AChR Clustering Research Articles

Stretched Fibroblasts Regulate Acetylcholine Receptor (AChR) Expression and Clustering on Skeletal Muscle

Repetitive tasks reduce fine motor control and manipulative therapy improves physical performance. Stretched muscle fascia may influence the contraction of the underlying skeletal muscle by secreting cytokines that regulate AChRs. A co‐culture was setup by seeding fibroblasts on deformable membranes and C2C12 myoblasts on non‐deformable coverslips for 24hrs before transferring the C2C12s two mm above the fibroblasts, within diffusible range of secreted cytokines. Fibroblasts were then subjected to repetitive stretch and/or manipulative therapy. Agrin‐induced myotubes in 2% horse serum and non‐strained C2C12s in co‐culture served as positive and negative controls. After 96hrs, C2C12s were analyzed for AChR expression by Western Blot (N=7) and for clustering by fluorescent imaging (130 images/treatment). Data were analyzed via ANOVA, p<0.05. AChR clusters numbered 196±21 for agrin and 65±16 for non‐strain co‐culture. Manipulative therapy did not affect cluster number 56±12, but repetitive stretch attenuated clusters 38±6. Inversely we found repetitive strain increased AChR expression by >;2‐fold vs. non‐strain, while manipulative therapy did not increase expression. Stretched fibroblasts differentially regulate AChRs on skeletal muscle cells through paracrine mediators. Opposite AChR expression and clustering results suggests that unique extrinsic factors regulate each outcome.AOA

Induced Formation and Maturation of Acetylcholine Receptor Clusters in a Defined 3D Bio-Artificial Muscle

Dysfunction of the neuromuscular junction is involved in a wide range of muscular diseases. The development of neuromuscular junction through which skeletal muscle is innervated requires the functional modulation of acetylcholine receptor (AchR) clustering on myofibers. However, studies on AchR clustering in vitro are mostly done on monolayer muscle cell culture, which lacks a three-dimensional (3D) structure, a prominent limitation of the two-dimensional (2D) system. To enable a better understanding on the structure-function correlation underlying skeletal muscle innervation, a muscle system with a well-defined geometry mimicking the in vivo muscular setting is needed. Here, we report a 3D bio-artificial muscle (BAM) bioengineered from green fluorescent protein-transduced C3H murine myoblasts as a novel in vitro tissue-based model for muscle innervation studies. Our cell biological and molecular analysis showed that this BAM is structurally similar to in vivo muscle tissue and can reach the perinatal differentiation stage, higher than does 2D culture. Effective clustering and morphological maturation of AchRs on BAMs induced by agrin and laminin indicate the functional activity and plasticity of this BAM system toward innervation. Taken together, our results show that the BAM provides a favorable 3D environment that at least partially recapitulates real physiological skeletal muscle with regard to innervation. With a convenience of fabrication and manipulation, this 3D in vitro system offers a novel model for studying mechanisms underlying skeletal muscle innervation and testing therapeutic strategies for relevant nervous and muscular diseases.

DOK7 congenital myasthenic syndrome

Despite being a fairly recent discovery, DOK7 congenital myasthenic syndrome (CMS) is the third most common form of CMS in the United Kingdom. DOK7 is a postsynaptic protein associated with the AChR clustering pathway. In contrast to AChR deficiency due to epsilon subunit mutations, onset of DOK7 CMS tends to be later--ages two to three years--and in DOK7 CMS eye movements are usually spared and anticholinesterases can exacerbate the weakness. The typical phenotype of DOK7 CMS is of a limb girdle weakness with associated nonspecific myopathic features. The presence of stridor in early onset cases and the observation of tongue wasting may be specific clues. Worsening in adulthood is common, particularly affecting bulbar and respiratory function. Treatment with ephedrine or oral salbutamol can result in a slow, steady, and often dramatic improvement over months.

