Postsynaptic Membrane Of Neuromuscular Junction Research Articles

Serum pentraxin 3 concentration correlates with disease severity in patients with myasthenia gravis

ObjectiveMyasthenia gravis (MG) is an antibody-mediated inflammatory disease affecting post-synaptic membranes of neuromuscular junctions, and objective biomarkers of MG disease activity are lacking. Pentraxin 3 (PTX3) is an acute-phase inflammatory glycoprotein in the same family as C-reactive protein that is associated with disease activity in several autoimmune disorders. Thus, we investigated whether circulating PTX3 is a useful biomarker of MG activity. MethodsSerum PTX3 was measured in 40 patients with MG who were positive for anti-acetylcholine receptor antibody, and in 30 healthy and disease controls, using a commercial enzyme-linked immunosorbent assay kit. In patients with MG, the correlation of serum PTX3 levels with disease severity scales at serum sampling, including MG Foundation of America (MGFA) classification, MG activity of daily living (MG-ADL) score, and quantitative MG (QMG) score, were investigated. ResultsAlthough there was no significant difference in serum PTX3 between the MG and control groups (mean, 3346 pg/mL in MG group vs. 2870 pg/mL in control group, P = 0.56), serum PTX3 moderately correlated with all disease severity scores (MGFA classification: Spearman’s ρ = 0.53, P = 0.0004; MG-ADL score: Spearman’s ρ = 0.45, P = 0.004; QMG score: Spearman’s ρ = 0.50, P = 0.004). ConclusionOur results suggest that circulating PTX3 may reflect the extent of neuromuscular junction damage and might be involved in the pathogenesis of MG.

Lack of Desmin in Mice Causes Structural and Functional Disorders of Neuromuscular Junctions.

Desmin, the major intermediate filament (IF) protein in muscle cells, interlinks neighboring myofibrils and connects the whole myofibrillar apparatus to myonuclei, mitochondria, and the sarcolemma. However, desmin is also known to be enriched at postsynaptic membranes of neuromuscular junctions (NMJs). The pivotal role of the desmin IF cytoskeletal network is underscored by the fact that over 120 mutations of the human DES gene cause hereditary and sporadic myopathies and cardiomyopathies. A subgroup of human desminopathies comprises autosomal recessive cases resulting in the complete abolition of desmin protein. In these patients, who display a more severe phenotype than the autosomal dominant cases, it has been reported that some individuals also suffer from a myasthenic syndrome in addition to the classical occurrence of myopathy and cardiomyopathy. Since further studies on the NMJ pathology are hampered by the lack of available human striated muscle biopsy specimens, we exploited homozygous desmin knock-out mice which closely mirror the striated muscle pathology of human patients lacking desmin protein. Here, we report on the impact of the lack of desmin on the structure and function of NMJs and the transcription of genes coding for postsynaptic proteins. Desmin knock-out mice display a fragmentation of NMJs in soleus, but not in the extensor digitorum longus muscle. Moreover, soleus muscle fibers show larger NMJs. Further, transcription levels of acetylcholine receptor (AChR) genes are increased in muscles from desmin knock-out mice, especially of the AChRγ subunit, which is known as a marker of muscle fiber regeneration. Electrophysiological recordings depicted a pathological decrement of nerve-dependent endplate potentials and an increased rise time of the nerve-independent miniature endplate potentials. The latter appears related to the fragmentation of NMJs in desmin knockout mice. Our study highlights the essential role of desmin for the structural and functional integrity of mammalian NMJs.

