Aggregation Of Acetylcholine Receptors Research Articles

Direct electrical stimulation impacts on neuromuscular junction morphology on both stimulated and unstimulated contralateral soleus.

There is increasing evidence of crosstalk between organs. The neuromuscular junction (NMJ) is a peripheral chemical synapse whose function and morphology are sensitive to acetylcholine (ACh) release and muscle depolarization. In an attempt to improve our understanding of NMJ plasticity and muscle crosstalk, the effects of unilateral direct electrical stimulation of a hindlimb muscle on the NMJ were investigated in rats exposed long-term post-synaptic neuromuscular blockade. Sprague Dawley rats were subjected to post-synaptic blockade of neuromuscular transmission by systemic administration of α-cobrotoxin and mechanically ventilated for up to 8days and compared with untreated sham operated controls and animals exposed to unilateral chronic electrical stimulation 12h/day for 5 or 8days. NMJs produced axonal and glial sprouts (growth of processes that extend beyond the confines of the synapse defined by high-density aggregates of acetylcholine receptors [AChRs]) in response to post-synaptic neuromuscular blockade, but less than reported after peripheral denervation or pre-synaptic blockade. Direct electrical soleus muscle stimulation reduced the terminal Schwann cell (tSC) and axonal sprouting in both stimulated and non-stimulated contralateral soleus. Eight days chronic stimulation reduced (P<0.001) the number of tSC sprouts on stimulated and non-stimulated soleus from 6.7±0.5 and 6.9±0.5 sprouts per NMJ, respectively, compared with 10.3±0.9 tSC per NMJ (P<0.001) in non-stimulated soleus from rats immobilized for 8days. A similar reduction of axonal sprouts (P<0.001) was observed in stimulated and non-stimulated contralateral soleus in response to chronic electrical stimulation. RNAseq-based gene expression analyses confirmed a restoring effect on both stimulated and unstimulated contralateral muscle. The cross-over effect was paralleled by increased cytokine/chemokine levels in stimulated and contralateral unstimulated muscle as well as in plasma. Motor axon terminals and terminal Schwann cells at NMJs of rats subjected to post-synaptic neuromuscular blockade exhibited sprouting responses. These axonal and glial responses were likely dampened by a muscle-derived myokines released in an activity-dependent manner with both local and systemic effects.

Sorting nexin 17 increases low-density lipoprotein receptor-related protein 4 membrane expression: A novel mechanism of acetylcholine receptor aggregation in myasthenia gravis.

Myasthenia gravis (MG) is characterized by autoimmune damage to the postsynaptic membrane of the neuromuscular junction (NMJ) with impaired postsynaptic acetylcholine receptor (AChR) aggregation. Low-density lipoprotein receptor-related protein 4 (LRP4) plays an important role in AChR aggregation at endplate membranes via the Agrin–LRP4–muscle-specific receptor tyrosine kinase (MuSK) cascade. Sorting nexin 17 (SNX17) regulates the degradation and recycling of various internalized membrane proteins. However, whether SNX17 regulates LRP4 remains unclear. Therefore, we examined the regulatory effects of SNX17 on LRP4 and its influence on AChR aggregation in MG. We selected C2C12 myotubes and induced LRP4 internalization via stimulation with anti-LRP4 antibody and confirmed intracellular interaction between SNX17 and LRP4. SNX17 knockdown and overexpression confirmed that SNX17 promoted MuSK phosphorylation and AChR aggregation by increasing cell surface LRP4 expression. By establishing experimental autoimmune MG (EAMG) mouse models, we identified that SNX17 upregulation improved fragmentation of the AChR structure at the NMJ and alleviated leg weakness in EAMG mice. Thus, these results reveal that SNX17 may be a novel target for future MG therapy.