IgG anti‐Galnac‐GD1a antibodies bind to neuromuscular junctions of rat hemidiaphragm

We investigated the localization of a ganglioside, N-acetylgalactosaminyl GD1a (GalNAc-GD1a), in peripheral nerves with an IgG anti-GalNAc-GD1a antibody, which was produced in rabbits immunized with GalNAc-GD1a. Teased fibers from ventral and dorsal roots and hemidiaphragm sections of rats were assessed using fluorescent double- and triple-labeling methods. The nodal and paranodal regions of teased fibers from ventral roots were immunostained with IgG anti-GalNAc-GD1a antibodies. After collagenase treatment, no staining was seen with IgG anti-GalNAc-GD1a or anti-NF200 antibodies, whereas α-bungarotoxin selectively stained nerve terminals. In cross-sectional and longitudinal sections of rat hemidiaphragm, IgG anti-GalNAc-GD1a antibodies overlapped with α-BuTx and anti-NF200 antibodies, indicating that GalNAc-GD1a is localized to the nerve terminal. IgG anti-GalNAc-GD1a antibody staining also overlapped with that of AChR clusters and syntaxin-positive presynaptic nerve terminals. GalNAc-GD1 is localized in both pre- and postsynaptic nerve terminals of neuromuscular junctions.

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.

β-Catenin gain of function in muscles impairs neuromuscular junction formation

Neuromuscular junction (NMJ) formation requires proper interaction between motoneurons and muscle cells. β-Catenin is required in muscle cells for NMJ formation. To understand underlying mechanisms, we investigated the effect of β-catenin gain of function (GOF) on NMJ development. In HSA-β-cat(flox(ex3)/+) mice, which express stable β-catenin specifically in muscles, motor nerve terminals became extensively defasciculated and arborized. Ectopic muscles were observed in the diaphragm and were innervated by ectopic phrenic nerve branches. Moreover, extensive outgrowth and branching of spinal axons were evident in the GOF mice. These results indicate that increased β-catenin in muscles alters presynaptic differentiation. Postsynaptically, AChR clusters in HSA-β-cat(flox(ex3)/+) diaphragms were distributed in a wider region, suggesting that muscle β-catenin GOF disrupted the signal that restricts AChR clustering to the middle region of muscle fibers. Expression of stable β-catenin in motoneurons, however, had no effect on NMJ formation. These observations provide additional genetic evidence that pre- and postsynaptic development of the NMJ requires an intricate balance of β-catenin activity in muscles.

Distinct Roles of Muscle and Motoneuron LRP4 in Neuromuscular Junction Formation

Neuromuscular junction (NMJ) formation requires precise interaction between motoneurons and muscle fibers. LRP4 is a receptor of agrin that is thought to act in cis to stimulate MuSK in muscle fibers for postsynaptic differentiation. Here we dissected the roles of LRP4 in muscle fibers and motoneurons in NMJ formation by cell-specific mutation. Studies of muscle-specific mutants suggest that LRP4 is involved in deciding where to form AChR clusters in muscle fibers, postsynaptic differentiation, and axon terminal development. LRP4 in HEK293 cells increased synapsin or SV2 puncta in contacting axons of cocultured neurons, suggesting a synaptogenic function. Analysis of LRP4 muscle and motoneuron double mutants and mechanistic studies suggest that NMJ formation may also be regulated by LRP4 in motoneurons, which could serve as agrin's receptor in trans to induce AChR clusters. These observations uncovered distinct roles of LRP4 in motoneurons and muscles in NMJ development.

β-Catenin gain of function in muscles impairs neuromuscular junction formation

Neuromuscular junction (NMJ) formation requires proper interaction between motoneurons and muscle cells. β-Catenin is required in muscle cells for NMJ formation. To understand underlying mechanisms, we investigated the effect of β-catenin gain of function (GOF) on NMJ development. In HSA-β-catflox(ex3)/+ mice, which express stable β-catenin specifically in muscles, motor nerve terminals became extensively defasciculated and arborized. Ectopic muscles were observed in the diaphragm and were innervated by ectopic phrenic nerve branches. Moreover, extensive outgrowth and branching of spinal axons were evident in the GOF mice. These results indicate that increased β-catenin in muscles alters presynaptic differentiation. Postsynaptically, AChR clusters in HSA-β-catflox(ex3)/+ diaphragms were distributed in a wider region, suggesting that muscle β-catenin GOF disrupted the signal that restricts AChR clustering to the middle region of muscle fibers. Expression of stable β-catenin in motoneurons, however, had no effect on NMJ formation. These observations provide additional genetic evidence that pre- and postsynaptic development of the NMJ requires an intricate balance of β-catenin activity in muscles.

