Impaired Neuromuscular Transmission Research Articles

A novel mutation in the TPR6 domain of the RAPSN gene associated with congenital myasthenic syndrome

Congenital myasthenic syndromes (CMS) are rare genetic disorders characterized by impaired neuromuscular transmission. They are caused by mutations in synaptic, presynaptic and post synaptic proteins. Rapsyn is a postsynaptic peripheral membrane protein that anchors the nicotinic acetylcholine receptor to the motor endplate. CMS patients of Iraqi and Persian Jewish origin, carry a common founder mutation in the E box of the RAPSN promoter region (−38A-G) that causes impaired transcriptional activities of the promoter region.We describe a Persian Jewish family with two siblings affected with typical CMS, harboring the common heterozygous (−38A-G) E-box mutation associated with a previously unreported heterozygous p.224 insT causing an insertion of Threonine in the TPR6 domain. To the best of our knowledge, this is the first mutation in the TPR6 domain and might give supportive evidence to the role of this domain in rapsyn self association and consequently co-clustering with AchR in the post synaptic membrane.

3,4-diaminopyridine Phosphate in the Treatment of Lambert-Eaton Myasthenic Syndrome

3,4-diaminopyridine (3,4-DAP, amifampridine) is the leading treatment for Lambert–Eaton myasthenic syndrome (LEMS), an autoimmune disorder with impaired neuromuscular transmission, for which few effective medications are currently available. 3,4-DAP has been available as a therapy for LEMS in special treatment programmes for approximately 25 years. As an unlicensed drug, doses for oral administration are required to be compounded by local, hospital or other compounding pharmacies from the base chemical. Administering the correct dose of 3,4-DAP is critical; overdosing can increase the risk of seizures and other adverse events, while underdosing can result in a substantial loss of efficacy or even treatment failure. Two recent studies, have shown a wide variation in the 3,4-DAP content of compounded preparations (22.2–125.2 %, n=9) and (53.5–128.5 %, n=21), thereby reflecting the possibility of patients receiving dosages that might be above safety limits or even markedly below efficacy limits. This inconsistency results from the variable quality and instability of the base chemical and compounding errors. A formulation of 3,4-DAP phosphate salt has now been licensed in Europe and the US with orphan medicinal product status and appears to be as efficacious as the base in relieving the symptoms of LEMS.

Long-term follow-up of patients with congenital myasthenic syndrome caused by COLQ mutations

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous inherited disorders characterized by impaired neuromuscular transmission. Mutations in the acetylcholinesterase (AChE) collagen-like tail subunit gene (COlQ) cause recessive forms of synaptic CMS with end plate AChE deficiency. We present data on 15 COLQ -mutant CMS carrying 16 different mutations (9 novel ones identified) followed-up for an average period of 10years. The mean age at the first examination was 19years old (range from 3 to 48). We report relapses during short or long-term periods characterized by worsening of muscle weakness sometimes associated with respiratory crises. All the relapses ended spontaneously or with 3–4 DAP or ephedrine with no residual impairment. The triggering factors identified were esterase inhibitors, effort, puberty or pregnancy highlighting the importance of hormonal factors. There was no genotype–phenotype correlation. At the end of the follow-up, 80% of patients were ambulant and 87% of patients had no respiratory trouble in spite of severe relapses.

Concurrent Chronic Motor Axonal Polyneuropathy and Synaptic Impairment of Neuromuscular Junction

Polyneuropathies may exhibits clinical, electrophysiologic signs of neuromuscular junction impairment. Distal motor nerve terminals and neuromuscular junction contain pre or postsynaptically specific targets for circulating autoantibodies, if present in neuropathies. Motor nerve terminal blockade either reversible or permanent is a putative factor of muscle weakness. A 59-year-old patient exhibited oropharyngeal, facial, extremity weakness, fluctuating fatigability, and areflexia. Elecectrophysiologic studies showed purely motor axonal polyneuropathy. Thenar, facial slow rate repetitive stimulation revealed up to 47% decrement of compound muscle action potential size. Single fiber electromyography on voluntary activation confirmed increased jitter and impulse blocking in all muscles examined in one third of the fibers. Repeated testings for antibodies to gangliosides, acetylcholine, muscle tyrosine kinase receptors, voltage-gated calcium channels were negative. Oral pyridostigmine bromide improved bulbar symptoms. Pulse intravenous immunoglobulin, oral steroids, and azathioprine had steady benefit. Impairment of neuromuscular transmission if occurring in chronic axonal neuropathies highlights mechanisms and significance of neuromuscular chronic "synaptopathies."

