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Role of ColQ, a specific collagen in the functionnal organisation of the neuromuscular junction

Final Report Summary - COLQ AND THE NMJ (Role of ColQ, a specific collagen in the functionnal organisation of the neuromuscular junction)

Project context and objectives

At the neuromuscular junction (NMJ), the release of acetylcholine (ACh) induces the opening of the acetylcholine receptor (AChR) and the depolarisation of the postsynaptic membrane that activates voltage-gated Na+ channels. As a result, an action potential is produced that triggers muscle contraction. Normal neural transmission requires apposed and highly differentiated domains of the nerve, the muscle and the Schwann cell at the synapse. In these domains, many proteins are accumulated through molecular scaffolds to ensure the physiological process. One of the major specificities of this synapse is the existence of a large synaptic space called the basal lamina. This space is highly organised and contains specific molecules which are absent or expressed at much lower levels in extrasynaptic regions. How do the proteins contained in this synaptic basal lamina participate in the formation and the plasticity of the synapse? A first clue for this process comes from the demonstration of direct interactions between proteins from the basal lamina and pre- and/or postsynaptic proteins. Moreover, the studies of myasthenic patients bearing mutations in some of these molecules of the synaptic basal lamina or animal models for these pathologies have shown disorganisation of the pre- and/or postsynaptic elements. However, it remains to discover the mechanisms used by these molecules to control the functional organisation of pre- and postsynaptic domains.

Prof Legay's group has worked extensively on ColQ, a specific collagen that anchors acetylcholinesterase (AChE) in the synaptic cleft. The function of ColQ is to tether AChE in the basal lamina. ColQ binds the muscle-specific kinase (MuSK) through its C-terminus peptide. MuSK, a central player in the formation of the NMJ, is a tyrosine kinase receptor activated by the nerve neurotrophic factor agrin. This activation induces several signal transduction pathways that lead to AChR aggregation and stabilisation, as well as an increase in synaptic genes expression. Since MuSK is only expressed on postsynaptic membranes, this interaction dictates the synaptic localisation of AChE-ColQ hetero-oligomers.

The goal of my research project was to show that ColQ interactions in the basal lamina control the postsynaptic organisation independently of AChE. We have demonstrated that ColQ, through MuSK interaction, regulates not only synaptic gene expression but also AChR clustering at the NMJ. In addition, we uncovered a new role for the protein named Dcx as a microtubule-binding molecule that is involved in the formation of NMJ.

This study has shed light on the molecular mechanisms involved in the formation of the NMJ and has contributed to a better understanding of the specific traits of myasthenic syndromes. Most of the results obtained here have been published.

Project objectives

Four main objectives have been defined in this project:

1. to confirm the microarray data;
2. to discover which pathway is driving ColQ effect on AChR;
3. to identify the ColQ domain of interaction on MuSK;
4. to determine the function of Doublecortin, a new player in the muscle.

Work performed

Confirmation of the microarray data

In order to investigate the role of ColQ on postsynaptic gene expression, Prof. Legay used a global genomic approach to compare gene expression in two muscle cell lines created by her group: wild-type (wt) and ColQ deficient muscle cell lines. They used the technique of the microarrays in collaboration with Jean Léger (Transcriptome facilities, INSERM U533, Nantes). The effect of the absence of ColQ on muscle differentiation was tested at three different stages of muscle cell differentiation using mouse pangenomic chips probed with 33 000 genes (Applied Biosystems). The different time points that were examined (T1, T2 and T3) were chosen according to results previously obtained by the group. T1 is the time at which all the myoblasts are fused into myotubes; T2 represents the time at which AChR clusters are detected (three days after T1); T3 corresponds to the first visualisation of AChE clusters in wt cells and the beginning of cell contraction (average six days after T2). It should be noted that T2 is the time of muscle cell differentiation that is mostly used for the studies of AChR expression in the literature. A large number of genes were differentially expressed between the two cell lines, mainly at T1 between the two cell lines (604 genes) and during differentiation of ColQ deficient cells (1 508 genes). A global analysis of the classes of genes differentially expressed during wt muscle cell differentiation and between wt and ColQ deficient cells at the three time points has been realised.

In order to confirm the data obtained with the microarrays, a subset of differentially expressed genes revealed by the microarrays has been quantified by real-time polymerase chain reaction (PCR) using a system that allows large-scale real-time PCR and which is commercialised by Applied Biosystems (Taqman Low Density Arrays). We selected around 100 genes that fall into three classes: synaptic genes, cytoskeletal genes and signalling genes. The quantification was made from the same batches of Messenger Ribonucleic Acids (mRNAs) used for the microarrays. The results show that 80% of the genes differentially expressed by microarrays are also found to be regulated the same way by real-time PCR.