Biglycan Is an Extracellular MuSK Binding Protein Important for Synapse Stability

The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for prepatterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn(-/o)) muscle is indistinguishable from wild-type controls. However, by 5 weeks after birth, nerve-muscle synapses in bgn(-/o) mice are abnormal as judged by the presence of perijunctional folds, increased segmentation, and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied presynaptic and postsynaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn(-/o) synapses. In bgn(-/o) myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability.

Wnt4 Participates in the Formation of Vertebrate Neuromuscular Junction

Neuromuscular junction (NMJ) formation requires the highly coordinated communication of several reciprocal signaling processes between motoneurons and their muscle targets. Identification of the early, spatially restricted cues in target recognition at the NMJ is still poorly documented, especially in mammals. Wnt signaling is one of the key pathways regulating synaptic connectivity. Here, we report that Wnt4 contributes to the formation of vertebrate NMJ in vivo. Results from a microarray screen and quantitative RT-PCR demonstrate that Wnt4 expression is regulated during muscle cell differentiation in vitro and muscle development in vivo, being highly expressed when the first synaptic contacts are formed and subsequently downregulated. Analysis of the mouse Wnt4−/− NMJ phenotype reveals profound innervation defects including motor axons overgrowing and bypassing AChR aggregates with 30% of AChR clusters being unapposed by nerve terminals. In addition, loss of Wnt4 function results in a 35% decrease of the number of prepatterned AChR clusters while Wnt4 overexpression in cultured myotubes increases the number of AChR clusters demonstrating that Wnt4 directly affects postsynaptic differentiation. In contrast, muscle structure and the localization of several synaptic proteins including acetylcholinesterase, MuSK and rapsyn are not perturbed in the Wnt4 mutant. Finally, we identify MuSK as a Wnt4 receptor. Wnt4 not only interacts with MuSK ectodomain but also mediates MuSK activation. Taken together our data reveal a new role for Wnt4 in mammalian NMJ formation that could be mediated by MuSK, a key receptor in synaptogenesis.

3PS2.1 Inherited Ataxia Update

acting on enzyme stability at presynaptic level. Transmission is compromised by desensitation and depolarization block at AChR at physiologic rate of stimulation in synaptic defects. Agrin stabilize laminin network being important for acetylcholinesterase (AChE) function, AChR clustering, and muscle-specific kinase (MuSK) autophosphorylation. Mutations in AChR impair ion channel gating, reducing number of AChR and cause slow channel, fast channel and AChR deficiency syndrome. AChR channel gating is impaired based probably on conformational transition by changing isomerisation between open and closed conformation. Dok7 mutations result in impaired activation of MuSK and rapsyn to concentrate AChR on junctional folds which cause small simplified and unstable neuromuscular junctions affecting both presynaptic and postsynaptic structures. MuSK mutations result in deficiency of presynaptic differentiation and postsynaptic development. Conclusions: There is no correlation of genotype and phenotype and there is no possibility to predict the severity of CMS according to known mutation. Molecular genetics is essential in differential diagnosis and understanding of the patomechanisms inducing CMS and its effective therapy.

