Postsynaptic Neuromuscular Junction Research Articles

Effective treatment with oral Salbutamol on late onset respiratory impairment in a DOK7 Congenital Myasthenia Syndrome: a case report.

DOK7 gene deficiency is a neuromuscular disease with an alteration in post-synaptic neuromuscular junction, leading to progressive respiratory impairment. Although, the therapy is not standardized, adrenergic agonists are suggested as first-line treatment. Case presentation: Our patient had an ambiguous late childhood-onset and had a generalized muscle weakness free of respiratory symptoms during the early phase of the disease. Subsequently, when the respiratory muscle and the diaphragm involvement was impaired, a substantial loss of respiratory function with hypopneas and severe desaturation was detected. It was noteworthy the striking respiratory beneficial impact of oral salbutamol in the resolution of symptoms and functional impairments, leading to a remarkable respiratory improvement and a better quality of life. Conclusion: Oral salbutamol treatment combined to a timely clinical recognition led to an outstanding respiratory improvement.

Single Nuclear Analyses Reveal Compromised Integrity of Neuromuscular Junction Myonuclei Following Joint Injury

The neuromuscular junction is an essential structure that links electrical signals from the nervous system to contraction of skeletal muscle. The post synaptic region of a muscle cell is maintained by a subset of specialized myonuclei responsible for clustering of post synaptic structural proteins and acetylcholine receptors. Disruptions in the transcriptional program of these myonuclei may result in ineffective myofiber recruitment, slowed force generation, and muscle weakness. We have shown that traumatic joint injury results in protracted deficits in muscle strength and power, but the peripheral mechanistic underpinnings remain unclear. Phenotypically, muscle pathology occurring after traumatic joint injury resembles that of neuromuscular destabilization, yet molecularly, this remains uninvestigated. Therefore, we hypothesize that disruption of neuromuscular regulatory gene programs and destabilization of the neuromuscular junction following joint injury will result in rapid muscle functional deficits. Human muscle biopsies were obtained from the vastus lateralis of anterior cruciate ligament (ACL) injured and healthy limbs of 26 participants. Injured biopsies were taken before and 7 days following reconstruction surgery. Bulk tissue RNA-seq analysis was performed on all participants and single-nucleus RNAseq (snRNAseq) was performed on a subset of participants. Briefly, for snRNAseq, muscle was dounce homogenized, filtered, and nuclei isolated via fluorescent activated cell sorting. Following sequencing, CellRanger output files containing count matrices were filtered, and analyzed using Partek Flow and Seurat. Biopsy sections were processed for histochemistry to analyze fiber size and motor end plate innervation. All participants underwent strength testing on a biodex to assess isometric quadriceps torque and rate of torque development. RNA sequencing displayed >3 fold upregulation of CHRNA1 (P< 0.001) after injury alone and >18 fold upregulation 7 days following reconstruction surgery (P< 0.001). This was accompanied by a host of other neuromuscular regulatory genes including HDAC4 (P< 0.001), GDNF (P< 0.001), and RUNX1 (P< 0.001). Single nucleus sequencing revealed a complete disappearance of neuromuscular junction regulatory myonuclei following reconstruction surgery coupled with delocalization of acetylcholine receptor expression. These findings were confirmed with fluorescent in situ hybridization of CHRNA1 RNA on biopsy sections. Functionally, CHRNA1 RNA expression strongly correlates with deficits in rate of torque development following injury (R^2 = 0.85, P< 0.001). Our results display rapid remodeling of the postsynaptic neuromuscular junction following joint injury that underscores sustained deficits in strength and power. Interventions targeting the preservation of peripheral innervation may be warranted to enhance outcomes following joint injury. R01AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial targeted catalase improves muscle strength following arteriovenous fistula creation in mice with chronic kidney disease