Dynamin-2 Regulates Postsynaptic Cytoskeleton Organization and the Maturation of Neuromuscular Junction

The neuromuscular junction (NMJ) governs rapid and efficient neuronal communication with muscle cells, which relies on the proper activity of postsynaptic compartment specialized with unique membrane and actin-dependent cytoskeleton organization. However, the intrinsic mechanism in muscle cells that contributes to the development and maintenance of the elaborate NMJ morphology is unclear. In this study, we discover that the large GTPase, dynamin, best-known for catalyzing synaptic vesicle endocytosis at the presynaptic membrane, is also involved in postsynaptic morphogenesis. By using cultured mouse myotube, Drosophila, and in vitro reconstitution assay, we demonstrate that dynamin-2 (Dyn2) is enriched in the postsynaptic membrane of muscle cells and is involved in the maturation of neurotransmitter receptor clusters and the functional integrity of NMJ. During the early stage of NMJ development, Dyn2 functions as a molecular girdle to regulate synaptic podosome turnover and promote the maturation of postsynaptic apparatus. After the maturation, Dyn2 remains enriched at NMJ to maintain proper actin cytoskeleton organization and its electrophysiological function. The self-assembly, GTP hydrolysis ability and Y597 phosphorylation of Dyn2 determine its localization at podosome and the actin bundling activity. Together, our results uncover a new function of Dyn2 on cytoskeleton remodeling and organization at the postsynaptic membrane of NMJ.

Myasthenia gravis and specific immunotherapy: monoclonal antibodies.

Myasthenia gravis (MG) is an acquired autoimmune disease affecting the postsynaptic membrane of neuromuscular junctions and characterized by antibody-mediated T cell dependence and complement involvement. Cholinesterase inhibitors (e.g., pyridostigmine bromide), glucocorticoids, and azathioprine are currently recommended as first-line treatments for MG, though they have limitations, including potential toxicity and ineffectiveness in patients with refractory MG. In recent years, owing to an increasing understanding of MG pathogenesis the development and execution of clinical trials with novel biologics, including monoclonal antibodies (mAbs) that have demonstrated higher safety and more specificity, provide new opportunities for the treatment of MG. In this article, we review recent advances in MG pathogenesis and the mAbs that have been used for target-specific MG therapy.

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.

Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines.

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca2+ concentration in sarcoplasm, while electrical stimulation evoked Ca2+ transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

C. elegans Punctin Clusters GABAA Receptors via Neuroligin Binding and UNC-40/DCC Recruitment

Positioning type A GABA receptors (GABA(A)Rs) in front of GABA release sites sets the strength of inhibitory synapses. The evolutionarily conserved Ce-Punctin/MADD-4 is an anterograde synaptic organizer that specifies GABAergic versus cholinergic identity of postsynaptic domains at the C. elegans neuromuscular junctions (NMJs). Here we show that the Ce-Punctin secreted by GABAergic motor neurons controls the clustering of GABA(A)Rs through the synaptic adhesion molecule neuroligin (NLG-1) and the netrin receptor UNC-40/DCC. The short isoform of Ce-Punctin binds and clusters NLG-1 postsynaptically at GABAergic NMJs. NLG-1 disruption causes a strong reduction of GABA(A)R content at GABAergic synapses. Ce-Punctin also binds and localizes UNC-40 receptors in the postsynaptic membrane of NMJs, which promotes the recruitment of GABA(A)Rs by NLG-1. Since the mammalian orthologs of these genes are expressed in the central nervous system and their mutations are implicated in neuropsychiatric diseases, this molecular pathway might have been evolutionarily conserved.

Myasthenia gravis with antibodies to MuSK

It has been 10 years since the first report of serum immunoglobulin G (IgG) against the muscle-specific tyrosine kinase (MuSK) in a subgroup of patients with myasthenia gravis (MG). From the initial clinical observations, it soon became evident that these antibodies (Abs) identify a clinical entity that differs in several aspects from MG with Abs to the acetylcholine receptor (AChR). MuSK is a 100-kDa protein, specifically expressed at the postsynaptic membrane of neuromuscular junction where it colocalizes with AChR.1 MuSK was an ideal candidate antigen in anti-AChR negative MG, as it is a transmembrane protein (thus accessible to circulating Abs) that plays a crucial role in neuromuscular junction development and maintenance. MuSK had long been known to be essential for AChR clustering; further studies have shed light on interacting proteins and molecular pathways involved in this process. At the postsynaptic membrane, MuSK is associated with the low-density lipoprotein receptor-related protein 4 (Lrp4). Binding of nerve-secreted agrin to Lrp4 activates MuSK by phosphorylation, and phosphorylated MuSK triggers a complex intracellular signaling pathway that, through Dok-7 recruitment and activation of nonreceptor tyrosine kinases and GTPases, leads to AChR assembly and stabilization.1 Another important, and relatively newly …

