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.

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.

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.

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.

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.

Analysis of combinatorial variability reveals selective accumulation of the fibronectin type III domains B and D of tenascin-C in injured brain.

Tenascin-C (Tnc) is a multimodular extracellular matrix glycoprotein that is markedly upregulated in CNS injuries where it is primarily secreted by reactive astrocytes. Different Tnc isoforms can be generated by the insertion of variable combinations of one to seven (in rats) alternatively spliced distinct fibronectin type III (FnIII) domains to the smallest variant. Each spliced FnIII repeat mediates specific actions on neurite outgrowth, neuron migration or adhesion. Hence, different Tnc isoforms might differentially influence CNS repair. We explored the expression pattern of Tnc variants after cortical lesions and after treatment of astrocytes with various cytokines. Using RT-PCR, we observed a strong upregulation of Tnc transcripts containing the spliced FnIII domains B or D in injured tissue at 2-4 days post-lesion (dpl). Looking at specific combinations, we showed a dramatic increase of Tnc isoforms harboring the neurite outgrowth-promoting BD repeat with both the B and D domains being adjacent to each other. Isoforms containing only the axon growth-stimulating spliced domain D were also dramatically enhanced after injury. Injury-induced increase of Tnc proteins comprising the domain D was confirmed by Western Blotting and immunostaining of cortical lesions. In contrast, the FnIII modules C and AD1 were weakly modulated after injury. The growth cone repulsive A1A2A4 domains were poorly expressed in normal and injured tissue but the smallest isoform, which is also repellant, was highly expressed after injury. Expression of the shortest Tnc isoform and of variants containing B, D or BD, was strongly upregulated in cultured astrocytes after TGFbeta1 treatment, suggesting that TGFbeta1 could mediate, at least in part, the injury-induced upregulation of these isoforms. We identified complex injury-induced differential regulations of Tnc isoforms that may well influence axonal regeneration and repair processes in the damaged CNS.

Contributions of selective knockout studies to understanding cholinesterase disposition and function.

The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.

Targeting of acetylcholinesterase in neurons in vivo: a dual processing function for the proline-rich membrane anchor subunit and the attachment domain on the catalytic subunit.

Acetylcholinesterase (AChE) accumulates on axonal varicosities and is primarily found as tetramers associated with a proline-rich membrane anchor (PRiMA). PRiMA is a small transmembrane protein that efficiently transforms secreted AChE to an enzyme anchored on the outer cell surface. Surprisingly, in the striatum of the PRiMA knock-out mouse, despite a normal level of AChE mRNA, we find only 2-3% of wild type AChE activity, with the residual AChE localized in the endoplasmic reticulum, demonstrating that PRiMA in vivo is necessary for intracellular processing of AChE in neurons. Moreover, deletion of the retention signal of the AChE catalytic subunit in mice, which is the domain of interaction with PRiMA, does not restore AChE activity in the striatum, establishing that PRiMA is necessary to target and/or to stabilize nascent AChE in neurons. These unexpected findings open new avenues to modulating AChE activity and its distribution in CNS disorders.

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.

Evidence for distinct leptomeningeal cell-dependent paracrine and EGF-linked autocrine regulatory pathways for suppression of fibrillar collagens in astrocytes.

A unique and unresolved property of the central nervous system is that its extracellular matrix lacks fibrillar elements. In the present report, we show that astrocytes secrete triple helices of fibrillar collagens type I, III and V in culture, while no astroglial collagen expression could be detected in vivo. We discovered two inhibitory mechanisms that could underlie this apparent discrepancy. Thus, we uncover a strong inhibitory effect of meningeal cells on astrocytic collagen expression in coculture assays. Furthermore, we present evidence that EGF-receptor activation downregulates collagen expression in astrocytes via an autocrine loop. These investigations provide a rational framework to explain why the brain is devoid of collagen fibers, which is a unique feature that characterizes the structure of the neural extracellular matrix. Moreover, fibrillar collagens were found transiently upregulated in a laser-induced cortical lesion, suggesting that these could contribute to the glial scar that inhibits axonal regeneration.

DSD-1-Proteoglycan/Phosphacan and receptor protein tyrosine phosphatase-beta isoforms during development and regeneration of neural tissues.

Interactions between neurons and glial cells play important roles in regulating key events of development and regeneration of the CNS. Thus, migrating neurons are partly guided by radial glia to their target, and glial scaffolds direct the growth and directional choice of advancing axons, e.g., at the midline. In the adult, reactive astrocytes and myelin components play a pivotal role in the inhibition of regeneration. The past years have shown that astrocytic functions are mediated on the molecular level by extracellular matrix components, which include various glycoproteins and proteoglycans. One important, developmentally regulated chondroitin sulfate proteoglycan is DSD-1-PG/phosphacan, a glial derived proteoglycan which represents a splice variant of the receptor protein tyrosine phosphatase (RPTP)-beta (also known as PTP-zeta). Current evidence suggests that this proteoglycan influences axon growth in development and regeneration, displaying inhibitory or stimulatory effects dependent on the mode of presentation, and the neuronal lineage. These effects seem to be mediated by neuronal receptors of the Ig-CAM superfamily.

Cholinesterases and the resistance of the mouse diaphragm to the effect of tubocurarine.

