Salbutamol modifies the neuromuscular junction in a mouse model of ColQ myasthenic syndrome.

The ß-adrenergic agonists salbutamol and ephedrine have proven to be effective as therapies for human disorders of the neuromuscular junction, in particular many subsets of congenital myasthenic syndromes. However, the mechanisms underlying this clinical benefit are unknown and improved understanding of the effect of adrenergic signalling on the neuromuscular junction is essential to facilitate the development of more targeted therapies. Here, we investigated the effect of salbutamol treatment on the neuromuscular junction in the ColQ deficient mouse, a model of end-plate acetylcholinesterase deficiency. ColQ-/- mice received 7 weeks of daily salbutamol injection, and the effect on muscle strength and neuromuscular junction morphology was analysed. We show that salbutamol leads to a gradual improvement in muscle strength in ColQ-/- mice. In addition, the neuromuscular junctions of salbutamol treated mice showed significant improvements in several postsynaptic morphological defects, including increased synaptic area, acetylcholine receptor area and density, and extent of postjunctional folds. These changes occurred without alterations in skeletal muscle fibre size or type. These findings suggest that ß-adrenergic agonists lead to functional benefit in the ColQ-/- mouse and to long-term structural changes at the neuromuscular junction. These effects are primarily at the postsynaptic membrane and may lead to enhanced neuromuscular transmission.

GFPT1 deficiency in muscle leads to myasthenia and myopathy in mice.

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway which yields precursors required for protein and lipid glycosylation. Mutations in GFPT1 and other genes downstream of this pathway cause congenital myasthenic syndrome (CMS) characterized by fatigable muscle weakness owing to impaired neurotransmission. The precise pathomechanisms at the neuromuscular junction (NMJ) owing to a deficiency in GFPT1 is yet to be discovered. One of the challenges we face is the viability of Gfpt1-/- knockout mice. In this study, we use Cre/LoxP technology to generate a muscle-specific GFPT1 knockout mouse model, Gfpt1tm1d/tm1d, characteristic of the human CMS phenotype. Our data suggest a critical role for muscle derived GFPT1 in the development of the NMJ, neurotransmission, skeletal muscle integrity and highlight that a deficiency in skeletal muscle alone is sufficient to cause morphological postsynaptic NMJ changes that are accompanied by presynaptic alterations despite the conservation of neuronal GFPT1 expression. In addition to the conventional morphological NMJ changes and fatigable muscle weakness, Gfpt1tm1d/tm1d mice display a progressive myopathic phenotype with the presence of tubular aggregates in muscle, characteristic of the GFPT1-CMS phenotype. We further identify an upregulation of skeletal muscle proteins glypican-1, farnesyltransferase/geranylgeranyltransferase type-1 subunit α and muscle-specific kinase, which are known to be involved in the differentiation and maintenance of the NMJ. The Gfpt1tm1d/tm1d model allows for further investigation of pathophysiological consequences on genes and pathways downstream of GFPT1 likely to involve misglycosylation or hypoglycosylation of NMJs and muscle targets.

The Structure of Human Neuromuscular Junctions: Some Unanswered Molecular Questions.

The commands that control animal movement are transmitted from motor neurons to their target muscle cells at the neuromuscular junctions (NMJs). The NMJs contain many protein species whose role in transmission depends not only on their inherent properties, but also on how they are distributed within the complex structure of the motor nerve terminal and the postsynaptic muscle membrane. These molecules mediate evoked chemical transmitter release from the nerve and the action of that transmitter on the muscle. Human NMJs are among the smallest known and release the smallest number of transmitter "quanta". By contrast, they have the most deeply infolded postsynaptic membranes, which help to amplify transmitter action. The same structural features that distinguish human NMJs make them particularly susceptible to pathological processes. While much has been learned about the molecules which mediate transmitter release and action, little is known about the molecular processes that control the growth of the cellular and subcellular components of the NMJ so as to give rise to its mature form. A major challenge for molecular biologists is to understand the molecular basis for the development and maintenance of functionally important aspects of NMJ structure, and thereby to point to new directions for treatment of diseases in which neuromuscular transmission is impaired.

Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates.

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission.

Glutamatergic modulation of synaptic-like vesicle recycling in mechanosensory lanceolate nerve terminals of mammalian hair follicles.

Our aim in the present study was to determine whether a glutamatergic modulatory system involving synaptic-like vesicles (SLVs) is present in the lanceolate ending of the mouse and rat hair follicle and, if so, to assess its similarity to that of the rat muscle spindle annulospiral ending we have described previously. Both types of endings are formed by the peripheral sensory terminals of primary mechanosensory dorsal root ganglion cells, so the presence of such a system in the lanceolate ending would provide support for our hypothesis that it is a general property of fundamental importance to the regulation of the responsiveness of the broad class of primary mechanosensory endings. We show not only that an SLV-based system is present in lanceolate endings, but also that there are clear parallels between its operation in the two types of mechanosensory endings. In particular, we demonstrate that, as in the muscle spindle: (i) FM1-43 labels the sensory terminals of the lanceolate ending, rather than the closely associated accessory (glial) cells; (ii) the dye enters and leaves the terminals primarily by SLV recycling; (iii) the dye does not block the electrical response to mechanical stimulation, in contrast to its effect on the hair cell and dorsal root ganglion cells in culture; (iv) SLV recycling is Ca(2+) sensitive; and (v) the sensory terminals are enriched in glutamate. Thus, in the lanceolate sensory ending SLV recycling is itself regulated, at least in part, by glutamate acting through a phospholipase D-coupled metabotropic glutamate receptor.

Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin.

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066-1315 (emGFP-1066:TetC), 1093-1315 (emGFP-1093:TetC) and 1109-1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

'Fragmentation' of NMJs: a sign of degeneration or regeneration? A long journey with many junctions.

Mammalian neuromuscular junctions (NMJs) often consist of curved bands of synaptic contact, about 3-6 µm wide, which resemble pretzels. This contrasts with the NMJs of most animal species which consist of a cluster of separate synaptic spots, each of which is also about 3-6 µm across. In a number of situations, including a variety of disease states as well as normal ageing, mammalian NMJs acquire a more 'fragmented' appearance that resembles somewhat that of other species. This 'fragmentation' of the NMJ has sometimes been interpreted as a 'disintegration' or 'degeneration', with the suggestion that it might be associated with impaired neuromuscular transmission. An alternative view is that NMJ fragmentation is the outcome of a normal process by which the NMJ is maintained in an effective state. In this highly personal commentary, I cite a number of examples of this and point out that although the 'pretzel' form arises during normal development as a result of the sculpting of an immature synaptic 'plaque', in virtually all situations where new synaptic contact is established in adult mammals this occurs by the addition of new synaptic 'spots' rather than by the extension, or neoformation, of 'pretzels'. Further, where appropriate studies have been performed, no evidence of a correlation between the degree of fragmentation and the efficacy of transmission has emerged. It may therefore be more appropriate to consider NMJ 'fragmentation' as a form of regeneration, rather than of degeneration. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Modulation of the Acetylcholine Receptor Clustering Pathway Improves Neuromuscular Junction Structure and Muscle Strength in a Mouse Model of Congenital Myasthenic Syndrome.

Introduction: Congenital myasthenic syndromes (CMS) are a diverse group of inherited neuromuscular disorders characterized by a failure of synaptic transmission at the neuromuscular junction (NMJ). CMS often present early with fatigable weakness and can be fatal through respiratory complications. The AGRN gene is one of over 30 genes known to harbor mutations causative for CMS. In this study, we aimed to determine if a compound (NT1654), developed to stimulate the acetylcholine receptor (AChR) clustering pathway, would benefit a mouse model of CMS caused by a loss-of-function mutation in Agrn (Agrn nmf380 mouse). Methods: Agrn nmf380 mice received an injection of either NT1654 or vehicle compound daily, with wild-type litter mates used for comparison. Animals were weighed daily and underwent grip strength assessments. After 30 days of treatment animals were sacrificed, and muscles collected. Investigations into NMJ and muscle morphology were performed on collected tissue. Results: While minimal improvements in NMJ ultrastructure were observed with electron microscopy, gross NMJ structure analysis using fluorescent labelling and confocal microscopy revealed extensive postsynaptic improvements in Agrn nmf380 mice with NT1654 administration, with variables frequently returning to wild type levels. An improvement in muscle weight and myofiber characteristics helped increase forelimb grip strength and body weight. Conclusions: We conclude that NT1654 restores NMJ postsynaptic structure and improves muscle strength through normalization of muscle fiber composition and the prevention of atrophy. We hypothesize this occurs through the AChR clustering pathway in Agrn nmf380 mice. Future studies should investigate if this may represent a viable treatment option for patients with CMS, especially those with mutations in proteins of the AChR clustering pathway.