Hand dysfunction is a common observation after arteriovenous fistula (AVF) creation for hemodialysis access and has a variable clinical phenotype; however, the underlying mechanism responsible is unclear. Grip strength changes are a common metric used to assess AVF-associated hand disability but has previously been found to poorly correlate with the hemodynamic perturbations post-AVF placement implicating other tissue-level factors as drivers of hand outcomes. In this study, we sought to test if expression of a mitochondrial targeted catalase (mCAT) in skeletal muscle could reduce AVF-related limb dysfunction in mice with chronic kidney disease (CKD). Male and female C57BL/6J mice were fed an adenine-supplemented diet to induce CKD prior to placement of an AVF in the iliac vascular bundle. Adeno-associated virus was used to drive expression of either a green fluorescent protein (control) or mCAT using the muscle-specific human skeletal actin (HSA) gene promoter prior to AVF creation. As expected, the muscle-specific AAV-HSA-mCAT treatment did not impact blood urea nitrogen levels (P = 0.72), body weight (P = 0.84), or central hemodynamics including infrarenal aorta and inferior vena cava diameters (P > 0.18) or velocities (P > 0.38). Hindlimb perfusion recovery and muscle capillary densities were also unaffected by AAV-HSA-mCAT treatment. In contrast to muscle mass and myofiber size which were not different between groups, both absolute and specific muscle contractile forces measured via a nerve-mediated in-situ preparation were significantly greater in AAV-HSA-mCAT treated mice (P = 0.0012 and P = 0.0002). Morphological analysis of the post-synaptic neuromuscular junction uncovered greater acetylcholine receptor cluster areas (P = 0.0094) and lower fragmentation (P = 0.0010) in AAV-HSA-mCAT treated mice. Muscle mitochondrial oxidative phosphorylation was not different between groups, but AAV-HSA-mCAT treated mice had lower succinate-fueled mitochondrial hydrogen peroxide emission compared to AAV-HSA-GFP mice (P < 0.001). In summary, muscle-specific scavenging of mitochondrial hydrogen peroxide significantly improves neuromotor function in mice with CKD following AVF creation.

Postsynaptic β1spectrin maintains Na+ channels at the neuromuscular junction.

Spectrins function together with actin as obligatory subunits of the submembranous cytoskeleton. Spectrins maintain cell shape, resist mechanical forces, and stabilize ion channel and transporter protein complexes through binding to scaffolding proteins. Recently, pathogenic variants of SPTBN4 (β4spectrin) were reported to cause both neuropathy and myopathy. Although the role of β4spectrin in neurons is mostly understood, its function in skeletal muscle, another excitable tissue subject to large forces, is unknown. Here, using a muscle specific β4spectrin conditional knockout mouse, we show that β4spectrin does not contribute to muscle function. In addition, we show β4spectrin is not present in muscle, indicating the previously reported myopathy associated with pathogenic SPTBN4 variants is neurogenic in origin. More broadly, we show that α2, β1 and β2spectrins are found in skeletal muscle, with α2 and β1spectrins being enriched at the postsynaptic neuromuscular junction (NMJ). Surprisingly, using muscle specific conditional knockout mice, we show that loss of α2 and β2spectrins had no effect on muscle health, function or the enrichment of β1spectrin at the NMJ. Muscle specific deletion of β1spectrin also had no effect on muscle health, but, with increasing age, resulted in the loss of clustered NMJ Na+ channels. Together, our results suggest that muscle β1spectrin functions independently of an associated α spectrin to maintain Na+ channel clustering at the postsynaptic NMJ. Furthermore, despite repeated exposure to strong forces and in contrast to neurons, muscles do not require spectrin cytoskeletons to maintain cell shape or integrity. KEY POINTS: The myopathy found in pathogenic human SPTBN4 variants (where SPTBN4 is the gene encoding β4spectrin) is neurogenic in origin. β1spectrin plays essential roles in maintaining the density of neuromuscular junction Nav1.4 Na+ channels. By contrast to the canonical view of spectrin organization and function, we show that β1spectrin can function independently of an associated α spectrin. Despite the large mechanical forces experienced by muscle, we show that spectrins are not required for muscle cell integrity. This is in stark contrast to red blood cells and the axons of neurons.

How should newer therapeutic agents be incorporated into the treatment of patients with myasthenia gravis?