Association of a newly identified lncRNA LNC_000280 with the formation of acetylcholine receptor clusters in vitro

Peripheral nerve injury (PNI) can disintegrate acetylcholine receptor (AChR) clusters in the postsynaptic membrane. In our previous research, lncRNAs that were differentially expressed in the whole transcriptome sequencing of denervated muscle atrophy after PNI were screened. By utilizing Gene Ontology (GO) analysis and protein-protein interaction (PPI) networks, a novel lncRNA LNC_000280 was predicted to be associated with neuromuscular junction (NMJ). The myotubes were used to assess the connection between LNC_000280 and AChR cluster formation in vitro by overexpression and knockdown of LNC_000280 in the C2C12 cell line. Our findings demonstrated that the overexpression of LNC_000280 repressed the gene expression and protein level of AChR subunits in myotubes and further reduced the area of AChR aggregates on the cell membrane. In contrast, the knockdown of LNC_000280 brought about opposite results. In addition, the transcriptional level of Sorbs2 changed inversely with the quantity change of LNC_000280. In conclusion, LNC_000280 may associate with the formation of AChR clusters.

Maturation of a postsynaptic domain: Role of small Rho GTPases in organising nicotinic acetylcholine receptor aggregates at the vertebrate neuromuscular junction.

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor axon and a skeletal muscle fibre that allows muscle contraction and the coordinated movement in many species. A main hallmark of the mature NMJ is the assembly of nicotinic acetylcholine receptor (nAChR) aggregates in the muscle postsynaptic domain, that distributes in perfect apposition to presynaptic motor terminals. To assemble its unique functional architecture, initial embryonic NMJs undergo an early postnatal maturation process characterised by the transformation of homogenous nAChR-containing plaques to elaborate and branched pretzel-like structures. In spite of a detailed morphological characterisation, the molecular mechanisms controlling the intracellular scaffolding that organises a postsynaptic domain at the mature NMJ have not been fully elucidated. In this review, we integrate evidence of key processes and molecules that have shed light on our current understanding of the NMJ maturation process. On the one hand, we consider in vitro studies revealing the potential role of podosome-like structures to define discrete low nAChR-containing regions to consolidate a plaque-to-pretzel transition at the NMJ. On the other hand, we focus on in vitro and in vivo evidence demonstrating that members of the Ras homologous (Rho) protein family of small GTPases (small Rho GTPases) play indispensable roles on NMJ maturation by regulating the stability of nAChR aggregates. We combine this evidence to propose that small Rho GTPases are key players in the assembly of podosome-like structures that drive the postsynaptic maturation of vertebrate NMJs.

HDAC6 regulates microtubule stability and clustering of AChRs at neuromuscular junctions.

Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NMJ), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.

Sepsis impairs aggregation of nicotinic acetylcholine receptors on murine skeletal muscle cell membranes by inhibiting AKT/GSK3β phosphorylation

To investigate the role of the protein-serine-threonine kinase (AKT)/glycogen synthase kinase 3β (GSK3β) signaling pathway in nicotinic acetylcholine receptors (nAChRs) aggregation disorder on skeletal muscle cell membranes induced by sepsis. Mouse C2C12 myoblasts were differentiated into myotubes by horse serum, and then C2C12 myotubes were randomly divided into four groups: the Sham group treated with serum from sham-operated mice, the Sepsis group treated with serum from septic mice, the Sepsis+D group treated with serum from septic mice and dimethyl sulfoxide (DMSO), the Sepsis+SB group treated with serum from septic mice and GSK3β inhibitor SB216763. Agrin was added into the cell culture to induce nAChRs aggregation before the treatment. After serum treatment for 5.5 h, the myotubes were examined for nAChRs clusters using Alexa Fluor 594-conjugated α-bungarotoxin (α- BTX). The expression levels of AKT, GSK3β and CLIP- associated protein 2 (CLASP2) and the phosphorylation of AKT, GSK3β were examined with Western blotting. The phosphorylation of CLASP2 and the interaction between CLASP2 and α-tubulin were detected with co-immunoprecipitation (Co-IP) assay. Compared with the serum from sham-operated mice, the serum from septic mice caused significant reduction in the area and density of nAChRs clusters on C2C12 myotubes, lowered the levels of phosphorylated AKT (p-AKT) and phosphorylated GSK3β (p-GSK3β), increased the expression of phosphorylated CLASP2 (p-CLASP2), and obviously reduced the binding between CLASP2 and α-tubulin. Compared with DMSO, SB216763 significantly increased the area and density of nAChRs clusters on C2C12 myotubes treated with serum from septic mice, decreased the expression of p-CLASP2, and enhanced the interaction between CLASP2 and α-tubulin. Septic mouse serum impairs nAChRs aggregation on C2C12 myotubes possibly by suppressing AKT/GSK3β phosphorylation to cause reduced interaction between CLASP2 and α-tubulin.

