Neuromuscular Junction Assembly Research Articles

Dpr10 and Nocte are required for Drosophila motor axon pathfinding

The paths axons travel to reach their targets and the subsequent synaptic connections they form are highly stereotyped. How cell surface proteins (CSPs) mediate these processes is not completely understood. The Drosophila neuromuscular junction (NMJ) is an ideal system to study how pathfinding and target specificity are accomplished, as the axon trajectories and innervation patterns are known and easily visualized. Dpr10 is a CSP required for synaptic partner choice in the neuromuscular and visual circuits and for axon pathfinding in olfactory neuron organization. In this study, we show that Dpr10 is also required for motor axon pathfinding. To uncover how Dpr10 mediates this process, we used immunoprecipitation followed by mass spectrometry to identify Dpr10 associated proteins. One of these, Nocte, is an unstructured, intracellular protein implicated in circadian rhythm entrainment. We mapped nocte expression in larvae and found it widely expressed in neurons, muscles, and glia. Cell-specific knockdown suggests nocte is required presynaptically to mediate motor axon pathfinding. Additionally, we found that nocte and dpr10 genetically interact to control NMJ assembly, suggesting that they function in the same molecular pathway. Overall, these data reveal novel roles for Dpr10 and its newly identified interactor, Nocte, in motor axon pathfinding and provide insight into how CSPs regulate circuit assembly.

The cell polarity protein Vangl2 in the muscle shapes the neuromuscular synapse by binding to and regulating the tyrosine kinase MuSK.

The development of the neuromuscular junction (NMJ) requires dynamic trans-synaptic coordination orchestrated by secreted factors, including Wnt family morphogens. To investigate how these synaptic cues in NMJ development are transduced, particularly in the regulation of acetylcholine receptor (AChR) accumulation in the postsynaptic membrane, we explored the function of Van Gogh-like protein 2 (Vangl2), a core component of Wnt planar cell polarity signaling. We found that conditional, muscle-specific ablation of Vangl2 in mice reproduced the NMJ differentiation defects seen in mice with global Vangl2 deletion. These alterations persisted into adulthood and led to NMJ disassembly, impaired neurotransmission, and deficits in motor function. Vangl2 and the muscle-specific receptor tyrosine kinase MuSK were functionally associated in Wnt signaling in the muscle. Vangl2 bound to and promoted the signaling activity of MuSK in response to Wnt11. The loss of Vangl2 impaired RhoA activation in cultured mouse myotubes and caused dispersed, rather than clustered, organization of AChRs at the postsynaptic or muscle cell side of NMJs in vivo. Our results identify Vangl2 as a key player of the core complex of molecules shaping neuromuscular synapses and thus shed light on the molecular mechanisms underlying NMJ assembly.

Dystrophic Muscle Affects Motoneuron Axon Outgrowth and NMJ Assembly

AbstractThe Neuromuscular Junction (NMJ) is a chemical synapse localized between the terminal branches of the spinal motor neurons and myofibers. In the past two decades coculture systems to generate the NMJ in culture are developed to address concerns about animal models, despite the complexity of its highly specialized structure makes the in vitro modeling a challenging task. Microfluidics, unlike mass cocultures, allow spatial and temporal control over different microenvironment by manipulating either neural cells or muscle cell populations independently. Therefore, exploiting an organ‐on‐a‐chip approach, the aim is to obtain a reliable and predictive in vitro human model of NMJ in physiological and pathological conditions, to investigate the occurrence of synapse detriment in α‐sarcoglycanopathy, a subtype of limb‐girdle muscular dystrophy. For this purpose, motor neurons derived from human induced pluripotent stem cells (hiPSCs) and either healthy or α‐sarcoglycan mutant human myogenic progenitors are seeded in two separated chambers of a microfluidic device. Differentiated myotubes and hiPSCs‐derived motor neurons on‐chip are able to establish points of interaction where pre‐ and postsynaptic structures colocalize. Moreover, the attraction of motor neurons axons by muscle fibers and the NMJ maturation appear to be affected by the muscular compartment, being impaired by the dystrophic cellular component.

