Search

Muscle‐specific kinase myasthenia gravis (MuSK MG) is caused by autoantibodies against MuSK in the neuromuscular junction (NMJ). MuSK MG patients have fluctuating, fatigable skeletal muscle weakness, in particular of bulbar muscles. Severity differs greatly between patients, in spite of comparable autoantibody levels. One explanation for inter‐patient and inter‐muscle variability in sensitivity might be variations in compensatory muscle responses. Previously, we developed a passive transfer mouse model for MuSK MG. In preliminary ex vivo experiments, we observed that muscle contraction of some mice, in particular those with milder myasthenia, had become partially insensitive to inhibition by μ‐Conotoxin‐GIIIB, a blocker of skeletal muscle NaV1.4 voltage‐gated sodium channels. We hypothesised that changes in NaV channel expression profile, possibly co‐expression of (μ‐Conotoxin‐GIIIB insensitive) NaV1.5 type channels, might lower the muscle fibre's firing threshold and facilitate neuromuscular synaptic transmission. To test this hypothesis, we here performed passive transfer in immuno‐compromised mice, using 'high', 'intermediate' and 'low' dosing regimens of purified MuSK MG patient IgG4. We compared myasthenia levels, μ‐Conotoxin‐GIIIB resistance and muscle fibre action potential characteristics and firing thresholds. High‐ and intermediate‐dosed mice showed clear, progressive myasthenia, not seen in low‐dosed animals. However, diaphragm NMJ electrophysiology demonstrated almost equal myasthenic severities amongst all regimens. Nonetheless, low‐dosed mouse diaphragms showed a much higher degree of μ‐Conotoxin‐GIIIB resistance. This was not explained by upregulation of Scn5a (the NaV1.5 gene), lowered muscle fibre firing thresholds or histologically detectable upregulated NaV1.5 channels. It remains to be established which factors are responsible for the observed μ‐Conotoxin‐GIIIB insensitivity and whether the NaV repertoire change is compensatory beneficial or a bystander effect. [ABSTRACT FROM AUTHOR]

Abstract: Immunoglobulin 4 (IgG4) is one of four human IgG subclasses and has several unique functional characteristics. It exhibits low affinity for complement and for most Fc receptors. It furthermore has generally high affinity for its antigen, with binding occurring in a monovalent fashion, as IgG4 can exchange Fab‐arms with other IgG4 molecules. Because of these characteristics, IgG4 is believed to block its targets and prevent inflammation, which, depending on the setting, can have a protective or pathogenic effect. One example of IgG4 pathogenicity is muscle‐specific kinase (MuSK) myasthenia gravis (MG), in which patients develop IgG4 MuSK autoantibodies, resulting in muscle weakness. As a consequence of the distinct IgG4 characteristics, the pathomechanism of MuSK MG is very different from IgG1‐and IgG3‐mediated autoimmune diseases, such as acetylcholine receptor MG. In recent years, new autoantibodies in a spectrum of autoimmune diseases have been discovered. Interestingly, some were found to be predominantly IgG4. These IgG4‐mediated autoimmune diseases share many pathomechanistic aspects with MuSK MG, suggesting that IgG4‐mediated autoimmunity forms a separate niche among the antibody‐mediated disorders. In this review, we summarize the group of IgG4‐mediated autoimmune diseases, discuss the role of IgG4 in MuSK MG, and highlight interesting future research questions for IgG4‐mediated autoimmunity. [ABSTRACT FROM AUTHOR]

Abstract: Myasthenia gravis (MG) with antibodies to muscle‐specific kinase (MuSK) is characterized by fluctuating fatigable weakness. In MuSK MG, involvement of bulbar muscles, neck, and shoulder and respiratory weakness are more prominent than in acetylcholine receptor (AChR) MG. MuSK autoantibodies are mainly of the IgG4 subclass, and as such are unable to activate complement, have low affinity for Fc receptors, and are functionally monovalent. Therefore, the pathogenicity of IgG4 MuSK autoantibodies was initially questioned. A broad collection of in vitro active immunization and passive transfer models has been developed that have shed light on the pathogenicity of MuSK autoantibodies. Passive transfer studies with purified IgG4 from MuSK MG patients confirmed that IgG4 is sufficient to reproduce clear clinical, electrophysiological, and histological signs of myasthenia. In vitro experiments revealed that MuSK IgG4 autoantibodies preferably bind the first Ig‐like domain of MuSK, correlate with disease severity, and interfere with the association between MuSK and low‐density lipoprotein receptor–related protein 4 and collagen Q. Some patients have additional IgG1 MuSK autoantibodies, but their role in the disease is unclear. Altogether, this provides a rationale for epitope‐specific or IgG4‐specific treatment strategies for MuSK MG and emphasizes the importance of the development of different experimental models. [ABSTRACT FROM AUTHOR]

