Presynaptic Membrane Receptors Modulate ACh Release, Axonal Competition and Synapse Elimination during Neuromuscular Junction Development.

PubMed

Tomàs, Josep; Garcia, Neus; Lanuza, Maria A; Santafé, Manel M; Tomàs, Marta; Nadal, Laura; Hurtado, Erica; Simó, Anna; Cilleros, Víctor

2017-01-01

During the histogenesis of the nervous system a lush production of neurons, which establish an excessive number of synapses, is followed by a drop in both neurons and synaptic contacts as maturation proceeds. Hebbian competition between axons with different activities leads to the loss of roughly half of the neurons initially produced so connectivity is refined and specificity gained. The skeletal muscle fibers in the newborn neuromuscular junction (NMJ) are polyinnervated but by the end of the competition, 2 weeks later, the NMJ are innervated by only one axon. This peripheral synapse has long been used as a convenient model for synapse development. In the last few years, we have studied transmitter release and the local involvement of the presynaptic muscarinic acetylcholine autoreceptors (mAChR), adenosine autoreceptors (AR) and trophic factor receptors (TFR, for neurotrophins and trophic cytokines) during the development of NMJ and in the adult. This review article brings together previously published data and proposes a molecular background for developmental axonal competition and loss. At the end of the first week postnatal, these receptors modulate transmitter release in the various nerve terminals on polyinnervated NMJ and contribute to axonal competition and synapse elimination.

Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail.

PubMed

Tokuyama, Minami A; Xu, Cindy; Fisher, Rebecca E; Wilson-Rawls, Jeanne; Kusumi, Kenro; Newbern, Jason M

2018-01-15

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response. Copyright © 2017 Elsevier Inc. All rights reserved.

LRP4 is critical for neuromuscular junction maintenance.

PubMed

Barik, Arnab; Lu, Yisheng; Sathyamurthy, Anupama; Bowman, Andrew; Shen, Chengyong; Li, Lei; Xiong, Wen-cheng; Mei, Lin

2014-10-15

The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms. Copyright © 2014 the authors 0270-6474/14/3413892-14$15.00/0.

Participation of Myosin Va and Pka Type I in the Regeneration of Neuromuscular Junctions

PubMed Central

Röder, Ira Verena; Strack, Siegfried; Reischl, Markus; Dahley, Oliver; Khan, Muzamil Majid; Kassel, Olivier; Zaccolo, Manuela; Rudolf, Rüdiger

2012-01-01

Background The unconventional motor protein, myosin Va, is crucial for the development of the mouse neuromuscular junction (NMJ) in the early postnatal phase. Furthermore, the cooperative action of protein kinase A (PKA) and myosin Va is essential to maintain the adult NMJ. We here assessed the involvement of myosin Va and PKA in NMJ recovery during muscle regeneration. Methodology/Principal Findings To address a putative role of myosin Va and PKA in the process of muscle regeneration, we used two experimental models the dystrophic mdx mouse and Notexin-induced muscle degeneration/regeneration. We found that in both systems myosin Va and PKA type I accumulate beneath the NMJs in a fiber maturation-dependent manner. Morphologically intact NMJs were found to express stable nicotinic acetylcholine receptors and to accumulate myosin Va and PKA type I in the subsynaptic region. Subsynaptic cAMP signaling was strongly altered in dystrophic muscle, particularly in fibers with severely subverted NMJ morphology. Conclusions/Significance Our data show a correlation between the subsynaptic accumulation of myosin Va and PKA type I on the one hand and NMJ regeneration status and morphology, AChR stability and specificity of subsynaptic cAMP handling on the other hand. This suggests an important role of myosin Va and PKA type I for the maturation of NMJs in regenerating muscle. PMID:22815846

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila.

PubMed

Mallik, Bhagaban; Dwivedi, Manish Kumar; Mushtaq, Zeeshan; Kumari, Manisha; Verma, Praveen Kumar; Kumar, Vimlesh

2017-06-01

The mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. We performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (ICA69) as one of the key regulators of morphological differentiation of the larval neuromuscular junction (NMJ). We show that Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of ICA69 deforms liposomes in vitro Full-length ICA69 and the ICAC but not the N-BAR domain of ICA69 induce filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, ICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of ICA69 or its interactor PICK1 alters the synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing PICK1 or Rab2 levels phenocopies ICA69 mutation. Interestingly, Rab2 regulates not only synaptic iGluR but also ICA69 levels. Thus, our data suggest that: (1) ICA69 regulates NMJ organization through a pathway that involves PICK1 and Rab2, and (2) Rab2 functions genetically upstream of ICA69 and regulates NMJ organization and targeting/retention of iGluRs by regulating ICA69 levels. © 2017. Published by The Company of Biologists Ltd.

The Scaffolding Protein, Grb2-associated Binder-1, in Skeletal Muscles and Terminal Schwann Cells Regulates Postnatal Neuromuscular Synapse Maturation

PubMed Central

Park, So Young; Jang, So Young; Shin, Yoon Kyoung; Jung, Dong Keun; Yoon, Byeol A; Kim, Jong Kook; Jo, Young Rae; Lee, Hye Jeong

2017-01-01

The vertebrate neuromuscular junction (NMJ) is considered as a “tripartite synapse” consisting of a motor axon terminal, a muscle endplate, and terminal Schwann cells that envelope the motor axon terminal. The neuregulin 1 (NRG1)-ErbB2 signaling pathway plays an important role in the development of the NMJ. We previously showed that Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling, is required for NRG1-induced peripheral nerve myelination. Here, we determined the role of Gab1 in the development of the NMJ using muscle-specific conditional Gab1 knockout mice. The mutant mice showed delayed postnatal maturation of the NMJ. Furthermore, the selective loss of the gab1 gene in terminal Schwann cells produced delayed synaptic elimination with abnormal morphology of the motor endplate, suggesting that Gab1 in both muscles and terminal Schwann cells is required for proper NMJ development. Gab1 in terminal Schwann cells appeared to regulate the number and process elongation of terminal Schwann cells during synaptic elimination. However, Gab2 knockout mice did not show any defects in the development of the NMJ. Considering the role of Gab1 in postnatal peripheral nerve myelination, our findings suggest that Gab1 is a pleiotropic and important component of NRG1 signals during postnatal development of the peripheral neuromuscular system. PMID:28680299

The Scaffolding Protein, Grb2-associated Binder-1, in Skeletal Muscles and Terminal Schwann Cells Regulates Postnatal Neuromuscular Synapse Maturation.

PubMed

Park, So Young; Jang, So Young; Shin, Yoon Kyoung; Jung, Dong Keun; Yoon, Byeol A; Kim, Jong Kook; Jo, Young Rae; Lee, Hye Jeong; Park, Hwan Tae

2017-06-01

The vertebrate neuromuscular junction (NMJ) is considered as a "tripartite synapse" consisting of a motor axon terminal, a muscle endplate, and terminal Schwann cells that envelope the motor axon terminal. The neuregulin 1 (NRG1)-ErbB2 signaling pathway plays an important role in the development of the NMJ. We previously showed that Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling, is required for NRG1-induced peripheral nerve myelination. Here, we determined the role of Gab1 in the development of the NMJ using muscle-specific conditional Gab1 knockout mice. The mutant mice showed delayed postnatal maturation of the NMJ. Furthermore, the selective loss of the gab1 gene in terminal Schwann cells produced delayed synaptic elimination with abnormal morphology of the motor endplate, suggesting that Gab1 in both muscles and terminal Schwann cells is required for proper NMJ development. Gab1 in terminal Schwann cells appeared to regulate the number and process elongation of terminal Schwann cells during synaptic elimination. However, Gab2 knockout mice did not show any defects in the development of the NMJ. Considering the role of Gab1 in postnatal peripheral nerve myelination, our findings suggest that Gab1 is a pleiotropic and important component of NRG1 signals during postnatal development of the peripheral neuromuscular system.

Muscle Mitochondrial Uncoupling Dismantles Neuromuscular Junction and Triggers Distal Degeneration of Motor Neurons

PubMed Central

Dupuis, Luc; Gonzalez de Aguilar, Jose-Luis; Echaniz-Laguna, Andoni; Eschbach, Judith; Rene, Frédérique; Oudart, Hugues; Halter, Benoit; Huze, Caroline; Schaeffer, Laurent; Bouillaud, Frédéric; Loeffler, Jean-Philippe

2009-01-01

Background Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS. Methodology/Principal Findings We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model. Conclusions/Significance These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases. PMID:19404401

Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics.

PubMed

Santhanam, Navaneetha; Kumanchik, Lee; Guo, Xiufang; Sommerhage, Frank; Cai, Yunqing; Jackson, Max; Martin, Candace; Saad, George; McAleer, Christopher W; Wang, Ying; Lavado, Andrea; Long, Christopher J; Hickman, James J

2018-06-01

There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle's contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX ® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs. Copyright © 2018 Elsevier Ltd. All rights reserved.

Neuromuscular Junction Formation between Human Stem cell-derived Motoneurons and Human Skeletal Muscle in a Defined System

PubMed Central

Guo, Xiufang; Gonzalez, Mercedes; Stancescu, Maria; Vandenburgh, Herman; Hickman, James

2011-01-01

Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time lapse recordings and their subsequent quenching by D-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair. PMID:21944471

Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system.

PubMed

Guo, Xiufang; Gonzalez, Mercedes; Stancescu, Maria; Vandenburgh, Herman H; Hickman, James J

2011-12-01

Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time-lapse recordings and their subsequent quenching by d-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair. Copyright © 2011 Elsevier Ltd. All rights reserved.

Muscle-derived collagen XIII regulates maturation of the skeletal neuromuscular junction.

PubMed

Latvanlehto, Anne; Fox, Michael A; Sormunen, Raija; Tu, Hongmin; Oikarainen, Tuomo; Koski, Anu; Naumenko, Nikolay; Shakirzyanova, Anastasia; Kallio, Mika; Ilves, Mika; Giniatullin, Rashid; Sanes, Joshua R; Pihlajaniemi, Taina

2010-09-15

Formation, maturation, stabilization, and functional efficacy of the neuromuscular junction (NMJ) are orchestrated by transsynaptic and autocrine signals embedded within the synaptic cleft. Here, we demonstrate that collagen XIII, a nonfibrillar transmembrane collagen, is another such signal. We show that collagen XIII is expressed by muscle and its ectodomain can be proteolytically shed into the extracellular matrix. The collagen XIII protein was found present in the postsynaptic membrane and synaptic basement membrane. To identify a role for collagen XIII at the NMJ, mice were generated lacking this collagen. Morphological and ultrastructural analysis of the NMJ revealed incomplete adhesion of presynaptic and postsynaptic specializations in collagen XIII-deficient mice of both genders. Strikingly, Schwann cells erroneously enwrapped nerve terminals and invaginated into the synaptic cleft, resulting in a decreased contact surface for neurotransmission. Consistent with morphological findings, electrophysiological studies indicated both postsynaptic and presynaptic defects in Col13a1(-/-) mice, such as decreased amplitude of postsynaptic potentials, diminished probabilities of spontaneous release and reduced readily releasable neurotransmitter pool. To identify the role of collagen XIII at the NMJ, shed ectodomain of collagen XIII was applied to cultured myotubes, and it was found to advance acetylcholine receptor (AChR) cluster maturation. Together with the delay in AChR cluster development observed in collagen XIII-deficient mutants in vivo, these results suggest that collagen XIII plays an autocrine role in postsynaptic maturation of the NMJ. Altogether, the results presented here reveal that collagen XIII is a novel muscle-derived cue necessary for the maturation and function of the vertebrate NMJ.

Degeneration and regeneration of neuromuscular junction architecture in rat skeletal muscle fibers damaged by bupivacaine hydrochloride.

PubMed

Nishizawa, Tomie; Tamaki, Hiroyuki; Kasuga, Norikatsu; Takekura, Hiroaki

2003-01-01

We evaluated the degeneration and regeneration of neuromuscular junctions (NMJs) on the extensor digitorum longus muscle of Fischer 344 rats between 4 h and 3 weeks after bupivacaine hydrochloride (BPVC) injection, which induces muscle fiber necrosis, using histochemical staining by acetylcholine esterase (AchE)-silver and electron microscopy. Degeneration of muscle fibers and NMJs was observed 4 h after BPVC injection. One week after BPVC injection, some terminal axons were almost completely retracted, and the level of basal lamina-associated AchE in some NMJ regions had gradually disappeared. At that time, the depression contained a few, mostly pit-like or elongated oval invaginations: the incipient junctional folds and some NMJs did not have any secondary junctional fold. By 2 weeks after the BPVC injection, secondary junctional folds began to develop: however, the number of secondary junctional folds was clearly less than that in normal NMJs. At 3 weeks when regeneration of muscle fibers was well advanced, the staining for AchE at the end-plates became stronger and better-defined. The volume density of mitochondria in the terminal area of the terminal significantly decreased upon BPVC-induced destruction of the NMJ, and the density reached the lowest value 24 h after BPVC injection. Significant changes in the ultrastructural features of the architecture of NMJs occurred in skeletal muscle fibers damaged by BPVC during both the degeneration and regeneration processes. The changes in the ultrastructural and morphological features of the NMJ architecture during the regeneration of degenerated muscle fibers resembled those that occur during the differentiation of normal muscle fibers.

Role of exercise in maintaining the integrity of the neuromuscular junction

PubMed Central

Nishimune, Hiroshi; Stanford, John A.; Mori, Yasuo

2014-01-01

Physical activity plays an important role in preventing chronic disease in adults and the elderly. Exercise has beneficial effects on the nervous system, including at the neuromuscular junction (NMJ). Exercise causes hypertrophy of NMJs and improves recovery from peripheral nerve injuries, whereas decreased physical activity causes degenerative changes in NMJs. Recent studies have begun to elucidate molecular mechanisms underlying the beneficial effects of exercise. These mechanisms involve Bassoon, neuregulin-1, peroxisome proliferator-activated receptor gamma coactivator 1α, Insulin-like growth factor-1, glial cell line-derived neurotrophic factor, neurotrophin 4, Homer, and nuclear factor of activated T cells c1. For example, NMJ denervation and active zone decreases have been observed in aged NMJs, but these age-dependent degenerative changes can be ameliorated by exercise. This review will discuss the effects of exercise on the maintenance and regeneration of NMJs and will highlight recent insights into the molecular mechanisms underlying these exercise effects. PMID:24122772

The glial cell line-derived neurotrophic factor (GDNF) does not acutely change acetylcholine release in developing and adult neuromuscular junction.

PubMed

Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Lanuza, Maria A; Besalduch, Nuria; Priego, Merche; Tomàs, Josep

2010-08-16

We use immunocytochemistry to show that the trophic molecule glial cell line-derived neurotrophic factor (GDNF) and its receptor GDNF family receptor alpha-1 (GFRalpha-1) are present in both neonatal (P6) and adult (P45) rodent neuromuscular junctions (NMJ) colocalized with several synaptic markers. However, incubation with exogenous GDNF (10-200ng/ml, 1-3h), does not affect spontaneous ACh release. Moreover, GDNF does not change the size of the evoked ACh release from the weak and the strong axonal inputs on dually innervated postnatal endplates nor in the most developed singly-innervated synapses at P6 and P45. Our findings indicate that GDNF (unlike neurotrophins) does not acutely modulate transmitter release during the developmental process of synapse elimination nor as the NMJ matures. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Autophagy promotes synapse development in Drosophila.

PubMed

Shen, Wei; Ganetzky, Barry

2009-10-05

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH(2)-terminal kinase (bsk(DN)). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin-proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.

Integrated genomics and proteomics of the Torpedo californica electric organ: concordance with the mammalian neuromuscular junction

PubMed Central

2011-01-01

Background During development, the branchial mesoderm of Torpedo californica transdifferentiates into an electric organ capable of generating high voltage discharges to stun fish. The organ contains a high density of cholinergic synapses and has served as a biochemical model for the membrane specialization of myofibers, the neuromuscular junction (NMJ). We studied the genome and proteome of the electric organ to gain insight into its composition, to determine if there is concordance with skeletal muscle and the NMJ, and to identify novel synaptic proteins. Results Of 435 proteins identified, 300 mapped to Torpedo cDNA sequences with ≥2 peptides. We identified 14 uncharacterized proteins in the electric organ that are known to play a role in acetylcholine receptor clustering or signal transduction. In addition, two human open reading frames, C1orf123 and C6orf130, showed high sequence similarity to electric organ proteins. Our profile lists several proteins that are highly expressed in skeletal muscle or are muscle specific. Synaptic proteins such as acetylcholinesterase, acetylcholine receptor subunits, and rapsyn were present in the electric organ proteome but absent in the skeletal muscle proteome. Conclusions Our integrated genomic and proteomic analysis supports research describing a muscle-like profile of the organ. We show that it is a repository of NMJ proteins but we present limitations on its use as a comprehensive model of the NMJ. Finally, we identified several proteins that may become candidates for signaling proteins not previously characterized as components of the NMJ. PMID:21798097

Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Tae Hee; Dharkar, Poorva; Mayer, Mark L.

The Drosophila larval neuromuscular junction (NMJ), at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute NMJ glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. In this paper, we find that Drosophila Neto functions as an essential component required for the function of NMJ glutamate receptors, permitting analysis of glutamate receptor responses in Xenopus oocytes. Finally, in combinationmore » with a crystallographic analysis of the GluRIIB ligand binding domain, we use this system to characterize the subunit dependence of assembly, channel block, and ligand selectivity for Drosophila NMJ glutamate receptors.« less

Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors

DOE PAGES

Han, Tae Hee; Dharkar, Poorva; Mayer, Mark L.; ...

2015-04-27

The Drosophila larval neuromuscular junction (NMJ), at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute NMJ glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. In this paper, we find that Drosophila Neto functions as an essential component required for the function of NMJ glutamate receptors, permitting analysis of glutamate receptor responses in Xenopus oocytes. Finally, in combinationmore » with a crystallographic analysis of the GluRIIB ligand binding domain, we use this system to characterize the subunit dependence of assembly, channel block, and ligand selectivity for Drosophila NMJ glutamate receptors.« less

Congenital Myasthenic Syndromes or Inherited Disorders of Neuromuscular Transmission: Recent Discoveries and Open Questions

PubMed Central

Nicole, Sophie; Azuma, Yoshiteru; Bauché, Stéphanie; Eymard, Bruno; Lochmüller, Hanns; Slater, Clarke

2017-01-01

Congenital myasthenic syndromes (CMS) form a heterogeneous group of rare diseases characterized by fatigable muscle weakness. They are genetically-inherited and caused by defective synaptic transmission at the cholinergic neuromuscular junction (NMJ). The number of genes known to cause CMS when mutated is currently 30, and the relationship between fatigable muscle weakness and defective functions is quite well-understood for many of them. However, some of the most recent discoveries in individuals with CMS challenge our knowledge of the NMJ, where the basis of the pathology has mostly been investigated in animal models. Frontier forms between CMS and congenital myopathy, which have been genetically and clinically identified, underline the poorly understood interplay between the synaptic and extrasynaptic molecules in the neuromuscular system. In addition, precise electrophysiological and histopathological investigations of individuals with CMS suggest an important role of NMJ plasticity in the response to CMS pathogenesis. While efficient drug-based treatments are already available to improve neuromuscular transmission for most forms of CMS, others, as well as neurological and muscular comorbidities, remain resistant. Taken together, the available pathological data point to physiological issues which remain to be understood in order to achieve precision medicine with efficient therapeutics for all individuals suffering from CMS. PMID:29125502

Role of exercise in maintaining the integrity of the neuromuscular junction.

PubMed

Nishimune, Hiroshi; Stanford, John A; Mori, Yasuo

2014-03-01

Physical activity plays an important role in preventing chronic disease in adults and the elderly. Exercise has beneficial effects on the nervous system, including at the neuromuscular junction (NMJ). Exercise causes hypertrophy of NMJs and improves recovery from peripheral nerve injuries, whereas decreased physical activity causes degenerative changes in NMJs. Recent studies have begun to elucidate molecular mechanisms underlying the beneficial effects of exercise. These mechanisms involve Bassoon, neuregulin-1, peroxisome proliferator-activated receptor gamma coactivator 1α, insulin-like growth factor-1, glial cell line-derived neurotrophic factor, neurotrophin 4, Homer, and nuclear factor of activated T cells c1. For example, NMJ denervation and active zone decreases have been observed in aged NMJs, but these age-dependent degenerative changes can be ameliorated by exercise. In this review we assess the effects of exercise on the maintenance and regeneration of NMJs and highlight recent insights into the molecular mechanisms underlying these exercise effects. Copyright © 2013 Wiley Periodicals, Inc.

Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

PubMed

Wang, Simon; Yoo, SooHyun; Kim, Hae-Yoon; Wang, Mannan; Zheng, Clare; Parkhouse, Wade; Krieger, Charles; Harden, Nicholas

2015-01-20

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development. Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Remodeling of the neuromuscular junction precedes sarcopenia related alterations in myofibers.

PubMed

Deschenes, Michael R; Roby, Mackenzie A; Eason, Margaret K; Harris, M Brennan

2010-05-01

Several mechanisms contributing to the etiology of sarcopenia (age-related loss of muscle size) have been postulated. One of these attributes the loss of muscle mass to a preceding age-related denervation of myofibers. The aim of this study was to determine if signs of denervation were apparent at the neuromuscular junction (NMJ) before fiber atrophy, or fiber type conversion could be documented, and to reveal if a muscle's activity level impacts its sensitivity to age-related denervation. Plantaris and soleus muscles were obtained from young adult (10 months) and early aged (21 months) rats. Pre- and post-synaptic NMJ morphology was quantified with cytofluorescent staining of nerve terminal branches and endplate regions, respectively. Myofiber profiles (fiber size and fiber type composition) were assessed with histochemical procedures. Results show that in the lightly recruited plantaris, significant (P<0.05) signs of denervation were noted in aged rats, while the same muscles displayed no change in myofiber profile. In the heavily recruited soleus, however, there was little evidence of denervation, and again no alterations in myofiber profile. These results indicate that age-related denervation occurs before myofiber atrophy, and that high amounts of neuromuscular activity may delay the onset of age-related denervation and sarcopenia.

Regulation of WNT Signaling at the Neuromuscular Junction by the Immunoglobulin Superfamily Protein RIG-3 in Caenorhabditis elegans

PubMed Central

Pandey, Pratima; Bhardwaj, Ashwani; Babu, Kavita

2017-01-01

Perturbations in synaptic function could affect the normal behavior of an animal, making it important to understand the regulatory mechanisms of synaptic signaling. Previous work has shown that in Caenorhabditis elegans an immunoglobulin superfamily protein, RIG-3, functions in presynaptic neurons to maintain normal acetylcholine receptor levels at the neuromuscular junction (NMJ). In this study, we elucidate the molecular and functional mechanism of RIG-3. We demonstrate by genetic and BiFC (Bi-molecular Fluorescence Complementation) assays that presynaptic RIG-3 functions by directly interacting with the immunoglobulin domain of the nonconventional Wnt receptor, ROR receptor tyrosine kinase (RTK), CAM-1, which functions in postsynaptic body-wall muscles. This interaction in turn inhibits Wnt/LIN-44 signaling through the ROR/CAM-1 receptor, and allows for maintenance of normal acetylcholine receptor, AChR/ACR-16, levels at the neuromuscular synapse. Further, this work reveals that RIG-3 and ROR/CAM-1 function through the β-catenin/HMP-2 at the NMJ. Taken together, our results demonstrate that RIG-3 functions as an inhibitory molecule of the Wnt/LIN-44 signaling pathway through the RTK, CAM-1. PMID:28515212

Neurotrophin-4 couples to locally modulated ACh release at the end of neuromuscular synapse maturation.

PubMed

Garcia, N; Santafe, M M; Tomas, M; Lanuza, M A; Besalduch, N; Tomas, J

2010-01-01

We use immunocytochemistry to show that neurotrophin-4 (NT-4) and its receptor proteins (p75(NTR) and tropomyosin-related tyrosine kinase B) are present in neonatal neuromuscular junctions (NMJ) colocalized with several synaptic markers. NT-4 incubation (1h, in the range 2-12 nM) does not change the size of the endplate potential between P6 and P45. However, extended exposure (3h) to a relatively low dose of NT-4 (2 nM) potentiates ACh release (approx. 70%) in adult but not in neonatal muscles. The present results suggest that the developmental mechanism of axonal competition and neonatal elimination of redundant synapses cannot be modulated by added NT-4. However, this neurotrophin was able to modulate synaptic transmission locally in the adult NMJ.

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

PubMed Central

Senderek, Jan; Müller, Juliane S.; Dusl, Marina; Strom, Tim M.; Guergueltcheva, Velina; Diepolder, Irmgard; Laval, Steven H.; Maxwell, Susan; Cossins, Judy; Krause, Sabine; Muelas, Nuria; Vilchez, Juan J.; Colomer, Jaume; Mallebrera, Cecilia Jimenez; Nascimento, Andres; Nafissi, Shahriar; Kariminejad, Ariana; Nilipour, Yalda; Bozorgmehr, Bita; Najmabadi, Hossein; Rodolico, Carmelo; Sieb, Jörn P.; Steinlein, Ortrud K.; Schlotter, Beate; Schoser, Benedikt; Kirschner, Janbernd; Herrmann, Ralf; Voit, Thomas; Oldfors, Anders; Lindbergh, Christopher; Urtizberea, Andoni; von der Hagen, Maja; Hübner, Angela; Palace, Jacqueline; Bushby, Kate; Straub, Volker; Beeson, David; Abicht, Angela; Lochmüller, Hanns

2011-01-01

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. PMID:21310273

Effects of aging and Parkinson's disease on motor unit remodeling: influence of resistance exercise training.

PubMed

Kelly, Neil A; Hammond, Kelley G; Bickel, C Scott; Windham, Samuel T; Tuggle, S Craig; Bamman, Marcas M

2018-04-01

Aging muscle atrophy is in part a neurodegenerative process revealed by denervation/reinnervation events leading to motor unit remodeling (i.e., myofiber type grouping). However, this process and its physiological relevance are poorly understood, as is the wide-ranging heterogeneity among aging humans. Here, we attempted to address 1) the relation between myofiber type grouping and molecular regulators of neuromuscular junction (NMJ) stability; 2) the impact of motor unit remodeling on recruitment during submaximal contractions; 3) the prevalence and impact of motor unit remodeling in Parkinson's disease (PD), an age-related neurodegenerative disease; and 4) the influence of resistance exercise training (RT) on regulators of motor unit remodeling. We compared type I myofiber grouping, molecular regulators of NMJ stability, and the relative motor unit activation (MUA) requirement during a submaximal sit-to-stand task among untrained but otherwise healthy young (YA; 26 yr, n = 27) and older (OA; 66 yr, n = 91) adults and OA with PD (PD; 67 yr, n = 19). We tested the effects of RT on these outcomes in OA and PD. PD displayed more motor unit remodeling, alterations in NMJ stability regulation, and a higher relative MUA requirement than OA, suggesting PD-specific effects. The molecular and physiological outcomes tracked with the severity of type I myofiber grouping. Together these findings suggest that age-related motor unit remodeling, manifested by type I myofiber grouping, 1) reduces MUA efficiency to meet submaximal contraction demand, 2) is associated with disruptions in NMJ stability, 3) is further impacted by PD, and 4) may be improved by RT in severe cases. NEW & NOTEWORTHY Because the physiological consequences of varying amounts of myofiber type grouping are unknown, the current study aims to characterize the molecular and physiological correlates of motor unit remodeling. Furthermore, because exercise training has demonstrated neuromuscular benefits in aged humans and improved innervation status and neuromuscular junction integrity in animals, we provide an exploratory analysis of the effects of high-intensity resistance training on markers of neuromuscular degeneration in both Parkinson's disease (PD) and age-matched older adults.

Milk fat globule membrane supplementation with voluntary running exercise attenuates age-related motor dysfunction by suppressing neuromuscular junction abnormalities in mice.

PubMed

Yano, Michiko; Minegishi, Yoshihiko; Sugita, Satoshi; Ota, Noriyasu

2017-10-15

Age-related loss of skeletal muscle mass and function attenuates physical performance, and maintaining fine muscle innervation is known to play an important role in its prevention. We had previously shown that consumption of milk fat globule membrane (MFGM) with habitual exercise improves the muscle mass and motor function in humans and mice. Improvement of neuromuscular junction (NMJ) was suggested as one of the mechanisms underlying these effects. In this study, we evaluated the effect of MFGM intake combined with voluntary running (MFGM-VR) on morphological changes of NMJ and motor function in aging mice. Seven months following the intervention, the MFGM-VR group showed a significantly improved motor coordination in the rotarod test and muscle force in the grip strength test compared with the control group at 13 and 14months of age, respectively. In 14-month old control mice, the extensor digitorum longus muscle showed increased abnormal NMJs, such as fragmentation and denervation, compared with 6-month old young mice. However, such age-related deteriorations of NMJs were significantly suppressed in the MFGM-VR group. Increase in the expression of NMJ formation-related genes, such as agrin and LDL Receptor Related Protein 4 (LRP4), might contribute to this beneficial effect. Rotarod performance and grip strength showed significant negative correlation with the status of denervation and fragmentation of NMJs. These results suggest that MFGM intake with voluntary running exercise effectively suppresses age-related morphological deterioration of NMJ, thus contributing to improvement of motor function. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Age-related Differences in Dystrophin: Impact on Force Transfer Proteins, Membrane Integrity, and Neuromuscular Junction Stability.

PubMed

Hughes, David C; Marcotte, George R; Marshall, Andrea G; West, Daniel W D; Baehr, Leslie M; Wallace, Marita A; Saleh, Perrie M; Bodine, Sue C; Baar, Keith

2017-05-01

The loss of muscle strength with age has been studied from the perspective of a decline in muscle mass and neuromuscular junction (NMJ) stability. A third potential factor is force transmission. The purpose of this study was to determine the changes in the force transfer apparatus within aging muscle and the impact on membrane integrity and NMJ stability. We measured an age-related loss of dystrophin protein that was greatest in the flexor muscles. The loss of dystrophin protein occurred despite a twofold increase in dystrophin mRNA. Importantly, this disparity could be explained by the four- to fivefold upregulation of the dystromir miR-31. To compensate for the loss of dystrophin protein, aged muscle contained increased α-sarcoglycan, syntrophin, sarcospan, laminin, β1-integrin, desmuslin, and the Z-line proteins α-actinin and desmin. In spite of the adaptive increase in other force transfer proteins, over the 48 hours following lengthening contractions, the old muscles showed more signs of impaired membrane integrity (fourfold increase in immunoglobulin G-positive fibers and 70% greater dysferlin mRNA) and NMJ instability (14- to 96-fold increases in Runx1, AchRδ, and myogenin mRNA). Overall, these data suggest that age-dependent alterations in dystrophin leave the muscle membrane and NMJ more susceptible to contraction-induced damage even before changes in muscle mass are obvious. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction.

PubMed

Hurtado, Erica; Cilleros, Víctor; Just, Laia; Simó, Anna; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A; Tomàs, Josep

2017-01-01

Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release.

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

PubMed Central

Hurtado, Erica; Cilleros, Víctor; Just, Laia; Simó, Anna; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A.; Tomàs, Josep

2017-01-01

Conventional protein kinase C βI (cPKCβI) is a conventional protein kinase C (PKC) isoform directly involved in the regulation of neurotransmitter release in the neuromuscular junction (NMJ). It is located exclusively at the nerve terminal and both synaptic activity and muscle contraction modulate its protein levels and phosphorylation. cPKCβI molecular maturation includes a series of phosphorylation steps, the first of which is mediated by phosphoinositide-dependent kinase 1 (PDK1). Here, we sought to localize PDK1 in the NMJ and investigate the hypothesis that synaptic activity and muscle contraction regulate in parallel PDK1 and cPKCβI phosphorylation in the membrane fraction. To differentiate the presynaptic and postsynaptic activities, we abolished muscle contraction with μ-conotoxin GIIIB (μ-CgTx-GIIIB) in some experiments before stimulation of the phrenic nerve (1 Hz, 30 min). Then, we analyzed total and membrane/cytosol fractions of skeletal muscle by Western blotting. Results showed that PDK1 is located exclusively in the nerve terminal of the NMJ. After nerve stimulation with and without coincident muscle contraction, total PDK1 and phosphorylated PDK1 (pPDK1) protein levels remained unaltered. However, synaptic activity specifically enhanced phosphorylation of PDK1 in the membrane, an important subcellular location for PDK1 function. This increase in pPDK1 coincides with a significant increase in the phosphorylation of its substrate cPKCβI also in the membrane fraction. Moreover, muscle contraction maintains PDK1 and pPDK1 but increases cPKCβI protein levels and its phosphorylation. Thus, even though PDK1 activity is maintained, pcPKCβI levels increase in concordance with total cPKCβI. Together, these results indicate that neuromuscular activity could induce the membrane targeting of pPDK1 in the nerve terminal of the NMJ to promote the phosphorylation of the cPKCβI, which is involved in ACh release. PMID:28890686

Genetic and evolutionary analysis of the Drosophila larval neuromuscular junction

NASA Astrophysics Data System (ADS)

Campbell, Megan

Although evolution of brains and behaviors is of fundamental biological importance, we lack comprehensive understanding of the general principles governing these processes or the specific mechanisms and molecules through which the evolutionary changes are effected. Because synapses are the basic structural and functional units of nervous systems, one way to address these problems is to dissect the genetic and molecular pathways responsible for morphological evolution of a defined synapse. I have undertaken such an analysis by examining morphology of the larval neuromuscular junction (NMJ) in wild caught D. melanogaster as well as in over 20 other species of Drosophila. Whereas variation in NMJ morphology within a species is limited, I discovered a surprisingly extensive variation among different species. Compared with evolution of other morphological traits, NMJ morphology appears to be evolving very rapidly. Moreover, my data indicate that natural selection rather than genetic drift is primarily responsible for evolution of NMJ morphology. To dissect underlying molecular mechanisms that may govern NMJ growth and evolutionary divergence, I focused on a naturally occurring variant in D. melanogaster that causes NMJ overgrowth. I discovered that the variant mapped to Mob2, a gene encoding a kinase adapter protein originally described in yeast as a member of the Mitotic Exit Network (MEN). I have subsequently examined mutations in the Drosophila orthologs of all the core components of the yeast MEN and found that all of them function as part of a common pathway that acts presynaptically to negatively regulate NMJ growth. As in the regulation of yeast cytokinesis, these components of the MEN appear to act ultimately by regulating actin dynamics during the process of bouton growth and division. These studies have thus led to the discovery of an entirely new role for the MEN---regulation of synaptic growth---that is separate from its function in cell division. This work has identified a rich source of material for discovery of novel genes and mechanisms that regulate synaptic growth and development, and has also provided new insights into the mechanisms that underlie morphological evolution of nervous systems.

Myasthenia gravis: the role of complement at the neuromuscular junction.

PubMed

Howard, James F

2018-01-01

Generalized myasthenia gravis (gMG) is a rare autoimmune disorder characterized by skeletal muscle weakness caused by disrupted neurotransmission at the neuromuscular junction (NMJ). Approximately 74-88% of patients with gMG have acetylcholine receptor (AChR) autoantibodies. Complement plays an important role in innate and antibody-mediated immunity, and activation and amplification of complement results in the formation of membrane attack complexes (MACs), lipophilic proteins that damage cell membranes. The role of complement in gMG has been demonstrated in animal models and patients. Studies in animals lacking specific complement proteins have confirmed that MAC formation is required to induce experimental autoimmune MG (EAMG) and NMJ damage. Complement inhibition in EAMG models can prevent disease induction and reverse its progression. Patients with anti-AChR + MG have autoantibodies and MACs present at NMJs. Damaged NMJs are associated with more severe disease, fewer AChRs, and MACs in synaptic debris. Current MG therapies do not target complement directly. Eculizumab is a humanized monoclonal antibody that inhibits cleavage of complement protein C5, preventing MAC formation. Eculizumab treatment improved symptoms compared with placebo in a phase II study in patients with refractory gMG. Direct complement inhibition could preserve NMJ physiology and muscle function in patients with anti-AChR + gMG. © 2017 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

The beta-adrenergic agonist salbutamol modulates neuromuscular junction formation in zebrafish models of human myasthenic syndromes.

PubMed

McMacken, Grace; Cox, Dan; Roos, Andreas; Müller, Juliane; Whittaker, Roger; Lochmüller, Hanns

2018-05-01

Inherited defects of the neuromuscular junction (NMJ) comprise an increasingly diverse range of disorders, termed congenital myasthenic syndromes (CMS). Therapies acting on the sympathetic nervous system, including the selective β2 adrenergic agonist salbutamol and the α and β adrenergic agonist ephedrine, have become standard treatment for several types of CMS. However, the mechanism of the therapeutic effect of sympathomimetics in these disorders is not understood. Here, we examined the effect of salbutamol on NMJ development using zebrafish with deficiency of the key postsynaptic proteins Dok-7 and MuSK. Treatment with salbutamol reduced motility defects in zebrafish embryos and larvae. In addition, salbutamol lead to morphological improvement of postsynaptic acetycholine receptor (AChR) clustering and size of synaptic contacts in Dok-7-deficient zebrafish. In MuSK-deficient zebrafish, salbutamol treatment reduced motor axon pathfinding defects and partially restored the formation of aneural prepatterned AChRs. In addition, the effects of salbutamol treatment were prevented by pre-treatment with a selective β2 antagonist. Treatment with the cyclic adenosine monophosphate (cAMP) activator forskolin, replicated the effects of salbutamol treatment. These results suggest that sympathomimetics exert a direct effect on neuromuscular synaptogenesis and do so via β2 adrenoceptors and via a cAMP-dependent pathway.

The beta-adrenergic agonist salbutamol modulates neuromuscular junction formation in zebrafish models of human myasthenic syndromes

PubMed Central

McMacken, Grace; Cox, Dan; Roos, Andreas; Müller, Juliane; Whittaker, Roger; Lochmüller, Hanns

2018-01-01

Abstract Inherited defects of the neuromuscular junction (NMJ) comprise an increasingly diverse range of disorders, termed congenital myasthenic syndromes (CMS). Therapies acting on the sympathetic nervous system, including the selective β2 adrenergic agonist salbutamol and the α and β adrenergic agonist ephedrine, have become standard treatment for several types of CMS. However, the mechanism of the therapeutic effect of sympathomimetics in these disorders is not understood. Here, we examined the effect of salbutamol on NMJ development using zebrafish with deficiency of the key postsynaptic proteins Dok-7 and MuSK. Treatment with salbutamol reduced motility defects in zebrafish embryos and larvae. In addition, salbutamol lead to morphological improvement of postsynaptic acetycholine receptor (AChR) clustering and size of synaptic contacts in Dok-7-deficient zebrafish. In MuSK-deficient zebrafish, salbutamol treatment reduced motor axon pathfinding defects and partially restored the formation of aneural prepatterned AChRs. In addition, the effects of salbutamol treatment were prevented by pre-treatment with a selective β2 antagonist. Treatment with the cyclic adenosine monophosphate (cAMP) activator forskolin, replicated the effects of salbutamol treatment. These results suggest that sympathomimetics exert a direct effect on neuromuscular synaptogenesis and do so via β2 adrenoceptors and via a cAMP-dependent pathway. PMID:29462491

Stimulated single fiber electromyography in the mouse: techniques and normative data

NASA Technical Reports Server (NTRS)

Gooch, C. L.; Mosier, D. R.

2001-01-01

As the number of new transgenic mouse models of human neuromuscular disease continues to increase, the development of sophisticated electrophysiologic techniques for assessing the peripheral nervous system in these models has become important. Neuromuscular junction (NMJ) dysfunction, in particular, is often not detectable by morphologic or other techniques. To enable sensitive testing of murine NMJ function, we developed and tested a method for stimulated single fiber electromyography (S-SFEMG) in the gastrocnemius muscles of anesthetized mice. Jitter was assessed by measuring the mean consecutive latency difference (MCD) of single fiber responses to sciatic nerve stimulation at 2 HZ. Mean MCD values in normothermic mice were in the range of 6-8 micros for different strains, with no MCD values exceeding 25 micros. Reduced core temperature (to 29 degrees--30 degrees C) resulted in increased jitter, whereas intubation and mechanical ventilation of mice did not alter these values. Intraperitoneal and intravenous injection of vecuronium, however, resulted in progressively increased jitter followed by blocking in continuously monitored fibers. These observations validate the utility of S-SFEMG in mice as an index of NMJ function under a variety of physiologic conditions, and suggest that a high safety factor for neuromuscular transmission exists at mouse NMJs. Copyright 2001 John Wiley & Sons, Inc.

The Maintenance of Synaptic Homeostasis at the Drosophila Neuromuscular Junction Is Reversible and Sensitive to High Temperature.

PubMed

Yeates, Catherine J; Zwiefelhofer, Danielle J; Frank, C Andrew

2017-01-01

Homeostasis is a vital mode of biological self-regulation. The hallmarks of homeostasis for any biological system are a baseline set point of physiological activity, detection of unacceptable deviations from the set point, and effective corrective measures to counteract deviations. Homeostatic synaptic plasticity (HSP) is a form of neuroplasticity in which neurons and circuits resist environmental perturbations and stabilize levels of activity. One assumption is that if a perturbation triggers homeostatic corrective changes in neuronal properties, those corrective measures should be reversed upon removal of the perturbation. We test the reversibility and limits of HSP at the well-studied Drosophila melanogaster neuromuscular junction (NMJ). At the Drosophila NMJ, impairment of glutamate receptors causes a decrease in quantal size, which is offset by a corrective, homeostatic increase in the number of vesicles released per evoked presynaptic stimulus, or quantal content. This process has been termed presynaptic homeostatic potentiation (PHP). Taking advantage of the GAL4/GAL80 TS /UAS expression system, we triggered PHP by expressing a dominant-negative glutamate receptor subunit at the NMJ. We then reversed PHP by halting expression of the dominant-negative receptor. Our data show that PHP is fully reversible over a time course of 48-72 h after the dominant-negative glutamate receptor stops being genetically expressed. As an extension of these experiments, we find that when glutamate receptors are impaired, neither PHP nor NMJ growth is reliably sustained at high culturing temperatures (30-32°C). These data suggest that a limitation of homeostatic signaling at high temperatures could stem from the synapse facing a combination of challenges simultaneously.

The Maintenance of Synaptic Homeostasis at the Drosophila Neuromuscular Junction Is Reversible and Sensitive to High Temperature

PubMed Central

Zwiefelhofer, Danielle J.

2017-01-01

Abstract Homeostasis is a vital mode of biological self-regulation. The hallmarks of homeostasis for any biological system are a baseline set point of physiological activity, detection of unacceptable deviations from the set point, and effective corrective measures to counteract deviations. Homeostatic synaptic plasticity (HSP) is a form of neuroplasticity in which neurons and circuits resist environmental perturbations and stabilize levels of activity. One assumption is that if a perturbation triggers homeostatic corrective changes in neuronal properties, those corrective measures should be reversed upon removal of the perturbation. We test the reversibility and limits of HSP at the well-studied Drosophila melanogaster neuromuscular junction (NMJ). At the Drosophila NMJ, impairment of glutamate receptors causes a decrease in quantal size, which is offset by a corrective, homeostatic increase in the number of vesicles released per evoked presynaptic stimulus, or quantal content. This process has been termed presynaptic homeostatic potentiation (PHP). Taking advantage of the GAL4/GAL80TS/UAS expression system, we triggered PHP by expressing a dominant-negative glutamate receptor subunit at the NMJ. We then reversed PHP by halting expression of the dominant-negative receptor. Our data show that PHP is fully reversible over a time course of 48–72 h after the dominant-negative glutamate receptor stops being genetically expressed. As an extension of these experiments, we find that when glutamate receptors are impaired, neither PHP nor NMJ growth is reliably sustained at high culturing temperatures (30–32°C). These data suggest that a limitation of homeostatic signaling at high temperatures could stem from the synapse facing a combination of challenges simultaneously. PMID:29255795

Involvement of neurotrophin-3 (NT-3) in the functional elimination of synaptic contacts during neuromuscular development.

PubMed

Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

2010-04-05

Confocal immunohistochemistry shows that neurotrophin-3 (NT-3) and its receptor tropomyosin-related tyrosin kinase C (trkC) are present in both neonatal (P6) and adult (P45) mouse motor nerve terminals in neuromuscular junctions (NMJ) colocalized with several synaptic proteins. NT-3 incubation (1-3h, in the range 10-200ng/ml) does not change the size of the evoked and spontaneous endplate potentials at P45. However, NT-3 (1h, 100ng/ml) strongly potentiates evoked ACh release from the weak (70%) and the strong (50%) axonal inputs on dually innervated postnatal endplates (P6) but not in the most developed postnatal singly innervated synapses at P6. The present results indicate that NT-3 has a role in the developmental mechanism that eliminates redundant synapses though it cannot modulate synaptic transmission locally as the NMJ matures.

Myasthenia gravis: past, present, and future

PubMed Central

Conti-Fine, Bianca M.; Milani, Monica; Kaminski, Henry J.

2006-01-01

Myasthenia gravis (MG) is an autoimmune syndrome caused by the failure of neuromuscular transmission, which results from the binding of autoantibodies to proteins involved in signaling at the neuromuscular junction (NMJ). These proteins include the nicotinic AChR or, less frequently, a muscle-specific tyrosine kinase (MuSK) involved in AChR clustering. Much is known about the mechanisms that maintain self tolerance and modulate anti-AChR Ab synthesis, AChR clustering, and AChR function as well as those that cause neuromuscular transmission failure upon Ab binding. This insight has led to the development of improved diagnostic methods and to the design of specific immunosuppressive or immunomodulatory treatments. PMID:17080188

Structural basis of agrin-LRP4-MuSK signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zong, Yinong; Zhang, Bin; Gu, Shenyan

Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation ofmore » the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.« less

Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using /sup 125/I-alpha scorpion toxin. I. Preferential labeling of glial cells on the presynaptic side

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boudier, J.L.; Jover, E.; Cau, P.

1988-05-01

Alpha-scorpion toxins bind specifically to the voltage-sensitive sodium channel in excitable membranes, and binding is potential-dependent. The radioiodinated toxin II from the scorpion Androctonus australis Hector (alpha ScTx) was used to localize voltage-sensitive sodium channels on the presynaptic side of mouse neuromuscular junctions (NMJ) by autoradiography using both light and electron microscopy. Silver grain localization was analyzed by the cross-fire method. At the light-microscopic level, grain density over NMJ appeared 6-8x higher than over nonjunctional muscle membrane. The specificity of labeling was verified by competition/displacement with an excess of native alpha ScTx. Labeling was also inhibited by incubation in depolarizingmore » conditions, showing its potential-dependence. At the electron-microscopic level, analysis showed that voltage-sensitive sodium channels labeled with alpha ScTx were almost exclusively localized on membranes, as expected. Due to washout after incubation, appreciable numbers of binding sites were not found on the postsynaptic membranes. However, on the presynaptic side, alpha ScTx-labeled voltage-sensitive sodium channels were localized on the membrane of non-myelin-forming Schwann cells covering NMJ. The axonal presynaptic membrane was not labeled. These results show that voltage-sensitive sodium channels are present on glial cells in vivo, as already demonstrated in vitro. It is proposed that these glial channels could be indirectly involved in the ionic homeostasis of the axonal environment.« less

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation.

PubMed

Kaplan, Mehmet Mahsum; Sultana, Nasreen; Benedetti, Ariane; Obermair, Gerald J; Linde, Nina F; Papadopoulos, Symeon; Dayal, Anamika; Grabner, Manfred; Flucher, Bernhard E

2018-06-26

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (Ca V 1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

PubMed

Itoh, Kazuyoshi; Akimoto, Yoshihiro; Kondo, Shu; Ichimiya, Tomomi; Aoki, Kazuhiro; Tiemeyer, Michael; Nishihara, Shoko

2018-04-15

T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles. Copyright © 2018 Elsevier Ltd. All rights reserved.

Bioinspired Three-Dimensional Human Neuromuscular Junction Development in Suspended Hydrogel Arrays.