Modulation of Agrin and RhoA Pathways Ameliorates Movement Defects and Synapse Morphology in MYO9A-Depleted Zebrafish.

Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterised by impaired function of the neuromuscular junction (NMJ). This is due to defects in one of the many proteins associated with the NMJ. In three patients with CMS, missense mutations in a gene encoding an unconventional myosin protein, MYO9A, were identified as likely causing their disorder. Preliminary studies revealed a potential involvement of the RhoA/ROCK pathway and of a key NMJ protein, agrin, in the pathophysiology of MYO9A-depletion. In this study, a CRISPR/Cas9 approach was used to generate genetic mutants of MYO9A zebrafish orthologues, myo9aa/ab, to expand and refine the morphological analysis of the NMJ. Injection of NT1654, a synthetic agrin fragment compound, improved NMJ structure and zebrafish movement in the absence of Myo9aa/ab. In addition, treatment of zebrafish with fasudil, a ROCK inhibitor, also provided improvements to the morphology of NMJs in early development, as well as rescuing movement defects, but not to the same extent as NT1654 and not at later time points. Therefore, this study highlights a role for MYO9A at the NMJ, the first unconventional myosin motor protein associated with a neuromuscular disease, and provides a potential mechanism of action of MYO9A-pathophysiology.

Structural factors influencing the efficacy of neuromuscular transmission.

Neuromuscular junctions (NMJs) in different species share many features of structure and function. At the same time, important differences distinguish, for example, human NMJs from those in other species. An understanding of the biological context of the human NMJ helps in the interpretation of the effects of disease on the biophysical properties of neuromuscular transmission. Many NMJs consist of a number of spot-like synaptic regions 1-5 microm across. Usually only a few multimolecular "quanta" of transmitter are released from each presynaptic "bouton" by a single nerve impulse. The total number of quanta released from an NMJ is roughly proportional to its total area. For example, human NMJs are about 10-fold smaller than those in frogs and release about 20 quanta/impulse versus 100-200 in frog NMJ. Although human NMJs release relatively few quanta, the effect of the transmitter is amplified by the high density of voltage-gated sodium channels (Na(V)1.4) in the highly folded postsynaptic membrane. A genetic influence on NMJ size has recently been discovered in some patients with limb-girdle myasthenia (LGM). Mutations of the gene encoding Dok-7, an essential component of the agrin-muscle-specific kinase pathway that controls postsynaptic differentiation at the mammalian NMJ, results in impaired transmission because the NMJs are abnormally small and have reduced folding but have a normal local density of normal acetylcholine receptors. This condition emphasizes the importance of structural features in achieving reliability of neuromuscular transmission.

Congenital myasthenic syndromes and the formation of the neuromuscular junction.

The congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders affecting neuromuscular transmission. Underlying mutations have been identified in at least 11 different genes. The majority of CMS patients have disorders due to mutations in postsynaptic proteins. Initial studies focused on dysfunction of the acetylcholine receptor (AChR) itself as the major cause of CMS. However, it is becoming apparent that mutations of proteins involved in clustering the AChR and maintaining neuromuscular junction structure form important subgroups. Analysis of the mutations in the AChR-clustering protein, rapsyn, show diverse causes for defective AChR localization and suggest that the common mutation rapsyn-N88K results in AChR clusters that are less stable than those generated by wild-type rapsyn. More recently, mutations in the newly identified endplate protein Dok-7 have been shown to affect AChR clustering and the generation and maintenance of specialized structures at the endplate. Dok-7 binds MuSK and many of the mutations of DOK7 impair the MuSK signaling pathway. Components of this pathway will provide attractive gene candidates for additional forms of CMS. The phenotypic characteristics of the different CMS in which muscle groups may be differentially affected not only provide clues for targeted genetic screening, but also pose further intriguing questions about underlying molecular mechanisms.

