Human Neuromuscular Junction Research Articles

Inherited disorders of the neuromuscular junction: an update.

Congenital myasthenic syndromes (CMSs) are a group of heterogeneous inherited disorders caused by mutations in genes affecting the function and structure of the neuromuscular junction. This review updates the reader on established and novel subtypes of congenital myasthenia, and the treatment strategies for these increasingly heterogeneous disorders. The discovery of mutations associated with the N-glycosylation pathway and in the family of serine peptidases has shown that causative genes encoding ubiquitously expressed molecules can produce defects at the human neuromuscular junction. By contrast, mutations in lipoprotein-like receptor 4 (LRP4), a long-time candidate gene for congenital myasthenia, and a novel phenotype of myasthenia with distal weakness and atrophy due to mutations in AGRN have now been described. In addition, a pathogenic splicing mutation in a nonfunctional exon of CHRNA1 has been reported emphasizing the importance of analysing nonfunctional exons in genetic analysis. The benefit of salbutamol and ephedrine alone or combined with pyridostigmine or 3,4-DAP is increasingly being reported for particular subtypes of CMS.

Congenital myasthenic syndromes and the neuromuscular junction.

Congenital myasthenic syndromes (CMS) are a group of heterogeneous inherited disorders caused by mutations in genes encoding proteins essential for the integrity of neuromuscular transmission. This review updates the reader on the new mutations of known CMS genes, new causative genes and the treatment strategies for these increasingly heterogeneous disorders. It also provides a brief summary of the congenital myopathies with myasthenic features. The discovery of causative genes encoding for ubiquitously expressed and extrajunctional molecules has changed our previous view of congenital myasthenia. Mutations associated with the N-glycosylation pathway and in the family of serine peptidases have shown that abnormalities in the post-translational modification of proteins can produce defects at the human neuromuscular junction. However, mutations in lipoprotein-like receptor 4, a long-time candidate gene for congenital myasthenia, have now been described and a new pathogenic splicing mutation in the nonfunctional exon of CHRNA1 has been reported. The use of salbutamol and ephedrine alone or combined with pyridostigmine or 3,4-DAP is increasingly being reported in different CMS subtypes with significant benefit. Recent studies of the CMS illustrate the increasing complexity of the genetics, pathophysiological mechanisms and therapy of impaired synaptic transmission at the neuromuscular junction.

Different types of botulinum neurotoxins in mouse and human neuromuscular junction

Different types of botulinum neurotoxins in mouse and human neuromuscular junction

Simulation of the Kinetics of Neuromuscular Block

The onset time for paralysis varies 3-fold among nondepolarizing muscle relaxants. Possible explanations include: (a) pharmacokinetic differences among drugs and (b) buffering of drug molecules by acetylcholine receptors as they diffuse into the neuromuscular junction. Although some pharmacokinetic models consider buffered diffusion, these models do not account for either the high density of receptors or synapse geometry. Here, I used computer simulations to calculate the kinetics of buffered diffusion. The goal was to determine the conditions under which buffered diffusion could account for differences in onset time among nondepolarizing muscle relaxants. Monte Carlo simulation was used along with a realistic 3-dimensional model of the rat neuromuscular junction. Simulations determined the time dependence of the number of drug-bound receptors. A 1000-fold range of drug potency was examined. In some simulations, the drug concentration outside the junction was changed instantaneously. In other simulations, the concentration changed according to predictions of pharmacokinetic models assuming time-dependent changes in plasma drug concentration. The rate constant for equilibration of drug between plasma and muscle, keo, was varied between 0.15 and 0.6 min(-1). Twitch amplitude was calculated from receptor occupancy assuming a high safety margin for neuromuscular transmission. Some simulations used a synaptic model with an increased nerve-muscle contact width. Simulations with instantaneous changes in drug concentration at the synapse, indicated that the time to 50% twitch depression (onset time) was 0.1 to 30 seconds and was proportional to drug potency. This corresponds to iontophoretic application of drug to isolated neuromuscular junctions, but is too fast to explain onset times in humans. When pharmacokinetic models were used to calculate the drug concentration outside the synapse, buffered diffusion increased onset times of potent drugs (drugs for which the effective concentration at 50% twitch height is <600 nM). Simulations using keo = 0.6 min(-1) and a model with a 2- to 3-fold wider nerve-muscle contact width indicated that buffered diffusion could account for the differences in clinical onset times among the nondepolarizing muscle relaxants. Monte Carlo simulation provides a biophysically appropriate way to incorporate buffered diffusion into pharmacokinetic modeling. The simulations indicated that buffered diffusion could account for differences in onset time among drugs. However, a better understanding of the geometry of the human neuromuscular junction is needed before the magnitude of the effect of buffered diffusion can be quantified.