PubMed

Dixon, Thomas Anthony; Cohen, Eliad; Cairns, Dana M; Rodriguez, Maria; Mathews, Juanita; Jose, Rod R; Kaplan, David L

2018-06-01

The physical connection between motoneurons and skeletal muscle targets is responsible for the creation of neuromuscular junctions (NMJs), which allow electrical signals to be translated to mechanical work. NMJ pathology contributes to the spectrum of neuromuscular, motoneuron, and dystrophic disease. Improving in vitro tools that allow for recapitulation of the physiology of the neuromuscular connection will enable researchers to better understand the development and maturation of NMJs, and will help to decipher mechanisms leading to NMJ degeneration. In this work, we first describe robust differentiation of bungarotoxin-positive human myotubes, as well as a reproducible method for encapsulating and aligning human myoblasts in three-dimensional (3D) suspended culture using bioprinted silk fibroin cantilevers as cell culture supports. Further analysis with coculture of motoneuron-like cells demonstrates feasibility of fully human coculture using two-dimensional and 2.5-dimensional culture methods, with appropriate differentiation of both cell types. Using these coculture differentiation conditions with motoneuron-like cells added to monocultures of 3D suspended human myotubes, we then demonstrate synaptic colocalization in coculture as well as acetylcholine and glutamic acid stimulation of human myocytes. This method represents a unique platform to coculture suspended human myoblast-seeded 3D hydrogels with integrated motoneuron-like cells derived from human induced neural stem cells. The platform described is fully customizable using 3D freeform printing into standard laboratory tissue culture materials, and allows for human myoblast alignment in 3D with precise motoneuron integration into preformed myotubes. The coculture method will ideally be useful in observation and analysis of neurite outgrowth and myogenic differentiation in 3D with quantification of several parameters of muscle innervation and function.

The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.

PubMed

Obis, Teresa; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Priego, Mercedes; Simon, Anna; Garcia, Neus; Santafe, Manel M; Lanuza, Maria A; Tomàs, Josep

2015-12-01

Various protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction. We use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca(2+), stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3',5'-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion. Together, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.

Alterations in the Local Axonal Environment Influence Target Reinnervation and Neuronal Survival After PostnataI Axotomy

DTIC Science & Technology

2000-06-21

2000 Dissertation directed by: Rosemary C. Borke, Ph.D. Professor Department of Anatomy and Cell Biology Following peripheral nerve injury in adult...augmented at the injury site and the neuromuscular junction (NMJ) following sciatic nerve axotomy. The current work determined that SC apoptosis occurs in...related and location-specific response to peripheral nerve injury . Apoptotic SC were found in two strategic locations for guiding axonal outgrowth during

Cardiac troponin T and fast skeletal muscle denervation in ageing

PubMed Central

Xu, Zherong; Feng, Xin; Dong, Juan; Wang, Zhong‐Min; Lee, Jingyun; Furdui, Cristina; Files, Daniel Clark; Beavers, Kristen M.; Kritchevsky, Stephen; Milligan, Carolanne; Jin, Jian‐Ping; Delbono, Osvaldo

2017-01-01

Abstract Background Ageing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast‐twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow‐twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre‐type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle‐specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown. Methods Studies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real‐time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ. Results Levels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region—but mainly in the fast‐twitch, not the slow‐twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIβ are enriched at, the NMJ—again, preferentially in fast‐twitch but not slow‐twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ. Conclusions Cardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast‐twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age‐related decline in muscle function. PMID:28419739

Crystal Structure of the Frizzled-Like Cysteine-Rich Domain of the Receptor Tyrosine Kinase MuSK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stiegler, A.; Burden, S; Hubbard, S

Muscle-specific kinase (MuSK) is an essential receptor tyrosine kinase for the establishment and maintenance of the neuromuscular junction (NMJ). Activation of MuSK by agrin, a neuronally derived heparan-sulfate proteoglycan, and LRP4 (low-density lipoprotein receptor-related protein-4), the agrin receptor, leads to clustering of acetylcholine receptors on the postsynaptic side of the NMJ. The ectodomain of MuSK comprises three immunoglobulin-like domains and a cysteine-rich domain (Fz-CRD) related to those in Frizzled proteins, the receptors for Wnts. Here, we report the crystal structure of the MuSK Fz-CRD at 2.1 {angstrom} resolution. The structure reveals a five-disulfide-bridged domain similar to CRDs of Frizzled proteinsmore » but with a divergent C-terminal region. An asymmetric dimer present in the crystal structure implicates surface hydrophobic residues that may function in homotypic or heterotypic interactions to mediate co-clustering of MuSK, rapsyn, and acetylcholine receptors at the NMJ.« less

The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction

PubMed Central

Machamer, James B.; Collins, Sarah E.; Lloyd, Thomas E.

2014-01-01

Mutations in the RNA binding protein Fused in sarcoma (FUS) are estimated to account for 5–10% of all inherited cases of amyotrophic lateral sclerosis (ALS), but the function of FUS in motor neurons is poorly understood. Here, we investigate the early functional consequences of overexpressing wild-type or ALS-associated mutant FUS proteins in Drosophila motor neurons, and compare them to phenotypes arising from loss of the Drosophila homolog of FUS, Cabeza (Caz). We find that lethality and locomotor phenotypes correlate with levels of FUS transgene expression, indicating that toxicity in developing motor neurons is largely independent of ALS-linked mutations. At the neuromuscular junction (NMJ), overexpression of either wild-type or mutant FUS results in decreased number of presynaptic active zones and altered postsynaptic glutamate receptor subunit composition, coinciding with a reduction in synaptic transmission as a result of both reduced quantal size and quantal content. Interestingly, expression of human FUS downregulates endogenous Caz levels, demonstrating that FUS autoregulation occurs in motor neurons in vivo. However, loss of Caz from motor neurons increases synaptic transmission as a result of increased quantal size, suggesting that the loss of Caz in animals expressing FUS does not contribute to motor deficits. These data demonstrate that FUS/Caz regulates NMJ development and plays an evolutionarily conserved role in modulating the strength of synaptic transmission in motor neurons. PMID:24569165

Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction

PubMed Central

Ramos, Cathy I.; Igiesuorobo, Oghomwen; Wang, Qi; Serpe, Mihaela

2015-01-01

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes. PMID:25905467

Cardiac troponin T and fast skeletal muscle denervation in ageing.

PubMed

Xu, Zherong; Feng, Xin; Dong, Juan; Wang, Zhong-Min; Lee, Jingyun; Furdui, Cristina; Files, Daniel Clark; Beavers, Kristen M; Kritchevsky, Stephen; Milligan, Carolanne; Jin, Jian-Ping; Delbono, Osvaldo; Zhang, Tan

2017-10-01

Ageing skeletal muscle undergoes chronic denervation, and the neuromuscular junction (NMJ), the key structure that connects motor neuron nerves with muscle cells, shows increased defects with ageing. Previous studies in various species have shown that with ageing, type II fast-twitch skeletal muscle fibres show more atrophy and NMJ deterioration than type I slow-twitch fibres. However, how this process is regulated is largely unknown. A better understanding of the mechanisms regulating skeletal muscle fibre-type specific denervation at the NMJ could be critical to identifying novel treatments for sarcopenia. Cardiac troponin T (cTnT), the heart muscle-specific isoform of TnT, is a key component of the mechanisms of muscle contraction. It is expressed in skeletal muscle during early development, after acute sciatic nerve denervation, in various neuromuscular diseases and possibly in ageing muscle. Yet the subcellular localization and function of cTnT in skeletal muscle is largely unknown. Studies were carried out on isolated skeletal muscles from mice, vervet monkeys, and humans. Immunoblotting, immunoprecipitation, and mass spectrometry were used to analyse protein expression, real-time reverse transcription polymerase chain reaction was used to measure gene expression, immunofluorescence staining was performed for subcellular distribution assay of proteins, and electromyographic recording was used to analyse neurotransmission at the NMJ. Levels of cTnT expression in skeletal muscle increased with ageing in mice. In addition, cTnT was highly enriched at the NMJ region-but mainly in the fast-twitch, not the slow-twitch, muscle of old mice. We further found that the protein kinase A (PKA) RIα subunit was largely removed from, while PKA RIIα and RIIβ are enriched at, the NMJ-again, preferentially in fast-twitch but not slow-twitch muscle in old mice. Knocking down cTnT in fast skeletal muscle of old mice: (i) increased PKA RIα and reduced PKA RIIα at the NMJ; (ii) decreased the levels of gene expression of muscle denervation markers; and (iii) enhanced neurotransmission efficiency at NMJ. Cardiac troponin T at the NMJ region contributes to NMJ functional decline with ageing mainly in the fast-twitch skeletal muscle through interfering with PKA signalling. This knowledge could inform useful targets for prevention and therapy of age-related decline in muscle function. © 2017 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.

The novel protein kinase C epsilon isoform at the adult neuromuscular synapse: location, regulation by synaptic activity-dependent muscle contraction through TrkB signaling and coupling to ACh release.

PubMed

Obis, Teresa; Besalduch, Núria; Hurtado, Erica; Nadal, Laura; Santafe, Manel M; Garcia, Neus; Tomàs, Marta; Priego, Mercedes; Lanuza, Maria A; Tomàs, Josep

2015-02-10

Protein kinase C (PKC) regulates a variety of neural functions, including neurotransmitter release. Although various PKC isoforms can be expressed at the synaptic sites and specific cell distribution may contribute to their functional diversity, little is known about the isoform-specific functions of PKCs in neuromuscular synapse. The present study is designed to examine the location of the novel isoform nPKCε at the neuromuscular junction (NMJ), their synaptic activity-related expression changes, its regulation by muscle contraction, and their possible involvement in acetylcholine release. We use immunohistochemistry and confocal microscopy to demonstrate that the novel isoform nPKCε is exclusively located in the motor nerve terminals of the adult rat NMJ. We also report that electrical stimulation of synaptic inputs to the skeletal muscle significantly increased the amount of nPKCε isoform as well as its phosphorylated form in the synaptic membrane, and muscle contraction is necessary for these nPKCε expression changes. The results also demonstrate that synaptic activity-induced muscle contraction promotes changes in presynaptic nPKCε through the brain-derived neurotrophic factor (BDNF)-mediated tyrosine kinase receptor B (TrkB) signaling. Moreover, nPKCε activity results in phosphorylation of the substrate MARCKS involved in actin cytoskeleton remodeling and related with neurotransmission. Finally, blocking nPKCε with a nPKCε-specific translocation inhibitor peptide (εV1-2) strongly reduces phorbol ester-induced ACh release potentiation, which further indicates that nPKCε is involved in neurotransmission. Together, these results provide a mechanistic insight into how synaptic activity-induced muscle contraction could regulate the presynaptic action of the nPKCε isoform and suggest that muscle contraction is an important regulatory step in TrkB signaling at the NMJ.

Recovery of whisking function promoted by manual stimulation of the vibrissal muscles after facial nerve injury requires insulin-like growth factor 1 (IGF-1).

PubMed

Kiryakova, S; Söhnchen, J; Grosheva, M; Schuetz, U; Marinova, Ts; Dzhupanova, R; Sinis, N; Hübbers, C U; Skouras, E; Ankerne, J; Fries, J W U; Irintchev, A; Dunlop, S A; Angelov, D N

2010-04-01

Recently, we showed that manual stimulation (MS) of denervated vibrissal muscles enhanced functional recovery following facial nerve cut and suture (FFA) by reducing poly-innervation at the neuro-muscular junctions (NMJ). Although the cellular correlates of poly-innervation are established, with terminal Schwann cells (TSC) processes attracting axon sprouts to "bridge" adjacent NMJ, molecular correlates are poorly understood. Since quantitative RT-PCR revealed a rapid increase of IGF-1 mRNA in denervated muscles, we examined the effect of daily MS for 2 months after FFA in IGF-1(+/-) heterozygous mice; controls were wild-type (WT) littermates including intact animals. We quantified vibrissal motor performance and the percentage of NMJ bridged by S100-positive TSC. There were no differences between intact WT and IGF-1(+/-) mice for vibrissal whisking amplitude (48 degrees and 49 degrees ) or the percentage of bridged NMJ (0%). After FFA and handling alone (i.e. no MS) in WT animals, vibrissal whisking amplitude was reduced (60% lower than intact) and the percentage of bridged NMJ increased (42% more than intact). MS improved both the amplitude of vibrissal whisking (not significantly different from intact) and the percentage of bridged NMJ (12% more than intact). After FFA and handling in IGF-1(+/-) mice, the pattern was similar (whisking amplitude 57% lower than intact; proportion of bridged NMJ 42% more than intact). However, MS did not improve outcome (whisking amplitude 47% lower than intact; proportion of bridged NMJ 40% more than intact). We conclude that IGF-I is required to mediate the effects of MS on target muscle reinnervation and recovery of whisking function. Copyright 2010 Elsevier Inc. All rights reserved.

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction.

PubMed

Valadão, Priscila Aparecida Costa; Naves, Lígia Araújo; Gomez, Renato Santiago; Guatimosim, Cristina

2013-11-01

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na(+) channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca(2+) because its effect was abolished in Ca(2+)-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca(2+) channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca(2+) channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent. Copyright © 2013 Elsevier Ltd. All rights reserved.

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation.

PubMed

Kopke, Danielle L; Broadie, Kendal

2018-05-24

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K + ] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Transmission, Development, and Plasticity of Synapses

PubMed Central

Harris, Kathryn P.

2015-01-01

Chemical synapses are sites of contact and information transfer between a neuron and its partner cell. Each synapse is a specialized junction, where the presynaptic cell assembles machinery for the release of neurotransmitter, and the postsynaptic cell assembles components to receive and integrate this signal. Synapses also exhibit plasticity, during which synaptic function and/or structure are modified in response to activity. With a robust panel of genetic, imaging, and electrophysiology approaches, and strong evolutionary conservation of molecular components, Drosophila has emerged as an essential model system for investigating the mechanisms underlying synaptic assembly, function, and plasticity. We will discuss techniques for studying synapses in Drosophila, with a focus on the larval neuromuscular junction (NMJ), a well-established model glutamatergic synapse. Vesicle fusion, which underlies synaptic release of neurotransmitters, has been well characterized at this synapse. In addition, studies of synaptic assembly and organization of active zones and postsynaptic densities have revealed pathways that coordinate those events across the synaptic cleft. We will also review modes of synaptic growth and plasticity at the fly NMJ, and discuss how pre- and postsynaptic cells communicate to regulate plasticity in response to activity. PMID:26447126

Mutations in GFPT1-related congenital myasthenic syndromes are associated with synaptic morphological defects and underlie a tubular aggregate myopathy with synaptopathy.

PubMed

Bauché, Stéphanie; Vellieux, Geoffroy; Sternberg, Damien; Fontenille, Marie-Joséphine; De Bruyckere, Elodie; Davoine, Claire-Sophie; Brochier, Guy; Messéant, Julien; Wolf, Lucie; Fardeau, Michel; Lacène, Emmanuelle; Romero, Norma; Koenig, Jeanine; Fournier, Emmanuel; Hantaï, Daniel; Streichenberger, Nathalie; Manel, Veronique; Lacour, Arnaud; Nadaj-Pakleza, Aleksandra; Sukno, Sylvie; Bouhour, Françoise; Laforêt, Pascal; Fontaine, Bertrand; Strochlic, Laure; Eymard, Bruno; Chevessier, Frédéric; Stojkovic, Tanya; Nicole, Sophie

2017-08-01

Mutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients' muscle biopsies. We report here the first long-term clinical follow-up of 11 French individuals suffering from LG-CMS with TAs due to GFPT1 mutations, of which nine are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from three unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation-reinnervation processes affecting the three main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extra-junctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a "tubular aggregates myopathy with synaptopathy".

Terminal Schwann Cells Participate in Neuromuscular Synapse Remodeling during Reinnervation following Nerve Injury

PubMed Central

Kang, Hyuno; Tian, Le; Mikesh, Michelle; Lichtman, Jeff W.

2014-01-01

Schwann cells (SCs) at neuromuscular junctions (NMJs) play active roles in synaptic homeostasis and repair. We have studied how SCs contribute to reinnervation of NMJs using vital imaging of mice whose motor axons and SCs are transgenically labeled with different colors of fluorescent proteins. Motor axons most commonly regenerate to the original synaptic site by following SC-filled endoneurial tubes. During the period of denervation, SCs at the NMJ extend elaborate processes from the junction, as shown previously, but they also retract some processes from territory they previously occupied within the endplate. The degree of this retraction depends on the length of the period of denervation. We show that the topology of the remaining SC processes influences the branching pattern of regenerating axon terminals and the redistribution of acetylcholine receptors (AChRs). Upon arriving at the junction, regenerating axons follow existing SC processes within the old synaptic site. Some of the AChR loss that follows denervation is correlated with failure of portions of the old synaptic site that lack SC coverage to be reinnervated. New AChR clustering is also induced by axon terminals that follow SC processes extended during denervation. These observations show that SCs participate actively in the remodeling of neuromuscular synapses following nerve injury by their guidance of axonal reinnervation. PMID:24790203

Severe neuromuscular denervation of clinically relevant muscles in a mouse model of spinal muscular atrophy

PubMed Central

Ling, Karen K. Y.; Gibbs, Rebecca M.; Feng, Zhihua; Ko, Chien-Ping

2012-01-01

Spinal muscular atrophy (SMA), a motoneuron disease caused by a deficiency of the survival of motor neuron (SMN) protein, is characterized by motoneuron loss and muscle weakness. It remains unclear whether widespread loss of neuromuscular junctions (NMJs) is involved in SMA pathogenesis. We undertook a systematic examination of NMJ innervation patterns in >20 muscles in the SMNΔ7 SMA mouse model. We found that severe denervation (<50% fully innervated endplates) occurs selectively in many vulnerable axial muscles and several appendicular muscles at the disease end stage. Since these vulnerable muscles were located throughout the body and were comprised of varying muscle fiber types, it is unlikely that muscle location or fiber type determines susceptibility to denervation. Furthermore, we found a similar extent of neurofilament accumulation at NMJs in both vulnerable and resistant muscles before the onset of denervation, suggesting that neurofilament accumulation does not predict subsequent NMJ denervation. Since vulnerable muscles were initially innervated, but later denervated, loss of innervation in SMA may be attributed to defects in synapse maintenance. Finally, we found that denervation was amendable by trichostatin A (TSA) treatment, which increased innervation in clinically relevant muscles in TSA-treated SMNΔ7 mice. Our findings suggest that neuromuscular denervation in vulnerable muscles is a widespread pathology in SMA, and can serve as a preparation for elucidating the biological basis of synapse loss, and for evaluating therapeutic efficacy. PMID:21968514

A New Fiji-Based Algorithm That Systematically Quantifies Nine Synaptic Parameters Provides Insights into Drosophila NMJ Morphometry

PubMed Central

Wolf, Louis; Scheffer-de Gooyert, Jolanda M.; Monedero, Ignacio; Torroja, Laura; Coromina, Lluis; van der Laak, Jeroen A. W. M.; Schenck, Annette

2016-01-01

The morphology of synapses is of central interest in neuroscience because of the intimate relation with synaptic efficacy. Two decades of gene manipulation studies in different animal models have revealed a repertoire of molecules that contribute to synapse development. However, since such studies often assessed only one, or at best a few, morphological features at a given synapse, it remained unaddressed how different structural aspects relate to one another. Furthermore, such focused and sometimes only qualitative approaches likely left many of the more subtle players unnoticed. Here, we present the image analysis algorithm ‘Drosophila_NMJ_Morphometrics’, available as a Fiji-compatible macro, for quantitative, accurate and objective synapse morphometry of the Drosophila larval neuromuscular junction (NMJ), a well-established glutamatergic model synapse. We developed this methodology for semi-automated multiparametric analyses of NMJ terminals immunolabeled for the commonly used markers Dlg1 and Brp and showed that it also works for Hrp, Csp and Syt. We demonstrate that gender, genetic background and identity of abdominal body segment consistently and significantly contribute to variability in our data, suggesting that controlling for these parameters is important to minimize variability in quantitative analyses. Correlation and principal component analyses (PCA) were performed to investigate which morphometric parameters are inter-dependent and which ones are regulated rather independently. Based on nine acquired parameters, we identified five morphometric groups: NMJ size, geometry, muscle size, number of NMJ islands and number of active zones. Based on our finding that the parameters of the first two principal components hardly correlated with each other, we suggest that different molecular processes underlie these two morphometric groups. Our study sets the stage for systems morphometry approaches at the well-studied Drosophila NMJ. PMID:26998933

The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila.

PubMed

Deshpande, Mugdha; Rodal, Avital A

2016-02-01

Neurons require target-derived autocrine and paracrine growth factors to maintain proper identity, innervation, homeostasis and survival. Neuronal growth factor signaling is highly dependent on membrane traffic, both for the packaging and release of the growth factors themselves, and for regulation of intracellular signaling by their transmembrane receptors. Here, we review recent findings from the Drosophila larval neuromuscular junction (NMJ) that illustrate how specific steps of intracellular traffic and inter-organelle interactions impinge on signaling, particularly in the bone morphogenic protein, Wingless and c-Jun-activated kinase pathways, regulating elaboration and stability of NMJ arbors, construction of synapses and synaptic transmission and homeostasis. These membrane trafficking and signaling pathways have been implicated in human motor neuron diseases including amyotrophic lateral sclerosis and hereditary spastic paraplegia, highlighting their importance for neuronal health and survival. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Pathogenesis of myasthenia gravis: update on disease types, models, and mechanisms.

PubMed

Phillips, William D; Vincent, Angela

2016-01-01

Myasthenia gravis is an autoimmune disease of the neuromuscular junction (NMJ) caused by antibodies that attack components of the postsynaptic membrane, impair neuromuscular transmission, and lead to weakness and fatigue of skeletal muscle. This can be generalised or localised to certain muscle groups, and involvement of the bulbar and respiratory muscles can be life threatening. The pathogenesis of myasthenia gravis depends upon the target and isotype of the autoantibodies. Most cases are caused by immunoglobulin (Ig)G1 and IgG3 antibodies to the acetylcholine receptor (AChR). They produce complement-mediated damage and increase the rate of AChR turnover, both mechanisms causing loss of AChR from the postsynaptic membrane. The thymus gland is involved in many patients, and there are experimental and genetic approaches to understand the failure of immune tolerance to the AChR. In a proportion of those patients without AChR antibodies, antibodies to muscle-specific kinase (MuSK), or related proteins such as agrin and low-density lipoprotein receptor-related protein 4 (LRP4), are present. MuSK antibodies are predominantly IgG4 and cause disassembly of the neuromuscular junction by disrupting the physiological function of MuSK in synapse maintenance and adaptation. Here we discuss how knowledge of neuromuscular junction structure and function has fed into understanding the mechanisms of AChR and MuSK antibodies. Myasthenia gravis remains a paradigm for autoantibody-mediated conditions and these observations show how much there is still to learn about synaptic function and pathological mechanisms.

Anterograde Jelly belly ligand to Alk receptor signaling at developing synapses is regulated by Mind the gap.

PubMed

Rohrbough, Jeffrey; Broadie, Kendal

2010-10-01

Bidirectional trans-synaptic signals induce synaptogenesis and regulate subsequent synaptic maturation. Presynaptically secreted Mind the gap (Mtg) molds the synaptic cleft extracellular matrix, leading us to hypothesize that Mtg functions to generate the intercellular environment required for efficient signaling. We show in Drosophila that secreted Jelly belly (Jeb) and its receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) are localized to developing synapses. Jeb localizes to punctate aggregates in central synaptic neuropil and neuromuscular junction (NMJ) presynaptic terminals. Secreted Jeb and Mtg accumulate and colocalize extracellularly in surrounding synaptic boutons. Alk concentrates in postsynaptic domains, consistent with an anterograde, trans-synaptic Jeb-Alk signaling pathway at developing synapses. Jeb synaptic expression is increased in Alk mutants, consistent with a requirement for Alk receptor function in Jeb uptake. In mtg null mutants, Alk NMJ synaptic levels are reduced and Jeb expression is dramatically increased. NMJ synapse morphology and molecular assembly appear largely normal in jeb and Alk mutants, but larvae exhibit greatly reduced movement, suggesting impaired functional synaptic development. jeb mutant movement is significantly rescued by neuronal Jeb expression. jeb and Alk mutants display normal NMJ postsynaptic responses, but a near loss of patterned, activity-dependent NMJ transmission driven by central excitatory output. We conclude that Jeb-Alk expression and anterograde trans-synaptic signaling are modulated by Mtg and play a key role in establishing functional synaptic connectivity in the developing motor circuit.

Cellular localization of the atypical isoforms of protein kinase C (aPKCζ/PKMζ and aPKCλ/ι) on the neuromuscular synapse.

PubMed

Besalduch, Núria; Lanuza, Maria A; Garcia, Neus; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Priego, Mercedes; Tomàs, Josep

2013-11-27

Several classic and novel protein kinase C (PKC) isoforms are selectively distributed in specific cell types of the adult neuromuscular junction (NMJ), in the neuron, glia and muscle components, and are involved in many functions, including neurotransmission. Here, we investigate the presence in this paradigmatic synapse of atypical PKCs, full-length atypical PKC zeta (aPKCζ), its separated catalytic part (PKMζ) and atypical lambda-iota PKC (aPKCλ/ι). High resolution immunohistochemistry was performed using a pan-atypical PKC antibody. Our results show moderate immunolabeling on the three cells (presynaptic motor nerve terminal, teloglial Schwann cell and postsynaptic muscle cell) suggesting the complex involvement of atypical PKCs in synaptic function. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non-Cell-Autonomous Mechanism in ALS.

PubMed

Maimon, Roy; Ionescu, Ariel; Bonnie, Avichai; Sweetat, Sahar; Wald-Altman, Shane; Inbar, Shani; Gradus, Tal; Trotti, Davide; Weil, Miguel; Behar, Oded; Perlson, Eran

2018-06-13

Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease's etiology is not fully understood, it is thought to involve a non-cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalized in vitro cocultures, we demonstrated that myocytes expressing diverse ALS-causing mutations promote axon degeneration and NMJ dysfunction, which were inhibited by applying Neuropilin1 blocking antibody. Finally, overexpressing miR126-5p is sufficient to transiently rescue axon degeneration and NMJ disruption both in vitro and in vivo Thus, we demonstrate a novel mechanism underlying ALS pathology, in which alterations in miR126-5p facilitate a non-cell-autonomous mechanism of motor neuron degeneration in ALS. SIGNIFICANCE STATEMENT Despite some progress, currently no effective treatment is available for amyotrophic lateral sclerosis (ALS). We suggest a novel regulatory role for miR126-5p in ALS and demonstrate, for the first time, a mechanism by which alterations in miR126-5p contribute to axon degeneration and NMJ disruption observed in ALS. We show that miR126-5p is altered in ALS models and that it can modulate Sema3 and NRP protein expression. Furthermore, NRP1 elevations in motor neurons and muscle secretion of Sema3A contribute to axon degeneration and NMJ disruption in ALS. Finally, overexpressing miR126-5p is sufficient to transiently rescue NMJ disruption and axon degeneration both in vitro and in vivo . Copyright © 2018 Maimon et al.

Drosophila Spastin Regulates Synaptic Microtubule Networks and Is Required for Normal Motor Function

PubMed Central

Sherwood, Nina Tang; Sun, Qi; Xue, Mingshan; Zhang, Bing

2004-01-01

The most common form of human autosomal dominant hereditary spastic paraplegia (AD-HSP) is caused by mutations in the SPG4 (spastin) gene, which encodes an AAA ATPase closely related in sequence to the microtubule-severing protein Katanin. Patients with AD-HSP exhibit degeneration of the distal regions of the longest axons in the spinal cord. Loss-of-function mutations in the Drosophila spastin gene produce larval neuromuscular junction (NMJ) phenotypes. NMJ synaptic boutons in spastin mutants are more numerous and more clustered than in wild-type, and transmitter release is impaired. spastin-null adult flies have severe movement defects. They do not fly or jump, they climb poorly, and they have short lifespans. spastin hypomorphs have weaker behavioral phenotypes. Overexpression of Spastin erases the muscle microtubule network. This gain-of-function phenotype is consistent with the hypothesis that Spastin has microtubule-severing activity, and implies that spastin loss-of-function mutants should have an increased number of microtubules. Surprisingly, however, we observed the opposite phenotype: in spastin-null mutants, there are fewer microtubule bundles within the NMJ, especially in its distal boutons. The Drosophila NMJ is a glutamatergic synapse that resembles excitatory synapses in the mammalian spinal cord, so the reduction of organized presynaptic microtubules that we observe in spastin mutants may be relevant to an understanding of human Spastin's role in maintenance of axon terminals in the spinal cord. PMID:15562320

Blocking p75 (NTR) receptors alters polyinnervationz of neuromuscular synapses during development.

PubMed

Garcia, Neus; Tomàs, Marta; Santafe, Manel M; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

2011-09-01

High-resolution immunohistochemistry shows that the receptor protein p75(NTR) is present in the nerve terminal, muscle cell, and glial Schwann cell at the neuromuscular junction (NMJ) of postnatal rats (P4-P6) during the synapse elimination period. Blocking the receptor with the antibody anti-p75-192-IgG (1-5 μg/ml, 1 hr) results in reduced endplate potentials (EPPs) in mono- and polyinnervated synapses ex vivo, but the mean number of functional inputs per NMJ does not change for as long as 3 hr. Incubation with exogenous brain-derived neurotrophic factor (BDNF) for 1 hr (50 nM) resulted in a significant increase in the size of the EPPs in all nerve terminals, and preincubation with anti-p75-192-IgG prevented this potentiation. Long exposure (24 hr) in vivo of the NMJs to the antibody anti-p75-192-IgG (1-2 μg/ml) results in a delay of postnatal synapse elimination and even some regrowth of previously withdrawn axons, but also in some acceleration of the morphologic maturation of the postsynaptic nicotinic acetylcholine receptor (nAChR) clusters. The results indicate that p75(NTR) is involved in both ACh release and axonal retraction during postnatal axonal competition and synapse elimination. Copyright © 2011 Wiley-Liss, Inc.

Evidence for mast cells contributing to neuromuscular pathology in an inherited model of ALS

PubMed Central

Trias, Emiliano; Ibarburu, Sofía; Barreto-Núñez, Romina; Varela, Valentina; Moura, Ivan C.; Hermine, Olivier

2017-01-01

Evidence indicates that neuroinflammation contributes to motor neuron degeneration in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease leading to progressive muscular paralysis. However, it remains elusive whether inflammatory cells can interact with degenerating distal motor axons, influencing the progressive denervation of neuromuscular junctions (NMJs). By analyzing the muscle extensor digitorum longus (EDL) following paralysis onset in the SOD1G93A rat model, we have observed a massive infiltration and degranulation of mast cells, starting after paralysis onset and correlating with progressive NMJ denervation. Remarkably, mast cells accumulated around degenerating motor axons and NMJs, and were also associated with macrophages. Mast cell accumulation and degranulation in paralytic EDL muscle was prevented by systemic treatment over 15 days with masitinib, a tyrosine kinase inhibitor currently in clinical trials for ALS exhibiting pharmacological activity affecting mast cells and microglia. Masitinib-induced mast cell reduction resulted in a 35% decrease in NMJ denervation and reduced motor deficits as compared with vehicle-treated rats. Masitinib also normalized macrophage infiltration, as well as regressive changes in Schwann cells and capillary networks observed in advanced paralysis. These findings provide evidence for mast cell contribution to distal axonopathy and paralysis progression in ALS, a mechanism that can be therapeutically targeted by masitinib. PMID:29046475

Pathogenesis of myasthenia gravis: update on disease types, models, and mechanisms

PubMed Central

Phillips, William D.; Vincent, Angela

2016-01-01

Myasthenia gravis is an autoimmune disease of the neuromuscular junction (NMJ) caused by antibodies that attack components of the postsynaptic membrane, impair neuromuscular transmission, and lead to weakness and fatigue of skeletal muscle. This can be generalised or localised to certain muscle groups, and involvement of the bulbar and respiratory muscles can be life threatening. The pathogenesis of myasthenia gravis depends upon the target and isotype of the autoantibodies. Most cases are caused by immunoglobulin (Ig)G1 and IgG3 antibodies to the acetylcholine receptor (AChR). They produce complement-mediated damage and increase the rate of AChR turnover, both mechanisms causing loss of AChR from the postsynaptic membrane. The thymus gland is involved in many patients, and there are experimental and genetic approaches to understand the failure of immune tolerance to the AChR. In a proportion of those patients without AChR antibodies, antibodies to muscle-specific kinase (MuSK), or related proteins such as agrin and low-density lipoprotein receptor-related protein 4 (LRP4), are present. MuSK antibodies are predominantly IgG4 and cause disassembly of the neuromuscular junction by disrupting the physiological function of MuSK in synapse maintenance and adaptation. Here we discuss how knowledge of neuromuscular junction structure and function has fed into understanding the mechanisms of AChR and MuSK antibodies. Myasthenia gravis remains a paradigm for autoantibody-mediated conditions and these observations show how much there is still to learn about synaptic function and pathological mechanisms. PMID:27408701

The interaction between tropomyosin-related kinase B receptors and serine kinases modulates acetylcholine release in adult neuromuscular junctions.

PubMed

Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Obis, Teresa; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

2014-02-21

We conducted an electrophysiological study of the functional link between the tropomyosin-related kinase B (trkB) receptor signaling mechanism and serine-threonine kinases, both protein kinase C (PKC) and protein kinase A (PKA). We describe their coordinated role in transmitter release at the neuromuscular junction (NMJ) of the Levator auris longus muscle of the adult mouse. The trkB receptor normally seems to be coupled to stimulate ACh release because inhibiting the trkB receptor with K-252a results in a significant reduction in the size of EPPs. We found that the intracellular PKC pathway can operate as in basal conditions (to potentiate ACh release) without the involvement of the trkB receptor function, although the trkB pathway needs an operative PKC pathway if it is to couple to the release mechanism and potentiate it. To actively stimulate PKA (which also results in ACh release potentiation), the operativity of trkB is a necessary condition, and one effect of trkB may be PKA stimulation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise, Neuromuscular Junctions, and Brains

PubMed Central

Nichols, Bailey; Takeda, Shin’ichi; Yokota, Toshifumi

2015-01-01

Dystrophin-glycoprotein complex (DGC) is an important structural unit in skeletal muscle that connects the cytoskeleton (f-actin) of a muscle fiber to the extracellular matrix (ECM). Several muscular dystrophies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophies (dystroglycanopathies), and limb-girdle muscular dystrophies (sarcoglycanopathies), are caused by mutations in the different DGC components. Although many early studies indicated DGC plays a crucial mechanical role in maintaining the structural integrity of skeletal muscle, recent studies identified novel roles of DGC. Beyond a mechanical role, these DGC members play important signaling roles and act as a scaffold for various signaling pathways. For example, neuronal nitric oxide synthase (nNOS), which is localized at the muscle membrane by DGC members (dystrophin and syntrophins), plays an important role in the regulation of the blood flow during exercise. DGC also plays important roles at the neuromuscular junction (NMJ) and in the brain. In this review, we will focus on recently identified roles of DGC particularly in exercise and the brain. PMID:26230713

Congenital Myasthenic Syndrome Type 19 Is Caused by Mutations in COL13A1, Encoding the Atypical Non-fibrillar Collagen Type XIII α1 Chain.

PubMed

Logan, Clare V; Cossins, Judith; Rodríguez Cruz, Pedro M; Parry, David A; Maxwell, Susan; Martínez-Martínez, Pilar; Riepsaame, Joey; Abdelhamed, Zakia A; Lake, Alice V R; Moran, Maria; Robb, Stephanie; Chow, Gabriel; Sewry, Caroline; Hopkins, Philip M; Sheridan, Eamonn; Jayawant, Sandeep; Palace, Jacqueline; Johnson, Colin A; Beeson, David

2015-12-03

The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs(∗)71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Crimpy enables discrimination of pre and postsynaptic pools of a BMP at the Drosophila NMJ

PubMed Central

James, Rebecca E.; Hoover, Kendall M.; Bulgari, Dinara; McLaughlin, Colleen N.; Wilson, Christopher G.; Wharton, Kristi A.; Levitan, Edwin S.; Broihier, Heather T.

2014-01-01

Summary Distinct pools of the BMP Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, while muscle-derived Gbb regulates NMJ growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's pro-neurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy co-release from presynaptic terminals defines a neuronal pro-transmission signal. PMID:25453556

Na+/K+ ATPase regulates the expression and localization of acetylcholine receptors in a pump activity-independent manner

PubMed Central

Doi, Motomichi; Iwasaki, Kouichi

2008-01-01

Na+/K+ ATPase is a plasma membrane-localized sodium pump that maintains the ion gradients between the extracellular and intracellular environments, which in turn controls the cellular resting membrane potential. Recent evidence suggests that the pump is also localized at synapses and regulates synaptic efficacy. However, its precise function at the synapse is unknown. Here we show that two mutations in the α subunit of the eat-6 Na+/K+ ATPase in Caenorhabditis elegans dramatically increase the sensitivity to acetylcholine (Ach) agonists and alter the localization of nicotinic Ach receptors at the neuromuscular junction (NMJ). These defects can be rescued by mutated EAT-6 proteins which lack its pump activity, suggesting the presence of a novel function for Ach signaling. The Na+/K+ ATPase accumulates at postsynaptic sites and appears to surround Ach receptors to maintain rigid clusters at the NMJ. Our findings suggest a critical pump activity-independent, allele –specific role for Na+/K+ ATPase on postsynaptic organization and synaptic efficacy. PMID:18599311

Homeostatic synaptic depression is achieved through a regulated decrease in presynaptic calcium channel abundance

PubMed Central

Gaviño, Michael A; Ford, Kevin J; Archila, Santiago; Davis, Graeme W

2015-01-01

Homeostatic signaling stabilizes synaptic transmission at the neuromuscular junction (NMJ) of Drosophila, mice, and human. It is believed that homeostatic signaling at the NMJ is bi-directional and considerable progress has been made identifying mechanisms underlying the homeostatic potentiation of neurotransmitter release. However, very little is understood mechanistically about the opposing process, homeostatic depression, and how bi-directional plasticity is achieved. Here, we show that homeostatic potentiation and depression can be simultaneously induced, demonstrating true bi-directional plasticity. Next, we show that mutations that block homeostatic potentiation do not alter homeostatic depression, demonstrating that these are genetically separable processes. Finally, we show that homeostatic depression is achieved by decreased presynaptic calcium channel abundance and calcium influx, changes that are independent of the presynaptic action potential waveform. Thus, we identify a novel mechanism of homeostatic synaptic plasticity and propose a model that can account for the observed bi-directional, homeostatic control of presynaptic neurotransmitter release. DOI: http://dx.doi.org/10.7554/eLife.05473.001 PMID:25884248

Muscle Contraction Regulates BDNF/TrkB Signaling to Modulate Synaptic Function through Presynaptic cPKCα and cPKCβI.

PubMed

Hurtado, Erica; Cilleros, Víctor; Nadal, Laura; Simó, Anna; Obis, Teresa; Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Halievski, Katherine; Jordan, Cynthia L; Lanuza, Maria A; Tomàs, Josep

2017-01-01

The neurotrophin brain-derived neurotrophic factor (BDNF) acts via tropomyosin-related kinase B receptor (TrkB) to regulate synapse maintenance and function in the neuromuscular system. The potentiation of acetylcholine (ACh) release by BDNF requires TrkB phosphorylation and Protein Kinase C (PKC) activation. BDNF is secreted in an activity-dependent manner but it is not known if pre- and/or postsynaptic activities enhance BDNF expression in vivo at the neuromuscular junction (NMJ). Here, we investigated whether nerve and muscle cell activities regulate presynaptic conventional PKC (cPKCα and βI) via BDNF/TrkB signaling to modulate synaptic strength at the NMJ. To differentiate the effects of presynaptic activity from that of muscle contraction, we stimulated the phrenic nerve of rat diaphragms (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Then, we performed ELISA, Western blotting, qRT-PCR, immunofluorescence and electrophysiological techniques. We found that nerve-induced muscle contraction: (1) increases the levels of mature BDNF protein without affecting pro-BDNF protein or BDNF mRNA levels; (2) downregulates TrkB.T1 without affecting TrkB.FL or p75 neurotrophin receptor (p75) levels; (3) increases presynaptic cPKCα and cPKCβI protein level through TrkB signaling; and (4) enhances phosphorylation of cPKCα and cPKCβI. Furthermore, we demonstrate that cPKCβI, which is exclusively located in the motor nerve terminals, increases activity-induced acetylcholine release. Together, these results show that nerve-induced muscle contraction is a key regulator of BDNF/TrkB signaling pathway, retrogradely activating presynaptic cPKC isoforms (in particular cPKCβI) to modulate synaptic function. These results indicate that a decrease in neuromuscular activity, as occurs in several neuromuscular disorders, could affect the BDNF/TrkB/PKC pathway that links pre- and postsynaptic activity to maintain neuromuscular function.

Muscle Contraction Regulates BDNF/TrkB Signaling to Modulate Synaptic Function through Presynaptic cPKCα and cPKCβI

PubMed Central

Hurtado, Erica; Cilleros, Víctor; Nadal, Laura; Simó, Anna; Obis, Teresa; Garcia, Neus; Santafé, Manel M.; Tomàs, Marta; Halievski, Katherine; Jordan, Cynthia L.; Lanuza, Maria A.; Tomàs, Josep

2017-01-01

The neurotrophin brain-derived neurotrophic factor (BDNF) acts via tropomyosin-related kinase B receptor (TrkB) to regulate synapse maintenance and function in the neuromuscular system. The potentiation of acetylcholine (ACh) release by BDNF requires TrkB phosphorylation and Protein Kinase C (PKC) activation. BDNF is secreted in an activity-dependent manner but it is not known if pre- and/or postsynaptic activities enhance BDNF expression in vivo at the neuromuscular junction (NMJ). Here, we investigated whether nerve and muscle cell activities regulate presynaptic conventional PKC (cPKCα and βI) via BDNF/TrkB signaling to modulate synaptic strength at the NMJ. To differentiate the effects of presynaptic activity from that of muscle contraction, we stimulated the phrenic nerve of rat diaphragms (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Then, we performed ELISA, Western blotting, qRT-PCR, immunofluorescence and electrophysiological techniques. We found that nerve-induced muscle contraction: (1) increases the levels of mature BDNF protein without affecting pro-BDNF protein or BDNF mRNA levels; (2) downregulates TrkB.T1 without affecting TrkB.FL or p75 neurotrophin receptor (p75) levels; (3) increases presynaptic cPKCα and cPKCβI protein level through TrkB signaling; and (4) enhances phosphorylation of cPKCα and cPKCβI. Furthermore, we demonstrate that cPKCβI, which is exclusively located in the motor nerve terminals, increases activity-induced acetylcholine release. Together, these results show that nerve-induced muscle contraction is a key regulator of BDNF/TrkB signaling pathway, retrogradely activating presynaptic cPKC isoforms (in particular cPKCβI) to modulate synaptic function. These results indicate that a decrease in neuromuscular activity, as occurs in several neuromuscular disorders, could affect the BDNF/TrkB/PKC pathway that links pre- and postsynaptic activity to maintain neuromuscular function. PMID:28572757

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells

PubMed Central

Gonzalez Porras, Maria A.; Durfee, Paul N.; Gregory, Ashley M.; Sieck, Gary C.; Brinker, C. Jeffrey; Mantilla, Carlos B.

2017-01-01

Background Trophic interactions between muscle fibers and motoneurons at the neuromuscular junction (NMJ) play a critical role in determining motor function throughout development, ageing, injury, or disease. Treatment of neuromuscular disorders is hindered by the inability to selectively target motoneurons with pharmacological and genetic interventions. New method We describe a novel delivery system to motoneurons using mesoporous silica nanoparticles encapsulated within a lipid bilayer (protocells) and modified with the atoxic subunit B of the cholera toxin (CTB) that binds to gangliosides present on neuronal membranes. Results CTB modified protocells showed significantly greater motoneuron uptake compared to unmodified protocells after 24 h of treatment (60% vs. 15%, respectively). CTB-protocells showed specific uptake by motoneurons compared to muscle cells and demonstrated cargo release of a surrogate drug. Protocells showed a lack of cytotoxicity and unimpaired cellular proliferation. In isolated diaphragm muscle-phrenic nerve preparations, preferential axon terminal uptake of CTB-modified protocells was observed compared to uptake in surrounding muscle tissue. A larger proportion of axon terminals displayed uptake following treatment with CTB-protocells compared to unmodified protocells (40% vs. 6%, respectively). Comparison with existing method(s) Current motoneuron targeting strategies lack the functionality to load and deliver multiple cargos. CTB-protocells capitalizes on the advantages of liposomes and mesoporous silica nanoparticles allowing a large loading capacity and cargo release. The ability of CTB-protocells to target motoneurons at the NMJ confers a great advantage over existing methods. Conclusions CTB-protocells constitute a viable targeted motoneuron delivery system for drugs and genes facilitating various therapies for neuromuscular diseases. PMID:27641118

A novel approach for targeted delivery to motoneurons using cholera toxin-B modified protocells.

PubMed

Gonzalez Porras, Maria A; Durfee, Paul N; Gregory, Ashley M; Sieck, Gary C; Brinker, C Jeffrey; Mantilla, Carlos B

2016-11-01

Trophic interactions between muscle fibers and motoneurons at the neuromuscular junction (NMJ) play a critical role in determining motor function throughout development, ageing, injury, or disease. Treatment of neuromuscular disorders is hindered by the inability to selectively target motoneurons with pharmacological and genetic interventions. We describe a novel delivery system to motoneurons using mesoporous silica nanoparticles encapsulated within a lipid bilayer (protocells) and modified with the atoxic subunit B of the cholera toxin (CTB) that binds to gangliosides present on neuronal membranes. CTB modified protocells showed significantly greater motoneuron uptake compared to unmodified protocells after 24h of treatment (60% vs. 15%, respectively). CTB-protocells showed specific uptake by motoneurons compared to muscle cells and demonstrated cargo release of a surrogate drug. Protocells showed a lack of cytotoxicity and unimpaired cellular proliferation. In isolated diaphragm muscle-phrenic nerve preparations, preferential axon terminal uptake of CTB-modified protocells was observed compared to uptake in surrounding muscle tissue. A larger proportion of axon terminals displayed uptake following treatment with CTB-protocells compared to unmodified protocells (40% vs. 6%, respectively). Current motoneuron targeting strategies lack the functionality to load and deliver multiple cargos. CTB-protocells capitalizes on the advantages of liposomes and mesoporous silica nanoparticles allowing a large loading capacity and cargo release. The ability of CTB-protocells to target motoneurons at the NMJ confers a great advantage over existing methods. CTB-protocells constitute a viable targeted motoneuron delivery system for drugs and genes facilitating various therapies for neuromuscular diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Tissue inhibitor of metalloproteinase-2(TIMP-2)-deficient mice display motor deficits.

PubMed

Jaworski, Diane M; Soloway, Paul; Caterina, John; Falls, William A

2006-01-01

The degradation of the extracellular matrix is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Matrix components of the basement membrane play critical roles in the development and maintenance of the neuromuscular junction (NMJ), yet almost nothing is known about the regulation of MMP and TIMP expression in either the pre- or postsynaptic compartments. Here, we demonstrate that TIMP-2 is expressed by both spinal motor neurons and skeletal muscle. To determine whether motor function is altered in the absence of TIMP-2, motor behavior was assessed using a battery of tests (e.g., RotaRod, balance beam, hindlimb extension, grip strength, loaded grid, and gait analysis). TIMP-2(-/-) mice fall off the RotaRod significantly faster than wild-type littermates. In addition, hindlimb extension is reduced and gait is both splayed and lengthened in TIMP-2(-/-) mice. Motor dysfunction is more pronounced during early postnatal development. A preliminary analysis revealed NMJ alterations in TIMP-2(-/-) mice. Juvenile TIMP-2(-/-) mice have increased nerve branching and acetylcholine receptor expression. Adult TIMP-2(-/-) endplates are enlarged and more complex. This suggests a role for TIMP-2 in NMJ sculpting during development. In contrast to the increased NMJ nerve branching, cerebellar Purkinje cells have decreased neurite outgrowth. Thus, the TIMP-2(-/-) motor phenotype is likely due to both peripheral and central defects. The tissue specificity of the nerve branching phenotype suggests the involvement of different MMPs and/or extracellular matrix molecules underlying the TIMP-2(-/-) motor phenotype.