Age-related changes in the structure and function of mammalian neuromuscular junctions.

As mammals age, their neuromuscular junctions (NMJs) change their form, with an increasingly complex system of axonal branches innervating increasingly fragmented regions of postsynaptic differentiation. It has been suggested that this remodeling is associated with impairment of neuromuscular transmission and that this contributes to age-related muscle weakness in mammals, including humans. Here, we review previous work on NMJ aging, most of which has focused on either structure or function, as well as a new study aimed at seeking correlation between the structure and function of individual NMJs. While it is clear that extensive structural changes occur as part of the aging process, it is much less certain how, if at all, these are correlated with an impairment of function. This leaves open the question of whether loss of NMJ function is a significant cause of age-related muscle weakness.

Congenital Myasthenic Syndromes or Inherited Disorders of Neuromuscular Transmission: Recent Discoveries and Open Questions.

Congenital myasthenic syndromes (CMS) form a heterogeneous group of rare diseases characterized by fatigable muscle weakness. They are genetically-inherited and caused by defective synaptic transmission at the cholinergic neuromuscular junction (NMJ). The number of genes known to cause CMS when mutated is currently 30, and the relationship between fatigable muscle weakness and defective functions is quite well-understood for many of them. However, some of the most recent discoveries in individuals with CMS challenge our knowledge of the NMJ, where the basis of the pathology has mostly been investigated in animal models. Frontier forms between CMS and congenital myopathy, which have been genetically and clinically identified, underline the poorly understood interplay between the synaptic and extrasynaptic molecules in the neuromuscular system. In addition, precise electrophysiological and histopathological investigations of individuals with CMS suggest an important role of NMJ plasticity in the response to CMS pathogenesis. While efficient drug-based treatments are already available to improve neuromuscular transmission for most forms of CMS, others, as well as neurological and muscular comorbidities, remain resistant. Taken together, the available pathological data point to physiological issues which remain to be understood in order to achieve precision medicine with efficient therapeutics for all individuals suffering from CMS.

Age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission in the mouse diaphragm.

As mammals age, their neuromuscular junctions (NMJs) gradually change their form, acquiring an increasingly fragmented appearance consisting of numerous isolated regions of synaptic differentiation. It has been suggested that this remodelling is associated with impairment of neuromuscular transmission, and that this contributes to age-related muscle weakness in mammals, including humans. The underlying hypothesis, that increasing NMJ fragmentation is associated with impaired transmission, has never been directly tested. Here, by comparing the structure and function of individual NMJs, we show that neuromuscular transmission at the most highly fragmented NMJs in the diaphragms of old (26-28 months) mice is, if anything, stronger than in middle-aged (12-14 months) mice. We suggest that NMJ fragmentation per se is not a reliable indicator of impaired neuromuscular transmission.

Voltage-gated sodium channels and ankyrinG occupy a different postsynaptic domain from acetylcholine receptors from an early stage of neuromuscular junction maturation in rats.