Using induced pluripotent stem cells (iPSC) to model human neuromuscular connectivity: promise or reality?

Motor neuron diseases (MND) such as amyotrophic lateral sclerosis and spinal muscular atrophy are devastating, progressive and ultimately fatal diseases for which there are no effective treatments. Recent evidence from systematic studies of animal models and human patients suggests that the neuromuscular junction (NMJ) is an important early target in MND, demonstrating functional and structural abnormalities in advance of pathological changes occurring in the motor neuron cell body. The ability to study pathological changes occurring at the NMJ in humans is therefore likely to be important for furthering our understanding of disease pathogenesis, and also for designing and testing new therapeutics. However, there are many practical and technical reasons why it is not possible to visualise or record from NMJs in pre- and early-symptomatic MND patients in vivo. Other approaches are therefore required. The development of stem cell technologies has opened up the possibility of creating human NMJs in vitro, using pluripotent cells generated from healthy individuals and patients with MND. This review covers historical attempts to develop mature and functional NMJs in vitro, using co-cultures of muscle and nerve from animals, and discusses how recent developments in the generation and specification of human induced pluripotent stem cells provides an opportunity to build on these previous successes to recapitulate human neuromuscular connectivity in vitro.

Evolution and comparative genomics of subcellular specializations: EST sequencing of Torpedo electric organ

Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (> 63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes ( C19orf29 [ Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specialization using evolutionary diversity of organ function and cross-species mapping.

Mutations in LAMB2 causing a severe form of synaptic congenital myasthenic syndrome

Background:We describe a severe form of congenital myasthenic syndrome (CMS) associated with congenital nephrosis and ocular malformations caused by two truncating mutations in the gene encoding the laminin β2 subunit...

Expression of platelet-derived growth factor and its receptor at human and mouse neuromuscular junctions

Objective To investigate the immunoreactivity of platelet-derived growth factor-A (PDGF-A) and its receptor at human and mouse neuromuscular junctions and to explore their regulatory effects on neuromuscular junctions. Methods Specific polyclonal antibodies were used to detect PDGF-A and PDGF a-receptor expression. Double immunohistochemistry for platelet-derived growth factor and the acetylcholine receptor was performed on normal human muscle biopsy specimens. Double fluorescence labeling was applied to detect immunoreactivity for PDGF-A, its receptor and acetylcholine receptors. Results PDGF-A and its receptor was closely co-localized with acetylcholine receptors at human and mouse neuromuscular junctions. Conclusion PDGF-A and its receptor concentrated at human and mouse neuromuscular junctions. PDGF might be involved in the interaction between the presynaptic and postsynaptic components, PDGF-A and its receptor might play regulatory role in signaling at neuromuscular junctions in normal muscle. Key words: Platelet-derived growth factor; Receptor; Neuromuscular junction

Venom of Collett's snake ( Pseudechis colletti) blocks the binding of α-bungarotoxin to acetylcholine receptors at chick but not human neuromuscular junctions: A histochemical study

Transverse cryosections, 6–8 μm thick, were cut from unfixed biventer cervicis muscles of chicks and quadriceps muscles of humans, mounted on glass slides and incubated for 1 h in either isotonic phosphate buffered saline, pH 7.3 (PBS), or crude venom of venom of Pseudechis colletti at concentrations between 2.1 and 210 μg ml −1 in PBS. They were then exposed to a fluoresceine-conjugated α-bungarotoxin to label ACh receptor sites. Exposure to the crude venom of P. colletti prevented the labelling of acetylcholine (ACh) receptors in chick muscle in a dose-dependant manner; at a concentration of 2.1 μg ml −1 labelling fell by 20% and at a concentration of 21 μg ml −1 by more than 90%. In contrast, exposure to the venom at concentrations as high as 210 μg ml −1 had no effect on receptor labelling in human skeletal muscle. The results suggest that ACh receptors in human skeletal muscle are relatively resistant to the postsynaptically active neurotoxins in the venom of P. colletti. The data explain the apparent anomaly that the venom blocks neuromuscular transmission in isolated nerve-muscle preparations of the chick whilst human subjects of envenoming bites by P. colletti exhibit no overt signs of neurotoxicity.