GFPT1 deficiency in muscle leads to myasthenia and myopathy in mice.

PubMed

Issop, Yasmin; Hathazi, Denisa; Khan, Muzamil Majid; Rudolf, Rüdiger; Weis, Joachim; Spendiff, Sally; Slater, Clarke R; Roos, Andreas; Lochmüller, Hanns

2018-06-14

Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway which yields precursors required for protein and lipid glycosylation. Mutations in GFPT1 and other genes downstream of this pathway cause congenital myasthenic syndrome (CMS) characterised by fatigable muscle weakness due to impaired neurotransmission. The precise pathomechanisms at the neuromuscular junction (NMJ) due to a deficiency in GFPT1 is yet to be discovered. One of the challenges we face is the viability of Gfpt1 -/- knockout mice. In this study, we use Cre/LoxP technology to generate a muscle-specific GFPT1 knockout mouse model, Gfpt1tm1d/tm1d, characteristic of the human CMS phenotype. Our data suggests a critical role for muscle derived GFPT1 in the development of the NMJ, neurotransmission, skeletal muscle integrity, and highlights that a deficiency in skeletal muscle alone is sufficient to cause morphological postsynaptic NMJ changes that are accompanied by presynaptic alterations despite the conservation of neuronal GFPT1 expression. In addition to the conventional morphological NMJ changes and fatigable muscle weakness, Gfpt1tm1d/tm1d mice display a progressive myopathic phenotype with the presence of tubular aggregates in muscle, characteristic of the GFPT1-CMS phenotype. We further identify an upregulation of skeletal muscle proteins glypican-1, farnesyltransferase/geranylgeranyltransferase type-1 subunit alpha and Muscle-specific kinase which are known to be involved in the differentiation and maintenance of the NMJ. The Gfpt1tm1d/tm1d model allows for further investigation of pathophysiological consequences on genes and pathways downstream of GFPT1 likely to involve misglycosylation or hypoglycosylation of NMJs and muscle targets.

Endoscopic thymectomy: a neurologist’s perspective

PubMed Central

Melfi, Franca; Maestri, Michelangelo; De Rosa, Anna; Petsa, Afroditi; Lucchi, Marco; Mussi, Alfredo

2016-01-01

Myasthenia gravis (MG) is an autoimmune neuromuscular disease characterized by the presence of antibodies interacting at the neuromuscular junction (NMJ), resulting in loss of strength and severe exhaustibility of striated muscles. The abnormal production of these antibodies is triggered mainly in the thymus, and hence thymectomy in MG is considered a universally recommended treatment in order to improve the symptomatologic condition of this pathology. Currently, minimally invasive thymectomy using the Da Vinci robot system is certainly one of the most innovative techniques, performed in Pisa since 2001. This approach provides a valuable alternative to the traditional thymectomy through median sternotomy. The contribution of a neurologist is fundamental for preoperative patient selection and for the peri-operative clinical assistance in both approaches. We believe that in the robotic approach, the multidisciplinary collaboration between the neurologist, thoracic surgeon and anesthetist is important in reducing perioperative complications and ensuring a higher rate of complete remission or stable clinical improvement of MG. PMID:26904430

Dbo/Henji Modulates Synaptic dPAK to Gate Glutamate Receptor Abundance and Postsynaptic Response.

PubMed

Wang, Manyu; Chen, Pei-Yi; Wang, Chien-Hsiang; Lai, Tzu-Ting; Tsai, Pei-I; Cheng, Ying-Ju; Kao, Hsiu-Hua; Chien, Cheng-Ting

2016-10-01

In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. Here, we analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. We propose that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK.

Dbo/Henji Modulates Synaptic dPAK to Gate Glutamate Receptor Abundance and Postsynaptic Response

PubMed Central

Wang, Manyu; Chen, Pei-Yi; Wang, Chien-Hsiang; Lai, Tzu-Ting; Tsai, Pei-I; Cheng, Ying-Ju; Kao, Hsiu-Hua; Chien, Cheng-Ting

2016-01-01

In response to environmental and physiological changes, the synapse manifests plasticity while simultaneously maintains homeostasis. Here, we analyzed mutant synapses of henji, also known as dbo, at the Drosophila neuromuscular junction (NMJ). In henji mutants, NMJ growth is defective with appearance of satellite boutons. Transmission electron microscopy analysis indicates that the synaptic membrane region is expanded. The postsynaptic density (PSD) houses glutamate receptors GluRIIA and GluRIIB, which have distinct transmission properties. In henji mutants, GluRIIA abundance is upregulated but that of GluRIIB is not. Electrophysiological results also support a GluR compositional shift towards a higher IIA/IIB ratio at henji NMJs. Strikingly, dPAK, a positive regulator for GluRIIA synaptic localization, accumulates at the henji PSD. Reducing the dpak gene dosage suppresses satellite boutons and GluRIIA accumulation at henji NMJs. In addition, dPAK associated with Henji through the Kelch repeats which is the domain essential for Henji localization and function at postsynapses. We propose that Henji acts at postsynapses to restrict both presynaptic bouton growth and postsynaptic GluRIIA abundance by modulating dPAK. PMID:27736876

Optogenetics in the teaching laboratory: using channelrhodopsin-2 to study the neural basis of behavior and synaptic physiology in Drosophila

PubMed Central

Hornstein, Nicholas J.; Land, Bruce L.; Johnson, Bruce R.

2011-01-01

Here we incorporate recent advances in Drosophila neurogenetics and “optogenetics” into neuroscience laboratory exercises. We used the light-activated ion channel channelrhodopsin-2 (ChR2) and tissue-specific genetic expression techniques to study the neural basis of behavior in Drosophila larvae. We designed and implemented exercises using inexpensive, easy-to-use systems for delivering blue light pulses with fine temporal control. Students first examined the behavioral effects of activating glutamatergic neurons in Drosophila larvae and then recorded excitatory junctional potentials (EJPs) mediated by ChR2 activation at the larval neuromuscular junction (NMJ). Comparison of electrically and light-evoked EJPs demonstrates that the amplitudes and time courses of light-evoked EJPs are not significantly different from those generated by electrical nerve stimulation. These exercises introduce students to new genetic technology for remotely manipulating neural activity, and they simplify the process of recording EJPs at the Drosophila larval NMJ. Relatively little research work has been done using ChR2 in Drosophila, so students have opportunities to test novel hypotheses and make tangible contributions to the scientific record. Qualitative and quantitative assessment of student experiences suggest that these exercises help convey principles of synaptic transmission while also promoting integrative and inquiry-based studies of genetics, cellular physiology, and animal behavior. PMID:21386006

Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.

PubMed

Groffen, A J; Ruegg, M A; Dijkman, H; van de Velden, T J; Buskens, C A; van den Born, J; Assmann, K J; Monnens, L A; Veerkamp, J H; van den Heuvel, L P

1998-01-01

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)

C-terminal Agrin Fragment as a potential marker for sarcopenia caused by degeneration of the neuromuscular junction.

PubMed

Drey, M; Sieber, C C; Bauer, J M; Uter, W; Dahinden, P; Fariello, R G; Vrijbloed, J W

2013-01-01

Sarcopenia is considered to be an enormous burden for both the individuals affected and for society at large. A multifactorial aetiology of this geriatric syndrome has been discussed. Amongst other pathomechanisms, the degeneration of the neuromuscular junction (NMJ) may be of major relevance. The intact balance between the pro-synaptic agent agrin and the anti-synaptic agent neurotrypsin ensures a structurally and functionally intact NMJ. Excessive cleavage of the native motoneuron-derived agrin by neurotrypsin into a C-terminal Agrin Fragment (CAF) leads to functional disintegration at the NMJ and may consecutively cause sarcopenia. The present study evaluates the hypothesis that CAF serum concentration is a potential marker for the loss of appendicular lean mass in older adults. It also explores how CAF concentration is influenced by vitamin D supplementation and physical exercise. Serum was taken from 69 (47 female) prefrail community-dwelling older adults participating in a training intervention study to measure the CAF concentration using the Western blot technique. All participants were supplemented orally with vitamin D3 before the training intervention period commenced. Appendicular lean mass (aLM) was evaluated by dual energy X-ray absorptiometry. Multiple linear regression models were used to identify factors significantly associated with CAF concentration. Appendicular lean mass, age and sex were identified as significant explanatory factors for CAF concentration. Gait speed and hand grip strength were not associated with CAF concentration. Male participants showed a strong correlation (r=-0.524) between CAF serum concentration and aLM, whereas this was not the case (r=-0.219) in females. Vitamin D supplementation and physical exercise were significantly associated with a reduction in CAF concentration, especially in participants with initially high CAF concentrations. C-terminal Agrin Fragment could be a potential marker for identifying sarcopenia in a subgroup of affected individuals in the future. The decline of muscle mass seems to be a CAF-associated process in males, whereas the situation in females may be more complex and multifactorial. CAF concentration is reduced by vitamin D supplementation and physical exercise and therefore suggests a potentially positive effect on NMJs. Further prospective studies of sarcopenic patients in addition to muscle biopsy and electromyographical investigations are planned to verify the external validity of the CAF concept. Copyright © 2012 Elsevier Inc. All rights reserved.

Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn–plectin 1f complexes

PubMed Central

Mihailovska, Eva; Raith, Marianne; Valencia, Rocio G.; Fischer, Irmgard; Banchaabouchi, Mumna Al; Herbst, Ruth; Wiche, Gerhard

2014-01-01

Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion. PMID:25318670

Neuromuscular transmission and muscle fatigue changes by nanostructured oxygen.

PubMed

Ivannikov, Maxim V; Sugimori, Mutsuyuki; Llinás, Rodolfo R

2017-04-01

Oxygen (O 2 ) nanobubbles offer a new method for tissue oxygenation. The effects of O 2 nanobubbles on transmission at neuromuscular junctions (NMJs) and muscle function were explored in murine diaphragm. Electrophysiological parameters, NMJ ultrastructure, muscle force, and muscle fatigue were studied during superfusion with solutions with different oxygen levels or oxygen nanobubbles. High frequency nerve stimulation of muscles superfused with O 2 nanobubble solution slowed neurotransmission decline over those with either control or hyperoxic solution. O 2 nanobubble solution increased the amplitude of evoked end plate potentials and quantal content but did not affect spontaneous activity. Electron microscopy of stimulated O 2 nanobubble treated NMJs showed accumulation of large synaptic vesicles and endosome-like structures. O 2 nanobubble solution had no effects on isometric muscle force, but it significantly decreased fatigability and maximum force recovery time in nerve stimulated muscles. O 2 nanobubbles increase neurotransmission and reduce the probability of neurotransmission failure in muscle fatigue. Muscle Nerve 55: 555-563, 2017. © 2016 Wiley Periodicals, Inc.

A stem-cell based bioassay to critically assess the pathology of dysfunctional neuromuscular junctions.

PubMed

Chipman, Peter H; Zhang, Ying; Rafuse, Victor F

2014-01-01

Pluripotent stem cells can be directed to differentiate into motor neurons and assessed for functionality in vitro. An emerging application of this technique is to model genetically inherited diseases in differentiated motor neurons and to screen for new therapeutic targets. The neuromuscular junction (NMJ) is essential to the functionality of motor neurons and its dysfunction is a primary hallmark of motor neuron disease. However, mature NMJs that possess the functional and morphological characteristics of those formed in vivo have so far not been obtained in vitro. Here we describe the generation and analysis of mature NMJs formed between embryonic stem cell-derived motor neurons (ESCMNs) and primary myotubes. We compared the formation and maturation of NMJs generated by wild-type (NCAM+/+) ESCMNs to those generated by neural cell adhesion molecule null (NCAM-/-) ESCMNs in order to definitively test the sensitivity of this assay to identify synaptic pathology. We find that co-cultures using NCAM-/- ESCMNs replicate key in vivo NCAM-/- phenotypes and reveal that NCAM influences neuromuscular synaptogenesis by controlling the mode of synaptic vesicle endocytosis. Further, we could improve synapse formation and function in NCAM-/- co-cultures by chronic treatment with nifedipine, which blocks an immature synaptic vesicle recycling pathway. Together, our results demonstrate that this ESCMN/myofiber co-culture system is a highly sensitive bioassay for examining molecules postulated to regulate synaptic function and for screening therapeutics that will improve the function of compromised NMJs.

Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction

PubMed Central

Gray, Lindsay; Tsurudome, Kazuya; El-Mounzer, Wassim; Elazzouzi, Fatima; Baim, Christopher; Calderon, Mario R.; Kauwe, Grant

2018-01-01

Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons. PMID:29373576

Repetitive nerve stimulation and single-fiber electromyography in the evaluation of patients with suspected myasthenia gravis or Lambert-Eaton myasthenic syndrome: Review of recent literature.

PubMed

Chiou-Tan, Faye Y; Gilchrist, James M

2015-09-01

Our aim in this study was to provide an updated literature review of electrodiagnostic testing in myasthenia gravis and Lambert-Eaton myasthenic syndrome. A systematic review of the recent literature was performed using the following key words: myasthenia gravis (MG); Lambert-Eaton myasthenic syndrome (LEMS); electromyography (EMG); repetitive nerve stimulation (RNS); single-fiber electromyography (SFEMG); nerve conduction study; and normative values. Several articles supported testing of facial, bulbar, and respiratory muscles in the diagnosis of neuromuscular junction (NMJ) disorders, including muscle-specific kinase antibody (MuSK)-seropositive MG. Several articles supported use of concentric needle EMG as an alternative to SFEMG jitter in disorders of neuromuscular transmission. A limited number of articles addressed measurement of area (vs. amplitude) decrement in RNS and decreasing the threshold of post-exercise facilitation. Electrodiagnostic testing continues to be useful for diagnosis of MG and LEMS, although the quality of the evidence is not great. This literature review summarizes RNS and jitter measurement of facial and respiratory muscles and use of concentric needle EMG for SFEMG. © 2015 American Association of Neuromuscular and Electrodiagnostic Medicine.

Sepsis Strengthens Antagonistic Actions of Neostigmine on Rocuronium in a Rat Model of Cecal Ligation and Puncture

PubMed Central

Wu, Jin; Jin, Tian; Wang, Hong; Li, Shi-Tong

2016-01-01

Background: The antagonistic actions of anticholinesterase drugs on non-depolarizing muscle relaxants are theoretically related to the activity of acetylcholinesterase (AChE) in the neuromuscular junction (NMJ). However, till date the changes of AChE activity in the NMJ during sepsis have not been directly investigated. We aimed to investigate the effects of sepsis on the antagonistic actions of neostigmine on rocuronium (Roc) and the underlying changes of AChE activity in the NMJ in a rat model of cecal ligation and puncture (CLP). Methods: A total of 28 male adult Sprague-Dawley rats were randomized to undergo a sham surgery (the sham group, n = 12) or CLP (the septic group, n = 16). After 24 h, the time-response curves of the antagonistic actions of 0.1 or 0.5 μmol/L of neostigmine on Roc (10 μmol/L)-depressed diaphragm twitch tension were measured. Meanwhile, the activity of AChE in the NMJ was detected using a modified Karnovsky and Roots method. The mRNA levels of the primary transcript and the type T transcript of AChE (AChET) in the diaphragm were determined by real-time reverse transcription-polymerase chain reaction. Results: Four of 16 rats in the septic group died within 24 h. The time-response curves of both two concentrations of neostigmine in the septic group showed significant upward shifts from those in the sham group (P < 0.001 for 0.1 μmol/L; P = 0.009 for 0.5 μmol/L). Meanwhile, the average optical density of AChE in the NMJ in the septic group was significantly lower than that in the sham group (0.517 ± 0.045 vs. 1.047 ± 0.087, P < 0.001). The AChE and AChET mRNA expression levels in the septic group were significantly lower than those in the sham group (P = 0.002 for AChE; P = 0.001 for AChET). Conclusions: Sepsis strengthened the antagonistic actions of neostigmine on Roc-depressed twitch tension of the diaphragm by inhibiting the activity of AChE in the NMJ. The reduced content of AChE might be one of the possible causes of the decreased AChE activity in the NMJ. PMID:27270546

Immunopathogenesis in Myasthenia Gravis and Neuromyelitis Optica

PubMed Central

Wang, Zhen; Yan, Yaping

2017-01-01

Myasthenia gravis (MG) and neuromyelitis optica (NMO) are autoimmune channelopathies of the peripheral neuromuscular junction (NMJ) and central nervous system (CNS) that are mainly mediated by humoral immunity against the acetylcholine receptor (AChR) and aquaporin-4 (AQP4), respectively. The diseases share some common features, including genetic predispositions, environmental factors, the breakdown of tolerance, the collaboration of T cells and B cells, imbalances in T helper 1 (Th1)/Th2/Th17/regulatory T cells, aberrant cytokine and antibody secretion, and complement system activation. However, some aspects of the immune mechanisms are unique. Both targets (AChR and AQP4) are expressed in the periphery and CNS, but MG mainly affects the NMJ in the periphery outside of CNS, whereas NMO preferentially involves the CNS. Inflammatory cells, including B cells and macrophages, often infiltrate the thymus but not the target—muscle in MG, whereas the infiltration of inflammatory cells, mainly polymorphonuclear leukocytes and macrophages, in NMO, is always observed in the target organ—the spinal cord. A review of the common and discrepant characteristics of these two autoimmune channelopathies may expand our understanding of the pathogenic mechanism of both disorders and assist in the development of proper treatments in the future. PMID:29312313

Decreased microRNA levels lead to deleterious increases in neuronal M2 muscarinic receptors in Spinal Muscular Atrophy models

PubMed Central

O'Hern, Patrick J; do Carmo G. Gonçalves, Inês; Brecht, Johanna; López Soto, Eduardo Javier; Simon, Jonah; Chapkis, Natalie; Lipscombe, Diane; Kye, Min Jeong; Hart, Anne C

2017-01-01

Spinal Muscular Atrophy (SMA) is caused by diminished Survival of Motor Neuron (SMN) protein, leading to neuromuscular junction (NMJ) dysfunction and spinal motor neuron (MN) loss. Here, we report that reduced SMN function impacts the action of a pertinent microRNA and its mRNA target in MNs. Loss of the C. elegans SMN ortholog, SMN-1, causes NMJ defects. We found that increased levels of the C. elegans Gemin3 ortholog, MEL-46, ameliorates these defects. Increased MEL-46 levels also restored perturbed microRNA (miR-2) function in smn-1(lf) animals. We determined that miR-2 regulates expression of the C. elegans M2 muscarinic receptor (m2R) ortholog, GAR-2. GAR-2 loss ameliorated smn-1(lf) and mel-46(lf) synaptic defects. In an SMA mouse model, m2R levels were increased and pharmacological inhibition of m2R rescued MN process defects. Collectively, these results suggest decreased SMN leads to defective microRNA function via MEL-46 misregulation, followed by increased m2R expression, and neuronal dysfunction in SMA. DOI: http://dx.doi.org/10.7554/eLife.20752.001 PMID:28463115

The Intercostal NMJ Assay: a new alternative to the conventional LD50 assay for the determination of the therapeutic potency of botulinum toxin preparations.

PubMed

Huber, Alexander; France, Richard M; Riccalton-Banks, Lisa; McLaren, Jane; Cox, Helen; Quirk, Robin A; Shakesheff, Kevin M; Thompson, David; Panjwani, Naveed; Shipley, Sarah; Pickett, Andy

2008-05-01

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.

VAChT overexpression increases acetylcholine at the synaptic cleft and accelerates aging of neuromuscular junctions.

PubMed

Sugita, Satoshi; Fleming, Leland L; Wood, Caleb; Vaughan, Sydney K; Gomes, Matheus P S M; Camargo, Wallace; Naves, Ligia A; Prado, Vania F; Prado, Marco A M; Guatimosim, Cristina; Valdez, Gregorio

2016-01-01

Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age- and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS). Chat-ChR2-EYFP (VAChT Hyp ) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1 G93A ), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and α-motor neurons' somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles. We show that VAChT is elevated in the spinal cord and at NMJs of VAChT Hyp mice. We also show that the amplitude of MEPPs is significantly higher in VAChT Hyp muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChT Hyp mice compared to control mice. While the development of NMJs was not affected in VAChT Hyp mice, NMJs prematurely acquired age-related structural alterations in adult VAChT Hyp mice. These structural changes at NMJs were accompanied by motor deficits in VAChT Hyp mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChT Hyp mice. In the SOD1 G93A mouse model for ALS, increasing synaptic ACh accelerated degeneration of NMJs caused motor deficits and resulted in premature death specifically in male mice. The data presented in this manuscript demonstrate that increasing levels of ACh at the synaptic cleft promote degeneration of adult NMJs, contributing to age- and disease-related motor deficits. We thus propose that maintaining normal cholinergic signaling in muscles will slow degeneration of NMJs and attenuate loss of motor function caused by aging and neuromuscular diseases.

Na+ current in presynaptic terminals of the crayfish opener cannot initiate action potentials.

PubMed

Lin, Jen-Wei

2016-01-01

Action potential (AP) propagation in presynaptic axons of the crayfish opener neuromuscular junction (NMJ) was investigated by simultaneously recording from a terminal varicosity and a proximal branch. Although orthodromically conducting APs could be recorded in terminals with amplitudes up to 70 mV, depolarizing steps in terminals to -20 mV or higher failed to fire APs. Patch-clamp recordings did detect Na(+) current (INa) in most terminals. The INa exhibited a high threshold and fast activation rate. Local perfusion of Na(+)-free saline showed that terminal INa contributed to AP waveform by slightly accelerating the rising phase and increasing the peak amplitude. These findings suggest that terminal INa functions to "touch up" but not to generate APs. Copyright © 2016 the American Physiological Society.

Acamprosate rescues neuronal defects in the Drosophila model of Fragile X Syndrome.

PubMed

Hutson, Russell L; Thompson, Rachel L; Bantel, Andrew P; Tessier, Charles R

2018-02-15

Several off-label studies have shown that acamprosate can provide some clinical benefits in youth with Fragile X Syndrome (FXS), an autism spectrum disorder caused by loss of function of the highly conserved FMR1 gene. This study investigated the ability of acamprosate to rescue cellular, molecular and behavioral defects in the Drosophila model of FXS. A high (100μM) and low (10μM) dose of acamprosate was fed to Drosophila FXS (dfmr1 null) or genetic control (w 1118 ) larvae and then analyzed in multiple paradigms. A larval crawling assay was used to monitor aberrant FXS behavior, overgrowth of the neuromuscular junction (NMJ) was quantified to assess neuronal development, and quantitative RT-PCR was used to evaluate expression of deregulated cbp53E mRNA. Acamprosate treatment partially or completely rescued all of the FXS phenotypes analyzed, according to dose. High doses rescued cellular overgrowth and dysregulated cbp53E mRNA expression, but aberrant crawling behavior was not affected. Low doses of acamprosate, however, did not affect synapse number at the NMJ, but could rescue NMJ overgrowth, locomotor defects, and cbp53E mRNA expression. This dual nature of acamprosate suggests multiple molecular mechanisms may be involved in acamprosate function depending on the dosage used. Acamprosate may be a useful therapy for FXS and potentially other autism spectrum disorders. However, understanding the molecular mechanisms involved with different doses of this drug will likely be necessary to obtain optimal results. Copyright © 2018 Elsevier Inc. All rights reserved.

Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice

PubMed Central

Cole, R N; Ghazanfari, N; Ngo, S T; Gervásio, O L; Reddel, S W; Phillips, W D

2010-01-01

The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day−1i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51–73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR β-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering. PMID:20603331

Presynaptic membrane receptors in acetylcholine release modulation in the neuromuscular synapse.

PubMed

Tomàs, Josep; Santafé, Manel M; Garcia, Neus; Lanuza, Maria A; Tomàs, Marta; Besalduch, Núria; Obis, Teresa; Priego, Mercedes; Hurtado, Erica

2014-05-01

Over the past few years, we have studied, in the mammalian neuromuscular junction (NMJ), the local involvement in transmitter release of the presynaptic muscarinic ACh autoreceptors (mAChRs), purinergic adenosine autoreceptors (P1Rs), and trophic factor receptors (TFRs; for neurotrophins and trophic cytokines) during development and in the adult. At any given moment, the way in which a synapse works is largely the logical outcome of the confluence of these (and other) metabotropic signalling pathways on intracellular kinases, which phosphorylate protein targets and materialize adaptive changes. We propose an integrated interpretation of the complementary function of these receptors in the adult NMJ. The activity of a given receptor group can modulate a given combination of spontaneous, evoked, and activity-dependent release characteristics. For instance, P1Rs can conserve resources by limiting spontaneous quantal leak of ACh (an A1 R action) and protect synapse function, because stimulation with adenosine reduces the magnitude of depression during repetitive activity. The overall outcome of the mAChRs seems to contribute to upkeep of spontaneous quantal output of ACh, save synapse function by decreasing the extent of evoked release (mainly an M2 action), and reduce depression. We have also identified several links among P1Rs, mAChRs, and TFRs. We found a close dependence between mAChR and some TFRs and observed that the muscarinic group has to operate correctly if the tropomyosin-related kinase B receptor (trkB) is also to operate correctly, and vice versa. Likewise, the functional integrity of mAChRs depends on P1Rs operating normally. Copyright © 2014 Wiley Periodicals, Inc.

Myopic (HD-PTP, PTPN23) selectively regulates synaptic neuropeptide release.

PubMed

Bulgari, Dinara; Jha, Anupma; Deitcher, David L; Levitan, Edwin S

2018-02-13

Neurotransmission is mediated by synaptic exocytosis of neuropeptide-containing dense-core vesicles (DCVs) and small-molecule transmitter-containing small synaptic vesicles (SSVs). Exocytosis of both vesicle types depends on Ca 2+ and shared secretory proteins. Here, we show that increasing or decreasing expression of Myopic (mop, HD-PTP, PTPN23), a Bro1 domain-containing pseudophosphatase implicated in neuronal development and neuropeptide gene expression, increases synaptic neuropeptide stores at the Drosophila neuromuscular junction (NMJ). This occurs without altering DCV content or transport, but synaptic DCV number and age are increased. The effect on synaptic neuropeptide stores is accounted for by inhibition of activity-induced Ca 2+ -dependent neuropeptide release. cAMP-evoked Ca 2+ -independent synaptic neuropeptide release also requires optimal Myopic expression, showing that Myopic affects the DCV secretory machinery shared by cAMP and Ca 2+ pathways. Presynaptic Myopic is abundant at early endosomes, but interaction with the endosomal sorting complex required for transport III (ESCRT III) protein (CHMP4/Shrub) that mediates Myopic's effect on neuron pruning is not required for control of neuropeptide release. Remarkably, in contrast to the effect on DCVs, Myopic does not affect release from SSVs. Therefore, Myopic selectively regulates synaptic DCV exocytosis that mediates peptidergic transmission at the NMJ.

RNAi-Mediated Reverse Genetic Screen Identified Drosophila Chaperones Regulating Eye and Neuromuscular Junction Morphology.

PubMed

Raut, Sandeep; Mallik, Bhagaban; Parichha, Arpan; Amrutha, Valsakumar; Sahi, Chandan; Kumar, Vimlesh

2017-07-05

Accumulation of toxic proteins in neurons has been linked with the onset of neurodegenerative diseases, which in many cases are characterized by altered neuronal function and synapse loss. Molecular chaperones help protein folding and the resolubilization of unfolded proteins, thereby reducing the protein aggregation stress. While most of the chaperones are expressed in neurons, their functional relevance remains largely unknown. Here, using bioinformatics analysis, we identified 95 Drosophila chaperones and classified them into seven different classes. Ubiquitous actin5C -Gal4-mediated RNAi knockdown revealed that ∼50% of the chaperones are essential in Drosophila Knocking down these genes in eyes revealed that ∼30% of the essential chaperones are crucial for eye development. Using neuron-specific knockdown, immunocytochemistry, and robust behavioral assays, we identified a new set of chaperones that play critical roles in the regulation of Drosophila NMJ structural organization. Together, our data present the first classification and comprehensive analysis of Drosophila chaperones. Our screen identified a new set of chaperones that regulate eye and NMJ morphogenesis. The outcome of the screen reported here provides a useful resource for further elucidating the role of individual chaperones in Drosophila eye morphogenesis and synaptic development. Copyright © 2017 Raut et al.

GaAs laser therapy reestablishes the morphology of the NMJ and nAChRs after injury due to bupivacaine.

PubMed

Pissulin, Cristiane Neves Alessi; de Souza Castro, Paula Aiello Tomé; Codina, Flávio; Pinto, Carina Guidi; Vechetti-Junior, Ivan Jose; Matheus, Selma Maria Michelin

2017-02-01

Local anesthetics are used to relieve pre- and postoperative pain, acting on both sodium channels and nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction (NMJ). Bupivacaine acts as a non-competitive antagonist and has limitations, such as myotoxicity, neurotoxicity, and inflammation. Low-level laser therapy (LLLT) has anti-inflammatory, regenerative, and analgesic effects. The aim of the present study was to evaluate the effects of a gallium arsenide laser (GaAs) on the morphology of the NMJ and nAChRs after application of bupivacaine in the sternomastoid muscle. Thirty-two adult male Wistar rats received injections of bupivacaine 0.5% (Bupi: right antimere) and 0.9% sodium chloride (Cl: left antimere). Next, the animals were divided into a Control group (C) and a Laser group (LLLT). The laser group received LLLT (GaAs 904nm, 50mW, 4,8J) in both antimeres for five consecutive days. After seven days, the animals were euthanized and the surface portion of the sternomastoid muscle was removed, frozen, and subjected to morphological and morphometric analyses of the NMJs (nonspecific esterase reaction), confocal laser scanning, and an ultrastructural analysis. The nAChRs were quantified by Western blotting. In the chloride group, the morphology and morphometry of the NMJs remained stable. The maximum diameters of the NMJs were lower in the Bupi (15.048±1.985) and LLLT/Bupi subgroups (15.456±1.983) compared to the Cl (18.502±2.058) and LLLT/Cl subgroups (19.356±2.522) (p<0.05). Ultrastructurally, LLLT reduced myonecrosis observed after application of bupivacaine, with recovery in the junctional folds and active zone. There was an increase in the perimeter of the LLLT/Bupi subgroup (150.33) compared to the Bupi subgroup (74.69) (p<0.01) observed by confocal microscopy. There was also an increase in the relative planar area of the NMJ after LBI (8.75) compared to CBupi (4.80) (p<0.01). An analysis of the protein expression of nAChRα1 showed no major differences in the groups studied. There was an increase in protein expression of the ε subunit after application of LLLT (13.055) compared to Bupi (0.251) (p<0.01). Taken together, the present experiments indicate that there was a positive association of the α and γ subunits (p<0.05). These results demonstrate that LLLT at the dose used in this study reduced structural alterations in the NMJ and molecular changes in nAChRs triggered by bupivacaine, providing important data supporting the use of LLLT in therapeutic protocols for injuries triggered by local anesthetics. Copyright © 2016 Elsevier B.V. All rights reserved.

The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.

PubMed

Voigt, Tilman; Neve, Anuja; Schümperli, Daniel

2014-06-01

As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis. © 2013 British Neuropathological Society.

A modified acetylcholine receptor δ-subunit enables a null mutant to survive beyond sexual maturation

PubMed Central

Epley, Kimberly E.; Urban, Jason M.; Ikenaga, Takanori; Ono, Fumihito

2008-01-01

The contraction of skeletal muscle is dependent upon synaptic transmission through acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). The lack of an AChR subunit causes a fetal akinesia in humans, leading to death in the first trimester and characteristic features of Fetal Akinesia Deformation Sequences (FADS). A corresponding null mutation of the δ-subunit in zebrafish (sofa potato; sop−/−) leads to the death of embryos around 5 days post-fertilization (dpf). In sop−/− mutants, we expressed modified δ-subunits, with one (δ1YFP) or two yellow fluorescent protein (δ2YFP) molecules fused at the intracellular loop, under the control of an α-actin promoter. AChRs containing these fusion proteins are fluorescent, assemble on the plasma membrane, make clusters under motor neuron endings, and generate synaptic current. We screened for germ-line transmission of the transgene and established a line of sop−/− fish stably expressing the δ2YFP. These δ2YFP/sop−/− embryos can mount escape behavior close to that of their wild type siblings. Synaptic currents in these embryos had a smaller amplitude, slower rise time, and slower decay when compared to wild type fish. Remarkably, these embryos grow to adulthood and display complex behaviors such as feeding and breeding. To the best of our knowledge, this is the first case of a mutant animal corresponding to first trimester lethality in human that has been rescued by a transgene and survived to adulthood. In the rescued fish, a foreign promoter drove the transgene expression and the NMJ had altered synaptic strength. The survival of the transgenic animal delineates requirements for gene therapies of NMJ. PMID:19052214

σ2-Adaptin Facilitates Basal Synaptic Transmission and Is Required for Regenerating Endo-Exo Cycling Pool Under High-Frequency Nerve Stimulation in Drosophila.

PubMed

Choudhury, Saumitra Dey; Mushtaq, Zeeshan; Reddy-Alla, Suneel; Balakrishnan, Sruthi S; Thakur, Rajan S; Krishnan, Kozhalmannom S; Raghu, Padinjat; Ramaswami, Mani; Kumar, Vimlesh

2016-05-01

The functional requirement of adapter protein 2 (AP2) complex in synaptic membrane retrieval by clathrin-mediated endocytosis is not fully understood. Here we isolated and functionally characterized a mutation that dramatically altered synaptic development. Based on the aberrant neuromuscular junction (NMJ) synapse, we named this mutation angur (a Hindi word meaning "grapes"). Loss-of-function alleles of angur show more than twofold overgrowth in bouton numbers and a dramatic decrease in bouton size. We mapped the angur mutation to σ2-adaptin, the smallest subunit of the AP2 complex. Reducing the neuronal level of any of the subunits of the AP2 complex or disrupting AP2 complex assembly in neurons phenocopied the σ2-adaptin mutation. Genetic perturbation of σ2-adaptin in neurons leads to a reversible temperature-sensitive paralysis at 38°. Electrophysiological analysis of the mutants revealed reduced evoked junction potentials and quantal content. Interestingly, high-frequency nerve stimulation caused prolonged synaptic fatigue at the NMJs. The synaptic levels of subunits of the AP2 complex and clathrin, but not other endocytic proteins, were reduced in the mutants. Moreover, bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ) signaling was altered in these mutants and was restored by normalizing σ2-adaptin in neurons. Thus, our data suggest that (1) while σ2-adaptin facilitates synaptic vesicle (SV) recycling for basal synaptic transmission, its activity is also required for regenerating SVs during high-frequency nerve stimulation, and (2) σ2-adaptin regulates NMJ morphology by attenuating TGFβ signaling. Copyright © 2016 by the Genetics Society of America.

Exogenous ciliary neurotrophic factor (CNTF) reduces synaptic depression during repetitive stimulation.

PubMed

Garcia, Neus; Santafé, Manel M; Tomàs, Marta; Priego, Mercedes; Obis, Teresa; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

2012-09-01

It has been shown that ciliary neurotrophic factor (CNTF) has trophic and maintenance effects on several types of peripheral and central neurons, glia, and cells outside the nervous system. Both CNTF and its receptor, CNTF-Rα, are expressed in the muscle. We use confocal immunocytochemistry to show that the trophic cytokine and its receptor are present in the pre- and post-synaptic sites of the neuromuscular junctions (NMJs). Applied CNTF (7.5-200 ng/ml, 60 min-3 h) does not acutely affect spontaneous potentials (size or frequency) or quantal content of the evoked acetylcholine release from post-natal (in weak or strong axonal inputs on dually innervated end plates or in the most mature singly innervated synapses at P6) or adult (P30) NMJ of Levator auris longus muscle of the mice. However, CNTF reduces roughly 50% the depression produced by repetitive stimulation (40 Hz, 2 min) on the adult NMJs. Our findings indicate that, unlike neurotrophins, exogenous CNTF does not acutely modulate transmitter release locally at the mammalian neuromuscular synapse but can protect mature end plates from activity-induced synaptic depression. © 2012 Peripheral Nerve Society.

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

NASA Astrophysics Data System (ADS)

Ehmann, Nadine; van de Linde, Sebastian; Alon, Amit; Ljaschenko, Dmitrij; Keung, Xi Zhen; Holm, Thorge; Rings, Annika; Diantonio, Aaron; Hallermann, Stefan; Ashery, Uri; Heckmann, Manfred; Sauer, Markus; Kittel, Robert J.

2014-08-01

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

A perisynaptic ménage à trois between Dlg, DLin-7, and Metro controls proper organization of Drosophila synaptic junctions.

PubMed

Bachmann, André; Kobler, Oliver; Kittel, Robert J; Wichmann, Carolin; Sierralta, Jimena; Sigrist, Stephan J; Gundelfinger, Eckart D; Knust, Elisabeth; Thomas, Ulrich

2010-04-28

Structural plasticity of synaptic junctions is a prerequisite to achieve and modulate connectivity within nervous systems, e.g., during learning and memory formation. It demands adequate backup systems that allow remodeling while retaining sufficient stability to prevent unwanted synaptic disintegration. The strength of submembranous scaffold complexes, which are fundamental to the architecture of synaptic junctions, likely constitutes a crucial determinant of synaptic stability. Postsynaptic density protein-95 (PSD-95)/ Discs-large (Dlg)-like membrane-associated guanylate kinases (DLG-MAGUKs) are principal scaffold proteins at both vertebrate and invertebrate synapses. At Drosophila larval glutamatergic neuromuscular junctions (NMJs) DlgA and DlgS97 exert pleiotropic functions, probably reflecting a few known and a number of yet-unknown binding partners. In this study we have identified Metro, a novel p55/MPP-like Drosophila MAGUK as a major binding partner of perisynaptic DlgS97 at larval NMJs. Based on homotypic LIN-2,-7 (L27) domain interactions, Metro stabilizes junctional DlgS97 in a complex with the highly conserved adaptor protein DLin-7. In a remarkably interdependent manner, Metro and DLin-7 act downstream of DlgS97 to control NMJ expansion and proper establishment of synaptic boutons. Using quantitative 3D-imaging we further demonstrate that the complex controls the size of postsynaptic glutamate receptor fields. Our findings accentuate the importance of perisynaptic scaffold complexes for synaptic stabilization and organization.

HERC1 Ubiquitin Ligase Is Required for Normal Axonal Myelination in the Peripheral Nervous System.

PubMed

Bachiller, Sara; Roca-Ceballos, María Angustias; García-Domínguez, Irene; Pérez-Villegas, Eva María; Martos-Carmona, David; Pérez-Castro, Miguel Ángel; Real, Luis Miguel; Rosa, José Luis; Tabares, Lucía; Venero, José Luis; Armengol, José Ángel; Carrión, Ángel Manuel; Ruiz, Rocío

2018-03-30

A missense mutation in HERC1 provokes loss of cerebellar Purkinje cells, tremor, and unstable gait in tambaleante (tbl) mice. Recently, we have shown that before cerebellar degeneration takes place, the tbl mouse suffers from a reduction in the number of vesicles available for release at the neuromuscular junction (NMJ). The aim of the present work was to study to which extent the alteration in HERC1 may affect other cells in the nervous system and how this may influence the motor dysfunction observed in these mice. The functional analysis showed a consistent delay in the propagation of the action potential in mutant mice in comparison with control littermates. Morphological analyses of glial cells in motor axons revealed signs of compact myelin damage as tomacula and local hypermyelination foci. Moreover, we observed an alteration in non-myelinated terminal Schwann cells at the level of the NMJ. Additionally, we found a significant increment of phosphorylated Akt-2 in the sciatic nerve. Based on these findings, we propose a molecular model that could explain how mutated HERC1 in tbl mice affects the myelination process in the peripheral nervous system. Finally, since the myelin abnormalities found in tbl mice are histological hallmarks of neuropathic periphery diseases, tbl mutant mice could be considered as a new mouse model for this type of diseases.

Active zone density is conserved during synaptic growth but impaired in aged mice

PubMed Central

Chen, Jie; Mizushige, Takafumi; Nishimune, Hiroshi

2013-01-01

Presynaptic active zones are essential structures for synaptic vesicle release, but the developmental regulation of their number and maintenance during aging at mammalian neuromuscular junctions (NMJs) remains unknown. Here, we analyzed the distribution of active zones in developing, mature, and aged mouse NMJs by immunohistochemical detection of the active zone-specific protein Bassoon. Bassoon is a cytosolic scaffolding protein essential for the active zone assembly in ribbon synapses and some brain synapses. Bassoon staining showed a punctate pattern in nerve terminals and axons at the nascent NMJ on embryonic days 16.5–18.5. Three-dimensional reconstruction of NMJs revealed that the majority of Bassoon puncta within an NMJ were attached to the presynaptic membrane from postnatal day 0 to adulthood, and colocalized with another active zone protein Piccolo. During postnatal development, the number of Bassoon puncta increased as the size of the synapses increased. Importantly, the density of Bassoon puncta remained relatively constant from postnatal day 0 to 54 at 2.3 puncta/μm2, while the synapse size increased 3.3-fold. However, Bassoon puncta density and signal intensity were significantly attenuated at the NMJs of 27-month-old aged mice. These results suggest that synapses maintain the density of synaptic vesicle release sites while the synapse size changes, but this density becomes impaired during aging. PMID:21935939

Mitochondrial ROS cause motor deficits induced by synaptic inactivity: Implications for synapse pruning.

PubMed

Sidlauskaite, Eva; Gibson, Jack W; Megson, Ian L; Whitfield, Philip D; Tovmasyan, Artak; Batinic-Haberle, Ines; Murphy, Michael P; Moult, Peter R; Cobley, James N

2018-06-01

Developmental synapse pruning refines burgeoning connectomes. The basic mechanisms of mitochondrial reactive oxygen species (ROS) production suggest they select inactive synapses for pruning: whether they do so is unknown. To begin to unravel whether mitochondrial ROS regulate pruning, we made the local consequences of neuromuscular junction (NMJ) pruning detectable as motor deficits by using disparate exogenous and endogenous models to induce synaptic inactivity en masse in developing Xenopus laevis tadpoles. We resolved whether: (1) synaptic inactivity increases mitochondrial ROS; and (2) chemically heterogeneous antioxidants rescue synaptic inactivity induced motor deficits. Regardless of whether it was achieved with muscle (α-bungarotoxin), nerve (α-latrotoxin) targeted neurotoxins or an endogenous pruning cue (SPARC), synaptic inactivity increased mitochondrial ROS in vivo. The manganese porphyrins MnTE-2-PyP 5+ and/or MnTnBuOE-2-PyP 5+ blocked mitochondrial ROS to significantly reduce neurotoxin and endogenous pruning cue induced motor deficits. Selectively inducing mitochondrial ROS-using mitochondria-targeted Paraquat (MitoPQ)-recapitulated synaptic inactivity induced motor deficits; which were significantly reduced by blocking mitochondrial ROS with MnTnBuOE-2-PyP 5+ . We unveil mitochondrial ROS as synaptic activity sentinels that regulate the phenotypical consequences of forced synaptic inactivity at the NMJ. Our novel results are relevant to pruning because synaptic inactivity is one of its defining features. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Identification of the Neuromuscular Junction Transcriptome of Extraocular Muscle by Laser Capture Microdissection

PubMed Central

Ketterer, Caroline; Zeiger, Ulrike; Budak, Murat T.; Rubinstein, Neal A.; Khurana, Tejvir S.

2010-01-01

Purpose. To examine and characterize the profile of genes expressed at the synapses or neuromuscular junctions (NMJs) of extraocular muscles (EOMs) compared with those expressed at the tibialis anterior (TA). Methods. Adult rat eyeballs with rectus EOMs attached and TAs were dissected, snap frozen, serially sectioned, and stained for acetylcholinesterase (AChE) to identify the NMJs. Approximately 6000 NMJs for rectus EOM (EOMsyn), 6000 NMJs for TA (TAsyn), equal amounts of NMJ-free fiber regions (EOMfib, TAfib), and underlying myonuclei and RNAs were captured by laser capture microdissection (LCM). RNA was processed for microarray-based expression profiling. Expression profiles and interaction lists were generated for genes differentially expressed at synaptic and nonsynaptic regions of EOM (EOMsyn versus EOMfib) and TA (TAsyn versus TAfib). Profiles were validated by using real-time quantitative polymerase chain reaction (qPCR). Results. The regional transcriptomes associated with NMJs of EOMs and TAs were identified. Two hundred seventy-five genes were preferentially expressed in EOMsyn (compared with EOMfib), 230 in TAsyn (compared with TAfib), and 288 additional transcripts expressed in both synapses. Identified genes included novel genes as well as well-known, evolutionarily conserved synaptic markers (e.g., nicotinic acetylcholine receptor (AChR) alpha (Chrna) and epsilon (Chrne) subunits and nestin (Nes). Conclusions. Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. The definition of the synaptic transcriptome provides insight into the mechanism of formation and functioning of the unique synapses of EOM and their differential involvement in diseases noted in the EOM allotype. PMID:20393109

Immunohistologic analysis of spontaneous recurrent laryngeal nerve reinnervation in a rat model.

PubMed

Rosko, Andrew J; Kupfer, Robbi A; Oh, Sang S; Haring, Catherine T; Feldman, Eva L; Hogikyan, Norman D

2018-03-01

After recurrent laryngeal nerve injury (RLN), spontaneous reinnervation of the larynx occurs with input from multiple sources. The purpose of this study was to determine the timing and efficiency of reinnervation across a resected RLN segment in a rat model of RLN injury. Animal study. Twelve male 60-day-old Sprague Dawley rats underwent resection of a 5-mm segment of the right RLN. Rats were sacrificed at 1, 2, 4, and 12 weeks after nerve injury to harvest the larynx and trachea for immunohistologic analysis. The distal RLN segment was stained with neurofilament, and axons were counted and compared to the nonoperated side. Thyroarytenoid (TA) muscles were stained with alpha-bungarotoxin, synaptophysin, and neurofilament to identify intact neuromuscular junctions (NMJ). The number of intact NMJs from the denervated side was compared to the nonoperated side. Nerve fibers regenerated across the resected RLN gap into the distal recurrent laryngeal nerve to innervate the TA muscle. The number of nerve fibers in the distal nerve segment increased over time and reached the normal number by 12 weeks postdenervation. Axons formed intact neuromuscular junctions in the TA, with 48.8% ± 16.7% of the normal number of intact NMJs at 4 weeks and 88.3% ± 30.1% of the normal number by 12 weeks. Following resection of an RLN segment in a rat model, nerve fibers spontaneously regenerate through the distal segment of the transected nerve and form intact NMJs in order to reinnervate the TA muscle. NA. Laryngoscope, 128:E117-E122, 2018. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

Adenosine A₁ and A₂A receptor-mediated modulation of acetylcholine release in the mice neuromuscular junction.

PubMed

Garcia, Neus; Priego, Mercedes; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Besalduch, Nuria; Lanuza, M Angel; Tomàs, Josep

2013-07-01

Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 μm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Age-related differences in synaptic plasticity following muscle unloading.

