To create functional neuronal circuit units during nervous system development and/or regeneration, axons are subjected to guidance signals. Expression of these signals occurs in spatiotemporal variations and is translated by the growth cone into a pathway to reach the connecting target which can be a neuron or a non-neuronal cell such as a muscle cell. This path is generated by interactions with the surrounding environment such as cells or the extracellular matrix, a complex molecular substrate. Understanding the interactions with this last component is essential to stimulate nerve regeneration in the context of motor peripheral nerve trauma, the most common source of disabilities, increasing with aging. The goal is to mimic its composition and specific characteristics using innovative biomaterials and/or implants. This review highlights some aspects of the recent findings in nerve repair. After an introduction to the peripheral nervous system, we present an overview of nerve degeneration and regeneration mechanisms before detailing the strategies used nowadays to optimize nerve (re)growth with a specific focus on the use of electric field. We discuss the advantages and limits of each option in terms of therapeutic applications.

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.

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.

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.

Ultrafast acoustic imaging experiments are a powerful tool to investigate, at the nanometer scale, cell mechanical properties such as stiffness, viscosity, and adhesion, properties that play some roles in the life and death of cells. However, due to cell complex structures, the ultrafast acoustic signal analysis is quite intricate and depends on multiple parameters. Complex data analysis with poorly known parameters can be handled by a data clustering method as already shown in particle physics and biology. In this work, ultrafast acoustic data analysis is tackled by a spectral clustering method followed by a hierarchical agglomerating method. Coupled to conventional microscopy performed on the very same cell, the clustered data can be assigned to inner-cell features such as the nucleus, the cytoplasm, and the cytoskeleton. The signal dependency on the cell thickness and stiffness is highlighted. Moreover, thanks to the improvement of the signal-to-noise ratio, the nature of the adhesion is also assessed through observation and characterization of a polymerlike layer as thin as a few nanometers.

Neuronal repair is promoted after a nerve injury by chemical and physical stimuli from their environment. Among them, local adhesion energy gradients generated on surfaces trigger cone formation and neurite outgrowth without any nerve growth factor (NGF) addition. In this study, the molecular mechanisms leading to neuronal differentiation after stimulation via energy gradients are investigated. For this purpose, PC12 cells are cultured on n ‐[3‐(trimethoxysilyl)propyl] ethylendiamine (EDA) and n ‐hexyl trimethoxysilane (HTMS), two functionalized surfaces possessing local energy gradients but chemically different. These surfaces similarly trigger neurite and growth cone formation after 3 days in culture. Gene expression of PI3/Akt quantified through a microarray and the action of a specific inhibitor of the corresponding pathway reveal that PI3/Akt signaling pathway is induced and activated on these surfaces in the same way as NGF does after binding on its TrkA receptor. The biological downstream effectors of this signaling pathway are also upregulated, thereby promoting cell survival, growth cone formation, and neurite outgrowth. EDA and HTMS surfaces act as an ongoing stimulus of the TrkA receptor as the addition of 100 nM K252a, its specific inhibitor, leads to the inhibition of neurite growth. Taken together, these results suggest that functionalized surface with energy surface gradients could be useful to promote nerve repair after an injury.

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.

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 β-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 β-subunit transcripts. This HuR/AChR transcript interaction induces AChR β-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 β-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.

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.

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 (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.