The difference in number and distribution of acetylcholine receptor in post-synaptic membrane of neuromuscular junction between young adult and elderly rats

Objective To examine the difference in the number and distribution of acetylcholine receptor(AChR)in post-synaptic membrane of neuromuscular junction between young adult and elderly rats.Methods Ten male Wistar rats were divided into 2 age groups(n = 5 each): group Ⅰ 4-6 months old and group Ⅱ 24-28 months old.The expression of nicotinic AChR in gastrocnemius muscle was detected by quantitative real-time PCR(qRT-PCR).Primers were designed according to the mRNA subsequence of e-subunit of AChR.The post-synaptic AChR in gastrocnemius muscle was labeled with tetramethyl-rhodamine-α-bungarotoxin.The distribution of post-synaptic AChR was investigated by confocal microscopy.The area,length and width of AChR region were calculated by computer.Results There was significant difference in AChR expression between the 2 groups.Furthermore a significant difference in distribution structure of AChR was found by confocal microscopy.There was no significant difference in the area,length and width of post-synaptic receptor region between the 2 groups.Conclusion Elderly rats have a low level of AChR expression but the area of neuro-muscular junction does not change significantly with age. Key words: Receptors,cholinergic ; Neuromuscular junction; Syuaptic membranes; Aged

Myasthenia Gravis Experimentally Induced with Muscle‐specific Kinase

Here we present the first evidence that muscle-specific kinase (MuSK) antigen can cause myasthenia in animals. MuSK is expressed at the postsynaptic membranes of neuromuscular junctions (NMJ) and forms complexes with acetylcholine receptors (AChR) and rapsyn. MuSK is activated by agrin, which is released from motoneurons, and induces AChR clustering and subsequent formation of NMJ in embryos. Notably, autoantibodies against MuSK were found in a proportion of patients with generalized myasthenia gravis (MG) but without the characteristic AChR autoantibodies. However, MuSK autoantibodies had no known pathogenic potential, and animals immunized with purified MuSK proteins did not develop MG in former studies. In contrast, we have now injected rabbits with MuSK ectodomain protein in vivo and evoked a MG-like muscle weakness with a reduction of AChR clustering at the NMJ. Our results showed that MuSK is required for maintenance of synapses and that interference with that function by MuSK antibodies causes myasthenic weakness. In vitro, AChR clustering in myotubes is induced by agrin and agrin-independent inducers, which do not activate MuSK. Neither the receptor nor the activation mechanisms of AChR clustering induced by agrin-independent inducers has been identified with certainty, but MuSK autoantibodies in myasthenic animals inhibited both agrin and agrin-independent AChR clustering. MuSK plays multiple roles in pre-patterning of the postsynaptic membrane before innervation and formation of NMJ in embryos. Some of these mechanisms may also participate in the maintenance of mature NMJ. This model system would provide new knowledge about the molecular pathogenesis of MG and MuSK functions in mature NMJ.

Amphiphysins: raising the BAR for synaptic vesicle recycling and membrane dynamics. Bin-Amphiphysin-Rvsp.

Amphiphysins, members of the BAR (Bin-Amphiphysin-Rvsp) protein super family, have been postulated to play a key role in clathrin-mediated endocytosis of synaptic vesicles (SVs). This review focuses on recent genetic studies of the role of amphiphysins in SV recycling and membrane morphogenesis. In the mouse, brain-specific amphiphysin I and II regulate, but are not essential for, SV recycling. The role of this regulation appears important, as mice deficient in these proteins have seizures and are deficient in learning and memory. In the fruit fly Drosophila melanogaster, amphiphysin is found in muscles and is enriched at postsynaptic membranes of neuromuscular junctions (NMJs); however, it does not play a role in SV recycling. Rather, amphiphysin in fly muscles appears to regulate the organization and structure of the muscle T-tubule system and possibly the subsynaptic reticulum. Amphiphysin is also involved in membrane organization in both neurons and non-neuronal cells in Drosophila. These studies reveal pleiotropic functions for amphiphysins in clathrin-mediated endocytosis and the regulation of membrane dynamics, perhaps through the actin cytoskeleton.