Abstract: The neuromuscular junction (NMJ) forms the synaptic connection between a motor neuron and a skeletal muscle fiber. In order to achieve a sustained muscle contraction, this synapse has to reliably transmit motor neuronal action potentials onto the muscle fiber. To guarantee successful transmission even during intense activation of the NMJ, a safety factor of neuromuscular transmission exists. In the neuromuscular disorder myasthenia gravis (MG), autoantibodies are directed against acetylcholine receptors or, in the rarer variants, against other postsynaptic NMJ proteins. This causes loss of functional acetylcholine receptors, which compromises the safety factor of neuromuscular transmission, leading to the typical fatigable muscle weakness of MG. With intracellular microelectrode measurement of (miniature) endplate potentials at NMJs in ex vivo nerve–muscle preparations from MG animal models, these functional synaptic defects have been determined in much detail. Here, we describe the electrophysiological events at the normal NMJ and the pathoelectrophysiology at NMJs of animal models for MG. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy ( DMD) is an X-linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin-associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction ( NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn+/− and mdx/utrn−/− mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn−/− mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation-evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high-rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn−/− mice. Overall, we showed mild 'myasthenia-like' neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration. [ABSTRACT FROM AUTHOR]

The pre-synaptic motor nerve terminal is a highly complex and dynamic compartment within the lower motor neuron responsible for converting electrical signals into secreted chemicals. This self-renewing process of synaptic transmission is accomplished by the calcium-triggered fusion of neurotransmitter-containing vesicles with the plasma membrane and the subsequent retrieval and recycling of vesicle components. Besides this conventional physiological role, the highly active process of vesicle fusion and re-uptake into endosomal sorting pathways acts as a conduit for entry of a range of substances into the intracellular compartment of the motor nerve terminal. Whilst this entry portal sub-serves many vital physiological processes, such as those mediated by neurotrophin trafficking, there is also the potential for substantial pathological consequences resulting from uptake of noxious agents, including autoantibodies, viruses and toxins. These may act locally to induce disease within the nerve terminal, or traffic beyond to the motor neuron cell body and central nervous system to exert their pathological effects. This review focuses on the recent evidence that the ganglioside-rich pre-synaptic membrane acts as a binding site for potentially neurotoxic serum autoantibodies that are present in human autoimmune motor neuropathies. Autoantibodies that bind surface antigens induce membrane lytic effects, whereas their uptake attenuates local injury and transfers any potential pathological consequences to the intracellular compartment. Herein the thesis is explored that a balance exists between local injury at the exofacial leaflet of the pre-synaptic membrane and antibody uptake, which dictates the overall level and site of motor nerve injury in this group of disorders. [ABSTRACT FROM AUTHOR]

Autoantibodies against muscle-specific kinase (MuSK), a protein essential for clustering of acetylcholine receptors at the neuromuscular junction (NMJ), are detected in the serum of a proportion of myasthenia gravis (MG) patients. In most MuSK MG patients the anti-MuSK activity resides in the IgG4 subclass, a minor IgG component without very well-defined, but presumably anti-inflammatory, roles in immunity. In recent years, several animal model studies showed that anti-MuSK autoantibodies can cause muscle weakness by directly affecting NMJ function and, therefore, are likely not simply bystander disease markers in MuSK MG patients. In passive transfer mice, we recently provided proof that MuSK MG patient IgG4 is severely myasthenogenic, causing functional defects at NMJs. Against the clinical, serological, and pharmacological background of MuSK MG, here we discuss the MuSK MG animal models generated by our laboratory and others that have been instrumental in elucidating the etiological and pathophysiological roles of anti-MuSK antibodies. [ABSTRACT FROM AUTHOR]