Spatial segregation of membrane proteins is a feature of many excitable cells. In skeletal muscle, clusters of acetylcholine receptors (AChRs) and voltage-gated sodium channels (Na(V)1s) occupy distinct domains at the neuromuscular junction (NMJ). We used quantitative immunolabeling of developing rat soleus muscles to study the mechanism of ion channel segregation and Na(V)1 clustering at NMJs. When Na(V)1s can first be detected, at birth, they already occupy a postsynaptic domain that is distinct from that occupied by AChRs. At this time, Na(V)1s are expressed only in a diffuse area that extends 50-100 microm from the immature NMJ. However, in the region of the high-density AChR cluster at NMJ itself, Na(V)1s are actually present in lower density than in the immediately surrounding membrane. These distinctive features of the Na(V)1 distribution at birth are closely correlated with the distribution of ankyrinG immunolabeling. This suggests that an interaction with ankyrinG plays a role in the initial segregation of Na(V)1s from AChRs. Both Na(V)1 and ankyrinG become clustered at the NMJ itself 1-2 weeks after birth, coincident with the formation of postsynaptic folds. Syntrophin immunolabeling codistributes with AChRs and never resembles that for Na(V)1 or ankyrinG. Therefore, syntrophin is unlikely to play an important part in the initial accumulation of Na(V)1 at the NMJ. These findings suggest that the segregation of Na(V)1 from AChRs begins early in NMJ formation and occurs as a result of the physical exclusion of Na(V)1 and ankyrinG from the region of nerve-muscle contact rather than by a process of active clustering.

The functional organization of motor nerve terminals.

Neuromuscular junctions (NMJs) have long been studied as particularly accessible examples of chemical synapses. Nonetheless, some important features of neuromuscular transmission are still poorly understood. One of these is the low statistical variability of the number of transmitter quanta released from motor nerve terminals by successive nerve impulses. This variability is well-described by a binomial distribution, suggesting that the quanta released are drawn, at high probability, from a small subset of those in the terminals. However, the nature of that subset remains unclear. In an effort to clarify what is understood, and what is not, about quantal release at NMJs, this review addresses the relationship between NMJ structure and function. After setting the biological context in which NMJs operate, key aspects of the variability of release and the structure of the motor nerve terminals are described. These descriptions are then used to explore the functional logic of motor nerve terminal organization and the structural basis of the low variability of release. This analysis supports the suggestion that the probability of release differs significantly at the different 'active zones' from which quanta are released. Finally, after a brief consideration of how release is maintained in the long term, a comparison is made of the features of NMJs with those of some well-studied neuronal synapses. An important conclusion is that NMJs share some important features with neuronal synapses, so continuing efforts to understand how motor nerve terminals work are likely to have much more general implications.

Structural abnormalities of the AChR caused by mutations underlying congenital myasthenic syndromes.

The objective was to define the molecular mechanisms underlying congenital myasthenic syndromes (CMS) by studying mutations within genes encoding the acetylcholine receptor (AChR) and related proteins at the neuromuscular junction. It was found that mutations within muscle AChRs are the most common cause of CMS. The majority are located within the epsilon-subunit gene and result in AChR deficiency.

Dok-7 mutations underlie a neuromuscular junction synaptopathy.

Congenital myasthenic syndromes (CMSs) are a group of inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. One major subgroup of patients shows a characteristic "limb girdle" pattern of muscle weakness, in which the muscles have small, simplified neuromuscular junctions but normal acetylcholine receptor and acetylcholinesterase function. We showed that recessive inheritance of mutations in Dok-7, which result in a defective structure of the neuromuscular junction, is a cause of CMS with proximal muscle weakness.

Accumulation of Nav1 mRNAs at differentiating postsynaptic sites in rat soleus muscles.

Acetylcholine receptors (AChRs) and voltage-gated sodium channels (Na(V)1s) accumulate at different times in the development of the murine neuromuscular junction (NMJ). We used in situ hybridization to study the relationship of Na(V)1 mRNA accumulation to this difference. mRNAs encoding both muscle Na(V)1 isoforms, Na(v)1.4 and Na(v)1.5, were first concentrated at NMJs at birth, when the proteins start to accumulate. Within 4 weeks, Na(v)1.4 mRNA increased 5-fold at the NMJ while Na(v)1.5 mRNA became undetectable. Na(V)1 mRNA accumulation occurred even if the nerve was cut at birth. Like AChR mRNA, Na(V)1 mRNA accumulated at denervated synaptic sites on regenerating muscles and in response to ectopically expressed neural agrin. Clustering of Na(V)1 at the NMJ follows that of its mRNA while AChR clustering precedes its mRNA clustering by several days. This suggests that factors other than local mRNA upregulation determine the timing of clustering of these two important postsynaptic ion channels.