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.

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.

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.

Molecular cloning and expression pattern of the Fkbp25 gene during cerebral cortical neurogenesis.

Neocortical neurons are generated predominantly from the cells that proliferate in the ventricular zone of the telencephalon. In order to understand the nature of these expanding cortical neuronal progenitor cells, we selected by differential display some transcripts that were enriched in the telencephalon as compared to the more caudal regions (diencephalon/mesencephalon). This systematic screening revealed one of the differentially expressed transcripts, namely the Fkbp25 mRNA that encodes a member of the FK506 binding proteins (FKBPs). Northern blot analysis showed that the expression of the single 1.4kb Fkbp25 transcript reached a maximum level on embryonic day 11.5 at the start of cortical neurogenesis in the mouse and was followed by a weak basal expression in the adult brain. In the embryo, Fkbp25 gene was strongly expressed in the telencephalon ventricular zone but also in areas active in myogenesis (walls of the ventricle and the atrium) and chondrogenesis (the cartilage of the rib and the hindlimb). An increase in the transcript levels of the Fkbp25 gene was also observed during the two successive proliferation waves of the cerebellum development. Immunostaining on primary cultures of embryonic day 10.5 telencephalon stem cells showed that the Fkbp25 protein was present in the cytoplasm and nuclei of cells cultured for 6h but exclusively in the nuclei of the Tuj-1 immunoreactive neurons obtained after 3 days of culture (The sequence data reported here have been submitted to GenBank under accession no. AF135595.).

Identification of a novel brain-specific and Reelin-regulated gene that encodes a protein colocalized with synapsin.

We carried out a screening of genes that are differentially expressed in normal mice and reeler mutants and are characterized by abnormal neuronal migration and neurite deployment due to defective Reelin signalling. A novel gene, provisionally named C61, was overexpressed in Reelin-deficient embryonic mouse brain RNA. C61 encodes a 3.7 kb mRNA that is brain specific and developmentally regulated, with predominant expression in differentiating neurons. The predicted protein is 664 amino acids long, and contains LAG1 and Ezrin/Radixin/Moesin-Myosin-Filament motifs, suggesting that it may function as an intracellular adaptor. From E14.5 to birth, C61 was highly expressed in all neuronal differentiation fields, with the highest signal in the telencephalic cortical plate and mitral cells in the olfactory bulb. When expressed as a GFP fusion protein in transfected non-neuronal cells and primary neurons, this protein localizes, respectively, to the nuclear membrane or axonal outgrowths, indicating a function in axonal traffic or signalling.