Autoantibodies to muscle-specific kinase (MuSK) can cause myasthenia gravis (MG). The pathophysiological mechanism remains unknown. We report in vitro electrophysiological and histological studies of the neuromuscular junction in a MuSK MG patient. Low levels of presynaptic acetylcholine release and small miniature endplate potentials were found. This combination of pre- and postsynaptic abnormalities was supported by histology, revealing partially denervated postsynaptic areas, and some degeneration of postsynaptic folds. Results suggest that anti-MuSK antibodies reduce the stability of muscle-nerve contact. Muscle Nerve, 2010 [ABSTRACT FROM AUTHOR]

Neuronal membrane gangliosides, forming a large family of sialylated glycosphingolipids, have been hypothesized to play important roles in synaptic transmission. We studied the ex vivo electrophysiological function of neuromuscular junctions of GM2/GD2-synthase*GD3-synthase compound null-mutant mice after acute removal of GM3, the only remaining ganglioside in this mouse, by in vitro treatment with neuraminidase. We found 16% enhancement of the acetylcholine release per nerve impulse at low-rate (0.3 Hz) nerve stimulation. Conversely, the treatment reduced the acetylcholine release evoked by high-rate (40 Hz) nerve stimulation. Also, 25 ms paired-pulse facilitation of endplate potentials was reduced by the neuraminidase-treatment. These effects may indicate a modest modulatory influence of the negative electrical charges carried by the sialic acid molecules of gangliosides on the function of presynaptic Cav2.1 channels, affecting the magnitude and kinetics of the Ca2+ influx that induces neurotransmitter release from the motor nerve terminal. Our results show that gangliosides are to some extent involved in neurotransmission at the neuromuscular junction, but that their presence is not an absolute requirement in this process. Synapse 64:335-338, 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Objective The CACNA1A gene encodes the pore-forming subunit of neuronal CaV2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. Methods We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. Results Cacna1aS218L mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage-dependent negative shift in voltage dependence of CaV2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1aS218L mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. Interpretation The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema. ANN NEUROL 2010;67:85-98 [ABSTRACT FROM AUTHOR]

The outer leaflet of neuronal membranes is highly enriched in gangliosides. Therefore, specific neuronal roles have been attributed to this family of sialylated glycosphingolipids, e.g. in modulation of ion channels and transporters, neuronal interaction and recognition, temperature adaptation, Ca2+ homeostasis, axonal growth, (para)node of Ranvier stability and synaptic transmission. Recent developmental, ageing and injury studies on transgenic mice lacking subsets of gangliosides indicate that gangliosides are involved in maintenance rather than development of the nervous system and that ganglioside family members are able to act in a mutually compensatory manner. Besides having physiological functions, gangliosides are the likely antigenic targets of autoantibodies present in Guillain-Barré syndrome (GBS), a group of neuropathies with clinical symptoms of motor- and/or sensory peripheral nerve dysfunction. Antibody binding to peripheral nerves is thought to either interfere with ganglioside function or activate complement, causing axonal damage and thereby disturbed action potential conduction. The presynaptic motor nerve terminal at the neuromuscular junction (NMJ) may be a prominent target because it is highly enriched in gangliosides and lies outside the blood–nerve barrier, allowing antibody access. The ensuing neuromuscular synaptopathy might contribute to the muscle weakness in GBS patients. Several groups, including our own, have studied the effects of anti-ganglioside antibodies in ex vivo and in vivo experimental settings at mouse NMJs. Here, after providing a background overview on ganglioside synthesis, localization and physiology, we will review those studies, which clearly show that anti-ganglioside antibodies are capable of binding to NMJs and thereby can exert a variety of pathophysiological effects. Furthermore, we will discuss the human clinical electrophysiological and histological evidence produced so far of the existence of a neuromuscular synaptopathy contributing to muscle weakness in GBS patients. [ABSTRACT FROM AUTHOR]