The modulation of myogenic cells differentiation using a semiconductor-muscle junction

The present study is aimed to design a prototype of hybrid silicon-muscle cell junction, analog to an artificial neuromuscular junction prototype and relevant to the development of advanced neuro-prostheses and bionic systems. The device achieves focal Electric Capacitive Stimulation (ECS) by coupling of single cells and semiconductors, without electrochemical reaction with the substrate. A voltage change applied to a stimulation spot beneath an electrogenic cell leads to a capacitive current (charge accumulation) that opens voltage-gated ion channels in the membrane and generates an action potential. The myo-electronic junction was employed to chronically stimulate muscle cells via ECS and to induce cytosolic calcium transients in myotubes, fibers isolated from mouse FDB (fast [Ca 2+] i transients) and surprisingly also in undifferentiated myoblasts (slow [Ca 2+] i waves). The hybrid junction elicited, via chronic ECS, a differential reprogramming of single muscle cells by inducing early muscle contraction maturation and plasticity effects, such as NFAT-C3 nuclear translocation. In addition, in the presence of agrin, chronic ECS induced a modulation of AchR clustering which simulates in vitro synaptogenesis. This methodology can coordinate the myogenic differentiation, thus offering direct but non-invasive single cell/wiring, providing a platform for regenerative medicine strategies.

Rescuing Z + agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing

Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z(+) agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z(+) agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z(+) agrin in motor neurons rescued AChR clustering. Surprisingly, however, these rescued mice remained paralyzed, while electrophysiologic studies demonstrated that the motor axon and synapse were functional-spontaneous and evoked recordings revealed synaptic transmission and muscle contraction. These results point to a proximal defect in motor neuron firing in the absence of Nova and reveal a previously unsuspected role for RNA regulation in the physiologic activation of motor neurons.

Alpha-Actinin interacts with rapsyn in agrin-stimulated AChR clustering

AChR is concentrated at the postjunctional membrane at the neuromuscular junction. However, the underlying mechanism is unclear. We show that α-actinin, a protein known to cross-link F-actin, interacts with rapsyn, a scaffold protein essential for neuromuscular junction formation. α-Actinin, rapsyn, and surface AChR form a ternary complex. Moreover, the rapsyn-α-actinin interaction is increased by agrin, a factor known to stimulate AChR clustering. Downregulation of α-actinin expression inhibits agrin-mediated AChR clustering. Furthermore, the rapsyn-α-actinin interaction can be disrupted by inhibiting Abl and by cholinergic stimulation. Together these results indicate a role for α-actinin in AChR clustering.

New Dogs in the Dogma: Lrp4 and Tid1 in Neuromuscular Synapse Formation

Two recent papers reported identification of a long-sought agrin coreceptor, Lrp4 (Kim et al. in Cell and Zhang et al. in Neuron). In this issue of Neuron, Linnoila et al. report the identification of a new player in the agrin-MuSK pathway, Tid1, which directly interacts with MuSK and is responsible for transducing signals from MuSK activation to AChR clustering, culminating in cross-linking to the subsynaptic cytoskeleton. These papers substantially reshape the agrin-MuSK-ACh hypothesis of neuromuscular synaptogenesis.

LRP4 Serves as a Coreceptor of Agrin

Neuromuscular junction (NMJ) formation requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. We report that LRP4, a low-density lipoprotein receptor (LDLR)-related protein, is expressed specifically in myotubes and binds to neuronal agrin. Its expression enables agrin binding and MuSK signaling in cells that otherwise do not respond to agrin. Suppression of LRP4 expression in muscle cells attenuates agrin binding, agrin-induced MuSK tyrosine phosphorylation, and AChR clustering. LRP4 also forms a complex with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations indicate that LRP4 is a coreceptor of agrin that is necessary for MuSK signaling and AChR clustering and identify a potential target protein whose mutation and/or autoimmunization may cause muscular dystrophies.

M.P.1.04 The effect of dok-7 on acetylcholine receptor clustering in C2C12 cells

Congenital myasthenic syndromes (CMS) are a group of rare inherited disorders that result in fatigable muscle weakness. Dok-7 is essential for synaptogenesis in vivo since mice lacking Dok-7 do not form AChR clusters or neuromuscular synapses. It is thought to interact with the juxtamembrane phosphotyrosine binding domain (PTB) motif of MuSK and amplify signalling in the MuSK/AChR clustering pathway. We screened CMS patient DNA for mutations in the DOK7 gene and identified 12 recessive mutations in 26 unrelated kinships.