Generalized myasthenia gravis (gMG) is a postsynaptic neuromuscular junction disorder that results in fatigable muscle weakness. The traditional treatment approach includes the use of acetylcholinesterase inhibitors, corticosteroids, and steroid-sparing immunosuppressant therapies (ISTs) for chronic management, whereas exacerbations and crises are managed with intravenous immunoglobulin (IVIg) and plasma exchange (PLEX). Over the past 6 years, four new therapeutic agents with novel immunological mechanisms of action-complement and neonatal Fc receptor (FcRn) inhibition-were approved as a result of clinically significant improvement in gMG symptoms of those treated with these newer agents in Phase 3 clinical trials. At present, it is unclear when and in whom to initiate these therapeutic agents and how to integrate them into the current treatment paradigm. When selecting a newer therapeutic agent, we use a simple equation: Value = Clinical Improvement/(Cost + Side Effects + Treatment Burden), which guides our decision-making. We consider using these novel therapeutic agents in two specific clinical situations. Firstly, the newer agents are fast-acting, suggesting they can be used in clinically unstable patients as "bridge therapy," and secondly, they provide additional options for those patients considered treatment-refractory. There are downsides, however, including treatment cost, unique side effect profiles, and intravenous and subcutaneous drug administration (though for some, this may be an advantage). As additional drugs enter the marketplace with unique mechanisms of action, routes of administration, and dosing schedules, the placement of the novel therapeutic agents in the gMG treatment algorithm will likely evolve.

Ravulizumab in Myasthenia Gravis: A Review of the Current Evidence.

The terminal complement C5 inhibitor ravulizumab was engineered from the humanized monoclonal antibody eculizumab to have an extended half-life and duration of action. It binds to human terminal complement protein C5, inhibiting its cleavage into C5a and C5b, thus preventing the cascade of events that lead to architectural destruction of the postsynaptic neuromuscular junction membrane by the membrane attack complex, and consequent muscle weakness in patients with anti-acetylcholine receptor (AChR) antibody-positive generalized myasthenia gravis (gMG). The 26-week randomized, placebo-controlled period (RCP) of the phase 3 CHAMPION MG study demonstrated the rapid efficacy of ravulizumab in reducing MG symptoms. Weight-based dosing of ravulizumab every 8 weeks provided sustained efficacy, in terms of patient-reported (Myasthenia Gravis-Activities of Daily Living) and clinician-reported (Quantitative Myasthenia Gravis) endpoints in patients with anti-AChR antibody-positive gMG. Pharmacokinetic and pharmacodynamic analyses showed therapeutic serum ravulizumab concentrations (>175 µg/mL) were achieved immediately after the first dose and were maintained throughout 26 weeks, irrespective of patient body weight; inhibition of serum free C5 was immediate, complete (<0.5 μg/mL), and sustained in all patients. Interim results from the open-label extension (OLE) showed that after 60 weeks, efficacy was maintained in patients continuing on ravulizumab. Rapid and sustained improvements in efficacy, similar to those seen in patients initiating ravulizumab in the RCP, were observed after initiation of ravulizumab treatment in patients who switched from placebo in the RCP to ravulizumab in the OLE. The findings from the RCP and OLE support ravulizumab's favorable safety profile. In conclusion, ravulizumab has a simple weight-based administration and long dosing interval. Its targeted mechanism of action without generalized immunosuppression is reflected in its rapid onset of symptom improvement, sustained efficacy and good safety profile in the treatment of patients with anti-AChR antibody-positive gMG.

Lack of Sod1 induces disruption of innervation accompanied by Schwann cell proliferation in skeletal muscle

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Myasthenia gravis in current status: epidemiology, types, etiology, pathophysiology, symptoms, diagnostic tests, prevention, treatment, and complications - correspondence.

Myasthenia gravis in current status: epidemiology, types, etiology, pathophysiology, symptoms, diagnostic tests, prevention, treatment, and complications - correspondence.

ROLE OF AYURVEDA IN THE MANAGEMENT OF MYASTHENIA GRAVIS: A CASE STUDY

Myasthenia Gravis (MG) is a neuromuscular junction disease that leads to varying degrees of skeletal muscle weakness with fatigue. The underlying defect in diseases is a decrease in the number of available acetylcholine receptors (AChRs) at postsynaptic neuromuscular junctions due to the binding of autoantibody at acetylcholine receptors. The most commonly and firstly affected muscles are those of the eyes, i.e., ocular muscles leading to drooping of eyelids because of more availability of receptors in less surface area of muscle, later leads to symptoms like difficulty in swallowing leading to (dysphagia), Difficulty to speak (dysarthria), other skeletal muscles fatigability etc. In Ayurveda, we cannot correlate to any particular diseases, but it can be understood in view of upahata dhatu ushma, srotas and marutha, respectively, leading to avarana to the normal functioning of vata. The present study was conducted to determine the efficacy of Ayurvedic management in Myasthenia gravis. In this paper, a case of 47 years old female who was diagnosed with myasthenia gravis (MG) with autoimmune hypothyroidism for 13 years was reported to complain of drooping of eyelids, pain and stiffness at the nape of the neck and bilateral shoulder joints, disturbed sleep, reduced appetite, with occasional diplopia, dysphagia, which got aggravated for six months, hence she approached OPD of SKAMCH&amp;RC, Bengaluru. Considering the signs and symptoms patient was treated on the line of kaphavruta vata, and by considering urdwajatrugata lakshana shirodhara, greevabasti along with nasya line of treatment was adopted, and when a patient got relief in her symptoms, and she was wanted to continue her treatment hence virechana was adopted for sroto shodhanartha. Treatment shows significant improvement in her symptoms without any side effects.