We also started to confirm these results in vivo using quantitative reverse transcription (RT)-PCR in muscle from mice that are deficient in the ColQ gene at two embryonic stages (E14 corresponding to T2 and E18 corresponding to T3) and after birth (P7 and P30). However, in contrast to our in vitro data, the results obtained from RT-PCR analysis of gene expression after birth (7 and 30 days) revealed that structural and extracellular matrix genes were not differentially expressed compared to wt, which suggests that compensatory mechanisms are in place after birth. In order to look at the NMJ phenotype and muscle structure during development, we started to analyse ColQ-deficient embryos compared to wt. Embryonic diaphragm muscles were dissected and stained with neurofilament and synaptophysin antibodies, together with alpha bungarotoxin to visualise nerve and AChR clusters respectively. This study is still ongoing, but preliminary data obtained at stage E18 indicate that the loss of ColQ in the embryos leads to severe defects of NMJ innervation (i.e. a decrease in the number and size of AChR clusters and a disorganised motor nerve terminal).

All these results, together with others, are included in an article in preparation, entitled 'Molecular and phenotypical analysis of ColQ deficient muscles in a model of myasthenic syndroms' (Sigoillot et al.,).

Study of the ColQ/perlecan/dystroglycan signalling pathway

Interestingly, the results obtained from in vitro RT-PCR analysis showed that the loss of ColQ function leads to drastic down-regulation of extracellular matrix genes. With ColQ being an extracellular matrix protein interacting indirectly with dystroglycan, we asked whether a loss of ColQ function would induce defects in the membrane cytoskeleton proteins, including dystrophin complex. In cultured muscle cells in vitro, ColQ deficiency leads to a 20 % decrease in the expression of dystroglycan protein labelled with antibodies at T2 and T3 compared to wt. However, no difference in dystroglycan expression could be detected in vivo (P7 and P30) compared to wt. In fact, we realised that most of the genes that were differentially expressed in ColQ deficient muscle compared to wt muscle in vivo were synaptic genes. The most striking observation concerned the genes involved in the MuSK pathway: AChE, AChR, MuSK and LRP4. All these genes were up-regulated in ColQ deficient muscle, both in vitro and in vivo (P30 muscles), suggesting that ColQ regulates this pathway.

Study of the ColQ/MuSK signalling pathway

We previously showed that AChR clusters are smaller and more densely packed in the absence of ColQ, both in vitro and in vivo. In order to investigate whether ColQ effects on AChR clustering are mediated by the MuSK signalling pathway, we analysed MuSK, rapsyn and AChR subunit genes and protein expressions, both in vitro in ColQ deficient muscle cells and in vivo in ColQ mutant muscles. Using quantitative RT-PCR and antibody staining, we found that most AChRs subunits and rapsyn mRNA levels and proteins were increased, both in ColQ deficient muscle cells in culture and in muscles in vivo. Surprisingly, MuSK mRNA levels and protein are increased in cultured cells but not in muscles lacking ColQ. We demonstrated that membrane-bound MuSK is decreased both in vitro and in vivo suggesting that ColQ not only controls synaptic gene expression but also MuSK sorting or stabilisation in the muscle membrane. Membrane-bound MuSK defect observed in the absence of ColQ may result in an alteration of agrin/MuSK signalling pathway leading to AChR clustering defects. Indeed, we show that agrin-induced AChR clustering is three times less effective in ColQ-deficient muscle cells in culture when compared to the control.

Since ColQ binds MuSK, which is involved in the control of gene expression at the synapse, we asked whether the effects of ColQ on synaptic mRNA levels are mediated by ColQ-MuSK interaction. We compared the mRNA levels of AChR a- and b-subunits in untransfected ColQ-/- cells and cells transfected with ColQ or a ColQ construct lacking the C-terminus domain or point mutated in the C-terminus, both of which impair ColQ-MuSK association. GFP-ColQ restoration in ColQ-/- cells led to a significant decrease in AChR subunit mRNA levels compared to untransfected cells. Importantly, impairing ColQ interaction with MuSK abolished the effect of ColQ on AChR subunit mRNA levels, thus indicating that ColQ-MuSK interaction is not only involved in MuSK sorting or stabilisation in the muscle membrane but also in the regulation of AChR expression via MuSK. Taken together, these results highlight a new role for ColQ in regulating AChR aggregation and transcription at the NMJ.

All these results are included in an article that was published in the Journal of Neuroscience (Sigoillot et al., 2010).

Identification of ColQ domain of interaction on MuSK
We obtained all the MuSK-tagged constructs deleted from the various extracellular domains. Preliminary results of immunoprecipitation of these MuSK constructs together with a ColQ-GFP construct in COS cells indicate that ColQ binds to more than one MuSK extracellular domains. These results need to be confirmed; however, we plan to perform a point-mutated MuSK construct within the extracellular domains of interest to fully clarify the binding between MuSK and ColQ.