Cytoskeletal architecture of neuromuscular junction: Localization of vinculin

Cytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick-freeze deep-etch method and immunoelectron microscopy of ultrathin frozen sections. In a quick-freeze deep-etched replica of fresh, unfixed muscles, 8.9 +/- 1.5-nm particles were present on the true postsynaptic membrane surface. Underneath this receptor-rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized--12 nm, 9 nm, and 7 nm in diameter. The 12-nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty-six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.

Contours and distribution of sites that react with antiacetylcholinesterase in chicken intrafusal fibers.

Serial cross and longitudinal sections of intrafusal fibers from the intracapsular portions of chicken tibialis anterior muscle spindles were incubated with a monoclonal antibody specific for chicken acetylcholinesterase (AchE) and examined by immunofluorescence for the presence of the enzyme on presynaptic and postsynaptic membranes of neuromuscular junctions. The midequatorial sensory region which lacks organized sarcomeres was negative, but immediately distal to it faintly staining regions of AchE localization were observed on intrafusal fibers. In cross sections at the juxtaequator, the outlines of areas that were positive for AchE were either thin and crescentlike or thick and compact. The distribution of both types of localization continued into the polar region. Toward the more distal polar region, the intensity of sites on the postsynaptic membrane that reacted with the anti-AchE progressively increased. In longitudinal sections, AchE localization was largely limited to two configurations. One was elongate, while the other was more round or oval and often also smaller. Both types might occur on the same, or on different, intrafusal fibers. Examination of silver-impregnated sections revealed the presence of platelike and of traillike axon terminals. The variety of shapes observed on presynaptic and postsynaptic membranes warrants further study to determine whether chicken muscle spindles are innervated by more than one type of motor neuron.

Direct structural localization of two toxin-recognition sites on an ACh receptor protein.

One of the most actively studied chemoreceptors is the acetylcholine receptor (AChR) in the postsynaptic membranes of neuromuscular junctions and electrocytes. The AChR from Torpedo is a membrane protein, each molecule comprising five polypeptide chains1,2: two α-chains of molecular weight (Mr) 40,000, and three homologous chains of Mr 50,000 (β), 60,000 (γ) and 65,000 (δ). Only the α-summits seem to carry the specific recognition sites for agonists and antagonists2. As snake α-neurotoxins seem to bind highly specifically and quasi-irreversibly to these sites we performed a structural analysis to localize the α-subunits, using as marker native α-bungarotoxin (α-BTX, Mr 8,000; ref. 3). We report here the results obtained at 20 A resolution by electron microscopy and single-particle image averaging4, which reveal two well-defined regions within the AChR structure5,6 where the mass increases significantly on binding of α-BTX. One of these (α1) is adjacent to the region where the δ-subunit has been located6; the second region (α2) is diametrically across the molecule, ∼50 A away from the δ-subunit and also from (α1). The topography revealed by direct structural analysis can explain the results of previous nearest-neighbour cross-linking studies2,7.

Turnover of acetylcholine receptors in skeletal muscle.

The acetylcholine receptor (AChR) is an integral membrane protein that opens a cation-selective ion channel in response to the binding of acetylcho­ line. The AChR is a major protein of the post-synaptic membranes of neuromuscular junctions and synapses in the electroplax. Here we review the mechanisms of synthesis and degradation of AChR and the mechanisms by which these processes are regulated to determine the number and loca­ tion of AChR. We emphasize data appearing since previous reviews (41, 47, 92).