PubMed

Deschenes, Michael R; Wilson, Meredith H

2003-12-01

The objective of the present investigation was to determine the effects of muscle unloading-a form of subtotal disuse- on the morphology of the neuromuscular junction (NMJ) in younger and aged animals. Sixteen aged (22 months) and 16 young adult (8 months) male Fischer 344 rats were assigned to control and hindlimb suspension (HS) conditions (n=8/group). At the conclusion of the 4 week experimental period, soleus muscles were collected, and immunofluorescent procedures were used to visualize acetylcholine (ACh) vesicles and receptors, nerve terminal branching, as well as NCAM and NT-4 expression. Quantitative analyses revealed that aged controls displayed significant (p<0.05) reductions in area and perimeter length of ACh vesicle and receptor regions, without affecting nerve terminal branch number or length. In contrast to younger NMJs, which were resilient to the effects of unloading, NMJs of aged HS rats demonstrated significant expansion of ACh vesicle and receptor dimensions compared to aged controls. Qualitative analyses of NCAM staining indicated that aging alone somewhat increased this molecule's expression (aged controls>young controls). Among the four groups, however, the greatest amount of NCAM content was detected among aged HS muscles, matching the degree of synaptic plasticity exhibited in those muscles. Unlike NCAM, the expression of NT-4 did not appear to differ among the treatment groups. These data suggest that although young adult muscle maintains normal NMJ structure during prolonged exposure to unloading, aged NMJs experience significant adaptation to that stimulus. Copyright 2003 Wiley Periodicals, Inc. J Neurobiol 57: 246-256, 2003

Viral delivery of C9orf72 hexanucleotide repeat expansions in mice leads to repeat-length-dependent neuropathology and behavioural deficits

PubMed Central

Herranz-Martin, Saul; Lewis, Katherine; Mulcahy, Padraig; Higginbottom, Adrian; Walker, Callum; Valenzuela, Isabel Martinez-Pena y; Coldicott, Ian; Shaw, Pamela J.

2017-01-01

ABSTRACT Intronic GGGGCC repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two major pathologies stemming from the hexanucleotide RNA expansions (HREs) have been identified in postmortem tissue: intracellular RNA foci and repeat-associated non-ATG dependent (RAN) dipeptides, although it is unclear how these and other hallmarks of disease contribute to the pathophysiology of neuronal injury. Here, we describe two novel lines of mice that overexpress either 10 pure or 102 interrupted GGGGCC repeats mediated by adeno-associated virus (AAV) and recapitulate the relevant human pathology and disease-related behavioural phenotypes. Similar levels of intracellular RNA foci developed in both lines of mice, but only mice expressing 102 repeats generated C9orf72 RAN pathology, neuromuscular junction (NMJ) abnormalities, dispersal of the hippocampal CA1, enhanced apoptosis, and deficits in gait and cognition. Neither line of mice, however, showed extensive TAR DNA-binding protein 43 (TDP-43) pathology or neurodegeneration. Our data suggest that RNA foci pathology is not a good predictor of C9orf72 RAN dipeptide formation, and that RAN dipeptides and NMJ dysfunction are drivers of C9orf72 disease pathogenesis. These AAV-mediated models of C9orf72-associated ALS/FTD will be useful tools for studying disease pathophysiology and developing new therapeutic approaches. PMID:28550099

Analysis of the fibroblast growth factor system reveals alterations in a mouse model of spinal muscular atrophy.

PubMed

Hensel, Niko; Ratzka, Andreas; Brinkmann, Hella; Klimaschewski, Lars; Grothe, Claudia; Claus, Peter

2012-01-01

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis.

Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy.

PubMed

Martinez, Tara L; Kong, Lingling; Wang, Xueyong; Osborne, Melissa A; Crowder, Melissa E; Van Meerbeke, James P; Xu, Xixi; Davis, Crystal; Wooley, Joe; Goldhamer, David J; Lutz, Cathleen M; Rich, Mark M; Sumner, Charlotte J

2012-06-20

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Immunization with Recombinantly Expressed LRP4 Induces Experimental Autoimmune Myasthenia Gravis in C57BL/6 Mice.

PubMed

Ulusoy, Canan; Çavuş, Filiz; Yılmaz, Vuslat; Tüzün, Erdem

2017-07-01

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), characterized with muscle weakness. While MG develops due to acetylcholine receptor (AChR) antibodies in most patients, antibodies to muscle-specific receptor tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4) may also be identified. Experimental autoimmune myasthenia gravis (EAMG) has been previously induced by both LRP4 immunization and passive transfer of LRP4 antibodies. Our aim was to confirm previous results and to test the pathogenic effects of LRP4 immunization in a commonly used mouse strain C57BL/6 (B6) using a recombinantly expressed human LRP4 protein. B6 mice were immunized with human LRP4 in CFA, Torpedo Californica AChR in CFA or only CFA. Clinical and pathogenic aspects of EAMG were compared among groups. LRP4- and AChR-immunized mice showed comparable EAMG clinical severity. LRP4-immunized mice displayed serum antibodies to LRP4 and NMJ IgG and complement factor C3 deposits. IgG2 was the dominant anti-LRP4 isotype. Cultured lymph node cells of LRP4- and AChR-immunized mice gave identical pro-inflammatory cytokine (IL-6, IFN-γ and IL-17) responses to LRP4 and AChR stimulation, respectively. Our results confirm the EAMG-inducing action of LRP4 immunization and identify B6 as a LRP4-EAMG-susceptible mouse strain. Demonstration of complement fixing anti-LRP4 antibodies in sera and complement/IgG deposits at the NMJ of LRP4-immunized mice indicates complement activation as a putative pathogenic mechanism. We have thus developed a practical LRP4-induced EAMG model using a non-conformational protein and a widely available mouse strain for future investigation of LRP4-related MG.

Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3β (GSK3β)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends.

PubMed

Basu, Sreya; Sladecek, Stefan; Pemble, Hayley; Wittmann, Torsten; Slotman, Johan A; van Cappellen, Wiggert; Brenner, Hans-Rudolf; Galjart, Niels

2014-10-31

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Poor functional recovery and muscle polyinnervation after facial nerve injury in fibroblast growth factor-2-/- mice can be improved by manual stimulation of denervated vibrissal muscles.

PubMed

Seitz, M; Grosheva, M; Skouras, E; Angelova, S K; Ankerne, J; Jungnickel, J; Grothe, C; Klimaschewski, L; Hübbers, C U; Dunlop, S A; Angelov, D N

2011-05-19

Functional recovery following facial nerve injury is poor. Adjacent neuromuscular junctions (NMJs) are "bridged" by terminal Schwann cells and numerous regenerating axonal sprouts. We have recently shown that manual stimulation (MS) restores whisking function and reduces polyinnervation of NMJs. Furthermore, MS requires both insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF). Here, we investigated whether fibroblast growth factor-2 (FGF-2) was also required for the beneficial effects of MS. Following transection and suture of the facial nerve (facial-facial anastomisis, FFA) in homozygous mice lacking FGF-2 (FGF-2(-/-)), vibrissal motor performance and the percentage of poly-innervated NMJ were quantified. In intact FGF-2(-/-) mice and their wildtype (WT) counterparts, there were no differences in amplitude of vibrissal whisking (about 50°) or in the percentage of polyinnervated NMJ (0%). After 2 months FFA and handling alone (i.e. no MS), the amplitude of vibrissal whisking in WT-mice decreased to 22±3°. In the FGF-2(-/-) mice, the amplitude was reduced further to 15±4°, that is, function was significantly poorer. Functional deficits were mirrored by increased polyinnervation of NMJ in WT mice (40.33±2.16%) with polyinnervation being increased further in FGF-2(-/-) mice (50.33±4.33%). However, regardless of the genotype, MS increased vibrissal whisking amplitude (WT: 33.9°±7.7; FGF-2(-/-): 33.4°±8.1) and concomitantly reduced polyinnervation (WT: 33.9%±7.7; FGF-2(-/-): 33.4%±8.1) to a similar extent. We conclude that, whereas lack of FGF-2 leads to poor functional recovery and target reinnervation, MS can nevertheless confer some functional benefit in its absence. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Acute Fasting Regulates Retrograde Synaptic Enhancement through a 4E-BP-Dependent Mechanism.

PubMed

Kauwe, Grant; Tsurudome, Kazuya; Penney, Jay; Mori, Megumi; Gray, Lindsay; Calderon, Mario R; Elazouzzi, Fatima; Chicoine, Nicole; Sonenberg, Nahum; Haghighi, A Pejmun

2016-12-21

While beneficial effects of fasting on organismal function and health are well appreciated, we know little about the molecular details of how fasting influences synaptic function and plasticity. Our genetic and electrophysiological experiments demonstrate that acute fasting blocks retrograde synaptic enhancement that is normally triggered as a result of reduction in postsynaptic receptor function at the Drosophila larval neuromuscular junction (NMJ). This negative regulation critically depends on transcriptional enhancement of eukaryotic initiation factor 4E binding protein (4E-BP) under the control of the transcription factor Forkhead box O (Foxo). Furthermore, our findings indicate that postsynaptic 4E-BP exerts a constitutive negative input, which is counteracted by a positive regulatory input from the Target of Rapamycin (TOR). This combinatorial retrograde signaling plays a key role in regulating synaptic strength. Our results provide a mechanistic insight into how cellular stress and nutritional scarcity could acutely influence synaptic homeostasis and functional stability in neural circuits. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiple roles of the Rho GEF ephexin1 in synapse remodeling

PubMed Central

Shi, Lei; Fu, Amy KY

2010-01-01

Synapse remodeling, which involves changes in the synaptic structure and their molecular composition, is required for the maturation and refinement of neural circuits. Although synapse remodeling is known to be tightly dependent on the assembly of local actin cytoskeleton, how actin directs the structural changes of synapse and targeting of synaptic proteins are not fully understood. Recently, we identified ephexin1, a Rho guanine nucleotide exchange factor (GEF) that regulates actin dynamics, to play an essential role in the maturation and functioning of the mammalian neuromuscular junction (NMJ). We showed that ephexin1 regulates the synaptic organization of the neurotransmitter receptor acetylcholine receptor (AChR) clusters through RhoA-dependent actin reorganization. Interestingly, ephexin1 has been implicated in the regulation of postsynaptic structure as well as the presynaptic vesicle release at various types of synapses. Our findings thus establish a novel function of ephexin1 in synapse remodeling through regulating the synaptic targeting of neurotransmitter receptors, revealing a versatile role of ephexin1 at synapses. PMID:21331259

Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes.

PubMed

Hoopmann, Peer; Rizzoli, Silvio O; Betz, William J

2012-01-01

The synaptic vesicle is the essential organelle of the synapse. Many approaches for studying synaptic vesicle recycling have been devised, one of which, the styryl (FM) dye, is well suited for this purpose. FM dyes reversibly stain, but do not permeate, membranes; hence they can specifically label membrane-bound organelles. Their quantum yield is drastically higher when bound to membranes than when in aqueous solution. This protocol describes the imaging of synaptic vesicle recycling by staining and destaining vesicles with FM dyes. Nerve terminals are stimulated (electrically or by depolarization with high K(+)) in the presence of dye, their vesicles are then allowed to recycle, and finally dye is washed from the chamber. In neuromuscular junction (NMJ) preparations, movements of the muscle must be inhibited if imaging during stimulation is desired (e.g., by application of curare, a potent acetylcholine receptor inhibitor). The main characteristics of FM dyes are also reviewed here, as are recent FM dye monitoring techniques that have been used to investigate the kinetics of synaptic vesicle fusion.

Role of BMP receptor traffic in synaptic growth defects in an ALS model.

PubMed

Deshpande, Mugdha; Feiger, Zachary; Shilton, Amanda K; Luo, Christina C; Silverman, Ethan; Rodal, Avital A

2016-10-01

TAR DNA-binding protein 43 (TDP-43) is genetically and functionally linked to amyotrophic lateral sclerosis (ALS) and regulates transcription, splicing, and transport of thousands of RNA targets that function in diverse cellular pathways. In ALS, pathologically altered TDP-43 is believed to lead to disease by toxic gain-of-function effects on RNA metabolism, as well as by sequestering endogenous TDP-43 and causing its loss of function. However, it is unclear which of the numerous cellular processes disrupted downstream of TDP-43 dysfunction lead to neurodegeneration. Here we found that both loss and gain of function of TDP-43 in Drosophila cause a reduction of synaptic growth-promoting bone morphogenic protein (BMP) signaling at the neuromuscular junction (NMJ). Further, we observed a shift of BMP receptors from early to recycling endosomes and increased mobility of BMP receptor-containing compartments at the NMJ. Inhibition of the recycling endosome GTPase Rab11 partially rescued TDP-43-induced defects in BMP receptor dynamics and distribution and suppressed BMP signaling, synaptic growth, and larval crawling defects. Our results indicate that defects in receptor traffic lead to neuronal dysfunction downstream of TDP-43 misregulation and that rerouting receptor traffic may be a viable strategy for rescuing neurological impairment. © 2016 Deshpande, Feiger, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Active zone protein Bassoon co-localizes with presynaptic calcium channel, modifies channel function, and recovers from aging related loss by exercise.

PubMed

Nishimune, Hiroshi; Numata, Tomohiro; Chen, Jie; Aoki, Yudai; Wang, Yonghong; Starr, Miranda P; Mori, Yasuo; Stanford, John A

2012-01-01

The P/Q-type voltage-dependent calcium channels (VDCCs) are essential for synaptic transmission at adult mammalian neuromuscular junctions (NMJs); however, the subsynaptic location of VDCCs relative to active zones in rodent NMJs, and the functional modification of VDCCs by the interaction with active zone protein Bassoon remain unknown. Here, we show that P/Q-type VDCCs distribute in a punctate pattern within the NMJ presynaptic terminals and align in three dimensions with Bassoon. This distribution pattern of P/Q-type VDCCs and Bassoon in NMJs is consistent with our previous study demonstrating the binding of VDCCs and Bassoon. In addition, we now show that the interaction between P/Q-type VDCCs and Bassoon significantly suppressed the inactivation property of P/Q-type VDCCs, suggesting that the Ca(2+) influx may be augmented by Bassoon for efficient synaptic transmission at NMJs. However, presynaptic Bassoon level was significantly attenuated in aged rat NMJs, which suggests an attenuation of VDCC function due to a lack of this interaction between VDCC and Bassoon. Importantly, the decreased Bassoon level in aged NMJs was ameliorated by isometric strength training of muscles for two months. The training increased Bassoon immunoreactivity in NMJs without affecting synapse size. These results demonstrated that the P/Q-type VDCCs preferentially accumulate at NMJ active zones and play essential role in synaptic transmission in conjunction with the active zone protein Bassoon. This molecular mechanism becomes impaired by aging, which suggests altered synaptic function in aged NMJs. However, Bassoon level in aged NMJs can be improved by muscle exercise.

cGMP-Dependent Protein Kinase Inhibition Extends the Upper Temperature Limit of Stimulus-Evoked Calcium Responses in Motoneuronal Boutons of Drosophila melanogaster Larvae.

PubMed

Krill, Jennifer L; Dawson-Scully, Ken

2016-01-01

While the mammalian brain functions within a very narrow range of oxygen concentrations and temperatures, the fruit fly, Drosophila melanogaster, has employed strategies to deal with a much wider range of acute environmental stressors. The foraging (for) gene encodes the cGMP-dependent protein kinase (PKG), has been shown to regulate thermotolerance in many stress-adapted species, including Drosophila, and could be a potential therapeutic target in the treatment of hyperthermia in mammals. Whereas previous thermotolerance studies have looked at the effects of PKG variation on Drosophila behavior or excitatory postsynaptic potentials at the neuromuscular junction (NMJ), little is known about PKG effects on presynaptic mechanisms. In this study, we characterize presynaptic calcium ([Ca2+]i) dynamics at the Drosophila larval NMJ to determine the effects of high temperature stress on synaptic transmission. We investigated the neuroprotective role of PKG modulation both genetically using RNA interference (RNAi), and pharmacologically, to determine if and how PKG affects presynaptic [Ca2+]i dynamics during hyperthermia. We found that PKG activity modulates presynaptic neuronal Ca2+ responses during acute hyperthermia, where PKG activation makes neurons more sensitive to temperature-induced failure of Ca2+ flux and PKG inhibition confers thermotolerance and maintains normal Ca2+ dynamics under the same conditions. Targeted motoneuronal knockdown of PKG using RNAi demonstrated that decreased PKG expression was sufficient to confer thermoprotection. These results demonstrate that the PKG pathway regulates presynaptic motoneuronal Ca2+ signaling to influence thermotolerance of presynaptic function during acute hyperthermia.

Mechanisms Regulating Neuromuscular Junction Development and Function and Causes of Muscle Wasting.

PubMed

Tintignac, Lionel A; Brenner, Hans-Rudolf; Rüegg, Markus A

2015-07-01

The neuromuscular junction is the chemical synapse between motor neurons and skeletal muscle fibers. It is designed to reliably convert the action potential from the presynaptic motor neuron into the contraction of the postsynaptic muscle fiber. Diseases that affect the neuromuscular junction may cause failure of this conversion and result in loss of ambulation and respiration. The loss of motor input also causes muscle wasting as muscle mass is constantly adapted to contractile needs by the balancing of protein synthesis and protein degradation. Finally, neuromuscular activity and muscle mass have a major impact on metabolic properties of the organisms. This review discusses the mechanisms involved in the development and maintenance of the neuromuscular junction, the consequences of and the mechanisms involved in its dysfunction, and its role in maintaining muscle mass during aging. As life expectancy is increasing, loss of muscle mass during aging, called sarcopenia, has emerged as a field of high medical need. Interestingly, aging is also accompanied by structural changes at the neuromuscular junction, suggesting that the mechanisms involved in neuromuscular junction maintenance might be disturbed during aging. In addition, there is now evidence that behavioral paradigms and signaling pathways that are involved in longevity also affect neuromuscular junction stability and sarcopenia. Copyright © 2015 the American Physiological Society.

BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction

PubMed Central

Simó, Anna; Just-Borràs, Laia; Cilleros-Mañé, Víctor; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A.; Tomàs, Josep

2018-01-01

Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se, and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity–induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity–induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling. PMID:29946239

BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction.

PubMed

Simó, Anna; Just-Borràs, Laia; Cilleros-Mañé, Víctor; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Garcia, Neus; Lanuza, Maria A; Tomàs, Josep

2018-01-01

Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se , and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity-induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity-induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling.

Large-Area, Ensemble Molecular Electronics: Motivation and Challenges.

PubMed

Vilan, Ayelet; Aswal, Dinesh; Cahen, David

2017-03-08

We review charge transport across molecular monolayers, which is central to molecular electronics (MolEl), using large-area junctions (NmJ). We strive to provide a wide conceptual overview of three main subtopics. First, a broad introduction places NmJ in perspective to related fields of research and to single-molecule junctions (1mJ) in addition to a brief historical account. As charge transport presents an ultrasensitive probe for the electronic perfection of interfaces, in the second part ways to form both the monolayer and the contacts are described to construct reliable, defect-free interfaces. The last part is dedicated to understanding and analyses of current-voltage (I-V) traces across molecular junctions. Notwithstanding the original motivation of MolEl, I-V traces are often not very sensitive to molecular details and then provide a poor probe for chemical information. Instead, we focus on how to analyze the net electrical performance of molecular junctions, from a functional device perspective. Finally, we point to creation of a built-in electric field as a key to achieve functionality, including nonlinear current-voltage characteristics that originate in the molecules or their contacts to the electrodes. This review is complemented by a another review that covers metal-molecule-semiconductor junctions and their unique hybrid effects.

Neural circuit architecture defects in a Drosophila model of Fragile X syndrome are alleviated by minocycline treatment and genetic removal of matrix metalloproteinase

PubMed Central

Siller, Saul S.; Broadie, Kendal

2011-01-01

SUMMARY Fragile X syndrome (FXS), caused by loss of the fragile X mental retardation 1 (FMR1) product (FMRP), is the most common cause of inherited intellectual disability and autism spectrum disorders. FXS patients suffer multiple behavioral symptoms, including hyperactivity, disrupted circadian cycles, and learning and memory deficits. Recently, a study in the mouse FXS model showed that the tetracycline derivative minocycline effectively remediates the disease state via a proposed matrix metalloproteinase (MMP) inhibition mechanism. Here, we use the well-characterized Drosophila FXS model to assess the effects of minocycline treatment on multiple neural circuit morphological defects and to investigate the MMP hypothesis. We first treat Drosophila Fmr1 (dfmr1) null animals with minocycline to assay the effects on mutant synaptic architecture in three disparate locations: the neuromuscular junction (NMJ), clock neurons in the circadian activity circuit and Kenyon cells in the mushroom body learning and memory center. We find that minocycline effectively restores normal synaptic structure in all three circuits, promising therapeutic potential for FXS treatment. We next tested the MMP hypothesis by assaying the effects of overexpressing the sole Drosophila tissue inhibitor of MMP (TIMP) in dfmr1 null mutants. We find that TIMP overexpression effectively prevents defects in the NMJ synaptic architecture in dfmr1 mutants. Moreover, co-removal of dfmr1 similarly rescues TIMP overexpression phenotypes, including cellular tracheal defects and lethality. To further test the MMP hypothesis, we generated dfmr1;mmp1 double null mutants. Null mmp1 mutants are 100% lethal and display cellular tracheal defects, but co-removal of dfmr1 allows adult viability and prevents tracheal defects. Conversely, co-removal of mmp1 ameliorates the NMJ synaptic architecture defects in dfmr1 null mutants, despite the lack of detectable difference in MMP1 expression or gelatinase activity between the single dfmr1 mutants and controls. These results support minocycline as a promising potential FXS treatment and suggest that it might act via MMP inhibition. We conclude that FMRP and TIMP pathways interact in a reciprocal, bidirectional manner. PMID:21669931

Collagen XIX Is Expressed by Interneurons and Contributes to the Formation of Hippocampal Synapses

PubMed Central

Su, Jianmin; Gorse, Karen; Ramirez, Francesco; Fox, Michael A.

2010-01-01

Extracellular matrix (ECM) molecules contribute to the formation and maintenance of synapses in the mammalian nervous system. We previously discovered a family of nonfibrillar collagens that organize synaptic differentiation at the neuromuscular junction (NMJ). Although many NMJ-organizing cues contribute to central nervous system (CNS) synaptogenesis, whether similar roles for collagens exist at central synapses remained unclear. In the present study we discovered that col19a1, the gene encoding nonfibrillar collagen XIX, is expressed by subsets of hippocampal neurons. Colocalization with the interneuron-specific enzyme glutamate decarboxylase 67 (Gad67), but not other cell-type-specific markers, suggests that hippocampal expression of col19a1 is restricted to interneurons. However, not all hippocampal interneurons express col19a1 mRNA; subsets of neuropeptide Y (NPY)-, somatostatin (Som)-, and calbindin (Calb)-immunoreactive interneurons express col19a1, but those containing parvalbumin (Parv) or calretinin (Calr) do not. To assess whether collagen XIX is required for the normal formation of hippocampal synapses, we examined synaptic morphology and composition in targeted mouse mutants lacking collagen XIX. We show here that subsets of synaptotagmin 2 (Syt2)-containing hippocampal nerve terminals appear malformed in the absence of collagen XIX. The presence of Syt2 in inhibitory hippocampal synapses, the altered distribution of Gad67 in collagen XIX-deficient subiculum, and abnormal levels of gephyrin in collagen XIX-deficient hippocampal extracts all suggest inhibitory synapses are affected by the loss of collagen XIX. Together, these data not only reveal that collagen XIX is expressed by central neurons, but show for the first time that a nonfibrillar collagen is necessary for the formation of hippocampal synapses. PMID:19937713

Single calcium channel domain gating of synaptic vesicle fusion at fast synapses; analysis by graphic modeling

PubMed Central

Stanley, Elise F

2015-01-01

At fast-transmitting presynaptic terminals Ca2+ enter through voltage gated calcium channels (CaVs) and bind to a synaptic vesicle (SV) -associated calcium sensor (SV-sensor) to gate fusion and discharge. An open CaV generates a high-concentration plume, or nanodomain of Ca2+ that dissipates precipitously with distance from the pore. At most fast synapses, such as the frog neuromuscular junction (NMJ), the SV sensors are located sufficiently close to individual CaVs to be gated by single nanodomains. However, at others, such as the mature rodent calyx of Held (calyx of Held), the physiology is more complex with evidence that CaVs that are both close and distant from the SV sensor and it is argued that release is gated primarily by the overlapping Ca2+ nanodomains from many CaVs. We devised a 'graphic modeling' method to sum Ca2+ from individual CaVs located at varying distances from the SV-sensor to determine the SV release probability and also the fraction of that probability that can be attributed to single domain gating. This method was applied first to simplified, low and high CaV density model release sites and then to published data on the contrasting frog NMJ and the rodent calyx of Held native synapses. We report 3 main predictions: the SV-sensor is positioned very close to the point at which the SV fuses with the membrane; single domain-release gating predominates even at synapses where the SV abuts a large cluster of CaVs, and even relatively remote CaVs can contribute significantly to single domain-based gating. PMID:26457441

Presynaptic Active Zone Density during Development and Synaptic Plasticity.

PubMed

Clarke, Gwenaëlle L; Chen, Jie; Nishimune, Hiroshi

2012-01-01

Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated.

Presynaptic Active Zone Density during Development and Synaptic Plasticity

PubMed Central

Clarke, Gwenaëlle L.; Chen, Jie; Nishimune, Hiroshi

2012-01-01

Neural circuits transmit information through synapses, and the efficiency of synaptic transmission is closely related to the density of presynaptic active zones, where synaptic vesicles are released. The goal of this review is to highlight recent insights into the molecular mechanisms that control the number of active zones per presynaptic terminal (active zone density) during developmental and stimulus-dependent changes in synaptic efficacy. At the neuromuscular junctions (NMJs), the active zone density is preserved across species, remains constant during development, and is the same between synapses with different activities. However, the NMJ active zones are not always stable, as exemplified by the change in active zone density during acute experimental manipulation or as a result of aging. Therefore, a mechanism must exist to maintain its density. In the central nervous system (CNS), active zones have restricted maximal size, exist in multiple numbers in larger presynaptic terminals, and maintain a constant density during development. These findings suggest that active zone density in the CNS is also controlled. However, in contrast to the NMJ, active zone density in the CNS can also be increased, as observed in hippocampal synapses in response to synaptic plasticity. Although the numbers of known active zone proteins and protein interactions have increased, less is known about the mechanism that controls the number or spacing of active zones. The following molecules are known to control active zone density and will be discussed herein: extracellular matrix laminins and voltage-dependent calcium channels, amyloid precursor proteins, the small GTPase Rab3, an endocytosis mechanism including synaptojanin, cytoskeleton protein spectrins and β-adducin, and a presynaptic web including spectrins. The molecular mechanisms that organize the active zone density are just beginning to be elucidated. PMID:22438837

Hypoglossal-facial anastomosis (HFA) over a 10 mm gap bridged by a Y-tube-conduit enhances neurite regrowth and reduces collateral axonal branching at the lesion site.

PubMed

Ozsoy, Umut; Demirel, Bahadir Murat; Hizay, Arzu; Ozsoy, Ozlem; Ankerne, Janina; Angelova, Srebrina; Sarikcioglu, Levent; Ucar, Yasar; Angelov, Doychin N

2011-01-01

The outcome of severe peripheral nerve injuries requiring surgical repair (transection and suture) is usually poor. Recent work suggests that direct suture of nerves increases collagen production and provides unfavourable conditions for a proper axonal regrowth. We tested whether entubulation of the hypoglossal nerve into a Y-tube conduit connecting it with the zygomatic and buccal facial nerve branches would improve axonal pathfinding at the lesion site, quality of muscle reinnervation and recovery of vibrissal whisking. For hypoglossal-facial anastomosis (HFA) over a Y-tube (HFA-Y-tube) the proximal stump of the hypoglossal nerve was entubulated and sutured into the long arm of a Y-tube (isogeneic abdominal aorta with its bifurcation). The zygomatic and buccal facial branches were entubulated and sutured to the short arms of the Y-tube. Restoration of vibrissal motor performance, degree of collateral axonal branching at the lesion site and quality of neuro-muscular junction (NMJ) reinnervation were compared to animals receiving HFA-Coaptation (no entubulation) after 4 months. HFA-Y-tube reduced collateral axonal branching. However it failed to reduce the proportion of polyinnervated NMJ and did not improve functional outcome when compared to HFA-Coaptation. Elimination of compression by tightly opposed nerve fragments improved axonal pathfinding. However, biometric analysis of vibrissae movements did not show positive effects suggesting that polyneuronal reinnervation - rather than collateral branching - may be the critical limiting factor. Since polyinnervation of muscle fibers is activity-dependent and can be manipulated, the present findings raise hopes that clinically feasible and effective therapies after HFA could be soon designed and tested.

A novel mouse model carrying a human cytoplasmic dynein mutation shows motor behavior deficits consistent with Charcot-Marie-Tooth type 2O disease.

PubMed

Sabblah, Thywill T; Nandini, Swaran; Ledray, Aaron P; Pasos, Julio; Calderon, Jami L Conley; Love, Rachal; King, Linda E; King, Stephen J

2018-01-29

Charcot-Marie-Tooth disease (CMT) is a peripheral neuromuscular disorder in which axonal degeneration causes progressive loss of motor and sensory nerve function. The loss of motor nerve function leads to distal muscle weakness and atrophy, resulting in gait problems and difficulties with walking, running, and balance. A mutation in the cytoplasmic dynein heavy chain (DHC) gene was discovered to cause an autosomal dominant form of the disease designated Charcot-Marie-Tooth type 2 O disease (CMT2O) in 2011. The mutation is a single amino acid change of histidine into arginine at amino acid 306 (H306R) in DHC. In order to understand the onset and progression of CMT2, we generated a knock-in mouse carrying the corresponding CMT2O mutation (H304R/+). We examined H304R/+ mouse cohorts in a 12-month longitudinal study of grip strength, tail suspension, and rotarod assays. H304R/+ mice displayed distal muscle weakness and loss of motor coordination phenotypes consistent with those of individuals with CMT2. Analysis of the gastrocnemius of H304R/+ male mice showed prominent defects in neuromuscular junction (NMJ) morphology including reduced size, branching, and complexity. Based on these results, the H304R/+ mouse will be an important model for uncovering functions of dynein in complex organisms, especially related to CMT onset and progression.

Perturbation to Cholesterol at the Neuromuscular Junction Confers Botulinum Neurotoxin A Sensitivity to Neonatal Mice.

PubMed

Thyagarajan, Baskaran; Potian, Joseph G; McArdle, Joseph J; Baskaran, Padmamalini

2017-09-01

Botulinum neurotoxin A (BoNT/A) cleaves SNAP25 at the motor nerve terminals and inhibits stimulus evoked acetylcholine release. This causes skeletal muscle paralysis. However, younger neonatal mice (P7) mice. However, neonatal mice younger than 7 days-age remained unaffected by BoNT/A injection. Also, BoNT/A inhibited stimulus evoked acetylcholine release and stimulus-evoked twitch tension of diaphragm nerve muscle preparations (NMPs) of adult mouse and >P7 neonates but not that of P7. However, cholesterol depletion using methyl-β-cyclodextrin (MβCD) sensitized

Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Santafé, Manel; Tomàs, Josep

2016-06-23

The development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses. Here we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses. The three receptor sets considered (mAChR, AR and TrkB receptors) intervene in modulating the conditions of the competition between nerve endings, possibly helping to determine the winner or the lossers but, thereafter, the final elimination would occur with some autonomy and independently of postsynaptic maturation.

Roles for the VCP co-factors Npl4 and Ufd1 in neuronal function in Drosophila melanogaster.

PubMed

Byrne, Dwayne J; Harmon, Mark J; Simpson, Jeremy C; Blackstone, Craig; O'Sullivan, Niamh C

2017-10-20

The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), and extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, we infer that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, we find that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Specific Retrograde Transduction of Spinal Motor Neurons Using Lentiviral Vectors Targeted to Presynaptic NMJ Receptors

PubMed Central

Eleftheriadou, I; Trabalza, A; Ellison, SM; Gharun, K; Mazarakis, ND

2014-01-01

To understand how receptors are involved in neuronal trafficking and to be able to utilize them for specific targeting via the peripheral route would be of great benefit. Here, we describe the generation of novel lentiviral vectors with tropism to motor neurons that were made by coexpressing onto the lentiviral surface a fusogenic glycoprotein (mutated sindbis G) and an antibody against a cell-surface receptor (Thy1.1, p75NTR, or coxsackievirus and adenovirus receptor) on the presynaptic terminal of the neuromuscular junction. These vectors exhibit binding specificity and efficient transduction of receptor positive cell lines and primary motor neurons in vitro. Targeting of each of these receptors conferred to these vectors the capability of being transported retrogradely from the axonal tip, leading to transduction of motor neurons in vitro in compartmented microfluidic cultures. In vivo delivery of coxsackievirus and adenovirus receptor-targeted vectors in leg muscles of mice resulted in predicted patterns of motor neuron labeling in lumbar spinal cord. This opens up the clinical potential of these vectors for minimally invasive administration of central nervous system-targeted therapeutics in motor neuron diseases. PMID:24670531

Transcriptomics of aged Drosophila motor neurons reveals a matrix metalloproteinase that impairs motor function.

PubMed

Azpurua, Jorge; Mahoney, Rebekah E; Eaton, Benjamin A

2018-04-01

The neuromuscular junction (NMJ) is responsible for transforming nervous system signals into motor behavior and locomotion. In the fruit fly Drosophila melanogaster, an age-dependent decline in motor function occurs, analogous to the decline experienced in mice, humans, and other mammals. The molecular and cellular underpinnings of this decline are still poorly understood. By specifically profiling the transcriptome of Drosophila motor neurons across age using custom microarrays, we found that the expression of the matrix metalloproteinase 1 (dMMP1) gene reproducibly increased in motor neurons in an age-dependent manner. Modulation of physiological aging also altered the rate of dMMP1 expression, validating dMMP1 expression as a bona fide aging biomarker for motor neurons. Temporally controlled overexpression of dMMP1 specifically in motor neurons was sufficient to induce deficits in climbing behavior and cause a decrease in neurotransmitter release at neuromuscular synapses. These deficits were reversible if the dMMP1 expression was shut off again immediately after the onset of motor dysfunction. Additionally, repression of dMMP1 enzymatic activity via overexpression of a tissue inhibitor of metalloproteinases delayed the onset of age-dependent motor dysfunction. MMPs are required for proper tissue architecture during development. Our results support the idea that matrix metalloproteinase 1 is acting as a downstream effector of antagonistic pleiotropy in motor neurons and is necessary for proper development, but deleterious when reactivated at an advanced age. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

The translational regulator Cup controls NMJ presynaptic terminal morphology.

PubMed

Menon, Kaushiki P; Carrillo, Robert A; Zinn, Kai

2015-07-01

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with two genes, EndoA and Dap160, that encode proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal. Copyright © 2015 Elsevier Inc. All rights reserved.

The translational regulator Cup controls NMJ presynaptic terminal morphology

PubMed Central

Menon, Kaushiki P.; Carrillo, Robert A.; Zinn, Kai

2015-01-01

During oogenesis and early embryonic development in Drosophila, translation of proteins from maternally deposited mRNAs is tightly controlled. We and others have previously shown that translational regulatory proteins that function during oogenesis also have essential roles in the nervous system. Here we examine the role of Cup in neuromuscular system development. Maternal Cup controls translation of localized mRNAs encoding the Oskar and Nanos proteins and binds to the general translation initiation factor eIF4E. In this paper, we show that zygotic Cup protein is localized to presynaptic terminals at larval neuromuscular junctions (NMJs). cup mutant NMJs have strong phenotypes characterized by the presence of small clustered boutons called satellite boutons. They also exhibit an increase in the frequency of spontaneous glutamate release events (mEPSPs). Reduction of eIF4E expression synergizes with partial loss of Cup expression to produce satellite bouton phenotypes. The presence of satellite boutons is often associated with increases in retrograde bone morphogenetic protein (BMP) signaling, and we show that synaptic BMP signaling is elevated in cup mutants. cup genetically interacts with four genes (EndoA, WASp, Dap160, and Synj) encoding proteins involved in endocytosis that are also neuronal modulators of the BMP pathway. Endophilin protein, encoded by the EndoA gene, is downregulated in a cup mutant. Our results are consistent with a model in which Cup and eIF4E work together to ensure efficient localization and translation of endocytosis proteins in motor neurons and control the strength of the retrograde BMP signal. PMID:26102195

Sequential photo-bleaching to delineate single Schwann cells at the neuromuscular junction.

PubMed

Brill, Monika S; Marinković, Petar; Misgeld, Thomas

2013-01-11

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP and Plp-GFP mice, respectively. Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact. Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species. In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation has proven useful for studies of NMJ development, physiology and pathology. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Drosophila fragile X mental retardation protein and metabotropic glutamate receptor A convergently regulate the synaptic ratio of ionotropic glutamate receptor subclasses.

PubMed

Pan, Luyuan; Broadie, Kendal S

2007-11-07

A current hypothesis proposes that fragile X mental retardation protein (FMRP), an RNA-binding translational regulator, acts downstream of glutamatergic transmission, via metabotropic glutamate receptor (mGluR) G(q)-dependent signaling, to modulate protein synthesis critical for trafficking ionotropic glutamate receptors (iGluRs) at synapses. However, direct evidence linking FMRP and mGluR function with iGluR synaptic expression is limited. In this study, we use the Drosophila fragile X model to test this hypothesis at the well characterized glutamatergic neuromuscular junction (NMJ). Two iGluR classes reside at this synapse, each containing common GluRIIC (III), IID and IIE subunits, and variable GluRIIA (A-class) or GluRIIB (B-class) subunits. In Drosophila fragile X mental retardation 1 (dfmr1) null mutants, A-class GluRs accumulate and B-class GluRs are lost, whereas total GluR levels do not change, resulting in a striking change in GluR subclass ratio at individual synapses. The sole Drosophila mGluR, DmGluRA, is also expressed at the NMJ. In dmGluRA null mutants, both iGluR classes increase, resulting in an increase in total synaptic GluR content at individual synapses. Targeted postsynaptic dmGluRA overexpression causes the exact opposite GluR phenotype to the dfmr1 null, confirming postsynaptic GluR subtype-specific regulation. In dfmr1; dmGluRA double null mutants, there is an additive increase in A-class GluRs, and a similar additive impact on B-class GluRs, toward normal levels in the double mutants. These results show that both dFMRP and DmGluRA differentially regulate the abundance of different GluR subclasses in a convergent mechanism within individual postsynaptic domains.

Comparison of trophic factors' expression between paralyzed and recovering muscles after facial nerve injury. A quantitative analysis in time course.

PubMed

Grosheva, Maria; Nohroudi, Klaus; Schwarz, Alisa; Rink, Svenja; Bendella, Habib; Sarikcioglu, Levent; Klimaschewski, Lars; Gordon, Tessa; Angelov, Doychin N

2016-05-01

After peripheral nerve injury, recovery of motor performance negatively correlates with the poly-innervation of neuromuscular junctions (NMJ) due to excessive sprouting of the terminal Schwann cells. Denervated muscles produce short-range diffusible sprouting stimuli, of which some are neurotrophic factors. Based on recent data that vibrissal whisking is restored perfectly during facial nerve regeneration in blind rats from the Sprague Dawley (SD)/RCS strain, we compared the expression of brain derived neurotrophic factor (BDNF), fibroblast growth factor-2 (FGF2), insulin growth factors 1 and 2 (IGF1, IGF2) and nerve growth factor (NGF) between SD/RCS and SD-rats with normal vision but poor recovery of whisking function after facial nerve injury. To establish which trophic factors might be responsible for proper NMJ-reinnervation, the transected facial nerve was surgically repaired (facial-facial anastomosis, FFA) for subsequent analysis of mRNA and proteins expressed in the levator labii superioris muscle. A complicated time course of expression included (1) a late rise in BDNF protein that followed earlier elevated gene expression, (2) an early increase in FGF2 and IGF2 protein after 2 days with sustained gene expression, (3) reduced IGF1 protein at 28 days coincident with decline of raised mRNA levels to baseline, and (4) reduced NGF protein between 2 and 14 days with maintained gene expression found in blind rats but not the rats with normal vision. These findings suggest that recovery of motor function after peripheral nerve injury is due, at least in part, to a complex regulation of lesion-associated neurotrophic factors and cytokines in denervated muscles. The increase of FGF-2 protein and concomittant decrease of NGF (with no significant changes in BDNF or IGF levels) during the first week following FFA in SD/RCS blind rats possibly prevents the distal branching of regenerating axons resulting in reduced poly-innervation of motor endplates. Copyright © 2016 Elsevier Inc. All rights reserved.

Membrane Receptor-Induced Changes of the Protein Kinases A and C Activity May Play a Leading Role in Promoting Developmental Synapse Elimination at the Neuromuscular Junction.

PubMed

Tomàs, Josep M; Garcia, Neus; Lanuza, Maria A; Nadal, Laura; Tomàs, Marta; Hurtado, Erica; Simó, Anna; Cilleros, Víctor

2017-01-01

Synapses that are overproduced during histogenesis in the nervous system are eventually lost and connectivity is refined. Membrane receptor signaling leads to activity-dependent mutual influence and competition between axons directly or with the involvement of the postsynaptic cell and the associated glial cell/s. Presynaptic muscarinic acetylcholine (ACh) receptors (subtypes mAChR; M 1 , M 2 and M 4 ), adenosine receptors (AR; A 1 and A 2A ) and the tropomyosin-related kinase B receptor (TrkB), among others, all cooperate in synapse elimination. Between these receptors there are several synergistic, antagonic and modulatory relations that clearly affect synapse elimination. Metabotropic receptors converge in a limited repertoire of intracellular effector kinases, particularly serine protein kinases A and C (PKA and PKC), to phosphorylate protein targets and bring about structural and functional changes leading to axon loss. In most cells A 1 , M 1 and TrkB operate mainly by stimulating PKC whereas A 2A , M 2 and M 4 inhibit PKA. We hypothesize that a membrane receptor-induced shifting in the protein kinases A and C activity (inhibition of PKA and/or stimulation of PKC) in some nerve endings may play an important role in promoting developmental synapse elimination at the neuromuscular junction (NMJ). This hypothesis is supported by: (i) the tonic effect (shown by using selective inhibitors) of several membrane receptors that accelerates axon loss between postnatal days P5-P9; (ii) the synergistic, antagonic and modulatory effects (shown by paired inhibition) of the receptors on axonal loss; (iii) the fact that the coupling of these receptors activates/inhibits the intracellular serine kinases; and (iv) the increase of the PKA activity, the reduction of the PKC activity or, in most cases, both situations simultaneously that presumably occurs in all the situations of singly and paired inhibition of the mAChR, AR and TrkB receptors. The use of transgenic animals and various combinations of selective and specific PKA and PKC inhibitors could help to elucidate the role of these kinases in synapse maturation.

A comparison between the blocking actions of 2-(4-phenylpiperidino) cyclohexanol (AH 5183) and its N-methyl quaternary analogue (AH 5954)

PubMed Central

Marshall, I. G.

1970-01-01

1. The neuromuscular blocking activities of AH 5183 (2-(4-phenylpiperidino) cyclohexanol) and its N-methyl quaternary analogue (AH 5954) were compared in rapidly stimulated nerve-skeletal muscle preparations of the rat, chicken and cat. 2. The evidence indicated that in isolated preparations the neuromuscular block produced by both AH 5183 and AH 5954 was primarily pre-junctional in origin. That produced by AH 5954 was readily reversible either by washing the tissue or by reducing the stimulation frequency, whereas that produced by AH 5183 was difficult to reverse in these ways. 3. Low doses of AH 5954 sensitized the rat hemidiaphragm preparation to the neuromuscular blocking action of choline. The neuromuscular block produced by choline was reversible by tetraethylammonium but not by neostigmine. This suggested that the blocking action of choline is at least partly pre-junctional in nature. 4. In anaesthetized cats AH 5954 possessed a biphasic neuromuscular blocking action. The initial phase was rapid in onset, suggestive of a post-junctional action, whereas the second phase was prolonged and reversible by choline, suggestive of a prejunctional inhibitory action on the choline transport mechanism. AH 5183 produced no initial blocking action and was irreversible by choline. 5. Both AH 5183 and AH 5954 possessed local anaesthetic and α-adrenoceptor blocking actions. These actions and the neuromuscular blocking action were affected to different degrees by quaternization, suggesting that the three main actions of the two drugs are independent. 6. It was concluded that AH 5954 and AH 5183 act at different pre-junctional sites at the neuromuscular junction, AH 5954 acting extraneuronally by inhibiting choline transport and AH 5183 intraneuronally at the level of the synaptic vesicle membrane. PMID:4395087

Protein kinase C isoforms at the neuromuscular junction: localization and specific roles in neurotransmission and development.

PubMed

Lanuza, Maria A; Santafe, Manel M; Garcia, Neus; Besalduch, Núria; Tomàs, Marta; Obis, Teresa; Priego, Mercedes; Nelson, Phillip G; Tomàs, Josep

2014-01-01

The protein kinase C family (PKC) regulates a variety of neural functions including neurotransmitter release. The selective activation of a wide range of PKC isoforms in different cells and domains is likely to contribute to the functional diversity of PKC phosphorylating activity. In this review, we describe the isoform localization, phosphorylation function, regulation and signalling of the PKC family at the neuromuscular junction. Data show the involvement of the PKC family in several important functions at the neuromuscular junction and in particular in the maturation of the synapse and the modulation of neurotransmission in the adult. © 2013 Anatomical Society.

Rolling blackout is required for synaptic vesicle exocytosis.

PubMed

Huang, Fu-De; Woodruff, Elvin; Mohrmann, Ralf; Broadie, Kendal

2006-03-01

Rolling blackout (RBO) is a putative transmembrane lipase required for phospholipase C-dependent phosphatidylinositol 4,5-bisphosphate-diacylglycerol signaling in Drosophila neurons. Conditional temperature-sensitive (TS) rbo mutants display complete, reversible paralysis within minutes, demonstrating that RBO is acutely required for movement. RBO protein is localized predominantly in presynaptic boutons at neuromuscular junction (NMJ) synapses and throughout central synaptic neuropil, and rbo TS mutants display a complete, reversible block of both central and peripheral synaptic transmission within minutes. This phenotype appears limited to adults, because larval NMJs do not manifest the acute blockade. Electron microscopy of adult rbo TS mutant boutons reveals an increase in total synaptic vesicle (SV) content, with a concomitant shrinkage of presynaptic bouton size and an accumulation of docked SVs at presynaptic active zones within minutes. Genetic tests reveal a synergistic interaction between rbo and syntaxin1A TS mutants, suggesting that RBO is required in the mechanism of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated SV exocytosis, or in a parallel pathway necessary for SV fusion. The rbo TS mutation does not detectably alter SNARE complex assembly, suggesting a downstream requirement in SV fusion. We conclude that RBO plays an essential role in neurotransmitter release, downstream of SV docking, likely mediating SV fusion.

Genomic and Proteomic Profiling Reveals Reduced Mitochondrial Function and Disruption of the Neuromuscular Junction Driving Rat Sarcopenia

PubMed Central

Ibebunjo, Chikwendu; Chick, Joel M.; Kendall, Tracee; Eash, John K.; Li, Christine; Zhang, Yunyu; Vickers, Chad; Wu, Zhidan; Clarke, Brian A.; Shi, Jun; Cruz, Joseph; Fournier, Brigitte; Brachat, Sophie; Gutzwiller, Sabine; Ma, QiCheng; Markovits, Judit; Broome, Michelle; Steinkrauss, Michelle; Skuba, Elizabeth; Galarneau, Jean-Rene; Gygi, Steven P.

2013-01-01

Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia. PMID:23109432

Dysfunction of the neuromuscular junction in spinal muscular atrophy types 2 and 3.

PubMed

Wadman, Renske I; Vrancken, Alexander F J E; van den Berg, Leonard H; van der Pol, W Ludo

2012-11-13

Spinal muscular atrophy (SMA) is pathologically characterized by degeneration of anterior horn cells. Recent observations in animal models of SMA and muscle tissue from patients with SMA suggest additional abnormalities in the development and maturation of the neuromuscular junction. We therefore evaluated neuromuscular junction function in SMA with repetitive nerve stimulation. In this case-control study, repetitive nerve stimulation was performed in 35 patients with SMA types 2, 3, and 4, 20 healthy controls, and 5 controls with motor neuron disease. Pathologic decremental responses (>10%) during 3-Hz repetitive nerve stimulation were observed in 17 of 35 patients (49%) with SMA types 2 and 3, but not in healthy controls or controls with motor neuron disease. None of the patients or controls had an abnormal incremental response of >60%. The presence of an abnormal decremental response was not specific for the type of SMA, nor was it associated with compound muscle action potential amplitude, clinical scores, or disease duration. Two of 4 patients with SMA type 3 who tried pyridostigmine reported increased stamina. These data suggest dysfunction of the neuromuscular junction in patients with SMA types 2 and 3. Therefore, drugs that facilitate neuromuscular transmission are candidate drugs for evaluation in carefully designed, placebo-controlled, clinical trials.