APP and APLP2 are essential at PNS and CNS synapses for transmission, spatial learning and LTP

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock-in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα-DM) by crossing APPsα-KI mice onto an APLP2-deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα-DM muscle showed fragmented post-synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABA(A) receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

Impaired neuromuscular transmission and response to acetylcholinesterase inhibitors in centronuclear myopathies

Many clinical features of autosomal centronuclear myopathies (CNM) and X-linked myotubular myopathy (XLMTM) are common to congenital myasthenic syndromes (CMS). We describe three children whose clinical and electrophysiological findings originally suggested CMS, in whom CNM was diagnosed pathologically, though not yet genetically characterised. A fourth case, with XLMTM, also showed electrophysiological features of a neuromuscular transmission defect. Three (including the XLMTM case) showed improved strength with acetylcholinesterase inhibitor treatment. We also studied neuromuscular junction structure and function in the MTM1 knockdown zebrafish model of XLMTM, demonstrating abnormal neuromuscular junction organization; anticholinesterase therapy resulted in marked clinical response. These observations suggest that a neuromuscular transmission defect may accompany CNM and contribute to muscle weakness. Muscle biopsy should be considered in infants suspected to have CMS, especially if treatment response is incomplete, or no CMS gene mutation is identified. Treatment with acetylcholinesterase inhibitors may benefit some CNM patients. This warrants further confirmation.

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.

Subclinical Myasthenia Gravis causing increased sensitivity to botulinum toxin therapy

In rare patients receiving botulinum toxin (BT) therapy the potency of the BT drug may be increased by pre-existent impairment of neuromuscular transmission (INT). Appropriate dose adjustment may correct for this. Subclinical INT has previously been described by us for the first time in a patient with Lambert-Eaton myasthenic syndrome. We are now describing subclinical INT for the first time in a patient with ocular myasthenia gravis. Due to its unpredictability subclinical INT may pose a risk to patients receiving BT therapy.

Antibody effector mechanisms in myasthenia gravis—Pathogenesis at the neuromuscular junction

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies that are either directed to the muscle nicotinic acetylcholine receptor (AChR) or to the muscle-specific tyrosine kinase (MuSK). These autoantibodies define two distinct subforms of the disease—AChR-MG and MuSK-MG. Both AChR and MuSK are expressed on the postsynaptic membrane of the neuromuscular junction (NMJ), which is a highly specialized region of the muscle dedicated to receive and process signals from the motor nerve. Autoantibody binding to proteins of the postsynaptic membrane leads to impaired neuromuscular transmission and muscle weakness. Pro-inflammatory antibodies of the human IgG1 and IgG3 subclass modulate the AChR, cause complement activation, and attract lymphocytes; together acting to decrease levels of the AChR and AChR-associated proteins and to reduce postsynaptic folding. In patients with anti-MuSK antibodies, there is no evidence of loss of junctional folds and no apparent loss of AChR density. Anti-MuSK antibodies are predominantly of the IgG4 isotype, which functionally differs from other IgG subclasses in its anti-inflammatory activity. Moreover, IgG4 undergoes a posttranslational modification termed Fab arm exchange that prevents cross-linking of antigens. These findings suggest that MuSK-MG may be different in etiological and pathological mechanisms from AChR-MG. The effector functions of IgG subclasses on synapse structure and function are discussed in this review.

Impairment of diaphragm muscle force and neuromuscular transmission after normothermic cardiopulmonary bypass: effect of low-dose inhaled CO

