The role of laminins in the organization and function of neuromuscular junctions.

The synapse between motor neurons and skeletal muscle is known as the neuromuscular junction (NMJ). Proper alignment of presynaptic and post-synaptic structures of motor neurons and muscle fibers, respectively, is essential for efficient motor control of skeletal muscles. The synaptic cleft between these two cells is filled with basal lamina. Laminins are heterotrimer extracellular matrix molecules that are key members of the basal lamina. Laminin α4, α5, and ß2 chains specifically localize to NMJs, and these laminin isoforms play a critical role in maintenance of NMJs and organization of synaptic vesicle release sites known as active zones. These individual laminin chains exert their role in organizing NMJs by binding to their receptors including integrins, dystroglycan, and voltage-gated calcium channels (VGCCs). Disruption of these laminins or the laminin-receptor interaction occurs in neuromuscular diseases including Pierson syndrome and Lambert-Eaton myasthenic syndrome (LEMS). Interventions to maintain proper level of laminins and their receptor interactions may be insightful in treating neuromuscular diseases and aging related degeneration of NMJs.

Lambert-Eaton syndrome IgG inhibits transmitter release via P/Q Ca2+ channels.

OBJECTIVE: To determine whether immunoglobulin G (IgG) from patients with Lambert-Eaton myasthenic syndrome (LEMS) decreases action potential­evoked synaptic vesicle exocytosis,and whether the effect is mediated by P/Q-type voltage-gated calcium channels (VGCCs). METHODS: IgG was obtained from 4 patients with LEMS (3 males, 1 female), including 2 patients with lung malignancy. Antibodies against P/Q-type VGCCs were detected in all 4 patients, and against N-type VGCCs in 2. We incubated neuronal cultures with LEMS IgG and determined the size of the total recycling pool of synaptic vesicles and the rate of action potential­evoked exocytosis using fluorescence imaging of the amphiphilic dye SynaptoRed C1. Pooled IgG from healthy volunteers was used as a control. We repeated the experiments on synapses lacking P/Q-type calcium channels from a Cacna1a knockout mouse to determine whether these channels account for the pathogenic effect of LEMS IgG. RESULTS: LEMS IgG had no effect on the total recycling pool size but significantly reduced the rate of action potential­evoked synaptic exocytosis in wild-type neurons when compared with neurons treated with control IgG. In contrast, LEMS IgG had no effect on the rate of synaptic vesicle exocytosis in neurons lacking P/Q-type channels. CONCLUSIONS: These data provide direct evidence that LEMS IgG inhibits neurotransmitter release by acting on P/Q-type VGCCs.

Myasthenia gravis and related disorders: Pathology and molecular pathogenesis.

Disorders affecting the presynaptic, synaptic, and postsynaptic portions of the neuromuscular junction arise from various mechanisms in children and adults, including acquired autoimmune or toxic processes as well as genetic mutations. Disorders include autoimmune myasthenia gravis associated with acetylcholine receptor, muscle specific kinase or Lrp4 antibodies, Lambert-Eaton myasthenic syndrome, nerve terminal hyperexcitability syndromes, Guillain Barré syndrome, botulism, organophosphate poisoning and a number of congenital myasthenic syndromes. This review focuses on the various molecular and pathophysiological mechanisms of these disorders, characterization of which has been crucial to the development of treatment strategies specific for each pathogenic mechanism. In the future, further understanding of the underlying processes may lead to more effective and targeted therapies of these disorders. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Complete reversal of Lambert-Eaton myasthenic syndrome synaptic impairment by the combined use of a K+ channel blocker and a Ca2+ channel agonist.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which a significant fraction of the presynaptic P/Q-type Ca(2+) channels critical to the triggering of neurotransmitter release at the neuromuscular junction (NMJ) are thought to be removed. There is no cure for LEMS, and the current most commonly used symptomatic treatment option is a potassium channel blocker [3,4-diaminopyridine (3,4-DAP)] that does not completely reverse symptoms and can have dose-limiting side-effects. We previously reported the development of a novel Ca(2+) channel agonist, GV-58, as a possible alternative treatment strategy for LEMS. In this study, we tested the hypothesis that the combination of GV-58 and 3,4-DAP will elicit a supra-additive increase in neurotransmitter release at LEMS model NMJs. First, we tested GV-58 in a cell survival assay to assess potential effects on cyclin-dependent kinases (Cdks) and showed that GV-58 did not affect cell survival at the relevant concentrations for Ca(2+) channel effects. Then, we examined the voltage dependence of GV-58 effects on Ca(2+) channels using patch clamp techniques; this showed the effects of GV-58 to be dependent upon Ca(2+) channel opening. Based on this mechanism, we predicted an interaction between 3,4-DAP and GV-58. We tested this hypothesis using a mouse passive transfer model of LEMS. Using intracellular electrophysiological ex vivo recordings, we demonstrated that a combined application of 3,4-DAP plus GV-58 had a supra-additive effect that completely reversed the deficit in neurotransmitter release magnitude at LEMS model NMJs. This reversal contrasts with the less significant improvement observed with either compound alone. Our data indicate that a combination of 3,4-DAP and GV-58 represents a promising treatment option for LEMS and potentially for other disorders of the NMJ.