Rapsyn facilitates recovery from desensitization in fetal and adult acetylcholine receptors expressed in a muscle cell line.

Key points The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied.Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform.Recovery from desensitization is determined by the AChR isoform‐specific cytoplasmic M3–M4 domain.The co‐expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering.These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions. The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle‐type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole‐cell currents in muscle‐type (TE671/CN21) and non‐muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P < 0.0001). Chimeric experiments showed that recovery from desensitization was determined by the M3–M4 cytoplasmic loops of the γ‐ and ε‐subunits. Expression of rapsyn in TE671/CN21 cells induced AChR aggregation and also, surprisingly, shortened recovery time constants in both fetal and adult AChRs. However, this was not dependent on clustering because rapsyn also facilitated recovery from desensitization in HEK293 cells expressing a δ‐R375H AChR mutant that did not form clusters in C2C12 myotubes. Thus, rapsyn interactions with AChRs lead not only to clustering, but also to a clustering independent faster recovery from desensitization. Both effects of rapsyn could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth.

Cycles of myofiber degeneration and regeneration lead to remodeling of the neuromuscular junction in two mammalian models of Duchenne muscular dystrophy.

Mice lacking the sarcolemmal protein dystrophin, designated mdx, have been widely used as a model of Duchenne muscular dystrophy. Dystrophic mdx mice as they mature develop notable morphological abnormalities to their neuromuscular junctions, the peripheral cholinergic synapses responsible for activating muscle fibers. Most obviously the acetylcholine receptor aggregates are fragmented into small non-continuous, islands. This contrasts with wild type mice whose acetylcholine receptor aggregates are continuous and pretzel-shaped in appearance. We show here that these abnormalities in mdx mice are also present in a canine model of Duchenne muscular dystrophy and provide additional evidence to support the hypothesis that NMJ remodeling occurs due to myofiber degeneration and regeneration. Using a method to investigate synaptic AChR replacement, we show that neuromuscular junction remodeling in mdx animals is caused by muscle fiber degeneration and regeneration at the synaptic site and is mimicked by deliberate myofiber injury in wild type mice. Importantly, the innervating motor axon plays a crucial role in directing the remodeling of the neuromuscular junction in dystrophy, as has been recorded in aging and deliberate muscle fiber injury in wild type mice. The remodeling occurs repetitively through the life of the animal and the changes in junctions become greater with age.

MyoScreen, a High-Throughput Phenotypic Screening Platform Enabling Muscle Drug Discovery

Despite the need for more effective drug treatments to address muscle atrophy and disease, physiologically accurate in vitro screening models and higher information content preclinical assays that aid in the discovery and development of novel therapies are lacking. To this end, MyoScreen was developed: a robust and versatile high-throughput high-content screening (HT/HCS) platform that integrates a physiologically and pharmacologically relevant micropatterned human primary skeletal muscle model with a panel of pertinent phenotypic and functional assays. MyoScreen myotubes form aligned, striated myofibers, and they show nerve-independent accumulation of acetylcholine receptors (AChRs), excitation–contraction coupling (ECC) properties characteristic of adult skeletal muscle and contraction in response to chemical stimulation. Reproducibility and sensitivity of the fully automated MyoScreen platform are highlighted in assays that quantitatively measure myogenesis, hypertrophy and atrophy, AChR clusterization, and intracellular calcium release dynamics, as well as integrating contractility data. A primary screen of 2560 compounds to identify stimulators of myofiber regeneration and repair, followed by further biological characterization of two hits, validates MyoScreen for the discovery and testing of novel therapeutics. MyoScreen is an improvement of current in vitro muscle models, enabling a more predictive screening strategy for preclinical selection of the most efficacious new chemical entities earlier in the discovery pipeline process.

Specific effects of neuregulin-1β on the communication between DRG neurons and skeletal muscle cells in vitro.

