient un effet positif sur le pré-patterning ou l'agrégation des AChR via MuSK (Barik et al., 2014;Strochlic et al., 2012;Zhang et al., 2012). Indépendamment de MuSK, les Wnt 7a, 8a et 3a réguleraient négativement l'agrégation des AChR et le pré-patterning (Henriquez et al., 2008;Barik et al., 2014). ...
A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.
Here, we investigated the role of the canonical Wnt signaling pathway transcriptional regulators at the neuromuscular junction. Upon applying a denervation paradigm, the transcription levels of Ctnnb1 , Tcf7l1 , Tle1 , Tle2 , Tle3 , and Tle4 were significantly downregulated. A significant decrease in canonical Wnt signaling activity was observed using the denervation paradigm in Axin2-lacZ reporter mice. Alterations in the transcriptional profile of the myogenic lineage in response to agrin (AGRN) suggested that TLE3 and TLE4, family members of groucho transducin-like enhancer of split 3 (TLE3), transcriptional repressors known to antagonize T cell factor/lymphoid enhancer factor (TCF)-mediated target gene activation, could be important regulators of canonical Wnt signaling activity at the postsynapse. Knockouts of these genes using CRISPR/Cas9 gene editing in primary skeletal muscle stem cells, called satellite cells, led to decreased AGRN-dependent acetylcholine receptor (CHRN) clustering and reduced synaptic gene transcription upon differentiation of these cells. Overall, our findings demonstrate that TLE3 and TLE4 participate in diminishing canonical Wnt signaling activity, supporting transcription of synaptic genes and CHRN clustering at the neuromuscular junction.
Background
Over the past few years, it has been established that wnt genes are involved in the regenerative processes of holothurians. The wnt4 gene was identified as one of the most active genes in Eupentacta fraudatrix regeneration using differential gene expression analysis and qPCR of individual genes. Also, the wntA gene was found in holothurians, which is present only in invertebrates and can perform unique functions.
Results
In this regard, both these genes and proteins were studied in this work. During regeneration, the Wnt4 protein is found in the cells of the coelomic and ambulacral epithelium, retractor muscles, and radial nerves. Single cells with this protein are also found in the connective tissue of the developing aquapharyngeal bulb and in the hypoderm of the body wall. Cells with WntA are found exclusively in the hypoderm of the body wall.
Conclusion
We assume that both genes are involved in regeneration, but Wnt4 coordinates the formation of the epithelial tissue structure, while WntA maintains the state of the intercellular substance of the body wall.
ColQ is a non-fibrillar collagen that plays a crucial role at the vertebrate neuromuscular junction (NMJ) by anchoring acetylcholinesterase (AChE) to the synapse. ColQ also functions in signaling, as it regulates acetylcholine receptor clustering and synaptic gene expression, in a manner dependent on Muscle-Specific Kinase (MuSK), a key protein in NMJ formation and maintenance. MuSK forms a complex with Low-density lipoprotein receptor-related protein 4 (LRP4), its co-receptor for the proteoglycan agrin at the NMJ. Previous studies suggested that ColQ also interacts with MuSK. However, the molecular mechanisms underlying ColQ functions and ColQ-MuSK interaction have not been fully elucidated. Here, we investigated whether ColQ binds directly to MuSK and/or LRP4 and whether it modulates agrin-mediated MuSK/LRP4 activation. Using co-immunoprecipitation, pull-down, plate binding assays and surface plasmon resonance, we show that ColQ binds directly to LRP4 but not to MuSK, and that ColQ interacts indirectly with MuSK through LRP4. In addition, we show the N-terminal region of LRP4, which contains the agrin binding sites, is also crucial for ColQ binding to LRP4. Moreover, ColQ-LRP4 interaction was reduced in the presence of agrin, suggesting that agrin and ColQ compete for binding to LRP4. Strikingly, we reveal ColQ has two opposing effects on agrin-induced MuSK/LRP4 signaling: it constitutively reduces MuSK phosphorylation levels in agrin-stimulated myotubes, but at the same time increases MuSK accumulation at the muscle cell surface. Together, our results identify LRP4 as a major receptor of ColQ and provide new insights into mechanisms of ColQ signaling and AChE anchoring at the NMJ.