Structure of the neuromuscular junction: function and cooperative mechanisms in the synapse.

As an overview of the structure of the neuromuscular junction, three items are described focusing on cooperative mechanisms involving the synapse and leading to muscle contraction: (1) presynaptic acetylcholine release regulated by vesicle cycling (exocytosis and endocytosis); the fast-mode of endocytosis requires a large influx of external Ca(2+) and is promoted by the activation of G protein-coupled receptors and receptor tyrosine kinases; (2) postsynaptic acetylcholine receptor clustering mediated by the muscle-specific, Dok7-stimulated tyrosine kinase (MuSK) through two signaling mechanisms: one via agrin-Lrp4-MuSK (Ig1/2 domains) and the second via Wnt-MuSK (Frizzled-like cysteine-rich domain)-adaptor Dishevelled; Wnts/MuSK and Lrp4 direct a retrograde signal to presynaptic differentiation; (3) muscle contractile machinery regulated by Ca(2+) -release and Ca(2+) -influx channels, including the depolarization-activated ryanodine receptor-1 and the receptor- and/or store-operated transient receptor potential canonical. The first mechanism is dysfunctional in Lambert-Eaton myasthenic syndrome, the second in anti-acetylcholine receptor-negative myasthenia gravis (MG), and the third in thymoma-associated MG.

Active zones of mammalian neuromuscular junctions: formation, density, and aging.

Presynaptic active zones are synaptic vesicle release sites that play essential roles in the function and pathology of mammalian neuromuscular junctions (NMJs). The molecular mechanisms of active zone organization use presynaptic voltage-dependent calcium channels (VDCCs) in NMJs as scaffolding proteins. VDCCs interact extracellularly with the muscle-derived synapse organizer, laminin ß2 and interact intracellularly with active zone-specific proteins, such as Bassoon, CAST/Erc2/ELKS2alpha, ELKS, Piccolo, and RIMs. These molecular mechanisms are supported by studies in P/Q- and N-type VDCCs double-knockout mice, and they are consistent with the pathological conditions of Lambert-Eaton myasthenic syndrome and Pierson syndrome, which are caused by autoantibodies against VDCCs or by a laminin ß2 mutation. During normal postnatal maturation, NMJs maintain the density of active zones, while NMJs triple their size. However, active zones become impaired during aging. Propitiously, muscle exercise ameliorates the active zone impairment in aged NMJs, which suggests the potential for therapeutic strategies.

[Histochemical findings of and fine structural changes in motor endplates in diseases with neuromuscular transmission abnormalities].

We herein review the histochemical findings and fine structural changes of motor endplates associated with diseases causing neuromuscular transmission abnormalities. In anti-acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG), type 2 fiber atrophy is observed, and the motor endplates show a reduction in the nerve terminal area, simplification of the postsynaptic membrane, decreased number of acetylcholine receptors, and deposition of immune complexes. In anti-MuSK antibody-positive MG, the fine structure shows a decrease in the postsynaptic membrane length, but the secondary synaptic cleft is preserved. There is no decrease in the number of AChRs, and there are no deposits of immune complexes at the motor endplates. Patients with Lambert-Eaton myasthenic syndrome show type 2 fiber atrophy, their motor endplates show a decrease in both the mean postsynaptic area and postsynaptic membrane length in the brachial biceps muscle. Congenital myasthenic syndrome with episodic apnea is characterized only by small-sized synaptic vesicles; the postsynaptic area is preserved. In subjects with congenital myasthenic syndrome with acetylcholinesterase deficiency, quantitative electron microscopy reveals a significant decrease in the nerve terminal size and presynaptic membrane length; further, the Schwann cell processes extend into the primary synaptic cleft, and partially or completely occlude the presynaptic membrane. The postsynaptic folds are degenerated, and associated with pinocytotic vesicles and labyrinthine membranous networks. Patients with slow-channel congenital myasthenia syndrome show type 1 fiber predominance, and their junctional folds are typically degenerated with widened synaptic space and loss of AChRs. Patients with AChR deficiency syndrome caused by recessive mutations in AChR subunits also show type 1 fiber predominance, and while most junctional folds are normal, some are simplified and have smaller than normal endplates. Rapsin and MuSK mutations cause type 1 fiber predominance, and the small postsynaptic area is associated with AChR decrease.