The communication between primary afferent neuron and skeletal muscle (SKM) is one of the important factors on maintaining the structure and function of SKM cells. Neuregulin-1β (NRG-1β) signaling is essential for regulating synaptic neurotransmission. Here, we established a neuromuscular coculture model of dorsal root ganglion (DRG) sensory neurons and SKM cells to explore the nerve-muscle communication in the presence of exogenous NRG-1β. The expression of three distinct subtypes (TrkA, TrkB, and TrkC) of tyrosine kinase receptors was monitored for the phenotypical alterations of the neurons. The aggregation extent of acetylcholine receptor (AChR) represents the specific changes of SKM cells after NRG-1β incubation in this neuromuscular coculture model. The results showed that NRG-1β not only enhanced neurite outgrowth of DRG neurons but also increased the length and branches of SKM cells. NRG-1β treatment not only induced expression of all the three subtypes of Trk receptors in neurons but also promoted AChR aggregation on the surface of SKM cells. The effects of NRG-1β could be blocked by administration of ERK1/2 inhibitor PD98059, PI3K inhibitor LY294002, and JAK2 inhibitor AG490, respectively. These data imply that NRG-1β is essential for the nerve-muscle communication by enhancing growth and modifying phenotypes of the two different kinds of cells. The specific effects produced by NRG-1β add novel interpretation for nerve-muscle communication between sensory neurons and SKM cells.

Diagnostic utility of cortactin antibodies in myasthenia gravis.

Patients with myasthenia gravis (MG) without antibodies to the acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK) have been classified as having double-seronegative myasthenia gravis (dSNMG). We used the sera from six dSNMG patients with positive immunohistochemistry assays in a protein array to screen reactivity with 9000 human proteins. We identified cortactin, an intracellular protein that interacts with agrin/MuSK favoring AChR aggregation, as a new antigen in dSNMG. We then designed an in-house enzyme-linked immunosorbent assay as a screening assay and confirmed these results by western blot. We found that 19.7% of dSNMG patients had anti-cortactin antibodies. In contrast, patients with AChR+ MG or other autoimmune disorders and healthy controls were positive at significantly lower rates. Five percent of healthy controls were positive. In a recent study, we screened sera from 250 patients (AChR+ MG, MuSK+ MG, dSNMG) and 29 healthy controls. Cortactin antibodies were identified in 23.7% of dSNMG and 9.5% AChR+ MG patients (P = 0.02). None of the MuSK+ MG patients, patients with other autoimmune disorders, or healthy controls had antibodies against cortactin. Patients with dSNMG cortactin+ MG were negative for anti-striated muscle and anti-LRP4 antibodies. Patients with dSNMG cortactin+ MG presented ocular or mild generalized MG without bulbar symptoms. We conclude that cortactin autoantibodies are biomarkers of MG that, when present, suggest that the disease will be mild.

Moving forward with the neuromuscular junction.

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 40years 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 invitro is within reach with major consequences for drug screening. Wnts are secreted neurogenic factors that have been involved invitro in acetylcholine receptor clustering, but their precise role invivo remains to be clarified. All these data are raising new and exciting perspectives in the field and are discussed in this Review. This is an article for thespecial issue XVth International Symposium on Cholinergic Mechanisms.

Cullin E3 Ligase Activity Is Required for Myoblast Differentiation

The role of cullin E3-ubiquitin ligases for muscle homeostasis is best known during muscle atrophy, as the cullin-1 substrate adaptor atrogin-1 is among the most well-characterized muscle atrogins. We investigated whether cullin activity was also crucial during terminal myoblast differentiation and aggregation of acetylcholine receptors for the establishment of neuromuscular junctions in vitro.The activity of cullin E3-ligases is modulated through post-translational modification with the small ubiquitin-like modifier nedd8. Using either the Nae1 inhibitor MLN4924 (Pevonedistat) or siRNA against nedd8 in early or late stages of differentiation on C2C12 myoblasts, and primary satellite cells from mouse and human, we show that cullin E3-ligase activity is necessary for each step of the muscle cell differentiation program in vitro. We further investigate known transcriptional repressors for terminal muscle differentiation, namely ZBTB38, Bhlhe41, and Id1. Due to their identified roles for terminal muscle differentiation, we hypothesize that the accumulation of these potential cullin E3-ligase substrates may be partially responsible for the observed phenotype.MLN4924 is currently undergoing clinical trials in cancer patients, and our experiments highlight concerns on the homeostasis and regenerative capacity of muscles in these patients who often experience cachexia.