Peripheral nerve injury (PNI) induces severe functional loss in extremities. Progressive denervation and atrophy occur in the muscles if the nerve repair is delayed for long periods of the time. To overcome these difficulties, detailed mechanisms should be determined for neuromuscular junction (NMJ) degeneration in target muscles after PNI and regeneration after nerve repair. We established two models of end-to-end neurorrhaphy and allogeneic nerve grafting in the chronic phase after common peroneal nerve injury in female mice (n=100 in total). We evaluated motor function, histology, and gene expression in the target muscles during their regeneration processes and compared the models. We found that the functional recovery with allogeneic nerve grafting was superior to that with end-to-end neurorrhaphy, and the number of reinnervated NMJs and Schwann cells was increased at 12 weeks after allograft. In addition, NMJ- and Schwann cell-related molecules showed high expression in the target muscle in the allograft model. These results suggest that Schwann cell migrating from the allograft might play a crucial role in nerve regeneration in the chronic phase after PNI. The relationship between the NMJ and Schwann cells should be further investigated in the target muscle.
It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.
The development of chemical synapses is regulated by interactions between pre- and postsynaptic cells. At the vertebrate skeletal neuromuscular junction, the organization of an acetylcholine receptor (AChR)-rich postsynaptic apparatus has been well studied. Much evidence suggests that the nerve-derived protein agrin activates muscle-specific kinase (MuSK) to cluster AChRs through the synapse-specific cytoplasmic protein rapsyn. But how postsynaptic differentiation is initiated, or why most synapses are restricted to an 'end-plate band' in the middle of the muscle remains unknown. Here we have used genetic methods to address these issues. We report that the initial steps in postsynaptic differentiation and formation of an end-plate band require MuSK and rapsyn, but are not dependent on agrin or the presence of motor axons. In contrast, the subsequent stages of synaptic growth and maintenance require nerve-derived agrin, and a second nerve-derived signal that disperses ectopic postsynaptic apparatus.
Synapses, as fundamental units of the neural circuitry, enable complex behaviors. The neuromuscular junction (NMJ) is a synapse type that forms between motoneurons and skeletal muscle fibers and that exhibits a high degree of subcellular specialization. Aided by genetic techniques and suitable animal models, studies in the past decade have brought significant progress in identifying NMJ components and assembly mechanisms. This review highlights recent advances in the study of NMJ development, focusing on signaling pathways that are activated by diffusible cues, which shed light on synaptogenesis in the brain and contribute to a better understanding of muscular dystrophy.
CollagenQ (ColQ) plays an important structural role at vertebrate neuromuscular junctions (NMJs) by anchoring and accumulating acetylcholinesterase (AChE) in the extracellular matrix (ECM). Moreover, ColQ interacts with perlecan/dystroglycan and the muscle-specific receptor tyrosine kinase (MuSK), key molecules in the NMJ formation. MuSK promotes acetylcholine receptor (AChR) clustering in a process mediated by rapsyn, a cytoplasmic protein that stimulates AChR packing in clusters and regulates synaptic gene transcription. Here, we investigated a regulatory role for ColQ by comparing the clustering and expression of synaptic proteins in wild type and ColQ-deficient muscle cells in culture and at NMJ. We show first that AChR clusters are smaller and more densely packed in the absence of ColQ both in vitro and in vivo. Second, we find that like AChRs and rapsyn, MuSK mRNA levels are increased in cultured cells but not in muscles lacking ColQ. However, membrane-bound MuSK is decreased both in vitro and in vivo suggesting that ColQ controls MuSK sorting or stabilization in the muscle membrane. In line with this, our data show that activation of the MuSK signaling pathway is altered in the absence of ColQ leading to (1) perturbation of AChR clustering and/or beta-AChR subunit phosphorylation and (2) modifications of AChR mRNA level due to the lack of ColQ-MuSK interaction. Together, our results demonstrate that ColQ, in addition to its structural role, has important regulatory functions at the synapse by controlling AChR clustering and synaptic gene expression through its interaction with MuSK.