IgG fc N-glycosylation changes in Lambert-Eaton myasthenic syndrome and myasthenia gravis.

N-glycosylation of the immunoglobulin Fc moiety influences its biological activity by, for example, modulating the interaction with Fc receptors. Changes in IgG glycosylation have been found to be associated with various inflammatory diseases. Here we evaluated for the first time IgG Fc N-glycosylation changes in well-defined antibody-mediated autoimmune diseases, that is, the neurological disorders Lambert-Eaton myasthenic syndrome and myasthenia gravis, with antibodies to muscle nicotinic acetylcholine receptors or muscle-specific kinase. IgGs were purified from serum or plasma by protein A affinity chromatography and digested with trypsin. Glycopeptides were purified and analyzed by MALDI-FTICR-MS. Glycoform distributions of both IgG1 and IgG2 were determined for 229 patients and 56 controls. We observed an overall age and sex dependency of IgG Fc N-glycosylation, which was in accordance with literature. All three disease groups showed lower levels of IgG2 galactosylation compared to controls. In addition, LEMS patients showed lower IgG1 galactosylation. Notably, the galactosylation differences were not paralleled by a difference in IgG sialylation. Moreover, the level of IgG core-fucosylation and bisecting N-acetylglucosamine were evaluated. The control and disease groups revealed similar levels of IgG Fc core-fucosylation. Interestingly, LEMS patients below 50 years showed elevated levels of bisecting N-acetylglucosamine on IgG1 and IgG2, demonstrating for the first time the link of changes in the level of bisecting N-acetylglucosamine with disease.

Anti-SOX1 antibodies in patients with paraneoplastic and non-paraneoplastic neuropathy.

Anti-SOX1 antibodies have been described to be positive in patients with paraneoplastic Lambert-Eaton myasthenic syndrome and, in a lower amount, in patients with anti-Hu positive paraneoplastic neurological syndromes, and with SCLC alone, respectively. We found 5/32 patients with paraneoplastic neuropathy and, surprisingly, 4/22 patients with neuropathy of unknown origin positive for anti-SOX1 antibodies, whereas no patient with inflammatory neuropathy and no healthy controls showed any reactivity (p=0.007). All patients with neuropathy of unknown origin where followed up for four years without diagnosis of a tumour so far. Anti-SOX1 antibodies are associated with paraneoplastic neuropathies and may define another group of non-paraneoplastic, immune-mediated neuropathies.

Lambert-Eaton myasthenic syndrome: search for alternative autoimmune targets and possible compensatory mechanisms based on presynaptic calcium homeostasis.

The Lambert-Eaton myasthenic syndrome (LEMS) is a disease of neuromuscular transmission in which autoantibodies against the P/Q-type voltage-gated calcium channel (VGCC) at the presynaptic nerve terminal play a major role in decreasing quantal release of acetylcholine (ACh), resulting in skeletal muscle weakness and autonomic symptoms. It is associated with cancer, particularly small-cell lung carcinoma (SCLC), in 50-60% of LEMS patients; the nerve terminal and carcinoma cells apparently share a common antigen (VGCC), suggesting an immunological cross-reactivity that may lead to the neurological abnormality. Non-tumor LEMS has a strong association with HLA-DR3-B8. In approximately 15% of LEMS patients, no anti-P/Q-type VGCC antibodies are found, suggesting recognition of other targets(s). The VGCC-associated protein synaptotagmin could be one candidate, because it acts as an exocytotic calcium receptor, is implicated in fast ACh release; its N-terminus is exposed extracellularly during exocytosis and it is expressed in SCLC. Antibodies against synaptotagmin-1 were detected in both anti-VGCC-positive and -negative LEMS patients (20%), and it can be immunogenic, allowing induction of an animal model of LEMS. Another candidate target is the M1-type presynaptic muscarinic ACh receptor (M1 mAChR), also expressed extracellularly on motor nerve terminals; it modulates cholinergic transmission, linking to P/Q-type VGCC. In our series of 25 LEMS patients with and without SCLC, anti-M1 mAChR antibodies were prevalent in both anti-VGCC-positive and -negative LEMS patients. Autonomic symptoms seemed more frequent in the latter; serum from one of them passively transferred LEMS-type electrophysiological defects to mice. As a compensatory mechanism, researchers in Oxford suggested a shift in the dependence of ACh release from the P/Q-type to other types of VGCC. We have also focused on G protein-coupled mAChRs and neurotrophins, which may affect both P/Q-type VGCC and clathrin-independent "kiss-and-run" synaptic vesicle recycling (fast-mode of endocytosis) via protein kinase C activation. We hypothesize that these signaling cascades help to compensate for the immune-mediated defects in calcium entry in LEMS, compensation that may frequently be restricted by the coincident anti-M1 mAChR antibodies in this disease.