Neurophysiological Strategies for the Diagnosis of Disorders of the Neuromuscular Junction in Children

ERIC Educational Resources Information Center

Pitt, Matthew

2008-01-01

The disorders of the neuromuscular junction seen in children, the congenital myasthenic syndromes and autoimmune myasthenia gravis, are very rare. Their clinical symptoms and signs may be variable, most notably in the neonate and infant. They should enter the differential diagnosis of many different clinical presentations, such as "floppy infant"…

Peripheral nerve hyperexcitability with preterminal nerve and neuromuscular junction remodeling is a hallmark of Schwartz-Jampel syndrome.

PubMed

Bauché, Stéphanie; Boerio, Delphine; Davoine, Claire-Sophie; Bernard, Véronique; Stum, Morgane; Bureau, Cécile; Fardeau, Michel; Romero, Norma Beatriz; Fontaine, Bertrand; Koenig, Jeanine; Hantaï, Daniel; Gueguen, Antoine; Fournier, Emmanuel; Eymard, Bruno; Nicole, Sophie

2013-12-01

Schwartz-Jampel syndrome (SJS) is a recessive disorder with muscle hyperactivity that results from hypomorphic mutations in the perlecan gene, a basement membrane proteoglycan. Analyses done on a mouse model have suggested that SJS is a congenital form of distal peripheral nerve hyperexcitability resulting from synaptic acetylcholinesterase deficiency, nerve terminal instability with preterminal amyelination, and subtle peripheral nerve changes. We investigated one adult patient with SJS to study this statement in humans. Perlecan deficiency due to hypomorphic mutations was observed in the patient biological samples. Electroneuromyography showed normal nerve conduction, neuromuscular transmission, and compound nerve action potentials while multiple measures of peripheral nerve excitability along the nerve trunk did not detect changes. Needle electromyography detected complex repetitive discharges without any evidence for neuromuscular transmission failure. The study of muscle biopsies containing neuromuscular junctions showed well-formed post-synaptic element, synaptic acetylcholinesterase deficiency, denervation of synaptic gutters with reinnervation by terminal sprouting, and long nonmyelinated preterminal nerve segments. These data support the notion of peripheral nerve hyperexcitability in SJS, which would originate distally from synergistic actions of peripheral nerve and neuromuscular junction changes as a result of perlecan deficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

Guidelines for standard preclinical experiments in the mouse model of myasthenia gravis induced by acetylcholine receptor immunization.

PubMed

Tuzun, Erdem; Berrih-Aknin, Sonia; Brenner, Talma; Kusner, Linda L; Le Panse, Rozen; Yang, Huan; Tzartos, Socrates; Christadoss, Premkumar

2015-08-01

Myasthenia gravis (MG) is an autoimmune disorder characterized by generalized muscle weakness due to neuromuscular junction (NMJ) dysfunction brought by acetylcholine receptor (AChR) antibodies in most cases. Although steroids and other immunosuppressants are effectively used for treatment of MG, these medications often cause severe side effects and a complete remission cannot be obtained in many cases. For pre-clinical evaluation of more effective and less toxic treatment methods for MG, the experimental autoimmune myasthenia gravis (EAMG) induced by Torpedo AChR immunization has become one of the standard animal models. Although numerous compounds have been recently proposed for MG mostly by using the active immunization EAMG model, only a few have been proven to be effective in MG patients. The variability in the experimental design, immunization methods and outcome measurements of pre-clinical EAMG studies make it difficult to interpret the published reports and assess the potential for application to MG patients. In an effort to standardize the active immunization EAMG model, we propose standard procedures for animal care conditions, sampling and randomization of mice, experimental design and outcome measures. Utilization of these standard procedures might improve the power of pre-clinical EAMG experiments and increase the chances for identifying promising novel treatment methods that can be effectively translated into clinical trials for MG. Copyright © 2015 Elsevier Inc. All rights reserved.

A Drosophila SNAP-25 null mutant reveals context-dependent redundancy with SNAP-24 in neurotransmission.

PubMed Central

Vilinsky, Ilya; Stewart, Bryan A; Drummond, James; Robinson, Iain; Deitcher, David L

2002-01-01

The synaptic protein SNAP-25 is an important component of the neurotransmitter release machinery, although its precise function is still unknown. Genetic analysis of other synaptic proteins has yielded valuable information on their role in synaptic transmission. In this study, we performed a mutagenesis screen to identify new SNAP-25 alleles that fail to complement our previously isolated recessive temperature-sensitive allele of SNAP-25, SNAP-25(ts). In a screen of 100,000 flies, 26 F(1) progeny failed to complement SNAP-25(ts) and 21 of these were found to be null alleles of SNAP-25. These null alleles die at the pharate adult stage and electroretinogram recordings of these animals reveal that synaptic transmission is blocked. At the third instar larval stage, SNAP-25 nulls exhibit nearly normal neurotransmitter release at the neuromuscular junction. This is surprising since SNAP-25(ts) larvae exhibit a much stronger synaptic phenotype. Our evidence indicates that a related protein, SNAP-24, can substitute for SNAP-25 at the larval stage in SNAP-25 nulls. However, if a wild-type or mutant form of SNAP-25 is present, then SNAP-24 does not appear to take part in neurotransmitter release at the larval NMJ. These results suggest that the apparent redundancy between SNAP-25 and SNAP-24 is due to inappropriate genetic substitution. PMID:12242238

Protein-anchoring therapy to target extracellular matrix proteins to their physiological destinations.

PubMed

Ito, Mikako; Ohno, Kinji

2018-02-20

Endplate acetylcholinesterase (AChE) deficiency is a form of congenital myasthenic syndrome (CMS) caused by mutations in COLQ, which encodes collagen Q (ColQ). ColQ is an extracellular matrix (ECM) protein that anchors AChE to the synaptic basal lamina. Biglycan, encoded by BGN, is another ECM protein that binds to the dystrophin-associated protein complex (DAPC) on skeletal muscle, which links the actin cytoskeleton and ECM proteins to stabilize the sarcolemma during repeated muscle contractions. Upregulation of biglycan stabilizes the DPAC. Gene therapy can potentially ameliorate any disease that can be recapitulated in cultured cells. However, the difficulty of tissue-specific and developmental stage-specific regulated expression of transgenes, as well as the difficulty of introducing a transgene into all cells in a specific tissue, prevents us from successfully applying gene therapy to many human diseases. In contrast to intracellular proteins, an ECM protein is anchored to the target tissue via its specific binding affinity for protein(s) expressed on the cell surface within the target tissue. Exploiting this unique feature of ECM proteins, we developed protein-anchoring therapy in which a transgene product expressed even in remote tissues can be delivered and anchored to a target tissue using specific binding signals. We demonstrate the application of protein-anchoring therapy to two disease models. First, intravenous administration of adeno-associated virus (AAV) serotype 8-COLQ to Colq-deficient mice, resulting in specific anchoring of ectopically expressed ColQ-AChE at the NMJ, markedly improved motor functions, synaptic transmission, and the ultrastructure of the neuromuscular junction (NMJ). In the second example, Mdx mice, a model for Duchenne muscular dystrophy, were intravenously injected with AAV8-BGN. The treatment ameliorated motor deficits, mitigated muscle histopathologies, decreased plasma creatine kinase activities, and upregulated expression of utrophin and DAPC component proteins. We propose that protein-anchoring therapy could be applied to hereditary/acquired defects in ECM and secreted proteins, as well as therapeutic overexpression of such factors. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

FIJI Macro 3D ART VeSElecT: 3D Automated Reconstruction Tool for Vesicle Structures of Electron Tomograms

PubMed Central

Kaltdorf, Kristin Verena; Schulze, Katja; Helmprobst, Frederik; Kollmannsberger, Philip; Stigloher, Christian

2017-01-01

Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i) in embryonic Danio rerio 4 and 8 days past fertilization (dpf) and (ii) to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ) wild-type and its septin mutant (unc-59(e261)). We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261) on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement). This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter). Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles) and specificity (true vesicles) as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual annotation. Both automatic and semi-automatic modes are explained including a tutorial. PMID:28056033

Stimulation Induced Changes in Frog Neuromuscular Junctions: A Quantitative Ultrastructural Comparison of Rapid-Frozen and Chemically Fixed Nerve Terminals

DTIC Science & Technology

1984-03-06

study was conducted to determine the presynaptic morphological changes due to neural activity in rapidly stimulated neuromuscular junctions...Control preparations were unstimulated and preserved either by chemical fixation or rapid-freezing. This study provides evidence that most of the...tissue. The rapid-frozen preparations in the present study showed, in addition, that rapid stimulation produces an increase in synaptic vesicle

Rapid synthesis of acetylcholine receptors at neuromuscular junctions.

PubMed

Ramsay, D A; Drachman, D B; Pestronk, A

1988-10-11

The rate of acetylcholine receptor (AChR) degradation in mature, innervated mammalian neuromuscular junctions has recently been shown to be biphasic; up to 20% are rapidly turned over (RTOs; half life less than 1 day) whereas the remainder are lost more slowly ('stable' AChRs; half life 10-12 days). In order to maintain normal junctional receptor density, synthesis and insertion of AChRs should presumably be sufficiently rapid to replace both the RTOs and the stable receptors. We have tested this prediction by blocking pre-existing AChRs in the mouse sternomastoid muscle with alpha-bungarotoxin (alpha-BuTx), and monitoring the subsequent appearance of 'new' junctional AChRs at intervals of 3 h to 20 days by labeling them with 125I-alpha-BuTx. The results show that new receptors were initially inserted rapidly (16% at 24 h and 28% at 48 h). The rate of increase of 'new' 125I-alpha-BuTx binding sites gradually slowed down during the remainder of the time period studied. Control observations excluded possible artifacts of the experimental procedure including incomplete blockade of AChRs, dissociation of toxin-receptor complexes, or experimentally induced alteration of receptor synthesis. The present demonstration of rapid synthesis and incorporation of AChRs at innervated neuromuscular junctions provides support for the concept of a subpopulation of rapidly turned over AChRs. The RTOs may serve as precursors for the larger population of stable receptors and have an important role in the metabolism of the neuromuscular synapse.

[Pharmacology of glutamate sensitive synapses (I). Glutamate agonists (author's transl)].

PubMed

Shinozaki, H

1982-04-01

The actions of kainic acid, quisqualic acid, and ibotenic acid on the crayfish neuromuscular junction were described, and it was particularly interesting that the discrepancy between glutamate responses and EJPs was revealed by the use of kainic acid. On the other hand, there is increasing evidence showing that glutamate is an excitatory transmitter at the crayfish neuromuscular junction. At this stage, we are unable as yet to definitively support or reject glutamate's candidacy as the excitatory transmitter at the crayfish neuromuscular junction. The discrepancy revealed by the use of kainic acid may bring up some questions. Certainly, the differential action of kainic acid on the glutamate current and the excitatory synaptic current opens to doubt the transmitter role of glutamate. In the case of the study on a transmitter role for a substance of doubt status, the value of pharmacological studies seems to be greater in disproving than in asserting such the role. However, we have to consider the matter of the extra-junctional receptor postulated on the crayfish postsynaptic membrane as one of the major problems for pharmacological identification.

Functional Roles of the Interaction of APP and Lipoprotein Receptors

PubMed Central

Pohlkamp, Theresa; Wasser, Catherine R.; Herz, Joachim

2017-01-01

The biological fates of the key initiator of Alzheimer’s disease (AD), the amyloid precursor protein (APP), and a family of lipoprotein receptors, the low-density lipoprotein (LDL) receptor-related proteins (LRPs) and their molecular roles in the neurodegenerative disease process are inseparably interwoven. Not only does APP bind tightly to the extracellular domains (ECDs) of several members of the LRP group, their intracellular portions are also connected through scaffolds like the one established by FE65 proteins and through interactions with adaptor proteins such as X11/Mint and Dab1. Moreover, the ECDs of APP and LRPs share common ligands, most notably Reelin, a regulator of neuronal migration during embryonic development and modulator of synaptic transmission in the adult brain, and Agrin, another signaling protein which is essential for the formation and maintenance of the neuromuscular junction (NMJ) and which likely also has critical, though at this time less well defined, roles for the regulation of central synapses. Furthermore, the major independent risk factors for AD, Apolipoprotein (Apo) E and ApoJ/Clusterin, are lipoprotein ligands for LRPs. Receptors and ligands mutually influence their intracellular trafficking and thereby the functions and abilities of neurons and the blood-brain-barrier to turn over and remove the pathological product of APP, the amyloid-β peptide. This article will review and summarize the molecular mechanisms that are shared by APP and LRPs and discuss their relative contributions to AD. PMID:28298885

Invaginating Structures in Mammalian Synapses

PubMed Central

Petralia, Ronald S.; Wang, Ya-Xian; Mattson, Mark P.; Yao, Pamela J.

2018-01-01

Invaginating structures at chemical synapses in the mammalian nervous system exist in presynaptic axon terminals, postsynaptic spines or dendrites, and glial processes. These invaginating structures can be divided into three categories. The first category includes slender protrusions invaginating into axonal terminals, postsynaptic spines, or glial processes. Best known examples of this category are spinules extending from postsynaptic spines into presynaptic terminals in forebrain synapses. Another example of this category are protrusions from inhibitory presynaptic terminals invaginating into postsynaptic neuronal somas. Regardless of the direction and location, the invaginating structures of the first category do not have synaptic active zones within the invagination. The second category includes postsynaptic spines invaginating into presynaptic terminals, whereas the third category includes presynaptic terminals invaginating into postsynaptic spines or dendrites. Unlike the first category, the second and third categories have active zones within the invagination. An example of the second category are mossy terminal synapses of the hippocampal CA3 region, in which enlarged spine-like structures invaginate partly or entirely into mossy terminals. An example of the third category is the neuromuscular junction (NMJ) where substantial invaginations of the presynaptic terminals invaginate into the muscle fibers. In the retina, rod and cone synapses have invaginating processes from horizontal and bipolar cells. Because horizontal cells act both as post and presynaptic structures, their invaginating processes represent both the second and third category. These invaginating structures likely play broad yet specialized roles in modulating neuronal cell signaling. PMID:29674962

Crimpy enables discrimination of presynaptic and postsynaptic pools of a BMP at the Drosophila neuromuscular junction.

PubMed

James, Rebecca E; Hoover, Kendall M; Bulgari, Dinara; McLaughlin, Colleen N; Wilson, Christopher G; Wharton, Kristi A; Levitan, Edwin S; Broihier, Heather T

2014-12-08

Distinct pools of the bone morphogenetic protein (BMP) Glass bottom boat (Gbb) control structure and function of the Drosophila neuromuscular junction. Specifically, motoneuron-derived Gbb regulates baseline neurotransmitter release, whereas muscle-derived Gbb regulates neuromuscular junction growth. Yet how cells differentiate between these ligand pools is not known. Here we present evidence that the neuronal Gbb-binding protein Crimpy (Cmpy) permits discrimination of pre- and postsynaptic ligand by serving sequential functions in Gbb signaling. Cmpy first delivers Gbb to dense core vesicles (DCVs) for activity-dependent release from presynaptic terminals. In the absence of Cmpy, Gbb is no longer associated with DCVs and is not released by activity. Electrophysiological analyses demonstrate that Cmpy promotes Gbb's proneurotransmission function. Surprisingly, the Cmpy ectodomain is itself released upon DCV exocytosis, arguing that Cmpy serves a second function in BMP signaling. In addition to trafficking Gbb to DCVs, we propose that Gbb/Cmpy corelease from presynaptic terminals defines a neuronal protransmission signal. Copyright © 2014 Elsevier Inc. All rights reserved.

Myasthenia gravis and amyotrophic lateral sclerosis: A pathogenic overlap.

PubMed

Gotaas, Håvard Torvik; Skeie, Geir Olve; Gilhus, Nils Erik

2016-06-01

The aim was to examine potential joint disease mechanisms for myasthenia gravis (MG) and amyotrophic lateral sclerosis (ALS) through the examination of long-term patient cohorts for comorbidity. Recent studies support early involvement of the neuromuscular junction in ALS patients with subsequent degeneration of motor neurons. Medical records at Haukeland University Hospital from 1987 to 2012 were examined for International Classification of Diseases diagnostic codes for MG and ALS. Sera were re-tested for antibodies to acetylcholine receptor, titin, MuSK and GM1. We report one patient with both MG and ALS, and another 3 patients with suggestive evidence of both conditions. This is far more than expected from prevalence and incidence figures in this area if the disorders were unrelated. Our data suggest that immunological mechanisms in the neuromuscular junction are relevant in ALS pathogenesis. Attention should be given to possible therapeutic targets in the neuromuscular junction and muscle in ALS patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Reversing the Outcome of Synapse Elimination at Developing Neuromuscular Junctions In Vivo: Evidence for Synaptic Competition and Its Mechanism

PubMed Central

Turney, Stephen G.; Lichtman, Jeff W.

2012-01-01

During mammalian development, neuromuscular junctions and some other postsynaptic cells transition from multiple- to single-innervation as synaptic sites are exchanged between different axons. It is unclear whether one axon invades synaptic sites to drive off other inputs or alternatively axons expand their territory in response to sites vacated by other axons. Here we show that soon-to-be-eliminated axons rapidly reverse fate and grow to occupy vacant sites at a neuromuscular junction after laser removal of a stronger input. This reversal supports the idea that axons take over sites that were previously vacated. Indeed, during normal development we observed withdrawal followed by takeover. The stimulus for axon growth is not postsynaptic cell inactivity because axons grow into unoccupied sites even when target cells are functionally innervated. These results demonstrate competition at the synaptic level and enable us to provide a conceptual framework for understanding this form of synaptic plasticity. PMID:22745601

Ultrastructure of identified fast excitatory, slow excitatory and inhibitory neuromuscular junctions in the locust.

PubMed

Titmus, M J

1981-06-01

The specialized jumping muscle of the locust, the metathoracic extensor tibiae (ETi), is innervated by four physiologically different motoneurons, including FETi, a phasic excitor, SETi, a tonic excitor, and CI, a tonic common inhibitor. FETi neuromuscular junctions were examined in three phasic ETi bundles innervated by FETi. FETi terminals were characterized by patchy contacts on to granular sarcoplasm. The ETi accessory extensor, innervated by both SETi and CI, contains two morphologically different types of axon ending. When this muscle was soaked in horseradish peroxidase, stimulation of SETi led to selective uptake in vesicles in terminals similar to those of FETi axons but containing smaller vesicles, while stimulation by CI caused increased uptake into terminals with more extensive contact directly on to fibrillar sarcoplasm. As has been observed in excitatory and inhibitory synapses in some crustacean and vertebrate nervous systems, the synaptic vesicles in the locust excitatory endings are round and electron-lucent while those in the inhibitory endings are more irregular in shape. The tonic neuromuscular junctions, SETi and CI, are more densely packed with vesicles, larger in cross-sectional area and appear to be of more complex shape than the smaller, vesicle-sparse, phasic FETi terminals. Following long duration stimulation at 10 Hz, the tonic neuromuscular junctions showed little morphological change. FETi endings, which fatigue within minutes at the same stimulation frequency, showed a 20% decrease in synaptic vesicle density and an increase in irregularly shaped membrane inclusions.

The Leu-Arg-Glu (LRE) adhesion motif in proteins of the neuromuscular junction with special reference to proteins of the carboxylesterase/cholinesterase family.

PubMed

Johnson, Glynis; Moore, Samuel W

2013-09-01

Short linear motifs confer evolutionary flexibility on proteins as they can be added with relative ease allowing the acquisition of new functions. Such motifs may mediate a variety of signalling functions. The adhesion-mediating Leu-Arg-Glu (LRE) motif is enriched in laminin beta 2, and has been observed in other proteins, including members of the carboxylesterase/cholinesterase family. It acts as a stop signal for growing axons in the developing neuromuscular junction, binding to the voltage-gated calcium channel. In this bioinformatic analysis, we have investigated the presence of the motif in proteins of the neuromuscular junction, and have also examined its structural position and potential for ligand interaction, as well as phylogenetic conservation, in the carboxylesterase/cholinesterase family. The motif was observed to occur with a significantly higher frequency than expected in the UniProt/Swiss-Prot database, as well as in four individual species (human, mouse, Caenorhabditis elegans and Drosophila melanogaster). Examination of its presence in neuromuscular junction proteins showed it to be enriched in certain proteins of the synaptic basement membrane, including laminin, agrin, acetylcholinesterase and tenascin. A highly significant enrichment was observed in cytoskeletal proteins, particularly intermediate filament proteins and members of the spectrin family. In the carboxylesterase/cholinesterase family, the motif was observed in four conserved positions in the protein structure. It is present in the majority of mammalian acetylcholinesterases, as well as acetylcholinesterases from electric fish and a number of invertebrates. In insects, it is present in the ace-2, rather than in the synaptic ace-1, enzyme. It is also observed in the cholinesterase-like adhesion molecules (neuroligins, neurotactin and glutactin). It is never seen in butyrylcholinesterases, which do not mediate cell adhesion. In conclusion, the significant enrichment of the motif in certain classes of protein, as well as its conserved presence and structural positioning in one protein family, suggests that it has specific functions both in cell adhesion in the neuromuscular junction and in maintaining the structural integrity of the cytoskeleton. Copyright © 2013 Elsevier Inc. All rights reserved.

Localization of survival motor neuron protein in human apoptotic-like and regenerating muscle fibers, and neuromuscular junctions.

PubMed

Broccolini, A; Engel, W K; Askanas, V

1999-06-03

Mutations in the gene encoding survival motor neuron (SMN) protein are found in > 98% of patients with autosomal-recessive spinal muscular atrophy. We investigated the possible role of SMN in normal and abnormal human muscle by immunostaining biopsies of 20 patients with various neuromuscular diseases using monoclonal antibodies against SMN. SMN was strongly expressed cytoplasmically in chronic peripheral neuropathies, in about 80% of chronically denervated, very atrophic muscle fibers containing clumps of TUNEL-positive pyknotic nuclei: about 60% of those fibers also had cytoplasmic Bcl-2 and Bax immunoreactivity. In regenerating muscle fibers of various myopathies SMN co-localized with desmin, Bcl-2 and Bax; it was also present at the postsynaptic domain of normal human neuromuscular junctions. Thus, SMN may play a role in normal and pathological processes of adult human muscle fibers.

Autoimmune paraneoplastic syndromes associated to lung cancer: A systematic review of the literature Part 4: Neurological paraneoplastic syndromes, involving the peripheral nervous system and the neuromuscular junction and muscles.

PubMed

Ruelle, Lucien; Bentea, Georgiana; Sideris, Spyridon; El Koulali, Mohamed; Holbrechts, Stéphane; Lafitte, Jean-Jacques; Grigoriu, Bogdan; Sculier, Claudine; Meert, Anne-Pascale; Durieux, Valérie; Berghmans, Thierry; Sculier, Jean-Paul

2017-09-01

The development of new immune treatment in oncology and particularly for lung cancer may induce new complications, particularly activation or reactivation of auto-immune diseases. In this context, a systematic review on the auto-immune paraneoplastic syndromes that can complicate lung cancer appears useful. This article is the fourth of a series of five and deals mainly with neurological paraneoplastic syndromes involving the peripheral nervous system and the neuromuscular junction and muscles. Copyright © 2017 Elsevier B.V. All rights reserved.

Factors that affect the onset of action of non-depolarizing neuromuscular blocking agents

PubMed Central

Kim, Yong Byum; Sung, Tae-Yun

2017-01-01

Neuromuscular blockade plays an important role in the safe management of patient airways, surgical field improvement, and respiratory care. Rapid-sequence induction of anesthesia is indispensable to emergency surgery and obstetric anesthesia, and its purpose is to obtain a stable airway, adequate depth of anesthesia, and appropriate respiration within a short period of time without causing irritation or damage to the patient. There has been a continued search for new neuromuscular blocking drugs (NMBDs) with a rapid onset of action. Factors that affect the onset time include the potency of the NMBDs, the rate of NMBDs reaching the effect site, the onset time by dose control, metabolism and elimination of NMBDs, buffered diffusion to the effect site, nicotinic acetylcholine receptor subunit affinity, drugs that affect acetylcholine (ACh) production and release at the neuromuscular junction, drugs that inhibit plasma cholinesterase, presynaptic receptors responsible for ACh release at the neuromuscular junction, anesthetics or drugs that affect muscle contractility, site and methods for monitoring neuromuscular function, individual variability, and coexisting disease. NMBDs with rapid onset without major adverse events are expected in the next few years, and the development of lower potency NMBDs will continue. Anesthesiologists should be aware of the use of NMBDs in the management of anesthesia. The choice of NMBD and determination of the appropriate dosage to modulate neuromuscular blockade characteristics such as onset time and duration of neuromuscular blockade should be considered along with factors that affect the effects of the NMBDs. In this review, we discuss the factors that affect the onset time of NMBDs. PMID:29046769

Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

PubMed Central

Kang, Min Jung; Hansen, Timothy J.; Mickiewicz, Monique; Kaczynski, Tadeusz J.; Fye, Samantha; Gunawardena, Shermali

2014-01-01

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer's disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases. PMID:25127478

Muscle autoantibodies in myasthenia gravis: beyond diagnosis?

PubMed Central

Meriggioli, Matthew N; Sanders, Donald B

2012-01-01

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction. A number of molecules, including ion channels and other proteins at the neuromuscular junction, may be targeted by autoantibodies leading to abnormal neuromuscular transmission. In approximately 85% of patients, autoantibodies, directed against the postsynaptic nicotinic acetylcholine receptor can be detected in the serum and confirm the diagnosis, but in general, do not precisely predict the degree of weakness or response to therapy. Antibodies to the muscle-specific tyrosine kinase are detected in approximately 50% of generalized myasthenia gravis patients who are seronegative for anti-acetylcholine receptor antibodies, and levels of anti-muscle-specific tyrosine kinase antibodies do appear to correlate with disease severity and treatment response. Antibodies to other muscle antigens may be found in the subsets of myasthenia gravis patients, potentially providing clinically useful diagnostic information, but their utility as relevant biomarkers (measures of disease state or response to treatment) is currently unclear. PMID:22882218

Localization of brain-derived neurotrophic factor, neurotrophin-4, tropomyosin-related kinase b receptor, and p75 NTR receptor by high-resolution immunohistochemistry on the adult mouse neuromuscular junction.

PubMed

Garcia, Neus; Tomàs, Marta; Santafe, Manel M; Lanuza, M Angel; Besalduch, Nuria; Tomàs, Josep

2010-03-01

Neurotrophins and their receptors, the trk receptor tyrosine kinases (trks) and p75(NTR), are differentially expressed among the cell types that make up synapses. It is important to determine the precise location of these molecules involved in neurotransmission. Here we use immunostaining and Western blotting to study the localization and expression of neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase b (trkB) and p75(NTR) at the adult neuromuscular junction. Our confocal immunofluorescence results on the whole mounts of the mouse Levator auris longus muscle and on semithin cross-sections showed that BDNF, NT-4, trkB, and p75(NTR) were localized on the three cells in the neuromuscular synapse (motor axons, post-synaptic muscle and Schwann cells).

In vivo imaging of skeletal muscle in mice highlights muscle defects in a model of myotubular myopathy

PubMed Central

Mercier, Luc; Böhm, Johann; Fekonja, Nina; Allio, Guillaume; Lutz, Yves; Koch, Marc; Goetz, Jacky G.; Laporte, Jocelyn

2016-01-01

ABSTRACT Skeletal muscle structure and function are altered in different myopathies. However, the understanding of the molecular and cellular mechanisms mainly rely on in vitro and ex vivo investigations in mammalian models. In order to monitor in vivo the intracellular structure of the neuromuscular system in its environment under normal and pathological conditions, we set-up and validated non-invasive imaging of ear and leg muscles in mice. This original approach allows simultaneous imaging of different cellular and intracellular structures such as neuromuscular junctions and sarcomeres, reconstruction of the 3D architecture of the neuromuscular system, and video recording of dynamic events such as spontaneous muscle fiber contraction. Second harmonic generation was combined with vital dyes and fluorescent-coupled molecules. Skin pigmentation, although limiting, did not prevent intravital imaging. Using this versatile toolbox on the Mtm1 knockout mouse, a model for myotubular myopathy which is a severe congenital myopathy in human, we identified several hallmarks of the disease such as defects in fiber size and neuromuscular junction shape. Intravital imaging of the neuromuscular system paves the way for the follow-up of disease progression or/and disease amelioration upon therapeutic tests. It has also the potential to reduce the number of animals needed to reach scientific conclusions. PMID:28243519

Functional maturation of motor nerve terminals in the avian iris: ultrastructure, transmitter metabolism and synaptic reliability

PubMed Central

Pilar, Guillermo; Tuttle, Jeremy; Vaca, Ken

1981-01-01

1. The transformation of easily fatigued embryonic neuromuscular junctions into highly reliable mature terminals was examined by studying functional and morphological changes during development of the avian iris. The mature ability to follow repetitive electrical nerve stimulation was correlated with the rate of acetylcholine (ACh) synthesis and choline uptake, and with the fine structure of the nerve terminals and the post-synaptic elements. 2. The terminals of the ciliary nerve of the chick initially form functional synaptic contacts with the iris muscle at embryonic St. 34-40. At the onset of this period, no Na+-dependent high affinity choline uptake can be demonstrated, and the low level of ACh synthesis present is sensitive to Na+ removal. At St. 36 [3H]ACh synthesis begins to increase, the increment being Na+-dependent. 3. ACh synthesis in the embryonic iris was insensitive to a conditioning [K+]o depolarization even as late as St. 43. Just before hatching, depolarization elicits some augmentation in synthesis, but by 2 days ex ovo this release-induced response has increased by an order of magnitude. 4. Concurrently with the acquisition of the ability to respond to depolarization with accelerated synthesis, neuromuscular transmission in the iris becomes reliable and secure during stimulation at 20 Hz. Embryonic junctions rapidly block during such stimulation, and the failure is shown to be presynaptic in origin, resulting most probably from failure to sustain adequate levels of transmitter release. 5. Ultrastructural examination of the developing ciliary terminals revealed few synaptic vesicles at early stages, and a dearth of other specializations. The sequence of development from these small structurally undistinguished endings to large en plaque junctions completely filled with vesicles was reconstructed and compared to other neuromuscular junctions. Morphological maturation appears progressive with little evidence of discontinuity signalling functional status, but it is only after the terminals enlarge and become closely packed with vesicles that mature synaptic reliability is found. 6. The temporal correlation between responsiveness of transmitter synthesis to depolarization and reliable neuromuscular transmission suggests that modulation of neurotransmitter metabolism in response to demand signals the achievement of junctional maturity. ImagesABPlate 2Plate 3Plate 4 PMID:6279822

Localization of the RNA-binding proteins Staufen1 and Staufen2 at the mammalian neuromuscular junction.

PubMed

Bélanger, Guy; Stocksley, Mark A; Vandromme, Marie; Schaeffer, Laurent; Furic, Luc; DesGroseillers, Luc; Jasmin, Bernard J

2003-08-01

Staufen is an RNA-binding protein, first identified for its role in oogenesis and CNS development in Drosophila. Two mammalian homologs of Staufen have been identified and shown to bind double-stranded RNA and tubulin, and to function in the somatodendritic transport of mRNA in neurons. Here, we examined whether Staufen proteins are expressed in skeletal muscle in relation to the neuromuscular junction. Immunofluorescence experiments revealed that Staufen1 (Stau1) and Staufen2 (Stau2) accumulate preferentially within the postsynaptic sarcoplasm of muscle fibers as well as at newly formed ectopic synapses. Western blot analyses showed that the levels of Stau1 and Stau2 are greater in slow muscles than in fast-twitch muscles. Muscle denervation induced a significant increase in the expression of Stau1 and Stau2 in the extrasynaptic compartment of both fast and slow muscles. Consistent with these observations, we also demonstrated that expression of Stau1 and Stau2 is increased during myogenic differentiation and that treatment of myotubes with agrin and neuregulin induces a further increase in the expression of both Staufen proteins. We propose that Stau1 and Stau2 are key components of the postsynaptic apparatus in muscle, and that they contribute to the maturation and plasticity of the neuromuscular junction.

Extraocular Muscles in Patients With Infantile Nystagmus

PubMed Central

Berg, Kathleen T.; Hunter, David G.; Bothun, Erick D.; Antunes-Foschini, Rosalia; McLoon, Linda K.

2013-01-01

Objective To test the hypothesis that the extraocular muscles (EOMs) of patients with infantile nystagmus have muscular and innervational adaptations that may have a role in the involuntary oscillations of the eyes. Methods Specimens of EOMs from 10 patients with infantile nystagmus and postmortem specimens from 10 control subjects were prepared for histologic examination. The following variables were quantified: mean myofiber cross-sectional area, myofiber central nucleation, myelinated nerve density, nerve fiber density, and neuromuscular junction density. Results In contrast to control EOMs, infantile nystagmus EOMs had significantly more centrally nucleated myofibers, consistent with cycles of degeneration and regeneration. The EOMs of patients with nystagmus also had a greater degree of heterogeneity in myofiber size than did those of controls, with no difference in mean myofiber cross-sectional area. Mean myelinated nerve density, nerve fiber density, and neuromuscular junction density were also significantly decreased in infantile nystagmus EOMs. Conclusions The EOMs of patients with infantile nystagmus displayed a distinct hypoinnervated phenotype. This represents the first quantification of changes in central nucleation and myofiber size heterogeneity, as well as decreased myelinated nerve, nerve fiber, and neuromuscular junction density. These results suggest that deficits in motor innervation are a potential basis for the primary loss of motor control. Clinical Relevance Improved understanding of the etiology of nystagmus may direct future diagnostic and treatment strategies. PMID:22411664

Inhibition of β-Glucocerebrosidase Activity Preserves Motor Unit Integrity in a Mouse Model of Amyotrophic Lateral Sclerosis.

PubMed

Henriques, Alexandre; Huebecker, Mylene; Blasco, Hélène; Keime, Céline; Andres, Christian R; Corcia, Philippe; Priestman, David A; Platt, Frances M; Spedding, Michael; Loeffler, Jean-Philippe

2017-07-12

Recent metabolomic reports connect dysregulation of glycosphingolipids, particularly ceramide and glucosylceramide, to neurodegeneration and to motor unit dismantling in amyotrophic lateral sclerosis at late disease stage. We report here altered levels of gangliosides in the cerebrospinal fluid of amyotrophic lateral sclerosis patients in early disease stage. Conduritol B epoxide is an inhibitor of acid beta-glucosidase, and lowers glucosylceramide degradation. Glucosylceramide is the precursor for all of the more complex glycosphingolipids. In SOD1 G86R mice, an animal model of amyotrophic lateral sclerosis, conduritol B epoxide preserved ganglioside distribution at the neuromuscular junction, delayed disease onset, improved motor function and preserved motor neurons as well as neuromuscular junctions from degeneration. Conduritol B epoxide mitigated gene dysregulation in the spinal cord and restored the expression of genes involved in signal transduction and axonal elongation. Inhibition of acid beta-glucosidase promoted faster axonal elongation in an in vitro model of neuromuscular junctions and hastened recovery after peripheral nerve injury in wild type mice. Here, we provide evidence that glycosphingolipids play an important role in muscle innervation, which degenerates in amyotrophic lateral sclerosis from the early disease stage. This is a first proof of concept study showing that modulating the catabolism of glucosylceramide may be a therapeutic target for this devastating disease.

Myasthenia and related disorders of the neuromuscular junction.

PubMed

Spillane, Jennifer; Beeson, David J; Kullmann, Dimitri M

2010-08-01

Our understanding of transmission at the neuromuscular junction has increased greatly in recent years. We now recognise a wide variety of autoimmune and genetic diseases that affect this specialised synapse, causing muscle weakness and fatigue. These disorders greatly affect quality of life and rarely can be fatal. Myasthenia gravis is the most common disorder and is most commonly caused by autoantibodies targeting postsynaptic acetylcholine receptors. Antibodies to muscle-specific kinase (MuSK) are detected in a variable proportion of the remainder. Treatment is symptomatic and immunomodulatory. Lambert-Eaton myasthenic syndrome is caused by antibodies to presynaptic calcium channels, and approximately 50% of cases are paraneoplastic, most often related to small cell carcinoma of the lung. Botulism is an acquired disorder caused by neurotoxins produced by Clostridium botulinum, impairing acetylcholine release into the synaptic cleft. In addition, several rare congenital myasthenic syndromes have been identified, caused by inherited defects in presynaptic, synaptic basal lamina and postsynaptic proteins necessary for neuromuscular transmission. This review focuses on recent advances in the diagnosis and treatment of these disorders.

Effects of nitric oxide on neuromuscular properties of developing zebrafish embryos.

PubMed

Jay, Michael; Bradley, Sophie; McDearmid, Jonathan Robert

2014-01-01

Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.

A role for solvents in the toxicity of agricultural organophosphorus pesticides

PubMed Central

Eddleston, Michael; Street, Jonathan M.; Self, Ian; Thompson, Adrian; King, Tim; Williams, Nicola; Naredo, Gregorio; Dissanayake, Kosala; Yu, Ly-Mee; Worek, Franz; John, Harald; Smith, Sionagh; Thiermann, Horst; Harris, John B.; Eddie Clutton, R.

2012-01-01

Organophosphorus (OP) insecticide self-poisoning is responsible for about one-quarter of global suicides. Treatment focuses on the fact that OP compounds inhibit acetylcholinesterase (AChE); however, AChE-reactivating drugs do not benefit poisoned humans. We therefore studied the role of solvent coformulants in OP toxicity in a novel minipig model of agricultural OP poisoning. Gottingen minipigs were orally poisoned with clinically relevant doses of agricultural emulsifiable concentrate (EC) dimethoate, dimethoate active ingredient (AI) alone, or solvents. Cardiorespiratory physiology and neuromuscular (NMJ) function, blood AChE activity, and arterial lactate concentration were monitored for 12 h to assess poisoning severity. Poisoning with agricultural dimethoate EC40, but not saline, caused respiratory arrest within 30 min, severe distributive shock and NMJ dysfunction, that was similar to human poisoning. Mean arterial lactate rose to 15.6 [SD 2.8] mM in poisoned pigs compared to 1.4 [0.4] in controls. Moderate toxicity resulted from poisoning with dimethoate AI alone, or the major solvent cyclohexanone. Combining dimethoate with cyclohexanone reproduced severe poisoning characteristic of agricultural dimethoate EC poisoning. A formulation without cyclohexanone showed less mammalian toxicity. These results indicate that solvents play a crucial role in dimethoate toxicity. Regulatory assessment of pesticide toxicity should include solvents as well as the AIs which currently dominate the assessment. Reformulation of OP insecticides to ensure that the agricultural product has lower mammalian toxicity could result in fewer deaths after suicidal ingestion and rapidly reduce global suicide rates. PMID:22365945

Delayed Disease Onset and Extended Survival in the SOD1G93A Rat Model of Amyotrophic Lateral Sclerosis after Suppression of Mutant SOD1 in the Motor Cortex

PubMed Central

Thomsen, Gretchen M.; Gowing, Genevieve; Latter, Jessica; Chen, Maximus; Vit, Jean-Philippe; Staggenborg, Kevin; Avalos, Pablo; Alkaslasi, Mor; Ferraiuolo, Laura; Likhite, Shibi; Kaspar, Brian K.

2014-01-01

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal disease with unknown etiology, characterized by a progressive loss of motor neurons leading to paralysis and death typically within 3–5 years of onset. Recently, there has been remarkable progress in understanding inherited forms of ALS in which well defined mutations are known to cause the disease. Rodent models in which the superoxide dismutase-1 (SOD1) mutation is overexpressed recapitulate hallmark signs of ALS in patients. Early anatomical changes in mouse models of fALS are seen in the neuromuscular junctions (NMJs) and lower motor neurons, and selective reduction of toxic mutant SOD1 in the spinal cord and muscle of these models has beneficial effects. Therefore, much of ALS research has focused on spinal motor neuron and NMJ aspects of the disease. Here we show that, in the SOD1G93A rat model of ALS, spinal motor neuron loss occurs presymptomatically and before degeneration of ventral root axons and denervation of NMJs. Although overt cell death of corticospinal motor neurons does not occur until disease endpoint, we wanted to establish whether the upper motor neuron might still play a critical role in disease progression. Surprisingly, the knockdown of mutant SOD1 in only the motor cortex of presymptomatic SOD1G93A rats through targeted delivery of AAV9–SOD1–shRNA resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of NMJs. This datum suggests an early dysfunction and thus an important role of the upper motor neuron in this animal model of ALS and perhaps patients with the disease. PMID:25411487

Temporal requirements of the fragile X mental retardation protein in the regulation of synaptic structure.

PubMed

Gatto, Cheryl L; Broadie, Kendal

2008-08-01

Fragile X syndrome (FraX), caused by the loss-of-function of one gene (FMR1), is the most common inherited form of both mental retardation and autism spectrum disorders. The FMR1 product (FMRP) is an mRNA-binding translation regulator that mediates activity-dependent control of synaptic structure and function. To develop any FraX intervention strategy, it is essential to define when and where FMRP loss causes the manifestation of synaptic defects, and whether the reintroduction of FMRP can restore normal synapse properties. In the Drosophila FraX model, dFMRP loss causes neuromuscular junction (NMJ) synapse over-elaboration (overgrowth, overbranching, excess synaptic boutons), accumulation of development-arrested satellite boutons, and altered neurotransmission. We used the Gene-Switch method to conditionally drive dFMRP expression to define the spatiotemporal requirements in synaptic mechanisms. Constitutive induction of targeted neuronal dFMRP at wild-type levels rescues all synaptic architectural defects in Drosophila Fmr1 (dfmr1)-null mutants, demonstrating a presynaptic requirement for synapse structuring. By contrast, presynaptic dFMRP expression does not ameliorate functional neurotransmission defects, indicating a postsynaptic dFMRP requirement. Strikingly, targeted early induction of dFMRP effects nearly complete rescue of synaptic structure defects, showing a primarily early-development role. In addition, acute dFMRP expression at maturity partially alleviates dfmr1-null defects, although rescue is not as complete as either early or constitutive dFMRP expression, showing a modest capacity for late-stage structural plasticity. We conclude that dFMRP predominantly acts early in synaptogenesis to modulate architecture, but that late dFMRP introduction at maturity can weakly compensate for early absence of dFMRP function.

Degeneration of Phrenic Motor Neurons Induces Long-Term Diaphragm Deficits following Mid-Cervical Spinal Contusion in Mice

PubMed Central

Nicaise, Charles; Putatunda, Rajarshi; Hala, Tamara J.; Regan, Kathleen A.; Frank, David M.; Brion, Jean-Pierre; Leroy, Karelle; Pochet, Roland; Wright, Megan C.

2012-01-01

Abstract A primary cause of morbidity and mortality following cervical spinal cord injury (SCI) is respiratory compromise, regardless of the level of trauma. In particular, SCI at mid-cervical regions targets degeneration of both descending bulbospinal respiratory axons and cell bodies of phrenic motor neurons, resulting in deficits in the function of the diaphragm, the primary muscle of inspiration. Contusion-type trauma to the cervical spinal cord is one of the most common forms of human SCI; however, few studies have evaluated mid-cervical contusion in animal models or characterized consequent histopathological and functional effects of degeneration of phrenic motor neuron–diaphragm circuitry. We have generated a mouse model of cervical contusion SCI that unilaterally targets both C4 and C5 levels, the location of the phrenic motor neuron pool, and have examined histological and functional outcomes for up to 6 weeks post-injury. We report that phrenic motor neuron loss in cervical spinal cord, phrenic nerve axonal degeneration, and denervation at diaphragm neuromuscular junctions (NMJ) resulted in compromised ipsilateral diaphragm function, as demonstrated by persistent reduction in diaphragm compound muscle action potential amplitudes following phrenic nerve stimulation and abnormalities in spontaneous diaphragm electromyography (EMG) recordings. This injury paradigm is reproducible, does not require ventilatory assistance, and provides proof-of-principle that generation of unilateral cervical contusion is a feasible strategy for modeling diaphragmatic/respiratory deficits in mice. This study and its accompanying analyses pave the way for using transgenic mouse technology to explore the function of specific genes in the pathophysiology of phrenic motor neuron degeneration and respiratory dysfunction following cervical SCI. PMID:23176637

Functional Motor Recovery from Motoneuron Axotomy Is Compromised in Mice with Defective Corticospinal Projections

PubMed Central

Ding, Yuetong; Qu, Yibo; Feng, Jia; Wang, Meizhi; Han, Qi; So, Kwok-Fai; Wu, Wutian; Zhou, Libing

2014-01-01

Brachial plexus injury (BPI) and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST) is genetically absent. In adult mice, we tore off right C5–C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP) in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs) and reduced peak-to-peak amplitudes in electromyogram (EMG), than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI. PMID:25003601

Unraveling Synaptic GCaMP Signals: Differential Excitability and Clearance Mechanisms Underlying Distinct Ca2+ Dynamics in Tonic and Phasic Excitatory, and Aminergic Modulatory Motor Terminals in Drosophila

PubMed Central

Xing, Xiaomin

2018-01-01

Abstract GCaMP is an optogenetic Ca2+ sensor widely used for monitoring neuronal activities but the precise physiological implications of GCaMP signals remain to be further delineated among functionally distinct synapses. The Drosophila neuromuscular junction (NMJ), a powerful genetic system for studying synaptic function and plasticity, consists of tonic and phasic glutamatergic and modulatory aminergic motor terminals of distinct properties. We report a first simultaneous imaging and electric recording study to directly contrast the frequency characteristics of GCaMP signals of the three synapses for physiological implications. Different GCaMP variants were applied in genetic and pharmacological perturbation experiments to examine the Ca2+ influx and clearance processes underlying the GCaMP signal. Distinct mutational and drug effects on GCaMP signals indicate differential roles of Na+ and K+ channels, encoded by genes including paralytic (para), Shaker (Sh), Shab, and ether-a-go-go (eag), in excitability control of different motor terminals. Moreover, the Ca2+ handling properties reflected by the characteristic frequency dependence of the synaptic GCaMP signals were determined to a large extent by differential capacity of mitochondria-powered Ca2+ clearance mechanisms. Simultaneous focal recordings of synaptic activities further revealed that GCaMPs were ineffective in tracking the rapid dynamics of Ca2+ influx that triggers transmitter release, especially during low-frequency activities, but more adequately reflected cytosolic residual Ca2+ accumulation, a major factor governing activity-dependent synaptic plasticity. These results highlight the vast range of GCaMP response patterns in functionally distinct synaptic types and provide relevant information for establishing basic guidelines for the physiological interpretations of presynaptic GCaMP signals from in situ imaging studies. PMID:29464198

Metabotropic glutamate receptor-mediated use-dependent down-regulation of synaptic excitability involves the fragile X mental retardation protein.

PubMed

Repicky, Sarah; Broadie, Kendal

2009-02-01

Loss of the mRNA-binding protein FMRP results in the most common inherited form of both mental retardation and autism spectrum disorders: fragile X syndrome (FXS). The leading FXS hypothesis proposes that metabotropic glutamate receptor (mGluR) signaling at the synapse controls FMRP function in the regulation of local protein translation to modulate synaptic transmission strength. In this study, we use the Drosophila FXS disease model to test the relationship between Drosophila FMRP (dFMRP) and the sole Drosophila mGluR (dmGluRA) in regulation of synaptic function, using two-electrode voltage-clamp recording at the glutamatergic neuromuscular junction (NMJ). Null dmGluRA mutants show minimal changes in basal synapse properties but pronounced defects during sustained high-frequency stimulation (HFS). The double null dfmr1;dmGluRA mutant shows repression of enhanced augmentation and delayed onset of premature long-term facilitation (LTF) and strongly reduces grossly elevated post-tetanic potentiation (PTP) phenotypes present in dmGluRA-null animals. Null dfmr1 mutants show features of synaptic hyperexcitability, including multiple transmission events in response to a single stimulus and cyclic modulation of transmission amplitude during prolonged HFS. The double null dfmr1;dmGluRA mutant shows amelioration of these defects but does not fully restore wildtype properties in dfmr1-null animals. These data suggest that dmGluRA functions in a negative feedback loop in which excess glutamate released during high-frequency transmission binds the glutamate receptor to dampen synaptic excitability, and dFMRP functions to suppress the translation of proteins regulating this synaptic excitability. Removal of the translational regulator partially compensates for loss of the receptor and, similarly, loss of the receptor weakly compensates for loss of the translational regulator.