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.

Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs

BackgroundThe Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown.ResultsUsing antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart.ConclusionThe localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.

Definition of pre- and postsynaptic forms of the CT carbohydrate antigen at the neuromuscular junction: ubiquitous expression of the CT antigens and the CT GalNAc transferase in mouse tissues

At the rodent neuromuscular junction, the synaptic expression of the CT carbohydrate antigens is defined by the binding of two monoclonal antibodies, CT1 and CT2. CT1 preferentially stains the presynaptic membrane, while CT2 preferentially stains the postsynaptic apparatus. Here we show that the differential subsynaptic distribution of these antigens is due to a preference of CT1 for structures containing N-acetyl neuraminic acid (NeuAc) and a preference of CT2 for structures containing N-glycolyl neuraminic acid (NeuGc). This was found to be the case both in binding to cultured myotubes, where NeuAc/NeuGc levels were manipulated by feeding acetylated N-acetyl mannosamine precursors, and in binding to purified GM2 ganglioside containing either NeuAc or NeuGc. At human neuromuscular junctions, where the enzymatic machinery to make NeuGc is absent [Proc. Natl. Acac. Sci. USA 95 (1998) 11751], CT1 and GM2(NeuAc) antibodies stained, while CT2 did not. Thus, the N-glycolyl modification of sialic acid helps to define the differential distribution of the CT antigens at the rodent neuromuscular junction, and this difference is lost in humans. In addition, sulfatase and 9- O-acetylesterase treatment of cells or tissues increased the amount of CT1 and CT2 antibody binding, with sulfatase differentially unmasking CT antigen expression on particular glycoproteins. Despite its uniquely synaptic localization in skeletal muscle, the CT antigens and the CT GalNAc transferase are ubiquitously expressed in other mouse tissues, including brain, spinal cord, and peripheral nerve. One of the proteins that can be co-purified with a CT-reactive glycoprotein is α dystroglycan. These data better define the sub-synaptic structures of the CT carbohydrate antigens at the neuromuscular junction and demonstrate their ubiquitous presence in mouse tissues, including the brain.

Book review

This ‘atlas of structural and molecular pathological changes’ is an edited English language edition of a German textbook on the pathology of peripheral nerves originally published as part of the series ‘Spezielle Pathologische Anatomie’, edited by Doerr, Siefert and Uehlinger and published by Springer-Verlag. It is exactly as the name implies – an atlas of over 1000 figures, supported by over 2000 references, largely derived from the collection of material assembled by the author during a professional life of more than 35 years. The atlas is beautifully produced on high quality paper and it contains many examples of superb microscopy. The atlas is organized into defined sections. The first chapters cover general topics. The introduction is a good summary of basic information on axonal growth, myelination, nodal and paranodal axon diameter and the relationships between these variables. Most data are drawn from measurements made on sural nerve biopsies. There follows a very brief chapter on the epidemiology and classification of peripheral nerve disorders and a more substantial chapter on the general reactions of peripheral nerves to a variety of lesions. Subsequent chapters deal with more specific topics: toxic neuropathies, paraneoplastic syndromes and tumours of the peripheral nervous system are typical. It is a complete compendium and could be considered complementary to the 2nd edition of Experimental and Clinical Neurotoxicology (Spencer, Schaumburg and Ludolph) and the new publication Medical Neurotoxicology (Blain and Harris). An investigator using this atlas to help in the interpretation of clinical pathological data will probably start by reading the introductory chapters. These chapters are accurate but they are curiously limited. ‘Peripheral nerve’ in the context of this book means axon. The cell body and the nerve terminal (motor or sensory) is ignored. This has probably arisen because the volume is based on the author's own collection of biopsies, and the majority of diagnostic nerve biopsies are made on sural nerve. However, there are quite distinct morphological differences between, for example, the motor end-plates of human muscle and those of laboratory mammals and there is a growing literature on the pathology and pathophysiology of the human neuromuscular junction. It is unfortunate that this important area of neuropathology is not covered at all in this book The subtitle of the book ‘An atlas of structural and molecular pathological changes’ is rather misleading. This is an atlas of structural changes of axons. Those changes are well illustrated and they are described in some detail in extensive legends to each illustration. The approach is not mechanistic in any way and contains very little molecular information at all. It neither addresses nor illustrates (except in a rather cursory way) the use of immunocytochemistry at light and electron microscopic level and I found no mention at all in situ hybridisaton. All of these techniques are now used in modern neuropathology laboratories, and they are essential techniques if any attempt is to be made to understand the relationships between genome, protein expression and structural pathology. There is also a lack of information on functional change. This is a significant gap if one believes that structure and function are essential components of the discipline of neuropathology. Despite these criticisms, this is an excellent atlas – without doubt the best available. No young clinical or experimental neuropathologist should fail to use it when trying to understand the mysteries of the organization of real tissue in health and disease. It is a book for regular consultation and enjoyment. John Harris Newcastle upon Tyne, UK