[Neurophysiological testing in myasthenia syndromes]. / Obraz elektrofizjologiczny zespolów miastenicznych.

Myasthenic syndromes are a heterogeneous group of congenital or acquired disorders of neuromuscular junction. Despite major advance in genetics and molecular biology of disorders of neuromuscular junction, clinical diagnosis and choice of treatment largely depends on results of neurophysiological tests. Different protocols of repetitive nerve stimulation and single fibre EMG are indispensable in confirming neuromuscular junction defect, they can also give additional information on the level of abnormality and differentiate myasthenia gravis from Lambert-Eaton syndrome (pre- or postsynaptic defect). Characteristic features of repetitive nerve stimulation test e.g. repetitive response allow diagnosing congenital myasthenic syndromes such as slow channel syndrome or acetylcholine deficiency. Patophysiological basis of neurophysiological tests of neuromuscular transmission is presented. Different neurophysiological findings in cases of Lambert-Eaton myasthenic syndrome and congenital myasthenic syndromes are presented.

Reduction of P/Q-type calcium channels in the postmortem cerebellum of paraneoplastic cerebellar degeneration with Lambert-Eaton myasthenic syndrome.

The aim of this study was to clarify whether autoimmunity against P/Q-type voltage-gated calcium channels (VGCCs) in the cerebellum was associated with the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome (LEMS). We used human autopsy cerebellar tissues from three PCD-LEMS patients and six other disease patients including one with LEMS as the controls. We compared cerebellar P/Q-type VGCC in these patients and controls for the amount and ratio of autoantibody-channel complex using an 125I-omega-conotoxin MVIIC-binding assay with Scatchard analysis, and their distribution using autoradiography. The quantity of cerebellar P/Q-type VGCC measured by Scatchard analysis were reduced in PCD-LEMS patients (63.0 +/- 7.0 fmol/mg, n = 3), compared with the controls (297.8 +/- 38.9 fmol/mg, n = 6). The ratio of autoantibody-VGCC complexes to total P/Q-type VGCCs measured by immunoprecipitation assay were increased in PCD-LEMS patients. We analysed cerebellar specimens by autoradiography using (125)I-omega-conotoxin MVIIC, which specifically binds to P/Q-type VGCCs. In PCD-LEMS cerebellum, the toxin binding sites of P/Q-type VGCCs were markedly reduced compared with controls, especially in the molecular layer, which is the richest area of P/Q-type VGCCs in the normal cerebellum. This suggests that P/Q-type VGCCs of the cerebellar molecular layer is the immunological target in developing PCD-LEMS.

Autonomic dysfunction in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder characterized by muscle weakness and autonomic dysfunction. Recent ex vivo and in vitro studies demonstrate that autoantibodies to the P/Q-subtype of voltage-gated calcium channel inhibit transmitter release from parasympathetic, sympathetic, and enteric neurons, a mechanism likely to underlie the widespread autonomic dysfunction in LEMS. This review summarizes clinical studies characterizing the autonomic symptoms and signs in LEMS and the effectiveness of treatment in alleviating these symptoms. Serological assays and in vitro pharmacologic and electrophysiologic studies are also discussed.

Semiquantitative measurement of acetylcholine receptor at the motor end-plate in myasthenia gravis.