Effects of tongue force training on bulbar motor function in the female SOD1-G93A rat model of Amyotrophic Lateral Sclerosis

PubMed Central

Ma, Delin; Shuler, Jeffrey M.; Kumar, Aishwarya; Stanford, Quincy R.; Tungtur, Sudheer; Nishimune, Hiroshi; Stanford, John A.

2016-01-01

Background The use of exercise in Amyotrophic Lateral Sclerosis (ALS) is controversial. Although moderate exercise appears to be beneficial for limb muscles in ALS, the effects of exercise on bulbar muscles such as the tongue have not been studied. Objective The aim of this study was to determine the effects of tongue force training on bulbar motor function in the SOD1-G93A rat model of ALS. Methods We compared the effects of tongue force training on bulbar motor function and neuromuscular junction (NMJ) innervation in female SOD1-G93A rats and age-matched female wild-type controls. Half of each group underwent afternoon tongue force training sessions, while all rats were tested under minimal force conditions in the mornings. Results Tongue force did not differ between the SOD1-G93A rats and healthy controls during the morning testing sessions, nor was it affected by training. Surprisingly, decreases in tongue motility, the number of licks per session, and body weight were greater in the tongue force-trained SOD1-G93A rats. Forelimb grip force, survival, and denervation of the genioglossus muscle did not differ between the trained and untrained SOD1-G93A rats. Genioglossus innervation was correlated with changes in tongue force but not tongue motility in SOD1-G93A rats at end stage. Conclusions The results indicate a potential deleterious effect of tongue force training on tongue motility in female SOD1-G93A rats. The lack of relationship between genioglossus innervation and tongue motility suggest that factors other than lower motor neuron integrity likely accounted for this effect. PMID:27573800

Non-Aggregating Tau Phosphorylation by Cyclin-Dependent Kinase 5 Contributes to Motor Neuron Degeneration in Spinal Muscular Atrophy

PubMed Central

Miller, Nimrod; Feng, Zhihua; Edens, Brittany M.; Yang, Ben; Shi, Han; Sze, Christie C.; Hong, Benjamin Taige; Su, Susan C.; Cantu, Jorge A.; Topczewski, Jacek; Crawford, Thomas O.; Ko, Chien-Ping; Sumner, Charlotte J.; Ma, Long

2015-01-01

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35−/− compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration. PMID:25878277

[Ocular disturbances in neuromuscular disorders].

PubMed

Pénisson-Besnier, I; Lamirel, C

2008-11-01

Compared with other skeletal muscles, extraocular muscles have fundamentally distinct properties that make them selectively vulnerable to certain neuromuscular disorders. When the oculomotor signs are predominant, their temporal progression allows the clinician to make the distinction between a muscular disease (mitochondrial disorder, oculopharyngeal muscular dystrophy...) and a disorder of the neuromuscular junction (myasthenia gravis, botulism...). In other instances, such as myotonic dystrophy or facioscapulohumeral dystrophy, the ocular signs are not in the forefront but must be recognized by the ophthalmologist as hallmarks of a muscular disorder. In all cases, the collaboration between the neurologist and the ophthalmologist is fruitful.

Serum matrix metalloproteinase-3 levels are elevated in myasthenia gravis.

PubMed

Romi, Fredrik R; Gilhus, Nils Erik; Luckman, Steven P

2008-03-01

MMP-3 is capable of degrading a variety of proteins, including agrin, which plays a critical role in neuromuscular signalling by controlling acetylcholine receptor clustering. The degradation of agrin by MMP-3 may disrupt the neuromuscular junction leading to a failure of neuromuscular transmission and muscle weakness. We have therefore examined the levels of MMP-3 in 116 patients with myasthenia gravis (MG) and 90 healthy controls. A significant elevation in MMP-3 levels was observed in 10% of seronegative and 17% of seropositive MG patients, indicating that MMP-3 may play a pathogenic role in a proportion of MG patients.

Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

PubMed

Garcia, Neus; Priego, Mercedes; Hurtado, Erica; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Lanuza, Maria Angel; Tomàs, Josep

2014-07-01

To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release. © 2014 Anatomical Society.

Progressive Motor Deficit is Mediated by the Denervation of Neuromuscular Junctions and Axonal Degeneration in Transgenic Mice Expressing Mutant (P301S) Tau Protein.

PubMed

Yin, Zhuoran; Valkenburg, Femke; Hornix, Betty E; Mantingh-Otter, Ietje; Zhou, Xingdong; Mari, Muriel; Reggiori, Fulvio; Van Dam, Debby; Eggen, Bart J L; De Deyn, Peter P; Boddeke, Erik

2017-01-01

Tauopathies include a variety of neurodegenerative diseases associated with the pathological aggregation of hyperphosphorylated tau, resulting in progressive cognitive decline and motor impairment. The underlying mechanism for motor deficits related to tauopathy is not yet fully understood. Here, we use a novel transgenic tau mouse line, Tau 58/4, with enhanced neuron-specific expression of P301S mutant tau to investigate the motor abnormalities in association with the peripheral nervous system. Using stationary beam, gait, and rotarod tests, motor deficits were found in Tau 58/4 mice already 3 months after birth, which deteriorated during aging. Hyperphosphorylated tau was detected in the cell bodies and axons of motor neurons. At the age of 9 and 12 months, significant denervation of the neuromuscular junction in the extensor digitorum longus muscle was observed in Tau 58/4 mice, compared to wild-type mice. Muscle hypotrophy was observed in Tau 58/4 mice at 9 and 12 months. Using electron microscopy, we observed ultrastructural changes in the sciatic nerve of 12-month-old Tau 58/4 mice indicative of the loss of large axonal fibers and hypomyelination (assessed by g-ratio). We conclude that the accumulated hyperphosphorylated tau in the axon terminals may induce dying-back axonal degeneration, myelin abnormalities, neuromuscular junction denervation, and muscular atrophy, which may be the mechanisms responsible for the deterioration of the motor function in Tau 58/4 mice. Tau 58/4 mice represent an interesting neuromuscular degeneration model, and the pathological mechanisms might be responsible for motor signs observed in some human tauopathies.

Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome

PubMed Central

O’Connor, Emily; Töpf, Ana; Müller, Juliane S.; Cox, Daniel; Evangelista, Teresinha; Colomer, Jaume; Abicht, Angela; Senderek, Jan; Hasselmann, Oswald; Yaramis, Ahmet; Laval, Steven H.

2016-01-01

Abstract Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes. PMID:27259756

ATF3 expression improves motor function in the ALS mouse model by promoting motor neuron survival and retaining muscle innervation.

PubMed

Seijffers, Rhona; Zhang, Jiangwen; Matthews, Jonathan C; Chen, Adam; Tamrazian, Eric; Babaniyi, Olusegun; Selig, Martin; Hynynen, Meri; Woolf, Clifford J; Brown, Robert H

2014-01-28

ALS is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons and atrophy of distal axon terminals in muscle, resulting in loss of motor function. Motor end plates denervated by axonal retraction of dying motor neurons are partially reinnervated by remaining viable motor neurons; however, this axonal sprouting is insufficient to compensate for motor neuron loss. Activating transcription factor 3 (ATF3) promotes neuronal survival and axonal growth. Here, we reveal that forced expression of ATF3 in motor neurons of transgenic SOD1(G93A) ALS mice delays neuromuscular junction denervation by inducing axonal sprouting and enhancing motor neuron viability. Maintenance of neuromuscular junction innervation during the course of the disease in ATF3/SOD1(G93A) mice is associated with a substantial delay in muscle atrophy and improved motor performance. Although disease onset and mortality are delayed, disease duration is not affected. This study shows that adaptive axonal growth-promoting mechanisms can substantially improve motor function in ALS and importantly, that augmenting viability of the motor neuron soma and maintaining functional neuromuscular junction connections are both essential elements in therapy for motor neuron disease in the SOD1(G93A) mice. Accordingly, effective protection of optimal motor neuron function requires restitution of multiple dysregulated cellular pathways.

Endomicroscopy and electromyography of neuromuscular junctions in situ

PubMed Central

Brown, Rosalind; Dissanayake, Kosala N; Skehel, Paul A; Ribchester, Richard R

2014-01-01

Objective Electromyography (EMG) is used routinely to diagnose neuromuscular dysfunction in a wide range of peripheral neuropathies, myopathies, and neuromuscular degenerative diseases including motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Definitive neurological diagnosis may also be indicated by the analysis of pathological neuromuscular innervation in motor-point biopsies. Our objective in this study was to preempt motor-point biopsy by combining live imaging with electrophysiological analysis of slow degeneration of neuromuscular junctions (NMJs) in vivo. Methods We combined conventional needle electromyography with fiber-optic confocal endomicroscopy (CEM), using an integrated hand-held, 1.5-mm-diameter probe. We utilized as a test bed, various axotomized muscles in the hind limbs of anaesthetized, double-homozygous thy1.2YFP16: WldS mice, which coexpress the Wallerian-degeneration Slow (WldS) protein and yellow fluorescent protein (YFP) in motor neurons. We also tested exogenous vital stains, including Alexa488-α-bungarotoxin; the styryl pyridinium dye 4-Di-2-Asp; and a GFP conjugate of botulinum toxin Type A heavy chain (GFP-HcBoNT/A). Results We show that an integrated EMG/CEM probe is effective in longitudinal evaluation of functional and morphological changes that take place over a 7-day period during axotomy-induced, slow neuromuscular synaptic degeneration. EMG amplitude declined in parallel with overt degeneration of motor nerve terminals. EMG/CEM was safe and effective when nerve terminals and motor endplates were selectively stained with vital dyes. Interpretation Our findings constitute proof-of-concept, based on live imaging in an animal model, that combining EMG/CEM may be useful as a minimally invasive precursor or alternative to motor-point biopsy in neurological diagnosis and for monitoring local administration of potential therapeutics. PMID:25540801

Pathophysiology and immunological profile of myasthenia gravis and its subgroups.

PubMed

Romi, Fredrik; Hong, Yu; Gilhus, Nils Erik

2017-12-01

Myasthenia gravis (MG) is an autoimmune antibody-mediated disease characterized by muscle weakness and fatigability. It is believed that the initial steps triggering humoral immunity in MG take place inside thymic tissue and thymoma. The immune response against one or several epitopes expressed on thymic tissue cells spills over to neuromuscular junction components sharing the same epitope causing humoral autoimmunity and antibody production. The main cause of MG is acetylcholine receptor antibodies. However, many other neuromuscular junction membrane protein targets, intracellular and extracellular proteins are suggested to participate in MG pathophysiology. MG should be divided into subgroups based on clinical presentation and immunology. This includes onset age, clinical characteristics, thymic pathology and antibody profile. The immunological profile of these subgroups is determined by the antibodies present. Copyright © 2017. Published by Elsevier Ltd.

Exercise and neuromodulators: choline and acetylcholine in marathon runners

NASA Technical Reports Server (NTRS)

Conlay, L. A.; Sabounjian, L. A.; Wurtman, R. J.

1992-01-01

Certain neurotransmitters (i.e., acetylcholine, catecholamines, and serotonin) are formed from dietary constituents (i.e., choline, tyrosine and tryptophan). Changing the consumption of these precursors alters release of their respective neurotransmitter products. The neurotransmitter acetylcholine is released from the neuromuscular junction and from brain. It is formed from choline, a common constituent in fish, liver, and eggs. Choline is also incorporated into cell membranes; membranes may likewise serve as an alternative choline source for acetylcholine synthesis. In trained athletes, running a 26 km marathon reduced plasma choline by approximately 40%, from 14.1 to 8.4 uM. Changes of similar magnitude have been shown to reduce acetylcholine release from the neuromuscular junction in vivo. Thus, the reductions in plasma choline associated with strenuous exercise may reduce acetylcholine release, and could thereby affect endurance or performance.

Bayesian analysis of the kinetics of quantal transmitter secretion at the neuromuscular junction.

PubMed

Saveliev, Anatoly; Khuzakhmetova, Venera; Samigullin, Dmitry; Skorinkin, Andrey; Kovyazina, Irina; Nikolsky, Eugeny; Bukharaeva, Ellya

2015-10-01

The timing of transmitter release from nerve endings is considered nowadays as one of the factors determining the plasticity and efficacy of synaptic transmission. In the neuromuscular junction, the moments of release of individual acetylcholine quanta are related to the synaptic delays of uniquantal endplate currents recorded under conditions of lowered extracellular calcium. Using Bayesian modelling, we performed a statistical analysis of synaptic delays in mouse neuromuscular junction with different patterns of rhythmic nerve stimulation and when the entry of calcium ions into the nerve terminal was modified. We have obtained a statistical model of the release timing which is represented as the summation of two independent statistical distributions. The first of these is the exponentially modified Gaussian distribution. The mixture of normal and exponential components in this distribution can be interpreted as a two-stage mechanism of early and late periods of phasic synchronous secretion. The parameters of this distribution depend on both the stimulation frequency of the motor nerve and the calcium ions' entry conditions. The second distribution was modelled as quasi-uniform, with parameters independent of nerve stimulation frequency and calcium entry. Two different probability density functions for the distribution of synaptic delays suggest at least two independent processes controlling the time course of secretion, one of them potentially involving two stages. The relative contribution of these processes to the total number of mediator quanta released depends differently on the motor nerve stimulation pattern and on calcium ion entry into nerve endings.

Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome.

PubMed

O'Connor, Emily; Töpf, Ana; Müller, Juliane S; Cox, Daniel; Evangelista, Teresinha; Colomer, Jaume; Abicht, Angela; Senderek, Jan; Hasselmann, Oswald; Yaramis, Ahmet; Laval, Steven H; Lochmüller, Hanns

2016-08-01

Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain.

Acetylcholine receptor distribution and synapse elimination at the developing neuromuscular junction of mdx mice.

PubMed

Minatel, Elaine; Neto, Humberto Santo; Marques, Maria Julia

2003-11-01

The pattern of innervation of the vertebrate neuromuscular junction is established during early development, when junctions go from multiple to single innervation in the phenomenon of synapse elimination, suggesting that changes at the molecular level in the postsynaptic cell lead to the removal of nerve terminals. The mdx mouse is deficient in dystrophin and associated proteins that are part of the postsynaptic cytoskeleton. We used rhodamine-alpha-bungarotoxin and anti-neurofilament IgG-FITC to stain acetylcholine receptors and nerve terminals of the sternomastoid muscle during postnatal development in mdx and control C57BL/10 mice. Using fluorescence confocal microscopy, we observed that, 7 days after birth, 86.7% of the endplates of mdx mice were monoinnervated (n = 200) compared with 41.4% in control mice (n = 200). By the end of the second postnatal week, all endplates were innervated singly (100% mdx and 94.7% controls, n = 200 per group). These results show that dystrophic fibers achieve single innervation earlier, perhaps because dystrophin or a normal cytoskeletal complex is implicated in this phenomenon.

Autoimmune Neuromuscular Disorders

PubMed Central

Kraker, Jessica; Živković, Saša A

2011-01-01

Autoimmune neuromuscular disorders affecting peripheral nerves, neuromuscular junction or muscle have a wide clinical spectrum with diverse pathogenetic mechanisms. Peripheral nervous system may be targeted in the context of complex immune reactions involving different cytokines, antigen-presenting cells, B cells and different types of T cells. Various immunomodulating and cytotoxic treatments block proliferation or activation of immune cells by different mechanisms attempting to control the response of the immune system and limit target organ injury. Most treatment protocols for autoimmune neuromuscular disorders are based on the use of corticosteroids, intravenous immunoglobulins and plasmapheresis, with cytotoxic agents mostly used as steroid-sparing medications. More recently, development of specific monoclonal antibodies targeting individual cell types allowed a different approach targeting specific immune pathways, but these new treatments are also associated with various adverse effects and their long-term efficacy is still unknown. PMID:22379454

Drosophila Neuronal Injury Follows a Temporal Sequence of Cellular Events Leading to Degeneration at the Neuromuscular Junction

PubMed Central

Lincoln, Barron L.; Alabsi, Sahar H.; Frendo, Nicholas; Freund, Robert; Keller, Lani C.

2015-01-01

Neurodegenerative diseases affect millions of people worldwide, and as the global population ages, there is a critical need to improve our understanding of the molecular and cellular mechanisms that drive neurodegeneration. At the molecular level, neurodegeneration involves the activation of complex signaling pathways that drive the active destruction of neurons and their intracellular components. Here, we use an in vivo motor neuron injury assay to acutely induce neurodegeneration in order to follow the temporal order of events that occur following injury in Drosophila melanogaster. We find that sites of injury can be rapidly identified based on structural defects to the neuronal cytoskeleton that result in disrupted axonal transport. Additionally, the neuromuscular junction accumulates ubiquitinated proteins prior to the neurodegenerative events, occurring at 24 hours post injury. Our data provide insights into the early molecular events that occur during axonal and neuromuscular degeneration in a genetically tractable model organism. Importantly, the mechanisms that mediate neurodegeneration in flies are conserved in humans. Thus, these studies have implications for our understanding of the cellular and molecular events that occur in humans and will facilitate the identification of biomedically relevant targets for future treatments. PMID:26512206

Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

PubMed

Malomouzh, Artem I; Petrov, Konstantin A; Nurullin, Leniz F; Nikolsky, Evgeny E

2015-12-01

Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release. © 2015 International Society for Neurochemistry.

Motor unit number estimates and neuromuscular transmission in the tibialis anterior of master athletes: evidence that athletic older people are not spared from age-related motor unit remodeling.

PubMed

Piasecki, Mathew; Ireland, Alex; Coulson, Jessica; Stashuk, Dan W; Hamilton-Wright, Andrew; Swiecicka, Agnieszka; Rutter, Martin K; McPhee, Jamie S; Jones, David A

2016-10-01

Muscle motor unit numbers decrease markedly in old age, while remaining motor units are enlarged and can have reduced neuromuscular junction transmission stability. However, it is possible that regular intense physical activity throughout life can attenuate this remodeling. The aim of this study was to compare the number, size, and neuromuscular junction transmission stability of tibialis anterior (TA) motor units in healthy young and older men with those of exceptionally active master runners. The distribution of motor unit potential (MUP) size was determined from intramuscular electromyographic signals recorded in healthy male Young (mean ± SD, 26 ± 5 years), Old (71 ± 4 years) and Master Athletes (69 ± 3 years). Relative differences between groups in numbers of motor units was assessed using two methods, one comparing MUP size and muscle cross-sectional area (CSA) determined with MRI, the other comparing surface recorded MUPs with maximal compound muscle action potentials and commonly known as a "motor unit number estimate (MUNE)". Near fiber (NF) jiggle was measured to assess neuromuscular junction transmission stability. TA CSA did not differ between groups. MUNE values for the Old and Master Athletes were 45% and 40%, respectively, of the Young. Intramuscular MUPs of Old and Master Athletes were 43% and 56% larger than Young. NF jiggle was slightly higher in the Master Athletes, with no difference between Young and Old. These results show substantial and similar motor unit loss and remodeling in Master Athletes and Old individuals compared with Young, which suggests that lifelong training does not attenuate the age-related loss of motor units. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis

PubMed Central

Patten, Shunmoogum A.; Aggad, Dina; Martinez, Jose; Tremblay, Elsa; Petrillo, Janet; Armstrong, Gary A.B.; Maios, Claudia; Liao, Meijiang; Ciura, Sorana; Wen, Xiao-Yan; Rafuse, Victor; Ichida, Justin; Zinman, Lorne; Julien, Jean-Pierre; Kabashi, Edor; Robitaille, Richard; Korngut, Lawrence; Parker, J. Alexander

2017-01-01

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease. PMID:29202456

Neuroleptics as therapeutic compounds stabilizing neuromuscular transmission in amyotrophic lateral sclerosis.

PubMed

Patten, Shunmoogum A; Aggad, Dina; Martinez, Jose; Tremblay, Elsa; Petrillo, Janet; Armstrong, Gary Ab; La Fontaine, Alexandre; Maios, Claudia; Liao, Meijiang; Ciura, Sorana; Wen, Xiao-Yan; Rafuse, Victor; Ichida, Justin; Zinman, Lorne; Julien, Jean-Pierre; Kabashi, Edor; Robitaille, Richard; Korngut, Lawrence; Parker, J Alexander; Drapeau, Pierre

2017-11-16

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, fatal disorder with no effective treatment. We used simple genetic models of ALS to screen phenotypically for potential therapeutic compounds. We screened libraries of compounds in C. elegans, validated hits in zebrafish, and tested the most potent molecule in mice and in a small clinical trial. We identified a class of neuroleptics that restored motility in C. elegans and in zebrafish, and the most potent was pimozide, which blocked T-type Ca2+ channels in these simple models and stabilized neuromuscular transmission in zebrafish and enhanced it in mice. Finally, a short randomized controlled trial of sporadic ALS subjects demonstrated stabilization of motility and evidence of target engagement at the neuromuscular junction. Simple genetic models are, thus, useful in identifying promising compounds for the treatment of ALS, such as neuroleptics, which may stabilize neuromuscular transmission and prolong survival in this disease.

Transmitter release in the neuromuscular synapse of the protein kinase C theta-deficient adult mouse.

PubMed

Besalduch, Núria; Santafé, Manel M; Garcia, Neus; Gonzalez, Carmen; Tomás, Marta; Tomás, Josep; Lanuza, Maria A

2011-04-01

We studied structural and functional features of the neuromuscular junction in adult mice (P30) genetically deficient in the protein kinase C (PKC) theta isoform. Confocal and electron microscopy shows that there are no differences in the general morphology of the endplates between PKC theta-deficient and wild-type (WT) mice. Specifically, there is no difference in the density of the synaptic vesicles. However, the myelin sheath is not as thick in the intramuscular nerve fibers of the PKC theta-deficient mice. We found a significant reduction in the size of evoked endplate potentials and in the frequency of spontaneous, asynchronous, miniature endplate potentials in the PKC theta-deficient neuromuscular preparations in comparison with the WT, but the mean amplitude of the spontaneous potentials is not different. These changes indicate that PKC theta has a presynaptic role in the function of adult neuromuscular synapses. Copyright © 2010 Wiley-Liss, Inc.

Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction

PubMed Central

Denker, Annette; Kröhnert, Katharina; Rizzoli, Silvio O

2009-01-01

The synaptic vesicles are organized in distinct populations or ‘pools’: the readily releasable pool (the first vesicles released upon stimulation), the recycling pool (which maintains release under moderate stimulation) and the reserve pool (which is called into action only upon strong, often unphysiological stimulation). A major question in the field is whether the pools consist of biochemically different vesicles or whether the pool tag is a spatial one (with the recycling vesicles found next to the release sites, and the reserve ones farther away). A strong and stable spatial segregation has been proposed in the last decade in the Drosophila larval neuromuscular junction – albeit based solely on light microscopy experiments. We have tested here this hypothesis using electron microscopy (EM) photoconversion. We found the recycling and reserve pools to be thoroughly intermixed at the EM level, indicating that spatial location is irrelevant for the functional properties of the vesicle. PMID:19403600

Simulation of miniature endplate potentials in neuromuscular junctions by using a cellular automaton

NASA Astrophysics Data System (ADS)

Avella, Oscar Javier; Muñoz, José Daniel; Fayad, Ramón

2008-01-01

Miniature endplate potentials are recorded in the neuromuscular junction when the acetylcholine contents of one or a few synaptic vesicles are spontaneously released into the synaptic cleft. Since their discovery by Fatt and Katz in 1952, they have been among the paradigms in neuroscience. Those potentials are usually simulated by means of numerical approaches, such as Brownian dynamics, finite differences and finite element methods. Hereby we propose that diffusion cellular automata can be a useful alternative for investigating them. To illustrate this point, we simulate a miniature endplate potential by using experimental parameters. Our model reproduces the potential shape, amplitude and time course. Since our automaton is able to track the history and interactions of each single particle, it is very easy to introduce non-linear effects with little computational effort. This makes cellular automata excellent candidates for simulating biological reaction-diffusion processes, where no other external forces are involved.

Presynaptic elements involved in the maintenance of the neuromuscular junction

NASA Technical Reports Server (NTRS)

Burrows, G. H.

1984-01-01

Alterations in the neuromuscular junction were observed in rats preceding loss of muscle mass. In view of the possibility that these alterations involve changes in the secretion of myotrophic agents by presynaptic motor neurons, an investigation was undertaken to characterize a neuronall factor which is thought to be involved in the initiation and maintenance of cholinergic synapses. This factor, which is secreted into the incubation medium by NG108-15 neuroblastoma x glioma hybrid cells, induces the aggregation of nicotinic acetylcholine receptors on primary cultures of rat hindlimb myotubes. Previous attempts to purify this factor failed. Extensive washing of the NG108-15 cells with hepes-buffered salt solution followed by short (4 hour) collection times resulted in the collection of incubation medium containing maximal aggregation activity with as little as 5 ug secreted protein per ml of fresh medium. A three-fold increase in specific activity was obtained after anion exchange chromatography.

The microRNA miR-1 regulates a MEF-2 dependent retrograde signal at neuromuscular junctions

PubMed Central

Simon, David J.; Madison, Jon M.; Conery, Annie L.; Thompson-Peer, Katherine L.; Soskis, Michael; Ruvkun, Gary B.; Kaplan, Joshua M.; Kim, John K.

2008-01-01

Summary We show that miR-1, a conserved muscle specific microRNA, regulates aspects of both pre- and post-synaptic function at C. elegans neuromuscular junctions. miR-1 regulates the expression level of two nicotinic acetylcholine receptor (nAChR) subunits (UNC-29 and UNC-63), thereby altering muscle sensitivity to acetylcholine (ACh). miR-1 also regulates the muscle transcription factor MEF-2, which results in altered pre-synaptic ACh secretion, suggesting that MEF-2 activity in muscles controls a retrograde signal. The effect of the MEF-2-dependent retrograde signal on secretion is mediated by the synaptic vesicle protein RAB-3. Finally, acute activation of levamisole-sensitive nAChRs stimulates MEF-2-dependent transcriptional responses, and induces the MEF-2-dependent retrograde signal. We propose that miR-1 refines synaptic function by coupling changes in muscle activity to changes in pre-synaptic function. PMID:18510933

Metabotropic and ionotropic glutamate receptors mediate the modulation of acetylcholine release at the frog neuromuscular junction.

PubMed

Tsentsevitsky, Andrei; Nurullin, Leniz; Nikolsky, Evgeny; Malomouzh, Artem

2017-07-01

There is some evidence that glutamate (Glu) acts as a signaling molecule at vertebrate neuromuscular junctions where acetylcholine (ACh) serves as a neurotransmitter. In this study, performed on the cutaneous pectoris muscle of the frog Rana ridibunda, Glu receptor mechanisms that modulate ACh release processes were analyzed. Electrophysiological experiments showed that Glu reduces both spontaneous and evoked quantal secretion of ACh and synchronizes its release in response to electrical stimulation. Quisqualate, an agonist of ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors and metabotropic Group I mGlu receptors, also exerted Glu-like inhibitory effects on the secretion of ACh but had no effect on the kinetics of quantal release. Quisqualate's inhibitory effect did not occur when a blocker of Group I mGlu receptors (LY 367385) or an inhibitor of phospholipase C (U73122) was present. An increase in the degree of synchrony of ACh quantal release, such as that produced by Glu, was obtained after application of N-methyl-D-aspartic acid (NMDA). The presence of Group I mGlu and NMDA receptors in the neuromuscular synapse was confirmed by immunocytochemistry. Thus, the data suggest that both metabotropic Group I mGlu receptors and ionotropic NMDA receptors are present at the neuromuscular synapse of amphibians, and that the activation of these receptors initiates different mechanisms for the regulation of ACh release from motor nerve terminals. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons.

PubMed

Höltje, Markus; Schulze, Sebastian; Strotmeier, Jasmin; Mahrhold, Stefan; Richter, Karin; Binz, Thomas; Bigalke, Hans; Ahnert-Hilger, Gudrun; Rummel, Andreas

2013-12-01

The modular four domain structure of clostridial neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial neurotoxins circumventing the passage of the blood-brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain HC. The corresponding exchange of the tetanus neurotoxin HC-fragment in botulinum neurotoxin A yielded the novel hybrid molecule AATT which displayed decreased potency at the neuromuscular junction like tetanus neurotoxin but exerted equal activity in cortical neurons compared to botulinum neurotoxin A wild-type. Minimizing the tetanus neurotoxin cell binding domain to its N- or C-terminal half drastically reduced the potencies of AATA and AAAT in cortical neurons indicating that the structural motif mediating sorting of tetanus neurotoxin is predominantly encoded within the entire HC-fragment. However, the reciprocal exchange resulted in TTAA which showed a similar potency as tetanus neurotoxin at the neuromuscular junction indicating that the tetanus neurotoxin portion prevents a high potency as observed for botulinum neurotoxins. In conclusion, clostridial neurotoxin based inactivated drug transporter for targeting central neurons should contain the cell binding domain of tetanus neurotoxin to exert its tropism for the central nervous system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transmitter release and presynaptic Ca2+ currents blocked by the spider toxin omega-Aga-IVA.

PubMed

Protti, D A; Uchitel, O D

1993-12-13

Mammalian neuromuscular transmission is resistant to L and N type calcium channel blockers but very sensitive to a low molecular weight funnel web spider venom toxin, FTX, which selectively blocks P type calcium channels. To further characterize the calcium channels involved in neuromuscular transmission we studied the effect of omega Agatoxin (omega-Aga-IVA) a polypeptide P type channel blocker from the same spider venom. We show that omega-Aga-IVA is a potent and irreversible inhibitor of the presynaptic Ca2+ currents and of acetylcholine release induced by electrical stimulation or by K+ depolarization. This provides further evidences that transmitter release at the mammalian neuromuscular junction is mediated by P type Ca2+ channels.

Treating pediatric neuromuscular disorders: The future is now

PubMed Central

D. Gonorazky, Hernan; Cohn, Ronald D.; Campbell, Craig

2017-01-01

Pediatric neuromuscular diseases encompass all disorders with onset in childhood and where the primary area of pathology is in the peripheral nervous system. These conditions are largely genetic in etiology, and only those with a genetic underpinning will be presented in this review. This includes disorders of the anterior horn cell (e.g., spinal muscular atrophy), peripheral nerve (e.g., Charcot–Marie–Tooth disease), the neuromuscular junction (e.g., congenital myasthenic syndrome), and the muscle (myopathies and muscular dystrophies). Historically, pediatric neuromuscular disorders have uniformly been considered to be without treatment possibilities and to have dire prognoses. This perception has gradually changed, starting in part with the discovery and widespread application of corticosteroids for Duchenne muscular dystrophy. At present, several exciting therapeutic avenues are under investigation for a range of conditions, offering the potential for significant improvements in patient morbidities and mortality and, in some cases, curative intervention. In this review, we will present the current state of treatment for the most common pediatric neuromuscular conditions, and detail the treatment strategies with the greatest potential for helping with these devastating diseases. PMID:28889642

Modeling and Identification of a Realistic Spiking Neural Network and Musculoskeletal Model of the Human Arm, and an Application to the Stretch Reflex.

PubMed

Sreenivasa, Manish; Ayusawa, Ko; Nakamura, Yoshihiko

2016-05-01

This study develops a multi-level neuromuscular model consisting of topological pools of spiking motor, sensory and interneurons controlling a bi-muscular model of the human arm. The spiking output of motor neuron pools were used to drive muscle actions and skeletal movement via neuromuscular junctions. Feedback information from muscle spindles were relayed via monosynaptic excitatory and disynaptic inhibitory connections, to simulate spinal afferent pathways. Subject-specific model parameters were identified from human experiments by using inverse dynamics computations and optimization methods. The identified neuromuscular model was used to simulate the biceps stretch reflex and the results were compared to an independent dataset. The proposed model was able to track the recorded data and produce dynamically consistent neural spiking patterns, muscle forces and movement kinematics under varying conditions of external forces and co-contraction levels. This additional layer of detail in neuromuscular models has important relevance to the research communities of rehabilitation and clinical movement analysis by providing a mathematical approach to studying neuromuscular pathology.

RESEARCH ON EFFECTS OF ACETYLCHOLINE ON THE MAMMALIAN MOTOR END-PLATE

DTIC Science & Technology

presumably through the release of a chemical mediator. (3) The chemical sensitivity of end-plate receptors in muscles from patients with myasthenia ... gravis is normal, suggesting that the neuromuscular defect in this disease is of pre-junctional origin.

SNT-1 functions as the Ca2+ sensor for tonic and evoked neurotransmitter release in C. elegans.

PubMed

Li, Lei; Liu, Haowen; Wang, Wei; Chandra, Mintu; Collins, Brett M; Hu, Zhitao

2018-05-14

Synaptotagmin-1 (Syt1) binds Ca 2+ through its tandem C2 domains (C2A and C2B) and triggers Ca 2+ -dependent neurotransmitter release. Here we show that snt-1 , the homolog of mammalian Syt1, functions as the Ca 2+ sensor for both tonic and evoked neurotransmitter release at the C. elegans neuromuscular junction. Mutations that disrupt Ca 2+ binding in double C2 domains of SNT-1 significantly impaired tonic release, whereas disrupting Ca 2+ binding in a single C2 domain had no effect, indicating that the Ca 2+ binding of the two C2 domains is functionally redundant for tonic release. Stimulus-evoked release was significantly reduced in snt-1 mutants, with prolonged release latency as well as faster rise and decay kinetics. Unlike tonic release, evoked release was triggered by Ca 2+ binding solely to the C2B domain. Moreover, we showed that SNT-1 plays an essential role in the priming process in different subpopulations of synaptic vesicles with tight or loose coupling to Ca 2+ entry. SIGNIFICANCE STATEMENT We showed that SNT-1 in C. elegans regulates evoked neurotransmitter release through Ca 2+ binding to its C2B domain, a similar way to Syt1 in the mouse CNS and the fly NMJ. However, the largely decreased tonic release in snt-1 mutants argues SNT-1 has a clamping function. Indeed, Ca 2+ -binding mutations in the C2 domains in SNT-1 significantly reduced the frequency of the miniature excitatory postsynaptic current (mEPSC), indicating that SNT-1 also acts as a Ca 2+ sensor for tonic release. Therefore, revealing the differential mechanisms between invertebrates and vertebrates will provide significant insights into our understanding how synaptic vesicle fusion is regulated. Copyright © 2018 the authors.

Delayed disease onset and extended survival in the SOD1G93A rat model of amyotrophic lateral sclerosis after suppression of mutant SOD1 in the motor cortex.

PubMed

Thomsen, Gretchen M; Gowing, Genevieve; Latter, Jessica; Chen, Maximus; Vit, Jean-Philippe; Staggenborg, Kevin; Avalos, Pablo; Alkaslasi, Mor; Ferraiuolo, Laura; Likhite, Shibi; Kaspar, Brian K; Svendsen, Clive N

2014-11-19

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal disease with unknown etiology, characterized by a progressive loss of motor neurons leading to paralysis and death typically within 3-5 years of onset. Recently, there has been remarkable progress in understanding inherited forms of ALS in which well defined mutations are known to cause the disease. Rodent models in which the superoxide dismutase-1 (SOD1) mutation is overexpressed recapitulate hallmark signs of ALS in patients. Early anatomical changes in mouse models of fALS are seen in the neuromuscular junctions (NMJs) and lower motor neurons, and selective reduction of toxic mutant SOD1 in the spinal cord and muscle of these models has beneficial effects. Therefore, much of ALS research has focused on spinal motor neuron and NMJ aspects of the disease. Here we show that, in the SOD1(G93A) rat model of ALS, spinal motor neuron loss occurs presymptomatically and before degeneration of ventral root axons and denervation of NMJs. Although overt cell death of corticospinal motor neurons does not occur until disease endpoint, we wanted to establish whether the upper motor neuron might still play a critical role in disease progression. Surprisingly, the knockdown of mutant SOD1 in only the motor cortex of presymptomatic SOD1(G93A) rats through targeted delivery of AAV9-SOD1-shRNA resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of NMJs. This datum suggests an early dysfunction and thus an important role of the upper motor neuron in this animal model of ALS and perhaps patients with the disease. Copyright © 2014 the authors 0270-6474/14/3415587-14$15.00/0.

The interaction between tropomyosin-related kinase B receptors and presynaptic muscarinic receptors modulates transmitter release in adult rodent motor nerve terminals.

PubMed

Garcia, Neus; Tomàs, Marta; Santafé, Manel M; Besalduch, Nuria; Lanuza, Maria A; Tomàs, Josep

2010-12-08

The neurotrophin brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase B (trkB) and p75(NTR) are present in the nerve terminals on the neuromuscular junctions (NMJs) of the levator auris longus muscle of the adult mouse. Exogenously added BDNF or NT-4 increased evoked ACh release after 3 h. This presynaptic effect (the size of the spontaneous potentials is not affected) is specific because it is not produced by neurotrophin-3 (NT-3) and is prevented by preincubation with trkB-IgG chimera or by pharmacological block of trkB [K-252a (C₂₇H₂₁N₃O₅)] or p75(NTR) [Pep5 (C₈₆H₁₁₁N₂₅O₁₉S₂] signaling. The effect of BDNF depends on the M₁ and M₂ muscarinic acetylcholine autoreceptors (mAChRs) because it is prevented by atropine, pirenzepine and methoctramine. We found that K-252a incubation reduces ACh release (~50%) in a short time (1 h), but the p75(NTR) signaling inhibitor Pep5 does not have this effect. The specificity of the K-252a blocking effect on trkB was confirmed with the anti-trkB antibody 47/trkB, which reduces evoked ACh release, like K-252a, whereas the nonpermeant tyrosine kinase blocker K-252b does not. Neither does incubation with the fusion protein trkB-IgG (to chelate endogenous BDNF/NT-4), anti-BDNF or anti-NT-4 change ACh release. Thus, the trkB receptor normally seems to be coupled to ACh release when there is no short-term local effect of neurotrophins at the NMJ. The normal function of the mAChR mechanism is a permissive prerequisite for the trkB pathway to couple to ACh release. Reciprocally, the normal function of trkB modulates M₁- and M₂-subtype muscarinic pathways.

GNX-4728, a novel small molecule drug inhibitor of mitochondrial permeability transition, is therapeutic in a mouse model of amyotrophic lateral sclerosis

PubMed Central

Martin, Lee J.; Fancelli, Daniele; Wong, Margaret; Niedzwiecki, Mark; Ballarini, Marco; Plyte, Simon; Chang, Qing

2014-01-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder in humans characterized by progressive degeneration of skeletal muscle and motor neurons in spinal cord, brainstem, and cerebral cortex causing skeletal muscle paralysis, respiratory insufficiency, and death. There are no cures or effective treatments for ALS. ALS can be inherited, but most cases are not associated with a family history of the disease. Mitochondria have been implicated in the pathogenesis but definitive proof of causal mechanisms is lacking. Identification of new clinically translatable disease mechanism-based molecular targets and small molecule drug candidates are needed for ALS patients. We tested the hypothesis in an animal model that drug modulation of the mitochondrial permeability transition pore (mPTP) is therapeutic in ALS. A prospective randomized placebo-controlled drug trial was done in a transgenic (tg) mouse model of ALS. We explored GNX-4728 as a therapeutic drug. GNX-4728 inhibits mPTP opening as evidenced by increased mitochondrial calcium retention capacity (CRC) both in vitro and in vivo. Chronic systemic treatment of G37R-human mutant superoxide dismutase-1 (hSOD1) tg mice with GNX-4728 resulted in major therapeutic benefits. GNX-4728 slowed disease progression and significantly improved motor function. The survival of ALS mice was increased significantly by GNX-4728 treatment as evidence by a nearly 2-fold extension of lifespan (360 days–750 days). GNX-4728 protected against motor neuron degeneration and mitochondrial degeneration, attenuated spinal cord inflammation, and preserved neuromuscular junction (NMJ) innervation in the diaphragm in ALS mice. This work demonstrates that a mPTP-acting drug has major disease-modifying efficacy in a preclinical mouse model of ALS and establishes mitochondrial calcium retention, and indirectly the mPTP, as targets for ALS drug development. PMID:25565966

Acute Respiratory Failure Induced by Magnesium Replacement in a 62-Year-Old Woman with Myasthenia Gravis.

PubMed

Singh, Paramveer; Idowu, Olakunle; Malik, Imrana; Nates, Joseph L

2015-10-01

Magnesium is known to act at the neuromuscular junction by inhibiting the presynaptic release of acetylcholine and desensitizing the postsynaptic membrane. Because of these effects, magnesium has been postulated to potentiate neuromuscular weakness. We describe the case of a 62-year-old woman with myasthenia gravis and a metastatic thymoma who was admitted to our intensive care unit for management of a myasthenic crisis. The patient's neuromuscular weakness worsened in association with standard intravenous magnesium replacement, and the exacerbated respiratory failure necessitated intubation, mechanical ventilation, and an extended stay in the intensive care unit. The effect of magnesium replacement on myasthenia gravis patients has not been well documented, and we present this case to increase awareness and stimulate research. In addition, we discuss the relevant medical literature.

Calcium channel blockers and transmitter release at the normal human neuromuscular junction.

PubMed

Protti, D A; Reisin, R; Mackinley, T A; Uchitel, O D

1996-05-01

Transmitter release evoked by nerve stimulation is highly dependent on Ca2+ entry through voltage-activated plasma membrane channels. Calcium influx may be modified in some neuromuscular diseases like Lambert-Eaton syndrome and amyotrophic lateral sclerosis. We studied the pharmacologic sensitivity of the transmitter release process to different calcium channel blockers in normal human muscles and found that funnel web toxin and omega-Agatoxin-IVA, both P-type calcium channel blockers, blocked nerve-elicited muscle action potentials and inhibited evoked synaptic transmission. The transmitter release was not affected either by nitrendipine, an L-type channel blocker, or omega-Conotoxin-GVIA, an N-type channel blocker. The pharmacologic profile of neuromuscular transmission observed in normal human muscles indicates that P-like channels mediate transmitter release at the motor nerve terminals.

The spectrum of mutations that underlie the neuromuscular junction synaptopathy in DOK7 congenital myasthenic syndrome.

PubMed

Cossins, Judith; Liu, Wei Wei; Belaya, Katsiaryna; Maxwell, Susan; Oldridge, Michael; Lester, Tracy; Robb, Stephanie; Beeson, David

2012-09-01

Congenital myasthenic syndromes (CMS) are a group of inherited diseases that affect synaptic transmission at the neuromuscular junction and result in fatiguable muscle weakness. A subgroup of CMS patients have a recessively inherited limb-girdle pattern of weakness caused by mutations in DOK7. DOK7 encodes DOK7, an adaptor protein that is expressed in the skeletal muscle and heart and that is essential for the development and maintenance of the neuromuscular junction. We have screened the DOK7 gene for mutations by polymerase chain reaction amplification and bi-directional sequencing of exonic and promoter regions and performed acetylcholine receptor (AChR) clustering assays and used exon trapping to determine the pathogenicity of detected variants. Approximately 18% of genetically diagnosed CMSs in the UK have mutations in DOK7, with mutations in this gene identified in more than 60 kinships to date. Thirty-four different pathogenic mutations were identified as well as 27 variants likely to be non-pathogenic. An exon 7 frameshift duplication c.1124_1127dupTGCC is commonly found in at least one allele. We analyse the effect of the common frameshift c.1124_1127dupTGCC and show that 10/11 suspected missense mutations have a deleterious effect on AChR clustering. We identify for the first time homozygous or compound heterozygous mutations that are localized 5' to exon 7. In addition, three silent variants in the N-terminal half of DOK7 are predicted to alter the splicing of the DOK7 RNA transcript. The DOK7 gene is highly polymorphic, and within these many variants, we define a spectrum of mutations that can underlie DOK7 CMS that will inform in managing this disorder.

Activity-Induced Synaptic Structural Modifications by an Activator of Integrin Signaling at the Drosophila Neuromuscular Junction.

PubMed

Lee, Joo Yeun; Geng, Junhua; Lee, Juhyun; Wang, Andrew R; Chang, Karen T

2017-03-22

Activity-induced synaptic structural modification is crucial for neural development and synaptic plasticity, but the molecular players involved in this process are not well defined. Here, we report that a protein named Shriveled (Shv) regulates synaptic growth and activity-dependent synaptic remodeling at the Drosophila neuromuscular junction. Depletion of Shv causes synaptic overgrowth and an accumulation of immature boutons. We find that Shv physically and genetically interacts with βPS integrin. Furthermore, Shv is secreted during intense, but not mild, neuronal activity to acutely activate integrin signaling, induce synaptic bouton enlargement, and increase postsynaptic glutamate receptor abundance. Consequently, loss of Shv prevents activity-induced synapse maturation and abolishes post-tetanic potentiation, a form of synaptic plasticity. Our data identify Shv as a novel trans-synaptic signal secreted upon intense neuronal activity to promote synapse remodeling through integrin receptor signaling. SIGNIFICANCE STATEMENT The ability of neurons to rapidly modify synaptic structure in response to neuronal activity, a process called activity-induced structural remodeling, is crucial for neuronal development and complex brain functions. The molecular players that are important for this fundamental biological process are not well understood. Here we show that the Shriveled (Shv) protein is required during development to maintain normal synaptic growth. We further demonstrate that Shv is selectively released during intense neuronal activity, but not mild neuronal activity, to acutely activate integrin signaling and trigger structural modifications at the Drosophila neuromuscular junction. This work identifies Shv as a key modulator of activity-induced structural remodeling and suggests that neurons use distinct molecular cues to differentially modulate synaptic growth and remodeling to meet synaptic demand. Copyright © 2017 the authors 0270-6474/17/373246-18$15.00/0.

Stabilization of acetylcholine receptors at the neuromuscular synapse: the role of the nerve.

PubMed

Ramsay, D A; Drachman, D B; Drachman, R J; Stanley, E F

1992-05-29

The majority of acetylcholine receptors (AChRs) at innervated neuromuscular junctions (NMJs) are stable, with half-lives averaging about 11 days in rodent muscles. In addition to the stable AChRs, approximately 18% of AChRs at these innervated junctions are rapidly turned over (RTOs), with half lives of less than 24 h. We have postulated that RTOs may be precursors of stable AChRs, and that the motor nerve may influence their stabilization. This hypothesis was tested by: (i) labeling AChRs in mouse sternomastoid (SM) muscles with 125I-alpha-BuTx; (ii) denervating one SM muscle in each mouse, and (iii) following the fate of the labeled AChRs through a 5-day period when RTOs were either stabilized or degraded. The hypothesis predicts that denervation should preclude stabilization of RTOs, resulting in a deficit of stable AChRs in denervated muscles. The results showed a highly significant (P less than 0.002) deficit of stable AChRs in denervated as compared with innervated muscles. Control experiments excluded the possibility that this deficit could be attributed to independent accelerated degradation of either RTOs or pre-existing stable AChRs. The observed deficit was quantitatively consistent with the deficit predicted by a mathematical model based on interruption of stabilization following denervation. We conclude that: (i) the observed deficit after denervation of NMJs is due to failure of stabilization of pre-existing RTOs; (ii) RTOs at normally innervated NMJs are precursors of stable AChRs; (iii) stabilization occurs after the insertion of AChRs at NMJs, and (iv) motor nerves play a key role in stabilization of RTOs. The concept of receptor stabilization has important implications for understanding the biology of the neuromuscular junction and post-synaptic plasticity.

LL5beta: a regulator of postsynaptic differentiation identified in a screen for synaptically enriched transcripts at the neuromuscular junction.

PubMed

Kishi, Masashi; Kummer, Terrance T; Eglen, Stephen J; Sanes, Joshua R

2005-04-25

In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5beta, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5beta is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in "corraling" AChRs. Consistent with this idea, perturbing LL5beta expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.