Dichotomous cis-regulatory motifs mediate the maturation of the neuromuscular junction by retrograde BMP signaling.

Retrograde bone morphogenetic protein (BMP) signaling at the Drosophila neuromuscular junction (NMJ) has served as a paradigm to study TGF-β-dependent synaptic function and maturation. Yet, how retrograde BMP signaling transcriptionally regulates these functions remains unresolved. Here, we uncover a gene network, enriched for neurotransmission-related genes, that is controlled by retrograde BMP signaling in motor neurons through two Smad-binding cis-regulatory motifs, the BMP-activating (BMP-AE) and silencer (BMP-SE) elements. Unpredictably, both motifs mediate direct gene activation, with no involvement of the BMP derepression pathway regulators Schnurri and Brinker. Genome editing of candidate BMP-SE and BMP-AE within the locus of the active zone gene bruchpilot, and a novel Ly6 gene witty, demonstrated the role of these motifs in upregulating genes required for the maturation of pre- and post-synaptic NMJ compartments. Our findings uncover how Smad-dependent transcriptional mechanisms specific to motor neurons directly orchestrate a gene network required for synaptic maturation by retrograde BMP signaling.

Pharmacotherapy of Generalized Myasthenia Gravis with Special Emphasis on Newer Biologicals.

Myasthenia gravis (MG) is a chronic, fluctuating, antibody-mediated autoimmune disorder directed against the post-synaptic neuromuscular junctions of skeletal muscles, resulting in a wide spectrum of manifestations ranging from mild to potentially fatal. Given its unique natural course, designing an ideal trial design for MG has been wrought with difficulties and evidence in favour of several of the conventional agents is weak as per current standards. Despite this, acetylcholinesterases and corticosteroids have remained the cornerstones of treatment for several decades with intravenous immunoglobulins (IVIG) and therapeutic plasma exchange (PLEX) offering rapid treatment response, especially in crises. However, the treatment of MG entails long-term immunosuppression and conventional agents are viable options but take longer to act and have a number of class-specific adverse effects. Advances in immunology, translational medicine and drug development have seen the emergence of several newer biological agents which offer selective, target-specific immunotherapy with fewer side effects and rapid onset of action. Eculizumab is one of the newer agents that belong to the class of complement inhibitors and has been approved for the treatment of refractory general MG. Zilucoplan and ravulizumab are other agents in this group in clinical trials. Neisseria meningitis is a concern with all complement inhibitors, mandating vaccination. Neonatal Fc receptor (FcRn) inhibitors prevent immunoglobulin recycling and cause rapid reduction in antibody levels. Efgartigimod is an FcRn inhibitor recently approved for MG treatment, and rozanolixizumab, nipocalimab and batoclimab are other agents in clinical trial development. Although lacking high quality evidence from randomized clinical trials, clinical experience with the use of anti-CD20 rituximab has led to its use in refractory MG. Among novel targets, interleukin 6 (IL6) inhibitors such as satralizumab are promising and currently undergoing evaluation. Cutting-edge therapies include genetically modifying T cells to recognise chimeric antigen receptors (CAR) and chimeric autoantibody receptors (CAAR). These may offer sustained and long-term remissions, but are still in very early stages of evaluation. Hematopoietic stem cell transplantation (HSCT) allows immune resetting and offers sustained remission, but the induction regimens often involve serious systemic toxicity. While MG treatment is moving beyond conventional agents towards target-specific biologicals, lack of knowledge as to the initiation, maintenance, switching, tapering and long-term safety profile necessitates further research. These concerns and the high financial burden of novel agents may hamper widespread clinical use in the near future.