OBJECTIVE: The purpose of this study was to investigate whether this semiquantitative measurement of the motor end-plate acetylcholine receptors (AChRs) can be used to confirm the diagnosis of myasthenia gravis (MG), and in particular ocular MG. METHODS: Motor point biopsies were performed from the biceps brachii muscles. Measurement of AChRs was made in peroxidase-labeled alpha-bungarotoxin stained muscle specimens. PATIENTS: Twenty patients with ocular MG, 37 with generalized MG, 5 with Lambert-Eaton myasthenic syndrome, 3 with botulism, 8 with amyotrophic lateral sclerosis, and 8 controls were included in this study. RESULTS: AChRs were decreased in all patients with generalized MG and in 80% of ocular MG including patients without detectable circulating anti-AChR antibodies, as compared with the control subjects. CONCLUSION: This method is useful to confirm the diagnosis of MG, in particular ocular MG without detectable anti-AChR antibodies.

Antibodies to calcium channel and synaptotagmin in Lambert-Eaton myasthenic syndrome.

In the Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease that is often associated with lung cancer and characterized by reduced quantal release of acetylcholine from the motor nerve terminal, our studies to search for the target of LEMS antibodies have brought the voltage-gated calcium channel (VGCC) into relief. Among multiple types of VGCCs, the P/Q-type was highly recognized by LEMS antibodies. Using synthetic peptides or recombinant proteins as antigens, the study specified the S5-S6 linker regions in 3 of 4 domains as immunodominant sites in the molecular structure of P/Q-type VGCC alpha1 subunit. Synaptotagmin, one of the functionally VGCC-associated synaptic proteins, was also found to be an immunogen in the pathogenesis of LEMS.

Lambert-Eaton syndrome antibodies inhibit acetylcholine release and P/Q-type Ca2+ channels in electric ray nerve endings.

1. The types of voltage-dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert-Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG. 2. In normal untreated synaptosomal preparations, maximal doses of N- and P and/or Q-type Ca2+ channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, inhibited depolarization-evoked ACh release by 47 % and 43 %, respectively. Calciseptine, an L-type VDCC antagonist, caused a 20 % reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N- and P/Q-type VDCCs. 3. LES IgG or sera caused an inhibition of ACh release by 39-45 % in comparison with the control antibody-treated preparations. The ionomycin-induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole-cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes. 4. The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. omega-Agatoxin IVA produced only an additional 3-5 % reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, omega-conotoxin GVIA and calciseptine inhibited the release to nearly their control levels. 5. These results indicate that LES antibodies mainly downregulate P/Q-type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ. 6. The present findings are consistent with the hypothesis that P/Q-type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease.

Functional evaluation of inhibition of autonomic transmitter release by autoantibody from Lambert-Eaton myasthenic syndrome.

The effects of the anti-voltage-gated Ca2+ channel (VGCC) antibody obtained from patients with Lambert-Eaton myasthenic syndrome (LEMS) on autonomic neurotransmission were studied in in-vitro experiments. The releases of acetylcholine (ACh) and norepinephrine from the autonomic nerves were evaluated by changes in the contractile responses of guinea pig taenia caeci and left atria to electric field stimulation, respectively. Incubations for 6 hours with LEMS serum and IgG, both of which contain anti-VGCC antibody, markedly suppressed the parasympathetic response but did not affect the sympathetic response. Pharmacological experiments with specific blockers to the VGCC subtypes showed that the Q-type VGCC is closely linked to the genesis of the parasympathetic response. We suggest that the anti-VGCC antibody from the LEMS patients specifically reduces the ACh release from the parasympathetic nerve by binding to the Q-type VGCC.

Down-regulation of non-L-, non-N-type (Q-like) Ca2+ channels by Lambert-Eaton myasthenic syndrome (LEMS) antibodies in rat insulinoma RINm5F cells.

The action exerted on non-L-, non-N-type (Q-like) Ca 2+ channels by immunoglobulins G (IgGs) obtained from two patients with Lambert-Eaton myasthenic syndrome (LEMS) was investigated in the rat insulinoma RINm5F cell line. LEMS IgGs reduced by 30-36% the whole-cell Ba2+ currents through Q-like Ca2+ channels at +10 mV without significantly modifying their voltage dependence and activation kinetics. Single- and multiple-channel recordings in cell-attached and outside-out patches of cells treated with LEMS IgGs showed no significant changes of the channel elementary properties but rather a decreased number of active channels per patch. This suggests that Q-like current depression by LEMS autoantibodies is mostly due to a down-regulation of functioning Ca2+ channels. In agreement with previous observations, LEMS IgGs also reduced by 20-33% the dihydropyridine-sensitive (L-type) Ba2+ current. The suggested down-regulation of Q-like channels by LEMS IgGs in RINm5F cells may have a functional correlation with the depressive action of LEMS autoantibodies on the P/Q-type Ca2+ channels controlling acetylcholine release from mammalian neuromuscular junctions.