Presynaptic active zones of mammalian neuromuscular junctions: Nanoarchitecture and selective impairments in aging.

PubMed

Badawi, Yomna; Nishimune, Hiroshi

2018-02-01

Neurotransmitter release occurs at active zones, which are specialized regions of the presynaptic membrane. A dense collection of proteins at the active zone provides a platform for molecular interactions that promote recruitment, docking, and priming of synaptic vesicles. At mammalian neuromuscular junctions (NMJs), muscle-derived laminin β2 interacts with presynaptic voltage-gated calcium channels to organize active zones. The molecular architecture of presynaptic active zones has been revealed using super-resolution microscopy techniques that combine nanoscale resolution and multiple molecular identification. Interestingly, the active zones of adult NMJs are not stable structures and thus become impaired during aging due to the selective degeneration of specific active zone proteins. This review will discuss recent progress in the understanding of active zone nanoarchitecture and the mechanisms underlying active zone organization in mammalian NMJs. Furthermore, we will summarize the age-related degeneration of active zones at NMJs, and the role of exercise in maintaining active zones. Copyright © 2017 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

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

PubMed

Nishimune, Hiroshi

2012-12-01

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 β2 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 β2 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. © 2012 New York Academy of Sciences.

ProBDNF and mature BDNF as punishment and reward signals for synapse elimination at mouse neuromuscular junctions.

PubMed

Je, H Shawn; Yang, Feng; Ji, Yuanyuan; Potluri, Srilatha; Fu, Xiu-Qing; Luo, Zhen-Ge; Nagappan, Guhan; Chan, Jia Pei; Hempstead, Barbara; Son, Young-Jin; Lu, Bai

2013-06-12

During development, mammalian neuromuscular junctions (NMJs) transit from multiple-innervation to single-innervation through axonal competition via unknown molecular mechanisms. Previously, using an in vitro model system, we demonstrated that the postsynaptic secretion of pro-brain-derived neurotrophic factor (proBDNF) stabilizes or eliminates presynaptic axon terminals, depending on its proteolytic conversion at synapses. Here, using developing mouse NMJs, we obtained in vivo evidence that proBDNF and mature BDNF (mBDNF) play roles in synapse elimination. We observed that exogenous proBDNF promoted synapse elimination, whereas mBDNF infusion substantially delayed synapse elimination. In addition, pharmacological inhibition of the proteolytic conversion of proBDNF to mBDNF accelerated synapse elimination via activation of p75 neurotrophin receptor (p75(NTR)). Furthermore, the inhibition of both p75(NTR) and sortilin signaling attenuated synapse elimination. We propose a model in which proBDNF and mBDNF serve as potential "punishment" and "reward" signals for inactive and active terminals, respectively, in vivo.

Cellular Uptake of Aminoglycosides

ERIC Educational Resources Information Center

Steyger, Peter S.

2005-01-01

Aminoglycosides exert their cytotoxic effect at three different locations: at the cell surface, in the cytosol, or in the nucleus. At the cell surface, aminoglycoside binding can cause temporary hearing loss, motor paralysis at the neuromuscular junction, ion wasting in kidneys, or analgesia in mechano- and nocioreceptors (touch and pain sensory…

A robust neuromuscular system protects rat and human skeletal muscle from sarcopenia.

PubMed

Pannérec, Alice; Springer, Margherita; Migliavacca, Eugenia; Ireland, Alex; Piasecki, Mathew; Karaz, Sonia; Jacot, Guillaume; Métairon, Sylviane; Danenberg, Esther; Raymond, Frédéric; Descombes, Patrick; McPhee, Jamie S; Feige, Jerome N

2016-04-01

Declining muscle mass and function is one of the main drivers of loss of independence in the elderly. Sarcopenia is associated with numerous cellular and endocrine perturbations, and it remains challenging to identify those changes that play a causal role and could serve as targets for therapeutic intervention. In this study, we uncovered a remarkable differential susceptibility of certain muscles to age-related decline. Aging rats specifically lose muscle mass and function in the hindlimbs, but not in the forelimbs. By performing a comprehensive comparative analysis of these muscles, we demonstrate that regional susceptibility to sarcopenia is dependent on neuromuscular junction fragmentation, loss of motoneuron innervation, and reduced excitability. Remarkably, muscle loss in elderly humans also differs in vastus lateralis and tibialis anterior muscles in direct relation to neuromuscular dysfunction. By comparing gene expression in susceptible and non-susceptible muscles, we identified a specific transcriptomic signature of neuromuscular impairment. Importantly, differential molecular profiling of the associated peripheral nerves revealed fundamental changes in cholesterol biosynthetic pathways. Altogether our results provide compelling evidence that susceptibility to sarcopenia is tightly linked to neuromuscular decline in rats and humans, and identify dysregulation of sterol metabolism in the peripheral nervous system as an early event in this process.

Age-related deficits in skeletal muscle recovery following disuse are associated with neuromuscular junction instability and ER stress, not impaired protein synthesis.

PubMed

Baehr, Leslie M; West, Daniel W D; Marcotte, George; Marshall, Andrea G; De Sousa, Luis Gustavo; Baar, Keith; Bodine, Sue C

2016-01-01

Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength.

Age-related deficits in skeletal muscle recovery following disuse are associated with neuromuscular junction instability and ER stress, not impaired protein synthesis

PubMed Central

Baehr, Leslie M.; West, Daniel W.D.; Marcotte, George; Marshall, Andrea G.; De Sousa, Luis Gustavo; Baar, Keith; Bodine, Sue C.

2016-01-01

Age-related loss of muscle mass and strength can be accelerated by impaired recovery of muscle mass following a transient atrophic stimulus. The aim of this study was to identify the mechanisms underlying the attenuated recovery of muscle mass and strength in old rats following disuse-induced atrophy. Adult (9 month) and old (29 month) male F344BN rats underwent hindlimb unloading (HU) followed by reloading. HU induced significant atrophy of the hindlimb muscles in both adult (17-38%) and old (8-29%) rats, but only the adult rats exhibited full recovery of muscle mass and strength upon reloading. Upon reloading, total RNA and protein synthesis increased to a similar extent in adult and old muscles. At baseline and upon reloading, however, proteasome-mediated degradation was suppressed leading to an accumulation of ubiquitin-tagged proteins and p62. Further, ER stress, as measured by CHOP expression, was elevated at baseline and upon reloading in old rats. Analysis of mRNA expression revealed increases in HDAC4, Runx1, myogenin, Gadd45a, and the AChRs in old rats, suggesting neuromuscular junction instability/denervation. Collectively, our data suggests that with aging, impaired neuromuscular transmission and deficits in the proteostasis network contribute to defects in muscle fiber remodeling and functional recovery of muscle mass and strength. PMID:26826670

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

USDA-ARS?s Scientific Manuscript database

Botulinum neurotoxins (BoNT) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family ...

Effect of Chlorpromazine on the Toxicity in Mice of the Venoms and Neurotoxins from Various Snakes

DTIC Science & Technology

1988-07-26

the neuromuscular junction and blocks muscle contraction stimulated by acetylcholine. P-Bungarotoxin is a presynaptic neurotoxin which inhibits the...release of acetylcholine from neurons, also blocking muscle contraction (Chang, 1985). The two toxins work in concert to cause respiratory failure

Acute lower motor neuron tetraparesis.

PubMed

Añor, Sònia

2014-11-01

Flaccid nonambulatory tetraparesis or tetraplegia is an infrequent neurologic presentation; it is characteristic of neuromuscular disease (lower motor neuron [LMN] disease) rather than spinal cord disease. Paresis beginning in the pelvic limbs and progressing to the thoracic limbs resulting in flaccid tetraparesis or tetraplegia within 24 to 72 hours is a common presentation of peripheral nerve or neuromuscular junction disease. Complete body flaccidity develops with severe decrease or complete loss of spinal reflexes in pelvic and thoracic limbs. Animals with acute generalized LMN tetraparesis commonly show severe motor dysfunction in all limbs and severe generalized weakness in all muscles. Copyright © 2014 Elsevier Inc. All rights reserved.

β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum

PubMed Central

Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J.; Mikami, Dean J.

2015-01-01

Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

Optimized Liposomal Dexamethasone Therapy Improves Functional Outcome of Post-Traumatic Skeletal Muscle and Neuromuscular Junction

DTIC Science & Technology

saline, tourniquet-induced IR+lipo-Dex, and tourniquet-induced IR+Dex groups. These experiments tested the protective effects of lipo-Dex and Dex on...skeletal muscle morphology and function in mice with tourniquet-induced IR. Lipo-Dex is liposome-encapsulated Dex. Our study found that lipo-Dex was

The effects of a sublethal dose of botulinum serotype E on the swimming performance of channel catfish fingerlings

USDA-ARS?s Scientific Manuscript database

Visceral toxicosis of catfish (VTC) is a disease of cultured Channel Catfish Ictalurus punctatus in the Mississippi Delta region and surrounding states. The etiology of VTC is associated with botulinum serotype E (BoNT/E), which causes blockage of acetylcholine release at the neuromuscular junction,...

The relationship of choline acetyltransferase activity at the neuromuscular junction to changes in muscle mass and function

PubMed Central

Diamond, Ivan; Franklin, Gary M.; Milfay, Dale

1974-01-01

1. The role of muscle mass and function in the regulation of choline acetyltransferase activity at the neuromuscular junction has been investigated in the rat. 2. Choline acetyltransferase (ChAc) is located in presynaptic nerve terminals and is a specific enzymatic marker of cholinergic innervation in muscle. 3. ChAc activity increased co-ordinately with developmental growth of the soleus muscle. However, another form of muscle growth, work hypertrophy, did not produce an increase in ChAc. 4. Growth arrest of muscle by hypophysectomy did not alter the normal development of ChAc activity, and cortisone-induced muscle atrophy did not reduce ChAc activity in the soleus or plantaris. 5. Tenotomy-induced muscle atrophy provoked a significant fall in ChAc in the soleus and plantaris. 6. The tonic soleus had significantly greater ChAc activity than the phasic plantaris. 7. These observations suggest that muscle mass per se does not influence the development and regulation of ChAc in muscle but that the quality of muscle contraction may modulate enzyme activity. PMID:4818500

Fatigue and neuromuscular diseases.

PubMed

Féasson, L; Camdessanché, J-P; El Mandhi, L; Calmels, P; Millet, G-Y

2006-07-01

To identify the role of fatigue, its evaluation and its causes in the pathophysiology context of acquired or hereditary neuromuscular diseases of the spinal anterior horn cell, peripheral nerve, neuromuscular junction and muscle. A literature review has been done on Medline with the following keywords: neuromuscular disease, peripheral neuropathy, myopathy, fatigue assessment, exercise intolerance, force assessment, fatigue scale and questionnaire, then with the terms: Fatigue Severity Scale, Chalder Fatigue Scale, Fatigue Questionnaire, Piper Fatigue Scale, electromyography and the combination of the word Fatigue with the following terms: Amyotrophic Lateral Sclerosis (ALS), Post-Polio Syndrome (PPS), Guillain-Barre Syndrome, Immune Neuropathy, Charcot-Marie-Tooth Disease, Myasthenia Gravis (MG), Metabolic Myopathy, Mitochondrial Myopathy, Muscular Dystrophy, Facioscapulohumeral Dystrophy, Myotonic Dystrophy. Fatigue is a symptom very frequently reported by patients. Fatigue is mainly evaluated by strength loss after an exercise, by change in electromyographic activity during a given exercise and by questionnaires that takes into account the subjective (psychological) part of fatigue. Due to the large diversity of motor disorders, there are multiple clinical expressions of fatigue that differ in their presentation, consequences and therapeutic approach. This review shows that fatigue has to be taken into account in patients with neuromuscular diseases. In this context, pathophysiology of fatigue often implies the motor component but the disease evolution and the physical obligates of daily life also induce an important psychological component.

Imaging three-dimensional innervation zone distribution in muscles from M-wave recordings

NASA Astrophysics Data System (ADS)

Zhang, Chuan; Peng, Yun; Liu, Yang; Li, Sheng; Zhou, Ping; Zev Rymer, William; Zhang, Yingchun

2017-06-01

Objective. To localize neuromuscular junctions in skeletal muscles in vivo which is of great importance in understanding, diagnosing and managing of neuromuscular disorders. Approach. A three-dimensional global innervation zone imaging technique was developed to characterize the global distribution of innervation zones, as an indication of the location and features of neuromuscular junctions, using electrically evoked high-density surface electromyogram recordings. Main results. The performance of the technique was evaluated in the biceps brachii of six intact human subjects. The geometric centers of the distributions of the reconstructed innervation zones were determined with a mean distance of 9.4  ±  1.4 cm from the reference plane, situated at the medial epicondyle of the humerus. A mean depth was calculated as 1.5  ±  0.3 cm from the geometric centers to the closed points over the skin. The results are consistent with those reported in previous histology studies. It was also found that the volumes and distributions of the reconstructed innervation zones changed as the stimulation intensities increased until the supramaximal muscle response was achieved. Significance. Results have demonstrated the high performance of the proposed imaging technique in noninvasively imaging global distributions of the innervation zones in the three-dimensional muscle space in vivo, and the feasibility of its clinical applications, such as guiding botulinum toxin injections in spasticity management, or in early diagnosis of neurodegenerative progression of amyotrophic lateral sclerosis.

Decreased phosphorylation of δ and ε subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC θ deficient mouse.

PubMed

Lanuza, Maria A; Besalduch, Núria; González, Carmen; Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Nelson, Phillip G; Tomàs, Josep

2010-09-01

Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCtheta) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCtheta deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCtheta deficient mice coincides with a reduction in the PKCtheta-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCtheta regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the theta isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCtheta and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters. Copyright 2010 Elsevier Inc. All rights reserved.

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction.

PubMed

Ohno, Kinji; Rahman, Mohammad Alinoor; Nazim, Mohammad; Nasrin, Farhana; Lin, Yingni; Takeda, Jun-Ichi; Masuda, Akio

2017-08-01

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChE T , AChE H , and AChE R are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

Early Functional Deficit and Microglial Disturbances in a Mouse Model of Amyotrophic Lateral Sclerosis

PubMed Central

Rabano, Miriam; Vivanco, Maria d M; Perrin, Florence Evelyne

2012-01-01

Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by selective motoneurons degeneration. There is today no clear-cut pathogenesis sequence nor any treatment. However growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. To better characterize the time course of pathological events in an animal model that recapitulates human ALS symptoms, we investigated functional and cellular characteristics of hSOD1G93A mice. Methods and Findings We have evaluated locomotor function of hSOD1G93A mice through dynamic walking patterns and spontaneous motor activity analysis. We detected early functional deficits that redefine symptoms onset at 60 days of age, i.e. 20 days earlier than previously described. Moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. To tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. We show that (1) decrease in neuromuscular junction's number correlates with motor impairment, (2) astrocytes number is not altered at pre- and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and (3) microglia modifications precede disease onset. At pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. Conclusions In conclusion, precise motor analysis updates the onset of the disease in hSOD1G93A mice and allows locomotor monitoring until the end stage of the disease. Early functional deficits coincide with alterations of neuromuscular junctions. Importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. This finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. PMID:22558300

Synaptic Failure: Focus in an Integrative View of ALS

PubMed Central

Casas, Caty; Manzano, Raquel; Vaz, Rita; Osta, Rosario; Brites, Dora

2015-01-01

From early description by Charcot, the classification of the Amyotrophic Lateral Sclerosis (ALS) is evolving from a subtype of Motor Neuron (MN) Disease to be considered rather a multi-systemic, non-cell autonomous and complex neurodegenerative disease. In the last decade, the huge amount of knowledge acquired has shed new insights on the pathological mechanisms underlying ALS from different perspectives. However, a whole vision on the multiple dysfunctional pathways is needed with the inclusion of information often excluded in other published revisions. We propose an integrative view of ALS pathology, although centered on the synaptic failure as a converging and crucial player to the etiology of the disease. Homeostasis of input and output synaptic activity of MNs has been proved to be severely and early disrupted and to definitively contribute to microcircuitry alterations at the spinal cord. Several cells play roles in synaptic communication across the MNs network system such as interneurons, astrocytes, microglia, Schwann and skeletal muscle cells. Microglia are described as highly dynamic surveying cells of the nervous system but also as determinant contributors to the synaptic plasticity linked to neuronal activity. Several signaling axis such as TNFα/TNFR1 and CX3CR1/CX3CL1 that characterize MN-microglia cross talk contribute to synaptic scaling and maintenance, have been found altered in ALS. The presence of dystrophic and atypical microglia in late stages of ALS, with a decline in their dynamic motility and phagocytic ability, together with less synaptic and neuronal contacts disrupts the MN-microglia dialogue, decreases homeostatic regulation of neuronal activity, perturbs “on/off” signals and accelerates disease progression associated to impaired synaptic function and regeneration. Other hotspot in the ALS affected network system is the unstable neuromuscular junction (NMJ) leading to distal axonal degeneration. Reduced neuromuscular spontaneous synaptic activity in ALS mice models was also suggested to account for the selective vulnerability of MNs and decreased regenerative capability. Synaptic destabilization may as well derive from increased release of molecules by muscle cells (e.g. NogoA) and by terminal Schwann cells (e.g. semaphorin 3A) conceivably causing nerve terminal retraction and denervation, as well as inhibition of re-connection to muscle fibers. Indeed, we have overviewed the alterations on the metabolic pathways and self-regenerative capacity presented in skeletal muscle cells that contribute to muscle wasting in ALS. Finally, a detailed footpath of pathologic changes on MNs and associated dysfunctional and synaptic alterations is provided. The oriented motivation in future ALS studies as outlined in the present article will help in fruitful novel achievements on the mechanisms involved and in developing more target-driven therapies that will bring new hope in halting or delaying disease progression in ALS patients. PMID:29765840

Neuromuscular disease and respiratory physiology in children: putting lung function into perspective.

PubMed

Fauroux, Brigitte; Khirani, Sonia

2014-08-01

Neuromuscular diseases represent a heterogeneous group of disorders of the muscle, nerve or neuromuscular junction. The respiratory muscles are rarely spared in neuromuscular diseases even if the type of muscle involvement, severity and time course greatly varies among the different diseases. Diagnosis of respiratory muscle weakness is crucial because of the importance of respiratory morbidity and mortality. Presently, routine respiratory evaluation is based on non-invasive volitional tests, such as the measurement of lung volumes, spirometry and the maximal static pressures, which may be difficult or impossible to obtain in some young children. Other tools or parameters are thus needed to assess the respiratory muscle weakness and its consequences in young children. The measurement of oesogastric pressures can be helpful as they allow the diagnosis and quantification of paradoxical breathing, as well as the assessment of the strength of the inspiratory and expiratory muscles by means of the oesophageal pressure during a maximal sniff and of the gastric pressure during a maximal cough. Sleep assessment should also be part of the respiratory evaluation of children with neuromuscular disease with at least the recording of nocturnal gas exchange if polysomnography is not possible or unavailable. This improvement in the assessment of respiratory muscle performance may increase our understanding of the respiratory pathophysiology of the different neuromuscular diseases, improve patient care, and guide research and innovative therapies by identifying and validating respiratory parameters. © 2014 Asian Pacific Society of Respirology.

Effect of complement and its regulation on myasthenia gravis pathogenesis

PubMed Central

Kusner, Linda L; Kaminski, Henry J; Soltys, Jindrich

2015-01-01

Myasthenia gravis (MG) is primarily caused by antibodies directed towards the skeletal muscle acetylcholine receptor, leading to muscle weakness. Although these antibodies may induce compromise of neuromuscular transmission by blocking acetylcholine receptor function or antigenic modulation, the predominant mechanism of injury to the neuromuscular junction is complement-mediated lysis of the postsynaptic membrane. The vast majority of data to support the role of complement derives from experimentally acquired MG (EAMG). In this article, we review studies that demonstrate the central role of complement in EAMG and MG pathogenesis along with the emerging role of complement in T- and B-cell function, as well as the potential for complement inhibitor-based therapy to treat human MG. PMID:20477586

Medical Administration: Patient Regulating To and Within the Continental United States

DTIC Science & Technology

1990-03-30

the muscles, neuromuscular junctions, peripheral nerves, spinal cord, and brain. APY Electroencephalography See EVALUATIONS. APM Electromyography See...Oxygen Ther- apy See EVALUATIONS. SSTX Kidney Transplants See SURGERY/Urology. SSF Morbid Obesity Surgery Patients requiring surgery for morbid obesity...Scans. APG Cytogenetics Patients having, or suspected of having, genetic abnormalities, or chromosomal study is indi- cated. APY

Fragile X mental retardation protein has a unique, evolutionarily conserved neuronal function not shared with FXR1P or FXR2P

PubMed Central

Coffee, R. Lane; Tessier, Charles R.; Woodruff, Elvin A.; Broadie, Kendal

2010-01-01

SUMMARY Fragile X syndrome (FXS), resulting solely from the loss of function of the human fragile X mental retardation 1 (hFMR1) gene, is the most common heritable cause of mental retardation and autism disorders, with syndromic defects also in non-neuronal tissues. In addition, the human genome encodes two closely related hFMR1 paralogs: hFXR1 and hFXR2. The Drosophila genome, by contrast, encodes a single dFMR1 gene with close sequence homology to all three human genes. Drosophila that lack the dFMR1 gene (dfmr1 null mutants) recapitulate FXS-associated molecular, cellular and behavioral phenotypes, suggesting that FMR1 function has been conserved, albeit with specific functions possibly sub-served by the expanded human gene family. To test evolutionary conservation, we used tissue-targeted transgenic expression of all three human genes in the Drosophila disease model to investigate function at (1) molecular, (2) neuronal and (3) non-neuronal levels. In neurons, dfmr1 null mutants exhibit elevated protein levels that alter the central brain and neuromuscular junction (NMJ) synaptic architecture, including an increase in synapse area, branching and bouton numbers. Importantly, hFMR1 can, comparably to dFMR1, fully rescue both the molecular and cellular defects in neurons, whereas hFXR1 and hFXR2 provide absolutely no rescue. For non-neuronal requirements, we assayed male fecundity and testes function. dfmr1 null mutants are effectively sterile owing to disruption of the 9+2 microtubule organization in the sperm tail. Importantly, all three human genes fully and equally rescue mutant fecundity and spermatogenesis defects. These results indicate that FMR1 gene function is evolutionarily conserved in neural mechanisms and cannot be compensated by either FXR1 or FXR2, but that all three proteins can substitute for each other in non-neuronal requirements. We conclude that FMR1 has a neural-specific function that is distinct from its paralogs, and that the unique FMR1 function is responsible for regulating neuronal protein expression and synaptic connectivity. PMID:20442204

Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications.

PubMed Central

Matthews-Bellinger, J; Salpeter, M M

1978-01-01

1. The distribution of acetylcholine receptors (AChR) at frog cutaneous pectoris neuromuscular junctions was studied quantitatively using [1125]alpha-bungarotoxin (alpha-BTX) labelling and EM autoradiography. 2. We found that, as in mouse end-plates, the AChR is localized uniformly along the thickened post-junctional membrane. In the frog muscle this specialized membrane constitutes approximately the top 50% of the junctional folds. 3. The receptor site density is approximately 26,000 +/- 6000 sites/micrometer2 on the thickened post-junctional membrane and falls sharply to approximately 50 sites/micrometer2 within 15 micrometer from the axon terminal. 4. alpha-BTX site density on the presynaptic axonal membrane was directly determined to be at most 5% of the value on the thickened post-junctional membrane. 5. The high post junctional AChR site density leads us to conclude that: (a) each quantum of ACh needs to spread only over a very small post-junctional area (to be called the 'critical area') before it encounters as many AChR (plus AchE) sites as there are ACh molecules in the quantum (for a packet of 10(4) ACh molecules this critical area is approximately 0.3 micrometer2), (b) the average concentration of ACh prevailing in the cleft over this critical area during a quantal response will be approximately 10(-3)M (independent of the size of the quantal packet), and (c) since 10(-3)M-ACh is large compared to any estimates of the dissociation constant Kd for ACh binding to the AChR, the ACh will essentially saturate the AChR within the critical area (provided the ACh binding rate is sufficiently faster than the ACh spreading rate). 6. The total receptive surface for a frog end-plate is calculated to be approximately 1500 micrometer2, and therefore an end-plate potential resulting from 300 quanta will be due to the activation of less than 10% of the total receptive area. 7. Free diffusion would allow each small post-junctional critical area to be reached in less than 15 musec. Therefore, either the recorded rise time of the miniature end-plate is not predominantly a function of ACh diffusion time, or, as suggested by Gage & McBurney (1975), the net rate of movement of ACh in the cleft is much slower than indicated by the free diffusion constant. Images Fig. 1a and b Fig. 2 Figs. 3, 5 Fig. 4 PMID:307600

Neurotoxins from venoms of the Hymenoptera--twenty-five years of research in Amsterdam.

PubMed

Piek, T

1990-01-01

1. In co-operation with colleagues in Europe, Japan and the U.S.A., 25 years of research in Amsterdam have provided new views on the way some hymenopteran insects incapacitate their prey by a diversity of neurotoxins, resulting in block of synaptic transmission in CNS or neuromuscular junctions, or affecting voltage dependent phenomena in nerve and muscle fibers. 2. Nicotinic synaptic transmission in the insect CNS is irreversibly blocked at the presynaptic side by kinins, or reversibly and postsynaptically blocked by philanthotoxins. 3. Glutamatergic neuromuscular transmission is reversibly blocked by philanthotoxins at the pre- and/or postsynaptic side. 4. A presynaptic block of neuromuscular transmission was found with the Microbracon toxins. 5. An irreversible deactivation, without paralysis, of cockroaches is caused by a sting of Ampulex compressa into the suboesophageal ganglion. 6. Poneratoxin, a 25 amino acid residue polypeptide, isolated from an ant venom, is the first described hymenopteran neurotoxin affecting excitability of nerve and muscle fibres by changing the kinetics of the voltage-dependent sodium channel.

Genome Editing of Monogenic Neuromuscular Diseases: A Systematic Review.

PubMed

Long, Chengzu; Amoasii, Leonela; Bassel-Duby, Rhonda; Olson, Eric N

2016-11-01

Muscle weakness, the most common symptom of neuromuscular disease, may result from muscle dysfunction or may be caused indirectly by neuronal and neuromuscular junction abnormalities. To date, more than 780 monogenic neuromuscular diseases, linked to 417 different genes, have been identified in humans. Genome-editing methods, especially the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) system, hold clinical potential for curing many monogenic disorders, including neuromuscular diseases such as Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1. To provide an overview of genome-editing approaches; to summarize published reports on the feasibility, efficacy, and safety of current genome-editing methods as they relate to the potential correction of monogenic neuromuscular diseases; and to highlight scientific and clinical opportunities and obstacles toward permanent correction of disease-causing mutations responsible for monogenic neuromuscular diseases by genome editing. PubMed and Google Scholar were searched for articles published from June 30, 1989, through June 9, 2016, using the following keywords: genome editing, CRISPR-Cas9, neuromuscular disease, Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1. The following sources were reviewed: 341 articles describing different approaches to edit mammalian genomes; 330 articles describing CRISPR-Cas9-mediated genome editing in cell culture lines (in vitro) and animal models (in vivo); 16 websites used to generate single-guide RNA; 4 websites for off-target effects; and 382 articles describing viral and nonviral delivery systems. Articles describing neuromuscular diseases, including Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1, were also reviewed. Multiple proof-of-concept studies reveal the feasibility and efficacy of genome-editing-meditated correction of monogenic neuromuscular diseases in cultured cells and animal models. Genome editing is a rapidly evolving technology with enormous translational potential once efficacy, delivery, and safety issues are addressed. The clinical impact of this technology is that genome editing can permanently correct disease-causing mutations and circumvent the hurdles of traditional gene- and cell-based therapies.

Genome Editing of Monogenic Neuromuscular Diseases

PubMed Central

Long, Chengzu; Amoasii, Leonela; Bassel-Duby, Rhonda; Olson, Eric N.

2017-01-01

IMPORTANCE Muscle weakness, the most common symptom of neuromuscular disease, may result from muscle dysfunction or may be caused indirectly by neuronal and neuromuscular junction abnormalities. To date, more than 780 monogenic neuromuscular diseases, linked to 417 different genes, have been identified in humans. Genome-editing methods, especially the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas9 (CRISPR-associated protein 9) system, hold clinical potential for curing many monogenic disorders, including neuromuscular diseases such as Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1. OBJECTIVES To provide an overview of genome-editing approaches; to summarize published reports on the feasibility, efficacy, and safety of current genome-editing methods as they relate to the potential correction of monogenic neuromuscular diseases; and to highlight scientific and clinical opportunities and obstacles toward permanent correction of disease-causing mutations responsible for monogenic neuromuscular diseases by genome editing. EVIDENCE REVIEW PubMed and Google Scholar were searched for articles published from June 30, 1989, through June 9, 2016, using the following keywords: genome editing, CRISPR-Cas9, neuromuscular disease, Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, andmyotonic dystrophy type 1. The following sources were reviewed: 341 articles describing different approaches to edit mammalian genomes; 330 articles describing CRISPR-Cas9–mediated genome editing in cell culture lines (in vitro) and animal models (in vivo); 16 websites used to generate single-guide RNA; 4 websites for off-target effects; and 382 articles describing viral and nonviral delivery systems. Articles describing neuromuscular diseases, including Duchenne muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis, and myotonic dystrophy type 1, were also reviewed. FINDINGS Multiple proof-of-concept studies reveal the feasibility and efficacy of genome-editing–meditated correction of monogenic neuromuscular diseases in cultured cells and animal models. CONCLUSIONS AND RELEVANCE Genome editing is a rapidly evolving technology with enormous translational potential once efficacy, delivery, and safety issues are addressed. The clinical impact of this technology is that genome editing can permanently correct disease-causing mutations and circumvent the hurdles of traditional gene- and cell-based therapies. PMID:27668807

Congenital myasthenic syndrome with tubular aggregates caused by GFPT1 mutations.

PubMed

Guergueltcheva, Velina; Müller, Juliane S; Dusl, Marina; Senderek, Jan; Oldfors, Anders; Lindbergh, Christopher; Maxwell, Susan; Colomer, Jaume; Mallebrera, Cecilia Jimenez; Nascimento, Andres; Vilchez, Juan J; Muelas, Nuria; Kirschner, Janbernd; Nafissi, Shahriar; Kariminejad, Ariana; Nilipour, Yalda; Bozorgmehr, Bita; Najmabadi, Hossein; Rodolico, Carmelo; Sieb, Jörn P; Schlotter, Beate; Schoser, Benedikt; Herrmann, Ralf; Voit, Thomas; Steinlein, Ortrud K; Najafi, Abdolhamid; Urtizberea, Andoni; Soler, Doriette M; Muntoni, Francesco; Hanna, Michael G; Chaouch, Amina; Straub, Volker; Bushby, Kate; Palace, Jacqueline; Beeson, David; Abicht, Angela; Lochmüller, Hanns

2012-05-01

Congenital myasthenic syndrome (CMS) is a clinically and genetically heterogeneous group of inherited disorders of the neuromuscular junction. A difficult to diagnose subgroup of CMS is characterised by proximal muscle weakness and fatigue while ocular and facial involvement is only minimal. DOK7 mutations have been identified as causing the disorder in about half of the cases. More recently, using classical positional cloning, we have identified mutations in a previously unrecognised CMS gene, GFPT1, in a series of DOK7-negative cases. However, detailed description of clinical features of GFPT1 patients has not been reported yet. Here we describe the clinical picture of 24 limb-girdle CMS (LG-CMS) patients and pathological findings of 18 of them, all carrying GFPT1 mutations. Additional patients with CMS, but without tubular aggregates, and patients with non-fatigable weakness with tubular aggregates were also screened. In most patients with GFPT1 mutations, onset of the disease occurs in the first decade of life with characteristic limb-girdle weakness and fatigue. A common feature was beneficial and sustained response to acetylcholinesterase inhibitor treatment. Most of the patients who had a muscle biopsy showed tubular aggregates in myofibers. Analysis of endplate morphology in one of the patients revealed unspecific abnormalities. Our study delineates the phenotype of CMS associated with GFPT1 mutations and expands the understanding of neuromuscular junction disorders. As tubular aggregates in context of a neuromuscular transmission defect appear to be highly indicative, we suggest calling this condition congenital myasthenic syndrome with tubular aggregates (CMS-TA).

Age-dependent synapse withdrawal at axotomised neuromuscular junctions in Wlds mutant and Ube4b/Nmnat transgenic mice

PubMed Central

Gillingwater, Thomas H; Thomson, Derek; Mack, Till G A; Soffin, Ellen M; Mattison, Richard J; Coleman, Michael P; Ribchester, Richard R

2002-01-01

Axons in WldS mutant mice are protected from Wallerian degeneration by overexpression of a chimeric Ube4b/Nmnat (Wld) gene. Expression of Wld protein was independent of age in these mice. However we identified two distinct neuromuscular synaptic responses to axotomy. In young adult Wlds mice, axotomy induced progressive, asynchronous synapse withdrawal from motor endplates, strongly resembling neonatal synapse elimination. Thus, five days after axotomy, 50–90 % of endplates were still partially or fully occupied and expressed endplate potentials (EPPs). By 10 days, fewer than 20 % of endplates still showed evidence of synaptic activity. Recordings from partially occupied junctions indicated a progressive decrease in quantal content in inverse proportion to endplate occupancy. In Wlds mice aged > 7 months, axons were still protected from axotomy but synapses degenerated rapidly, in wild-type fashion: within three days less than 5 % of endplates contained vestiges of nerve terminals. The axotomy-induced synaptic withdrawal phenotype decayed with a time constant of ∼30 days. Regenerated synapses in mature Wlds mice recapitulated the juvenile phenotype. Within 4–6 days of axotomy 30–50 % of regenerated nerve terminals still occupied motor endplates. Age-dependent synapse withdrawal was also seen in transgenic mice expressing the Wld gene. Co-expression of Wld protein and cyan fluorescent protein (CFP) in axons and neuromuscular synapses did not interfere with the protection from axotomy conferred by the Wld gene. Thus, Wld expression unmasks age-dependent, compartmentally organised programmes of synapse withdrawal and degeneration. PMID:12231635

Guidelines for pre-clinical animal and cellular models of MuSK-myasthenia gravis.

PubMed

Phillips, W D; Christadoss, P; Losen, M; Punga, A R; Shigemoto, K; Verschuuren, J; Vincent, A

2015-08-01

Muscle-specific tyrosine kinase (MuSK) autoantibodies are the hallmark of a form of myasthenia gravis (MG) that can challenge the neurologist and the experimentalist. The clinical disease can be difficult to treat effectively. MuSK autoantibodies affect the neuromuscular junction in several ways. When added to muscle cells in culture, MuSK antibodies disperse acetylcholine receptor clusters. Experimental animals actively immunized with MuSK develop MuSK autoantibodies and muscle weakness. Weakness is associated with reduced postsynaptic acetylcholine receptor numbers, reduced amplitudes of miniature endplate potentials and endplate potentials, and failure of neuromuscular transmission. Similar impairments have been found in mice injected with IgG from MG patients positive for MuSK autoantibody (MuSK-MG). The active and passive models have begun to reveal the mechanisms by which MuSK antibodies disrupt synaptic function at the neuromuscular junction, and should be valuable in developing therapies for MuSK-MG. However, translation into new and improved treatments for patients requires procedures that are not too cumbersome but suitable for examining different aspects of MuSK function and the effects of potential therapies. Study design, conduct and analysis should be carefully considered and transparently reported. Here we review what has been learnt from animal and culture models of MuSK-MG, and offer guidelines for experimental design and conduct of studies, including sample size determination, randomization, outcome parameters and precautions for objective data analysis. These principles may also be relevant to the increasing number of other antibody-mediated diseases that are now recognized. Copyright © 2014 Elsevier Inc. All rights reserved.

The Action of Botulinum Toxin at the Neuromuscular Junction

DTIC Science & Technology

1980-12-22

fast - twitch " (gastrocnemius) and " slow - twitch " (soleus) muscles ... muscle fibers -"_re not significantly affected by the toxin. It is interesting to note that, although fast - twitch and slow - twitch mucles were...Duchen LW: An electron microscopic study of the changes induced by borulinum o::in in the motor end-plates of slow and fast skeletal muscle fibres of

Channels Active in the Excitability of Nerves and Skeletal Muscles across the Neuromuscular Junction: Basic Function and Pathophysiology

ERIC Educational Resources Information Center

Goodman, Barbara E.

2008-01-01

Ion channels are essential for the basic physiological function of excitable cells such as nerve, skeletal, cardiac, and smooth muscle cells. Mutations in genes that encode ion channels have been identified to cause various diseases and disorders known as channelopathies. An understanding of how individual ion channels are involved in the…

Frontoxins, three-finger toxins from Micrurus frontalis venom, decrease miniature endplate potential amplitude at frog neuromuscular junction.

PubMed

Moreira, K G; Prates, M V; Andrade, F A C; Silva, L P; Beirão, P S L; Kushmerick, C; Naves, L A; Bloch, C

2010-08-01

Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I-VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain alpha-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain alpha-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors. Copyright 2010 Elsevier Ltd. All rights reserved.

Effect of oxygen at high pressure on spontaneous transmitter release.

PubMed

Colton, J S; Colton, C A

1978-11-01

The effect of oxygen at high pressure (OHP) on resting membrane properties (effective membrane resistance (Reff) and membrane potential (Vm)) and the spontaneous release of excitatory transmitter were examined at the lobster neuromuscular junction. Pressurization with 100% oxygen to 150 pounds per square inch gauge pressure (psig) or with nitrogen to 150 psig (7,000 mmHg nitrogen and 135 mmHg oxygen) produced a decrease in Reff associated with a hyperpolarization of Vm. These changes, however, returned to control values within 20--30 min after completion of pressurization. Spontaneous release of excitatory transmitter was shown to increase dramatically in the presence of 100% oxygen at 150 psig. The increase in miniature end-plate potential (MEPP) frequency persisted beyond the transient changes seen with Reff and Vm. This effect was selective to oxygen, as pressurization with nitrogen did not produce an increase in MEPP frequency. No change in average MEPP amplitude was seen with either OHP or pressure alone. An OHP-induced increase in MEPP frequency was also seen at the frog neuromuscular junction. The results indicate that both glutamate-mediated and acetylcholine-mediated synaptic transmission are altered by OHP.

Key changes in denervated muscles and their impact on regeneration and reinnervation

PubMed Central

Wu, Peng; Chawla, Aditya; Spinner, Robert J.; Yu, Cong; Yaszemski, Michael J.; Windebank, Anthony J.; Wang, Huan

2014-01-01

The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair. PMID:25422641

Parabrachial origin of calcitonin gene-related peptide-immunoreactive axons innervating Meynert's basal nucleus.

PubMed

Knyihár-Csillik, E; Boncz, I; Sáry, G; Nemcsók, J; Csillik, B

1999-06-01

Meynert's basal nucleus is innervated by calcitonin gene-related peptide (CGRP)-immunoreactive axons synapsing with cholinergic principal cells. Origin of CGRP-immunopositive axons was studied in the albino rat. Since beaded axons containing the nicotinic acetylcholine receptor (nAChR) are also present in the basal nucleus, the microstructural arrangement raises the question whether or not an interaction between CGRP and nAChR exists like in the neuromuscular junction. We found that electrolytic lesion of the parabrachial nucleus results in degeneration of CGRP-immunoreactive axons in the ipsilateral nucleus basalis and induces shrinkage of principal cholinergic neurons while the contralateral nucleus basalis remains intact. Electrolytic lesions in the thalamus, caudate-putamen, and hippocampus did not induce alterations in Meynert's basal nucleus. Disappearance of CGRP after lesions of the parabrachial nucleus does not impair presynaptic nAChR in the basal nucleus, suggesting that, unlike in the neuromuscular junction, CGRP is not involved in the maintenance of nAChR in the basal forebrain. It is concluded that the parabrachial nucleus is involved in the activation of the nucleus basalis-prefrontal cortex system, essential in gnostic and mnemonic functions. Copyright 1999 Academic Press.

A Presynaptic Regulatory System Acts Transsynaptically via Mon1 to Regulate Glutamate Receptor Levels in Drosophila.

PubMed

Deivasigamani, Senthilkumar; Basargekar, Anagha; Shweta, Kumari; Sonavane, Pooja; Ratnaparkhi, Girish S; Ratnaparkhi, Anuradha

2015-10-01

Mon1 is an evolutionarily conserved protein involved in the conversion of Rab5 positive early endosomes to late endosomes through the recruitment of Rab7. We have identified a role for Drosophila Mon1 in regulating glutamate receptor levels at the larval neuromuscular junction. We generated mutants in Dmon1 through P-element excision. These mutants are short-lived with strong motor defects. At the synapse, the mutants show altered bouton morphology with several small supernumerary or satellite boutons surrounding a mature bouton; a significant increase in expression of GluRIIA and reduced expression of Bruchpilot. Neuronal knockdown of Dmon1 is sufficient to increase GluRIIA levels, suggesting its involvement in a presynaptic mechanism that regulates postsynaptic receptor levels. Ultrastructural analysis of mutant synapses reveals significantly smaller synaptic vesicles. Overexpression of vglut suppresses the defects in synaptic morphology and also downregulates GluRIIA levels in Dmon1 mutants, suggesting that homeostatic mechanisms are not affected in these mutants. We propose that DMon1 is part of a presynaptically regulated transsynaptic mechanism that regulates GluRIIA levels at the larval neuromuscular junction. Copyright © 2015 by the Genetics Society of America.

A Co-operative Regulation of Neuronal Excitability by UNC-7 Innexin and NCA/NALCN Leak Channel

PubMed Central

2011-01-01

Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels. PMID:21489288

Pathogenic immune mechanisms at the neuromuscular synapse: the role of specific antibody-binding epitopes in myasthenia gravis.

PubMed

Huijbers, M G; Lipka, A F; Plomp, J J; Niks, E H; van der Maarel, S M; Verschuuren, J J

2014-01-01

Autoantibodies against three different postsynaptic antigens and one presynaptic antigen at the neuromuscular junction are known to cause myasthenic syndromes. The mechanisms by which these antibodies cause muscle weakness vary from antigenic modulation and complement-mediated membrane damage to inhibition of endogenous ligand binding and blocking of essential protein-protein interactions. These mechanisms are related to the autoantibody titre, specific epitopes on the target proteins and IgG autoantibody subclass. We here review the role of specific autoantibody-binding epitopes in myasthenia gravis, their possible relevance to the pathophysiology of the disease and potential implications of epitope mapping knowledge for new therapeutic strategies. © 2013 The Association for the Publication of the Journal of Internal Medicine.

AmeriFlux US-NMj Northern Michigan Jack Pine Stand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Jiquan

This is the AmeriFlux version of the carbon flux data for the site US-NMj Northern Michigan Jack Pine Stand. Site Description - The jack pine site is owned by Michigan Technological University. The stand is managed, and thus thinned and harvested depending on stand age. This jack pine site is naturally regenerating following a clearcut around 1989. Heavy snow in December 2001 c

Some effects of long chain polymethylene bisonium salts on junctional transmission in the peripheral nervous system

PubMed Central

Barlow, R. B.; Zoller, Anne

1964-01-01

A survey has been made of the effects on junctional transmission of the complete series of polymethylene bis-trimethylammonium (BTM) and bis-triethylammonium (BTE) salts from the decamethylene compounds (BTM 10 and BTE 10) to those with twenty-one methylene groups in the chain. These were tested for their ability to cause contracture of the isolated chick biventer cervicis preparation, and for their ability to block the twitch responses of this preparation, those of the rat isolated diaphragm preparation, and those of the cat tibialis anterior preparation. They were also tested for their ability to block transmission in the cat superior cervical ganglion, to block the actions of acetylcholine on the guinea-pig isolated ileum, and for ability to inhibit the hydrolysis of acetylcholine by acetylcholinesterase. Their electrical conductivity has been measured in aqueous solution. Ability to cause contracture of the chick biventer cervicis is confined to the compounds BTM 10 to 15; BTE 10, 11 and 12 have some weak activity but the other BTE compounds, and the BTM compounds with more than fifteen methylene groups, have virtually no activity. In the BTE series both neuromuscular blocking and ganglion-blocking activities increase with chain length up to a maximum in the region of BTE 15 to 17 and then decline. In the BTM series ganglion-blocking activity increases with chain length in much the same way as in the BTE series, though the maximum activity is at a slightly longer chain length. At the neuromuscular junction an increase in chain length beyond BTM 10 leads to a decline in activity but this returns to some extent at longer chain lengths, reaching a second maximum at BTM 18, above which it declines further. At the ganglion BTE 16 is only slightly more active than BTM 16 and about five-times as active as hexamethonium; at the neuromuscular junction in the cat BTE 16 is about five-times as active as BTM 16 and about eight-times as active as (+)-tubocurarine. The affinity of the BTE compounds for the postganglionic acetylcholine receptors of the guinea-pig ileum reaches a maximum at BTE 14 but does not decline significantly with further increase in chain length. Anticholinesterase activity, likewise, does not alter significantly between BTM 12 and BTM 21 and the activity of the compounds in the BTE series appears to be similar. This property could conceivably be modifying the actions of some of the intermediate compounds but is not likely to be affecting those of the more active ones. The conductivity experiments indicate that micelle formation could be limiting the actions of the compounds with 20 or 21 methylene groups, but is not likely to be affecting those of the other compounds. The results suggest that there is a regular increase with chain length of the affinity of these compounds for the receptors in the ganglia and at the neuromuscular junction but that efficacy in causing contracture is limited to compounds with three methyl groups in the cationic head and a chain of about ten methylene groups. The connexion between this ability to depolarize and the ability to block transmission by desensitization is discussed. PMID:14208190

Selective effects of an octopus toxin on action potentials

PubMed Central

Dulhunty, Angela; Gage, Peter W.

1971-01-01

1. A lethal, water soluble toxin (Maculotoxin, MTX) with a molecular weight less than 540, can be extracted from the salivary glands of an octopus (Hapalochlaena maculosa). 2. MTX blocks action potentials in sartorius muscle fibres of toads without affecting the membrane potential. Delayed rectification is not inhibited by the toxin. 3. At low concentrations (10-6-10-5 g/ml.) MTX blocks action potentials only after a certain number have been elicited. The number of action potentials, which can be defined accurately, depends on the concentration of MTX and the concentration of sodium ions in the extracellular solution. 4. The toxin has no post-synaptic effect at the neuromuscular junction and it is concluded that it blocks neuromuscular transmission by inhibiting action potentials in motor nerve terminals. PMID:4330930

Idiopathic Pulmonary Fibrosis and Myasthenia Gravis: An Unusual Association

PubMed Central

Chogtu, Bharti; Malik, Daliparty Vasudev

2016-01-01

Idiopathic Pulmonary Fibrosis (IPF) is a chronic fibrosing lung condition with high morbidity and mortality, accounting for about 25% of the cases of interstitial lung diseases. It usually has a progressive course resulting in death due to respiratory failure. Myasthenia Gravis (MG) is an autoimmune neuromuscular disease, caused by antibody mediated activity against acetylcholine receptor at the neuromuscular junction. It is characterized by fluctuating muscle weakness and fatigue. Extensive literature search did not reveal any case report of an association between these two conditions. Here we present a case of a patient with IPF who also developed MG. The diagnosis of IPF was based on High Resolution Computed Tomography (HRCT) of the lung and that of MG was based on clinical criteria and electrophysiological testing. The case was successfully managed. PMID:27190866

Tomosyn-2 is required for normal motor performance in mice and sustains neurotransmission at motor endplates.

PubMed

Geerts, Cornelia J; Plomp, Jaap J; Koopmans, Bastijn; Loos, Maarten; van der Pijl, Elizabeth M; van der Valk, Martin A; Verhage, Matthijs; Groffen, Alexander J A

2015-07-01

Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2(KO/KO)) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders.

[An update on myasthenia gravis].