Guillain-Barré syndrome and its clinical variants, including the anti-GQ1b ganglioside–mediated Miller Fisher syndrome (MFS), comprise the world’s leading cause of acute neuromuscular paralysis. Presently, no specific drug therapies exist. The complement cascade, which is activated in these patients, forms an attractive drug target. In this study, we tested whether the complement C5–inhibiting recombinant protein, rEV576, was able to prevent neural injury in a previously developed in vitro mouse model for MFS. Mouse hemidiaphragm preparations were treated with anti-GQ1b antibody and normal human serum as a source of complement with added rEV576 or control protein. Immunohistology in control tissue showed deposition of C3c and membrane attack complex at neuromuscular junctions (NMJs), along with terminal motor axonal neurofilament degradation as well as ethidium homodimer-2 staining showing perisynaptic Schwann cell (pSC) injury. Electrophysiological and functional analyses showed block of synaptic transmission at the NMJ after an initial period of a dramatically high level of asynchronous acetylcholine release. In tissue treated with rEV576, all these indicators of motor neuronal damage were absent, except for the presence of C3c, indicating effective inhibition of C5. These results demonstrate that rEV576 effectively prevents development of neuronal and pSC damage in experimental murine neuropathy. [ABSTRACT FROM AUTHOR]

The Guillain Barré syndromes (GBS) are the world's leading cause of acute autoimmune neuromuscular paralysis. Understanding the pathophysiological events of GBS, and improving immunotherapies are fundamental to improving the clinical outcome. Recent research into GBS and the Miller Fisher syndrome (MFS) variant has focused on the forms mediated by anti-ganglioside antibodies in which correlations have been established between anti-ganglioside antibodies and specific clinical phenotypes, notably between anti-GM1/GD1a antibodies and the acute motor axonal variant and anti-GQ1b/GT1a antibodies and MFS. Anti-ganglioside antibodies can arise through molecular mimicry with GBS-associated Campylobacter jejuni oligosaccharides. Our work has focused on axonal and glial components of the motor nerve terminal as a model site of injury, and through combined active and passive immunization paradigms in glycosyltransferase knockout mice we have developed murine neuropathy phenotypes mediated by anti-ganglioside antibodies. Several determinants influence disease expression including the level of immunological tolerance to microbial glycans that mimic self gangliosides, the degree of complement activation, and the ganglioside density in target tissue. Such studies provide us with clear information on an antibody-mediated pathogenesis model for GBS and should lead to rational therapeutic testing of agents that are potentially suitable for use in man. [ABSTRACT FROM AUTHOR]