Apoptosis in co-cultures of human muscle and rat spinal cord during the formation of the neuromuscular junction.

The motor neurons and skeletal muscle fibers they innervate are strongly dependent on each other. Important communication between both tissues is mediated through the neuromuscular junction. However, release and reception of various factors at other parts of both tissues must also be considered as means of mutual influences. In vitro innervated human muscle is the only experimental model to investigate nerve - muscle interactions during synaptogenesis of human neuromuscular junction. Here we studied the occurrence of programmed cell death in this model. In order to find out if mutual interactions between neurons and myotubes control the extent of apoptosis in both tissues, we compared the number of apoptotic cells in spinal cord explants and myotubes in co-cultures and mono-cultures of these tissues. Apoptotic cells were detected by TUNEL at selected time intervals (10, 21 and 30 days after co-culture) coresponding to various stages of synaptogenesis of neuromuscular junction. The number of apoptotic cells in spinal cord explants was higher at earlier (day 10) in comparison to later stages (days 21 and 30). Co-cultures and mono-cultures did not differ in this respect. TUNEL positive cells were not found in mono-cultured human myotubes under our experimental conditions. These results suggest that the process of apoptosis seems to be rather independent on neuron - muscle interactions at least at the time points examine.

Association of Active Extracellular Signal-Regulated Protein Kinase with Paired Helical Filaments of Inclusion-Body Myositis Muscle Suggests Its Role in Inclusion-Body Myositis Tau Phosphorylation

The possible role of extracellular signal-regulated kinase (ERK) in the pathogenesis of inclusion-body myositis (IBM) was investigated by immunostaining the active phosphorylated form of ERK in muscle biopsies of six IBM and 14 control patients. Between 80% and 90% of IBM vacuolated muscle fibers contained well-defined ERK-immunoreactive inclusions, which were co-localized by light microscopy, with phosphorylated tau in 70 to 80% of those fibers. Immunoelectronmicroscopy colocalized ERK to small amorphous tufts adjacent to the muscle fiber paired-helical filaments. Strong ERK immunoreactivity was also present at the postsynaptic domain of all human neuromuscular junctions. Our study suggests 1) that ERK, a signal transducer, might play a role in IBM pathogenesis, including participation in the pathological phosphorylation of IBM tau; and 2) that signal transduction abnormalities may be a component of the IBM pathogenic cascade. Our novel immunolocalization of ERK at the postsynaptic domain of human neuromuscular junctions supports a role in transcription of junctional-protein genes. The ERK localized in nonjunctional regions of IBM fibers may underlie the known pathological up-regulation of junctional proteins there.

