Neuromuscular Junction Formation Research Articles

IPSC-derived functional human neuromuscular junctions model the pathophysiology of neuromuscular diseases.

The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.

Glutamate at the Vertebrate Neuromuscular Junction: From Modulation to Neurotransmission.

Although acetylcholine is the major neurotransmitter operating at the skeletal neuromuscular junction of many invertebrates and of vertebrates, glutamate participates in modulating cholinergic transmission and plastic changes in the last. Presynaptic terminals of neuromuscular junctions contain and release glutamate that contribute to the regulation of synaptic neurotransmission through its interaction with pre- and post-synaptic receptors activating downstream signaling pathways that tune synaptic efficacy and plasticity. During vertebrate development, the chemical nature of the neurotransmitter at the vertebrate neuromuscular junction can be experimentally shifted from acetylcholine to other mediators (including glutamate) through the modulation of calcium dynamics in motoneurons and, when the neurotransmitter changes, the muscle fiber expresses and assembles new receptors to match the nature of the new mediator. Finally, in adult rodents, by diverting descending spinal glutamatergic axons to a denervated muscle, a functional reinnervation can be achieved with the formation of new neuromuscular junctions that use glutamate as neurotransmitter and express ionotropic glutamate receptors and other markers of central glutamatergic synapses. Here, we summarize the past and recent experimental evidences in support of a role of glutamate as a mediator at the synapse between the motor nerve ending and the skeletal muscle fiber, focusing on the molecules and signaling pathways that are present and activated by glutamate at the vertebrate neuromuscular junction.

Agrin-Lrp4-Ror2 signaling regulates adult hippocampal neurogenesis in mice.

Adult neurogenesis in the hippocampus may represent a form of plasticity in brain functions including mood, learning and memory. However, mechanisms underlying neural stem/progenitor cells (NSPCs) proliferation are not well understood. We found that Agrin, a factor critical for neuromuscular junction formation, is elevated in the hippocampus of mice that are stimulated by enriched environment (EE). Genetic deletion of the Agrn gene in excitatory neurons decreases NSPCs proliferation and increases depressive-like behavior. Low-density lipoprotein receptor-related protein 4 (Lrp4), a receptor for Agrin, is expressed in hippocampal NSPCs and its mutation blocked basal as well as EE-induced NSPCs proliferation and maturation of newborn neurons. Finally, we show that Lrp4 interacts with and activates receptor tyrosine kinase-like orphan receptor 2 (Ror2); and Ror2 mutation impairs NSPCs proliferation. Together, these observations identify a role of Agrin-Lrp4-Ror2 signaling for adult neurogenesis, uncovering previously unexpected functions of Agrin and Lrp4 in the brain.

Signal Exchange through Extracellular Vesicles in Neuromuscular Junction Establishment and Maintenance: From Physiology to Pathology.

Neuromuscular junction (NMJ) formation involves morphological changes both in motor terminals and muscle membrane. The molecular mechanisms leading to NMJ formation and maintenance have not yet been fully elucidated. During the last decade, it has become clear that virtually all cells release different types of extracellular vesicles (EVs), which can be taken up by nearby or distant cells modulating their activity. Initially, EVs were associated to a mechanism involved in the elimination of unwanted material; subsequent evidence demonstrated that exosomes, and more in general EVs, play a key role in intercellular communication by transferring proteins, lipids, DNA and RNA to target cells. Recently, EVs have emerged as potent carriers for Wnt, bone morphogenetic protein, miRNA secretion and extracellular traveling. Convincing evidence demonstrates that presynaptic terminals release exosomes that are taken up by muscle cells, and these exosomes can modulate synaptic plasticity in the recipient muscle cell in vivo. Furthermore, recent data highlighted that EVs could also be a potential cause of neurodegenerative disorders. Indeed, mutant SOD1, TDP-43 and FUS/TLS can be secreted by neural cells packaged into EVs and enter in neighboring neural cells, contributing to the onset and severity of the disease.