Ablation of Protein Kinase CK2β in Skeletal Muscle Fibers Interferes with Their Oxidative Capacity.

The tetrameric protein kinase CK2 was identified playing a role at neuromuscular junctions by studying CK2β-deficient muscle fibers in mice, and in cultured immortalized C2C12 muscle cells after individual knockdown of CK2α and CK2β subunits. In muscle cells, CK2 activity appeared to be at least required for regular aggregation of nicotinic acetylcholine receptors, which serves as a hallmark for the presence of a postsynaptic apparatus. Here, we set out to determine whether any other feature accompanies CK2β-deficient muscle fibers. Hind limb muscles gastrocnemius, plantaris, and soleus of adult wildtype and CK2β-deficient mice were dissected, cross-sectioned, and stained histochemically by Gomori trichrome and for nicotinamide adenine dinucleotide (NADH) dehydrogenase and succinate dehydrogenase (SDH) enzymatic activities. A reduction of oxidative enzymatic activity was determined for CK2β-deficient muscle fibers in comparison with wildtype controls. Importantly, the CK2β-deficient fibers, muscle fibers that typically exhibit high NADH dehydrogenase and SDH activities, like slow-type fibers, showed a marked reduction in these activities. Altogether, our data indicate additional impairments in the absence of CK2β in skeletal muscle fibers, pointing to an eventual mitochondrial myopathy.

Laminin is instructive and calmodulin dependent kinase II is non-permissive for the formation of complex aggregates of acetylcholine receptors on myotubes in culture

Previous work has shown that myotubes cultured on laminin-coated substrates form complex aggregates of synaptic proteins that are similar in shape and composition to neuromuscular junctions (NMJs). Here we show that laminin instructs the location of complex aggregates which form only on the lower surface when laminin is coated onto culture dishes but over the entire cell when laminin is added in solution. Silencing of myotubes by agents that block electrical activity (tetrodotoxin, verapamil) or by inhibitors of calmodulin dependent kinase (CaMKII) render the myotube permissive for the formation of complex aggregates. Treatment with laminin alone will facilitate the formation of complex aggregates hours later when myotubes are made permissive by inhibiting CaMKII. The AChR agonist carbachol disperses pre formed aggregates suggesting that non-permissiveness may involve active dispersal of AChRs. The permissive period requires ongoing protein synthesis. The latter may reflect a requirement for rapsyn, which turns over rapidly, and is necessary for aggregation. Consistent with this geldanamycin, an agent that increases rapsyn turnover disrupts complex aggregates. Agrin is well known to induce small clusters of AChRs but does not induce complex aggregates even though aggregate formation requires MuSK, a receptor tyrosine kinase activated by agrin. Dystroglycan (DG) is the major laminin receptor mediating complex aggregate formation with some contribution from β1 integrins. In addition, there is a pool of CaMKII associated with DG. We discuss how these permissive and instructive mechanisms bear on NMJ formation in vivo.

LAP proteins are localized at the post-synaptic membrane of neuromuscular junctions and appear to modulate synaptic morphology and transmission.

Erbin, Lano, Scribble, and Densin-180 belong to LAP (leucine-rich repeats and PDZ domain) adaptor proteins involved in cell signaling pathways. Previously, we identified Erbin, Lano, and Scribble, but not Densin-180, in muscle cells, where they are involved in regulating the aggregation of nicotinic acetylcholine receptors invitro. Here, we analyzed their cellular localization at the neuromuscular junction (NMJ) in skeletal muscles of mice. Erbin, Lano, and Scribble were significantly accumulated at NMJs and localized in different synaptic cells. Moreover, we used mouse mutants to analyze the role of Erbin at the NMJ. We used two Erbin mutant mouse strains that either completely lack Erbin protein (Erbinnull/null ) or express a truncated Erbin mutant where the carboxy-terminal PDZ domain is replaced by β-galactosidase (ErbinΔC/ΔC ) thereby abolishing its interaction with ErbB receptor tyrosine kinases. Neither the lack of the PDZ domain of Erbin, nor its complete absence interfered with the general localization of LAP proteins at NMJs, but Lano and Scribble transcript levels were up-regulated in homozygous Erbin-null muscles. Furthermore, grip strength was reduced and neural transmission impaired in homozygous aged Erbin-null but not Erbin-ΔC mice. Erbin-null skeletal muscles did not reveal any conspicuous impairment of the muscle fiber. Localization of other NMJ marker proteins was not affected either. Quantitative 3D morphometry showed that NMJs of Erbin-null muscles were significantly smaller and fragmented in the soleus. We speculate that Erbin, Lano, and Scribble act at the post-synaptic membrane of NMJs in a concerted fashion to regulate nicotinic acetylcholine receptors cluster morphology and neural transmission. Cover Image for this issue: doi: 10.1111/jnc.13340.