The formation of functional synapses requires a proper dialogue between incoming axons and their future synaptic targets. As axons approach their target, they are instructed to slow down and remodel to form proper presynaptic terminals. Although significant progress has been made in the identification of the mechanisms that control axon guidance, little is known about the mechanisms that regulate the conversion of actively growing axon into a presynaptic terminal. We found that Wnt secreted proteins are retrograde signals that regulate the terminal arborization of axons and synaptic differentiation. Wnts released from postsynaptic neurons induce extensive remodelling on incoming axons. This remodelling is manifested by a decrease in axon extension with a concomitant increase in growth-cone size. This morphological change is correlated with changes in the dynamics and organization of microtubules. Studies of a vertebrate synapse and the Drosophila neuromuscular junction suggest that a conserved Wnt signalling pathway modulates presynaptic microtubules as axons remodel during synapse formation. In this paper I discuss the role of the Wnt–Dvl (Dishevelled protein)–GSK-3β (glycogen synthase kinase-3β) signalling pathway in axon remodelling during synapse formation in the central nervous system.
THE kidney has been widely exploited as a model system for the study of tissue inductions regulating vertebrate organogenesis1,2. Kidney development is initiated by the ingrowth of the Wolfian duct-derived ureteric bud into the presumptive kidney mesenchyme. In response to a signal from the ureter, mesenchymal cells condense, aggregate into pretubular clusters and undergo an epithelial conversion generating a simple tubule. This then undergoes morphogenesis and is transformed into the excretory system of the kidney, the nephron. We report here that the expression of Wnt-4, which encodes a secreted glycoprotein, correlates with, and is required for, kidney tubulogenesis. Mice lacking Wnt-4 activity fail to form pretubular cell aggregates; however, other aspects of mesenchymal and ureteric development are unaffected. Thus, Wnt-4 appears to act as an autoinducer of the mesenchyme to epithelial transition that underlies nephron development.
We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse > human > bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., l M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent-soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and delergent-soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer' G4. Athough we cannot exclude the existence of significant differences belween the various molecular forms of acelylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by postlranslalional modifications.
Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six/sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1:Six4 mutants (SixdKO) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO. We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca(2+) homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development.
Wnt proteins play prominent roles in different aspects of neuronal development culminating with the formation of complex neuronal circuits. Here, we discuss new studies addressing the function of Wnt signalling at the peripheral neuromuscular junction (NMJ). In both, invertebrate and vertebrate organisms, Wnt signalling promotes and also inhibits the assembly of the neuromuscular synapse. Here, we focus our attention on recent studies at the vertebrate NMJ that demonstrate that some Wnt proteins collaborate with the Agrin-MuSK signalling to induce post-synaptic differentiation. In contrast, Wnts that activate the Wnt/β-catenin signalling inhibit post-synaptic differentiation. The dual function of different Wnts might finely modulate the proper apposition of the pre- and post-synaptic terminals during NMJ formation and growth.
The formation of synaptic connections requires a dialogue between pre and postsynaptic cells to coordinate the assembly of the presynaptic release machinery and the postsynaptic receptive complexes. Signaling molecules of the Wnt family of proteins are central to this trans-synaptic dialogue. At the neuromuscular junction and central synapses, Wnts promote synaptic assembly by signaling to the developing pre and postsynaptic compartments. In addition, new studies reveal that expression of Wnt proteins and localization of their Fz receptors are regulated by neuronal activity. Importantly, Wnts mediates the synaptic changes induced by patterned neuronal activity or sensory experience in mature neurons. Here we review recent findings into the function of Wnt signaling at the synapse and its link to activity-dependent synaptic growth and function.
