Molecular Analysis of a Congenital Myasthenic Syndrome Due to a Pathogenic Variant Affecting the C-Terminus of ColQ.

Congenital Myasthenic Syndromes (CMSs) are rare inherited diseases of the neuromuscular junction characterized by muscle weakness. CMSs with acetylcholinesterase deficiency are due to pathogenic variants in COLQ, a collagen that anchors the enzyme at the synapse. The two COLQ N-terminal domains have been characterized as being biochemical and functional. They are responsible for the structure of the protein in the triple helix and the association of COLQ with acetylcholinesterase. To deepen the analysis of the distal C-terminal peptide properties and understand the CMSs associated to pathogenic variants in this domain, we have analyzed the case of a 32 year old male patient bearing a homozygote splice site variant c.1281 C > T that changes the sequence of the last 28 aa in COLQ. Using COS cell and mouse muscle cell expression, we show that the COLQ variant does not impair the formation of the collagen triple helix in these cells, nor its association with acetylcholinesterase, and that the hetero-oligomers are secreted. However, the interaction of COLQ variant with LRP4, a signaling hub at the neuromuscular junction, is decreased by 44% as demonstrated by in vitro biochemical methods. In addition, an increase in all acetylcholine receptor subunit mRNA levels is observed in muscle cells derived from the patient iPSC. All these approaches point to pathophysiological mechanisms essentially characterized by a decrease in signaling and the presence of immature acetylcholine receptors.

The collagen ColQ binds to LRP4 and regulates the activation of the Muscle-Specific Kinase-LRP4 receptor complex by agrin at the neuromuscular junction.

Collagen Q (ColQ) is a nonfibrillar collagen that plays a crucial role at the vertebrate neuromuscular junction (NMJ) by anchoring acetylcholinesterase 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 coreceptor 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 coimmunoprecipitation, 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 that the LRP4 N-terminal region, 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 concomitantly increases MuSK accumulation at the muscle cell surface. Our results identify LRP4 as a major receptor of ColQ and provide new insights into mechanisms of ColQ signaling and acetylcholinesterase anchoring at the NMJ.

MBNL-dependent impaired development within the neuromuscular system in myotonic dystrophy type 1.

AIMS: Myotonic dystrophy type I (DM1) is one of the most frequent muscular dystrophies in adults. Although DM1 has long been considered mainly a muscle disorder, growing evidence suggests the involvement of peripheral nerves in the pathogenicity of DM1 raising the question of whether motoneurons (MNs) actively contribute to neuromuscular defects in DM1. METHODS: By using micropatterned 96-well plates as a coculture platform, we generated a functional neuromuscular model combining DM1 and muscleblind protein (MBNL) knock-out human-induced pluripotent stem cells-derived MNs and human healthy skeletal muscle cells. RESULTS: This approach led to the identification of presynaptic defects which affect the formation or stability of the neuromuscular junction at an early developmental stage. These neuropathological defects could be reproduced by the loss of RNA-binding MBNL proteins, whose loss of function in vivo is associated with muscular defects associated with DM1. These experiments indicate that the functional defects associated with MNs can be directly attributed to MBNL family proteins. Comparative transcriptomic analyses also revealed specific neuronal-related processes regulated by these proteins that are commonly misregulated in DM1. CONCLUSIONS: Beyond the application to DM1, our approach to generating a robust and reliable human neuromuscular system should facilitate disease modelling studies and drug screening assays.

Correlative Imaging of Motoneuronal Cell Elasticity by Pump and Probe Spectroscopy.

Because of their role of information transmitter between the spinal cord and the muscle fibers, motor neurons are subject to physical stimulation and mechanical property modifications. We report on motoneuron elasticity investigated by time-resolved pump and probe spectroscopy. A dual picosecond geometry simultaneously probing the acoustic impedance mismatch at the cell-titanium transducer interface and acoustic wave propagation inside the motoneuron is presented. Such noncontact and nondestructive microscopy, correlated to standard atomic force microscopy or a fluorescent labels approach, has been carried out on a single cell to address some physical properties such as bulk modulus of elasticity, dynamical longitudinal viscosity, and adhesion.

AChR ß-Subunit mRNAs Are Stabilized by HuR in a Mouse Model of Congenital Myasthenic Syndrome With Acetylcholinesterase Deficiency.