A CHRNB1 frameshift mutation is associated with familial arthrogryposis multiplex congenita in Red dairy cattle.

BackgroundBovine arthrogryposis multiplex congenita (AMC) is a syndromic term for a congenital condition characterized by multiple joint contractures. Rare inherited forms of bovine AMC have been reported in different breeds. For AMC in Angus cattle a causative genomic deletion encompassing the agrin (AGRN) gene, encoding an essential neural regulator that induces the aggregation of acetylcholine receptors (AChRs), is known. In 2015, three genetically related cases of generalized AMC affecting Red dairy calves were diagnosed in Denmark.ResultsThe family history of three affected calves suggested an autosomal recessive inheritance. Single nucleotide polymorphism (SNP) genotyping showed a single genomic region of extended homozygosity of 21.5 Mb on chromosome 19. Linkage analysis revealed a maximal parametric LOD score of 1.8 at this region. By whole genome re-sequencing of the three cases, two private homozygous non-synonymous variants were detected in the critical interval. Both variants, located in the myosin phosphatase Rho interacting protein (MPRIP) and the cholinergic receptor nicotinic beta 1 subunit gene (CHRNB1), were perfectly associated with the AMC phenotype. Previously described CHRNB1 variants in humans lead to a congenital myasthenic syndrome with impaired neuromuscular transmission. The cattle variant represents a single base deletion in the first exon of CHRNB1 (c.55delG) introducing a premature stop codon (p.Ala19Profs47*) in the second exon, truncating 96 % of the protein.ConclusionsThis study provides the first phenotypically and genetically characterized example of a bovine AMC phenotype that represents an inherited neuromuscular disorder corresponding to human congenital myasthenic syndrome. The identified CHRNB1 loss of function variant is predicted to have a deleterious effect on fetal AChR function, which could explain the lethal phenotype reported in this study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of CHRNB1 mutations and enables selection against this pathogenic variant in Red dairy cattle.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2832-x) contains supplementary material, which is available to authorized users.

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

Differential regulation of AChR clustering in the polar and equatorial region of murine muscle spindles.

Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.

Chemical chaperones exceed the chaperone effects of RIC-3 in promoting assembly of functional α7 AChRs.

Functional α7 nicotinic acetylcholine receptors (AChRs) do not assemble efficiently in cells transfected with α7 subunits unless the cells are also transfected with the chaperone protein RIC-3. Despite the presence of RIC-3, large amounts of these subunits remain improperly assembled. Thus, additional chaperone proteins are probably required for efficient assembly of α7 AChRs. Cholinergic ligands can act as pharmacological chaperones to promote assembly of mature AChRs and upregulate the amount of functional AChRs. In addition, we have found that the chemical chaperones 4-phenylbutyric acid (PBA) and valproic acid (VPA) greatly increase the amount of functional α7 AChRs produced in a cell line expressing both α7 and RIC-3. Increased α7 AChR expression allows assay of drug action using a membrane potential-sensitive fluorescent indicator. Both PBA and VPA also increase α7 expression in the SH-SY5Y neuroblastoma cell line that endogenously expresses α7 AChRs. VPA increases expression of endogenous α7 AChRs in hippocampal neurons but PBA does not. RIC-3 is insufficient for optimal assembly of α7 AChRs, but provides assay conditions for detecting additional chaperones. Chemical chaperones are a useful pragmatic approach to express high levels of human α7 AChRs for drug selection and characterization and possibly to increase α7 expression in vivo.