Distal oesophageal spasm as a manifestation of myasthenia gravis

Myasthenia gravis is an autoimmune neuromuscular junction disorder affecting skeletal muscles. It is characterized by muscle fatigability with fluctuating weakness and diurnal variations. Here, we present an elderly female presented with subacute onset dysphagia predominantly to solids with weight loss for one month. Upper gastrointestinal endoscopy showed a crico-esophageal spasm. Manometric studies showed distal esophageal spasm with normal pressures. Clinically, she had proximal muscle weakness of all four limbs with bulbar weakness. Nerve conduction studies done were suggestive of postsynaptic neuromuscular junction disorder. Serum acetylcholinesterase receptor antibodies were elevated. We started her on pyridostigmine and low dose steroids, following which her symptoms improved. Here smooth muscle involvement was considered secondary to myasthenia gravis, which recovered after treatment. Treating physicians should even think of neurological causes in patients presenting with dysphagia predominant to solids.

Variation in muscle and neuromuscular junction morphology between atrophy-resistant and atrophy-prone muscles supports failed re-innervation in aging muscle atrophy.

In advanced age, there is an accelerated decline in skeletal muscle mass that appears to be secondary to repeated cycles of denervation-reinnervation and eventually, failed reinnervation. However, whether variation in reinnervation capacity explains why some muscles are less vulnerable to age-related atrophy has not been addressed. In this study we examined changes in neuromuscular junction (NMJ) morphology, fiber cross-sectional area (CSA) and fiber type, accumulation of severely atrophied myofibers, and expression of a marker of denervation in four muscles that exhibit differences in the degree of age-related atrophy and which span the extremes of fiber type composition in 8 mo old (8M) and 34 mo old (34M) male Fischer 344 Brown Norway F1 hybrid rats. Aging muscle atrophy was most pronounced in the fast twitch gastrocnemius (Gas; 25%) and similar between extensor digitorum longus (EDL) and slow-twitch soleus (Sol) muscle (14-15%), whereas the slow-twitch adductor longus (AL) increased in mass by 21% between 8M and 34M (P<0.05 for all). Only the Sol exhibited significant alterations in fiber type with aging, and there was a decrease in fiber CSA in the Gas, EDL, and Sol (P<0.05) with aging that was not seen in the AL. Muscles that atrophied had an increased fraction of severely atrophic myofibers (P<0.05), but this was not observed in the AL. The Gas and EDL both demonstrated a similar degree of age-related remodeling of pre- and post-synaptic NMJ components. On the other hand, pre- and post-synaptic morphology underwent greater changes with aging in the AL, and many of these same morphological variables were already greater in the Sol vs AL at 8M, suggesting the Sol had already undergone substantial remodeling and may be nearing its adaptive limits. Consistent with this idea, analysis of NMJ morphology in Sol from 3M rats exhibited similar values as 8M AL, and the Sol demonstrated greater expression of the denervation marker neural cell adhesion molecule (NCAM) compared to the AL at 34M. Collectively, our results are consistent with NMJ remodeling capacity being finite with aging and that maintained remodeling potential confers atrophy protection in aging skeletal muscle by reducing the degree of persistent denervation.

Postsynaptic neuromuscular junction defect in a patient with miller fisher syndrome

Miller Fisher syndrome (MFS) is an acute immune-mediated neuropathy. To date only few studies reported a possible overlap of Myasthenia Gravis (MG) and MFS in patients with acute ophthalmoplegia. We report the case of a patient diagnosed as MFS with a coexistent neuromuscular junction defect.

Anesthetic implications in patients with myasthenia gravis undergoing spine surgery

Myasthenia gravis is a chronic autoimmune disorder affecting post-synaptic neuromuscular junction. It is characterized by skeletal muscle weakness and fatigability which improves with rest. The anesthesiologists may encounter these patients for thymectomy or sometimes for incidental surgeries. However, it is a real challenge for anesthesiologists because of the unpredictable response with muscle relaxants and analgesics and the possibility of ventilatory complications. In the present case report, we report the management of a 52-year-old female patient with myasthenia gravis who is posted for schwannoma surgery.

New recessive mutations in SYT2 causing severe presynaptic congenital myasthenic syndromes.