Collagen Q (COLQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction. So far, no mutation has been identified in the ACHE human gene but over 50 different mutations in the COLQ gene are causative for a congenital myasthenic syndrome (CMS) with AChE deficiency. Mice deficient for COLQ mimic most of the functional deficit observed in CMS patients. At the molecular level, a striking consequence of the absence of COLQ is an increase in the levels of acetylcholine receptor (AChR) mRNAs and proteins in vivo and in vitro in murine skeletal muscle cells. Here, we decipher the mechanisms that drive AChR mRNA upregulation in cultured muscle cells deficient for COLQ. We show that the levels of AChR ß-subunit mRNAs are post-transcriptionally regulated by an increase in their stability. We demonstrate that this process results from an activation of p38 MAPK and the cytoplasmic translocation of the nuclear RNA-binding protein human antigen R (HuR) that interacts with the AU-rich element located within AChR ß-subunit transcripts. This HuR/AChR transcript interaction induces AChR ß-subunit mRNA stabilization and occurs at a specific stage of myogenic differentiation. In addition, pharmacological drugs that modulate p38 activity cause parallel modifications of HuR protein and AChR ß-subunit levels. Thus, our study provides new insights into the signaling pathways that are regulated by ColQ-deficiency and highlights for the first time a role for HuR and p38 in mRNA stability in a model of congenital myasthenic syndrome.

Generation of a human induced pluripotent stem cell line (iPSC) from peripheral blood mononuclear cells of a patient with a myasthenic syndrome due to mutation in COLQ.

Congenital myasthenic syndromes (CMS) are a class of inherited disorders affecting the neuromuscular junction, a synapse whose activity is essential for movement. CMS with acetylcholinesterase (AChE) deficiency are caused by mutations in COLQ, a collagen that anchors AChE in the synapse. To study the pathophysiological mechanisms of the disease in human cells, we have generated iPSC from a patient's Peripheral Blood Mononuclear cells (PBMC) by reprogramming these cells using a non-integrative method using Sendai viruses bearing the four Yamanaka factors Oct3/4, Sox2, Klf4, and L-Myc.

Building neuromuscular junctions in vitro.

The neuromuscular junction (NMJ) has been the model of choice to understand the principles of communication at chemical synapses. Following groundbreaking experiments carried out over 60 years ago, many studies have focused on the molecular mechanisms underlying the development and physiology of these synapses. This Review summarizes the progress made to date towards obtaining faithful models of NMJs in vitro We provide a historical approach discussing initial experiments investigating NMJ development and function from Xenopus to mice, the creation of chimeric co-cultures, in vivo approaches and co-culture methods from ex vivo and in vitro derived cells, as well as the most recent developments to generate human NMJs. We discuss the benefits of these techniques and the challenges to be addressed in the future for promoting our understanding of development and human disease.

Collagens at the vertebrate neuromuscular junction, from structure to pathologies.

The extracellular matrix at the neuromuscular junction is built upon components secreted by the motoneuron, the muscle cell and terminal Schwann cells, the cells constituting this specific synapse. This compartment contains glycoproteins, proteoglycans and collagens that form a dense and specialized layer, the synaptic basal lamina. A number of these molecules are known to play a crucial role in anterograde and retrograde signalings that are active in neuromuscular junction formation, maintenance and function. Here, we focus on the isoforms of collagens which are enriched at the synapse. We summarize what we know of their structure, their function and their interactions with transmembrane receptors and other components of the synaptic basal lamina. A number of neuromuscular diseases, congenital myastenic syndromes and myasthenia gravis are caused by human mutations and autoantibodies against these proteins. Analysis of these diseases and of the specific collagen knock-out mice highlights the roles of some of these collagens in promoting a functional synapse.

Congenital myasthenic syndromes with acetylcholinesterase deficiency, the pathophysiological mechanisms.