The function of Doublecortin, a new player in the muscle

The cytoskeleton plays a vital role in NMJ formation but very little is known about the function of actin and microtubules in organising the postsynaptic density. AChRs associate with the actin cytoskeleton but how AChRs are tethered to the actin cytoskeleton remains unclear. Sparse data has reported an association between synaptic proteins and microtubules. Results from Prof. Legay's group suggest that Doublecortin (Dcx), a microtubule-associated protein in migrating neurons, is expressed in myotubes and is down-regulated in the absence of ColQ. Mutations in the Dcx gene lead to X-linked lissencephaly in humans. In these patients, neurons that migrated aberrantly are deposited in a broad band in subcortical layers and thus may cause severe mental retardation, seizures and decreased lifespan in affected individuals. We have started to investigate the function of Dcx in muscle. Results from the microarray screen in muscle cells in culture revealed that Dcx mRNA is regulated during muscle cell differentiation, being up-regulated during the transition T1 to T2 and down-regulated as the differentiation proceeds. Western blot analysis in muscle cells in culture using Dcx antibodies confirmed that the protein was expressed at the three muscle-differentiation stages (T1, T2 and T3) and up-regulated during the transition T1 to T2. Quantitative RT-PCR experiments performed in muscles from stage E14 and E18 mice embryos further showed that Dcx is expressed by muscle. The most straightforward strategy to understand the function of Dcx in muscle is to identify its partners. To identify direct partners of Dcx in muscle, I have immunoprecipitated Dcx from myotubes and, in collaboration with the group of Dr Rossier (ESPCI, France), analysed it using mass spectrometry on the proteins found in the immunoprecipitate. We have identified the protein called MAB1 (microtubule associated protein) as a potential partner of doublecortin and we are now exploring the functional role of this protein at the neuromuscular junction using in vivo knock-out mice and in vitro muscle cell cultures. In addition, Dcx is known to be essential for proper neurogenesis, neuronal migration and axonal wiring. Using my expertise in axon guidance, I have investigated the possibility that Dcx might play a role in motoneuron axon guidance and in the initial steps of the NMJ formation. We obtained from J. Chelly and F. Francis (UMR8104) Dcx-deficient mutant mice. An analysis of a NMJ phenotype of a diaphragm from E18-stage Dcx mutant embryos was performed using neurofilament and synaptophysin, together with alpha bungarotoxin to visualise nerve terminals and AChR clusters respectively. A 25 % increase in the endplate bandwidth was found in Dcx mutant compared to wt. In addition, the nerve terminal bypassed AChR clusters and grew aberrantly all over the muscle, indicating that the innervation pattern is affected in Dcx mutants. Interestingly, electron microscopy analysis of the Dcx mutant NMJ morphology revealed no defects in the muscle structure, which suggests that Dcx is required for the first step of NJ formation without affecting the proper muscle morphology. Taken together, these results demonstrate that Dcx is involved in NMJ formation and might play a role in vesicular trafficking by interacting with microtubule binding proteins. Further mechanistic studies are being realised in the laboratory to further elucidate a Dcx mode of action in muscle (for example, the use of drugs disturbing the microtubule network in muscle cells in vitro and in vivo in comparison with the NMJ phenotype of Dcx and of Tuba1 (tubulin) mutant mice embryos.

All these results are part of an article in preparation (Bourgeois et al., 2012).

The reintegration grant has allowed me to return to France after a postdoctoral position in the United Kingdom. The project subject was selected with a view to complement and diversify my scientific training. The goal for me was to integrate into the CNRS or INSERM research institutes as an independent scientist. This was achieved in 2010 and I am now an independent research scientist working (permanent position) for the INSERM institute. In parallel to this study, I have developed my own research project based on a new family of axon guidance molecules: the role of Wnt proteins during the formation of NMJ. Using my expertise in axon guidance and in the NMJ field, I developed new tools in the laboratory to unravel the role of new players in the early steps of NMJ formation, including in vivo analysis of mutant mice embryos and axon guidance assays. I developed several collaborations to extend my expertise (L. Schaeffer, Lyon; V. Castellani, Lyon, A. Swain, London, L. Legres, France, for example). This has led to a recent publication in PloS One (Strochlic et al., 2012). I have been awarded a Trampoline Grant from the French Association contre les myopathies (AFM). I am also supervising two doctoral students, S. Sigoillot, who finished her PhD in 2010 and is now starting a postdoctoral position in the College de France (Paris), and J. Messeant, who started his PhD in 2011 on the role of Wnt signalling in NMJ formation. I am also part of the imaging committee in charge of running the University Paris Descartes imaging facility and I am participating in teaching courses for Masters students (Neuroscience).