ObjectiveTo report the identification of 2 new homozygous recessive mutations in the synaptotagmin 2 (SYT2) gene as the genetic cause of severe and early presynaptic forms of congenital myasthenic syndromes (CMSs).MethodsNext-generation sequencing identified new homozygous intronic and frameshift mutations in the SYT2 gene as a likely cause of presynaptic CMS. We describe the clinical and electromyographic patient phenotypes, perform ex vivo splicing analyses to characterize the effect of the intronic mutation on exon splicing, and analyze the functional impact of this variation at the neuromuscular junction (NMJ).ResultsThe 2 infants presented a similar clinical phenotype evoking first a congenital myopathy characterized by muscle weakness and hypotonia. Next-generation sequencing allowed to the identification of 1 homozygous intronic mutation c.465+1G>A in patient 1 and another homozygous frameshift mutation c.328_331dup in patient 2, located respectively in the 5′ splice donor site of SYT2 intron 4 and in exon 3. Functional studies of the intronic mutation validated the abolition of the splice donor site of exon 4 leading to its skipping. In-frame skipping of exon 4 that encodes part of the C2A calcium-binding domain of SYT2 is associated with a loss-of-function effect resulting in a decrease of neurotransmitter release and severe pre- and postsynaptic NMJ defects.ConclusionsThis study identifies new homozygous recessive SYT2 mutations as the underlying cause of severe and early presynaptic form of CMS expanding the genetic spectrum of recessive SYT2-related CMS associated with defects in neurotransmitter release.

Myasthenia Gravis and its Association With Thyroid Diseases.

Myasthenia gravis (MG) is a rare autoimmune neuromuscular junction disorder, and thyroid disorder is a disorder involving the thyroid receptor, of which Graves' disease (GD) is the most common autoimmune thyroid disorder, in which antibodies develop against thyroid receptors. Both may have similar clinical features. In myasthenia gravis, autoimmune antibodies develop against postsynaptic neuromuscular junction disrupting the neuromuscular transmission, resulting in fluctuating muscle weakness and fatigue. It is a disease of young women and older men. The two pathologies may coexist in a patient or can precede one another. Graves' disease (GD) among thyroid diseases is most often associated with MG. Similarities in clinical features lead to difficulty in distinguishing MG and GD. Despite the standard treatment of myasthenia gravis, including steroids, acetylcholinesterases, rituximab, immunosuppressants, and thymectomy, there is still an increased number of relapses and myasthenia crisis. Eculizumab and plasmapheresis are the two new treatment options for MG, with supporting evidence of marked improvement in recent studies. Myasthenia gravis and Graves' disease have a see-saw relationship. Treating one pathology may worsen the other, so physicians should always consider MG as a differential in patients of hyperthyroidism presenting with new symptoms of fatigue or respiratory failure or neuromuscular weakness.In this comprehensive review article, we tried to establish an association between myasthenia gravis and Graves' disease (GD) by exploring currently available literature from PubMed. However, more studies need to be done to establish an association between pathologies.

Monoclonal antibodies to fetal bovine serum acetylcholinesterase distinguish between acetylcholinesterases from ruminant and non-ruminant species

Two types of cholinesterases (ChEs) are present in mammalian blood and tissues: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). While AChE regulates neurotransmission by hydrolyzing acetylcholine at the postsynaptic membranes and neuromuscular junctions, BChE in plasma has been suggested to be involved in detoxifying toxic compounds. This study was undertaken to establish the identity of circulating ChE activity in plasmas from domestic animals (bovine, ovine, caprine, porcine and equine) by assessing sensitivity to AChE-specific inhibitors (BW284c51 and edrophonium) and BChE-specific inhibitors (dibucaine, ethopropazine and Iso-OMPA) as well as binding to anti-FBS AChE monoclonal antibodies (MAbs). Based on the inhibition of ChE activity by ChE-specific inhibitors, it was determined that bovine, ovine and caprine plasma predominantly contain AChE, while porcine and equine plasma contain BChE. Three of the anti-FBS AChE MAbs, 4E5, 5E8 and 6H9, inhibited 85–98% of enzyme activity in bovine, ovine and caprine plasma, confirming that the esterase in these plasmas was AChE. These MAbs did not bind to purified recombinant human or mouse AChE, demonstrating that these MAbs were specific for AChEs from ruminant species. These MAbs did not inhibit the activity of purified human BChE, or ChE activity in porcine and equine plasma, confirming that the ChE in these plasmas was BChE. Taken together, these results demonstrate that anti-FBS AChE MAbs can serve as useful tools for distinguishing between AChEs from ruminant and non-ruminant species and BChEs.