Episodic ataxia type 2 (EA2) is an autosomal dominantly inherited neurological disorder. Patients have CACNA1A gene mutations resulting in truncation or single amino acid changes in the pore-forming subunit of Cav2.1 (P/Q-type) Ca2+ channels. These neuronal channels mediate synaptic neurotransmitter release. EA2 symptoms are thought to result from disturbed neurotransmission at cerebellar and neuromuscular synapses, caused by loss-of-function of Cav2.1 channels. Heterozygous leaner ( Ln/wt) mice, carrying a Cacna1a truncation mutation, as well as heterozygous Cav2.1 null-mutant (KO/wt) mice may model synaptic aspects of EA2. We studied Cav2.1-mediated acetylcholine (ACh) release at their neuromuscular junctions (NMJs) ex vivo. KO/wt mice did not show any ACh release abnormalities, not even at older age. However, Ln/wt mice had ∼25% reduced spontaneous uniquantal ACh release and ∼10% reduced nerve-stimulation evoked release, compared with wild-type. EA2 is treated with acetazolamide (AZA), but the pharmacotherapeutic mechanism is unknown. We tested the possibility of a direct influence on (mutant) presynaptic Cav2.1 channel function by studying the acute effect of 50 μM AZA on ACh release at ex vivo NMJs of wild-type, KO/wt, and Ln/wt mice. No changes were found in any of the release parameters. Our results indicate that Ln-mutated Cav2.1 channels at Ln/wt NMJs are either normally inserted in the presynaptic membrane but have reduced function, or that they inhibit wild-type channels by hampering their expression, trafficking, membrane insertion and/or function. In this respect Ln/wt NMJs may model EA2 synapses. Furthermore, AZA does not exert an acute, direct influence on the function of presynaptic (mutant) Cav2.1 channels. Synapse 62:337-344, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The ataxic mouse rolling Nagoya (RN) carries a missense mutation in the Cacna1a gene, encoding the pore-forming subunit of neuronal Cav2.1 (P/Q-type) Ca2+ channels. Besides being the predominant type of Cav channel in the cerebellum, Cav2.1 channels mediate acetylcholine (ACh) release at the peripheral neuromuscular junction (NMJ). Therefore, Cav2.1 dysfunction induced by the RN mutation may disturb ACh release at the NMJ. The dysfunction may resemble the situation in Lambert–Eaton myasthenic syndrome (LEMS), in which autoantibodies target Cav2.1 channels at NMJs, inducing severely reduced ACh release and resulting in muscle weakness. We tested neuromuscular function of RN mice and characterized transmitter release properties at their NMJs in diaphragm, soleus and flexor digitorum brevis muscles. Clinical muscle weakness and fatigue were demonstrated using repetitive nerve-stimulation electromyography, grip strength testing and an inverted grid hanging test. Muscle contraction experiments showed a compromised safety factor of neuromuscular transmission. In ex vivo electrophysiological experiments we found severely impaired ACh release. Compared to wild-type, RN NMJs had 50–75% lower nerve stimulation-evoked transmitter release, explaining the observed muscle weakness. Surprisingly, the reduction in evoked release was accompanied by an ∼ 3-fold increase in spontaneous ACh release. This synaptic phenotype suggests a complex effect of the RN mutation on different functional Cav2.1 channel parameters, presumably with a positive shift in activation potential as a prevailing feature. Taken together, our studies indicate that the gait abnormality of RN mice is due to a combination of ataxia and muscle weakness and that RN models aspects of the NMJ dysfunction in LEMS. [ABSTRACT FROM AUTHOR]

The article presents an abstract related to the study of the development of synapse in mouse brain in the absence of presynaptic signaling. Researchers generated mice defective in presynaptic signaling deleting the munc18-1 gene, which is necessary for regulated exocytosis. These mice show no evoked or spontaneous secretion throughout development and throughout the brain.

Duchenne muscular dystrophy (DMD) is an X-linked myopathy caused by dystrophin deficiency. Dystrophin is present intracellularly at the sarcolemma, connecting actin to the dystrophin-associated glycoprotein complex. Interestingly, it is enriched postsynaptically at the neuromuscular junction (NMJ), but its synaptic function is largely unknown. Utrophin, a dystrophin homologue, is also concentrated at the NMJ, and upregulated in DMD. It is possible that the absence of dystrophin at NMJs in DMD causes neuromuscular transmission defects that aggravate muscle weakness. We studied NMJ function in mdx mice (lacking dystrophin) and wild type mice. In addition, mdx/utrn(+/-) and mdx/utrn(-/-) mice (lacking utrophin) were used to investigate influences of utrophin levels. The three Duchenne mouse models showed muscle weakness when comparatively tested in vivo, with mdx/utrn(-/-) mice being weakest. Ex vivo muscle contraction and electrophysiological studies showed a reduced safety factor of neuromuscular transmission in all models. NMJs had ~ 40% smaller miniature endplate potential amplitudes compared with wild type, indicating postsynaptic sensitivity loss for the neurotransmitter acetylcholine. However, nerve stimulation-evoked endplate potential amplitudes were unchanged. Consequently, quantal content (i.e. the number of acetylcholine quanta released per nerve impulse) was considerably increased. Such a homeostatic compensatory increase in neurotransmitter release is also found at NMJs in myasthenia gravis, where autoantibodies reduce acetylcholine receptors. However, high-rate nerve stimulation induced exaggerated endplate potential rundown. Study of NMJ morphology showed that fragmentation of acetylcholine receptor clusters occurred in all models, being most severe in mdx/utrn(-/-) mice. Overall, we showed mild 'myasthenia-like' neuromuscular synaptic dysfunction in several Duchenne mouse models, which possibly affects muscle weakness and degeneration., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)