Ablation of All Synaptobrevin vSNAREs Blocks Evoked But Not Spontaneous Neurotransmitter Release at Neuromuscular Synapses.

Synaptic transmission occurs when an action potential triggers neurotransmitter release via the fusion of synaptic vesicles with the presynaptic membrane, driven by the formation of SNARE complexes composed of the vesicular (v)-SNARE synaptobrevin and the target (t)-SNAREs Snap-25 and syntaxin-1. Neurotransmitters are also released spontaneously, independent of an action potential, through the fusion of synaptic vesicles with the presynaptic membrane. The major neuronal vSNAREs, synaptobrevin-1 and synaptobrevin-2, are expressed at the developing neuromuscular junction (NMJ) in mice, but their specific roles in NMJ formation and function remain unclear. Here, we examine the NMJs in mutant mouse embryos lacking either synaptobrevin 1 (Syb1lew/lew ) or synaptobrevin 2 (Syb2-/-), and those lacking both (Syb1lew/lewSyb2-/-). We found that, compared with controls: (1) the number and size of NMJs was markedly increased in Syb2-/- and Syb1lew/lewSyb2-/- mice, but not in Syb1lew/lew mice; (2) synaptic vesicle density was markedly reduced in Syb1lew/lewSyb2-/- NMJs; and (3) evoked neurotransmission was markedly reduced in Syb2-/- NMJs and completely abolished in Syb1lew/lewSyb2-/- NMJs. Surprisingly, however, spontaneous neurotransmission persists in the absence of both Syb1 and Syb2. Furthermore, spontaneous neurotransmission remains constant in Syb1lew/lewSyb2-/- NMJs despite changing Ca2+ levels. These findings reveal an overlapping role for Syb1 and Syb2 (with Syb2 being dominant) in developing NMJs in mice. Moreover, because spontaneous release becomes Ca2+-insensitive in Syb1lew/lewSyb2-/- NMJs, our findings suggest that synaptobrevin-based SNARE complexes play a critical role in conferring Ca2+ sensitivity during spontaneous release.SIGNIFICANCE STATEMENT Neurotransmitters can be released at synapses with (evoked) or without (spontaneous) the influence of action potentials. Whereas evoked neurotransmission requires Ca2+ influx, those underlying the spontaneous neurotransmission may occur with or without Ca2+ Our findings show that, in the absence neuronal vSNARE synaptobrevin-1 and synaptobrevin-2, evoked neurotransmission is completely abolished; however, spontaneous synaptic transmission not only persists but even increased. Furthermore, spontaneous synaptic transmission that is normally highly Ca2+-sensitive became Ca2+-independent upon deletion of vSNARE synaptobrevin-1 and synaptobrevin-2. These findings reveal distinct mechanisms for evoked and spontaneous neurotransmitter release. Moreover, these findings suggest that synaptobrevin-based SNARE complexes play critical roles in conferring Ca2+ sensitivity during spontaneous neurotransmission at developing neuromuscular synapses in mice.

Differentiation of Motor Neuron-Like Cells from Tonsil-Derived Mesenchymal Stem Cells and Their Possible Application to Neuromuscular Junction Formation.

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.

Schwann cell-specific PTEN and EGFR dysfunctions affect neuromuscular junction development by impairing Agrin signaling and autophagy

The neuromuscular junction (NMJ) is formed by motor nerve terminals, post-junctional muscle membranes, and terminal Schwann cells (SCs). The formation of NMJ requires complex and dynamic molecular interactions. Nerve- and muscle-derived molecules have been well characterized but the mechanistic involvement of SC in NMJ development remains poorly understood. SC-specific phosphatase and tensin homolog (Pten) inactivation and epidermal growth factor receptor (EGFR) overexpression (Dhh-Cre; Cnp-EGFR; Ptenflox/flox or DET) mice were used and NMJ malformation was observed in these mice. Acetylcholine receptors (AChRs) were distorted and varicose presynaptic nerve terminals appeared in the tibialis anterior (TA) muscle of DET mice. Agrin signaling related to NMJ development, was downregulated in TA muscle. Both RAS/MEK/ERK and PI3K/AKT/mTOR signaling pathways were activated in the sciatic nerves of DET mice. In addition, autophagy was downregulated in these sciatic nerves. Interestingly, the use of Torin 2, an mTOR inhibitor, rescued the phenotype. The downregulated-autophagy might account for Agrin signaling abnormity, which induced NMJ malformation. Taken together, our results indicate that SCs-specific Pten and EGFR cooperation are essential for NMJ development.

Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, growth of an identified motor axon beyond an “en passant” synapse and later axon branching from synaptic points is impaired, leading to functional deficits. Mechanistically, motor neuron-autonomous Chodl function depends on the presence of intracellular phosphorylation sites and on binding muscle-derived Collagen XIXa1 via its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a “synapse-first” scenario of motor axon growth in zebrafish.

In Vitro Modelling of Nerve-Muscle Connectivity in a Compartmentalised Tissue Culture Device.

Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions. Studies of neuromuscular junction formation and homeostasis have been largely confined to in vivo models. In this study we have merged three powerful tools - pluripotent stem cells, optogenetics and microfabrication - and designed an open microdevice in which motor axons grow from a neural compartment containing embryonic stem cell-derived motor neurons and astrocytes through microchannels to form functional neuromuscular junctions with contractile myofibers in a separate compartment. Optogenetic entrainment of motor neurons in this reductionist neuromuscular circuit enhanced neuromuscular junction formation more than two-fold, mirroring the activity-dependence of synapse development in vivo. We incorporated an established motor neuron disease model into our system and found that coculture of motor neurons with SOD1G93A astrocytes resulted in denervation of the central compartment and diminished myofiber contractions, a phenotype which was rescued by the Receptor Interacting Serine/Threonine Kinase 1 (RIPK1) inhibitor Necrostatin. This coculture system replicates key aspects of nerve-muscle connectivity in vivo and represents a rapid and scalable alternative to animal models of neuromuscular function and disease.

Lipoprotein receptor‐related protein 4 autoantibodies in myasthenia gravis: Where are we and where are we going?

AbstractMyasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction that leads to fatigue of skeletal muscles and fluctuating weakness. The pathogenesis of MG is sufficiently diverse to include autoimmune mechanisms. In most patients with MG, autoantibodies against acetylcholine receptor can be detected. In a smaller proportion of patients without anti‐acetylcholine receptor antibodies, autoantibodies to muscle‐specific kinase and low‐density lipoprotein receptor‐related protein 4 (LRP4) are present. LRP4 is critical for neuromuscular junction formation and maintenance. A multimolecular complex is formed among agrin, LRP4 and muscle‐specific kinase that is critical for agrin signaling. The presence of anti‐LRP4 antibodies is associated with mild symptoms of MG, while thymomas are rarely observed in association with MG and LRP4 antibodies. We review recent findings on the pathogenesis, clinical features and treatment of LRP4 MG.

Open-Chamber Co-Culture Microdevices for Single-Cell Analysis of Skeletal Muscle Myotubes and Motor Neurons with Neuromuscular Junctions

Degeneration of motor neurons and skeletal muscles or the collapse of neuromuscular junctions (NMJs) causes progressive motility disturbances in many neuromuscular diseases. Although various microdevices for the co-culture of skeletal muscle myotubes and motor neurons have been developed to investigate neuromuscular diseases in vitro, it remains difficult to isolate single myotubes and motor neurons from the device for single-cell analyses, such as gene expression analysis. Here, we developed open chamber-coculture microdevices that contain cell culture chambers with narrow widths. Given the small chamber width (0.2 mm), the device significantly prevented the overlap among myotubes within the chamber. The percentage of non-overlapping was 95.6 ± 7.7% for the 0.2-mmwidth chamber and 11.8 ± 6.4% for the 7-mm-width chamber as a control. In addition, the device with the 0.2-mm chamber promoted myotube maturation, as indicated by the longer widths and lengths of the myotubes relative to those in the control chamber. Single C2C12 myotubes and human induced pluripotent stem cell (hiPSC)-derived motor neurons were successfully collected from the device with the 0.2-mm chamber using a micromanipulator equipped with a glass capillary. Furthermore, myotubes and hiPSC-derived motor neurons were co-cultured in the device with the 0.2- mm chamber, and the formation of NMJs were observed. Thus, the developed device is a useful tool for performing single-cell analysis for studying neuromuscular diseases in vitro.