Morphological variability is greater at developing than mature mouse neuromuscular junctions.

The neuromuscular junction (NMJ) is the highly specialised peripheral synapse formed between lower motor neuron terminals and muscle fibres. Post‐synaptic acetylcholine receptors (AChRs), which are found in high density in the muscle membrane, bind to acetylcholine released into the synaptic cleft of the NMJ, thereby enabling the conversion of motor action potentials to muscle contractions. NMJs have been studied for many years as a general model for synapse formation, development and function, and are known to be early sites of pathological changes in many neuromuscular diseases. However, information is limited on the diversity of NMJs in different muscles, how synaptic morphology changes during development, and the relevance of these parameters to neuropathology. Here, this crucial gap was addressed using a robust and standardised semi‐automated workflow called NMJ‐morph to quantify features of pre‐ and post‐synaptic NMJ architecture in an unbiased manner. Five wholemount muscles from wild‐type mice were dissected and compared at immature (post‐natal day, P7) and early adult (P31−32) timepoints. The inter‐muscular variability was greater in mature post‐synaptic AChR morphology than that of the pre‐synaptic motor neuron terminal. Moreover, the developing NMJ showed greater differences across muscles than the mature synapse, perhaps due to the observed distinctions in synaptic growth between muscles. Nevertheless, the amount of nerve to muscle contact was consistent, suggesting that pathological denervation can be reliably compared across different muscles in mouse models of neurodegeneration. Additionally, mature post‐synaptic endplate diameters correlated with fibre type, independently of muscle fibre diameter. Altogether, this work provides detailed information on healthy pre‐ and post‐synaptic NMJ morphology from five anatomically and functionally distinct mouse muscles, delivering useful reference data for future comparison with neuromuscular disease models.

Myasthenia Gravis: From the Viewpoint of Pathogenicity Focusing on Acetylcholine Receptor Clustering, Trans-Synaptic Homeostasis and Synaptic Stability.

Myasthenia gravis (MG) is a disease of the postsynaptic neuromuscular junction (NMJ) where nicotinic acetylcholine (ACh) receptors (AChRs) are targeted by autoantibodies. Search for other pathogenic antigens has detected the antibodies against muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein-related protein 4 (Lrp4), both causing pre- and post-synaptic impairments. Agrin is also suspected as a fourth pathogen. In a complex NMJ organization centering on MuSK: (1) the Wnt non-canonical pathway through the Wnt-Lrp4-MuSK cysteine-rich domain (CRD)-Dishevelled (Dvl, scaffold protein) signaling acts to form AChR prepatterning with axonal guidance; (2) the neural agrin-Lrp4-MuSK (Ig1/2 domains) signaling acts to form rapsyn-anchored AChR clusters at the innervated stage of muscle; (3) adaptor protein Dok-7 acts on MuSK activation for AChR clustering from “inside” and also on cytoskeleton to stabilize AChR clusters by the downstream effector Sorbs1/2; (4) the trans-synaptic retrograde signaling contributes to the presynaptic organization via: (i) Wnt-MuSK CRD-Dvl-β catenin-Slit 2 pathway; (ii) Lrp4; and (iii) laminins. The presynaptic Ca2+ homeostasis conditioning ACh release is modified by autoreceptors such as M1-type muscarinic AChR and A2A adenosine receptors. The post-synaptic structure is stabilized by: (i) laminin-network including the muscle-derived agrin; (ii) the extracellular matrix proteins (including collagen Q/perlecan and biglycan which link to MuSK Ig1 domain and CRD); and (iii) the dystrophin-associated glycoprotein complex. The study on MuSK ectodomains (Ig1/2 domains and CRD) recognized by antibodies suggested that the MuSK antibodies were pathologically heterogeneous due to their binding to multiple functional domains. Focussing one of the matrix proteins, biglycan which functions in the manner similar to collagen Q, our antibody assay showed the negative result in MG patients. However, the synaptic stability may be impaired by antibodies against MuSK ectodomains because of the linkage of biglycan with MuSK Ig1 domain and CRD. The pathogenic diversity of MG is discussed based on NMJ signaling molecules.