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.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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.
Myotonic dystrophy type 1 (DM1) is one of the most common hereditary muscular dystrophies in adult. In this study, we assessed the anterograde contribution of spinal motoneurons in the neuromuscular defects observed in DM1. By using micropatterned cocultures between human skeletal muscle cells and human‐induced pluripotent stem cells (hiPSC)‐spinal motoneurons derived from DM1 patients, our analysis revealed presynaptic defects including abnormal neuritogenesis, which affect the formation or stability of the neuromuscular junction. These defects can be reproduced by depleting muscleblind (MBNL) proteins whose loss‐of‐function is a key event in DM1 pathogenesis. Thus, our study highlighted the importance of considering the anterograde pathological contribution of spinal motoneurons in discovery future therapy for DM1.
Full text
Available for:
DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
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. FASEB J. 30, 2382–2399 (2016). www.fasebj.org
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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 Ca2+ 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.
► Genetic and embryologic origin of muscle fiber-type diversity remains unknown in mammals. ► We investigate the role of Six proteins in the genetic of muscle fiber-type diversity. ► We establish the gene network controlled by Six1/4 proteins in mouse E18 embryo. ► Six control crosstalk between slow and fast type determinant genes. ► Six are required for genesis of muscle fiber type diversity during embryogenesis.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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 β-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR β-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 β-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR β-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 β-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 β-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR β-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression.
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 β-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.
Acetylcholinesterase (AChE) possesses short C-terminal peptides that are not necessary for catalytic activity. These peptides belong to different classes (R, H, T, S) and define the ...post-translational processing and targeting of the enzyme. In vertebrates, subunits of type H (AChE
H) and of type T (AChE
T) are the most important: AChE
H subunits produced glycolipid (GPI)-anchored dimers and AChE
T subunits produce hetero-oligometric forms such as membrane-bound tetramer in the mammalian brain (containing a 20 kDa hydrophobic protein) and asymmetric collagen-tailed forms in neuromuscular junctions (containing a specific collagen, ColQ). The T peptide allows the formation of tetrameric assemblies with a proline-rich attachment domain (PRAD) of collagen ColQ. These complex molecular structures condition the functional localization of the enzyme in the supramolecular architecture of cholinegic synapses.
L'acétylcholinestérase (AChE) possède de courts peptides C-terminaux qui ne sont pas indispensables pour l'activité enzymatique. Ces peptides, classés en différents types (R, H, T, S), déterminent la maturation et le devenir de l'enzyme. Chez les vertébrés, les sous-unités de type H (AChE
H et de type T (AChE
T) sont les plus importantes, et produisent respectivement des dimères ancrés par un glycolipide (GPI) et des formes hétéro-oligométriques telles que les tétramères membranaires du cerveau des mammifères (comprenant une protéine hydrophobe de 20 kDa) et les formes asymétriques des jonctions neuromusculaires (comprenant un collagène spècifique, ColQ). Le peptide T permet la formation d'un assemblage tétramérique avec un domaine riche en prolines (‘proline-rich attachment domain’, PRAD) du collagène ColQ. Ces structures moléculaires complexes conditionnent la localisation fonctionnelle de l'enzyme dans l'architecture supramoléculaire des synapses cholinegiques.
Full text
Available for:
IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•Distinct branches of Wnt signaling regulate NMJ formation.•Genetic experiments support the functional role of Wnt/MuSK interaction in vivo.•A role for Wnt signaling in neuromuscular junction ...maintenance is proposed.
The development and maintenance of the neuromuscular connectivity relies on a temporally fine-tuned balance of distinct bi-directional communication between motor neurons and their muscle targets. Disruption of this communication leads to functional and structural defects that affect the motor function and causes severe neuromuscular pathologies. A limited number of secreted molecules exchanged across the synaptic cleft are critical for the development and maintenance of the nerve/muscle contact. Among those factors, members of the large family of Wnt molecules, best known for their role in numerous developmental processes have emerged as complex key players at the neuromuscular junction (NMJ), being possibly involved in multiple aspects of synapse positioning, differentiation and maintenance. Yet, their specific mode of action and downstream signaling in vivo remain controversial in mammals creating a certain degree of confusion as to the mechanistic frame of Wnt-elicited function at the NMJ. Recent advances in understanding the role of the Wnt signaling network during mammalian NMJ formation and maintenance is reviewed here.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
We studied the splicing and compartmentalization of acetylcholinesterase (AchE) mRNAs during muscle differentiation in the mouse, both in vitro and in vivo. We used the polymerase chain reaction ...(PCR) to analyse AChE mRNAs in cultures of the myogenic C2 and Sol8 cell lines, and in the developing diaphragm, from embryonic day 14 (E14). We characterized three types of alternatively spliced AChE mRNAs, encoding catalytic subunits that differ by their C-terminal regions (R, H and T). The T transcript is predominant in all cases and represents the only AChE mRNA in the adult muscle. We detected the presence of the minor R and H transcripts in the myogenic cell lines, both as myoblasts and differentiated myotubes, and also in the diaphragm from E14 until birth. At E14 the R transcript represents approximately 1% of AChE mRNA and the level of the H transcript is still lower. By in situ hybridization, we found that the T AChE mRNAs begin to preferentially accumulate at the level of the first neuromuscular contacts in the mouse diaphragm and other muscles as early as E14, e.g. concomitantly with mRNAs encoding the receptor subunits. This suggests that a common control mechanism ensures the synaptic focalization of mRNAs encoding the cholinergic proteins AChE and acetylcholine receptor during muscle development.
We report the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two ...heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) ε-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.
PDF