Cardiopulmonary bypass (CPB) is associated with significant postoperative morbidity, but its effects on the neuromuscular system are unclear. Recent studies indicate that even relatively short periods of mechanical ventilation result in significant neuromuscular effects. Carbon monoxide (CO) has gained recent attention as therapy to reduce the deleterious effects of CPB. We hypothesized that 1) CPB results in impaired neuromuscular transmission and reduced diaphragm force generation; and 2) CO treatment during CPB will mitigate these effects. In adult male Sprague-Dawley rats, diaphragm muscle-specific force and neuromuscular transmission properties were measured 90 min after weaning from normothermic CPB (1 h). During CPB, either low-dose inhaled CO (250 ppm) or air was administered. The short period of mechanical ventilation used in the present study ( approximately 3 h) did not adversely affect diaphragm muscle contractile properties or neuromuscular transmission. CPB elicited a significant decrease in isometric diaphragm muscle-specific force compared with time-matched, mechanically ventilated rats ( approximately 25% decline in both twitch and tetanic force). Diaphragm muscle fatigability to 40-Hz repetitive stimulation did not change significantly. Neuromuscular transmission failure during repetitive activation was 60 +/- 2% in CPB animals compared with 76 +/- 4% in mechanically ventilated rats (P < 0.05). CO treatment during CPB abrogated the neuromuscular effects of CPB, such that diaphragm isometric twitch force and neuromuscular transmission were no longer significantly different from mechanically ventilated rats. Thus, CPB has important detrimental effects on diaphragm muscle contractility and neuromuscular transmission that are largely mitigated by CO treatment. Further studies are needed to ascertain the underlying mechanisms of CPB-induced neuromuscular dysfunction and to establish the potential role of CO therapy.

Neural cell adhesion molecule is required for stability of reinnervated neuromuscular junctions

Studies examining the etiology of motoneuron diseases usually focus on motoneuron death as the defining pathophysiology of the disease. However, impaired neuromuscular transmission and synapse withdrawal often precede cell death, raising the possibility that abnormalities in synaptic function contribute to disease onset. Although little is known about the mechanisms maintaining the synaptic integrity of neuromuscular junctions (NMJs), Drosophila studies suggest that Fasciclin II plays an important role. Inspired by these studies we used a reinnervation model of synaptogenesis to analyze neuromuscular function in mice lacking neural cell adhesion molecule (NCAM), the Fasciclin II vertebrate homolog. Our results showed that the recovery of contractile force was the same in wild-type and NCAM-/- mice at 1 month after nerve injury, indicating that endplates were appropriately reformed. This normality was only transient because the contractile force and myofiber number decreased at 3 months after injury in NCAM-/- mice. Both declined further 3 months later. Myofibers degenerated, not because motoneurons died but because synapses were withdrawn. Although neurotransmission was initially normal at reinnervated NCAM-/- NMJs, it was significantly compromised 3 months later. Interestingly, the selective ablation of NCAM from motoneurons, or muscle fibers, did not mimic the deficits observed in reinnervated NCAM-/- mice. Taken together, these results indicate that NCAM is required to maintain normal synaptic function at reinnervated NMJs, although its loss pre-synaptically or post-synaptically is not sufficient to induce synaptic destabilization. Consideration is given to the role of NCAM in terminal Schwann cells for maintaining synaptic integrity and how NCAM dysfunction may contribute to motoneuron disorders.

Muscle strength and fatigue in patients with generalized myasthenia gravis

Myasthenia gravis (MG) is characterized by fatigue and fluctuating muscle weakness resulting from impaired neuromuscular transmission (NMT). The objective of this study was to quantify, by direct measurement of muscle force, the strength and fatigue of patients with MG. A maximal voluntary isometric contraction protocol of shoulder abductors was used in conjunction with conventional fatigue and disease-severity instruments. Results from patients with (D-MG) and without (ND-MG) decrement on repetitive nerve stimulation (RNS) of the spinal accessory and axillary nerves were compared with healthy controls. Patients with MG reported greater fatigue than controls. Muscle strength was lowest in the D-MG group, followed by the ND-MG group and controls. Normalized shoulder abduction fatigue and recovery values did not differ between the D-MG and ND-MG groups or controls. The RNS decrement appears to relate best to disease severity and muscle weakness but not to objective measures of fatigue in this population.

Myasthenia gravis precipitated by trauma: Latent myasthenia and the concept of ‘threshold’

Myasthenia gravis is caused by antibodies to post-synaptic neuromuscular junction proteins, most commonly nicotinic acetylcholine receptors. Evidence indicates that acetylcholine receptor antibodies must be present in the circulation prior to the development of clinical or neurophysiological manifestations of myasthenia. We describe the onset of seropositive myasthenia within minutes of minor trauma to the chest and neck in a previously asymptomatic 68-year-old man, followed by protracted disease with persistent antibodies. Such rapid evolution of myasthenia following an identifiable stimulus has not been reported previously and suggests a humoral mechanism. We speculate that the remote effects of autoinflammation secondary to tissue microtrauma led to a sudden increase in muscle permeability and greater exposure of receptors to antibody, with resulting acute impairment of neuromuscular transmission. We also suggest that the trauma might have resulted in increased antibody production by remnant thymic tissue, leading to chronic isease.

