Understanding the developmental steps that shape formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers is crucial. Wnt morphogens are key players in the ...formation of this specialized peripheral synapse, but their individual and collaborative functions and downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain-of-function studies in cell culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. By contrast, loss of Wnt11 or Wnt-dependent signaling
decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR mRNA levels and AChR clustering downstream of activation of the β-catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the planar cell polarity (PCP) pathway, which accumulates at embryonic NMJs. Moreover, mice bearing a
loss-of-function mutation (loop-tail) exhibit fewer AChR clusters and overgrowth of motor axons bypassing AChR clusters. Together, our results provide genetic and biochemical evidence that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways.
The muscle-specific kinase MuSK is one of the key molecules orchestrating neuromuscular junction (NMJ) formation. MuSK interacts with the Wnt morphogens, through its Frizzled-like domain ...(cysteine-rich domain CRD). Dysfunction of MuSK CRD in patients has been recently associated with the onset of myasthenia, common neuromuscular disorders mainly characterized by fatigable muscle weakness. However, the physiological role of Wnt-MuSK interaction in NMJ formation and function remains to be elucidated. Here, we demonstrate that the CRD deletion of MuSK in mice caused profound defects of both muscle prepatterning, the first step of NMJ formation, and synapse differentiation associated with a drastic deficit in AChR clusters and excessive growth of motor axons that bypass AChR clusters. Moreover, adult MuSKΔCRD mice developed signs of congenital myasthenia, including severe NMJs dismantlement, muscle weakness, and fatigability. We also report, for the first time, the beneficial effects of lithium chloride, a reversible inhibitor of the glycogen synthase kinase-3, that rescued NMJ defects in MuSKΔCRD mice and therefore constitutes a novel therapeutic reagent for the treatment of neuromuscular disorders linked to Wnt-MuSK signaling pathway deficiency. Together, our data reveal that MuSK CRD is critical for NMJ formation and plays an unsuspected role in NMJ maintenance in adulthood.
Most of these disorders are difficult to treat and can be life threatening. ...NMJ maintenance plays a key role in weakened muscle function during aging (Hunter et al., 2016). The authors found an ...enrichment of known and novel genes that are part of different networks such as diacylglycerol and phosphatidylinositol signaling or extracellular matrix components, thus expanding the group of molecular players involved in NMJ development. ...Spendiff et al. treated CMS model mice with a genetic defect in the AGRN gene using a small agrin compound, which restored post-synaptic structure and improved muscle strength through normalization of muscle fiber composition and the prevention of atrophy. Coppedè et al. investigated blood and thymoma tissues from 69 TAMG patients and studied the methylation pattern of tumor suppressor and DNA repair genes but did not observe any differences in the methylation between thymoma tissue and unaffected thymus tissue or any correlation between clinic, methylation and histopathology.
Congenital myasthenic syndromes (CMS) are a clinically and genetically heterogeneous group of rare diseases due to mutations in neuromuscular junction (NMJ) protein-coding genes. Until now, many ...mutations encoding postsynaptic proteins as Agrin, MuSK and LRP4 have been identified as responsible for increasingly complex CMS phenotypes. The majority of mutations identified in LRP4 gene causes bone diseases including CLS and sclerosteosis-2 and rare cases of CMS with mutations in LRP4 gene has been described so far. In the French cohort of CMS patients, we identified a novel LRP4 homozygous missense mutation (c.1820A > G; p.Thy607Cys) within the β1 propeller domain in a patient presenting CMS symptoms, including muscle weakness, fluctuating fatigability and a decrement in compound muscle action potential in spinal accessory nerves, associated with congenital agenesis of the hands and feet and renal malformation. Mechanistic expression studies show a significant decrease of AChR aggregation in cultured patient myotubes, as well as altered in vitro binding of agrin and Wnt11 ligands to the mutated β1 propeller domain of LRP4 explaining the dual phenotype characterized clinically and electoneuromyographically in the patient. These results expand the LRP4 mutations spectrum associated with a previously undescribed clinical association involving impaired neuromuscular transmission and limb deformities and highlighting the critical role of a yet poorly described domain of LRP4 at the NMJ. This study raises the question of the frequency of this rare neuromuscular form and the future diagnosis and management of these cases.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
At the neuromuscular junction, acetylcholinesterase (AChE) is mainly present as asymmetric forms in which tetramers of catalytic subunits are associated to a specific collagen, collagen Q (ColQ). The ...accumulation of the enzyme in the synaptic basal lamina strictly relies on ColQ. This has been shown to be mediated by interaction between ColQ and perlecan, which itself binds dystroglycan. Here, using transfected mutants of ColQ in a ColQ-deficient muscle cell line or COS-7 cells, we report that ColQ clusterizes through a more complex mechanism. This process requires two heparin-binding sites contained in the collagen domain as well as the COOH terminus of ColQ. Cross-linking and immunoprecipitation experiments in Torpedo postsynaptic membranes together with transfection experiments with muscle-specific kinase (MuSK) constructs in MuSK-deficient myotubes or COS-7 cells provide the first evidence that ColQ binds MuSK. Together, our data suggest that a ternary complex containing ColQ, perlecan, and MuSK is required for AChE clustering and support the notion that MuSK dictates AChE synaptic localization at the neuromuscular junction.
Understanding the developmental steps shaping the formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers, is critical. Wnt morphogens are key players in the ...formation of this specialized peripheral synapse. Yet, the individual and collaborative functions of Wnts as well as their downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain of function studies in culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. In contrast, loss of Wnt11 or Wnt-dependent signaling in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR clustering and mRNA downstream activation of the β-catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the Planar Cell Polarity (PCP) pathway, which accumulates at embryonic NMJ. Moreover, mice bearing a Vangl2 loss of function mutation (looptail) exhibit a decreased number of AChR clusters and overgrowth of motor axons bypassing AChR clusters. Taken together, our results provide genetic and biochemical evidences that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways.
Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and ...heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.
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