Abstract 136: Denervation Induced Degradation of Motor Endplates can be Delayed Using Local Supplementation of Agrin at the Time of Injury.

PURPOSE: Robust muscle atrophy and fibrosis are cited as the prime causes for the permanence of motor deficits following surgical intervention for a denervation injury. Following a peripheral nerve transection, regenerating axons traverse long distances to reinnervate their target organs. However, due to the slow rate of axonal regrowth and a large segmental defect, muscle atrophy and destabilization of the neuromuscular junction (NMJ) usually proceeds before reinnervation occurs. Agrin is characterized as an essential component of NMJ formation and synaptogenesis. Here, we utilize an in vivo agrin deficient mouse nerve denervation model to both characterize the morphology of agrin deficient endplates as well as the response of the NMJ to local delivery of agrin following a denervation event. We found the endplates of agrin-deficient mice in a more degraded states than the endplates of wildtype mice following denervation. We also found that local delivery of agrin following denervation serves to transiently improve motor endplate morphology. We thus demonstrate the efficacy of using a biomolecular therapeutic approach to maintain and preserve the denervated NMJ. MATERIALS AND METHODS: A denervation model was created in 6-week-old mice from WT and agrin deficient C57BL/6 strains by excising 10 mm right sciatic nerve segment from the mid-thigh of the mice and suturing the proximal nerve stump to the gluteal muscle with 9-0 suture so as to prevent regeneration. Agrin deficient mice were either injected with supplemental agrin or PBS as a control at the site of injury. The downstream denervated and contralateral control soleus, plantaris and gastrocnemius muscles were harvested for immunohistochemistry, cryo-sections with H&E staining, and quantitative western blots at the 1,2,4,8 and 16-week timepoints. Quantification of motor endplate morphology was done using Volocity 3-D image software. Quantification of muscle fiber diameter was done using ImageJ software. RESULTS: Fluorescence confocal imaging of harvested soleus muscles revealed that agrin supplemented animals retained superior motor endplate morphology over control animals in all timepoints. The average surface area of agrin supplemented endplates were significantly greater than control endplates in all analyzed timepoints (p <0.05) CONCLUSION: Highlighting the importance of agrin, we have shown that even a single supplemental dose of agrin delivered locally at the site of injury is effective in preserving motor endplates in denervated mice hindlimbs even at the latest time points. This is consistent with our previous work that detailed MMP3 knock down maintained agrin at the NMJ and thereby preserved the motor endplates so as to improve function recovery. Taken together, these current experiments support the strategy to preserve motor endplates so as to prolong the window of opportunity for surgical intervention after a traumatic nerve injury.

Regulation of Wnt signaling by physical exercise in the cell biological processes of the locomotor system.

In the past decade, researches on Wnt signaling in cell biology have made remarkable progress regarding our understanding of embryonic development, bone formation, muscle injury and repair, neurogenesis, and tumorigenesis. The study also showed that physical activity can reverse age-dependent decline in skeletal muscle, preventing osteoporosis, regenerative neurogenesis, hippocampal function, cognitive ability, and neuromuscular junction formation, and the age-dependent recession is highly correlated with Wnt signaling pathways. However, how the biological processes in cell and physical activity during/following exercise affect the Wnt signaling path of the locomotor system is largely unknown. In this study, we first briefly introduce the important features of the cellular biological processes of exercise in the locomotor system. Then, we discuss Wnt signaling and review the very few studies that have examined Wnt signaling pathways in cellular biological processes of the locomotor system during physical exercise.