Expression of human GFRα-1 (GDNF receptor) at the neuromuscular junction and myelinated nerves

Motor neurons have been known to require a wide variety of neurotrophic factors for their survival. As one of the target-derived trophic factors, glial cell line-derived neurotrophic factor (GDNF) has been shown to exert its effects on motor neurons via a receptor complex including GDNF receptor alpha 1 (GFRα-1). Immunoreactivity of GFRα-1 was observed at myelinated peripheral nerves and neuromuscular junction (NMJ) of human skeletal muscles. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed that mRNA of GFRα-1 existed in the ventral horn of human spinal cord, but not in the skeletal muscles. The results suggested that GFRα-1 might play a key role for uptake and internalization of GDNF at the human NMJ.

Localization of survival motor neuron protein in human apoptotic-like and regenerating muscle fibers, and neuromuscular junctions.

Mutations in the gene encoding survival motor neuron (SMN) protein are found in > 98% of patients with autosomal-recessive spinal muscular atrophy. We investigated the possible role of SMN in normal and abnormal human muscle by immunostaining biopsies of 20 patients with various neuromuscular diseases using monoclonal antibodies against SMN. SMN was strongly expressed cytoplasmically in chronic peripheral neuropathies, in about 80% of chronically denervated, very atrophic muscle fibers containing clumps of TUNEL-positive pyknotic nuclei: about 60% of those fibers also had cytoplasmic Bcl-2 and Bax immunoreactivity. In regenerating muscle fibers of various myopathies SMN co-localized with desmin, Bcl-2 and Bax; it was also present at the postsynaptic domain of normal human neuromuscular junctions. Thus, SMN may play a role in normal and pathological processes of adult human muscle fibers.

Ultrastructural localization of cellular prion protein (PrPc) at the neuromuscular junction

We examined the localization of the normal cellular isoform of prion protein (PrPc) in mammalian skeletal muscle. Using two anti-PrP antibodies, the neuromuscular junction (NMJ) was preferentially stained after immunohistofluorescence. The mouse, hamster, and human NMJ displayed a fluorescent signal specific for PrPc. Postembedding immunoelectron microscopy analysis performed in the mouse muscle showed that the PrPc-specific colloidal gold immunolabelling was concentrated over the sarcoplasmic cytoplasm. The membrane of the postsynaptic domain was devoid of gold particles, while a weak signal was occasionally observed close to the presynaptic vesicles of the terminal axons. These results indicate that the PrP gene is expressed in mammalian muscle at the NMJ. The subsynaptic sarcoplasm of the NMJ appears to be the privileged site where PrPc presumably associated with endosome membrane may play a role in either physiological activity or maintenance of the morphological integrity of the synapse. J. Neurosci. Res. 55:261–267, 1999. © 1999 Wiley-Liss, Inc.

Ultrastructural localization of cellular prion protein (PrPc) at the neuromuscular junction.

We examined the localization of the normal cellular isoform of prion protein (PrPc) in mammalian skeletal muscle. Using two anti-PrP antibodies, the neuromuscular junction (NMJ) was preferentially stained after immunohistofluorescence. The mouse, hamster, and human NMJ displayed a fluorescent signal specific for PrPc. Postembedding immunoelectron microscopy analysis performed in the mouse muscle showed that the PrPc-specific colloidal gold immunolabelling was concentrated over the sarcoplasmic cytoplasm. The membrane of the postsynaptic domain was devoid of gold particles, while a weak signal was occasionally observed close to the presynaptic vesicles of the terminal axons. These results indicate that the PrP gene is expressed in mammalian muscle at the NMJ. The subsynaptic sarcoplasm of the NMJ appears to be the privileged site where PrPc presumably associated with endosome membrane may play a role in either physiological activity or maintenance of the morphological integrity of the synapse.

Strong immunoreactivity of platelet-derived growth factor and its receptor at human and mouse neuromuscular junctions.

Platelet-derived growth factor (PDGF) and its alpha-receptor were localized at human and mouse neuromuscular junctions (NMJs) using specific polyclonal antibodies against each, anti-PDGF-A and anti-PDGF alpha-receptor, respectively. By applying double fluorescence labeling, immunoreactivity for PDGF and its receptor was closely co-localized with acetylcholine receptors, which were identified with alpha-bungarotoxin. PDGF might be involved in the interaction between the presynaptic and postsynaptic components. This is the first demonstration of PDGF and its receptor concentrated at human and mouse NMJs.