Optogenetic modeling of human neuromuscular circuits in Duchenne muscular dystrophy with CRISPR and pharmacological corrections.

Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abnormalities contribute to DMD pathogenesis remains unclear. Here, we combine transcriptome analysis and modeling of DMD patient-derived neuromuscular circuits with CRISPR-corrected isogenic controls in compartmentalized microdevices. We show that NMJ volumes and optogenetic motor neuron–stimulated myofiber contraction are compromised in DMD neuromuscular circuits, which can be rescued by pharmacological inhibition of TGFβ signaling, an observation validated in a 96-well human neuromuscular circuit coculture assay. These beneficial effects are associated with normalization of dysregulated gene expression in DMD myogenic transcriptomes affecting NMJ assembly (e.g., MUSK) and axon guidance (e.g., SLIT2 and SLIT3). Our study provides a new human microphysiological model for investigating NMJ defects in DMD and assessing candidate drugs and suggests that enhancing neuromuscular connectivity may be an effective therapeutic strategy.

Membraneless condensates by Rapsn phase separation as a platform for neuromuscular junction formation

Our daily life depends on muscle contraction, a process that is controlled by the neuromuscular junction (NMJ). However, the mechanisms of NMJ assembly remain unclear. Here we show that Rapsn, a protein critical for NMJ formation, undergoes liquid-liquid phase separation (LLPS) and condensates into liquid-like assemblies. Such assemblies can recruit acetylcholine receptors (AChRs), cytoskeletal proteins, and signaling proteins for postsynaptic differentiation. Rapsn LLPS requires multivalent binding of tetratricopeptide repeats (TPRs) and is increased by Musk signaling. The capacity of Rapsn to condensate and co-condensate with interaction proteins is compromised by mutations of congenital myasthenic syndromes (CMSs). NMJ formation is impaired in mutant mice carrying a CMS-associated, LLPS-deficient mutation. These results reveal a critical role of Rapsn LLPS in forming a synaptic semi-membraneless compartment for NMJ formation.

A Review of the Mechanisms Modulating Neuromuscular Junction Assembly

Synaptogenesis of the neuromuscular junction (NMJ) can be divided into 3 main processes - prepatterning, patterning of the pre- and postsynaptic membranes and maturation. Prepatterning, the formation of aneural acetylcholine receptor (AChR) clusters, is mediated by Wnt 11r that is secreted from the motor neuron. Later, patterning is guided by secreted and transmembrane signalling molecules. After release into the synaptic cleft, the former become integrated into the synaptic basal lamina. They include Agrin, fibroblast growth factor (FGF) 7/10/22 and laminin β2. Agrin is responsible for the formation of AChR neural clusters in the postsynaptic membrane. FGF and laminin β2 signalling are involved in the patterning of the presynaptic membrane as they are required for the clustering of synaptic vesicles and active zone maintenance/growth, respectively. A transmembrane adhesion molecule important for NMJ synaptogenesis is neural cell adhesion molecule (NCAM). Intracellular NCAM signalling supports the accumulation of synaptic vesicle proteins at the presynaptic terminal and confines vesicle cycling to the active zones by downregulating the immature vesicle-cycling-transmitter release system. Finally, maturation processes follow, one being the elimination of inactive synapses. The cytokine tumour necrosis factor α (TNFα) has been proposed as a retrograde signal involved in this process.