Targeting the Sigma-1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease affecting both the upper and lower motor neurons (MNs), with no effective treatment currently available. Early pathological events in ALS include perturbations in axonal transport (AT), formation of toxic protein aggregates and Neuromuscular Junction (NMJ) disruption, which all lead to axonal degeneration and motor neuron death. Pridopidine is a small molecule that has been clinically developed for Huntington disease. Here we tested the efficacy of pridopidine for ALS using in vitro and in vivo models. Pridopidine beneficially modulates AT deficits and diminishes NMJ disruption, as well as motor neuron death in SOD1G93A MNs and in neuromuscular co-cultures. Furthermore, we demonstrate that pridopidine activates the ERK pathway and mediates its beneficial effects through the sigma-1 receptor (S1R). Strikingly, in vivo evaluation of pridopidine in SOD1G93A mice reveals a profound reduction in mutant SOD1 aggregation in the spinal cord, and attenuation of NMJ disruption, as well as subsequent muscle wasting. Taken together, we demonstrate for the first time that pridopidine improves several cellular and histological hallmark pathologies of ALS through the S1R.

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells.

BackgroundNeuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function.MethodsThis report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development.ResultsThis unique serum- and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions.ConclusionThis coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs.

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

BackgroundAlthough considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model.ObjectiveWe aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration.MethodsImmortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons.ResultsNeuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin.ConclusionWe established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy.

Fabricating Muscle-Neuron Constructs with Improved Contractile Force Generation.

In this study, we fabricated innervated skeletal muscle tissue constructs comprising C2C12 myoblasts and PC12 neural cells using a magnetic force-based tissue engineering technique. We found that the C2C12/PC12 co-culture enhanced neural differentiation of PC12 cells and sarcomere formation of C2C12 myotubes, accompanying with neuromuscular junction formation. The innervated skeletal muscle tissue constructs generated significantly higher contractile forces compared with aneural (C2C12 monoculture) skeletal muscle tissue constructs. These innervated skeletal muscle tissue constructs can be a useful tool for drug testing and biological research for neuromuscular diseases.

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.

Deletion of Lrp4 increases the incidence of microphthalmia

Microphthalmia is a malformation that reduces the size of the ocular globe. The etiologies of this anomaly are various, but the genetic background appears to have a predominant influence on its development through mutations of genes controlling ocular developmental processes. LRP4 is a type I single transmembrane protein that is essential for the formation of neuromuscular junctions. We created and experimented on homozygous Lrp4-deficient mice and found the microphthalmia phenotype in their eyes. The loss of Lrp4 resulted in an elevated incidence of microphthalmia and affected the mRNA expression of the members of bone morphogenetic protein, fibroblast growth factor, Sonic hedgehog, and WNT signaling pathways and of several pathogenic genes for microphthalmia. Moreover, the loss of Lrp4 enhanced the incidence of aberrant retinal folds, which appeared pleated and corrugated in the eyeball.

Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction

We recently reported that R-spondin 2 (Rspo2), a secreted activator of Wnt/β-catenin signaling, promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor, Lgr5. Rspo2 is expressed highly in spinal motor neurons (SMNs) and marginally in the skeletal muscle, but the origin of Rspo2 at the NMJ remains elusive. We rescued Rspo2-deficient (Rspo2−/−) mice by specifically expressing Rspo2 in the skeletal muscle and SMNs. SMN-specific Rspo2 mitigated or over-corrected abnormal features of the NMJs and AChR clusters observed in Rspo2−/− mice including (i) abnormal broadening of enlarged AChR clusters, (ii) three of six abnormal ultrastructural features, and (iii) abnormal expression of nine genes in SMNs and the diaphragm. In contrast, muscle-specific Rspo2 normalized all six abnormal ultrastructural features, but it had no effect on AChR clustering and NMJ formation at the light microscopy level or on abnormal gene expression in SMNs and the diaphragm. These results suggest that SMN-derived Rspo2 plays a major role in AChR clustering and NMJ formation in the postsynaptic region, and muscle-derived Rspo2 also plays a substantial role in juxtaposition of the active zones and synaptic folds.