G.O.8 Impaired neuromuscular transmission and response to acetylcholinesterase inhibitors in centronuclear myopathy

The centronuclear myopathies (CNM) are genetically heterogeneous and phenotypically variable congenital myopathies, characterised by prominent, centrally located nuclei in a significant proportion of muscle fibres.

Transcription Profile Analysis of Vastus Lateralis Muscle from Patients with Chronic Fatigue Syndrome

Chronic fatigue syndrome (CFS) is a disabling condition characterized by unexplained chronic fatigue that impairs normal activities. Many body systems are affected and etiology has not yet been identified. In addition to immunological and psychological aspects, skeletal muscle symptoms are prominent in CFS patients. In an effort to establish which pathways might be involved in the onset and development of muscle symptoms, we used global transcriptome analysis to identify genes that were differentially expressed in the vastus lateralis muscle of female and male CFS patients. We found that the expression of genes that play key roles in mitochondrial function and oxidative balance, including superoxide dismutase 2, were altered, as were genes involved in energy production, muscular trophism and fiber phenotype determination. Importantly, the expression of a gene encoding a component of the nicotinic cholinergic receptor binding site was reduced, suggesting impaired neuromuscular transmission. We argue that these major biological processes could be involved in and/or responsible for the muscle symptoms of CFS.

A Mammalian Homolog of Drosophila Tumorous Imaginal Discs, Tid1, Mediates Agrin Signaling at the Neuromuscular Junction

Motoneuron-derived agrin clusters nicotinic acetylcholine receptors (AChRs) in mammalian muscle cells. We used two-hybrid screens to identify a protein, tumorous imaginal discs (Tid1), that binds to the cytoplasmic domain of muscle-specific kinase (MuSK), a major component of the agrin receptor. Like MuSK, Tid1 colocalizes with AChRs at developing, adult, and denervated motor endplates. Knockdown of Tid1 by short hairpin RNA (shRNA) in skeletal muscle fibers dispersed synaptic AChR clusters and impaired neuromuscular transmission. In cultured myotubes, Tid1 knockdown inhibited AChR clustering, as well as agrin-induced activation of the Rac and Rho small GTPases and tyrosine phosphorylation of the AChR, without affecting MuSK activation. Tid1 knockdown also decreased Dok-7-induced clustering of AChRs. Overexpression of the N-terminal half of Tid1 induced agrin- and MuSK-independent phosphorylation and clustering of AChRs. These results demonstrate that Tid1 is an essential component of the agrin signaling pathway, crucial for synaptic development.

Changes of surface and t-tubular membrane excitability during fatigue with repeated tetani in isolated mouse fast- and slow-twitch muscle

We investigated whether impaired sarcolemmal excitability causes severe fatigue during repeated tetani in isolated mouse skeletal muscle. Slow-twitch soleus or fast-twitch extensor digitorum longus (EDL) muscles underwent intensive stimulation (standard protocol: 125 Hz for 500 ms, every second, parallel plate electrodes, 20 V, 0.1-ms pulses). Interventions with altered stimulation characteristics were tested either on the entire fatigue profile or after 90- to 100-s stimulation. d-tubocurarine did not alter the fatigue profile in soleus thereby eliminating impaired neuromuscular transmission. Lower stimulation frequencies partially restored peak force, especially in soleus. The twitch force-stimulation strength relationship shifted towards higher voltages in both muscle types, with a much larger shift in EDL. Augmenting pulse strength restored tetanic force from 29% (4.4 V) to 79% (20 V), or slowed fatigue in soleus. Increasing pulse duration (0.1 to 1.0 ms) restored tetanic force from 8 to 46% in EDL and from 41 to 90% in soleus; 0.25-ms pulses restored tetanic force to 83% in soleus. Switching from transverse wire to parallel plate stimulation increased tetanic force from 34 to 63%, and fatigue was exacerbated with wires compared with plates in soleus. The combined data suggest that impaired excitability (disrupted action potential generation) within trains is the main contributor ( approximately 50% initial force) to severe fatigue in both muscle types, the surface rather than t-tubular membrane is the main site of impairment during wire stimulation, and extreme fatigue in EDL includes an increased action potential threshold leading to inexcitable fibers. Moreover, mathematical modeling discounts anoxia as the major contributor to fatigue during our stimulation regime in isolated muscles.