The neuromuscular junction (NMJ) is a cholinergic synapse in vertebrates. This synapse connects motoneurons to muscles and is responsible for muscle contraction, a physiological process that is essential for survival. A key factor for the normal functioning of this synapse is the regulation of acetylcholine (ACh) levels in the synaptic cleft. This is ensured by acetylcholinesterase (AChE), which degrades ACh. A number of mutations in synaptic genes expressed in motoneurons or muscle cells have been identified and are causative for a class of neuromuscular diseases called congenital myasthenic syndromes (CMSs). One of these CMSs is due to deficiency in AChE, which is absent or diffuse in the synaptic cleft. Here, I focus on the origins of the syndrome. The role of ColQ, a collagen that anchors AChE in the synaptic cleft, is discussed in this context. Studies performed on patient biopsies, transgenic mice, and muscle cultures have provided a more comprehensive view of the connectome at the NMJ that should be useful for understanding the differences in the symptoms observed in specific CMSs due to mutated proteins in the synaptic cleft.

Moving forward with the neuromuscular junction.

The neuromuscular junction (NMJ) is indispensable for survival. This synapse between motoneurons and skeletal muscle fibers allows posture, movement and respiration. Therefore, its dysfunction creates pathologies than can be lethal. The molecular mechanisms of NMJ development and maintenance are the subject of intensive studies. This mini-review focuses on some of the most recent discoveries. An unexpected role for a protein, rapsyn, which has been known for 40 years to aggregate acetylcholine receptors has emerged. A new cell partner at NMJ has been unmasked and is challenging our understanding of the functioning of this synapse. Toxins are now used as new tools to study degeneration/regeneration. The possibility of creating human NMJ in vitro is within reach with major consequences for drug screening. Wnts are secreted neurogenic factors that have been involved in vitro in acetylcholine receptor clustering, but their precise role in vivo remains to be clarified. All these data are raising new and exciting perspectives in the field and are discussed in this Review. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Wnt proteins contribute to neuromuscular junction formation through distinct signaling pathways.

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 in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR mRNA levels and AChR clustering downstream of activation of the ß-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 Vangl2 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.

Neuromuscular junction immaturity and muscle atrophy are hallmarks of the ColQ-deficient mouse, a model of congenital myasthenic syndrome with acetylcholinesterase deficiency.

The collagen ColQ anchors acetylcholinesterase (AChE) in the synaptic cleft of the neuromuscular junction (NMJ). It also binds MuSK and perlecan/dystroglycan, 2 signaling platforms of the postsynaptic domain. Mutations in ColQ cause a congenital myasthenic syndrome (CMS) with AChE deficiency. Because the absence of AChE does not fully explain the complexity of the syndrome and there is no curative treatment for the disease, we explored additional potential targets of ColQ by conducting a large genetic screening of ColQ-deficient mice, a model for CMS with AChE deficiency, and analyzed their NMJ and muscle phenotypes. We demonstrated that ColQ controls the development and the maturation of the postsynaptic domain by regulating synaptic gene expression. Notably, ColQ deficiency leads to an up-regulation of the 5 subunits of the nicotinic acetylcholine receptor (AChR), leading to mixed mature and immature AChRs at the NMJ of adult mice. ColQ also regulates the expression of extracellular matrix (ECM) components. However, whereas the ECM mRNAs were down-regulated in vitro, compensation seemed to occur in vivo to maintain normal levels of these mRNAs. Finally, ColQ deficiency leads to a general atrophic phenotype and hypoplasia that affect fast muscles. This study points to new specific hallmarks for this CMS.-Sigoillot, S. M., Bourgeois, F., Karmouch, J., Molgó, J., Dobbertin, A., Chevalier, C., Houlgatte, R., Léger, J., Legay, C. Neuromuscular junction immaturity and muscle atrophy are hallmarks of the ColQ-deficient mouse, a model of congenital myasthenic syndrome with acetylcholinesterase deficiency.

HuR Mediates Changes in the Stability of AChR ß-Subunit mRNAs after Skeletal Muscle Denervation.

Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate AChR ß-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR ß-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3'-untranslated region (UTR) of AChR ß-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR ß-subunit 3'UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR ß-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38 MAPK, a pathway known to activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR ß-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR ß-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression. SIGNIFICANCE STATEMENT: Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR ß-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.

MuSK frizzled-like domain is critical for mammalian neuromuscular junction formation and maintenance.

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.

Developmental consequences of the ColQ/MuSK interactions.