O-fucosylation of muscle agrin determines its ability to cluster acetylcholine receptors

Protein O-fucosyltransferase 1 (Pofut1) transfers fucose to serine or threonine on proteins, including Notch receptors, that contain EGF repeats with a particular consensus sequence. Here we demonstrate that agrin is O-fucosylated in a Pofut1-dependent manner, and that this glycosylation can regulate agrin function. Fucosylation of recombinant C45 agrin, both active (neural, z8) and inactive (muscle, z0) splice forms, was eliminated when agrin was overexpressed in Pofut1-deficient cells or by mutation of a consensus site for Pofut1 fucosylation (serine 1726 in the EGF4 domain). Loss of O-fucosylation caused a gain of function for muscle agrin such that it stimulated AChR clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form. Deletion of Pofut1 in cultured primary myotubes and in adult skeletal muscle increased AChR aggregation. In addition, Pofut1 gene and protein expression and Pofut1 activity of the EGF4 domain of agrin were modulated during neuromuscular development. These data are consistent with a role for Pofut1 in AChR aggregation during synaptogenesis via the regulation of the synaptogenic activity of muscle agrin.

Anti‐MuSK patient antibodies disrupt the mouse neuromuscular junction

A subset of myasthenia gravis patients that are seronegative for anti-acetylcholine receptor (anti-AChR) antibodies are instead seropositive for antibodies against the muscle-specific kinase (anti-MuSK-positive). Here, we test whether transfer of IgG from anti-MuSK-positive patients to mice confers impairment of the neuromuscular junction and muscle weakness. IgG from anti-MuSK-positive myasthenia gravis patients or control IgG (seronegative for AChR and MuSK) was injected intraperitoneally (45 mg daily for 14 days) into 6-week-old female FVB/NJ and C57BL/6J mice. Changes at neuromuscular junctions in the tibialis anterior and diaphragm muscles were assessed by confocal fluorescent imaging of AChRs stained with fluorescent-alpha-bungarotoxin. Loss of function was assessed by electromyography. In experimental mice injected with anti-MuSK-positive patient IgG, postsynaptic AChR staining was reduced to as little as 22% of that seen in control mice. Experimental mice showed reduced apposition of the nerve terminal (labeled with antibodies against synaptophysin and neurofilament) and the postsynaptic AChR cluster (labeled with fluorescent-alpha-bungarotoxin). Mice injected with IgG from two of three anti-MuSK-positive patients lost weight and developed muscle weakness associated with a decremental electromyographic trace on repetitive nerve stimulation. IgG from anti-MuSK-positive patients can cause myasthenia gravis when injected into mice. This may be explained by a progressive reduction in the density of postsynaptic AChR combined with changes in the nerve terminal and its relation to the postsynaptic structure.

The Ig1/2 Domain of MuSK Binds to Muscle Surface and Is Involved in Acetylcholine Receptor Clustering

The neuromuscular junction, the synapse between motor neurons and muscle cells, serves as an excellent model for studying synapse formation. Agrin is believed to be released by motor neurons to induce postsynaptic differentiation at the neuromuscular junction. MuSK, a receptor tyrosine kinase, appears to be a key component of the agrin receptor complex. However, how agrin activates MuSK remains unclear. To address this question, we characterized the binding of the MuSK extracellular region to the muscle cell surface. The MuSK ectodomain was found to bind to muscle cells in a manner dependent on stimulation with neural agrin. Moreover, the binding was myotube specific and appeared to be mediated by two regions in the MuSK: one region containing the first and second immunoglobin domains and the other containing the cysteine-rich domain. Importantly, recombinant proteins containing the binding activity can block full-length MuSK binding to muscle cells and agrin-induced AChR clustering. These results suggest that the Ig1/2 domain of MuSK is involved in AChR clustering by binding to the muscle surface.