Neuromuscular Junction Dismantling in Amyotrophic Lateral Sclerosis

Neuromuscular junction assembly and plasticity during embryonic, postnatal, and adult life are tightly regulated by the continuous cross-talk among motor nerve endings, muscle fibers, and glial cells. Altered communications among these components is thought to be responsible for the physiological age-related changes at this synapse and possibly for its destruction in pathological states. Neuromuscular junction dismantling plays a crucial role in the onset of Amyotrophic Lateral Sclerosis (ALS). ALS is characterized by the degeneration and death of motor neurons leading to skeletal muscle denervation, atrophy and, most often, death of the patient within five years from diagnosis. ALS is a non-cell autonomous disease as, besides motor neuron degeneration, glial cells, and possibly muscle fibers, play a role in its onset and progression. Here, we will review the recent literature regarding the mechanisms leading to neuromuscular junction disassembly and muscle denervation focusing on the role of the three players of this peripheral tripartite synapse.

Dominant, toxic gain-of-function mutations in gars lead to non-cell autonomous neuropathology.

Charcot–Marie–Tooth (CMT) neuropathies are collectively the most common hereditary neurological condition and a major health burden for society. Dominant mutations in the gene GARS, encoding the ubiquitous enzyme, glycyl-tRNA synthetase (GlyRS), cause peripheral nerve degeneration and lead to CMT disease type 2D. This genetic disorder exemplifies a recurring motif in neurodegeneration, whereby mutations in essential, widely expressed genes have selective deleterious consequences for the nervous system. Here, using novel Drosophila models, we show a potential solution to this phenomenon. Ubiquitous expression of mutant GlyRS leads to motor deficits, progressive neuromuscular junction (NMJ) denervation and pre-synaptic build-up of mutant GlyRS. Intriguingly, neuronal toxicity is, at least in part, non-cell autonomous, as expression of mutant GlyRS in mesoderm or muscle alone results in similar pathology. This mutant GlyRS toxic gain-of-function, which is WHEP domain-dependent, coincides with abnormal NMJ assembly, leading to synaptic degeneration, and, ultimately, reduced viability. Our findings suggest that mutant GlyRS gains access to ectopic sub-compartments of the motor neuron, providing a possible explanation for the selective neuropathology caused by mutations in a widely expressed gene.

Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction.

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes.

Differential regulation of axonal growth and neuromuscular junction assembly by HGF/c‐Met signaling

During vertebrate neuromuscular junction (NMJ) development, contact between motor axons and muscle fibers is followed by pre- and post-synaptic specialization. Using Xenopus nerve-muscle cocultures, we recently showed that spinal neurons initially contacted muscle cells by means of filopodial processes, and that muscle-derived basic fibroblast growth factor induced axonal filopodia and slowed axonal advance to promote nerve-muscle interaction and NMJ establishment. In contrast, neurotrophins enhanced axonal growth but suppressed the extension of axonal filopodia and blocked NMJ formation. Here we report that hepatocyte growth factor (HGF), which also supports motor neuron survival, was expressed by Xenopus muscle cells, and that forced expression of HGF in Xenopus spinal neurons inhibited the extension of axonal filopodia. Overexpression of the HGF-receptor c-Met in neurons also blocked the formation of axonal filopodia and furthermore sped up axonal growth, but a kinase-dead form of c-Met was unable to effect these changes. Importantly, treatment of nerve-muscle cocultures with recombinant HGF or the expression of HGF or active c-Met in neurons, or that of excess HGF in muscle, inhibited nerve-induced AChR clustering in muscle. Our results suggest that HGF/c-Met signaling in neurons promotes axonal growth but suppresses filopodial assembly in neurons and hinders NMJ establishment.