CollagenQ (ColQ) is a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic basal lamina of the neuromuscular junction (NMJ). Over 30 mutations in the COLQ gene have been identified that are responsible for a congenital myasthenic syndrome with AChE deficiency, highlighting the importance of this collagen in the physiology of the NMJ. The anchoring of AChE at the synapse requires the interaction of ColQ with MuSK (Muscle-Specific Kinase), a tyrosine kinase expressed on the muscle membrane that is necessary for the formation and the maintenance of the NMJ. MuSK forms with its co-receptor LRP4, a member of the Low-density Related Protein family, a receptor complex for agrin and Wnts, representing the core system from which the postsynaptic domain is built, the growth cone attracted and the presynaptic element instructed for some aspects of its differentiation. Therefore, the discovery that ColQ binds to MuSK prompted us to study a possible regulatory function of ColQ during NMJ development. In this review, after a brief survey on ColQ, we summarize our recent data demonstrating that ColQ, in addition to its anchoring role, exerts signaling functions and controls some aspects of postsynaptic differentiation such as the clustering of acetylcholine receptors. Our results also strengthen the hypothesis that the defects observed in synaptic congenital myasthenic syndromes might be linked, at least in part, to alterations of ColQ signaling functions and not only to AChE deficiency. Finally, we discuss future research directions to understand how ColQ may modulate the action of the other ligands of the MuSK/LRP4 complex and cooperate with them to coordinate the different steps of NMJ formation and maintenance.

Wnt4 participates in the formation of vertebrate neuromuscular junction.

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.

Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression.

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.

Cholinesterases regulation in the absence of ColQ.

Normal physiological activity of the neuromuscular junction (NMJ) requires that key molecules are clustered at the synapse. One of these molecules is acetylcholinesterase (AChE) that regulates acetylcholine levels. This enzyme exists under different isoforms but the predominant form at the NMJ is a collagen-tailed enzyme. The collagen associated to AChE (ColQ) fulfills two functions. It anchors and accumulates AChE in the extracellular matrix. Mutations in ColQ lead to faint or no activity of AChE in the synaptic cleft. As a consequence, normal NMJ functioning is impaired and myasthenic syndromes are observed in patients bearing these mutations. Here, we investigated the effects of ColQ deficiency on cholinesterases mRNA levels and cluster formation. We show that overexpression of AChE but not ColQ in muscle cells is sufficient to drive the formation of AChE clusters. The absence of ColQ in muscle cells in vitro and in vivo leads to an increase in AChE(R) and AChE(T) mRNAs, corresponding to two isoforms of AChE. However, AChE activity is decreased in the medium of ColQ-deficient cells suggesting that AChE secretion is impaired. Butyrylcholinesterase (BChE) mRNAs are also upregulated in vivo. Since AChE and BChE can associate with PRiMA, a membrane anchor, we explored the pattern of expression of PRiMA in vitro and in vivo. The level of PRiMA transcripts is downregulated in the absence of ColQ. Therefore, AChE, BChE and PRiMA mRNA level modifications found in the absence of ColQ cannot compensate for the physiological defects observed at the ColQ-deficient NMJs.

ColQ controls postsynaptic differentiation at the neuromuscular junction.

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.

Identification of cis-acting elements involved in acetylcholinesterase RNA alternative splicing.

The 3' end of Acetylcholinesterase (AChE) pre-mRNA is processed by a complex mechanism of alternative splicing. Three different transcripts are generated and called R, H and T according respectively to the intron (intron 4') or exons (5 or 6) retained in the mature RNA. The relative expression of the specific transcripts depends on cell type, developmental stage or pathophysiological conditions. The aim of our study was to identify sequences involved in AChE pre-mRNA splicing choices. For this purpose, we constructed a minigene in which the constitutive exons were fused and followed by the entire alternative domain without 3' UTR. We transfected the wild-type or minigene mutated in the alternative domain in muscle or COS-7 cells and identified the splicing products by RPA, RT-PCR and sedimentation coefficients of the enzymatic molecular forms. We find that the alternative splicing domain contains most of the necessary signals to control splicing choices in skeletal muscle cells with the coding sequences of the domain having little effect on the splicing outcome. A branch point at an unusual location 278 nt from the 3' acceptor site of exon 6 is characterized. We further identify several regulatory sequences in the non-coding sequence of exon 5 that regulate the splicing pattern. Sequences that control the splice to exon 5 and those which influence intron 4' retention or splicing to exon 6 appear to be distinct.