Perturbation of tissue rigidity is associated with different types of pathology. However, it is sometimes difficult to conclude if this change in stiffness of cells or tissue is the effect or the source of the pathologies. This is why the characterization of the mechanical properties of cells is essential to understand their behavior during mitosis, apoptosis, adhesion, mobility and disease development. However, the complexity of the inner cell composition and the intricate meshwork formed by molecular mediators of the transmembrane cell-substrate interactions requires non-invasive techniques to probe and quantify local mechanical properties of cells, including modulus of elasticity, viscoelastic properties, adhesion, and forces created at the single-cell scale. Several recent reviews describe tools used to study cell mechanics and to apply forces on them. Acoustic waves generated by lasers provide a very adequate tool for probing the mechanical properties of biological cells or tissues in a non-contact, non-invasive configuration. We report on detection of Brillouin (BO) signature used for mapping elastic properties in single neuron-like cells. To our knowledge, this is the first time this technique is used on this type of cells, in which the regeneration processes are closely related to cell elasticity. The elasticity contrasts thus revealed between the nucleus and the cytoskeleton in the reticulate cells also obviously exist in living cells for which the study is more subtle given the very specific conditions necessary for their maintenance . The frequencies and the lifetimes of the BOs are mapped across the cell using Pearson correlation method. The influence of hydration, i.e. a more biologically relevant conditions, on the cell elasticity is investigated. Finally a correlative microscopy coupling elastic mapping, atomic force microscopy and fluorescence will be presented in motoneuron single cells.

We report on elasticity measurements in neuronlike cells using picosecond acoustics pump and probe spectroscopy. The stimulated Brillouin oscillations were mapped in PC12 cells to reveal their internal elastic structure. Thanks to a Pearson correlation coefficient mapping, different areas could be distinguished. The nucleus material shows a bulk modulus equal to 12.9 GPa in the case of a dry cell. Attenuation of the Brillouin signature gives access to dynamical longitudinal viscosity equal to 10.6 mPa ⋅ s, one order of magnitude higher than that of water. The modulus considerably drops to 2.6 GPa in the most physiologically relevant case of a hydrated cell.

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.

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.

Understanding the developmental steps shaping the formation of the neuromuscular junction (NMJ) connecting motoneurons to skeletal muscle fibers, is critical. Wnt morphogens are key players in the formation of this specialized peripheral synapse. Yet, the individual and collaborative functions of Wnts as well as their downstream pathways remain poorly understood at the NMJ. Here, we demonstrate through Wnt4 and Wnt11 gain of function studies in culture or in mice that Wnts enhance acetylcholine receptor (AChR) clustering and motor axon outgrowth. In contrast, loss of Wnt11 or Wnt-dependent signaling in vivo decreases AChR clustering and motor nerve terminal branching. Both Wnt4 and Wnt11 stimulate AChR clustering and mRNA downstream activation of the β-catenin pathway. Strikingly, Wnt4 and Wnt11 co-immunoprecipitate with Vangl2, a core component of the Planar Cell Polarity (PCP) pathway, which accumulates at embryonic NMJ. Moreover, mice bearing a Vangl2 loss of function mutation (looptail) exhibit a decreased number of AChR clusters and overgrowth of motor axons bypassing AChR clusters. Taken together, our results provide genetic and biochemical evidences that Wnt4 and Wnt11 cooperatively contribute to mammalian NMJ formation through activation of both the canonical and Vangl2-dependent core PCP pathways.

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.

Mutations in the microtubule-associated protein doublecortin (DCX) cause type I (X-linked or XLIS) lissencephaly in hemizygous males and subcortical band heterotopia (SBH) in females, with defects in neuron migration during development affecting cortical lamination. We found that besides its well-established expression in migrating neurons of the brain, doublecortin (Dcx in mice) is also expressed in motor neurons and skeletal muscle in embryonic neuromuscular junctions (NMJs), raising the possibility of a role in synaptogenesis. Studies with whole-mount preparations of embryonic mouse diaphragm revealed that loss of Dcx leads to abnormal presynaptic arborization and a significantly increased incidence of short axonal extensions beyond innervated acetylcholine receptor (AChR) clusters in the developing NMJ. This phenotype, albeit relatively mild, suggests that Dcx contributes to a stop/stabilizing signal at the synapse, which normally limits further axonal growth following establishment of synaptic contact with the postsynaptic element. Importantly, we also identified abnormal and denervated NMJs in a muscle biopsy from a 16-year-old female patient with SBH, showing both profound presynaptic and postsynaptic morphological defects. Overall, these combined results point to a critical role of doublecortin in the formation of the NMJ.