Regulation of axonal growth and neuromuscular junction formation by neuronal phosphatase and tensin homologue signaling

During the development of the vertebrate neuromuscular junction (NMJ), motor axon tips stop growing after contacting muscle and transform into presynaptic terminals that secrete the neurotransmitter acetylcholine and activate postsynaptic ACh receptors (AChRs) to trigger muscle contraction. The neuron-intrinsic signaling that retards axonal growth to facilitate stable nerve-muscle interaction and synaptogenesis is poorly understood. In this paper, we report a novel function of presynaptic signaling by phosphatase and tensin homologue (PTEN) in mediating a growth-to-synaptogenesis transition in neurons. In Xenopus nerve-muscle cocultures, axonal growth speed was halved after contact with muscle, when compared with before contact, but when cultures were exposed to the PTEN blocker bisperoxo (1,10-phenanthroline) oxovanadate, axons touching muscle grew ~50% faster than their counterparts in control cultures. Suppression of neuronal PTEN expression using morpholinos or the forced expression of catalytically inactive PTEN in neurons also resulted in faster than normal axonal advance after contact with muscle cells. Significantly, interference with PTEN by each of these methods also led to reduced AChR clustering at innervation sites in muscle, indicating that disruption of neuronal PTEN signaling inhibited NMJ assembly. We thus propose that PTEN-dependent slowing of axonal growth enables the establishment of stable nerve-muscle contacts that develop into NMJs.

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

At the developing neuromuscular junction (NMJ), physical contact between motor axons and muscle cells initiates presynaptic and postsynaptic differentiation. Using Xenopus nerve-muscle cocultures, we previously showed that innervating axons induced muscle filopodia (myopodia), which facilitated interactions between the synaptic partners and promoted NMJ formation. The myopodia were generated by nerve-released signals through muscle p120 catenin (p120ctn), a protein of the cadherin complex that modulates the activity of Rho GTPases. Because axons also extend filopodia that mediate early nerve-muscle interactions, here we test p120ctn's function in the assembly of these presynaptic processes. Overexpression of wild-type p120ctn in Xenopus spinal neurons leads to an increase in filopodial growth and synaptic vesicle (SV) clustering along axons, whereas the development of these specializations is inhibited following the expression of a p120ctn mutant lacking sequences important for regulating Rho GTPases. The p120ctn mutant also inhibits the induction of axonal filopodia and SV clusters by basic fibroblast growth factor, a muscle-derived molecule that triggers presynaptic differentiation. Of importance, introduction of the p120ctn mutant into neurons hinders NMJ formation, which is observed as a reduction in the accumulation of acetylcholine receptors at innervation sites in muscle. Our results suggest that p120ctn signaling in motor neurons promotes nerve-muscle interaction and NMJ assembly.

Axonal filopodial asymmetry induced by synaptic target

During vertebrate neuromuscular junction (NMJ) assembly, motor axons and their muscle targets exchange short-range signals that regulate the subsequent steps of presynaptic and postsynaptic specialization. We report here that this interaction is in part mediated by axonal filopodia extended preferentially by cultured Xenopus spinal neurons toward their muscle targets. Immunoblotting and labeling experiments showed that basic fibroblast growth factor (bFGF) was expressed by muscle and associated with the cell surface, and treatment of cultured spinal neurons with recombinant bFGF nearly doubled the normal density of filopodia in neurites. This effect of bFGF was abolished by SU5402, a selective inhibitor of FGF-receptor 1 (FGFR1), and forced expression of wild-type or dominant-negative FGFR1 in neurons enhanced or suppressed the assembly of filopodia, respectively. Significantly, in nerve-muscle cocultures, knocking down bFGF in muscle decreased both the asymmetric extension of filopodia by axons toward muscle and the assembly of NMJs. In addition, neurons expressing dominant-negative FGFR1 less effectively triggered the aggregation of muscle acetylcholine receptors at innervation sites than did control neurons. These results suggest that bFGF activation of neuronal FGFR1 generates filopodial processes in neurons that promote nerve-muscle interaction and facilitate NMJ establishment.