BACKGROUND: The diaphragm is resistant to competitive neuromuscular blocking agents. Because of the competitive mechanism of action of tubocurarine, the rate of hydrolysis of acetylcholine at the neuromuscular junction may modulate its neuromuscular blocking effect. The authors compared the neuromuscular blocking effect of tubocurarine on isolated diaphragm and extensor digitorum longus (EDL) muscles and quantified the acetylcholinesterase activity in hetero-oligomers. METHODS: Adult Swiss-Webster and collagen Q-deficient (ColQ) mice were used. The blocking effect of tubocurarine on nerve-evoked muscle twitches was determined in isolated diaphragm and EDL muscles, after inhibition of acetylcholinesterase by fasciculin-1, butyrylcholinesterase by tetraisopropylpyro-phosphoramide, or both acetylcholinesterase and butyrylcholinesterase by neostigmine, and in acetylcholinesterase-deficient ColQ muscles. The different acetylcholinesterase oligomers extracted from diaphragm and EDL muscles were quantified in sucrose gradient. RESULTS: The EC50 for tubocurarine to decrease the nerve-evoked twitch response was four times higher in the diaphragm than in the EDL. The activity of the different acetylcholinesterase oligomers was lower in the diaphragm as compared with the EDL. Inhibition of acetylcholinesterase by antagonists resulted in an increased dose of tubocurarine but an unchanged resistance ratio between the diaphragm and the EDL. A similar diaphragmatic resistance was found in ColQ muscles. CONCLUSION: The current study indicates that, despite differences in acetylcholinesterase activity between the diaphragm and EDL, the diaphragmatic resistance to tubocurarine cannot be explained by the different rate of acetylcholine hydrolysis in the synaptic cleft.

The extracellular matrix glycoprotein Tenascin-C is expressed by oligodendrocyte precursor cells and required for the regulation of maturation rate, survival and responsiveness to platelet-derived growth factor.

Analysis of Tenascin-C (TN-C) knockout mice revealed novel roles for this extracellular matrix (ECM) protein in regulation of the developmental programme of oligodendrocyte precursor cells (OPCs), their maturation into myelinating oligodendrocytes and sensitivity to growth factors. A major component of the ECM of developing nervous tissue, TN-C was expressed in zones of proliferation, migration and morphogenesis. Examination of TN-C knockout mice showed roles for TN-C in control of OPC proliferation and migration towards zones of myelination [E. Garcion et al. (2001) Development, 128, 2485-2496]. Extending our studies of TN-C effects on OPC development we found that OPCs can endogenously express TN-C protein. This expression covered the whole range of possible TN-C isoforms and could be strongly up-regulated by leukaemia inhibitory factor and ciliary neurotrophic factor, cytokines known to modulate OPC proliferation and survival. Comparative analysis of TN-C knockout OPCs with wild-type OPCs reveals an accelerated rate of maturation in the absence of TN-C, with earlier morphological differentiation and precocious expression of myelin basic protein. TN-C knockout OPCs plated on poly-lysine displayed higher levels of apoptosis than wild-type OPCs and there was also an earlier loss of responsiveness to the protective effects of platelet-derived growth factor (PDGF), indicating that TN-C has anti-apoptotic effects that may be associated with PDGF signalling. The existence of mechanisms to compensate for the absence of TN-C in the knockout is indicated by the development of oligodendrocytes derived from TN-C knockout neurospheres. These were present in equivalent proportions to those found in wild-type neurospheres but displayed enhanced myelin membrane formation.

Regulation of RPTPbeta/phosphacan expression and glycosaminoglycan epitopes in injured brain and cytokine-treated glia.

Several chondroitin sulfate proteoglycans (CSPGs) are upregulated after CNS injury and are thought to limit axonal regeneration in the adult mammalian CNS. Therefore, we examined the expression of the CSPG, receptor protein tyrosine phosphatase beta (RPTPbeta)/phosphacan, after a knife lesion to the cerebral cortex and after treatment of glial cultures with regulatory factors. The three splice variants of this CSPG gene, the secreted isoform, phosphacan, and the two transmembrane isoforms, the long and short RPTPbeta, were examined. Western blot and immunostaining analysis of injured and uninjured tissue revealed a transient decrease of phosphacan protein levels, but not of short RPTPbeta, in the injured tissue from 1 to 7 days postlesion (dpl). By real time RT-PCR, we show that phosphacan and long RPTPbeta mRNA levels are transiently down-regulated at 2 dpl, unlike those of short RPTPbeta which increased after 4 dpl. In contrast to the core glycoprotein, the phosphacan chondroitin sulfate (CS) glycosaminoglycan epitope DSD-1 was up-regulated after 7 dpl. Phosphacan was expressed by cultivated astrocytes and oligodendrocyte precursors but was more glycanated in oligodendrocyte precursors, which produce more of DSD-1 epitope than astrocytes. Epidermal growth factor/transforming growth factor alpha strongly increased the astrocytic expression of long RPTPbeta and phosphacan and slightly the short RPTPbeta protein levels, while interferon gamma and tumor necrosis factor alpha reduced astrocytic levels of phosphacan, but not of the receptor forms. Examining the effects of phosphacan on axon growth from rat E17 cortical neurons, we found that phosphacan stimulates outgrowth in a largely CS dependent manner, while it blocks the outgrowth-promoting effects of laminin through an interaction that is not affected by removal of the CS chains. These results demonstrate complex injury-induced modifications in phosphacan expression and glycanation that may well influence axonal regeneration and repair processes in the damaged CNS.