A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in α-synuclein transgenic C . elegans

Mutations or multiplications in alpha-synuclein gene cause familial forms of Parkinson disease or dementia with Lewy bodies (LB), and the deposition of wild-type alpha-synuclein as LB occurs as a hallmark lesion of these disorders, collectively referred to as synucleinopathies, implicating alpha-synuclein in the pathogenesis of synucleinopathy. To identify modifier genes of alpha-synuclein-induced neurotoxicity, we conducted an RNAi screen in transgenic C. elegans (Tg worms) that overexpress human alpha-synuclein in a pan-neuronal manner. To enhance the RNAi effect in neurons, we crossed alpha-synuclein Tg worms with an RNAi-enhanced mutant eri-1 strain. We tested RNAi of 1673 genes related to nervous system or synaptic functions, and identified 10 genes that, upon knockdown, caused severe growth/motor abnormalities selectively in alpha-synuclein Tg worms. Among these were four genes (i.e. apa-2, aps-2, eps-8 and rab-7) related to the endocytic pathway, including two subunits of AP-2 complex. Consistent with the results by RNAi, crossing alpha-synuclein Tg worms with an aps-2 mutant resulted in severe growth arrest and motor dysfunction. alpha-Synuclein Tg worms displayed a decreased touch sensitivity upon RNAi of genes involved in synaptic vesicle endocytosis, and they also showed impaired neuromuscular transmission, suggesting that overexpression of alpha-synuclein caused a failure in uptake or recycling of synaptic vesicles. Furthermore, knockdown of apa-2, an AP-2 subunit, caused an accumulation of phosphorylated alpha-synuclein in neuronal cell bodies, mimicking synucleinopathy. Collectively, these findings raise a novel pathogenic link between endocytic pathway and alpha-synuclein-induced neurotoxicity in synucleinopathy.

Demyelinating disease in patients with myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease characterized by fluctuating muscle weakness, caused by impaired neuromuscular transmission. Patients with MG can present other autoimmune diseases in association, commonly hypo or hyperthyroidism. The association of MG to demyelinating disease is rare and has been described before. We report on three Brazilian patients with MG that presented distinct demyelinating diseases, two monophasic and one recurrent neuromyelitis optica, several years after the diagnosis of MG, and discuss their clinical courses.

Clinical and molecular genetic findings in COLQ-mutant congenital myasthenic syndromes

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous inherited disorders characterized by impaired neuromuscular transmission. Mutations in the acetylcholinesterase (AChE) collagen-like tail subunit gene (COLQ) cause synaptic basal-lamina associated CMS with end-plate AChE deficiency. Here we present the clinical and molecular genetic findings of 22 COLQ-mutant CMS patients, carrying a total of 20 different COLQ mutations, 11 of them had not previously been reported. Typically, patients with esterase deficiency suffer from a severe, progressive weakness with onset at birth or in early infancy. In addition, patients with a late onset showing a mild course of disease are described. AChE inhibitor therapy, beneficial for other forms of CMS, is of no effect in cases of esterase deficiency. The large cohort of COLQ patients studied here enabled us to define additional clinical presentations associated with COLQ mutations that differ from the 'classical' phenotypes: several patients with disease onset at birth or in early infancy presented an unexpected, mild disease course without significant progression of weakness. Moreover, many patients had clinical features reminiscent of limb-girdle CMS with mutations in the recently discovered DOK7 gene, including sparing of eye movements and a predominantly proximal muscle weakness. There was no long-term objective benefit from esterase inhibitors treatment in COLQ patients. Surprisingly, a short-term beneficial effect was observed in four patients and a Tensilon test was positive in two. Treatment with ephedrine was efficient in all five cases where it was administered. The variability of phenotypes caused by COLQ mutations, the divergence from the previously published classical clinical features and an initial positive response to esterase inhibitors in some patients may obscure AChE deficiency as the molecular cause of the disease and delay the start of appropriate therapy. Moreover, overlap with other CMS subtypes and potentially absence of a repetitive compound muscle action potential should be considered in the diagnosis of COLQ-mutated patients.