To build a synapse: signaling pathways in neuromuscular junction assembly

Synapses, as fundamental units of the neural circuitry, enable complex behaviors. The neuromuscular junction (NMJ) is a synapse type that forms between motoneurons and skeletal muscle fibers and that exhibits a high degree of subcellular specialization. Aided by genetic techniques and suitable animal models, studies in the past decade have brought significant progress in identifying NMJ components and assembly mechanisms. This review highlights recent advances in the study of NMJ development, focusing on signaling pathways that are activated by diffusible cues, which shed light on synaptogenesis in the brain and contribute to a better understanding of muscular dystrophy.

Mechanisms of TSC-mediated Control of Synapse Assembly and Axon Guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development.

LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction

Low-density lipoprotein receptor-related protein 4 (Lrp4) is a member of a family of structurally related, single-pass transmembrane proteins that carry out a variety of functions in development and physiology, including signal transduction and receptor-mediated endocytosis. Lrp4 is expressed in multiple tissues in the mouse, and is important for the proper development and morphogenesis of limbs, ectodermal organs, lungs and kidneys. We show that Lrp4 is also expressed in the post-synaptic endplate region of muscles and is required to form neuromuscular synapses. Lrp4-mutant mice die at birth with defects in both presynaptic and postsynaptic differentiation, including aberrant motor axon growth and branching, a lack of acetylcholine receptor and postsynaptic protein clustering, and a failure to express postsynaptic genes selectively by myofiber synaptic nuclei. Our data show that Lrp4 is required during the earliest events in postsynaptic neuromuscular junction (NMJ) formation and suggest that it acts in the early, nerveindependent steps of NMJ assembly. The identification of Lrp4 as a crucial factor for NMJ formation may have implications for human neuromuscular diseases such as myasthenia syndromes.

An Intrinsic Distinction in Neuromuscular Junction Assembly and Maintenance in Different Skeletal Muscles

We analyzed the formation of neuromuscular junctions (NMJs) in individual muscles of the mouse embryo. Skeletal muscles can be assigned to one of two distinct classes of muscles, termed “Fast Synapsing” (FaSyn) and “Delayed Synapsing” (DeSyn) muscles, which differ significantly with respect to the initial focal clustering of postsynaptic AChRs, the timing of presynaptic maturation, and the maintenance of NMJs in young adult mice. Differences between classes were intrinsic to the muscles and manifested in the absence of innervation or agrin. Paralysis or denervation of young adult muscles resulted in disassembly of AChR clusters on DeSyn muscles, whereas those on FaSyn muscles were preserved. Our results show that postsynaptic differentiation processes intrinsic to FaSyn and DeSyn muscles influence the formation of NMJs during development and their maintenance in the adult.

Receptor targeting and heterogeneity at interneuronal nicotinic cholinergic synapses in vivo.

Within a single neuron the correct targeting of the diverse neurotransmitter receptor types to discrete synaptic regions is crucial for proper function. However, the molecular mechanisms that underlie neuronal receptor clustering and targeting are still largely undefined. Here we report advances in defining the mechanisms that mediate nicotinic acetylcholine receptor (nAChR) targeting to interneuronal synapses. Recent in vivo studies have demonstrated that one subunit plays a critical role in the differentiation of nicotinic cholinergic synapses on vertebrate autonomic neurons. The major cytoplasmic loop of the alpha3 subunit targets specific nAChR subtypes to the synapse. In contrast, nAChR complexes that lack the alpha3 targeting domain are excluded and are perisynaptic. Additional studies have demonstrated a greater complexity to alpha3-nAChR targeting due to a unique postsynaptic receptor microheterogeneity - under one presynaptic terminal, alpha3-nAChR clusters are separate, but proximal to, glycine receptor (GlyR) clusters in discrete postsynaptic membrane microregions. The surprising coexistence under one nerve ending of separate clusters of receptors that respond to different fast-acting transmitters with opposing functions may represent a novel mechanism for modulating synaptic activity. Overall, the receptor targeting and clustering studies reviewed in this issue suggest that a common mechanism underlies the formation of the diverse types of interneuronal synapses but differs from that responsible for neuromuscular junction assembly in vertebrates.