PubMed

Martínez Torre, S; Gómez Molinero, I; Martínez Girón, R

2018-03-16

Myasthenia gravis is one of the most common disorders that affect neuromuscular transmission. It is currently one of the most understood and characterised autoimmune disorders Its typical symptoms are fluctuating weakness and fatigue that affects a combination of ocular muscles, bulbar functions, as well as limb and respiratory muscles, which are due to an immune attack against the postsynaptic membrane of the neuromuscular junction. The diagnosis of myasthenia gravis is based on clinical and serological test. It is a disease that can be effectively controlled with the current therapeutic lines, even achieving a complete remission. An update of this interesting disorder is now presented. Copyright © 2018 Sociedad Española de Médicos de Atención Primaria (SEMERGEN). Publicado por Elsevier España, S.L.U. All rights reserved.

Mutations in GMPPB cause congenital myasthenic syndrome and bridge myasthenic disorders with dystroglycanopathies

PubMed Central

Belaya, Katsiaryna; Rodríguez Cruz, Pedro M.; Liu, Wei Wei; Maxwell, Susan; McGowan, Simon; Farrugia, Maria E.; Petty, Richard; Walls, Timothy J.; Sedghi, Maryam; Basiri, Keivan; Yue, Wyatt W.; Sarkozy, Anna; Bertoli, Marta; Pitt, Matthew; Kennett, Robin; Schaefer, Andrew; Bushby, Kate; Parton, Matt; Lochmüller, Hanns; Palace, Jacqueline; Muntoni, Francesco

2015-01-01

Congenital myasthenic syndromes are inherited disorders that arise from impaired signal transmission at the neuromuscular junction. Mutations in at least 20 genes are known to lead to the onset of these conditions. Four of these, ALG2, ALG14, DPAGT1 and GFPT1, are involved in glycosylation. Here we identify a fifth glycosylation gene, GMPPB, where mutations cause congenital myasthenic syndrome. First, we identified recessive mutations in seven cases from five kinships defined as congenital myasthenic syndrome using decrement of compound muscle action potentials on repetitive nerve stimulation on electromyography. The mutations were present through the length of the GMPPB, and segregation, in silico analysis, exon trapping, cell transfection followed by western blots and immunostaining were used to determine pathogenicity. GMPPB congenital myasthenic syndrome cases show clinical features characteristic of congenital myasthenic syndrome subtypes that are due to defective glycosylation, with variable weakness of proximal limb muscle groups while facial and eye muscles are largely spared. However, patients with GMPPB congenital myasthenic syndrome had more prominent myopathic features that were detectable on muscle biopsies, electromyography, muscle magnetic resonance imaging, and through elevated serum creatine kinase levels. Mutations in GMPPB have recently been reported to lead to the onset of muscular dystrophy dystroglycanopathy. Analysis of four additional GMPPB-associated muscular dystrophy dystroglycanopathy cases by electromyography found that a defective neuromuscular junction component is not always present. Thus, we find mutations in GMPPB can lead to a wide spectrum of clinical features where deficit in neuromuscular transmission is the major component in a subset of cases. Clinical recognition of GMPPB-associated congenital myasthenic syndrome may be complicated by the presence of myopathic features, but correct diagnosis is important because affected individuals can respond to appropriate treatments. PMID:26133662

Effects of microgravity on muscle and cerebral cortex: a suggested interaction

NASA Astrophysics Data System (ADS)

D'Amelio, F.; Fox, R. A.; Wu, L. C.; Daunton, N. G.; Corcoran, M. L.

The ``slow'' antigravity muscle adductor longus was studied in rats after 14 days of spaceflight (SF). The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light and electron microscopy revealed myofiber atrophy, segmental necrosis and regenerative myofibers. Regenerative myofibers were N-CAM immunoreactive (N-CAM-IR). The neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles, degenerative changes, vacant axonal spaces and changes suggestive of axonal sprouting. No alterations of muscle spindles was seen either by light or electron microscopy. These observations suggest that muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight. In a separate study, GABA immunoreactivity (GABA-IR) was evaluated at the level of the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension (``simulated'' microgravity). A reduction in number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-IR terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system after spaceflight and hindlimb suspension it is suggested that after limb unloading there are alterations of afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the changes observed in GABA immunoreactivity of cells and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.

LRP4 third β-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner

PubMed Central

Ohkawara, Bisei; Cabrera-Serrano, Macarena; Nakata, Tomohiko; Milone, Margherita; Asai, Nobuyuki; Ito, Kenyu; Ito, Mikako; Masuda, Akio; Ito, Yasutomo; Engel, Andrew G.; Ohno, Kinji

2014-01-01

Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Using Sanger and exome sequencing in a CMS patient, we identified two heteroallelic mutations, p.Glu1233Lys and p.Arg1277His, in LRP4 coding for the postsynaptic low-density lipoprotein receptor-related protein 4. LRP4, expressed on the surface of the postsynaptic membrane of the neuromuscular junction, is a receptor for neurally secreted agrin, and LRP4 bound by agrin activates MuSK. Activated MuSK in concert with Dok-7 stimulates rapsyn to concentrate and anchor AChR on the postsynaptic membrane and interacts with other proteins implicated in the assembly and maintenance of the neuromuscular junction. LRP4 also functions as an inhibitor of Wnt/beta-catenin signaling. The identified mutations in LRP4 are located at the edge of its 3rd beta-propeller domain and decrease binding affinity of LRP4 for both MuSK and agrin. Mutations in the LRP4 3rd beta-propeller domain were previously reported to impair Wnt signaling and cause bone diseases including Cenani–Lenz syndactyly syndrome and sclerosteosis-2. By analyzing naturally occurring and artificially introduced mutations in the LRP4 3rd beta-propeller domain, we show that the edge of the domain regulates the MuSK signaling whereas its central cavity governs Wnt signaling. We conclude that LRP4 is a new CMS disease gene and that the 3rd beta propeller domain of LRP4 mediates the two signaling pathways in a position-specific manner. PMID:24234652

Actions of the selective inhibitor of cholinesterase tetramonoisopropyl pyrophosphortetramide on the rat phrenic nerve-diaphragm preparation

PubMed Central

Heffron, P. F.

1972-01-01

1. Tetramonoisopropyl pyrophosphortetramide (iso-OMPA) added for 15 min to the rat isolated phrenic nerve-diaphragm in a concentration of 30 μM, produced a complete selective and stable inhibition of cholinesterase. A concentration of 3 μM produced near complete inhibition of cholinesterase, and a concentration of 300 μM also inhibited acetylcholinesterase marginally. 2. Inhibition of cholinesterase was associated with a sustained increase in the neuromuscular blocking action of exogenous butyrylcholine but not of exogenous acetylcholine. Iso-OMPA, 300 μM, in addition caused transient increases in the sensitivity of the rat diaphragm to exogenous acetylcholine and butyrylcholine. In the same concentration, it had a curare-like action on the frog rectus abdominis muscle preparation. 3. Iso-OMPA, 30 μM, caused reversible increases in the amplitude of the twitch response and tetanic responses, which were of a similar magnitude in the indirectly stimulated preparation and the directly stimulated curarized preparation. Caffeine had a similar effect on the twitch response and its effectiveness was increased by iso-OMPA, and vice-versa. Amongst anticholinesterases, octamethyl pyrophosphortetramide and tetraethylpyrophosphate also enhanced the amplitude of the tetanic response, but paraoxon, dyflos, and mipafox did not. 4. It is concluded that iso-OMPA, in concentrations (3 and 30 μM) which in 15 min give near maximal or maximal selective inhibition of cholinesterase, has no effect on the transmission of nerve impulses at the neuromuscular junction, but enhances reversibly the amplitude of the contractile response to stimulation by a direct action upon the muscle fibre, which involves a mechanism related to but not identical with that by which caffeine potentiates twitch tension. In higher concentrations, iso-OMPA has a curare-like action at the neuromuscular junction. PMID:4347708

An electrophysiological study on the effects of Pa-1G (a phospholipase A(2)) from the venom of king brown snake, Pseudechis australis, on neuromuscular function.

PubMed

Fatehi, M; Rowan, E G; Harvey, A L

2002-01-01

The effects of Pa-1G, a phospholipase A(2) (PLA(2)) from the venom of the Australian king brown snake (Pseudechis australis) were determined on the release of acetylcholine, muscle resting membrane potential and motor nerve terminal action potential at mouse neuromuscular junction. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations revealed that Pa-1G (800 nM) significantly reduced the amplitude of endplate potentials within 10 min exposure. The quantal content of endplate potentials was decreased to 58+/-6% of control after 30 min exposure to 800 nM Pa-1G. The toxin also caused a partial depolarisation of mouse muscle fibres within 60 min exposure. Extracellular recording of action potentials at motor nerve terminals showed that Pa-1G reduced the waveforms associated with both sodium and potassium conductances. To investigate whether this was a direct or indirect effect of the toxin on these ionic currents, whole cell patch clamp experiments were performed using human neuroblastoma (SK-N-SH) cells and B82 mouse fibroblasts stably transfected with rKv1.2. Patch clamp recording experiments confirmed that potassium currents sensitive to alpha-dendrotoxin recorded from B82 cells and sodium currents in SK-N-SH cells were not affected by the toxin. Since neither facilitation of acetylcholine release at mouse neuromuscular junction nor depression of potassium currents in B82 cells has been observed, the apparent blockade of potassium currents at mouse motor nerve endings induced by the toxin is unlikely to be due to a selective block of potassium channels.

Duplication in the Microtubule-Actin Cross-linking Factor 1 gene causes a novel neuromuscular condition

PubMed Central

Jørgensen, Louise H.; Mosbech, Mai-Britt; Færgeman, Nils J.; Graakjaer, Jesper; Jacobsen, Søren V.; Schrøder, Henrik D.

2014-01-01

Spectrins and plakins are important communicators linking cytoskeletal components to each other and to cellular junctions. Microtubule-actin cross-linking factor 1 (MACF1) belongs to the spectraplakin family and is involved in control of microtubule dynamics. Complete knock out of MACF1 in mice is associated with developmental retardation and embryonic lethality. Here we present a family with a novel neuromuscular condition. Genetic analyses show a heterozygous duplication resulting in reduced MACF1 gene product. The functional consequence is affected motility observed as periodic hypotonia, lax muscles and diminished motor skills, with heterogeneous presentation among the affected family members. To corroborate these findings we used RNA interference to knock down the VAB-10 locus containing the MACF1 homologue in C. elegans, and we could show that this also causes movement disturbances. These findings suggest that changes in the MACF1 gene is implicated in this neuromuscular condition, which is an important observation since MACF1 has not previously been associated with any human disease and thus presents a key to understanding the essential nature of this gene. PMID:24899269

Duplication in the microtubule-actin cross-linking factor 1 gene causes a novel neuromuscular condition.

PubMed

Jørgensen, Louise H; Mosbech, Mai-Britt; Færgeman, Nils J; Graakjaer, Jesper; Jacobsen, Søren V; Schrøder, Henrik D

2014-06-05

Spectrins and plakins are important communicators linking cytoskeletal components to each other and to cellular junctions. Microtubule-actin cross-linking factor 1 (MACF1) belongs to the spectraplakin family and is involved in control of microtubule dynamics. Complete knock out of MACF1 in mice is associated with developmental retardation and embryonic lethality. Here we present a family with a novel neuromuscular condition. Genetic analyses show a heterozygous duplication resulting in reduced MACF1 gene product. The functional consequence is affected motility observed as periodic hypotonia, lax muscles and diminished motor skills, with heterogeneous presentation among the affected family members. To corroborate these findings we used RNA interference to knock down the VAB-10 locus containing the MACF1 homologue in C. elegans, and we could show that this also causes movement disturbances. These findings suggest that changes in the MACF1 gene is implicated in this neuromuscular condition, which is an important observation since MACF1 has not previously been associated with any human disease and thus presents a key to understanding the essential nature of this gene.

Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis

PubMed Central

Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Feng, Kuan; Zhang, Shuangyan; Huang, Jiefang; Miao, Xiang; Baggi, Fulvio; Ostrom, Rennolds S.; Zhang, Yanyun; Chen, Xiangjun; Xu, Congfeng

2017-01-01

ABSTRACT Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder. PMID:28933591

Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis.

PubMed

Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Feng, Kuan; Zhang, Shuangyan; Huang, Jiefang; Miao, Xiang; Baggi, Fulvio; Ostrom, Rennolds S; Zhang, Yanyun; Chen, Xiangjun; Xu, Congfeng

2017-01-01

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.

Botulinum Neurotoxin Type A in Neurology: Update.

PubMed

Orsini, Marco; Leite, Marco Antonio Araujo; Chung, Tae Mo; Bocca, Wladimir; de Souza, Jano Alves; de Souza, Olivia Gameiro; Moreira, Rayele Priscila; Bastos, Victor Hugo; Teixeira, Silmar; Oliveira, Acary Bulle; Moraes, Bruno da Silva; Matta, André Palma; Jacinto, Luis Jorge

2015-09-24

This paper reviews the current and most neurological (central nervous system, CNS) uses of the botulinum neurotoxin type A. The effect of these toxins at neuromuscular junction lends themselves to neurological diseases of muscle overactivity, particularly abnormalities of muscle control. There are seven serotypes of the toxin, each with a specific activity at the molecular level. Currently, serotypes A (in two preparations) and B are available for clinical purpose, and they have proved to be safe and effective for the treatment of dystonia, spasticity, headache, and other CNS disorders in which muscle hyperactivity gives rise to symptoms. Although initially thought to inhibit acetylcholine release only at the neuromuscular junction, botulinum toxins are now recognized to inhibit acetylcholine release at autonomic cholinergic nerve terminals, as well as peripheral release of neuro-transmitters involved in pain regulation. Its effects are transient and nondestructive, and largely limited to the area in which it is administered. These effects are also graded according to the dose, allowing individualized treatment of patients and disorders. It may also prove to be useful in the control of autonomic dysfunction and sialorrhea. In over 20 years of use in humans, botulinum toxin has accumulated a considerable safety record, and in many cases represents relief for thousands of patients unaided by other therapy.

Botulinum Neurotoxin Type A in Neurology: Update

PubMed Central

Orsini, Marco; Leite, Marco Antonio Araujo; Chung, Tae Mo; Bocca, Wladimir; de Souza, Jano Alves; de Souza, Olivia Gameiro; Moreira, Rayele Priscila; Bastos, Victor Hugo; Teixeira, Silmar; Oliveira, Acary Bulle; Moraes, Bruno da Silva; Matta, André Palma; Jacinto, Luis Jorge

2015-01-01

This paper reviews the current and most neurological (central nervous system, CNS) uses of the botulinum neurotoxin type A. The effect of these toxins at neuromuscular junction lends themselves to neurological diseases of muscle overactivity, particularly abnormalities of muscle control. There are seven serotypes of the toxin, each with a specific activity at the molecular level. Currently, serotypes A (in two preparations) and B are available for clinical purpose, and they have proved to be safe and effective for the treatment of dystonia, spasticity, headache, and other CNS disorders in which muscle hyperactivity gives rise to symptoms. Although initially thought to inhibit acetylcholine release only at the neuromuscular junction, botulinum toxins are now recognized to inhibit acetylcholine release at autonomic cholinergic nerve terminals, as well as peripheral release of neuro-transmitters involved in pain regulation. Its effects are transient and nondestructive, and largely limited to the area in which it is administered. These effects are also graded according to the dose, allowing individualized treatment of patients and disorders. It may also prove to be useful in the control of autonomic dysfunction and sialorrhea. In over 20 years of use in humans, botulinum toxin has accumulated a considerable safety record, and in many cases represents relief for thousands of patients unaided by other therapy. PMID:26487928

Neuromodulation of activity-dependent synaptic enhancement at crayfish neuromuscular junction.

PubMed

Qian, S M; Delaney, K R

1997-10-17

Action potential-evoked transmitter release is enhanced for many seconds after moderate-frequency stimulation (e.g. 15 Hz for 30 s) at the excitor motorneuron synapse of the crayfish dactyl opener muscle. Beginning about 1.5 s after a train, activity-dependent synaptic enhancement (ADSE) is dominated by a process termed augmentation (G.D. Bittner, D.A. Baxter, Synaptic plasticity at crayfish neuromuscular junctions: facilitation and augmentation, Synapse 7 (1991) 235-243'[4]; K.L. Magleby, Short-term changes in synaptic efficacy, in: G.M. Edelman, L.E. Gall, C.W. Maxwell (Eds.), Synaptic Function, John Wiley and Sons, New York, 1987, pp. 21-56; K.L. Magleby; J.E. Zengel, Augmentation: a process that acts to increase transmitter release at the frog neuromuscular junction, J. Physiol. (Lond.) 257 (1976) 449-470) which decays approximately exponentially with a time constant of about 10 s at 16 degrees C, reflecting the removal of Ca2+ which accumulates during the train in presynaptic terminals (K.R. Delaney, D.W. Tank, R.S. Zucker, Serotonin-mediated enhancement of transmission at crayfish neuromuscular junction is independent of changes in calcium, J. Neurosci. 11 (1991) 2631-2643). Serotonin (5-HT, 1 microM) increases evoked and spontaneous transmitter release several-fold (D. Dixon, H.L. Atwood, Crayfish motor nerve terminal's response to serotonin examined by intracellular microelectrode, J. Neurobiol. 16 (1985) 409-424; J. Dudel, Modulation of quantal synaptic release by serotonin and forskolin in crayfish motor nerve terminals, in: Modulation of Synaptic Transmission and Plasticity in Nervous Systems, G. Hertting, H.-C. Spatz (Eds.), Springer-Verlag, Berlin, 1988; S. Glusman, E.A. Kravitz. The action of serotonin on excitatory nerve terminals in lobster nerve-muscle preparations, J. Physiol. (Lond.) 325 (1982) 223-241). We found that ADSE persists about 2-3 times longer after moderate-frequency presynaptic stimulation in the presence of 5-HT. This slowing of the decay of ADSE by 5-HT was not accompanied by significant changes in the initial amplitude of activity-dependent components of enhancement 1.5 s after the train. Measurements of presynaptic [Ca2+] indicated that the time course of Ca2+ removal from the presynaptic terminals after trains was not altered by 5-HT. Changes in presynaptic action potential shape, resting membrane potential or postsynaptic impedance after trains cannot account for slower recovery of ADSE. Axonal injection of EDTA slows the removal of residual Ca2+ and the decay of synaptic augmentation after trains of action potentials (K.R. Delaney, D.W. Tank, A quantitative measure of the dependence of short-term synaptic enhancement on presynaptic residual calcium, J. Neurosci. 14 (1994) 5885-5902), but has little or no effect on the 5-HT-induced persistence of ADSE. This also suggests that the time course of ADSE in the presence of 5-HT is not determined primarily by residual Ca2+ removal kinetics. The slowing of ADSE recovery after trains by 5-HT reverses with washing in 5-HT-free saline along with the 5-HT-mediated enhancement of release.

Clinical and Genetic Features of Congenital Myasthenic Syndromes due to CHAT Mutations: Case Report and Literature Review.

PubMed

Arican, Pinar; Gencpinar, Pinar; Cavusoglu, Dilek; Olgac Dundar, Nihal

2018-05-21

Congenital myasthenic syndromes (CMS) are neuromuscular transmission disorders caused by mutations in genes encoding neuromuscular junction proteins. CMS due to choline acetyltransferase (CHAT) gene is characterized by episodic apnea. We report a case of a 12-month-old female patient presented with recurrent episodic apnea carrying a mutation in CHAT gene, p.I336T. Furthermore, we describe the genetic and clinical findings in 44 CMS patients due to CHAT mutations in the literature up to date. Episodes of apnea and respiratory insufficiency are the hallmarks of CHAT mutations. Clinical manifestations usually provoked by infections and fever. CMS due to CHAT mutations are rare, but it is important to diagnosis. Early diagnosis and appropriate treatment can improve morbidity and mortality. Georg Thieme Verlag KG Stuttgart · New York.

[Characteristics of neuromuscular scoliosis].

PubMed

Putzier, M; Groß, C; Zahn, R K; Pumberger, M; Strube, P

2016-06-01

Usually, neuromuscular scolioses become clinically symptomatic relatively early and are rapidly progressive even after the end of growth. Without sufficient treatment they lead to a severe reduction of quality of life, to a loss of the ability of walking, standing or sitting as well as to an impairment of the cardiopulmonary system resulting in an increased mortality. Therefore, an intensive interdisciplinary treatment by physio- and ergotherapists, internists, pediatricians, orthotists, and orthopedists is indispensable. In contrast to idiopathic scoliosis the treatment of patients with neuromuscular scoliosis with orthosis is controversially discussed, whereas physiotherapy is established and essential to prevent contractures and to maintain the residual sensorimotor function.Frequently, the surgical treatment of the scoliosis is indicated. It should be noted that only long-segment posterior correction and fusion of the whole deformity leads to a significant improvement of the quality of life as well as to a prevention of a progression of the scoliosis and the development of junctional problems. The surgical intervention is usually performed before the end of growth. A prolonged delay of surgical intervention does not result in an increased height but only in a deformity progression and is therefore not justifiable. In early onset neuromuscular scolioses guided-growth implants are used to guarantee the adequat development. Because of the high complication rates, further optimization of these implant systems with regard to efficiency and safety have to be addressed in future research.

Identification and Characterization of Novel FMRP-Associated miRNAs

DTIC Science & Technology

2013-10-01

Dependent Synaptic Structure at the Drosophila melanogaster Neuromuscular Junction. Plos One 8: e68385. 11. Adams CM, Anderson MG, Motto DG, Price MP...extension towards the end of the funding period in order to complete the all proposed experiments. Aim 1. Purification and deep sequencing of FMRP...First, when expression of transgenic PrA-tagged FMRP was driven in the adult Drosophila brain, we did not observe an expected shift in size when

MYONEURAL JUNCTIONS OF TWO ULTRASTRUCTURALLY DISTINCT TYPES IN EARTHWORM BODY WALL MUSCLE

PubMed Central

Rosenbluth, Jack

1972-01-01

The longitudinal muscle of the earthworm body wall is innervated by nerve bundles containing axons of two types which form two corresponding types of myoneural junction with the muscle fibers Type I junctions resemble cholinergic neuromuscular junctions of vertebrate skeletal muscle and are characterized by three features: (a) The nerve terminals contain large numbers of spherical, clear, ∼500 A vesicles plus a small number of larger dense-cored vesicles (b) The junctional gap is relatively wide (∼900 A), and it contains a basement membrane-like material, (c) The postjunctional membrane, although not folded, displays prominent specializations on both its external and internal surfaces The cytoplasmic surface is covered by a dense matrix ∼200 A thick which appears to be the site of insertion of fine obliquely oriented cytoplasmic filaments The external surface exhibits rows of projections ∼200 A long whose bases consist of hexagonally arrayed granules seated in the outer dense layer of the plasma membrane The concentration of these hexagonally disposed elements corresponds to the estimated concentration of both receptor sites and acetylcholinesterase sites at cholinergic junctions elsewhere. Type II junctions resemble the adrenergic junctions in vertebrate smooth muscle and exhibit the following structural characteristics: (a) The nerve fibers contain predominantly dense-cored vesicles ∼1000 A in diameter (b) The junctional gap is relatively narrow (∼150 A) and contains no basement membrane-like material, (c) Postjunctional membrane specialization is minimal. It is proposed that the structural differences between the two types of myoneural junction reflect differences in the respective transmitters and corresponding differences in the mechanisms of transmitter action and/or inactivation. PMID:5044759

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

PubMed Central

Sears, James C.; Broadie, Kendal

2018-01-01

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the activity-dependent repression of translation. In the well-characterized Drosophila neuromuscular junction (NMJ) model, developmental synaptogenesis and activity-dependent synaptic remodeling both require extracellular matrix metalloproteinase (MMP) enzymes interacting with the heparan sulfate proteoglycan (HSPG) glypican dally-like protein (Dlp) to restrict trans-synaptic Wnt signaling, with FXS synaptogenic defects alleviated by both MMP and HSPG reduction. This new mechanistic axis spanning from activity to FMRP to HSPG-dependent MMP regulation modulates activity-dependent synaptogenesis. We discuss future directions for these mechanisms, and intersecting research priorities for FMRP in glial and signaling interactions. PMID:29375303

Infant botulism following honey ingestion

PubMed Central

Abdulla, C O; Ayubi, A; Zulfiquer, F; Santhanam, G; Ahmed, M A S; Deeb, J

2012-01-01

An apparently well baby girl born at term was presented with signs and symptoms suggestive of acute onset of generalised floppiness at the age of 3 months. Clinically, the baby had lower motor neuron type of muscle weakness; detailed investigation lead to the diagnosis of neuromuscular junction disorder secondary to botulism toxicity. Further tests confirmed the botulism toxicity secondary to honey ingestion. The baby was treated with specific anticlostridium antibodies; she recovered remarkably, now growing and developing normally. PMID:22962382

Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Daunton, N. G.

1992-01-01

The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Possible mechanisms for sensitivity to organophosphorus and carbamate insecticides in eastern screech-owls and American kestrels.

PubMed

Vyas, N B; Thiele, L A; Garland, S C

1998-07-01

Effects of a single dietary exposure to fenthion and carbofuran on the survival, feeding behavior and brain ChE activity of eastern screech-owls, Otus asio and American kestrels, Falco sparverius, were evaluated. Birds were exposed to fenthion (23.6-189.0 ppm) or carbofuran (31.7-253.6 ppm) via meatballs. Carbofuran-exposed owls ate either < or = 10% or > or = 80% of the meatball whereas all kestrels ate < or = 10% of the meatball before exhibiting acute signs of toxicity. Fenthion-exposed owls and kestrels displayed a wide spectrum of meatball consumption (< 10-100%). Significant brain ChE inhibition was observed in dead and surviving kestrels exposed to fenthion and carbofuran and dead owls exposed to fenthion (P < 0.0001). Brain ChE activity of owls exposed to carbofuran that survived was not different from that of controls (P = 0.25). Data suggest: (1) slow feeding on a carbamate-contaminated item may provide limited protection from the toxicity of the chemical at certain rates of exposure; (2) the degree of ChE inhibition at neuromuscular junctions may be critical in determining the sensitivity of a species to a carbamate insecticide; (3) sensitivity may be a function of the ChE affinity for the carbamate inhibitor; and (4) the importance of neuromuscular junction ChE depression in determining the sensitivity of an animal may be species-specific.

Possible mechanisms for sensitivity to organophosphorus and carbamate insecticides in eastern screech-owls and American kestrels

USGS Publications Warehouse

Vyas, N.B.; Thiele, L.A.; Garland, S.C.

1998-01-01

Effects of a single dietary exposure to fenthion and carbofuran on the survival, feeding behavior and brain ChE activity of eastern screech-owls, Otus asio and American kestrels, Falco sparverius, were evaluated. Birds were exposed to fenthion (23.6–189.0 ppm) or carbofuran (31.7–253.6 ppm) via meatballs. Carbofuran-exposed owls ate either ≤10% or ≥80% of the meatball whereas all kestrels ate ≤10% of the meatball before exhibiting acute signs of toxicity. Fenthion-exposed owls and kestrels displayed a wide spectrum of meatball consumption (<10–100%). Significant brain ChE inhibition was observed in dead and surviving kestrels exposed to fenthion and carbofuran and dead owls exposed to fenthion (P<0.0001). Brain ChE activity of owls exposed to carbofuran that survived was not different from that of controls (P=0.25). Data suggest: (1) slow feeding on a carbamate-contaminated item may provide limited protection from the toxicity of the chemical at certain rates of exposure; (2) the degree of ChE inhibition at neuromuscular junctions may be critical in determining the sensitivity of a species to a carbamate insecticide; (3) sensitivity may be a function of the ChE affinity for the carbamate inhibitor; and (4) the importance of neuromuscular junction ChE depression in determining the sensitivity of an animal may be species-specific.

Novel immunolocalization of alpha-synuclein in human muscle of inclusion-body myositis, regenerating and necrotic muscle fibers, and at neuromuscular junctions.

PubMed

Askanas, V; Engel, W K; Alvarez, R B; McFerrin, J; Broccolini, A

2000-07-01

Alpha-synuclein (alpha-syn) is an important component of neuronal and glial inclusions in brains of patients with several neurodegenerative disorders. Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease of older patients. Its muscle phenotype shows several similarities with Alzheimer disease brain. A distinct feature of s-IBM pathology is specific vacuolar degeneration of muscle fibers characterized by intracellular amyloid inclusions formed by both amyloid-beta (Abeta) and paired-helical filaments composed of phosphorylated tau. We immunostained alpha-syn in muscle biopsies of s-IBM, disease-control, and normal patients. Approximately 60% of Abeta-positive vacuolated muscle fibers (VMF) contained well-defined inclusions immunoreactive with antibodies against alpha-syn. In those fibers. alpha-syn co-localized with Abeta, both by light microscopy, and ultrastructurally. Paired-helical filaments did not contain alpha-syn immunoreactivity. In all muscle biopsies, alpha-syn was strongly immunoreactive at the postsynaptic region of the neuromuscular junctions. alpha-syn immunoreactivity also occurred diffusely in regenerating and necrotic muscle fibers. In cultured human muscle fibers, alpha-syn and its mRNA were expressed by immunocytochemistry, immunoblots, and Northern blots. Our study provides the first demonstration that alpha-syn participates in normal and pathologic processes of human muscle. Therefore. its function is not exclusive to the brain and neurodegenerative diseases.

Spatacsin and spastizin act in the same pathway required for proper spinal motor neuron axon outgrowth in zebrafish.

PubMed

Martin, Elodie; Yanicostas, Constantin; Rastetter, Agnès; Alavi Naini, Seyedeh Maryam; Maouedj, Alissia; Kabashi, Edor; Rivaud-Péchoux, Sophie; Brice, Alexis; Stevanin, Giovanni; Soussi-Yanicostas, Nadia

2012-12-01

Hereditary spastic paraplegias (HSPs) are rare neurological conditions caused by degeneration of the long axons of the cerebrospinal tracts, leading to locomotor impairment and additional neurological symptoms. There are more than 40 different causative genes, 24 of which have been identified, including SPG11 and SPG15 mutated in complex clinical forms. Since the vast majority of the causative mutations lead to loss of function of the corresponding proteins, we made use of morpholino-oligonucleotide (MO)-mediated gene knock-down to generate zebrafish models of both SPG11 and SPG15 and determine how invalidation of the causative genes (zspg11 and zspg15) during development might contribute to the disease. Micro-injection of MOs targeting each gene caused locomotor impairment and abnormal branching of spinal cord motor neurons at the neuromuscular junction. More severe phenotypes with abnormal tail developments were also seen. Moreover, partial depletion of both proteins at sub-phenotypic levels resulted in the same phenotypes, suggesting for the first time, in vivo, a genetic interaction between these genes. In conclusion, the zebrafish orthologues of the SPG11 and SPG15 genes are important for proper development of the axons of spinal motor neurons and likely act in a common pathway to promote their proper path finding towards the neuromuscular junction. Copyright © 2012 Elsevier Inc. All rights reserved.

Mutational Analysis of Rab3 Function for Controlling Active Zone Protein Composition at the Drosophila Neuromuscular Junction

PubMed Central

Roche, John P.; Alsharif, Peter; Graf, Ethan R.

2015-01-01

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function. PMID:26317909

Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-β/BMP signaling and orphan nuclear receptors.

PubMed

Boulanger, Ana; Farge, Morgane; Ramanoudjame, Christophe; Wharton, Kristi; Dura, Jean-Maurice

2012-01-01

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. In this study, we describe the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, we show that phagocytes have a key role in this process. Importantly, we show the presence of peripheral glial cells close to the neuro-muscular junction that retracts before the motor neuron. We show also that in muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. We propose that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation.

Expression of synapsin I correlates with maturation of the neuromuscular synapse.

PubMed

Lu, B; Czernik, A J; Popov, S; Wang, T; Poo, M M; Greengard, P

1996-10-01

Synapsins are a family of neuron-specific phosphoproteins that are localized within the presynaptic terminals in adult brain. Previous work has demonstrated that introduction of exogenous synapsins I(a + b) or IIa into Xenopus spinal neurons promoted maturation of the neuromuscular synapse in a nerve-muscle co-culture system. We have now studied the expression of endogenous Xenopus synapsin I during synaptic maturation in vivo and in culture, using a polyclonal antibody raised against Xenopus synapsin I. Immunoprecipitation experiments indicated that synapsin I was not detectable during the early phase of synaptogenesis in vivo, and exhibited a marked increase during the period of synaptic maturation. In contrast, the expression of synaptophysin, another synaptic vesicle protein, was detected at the start of nervous system formation, and remained at a high level thereafter. Similar expression profiles for the two proteins were also observed in immunocytochemical studies of Xenopus spinal neurons in culture: intense staining of synaptophysin was found on the first day, while synapsin I was not detected until after three days in culture. The expression of synapsin I correlated very well with the appearance of a bell-shaped amplitude distribution of spontaneous synaptic currents, a physiological parameter which reflects functional maturation of the neuromuscular synapse. In one-day-old cultures grown in the absence of laminin, an extracellular matrix protein known to be present at the neuromuscular junction, the amplitude distribution of virtually all synapses was skewed towards smaller values. In contrast, when laminin was used as a culture substrate, many synapses exhibited a bell-shaped amplitude distribution. Laminin treatment also induced synapsin I expression in one-day-old cultures. These results suggest that the expression of endogenous synapsin I may regulate maturation at neuromuscular synapses.

The effect of ventilatory muscle training on respiratory function and capacity in ambulatory and bed-ridden patients with neuromuscular disease.

PubMed

Gross, D; Meiner, Z

1993-08-01

Most patients with neuromuscular disease develop muscle weakness, including the ventilatory muscles leading to respiratory difficulty and, at times, respiratory insufficiency. We studied the effect of ventilatory muscle training on the ventilatory function and capacity of patients with various types of neuromuscular disease. The ambulatory patients were divided into three major groups. Group I (n = 6) patients with motor neuron disease (MND), such as amyotrophic latera sclerosis; Group II (n = 11) patients with myoneural junction disease (MNJ), such as myasthenia gravis and: Group III (n = 7) patients with muscle diseases such as progressive muscular disease. Patients were evaluated for their neuromuscular diagnosis and status of the disease. A complete physical examination and the various neuromuscular tests were performed. A complete respiratory evaluation was applied: pulmonary function tests (PFT), maximum inspiratory pressure (MIP). Patients then started ventilatory muscle training by resistive breathing, as a prophylactic treatment, for 10 min, three times daily, with a resistance which would induce fatigue. All tests were repeated every six weeks, and the results were as follow: forced vital capacity (FVC) changed from 38.8 +/- 12.3 to 53.2 +/- 9.6% (NS) of predicted value in group I, from 49.8 +/- 8.7 to 66.1 +/- 7.5% (p < 0.002) in group II, and from 47.0 +/- 7.5 to 53.3 +/- 7.6% (p < 0.04) in group III. Forced expiratory volume in one second (FEV1) was 34.8 +/- 11.0, 46.3 +/- 5, and 45.1 +/- 9% for the three groups, respectively, and did not change with training.(ABSTRACT TRUNCATED AT 250 WORDS)

Myasthenia gravis: recent advances in immunopathology and therapy.

PubMed

Lee, John-Ih; Jander, Sebastian

2017-03-01

Myasthenia gravis is the most frequent acquired disorder of neuromuscular transmission. In the majority of cases, pathogenic antibodies against components of the postsynaptic muscle endplate membrane can be detected. In recent years there have been significant advances in the pathophysiological understanding and therapy of the disease. Areas covered: PubMed searches were conducted for the term 'myasthenia gravis' cross-referenced with the terms 'immunology', 'subgroups', 'antibody', 'ocular', 'thymoma', 'treatment' and 'thymectomy'. Additionally, we summarized the current state of immunopathology and therapy. Expert commentary: Immunological research defined new target antigens at the postsynaptic neuromuscular junction which along with clinical features allow a refined definition of disease subgroups. Overall the prognosis of myasthenia gravis with best possible symptomatic, immunosuppressive and supportive treatment is good but new immunomodulatory treatment options are developed for patients who do not respond well to the first line therapy. For most patients individually adapted long-term drug therapy is needed.

Myostatin-like proteins regulate synaptic function and neuronal morphology.

PubMed

Augustin, Hrvoje; McGourty, Kieran; Steinert, Joern R; Cochemé, Helena M; Adcott, Jennifer; Cabecinha, Melissa; Vincent, Alec; Halff, Els F; Kittler, Josef T; Boucrot, Emmanuel; Partridge, Linda

2017-07-01

Growth factors of the TGFβ superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. Here, we show that Myoglianin (MYO), the Drosophila homolog of myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. We also show that MYO acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth. © 2017. Published by The Company of Biologists Ltd.

Myasthenia gravis and related disorders: Pathology and molecular pathogenesis.

PubMed

Ha, James C; Richman, David P

2015-04-01

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Silent synapses in neuromuscular junction development.

PubMed

Tomàs, Josep; Santafé, Manel M; Lanuza, Maria A; García, Neus; Besalduch, Nuria; Tomàs, Marta

2011-01-01

In the last few years, evidence has been found to suggest that some synaptic contacts become silent but can be functionally recruited before they completely retract during postnatal synapse elimination in muscle. The physiological mechanism of developmental synapse elimination may be better understood by studying this synapse recruitment. This Mini-Review collects previously published data and new results to propose a molecular mechanism for axonal disconnection. The mechanism is based on protein kinase C (PKC)-dependent inhibition of acetylcholine (ACh) release. PKC activity may be stimulated by a methoctramine-sensitive M2-type muscarinic receptor and by calcium inflow though P/Q- and L-type voltage-dependent calcium channels. In addition, tropomyosin-related tyrosine kinase B (trkB) receptor-mediated brain-derived neurotrophic factor (BDNF) activity may oppose the PKC-mediated ACh release depression. Thus, a balance between trkB and muscarinic pathways may contribute to the final functional suppression of some neuromuscular synapses during development. © 2010 Wiley-Liss, Inc.

A General Model of Synaptic Transmission and Short-Term Plasticity

PubMed Central

Pan, Bin; Zucker, Robert S.

2011-01-01

SUMMARY Some synapses transmit strongly to action potentials (APs), but weaken with repeated activation; others transmit feebly at first, but strengthen with sustained activity. We measured synchronous and asynchronous transmitter release at “phasic” crayfish neuromuscular junctions (NMJs) showing depression and at facilitating “tonic” junctions, and define the kinetics of depression and facilitation. We offer a comprehensive model of presynaptic processes, encompassing mobilization of reserve vesicles, priming of docked vesicles, their association with Ca2+ channels, and refractoriness of release sites, while accounting for data on presynaptic buffers governing Ca2+ diffusion. Model simulations reproduce many experimentally defined aspects of transmission and plasticity at these synapses. Their similarity to vertebrate central synapses suggests that the model might be of general relevance to synaptic transmission. PMID:19477155

Peristalsis and propulsion of colonic content can occur after blockade of major neuroneuronal and neuromuscular transmitters in isolated guinea pig colon.

PubMed

Sia, T C; Brookes, S J; Dinning, P G; Wattchow, D A; Spencer, N J

2013-12-01

We recently identified hexamethonium-resistant peristalsis in the guinea pig colon. We showed that, following acute blockade of nicotinic receptors, peristalsis recovers, leading to normal propagation velocities of fecal pellets along the colon. This raises the fundamental question: what mechanisms underlie hexamethonium-resistant peristalsis? We investigated whether blockade of the major receptors that underlie excitatory neuromuscular transmission is required for hexamethonium-resistant peristalsis. Video imaging of colonic wall movements was used to make spatiotemporal maps and determine the velocity of peristalsis. Propagation of artificial fecal pellets in the guinea pig distal colon was studied in hexamethonium, atropine, ω-conotoxin (GVIA), ibodutant (MEN-15596), and TTX. Hexamethonium and ibodutant alone did not retard peristalsis. In contrast, ω-conotoxin abolished peristalsis in some preparations and reduced the velocity of propagation in all remaining specimens. Peristalsis could still occur in some animals in the presence of hexamethonium + atropine + ibodutant + ω-conotoxin. Peristalsis never occurred in the presence of TTX. The major finding of the current study is the unexpected observation that peristalsis can occur after blockade of the major excitatory neuroneuronal and neuromuscular transmitters. Also, the colon retained an intrinsic polarity in the presence of these antagonists and was only able to expel pellets in an aboral direction. The nature of the mechanism(s)/neurotransmitter(s) that generate(s) peristalsis and facilitate(s) natural fecal pellet propulsion, after blockade of major excitatory neurotransmitters, at the neuroneuronal and neuromuscular junction remains to be identified.

Adverse effects of neuromuscular blockers and their antagonists.

PubMed

Naguib, M; Magboul, M M

1998-02-01

Among all the drugs used for general anaesthesia, neuromuscular blockers appear to play a prominent role in the incidence of severe adverse reactions. It now seems likely that most serious adverse drug reactions occurring during anaesthesia are immunological in type. The frequency of life-threatening anaphylactic or anaphylactoid reactions occurring during anaesthesia has been estimated to be between 1 in 1000 and 1 in 25,000 anaesthetic procedures, with the neuromuscular blockers being involved in 80% of cases. The mortality from such serious reactions is reported to be in the range of 3.4 to 6%. The highly immunogenic drug, suxamethonium chloride (succinylcholine), was found to be the most hazardous agent. Drug-specific immunoglobulin E antibodies to suxamethonium chloride and other neuromuscular blockers have been demonstrated. This sensitivity to neuromuscular blockers seems to be a long-lasting phenomenon. During anaesthesia, the clinical features of an allergic reaction are often masked. Tachycardia and circulatory collapse may be the only signs of an allergic reaction, and they are easily misdiagnosed. Bronchospasm is reported to be present in about 40% of cases. Successful management of these patients includes stabilisation during the acute reaction and avoidance of future reactions. The latter is based on the identification of the causative drug and potentially cross-reacting compounds. The use of suxamethonium chloride is associated with many other adverse effects, such as fasciculations, myalgia, potassium release, changes in the heart rate, increases in intragastric and intraocular pressures, and malignant hyperthermia. Because of the dangers of hyperkalaemic cardiac arrest after suxamethonium chloride administration in children with unrecognised muscular dystrophy, there have now been moves to limit the use of this drug in children. Although neuromuscular blockers are designed to specifically block nicotinic cholinergic receptors at the neuromuscular junction, many bind to muscarinic cholinergic receptors on ganglia and smooth muscle, and alter parasympathetically mediated heart rate and airway calibre. Most benzylisoquinolinium muscle relaxants can induce histamine release, especially when they are administered rapidly, which can lead to disturbances of cardiovascular function. In addition, nondepolarising neuromuscular blockers have been implicated in causing generalised weakness following their long term administration to patients on an intensive care unit. The problem with these adverse drug reactions is their unpredictable nature. Therefore, prompt recognition with appropriate therapy can help to improve the outcome.

Adverse effects of neuromuscular blockers and their antagonists.

PubMed

Naguib, M; Magboul, M M

1998-06-01

Among all the drugs used for general anesthesia, neuromuscular blockers appear to play a prominent role in the incidence of severe adverse reactions. It now seems likely that most serious adverse drug reactions occurring during anesthesia are immunological in type. The frequency of life-threatening anaphylactic or anaphylactoid reactions occurring during anesthesia has been estimated to be between 1 in 1000 and 1 in 25,000 anesthetic procedures, with the neuromuscular blockers being involved in 80% of cases. The mortality from such serious reactions is reported to be in the range of 3.4 to 6%. The highly immunogenic drug, suxamethonium chloride (succinylcholine), was found to be the most hazardous agent. Drug-specific immunoglobulin E antibodies to suxamethonium chloride and other neuromuscular blockers have been demonstrated. This sensitivity to neuromuscular blockers seems to be a long-lasting phenomenon. During anesthesia, the clinical features of an allergic reaction are often masked. Tachycardia and circulatory collapse may be the only signs of an allergic reaction, and they are easily misdiagnosed. Bronchospasm is reported to be present in about 40% of cases. Successful management of these patients includes stabilisation during the acute reaction and avoidance of future reactions. The latter is based on the identification of the causative drug and potentially cross-reacting compounds. The use of suxamethonium chloride is associated with many other adverse effects, such as fasciculations, myalgia, potassium release, changes in the heart rate, increases in intragastric and intraocular pressures, and malignant hyperthermia. Because of the dangers of hyperkalemic cardiac arrest suxamethonium chloride administration in children with unrecognised muscular dystrophy, there have now been moves to limit the use of this drug in children. Although neuromuscular blockers are designed to specifically block nicotinic cholinergic receptors at the neuromuscular junction, many bind to muscarinic cholinergic receptors on ganglia and smooth muscle, and alter parasympathetically mediated heart rate and airway calibre. Most benzylisoquinolinium muscle relaxants can induce histamine release, especially when they are administered rapidly, which can lead to disturbances of cardiovascular function. In addition, nondepolarising neuromuscular blockers have been implicated in causing generalised weakness following their long term administration to patients on an intensive care unit. The problem with these adverse drug reactions is their upredictable nature. Therefore, prompt recognition with appropriate therapy can help to improve the outcome.

The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grandič, Marjana; Aráoz, Romulo; Molgó, Jordi

APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC{sub 50} = 0.74 μM), without affecting directly-elicited twitch tension up to 2.72 μM. The compound (0.007–3.40 μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 μM, without affecting their frequency. Full size endplate potentials,more » recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC{sub 50} = 0.36 μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1{sub 2}β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC{sub 50} = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1{sub 2}β1γδ) than for the mouse (α1{sub 2}β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction. -- Highlights: ► APS12-2 produces concentration-dependent inhibition of nerve-evoked muscle contraction in vitro. ► APS12-2 blocks MEPPs and EPPs at the neuromuscular junction. APS12-2 blocks ACh-activated current in Xenopus oocytes incorporated with Torpedo nAChRs.« less

Mutations in GMPPB cause congenital myasthenic syndrome and bridge myasthenic disorders with dystroglycanopathies.

PubMed

Belaya, Katsiaryna; Rodríguez Cruz, Pedro M; Liu, Wei Wei; Maxwell, Susan; McGowan, Simon; Farrugia, Maria E; Petty, Richard; Walls, Timothy J; Sedghi, Maryam; Basiri, Keivan; Yue, Wyatt W; Sarkozy, Anna; Bertoli, Marta; Pitt, Matthew; Kennett, Robin; Schaefer, Andrew; Bushby, Kate; Parton, Matt; Lochmüller, Hanns; Palace, Jacqueline; Muntoni, Francesco; Beeson, David

2015-09-01

Congenital myasthenic syndromes are inherited disorders that arise from impaired signal transmission at the neuromuscular junction. Mutations in at least 20 genes are known to lead to the onset of these conditions. Four of these, ALG2, ALG14, DPAGT1 and GFPT1, are involved in glycosylation. Here we identify a fifth glycosylation gene, GMPPB, where mutations cause congenital myasthenic syndrome. First, we identified recessive mutations in seven cases from five kinships defined as congenital myasthenic syndrome using decrement of compound muscle action potentials on repetitive nerve stimulation on electromyography. The mutations were present through the length of the GMPPB, and segregation, in silico analysis, exon trapping, cell transfection followed by western blots and immunostaining were used to determine pathogenicity. GMPPB congenital myasthenic syndrome cases show clinical features characteristic of congenital myasthenic syndrome subtypes that are due to defective glycosylation, with variable weakness of proximal limb muscle groups while facial and eye muscles are largely spared. However, patients with GMPPB congenital myasthenic syndrome had more prominent myopathic features that were detectable on muscle biopsies, electromyography, muscle magnetic resonance imaging, and through elevated serum creatine kinase levels. Mutations in GMPPB have recently been reported to lead to the onset of muscular dystrophy dystroglycanopathy. Analysis of four additional GMPPB-associated muscular dystrophy dystroglycanopathy cases by electromyography found that a defective neuromuscular junction component is not always present. Thus, we find mutations in GMPPB can lead to a wide spectrum of clinical features where deficit in neuromuscular transmission is the major component in a subset of cases. Clinical recognition of GMPPB-associated congenital myasthenic syndrome may be complicated by the presence of myopathic features, but correct diagnosis is important because affected individuals can respond to appropriate treatments. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.

Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

PubMed

Santafe, M M; Priego, M; Obis, T; Garcia, N; Tomàs, M; Lanuza, M A; Tomàs, J

2015-07-01

Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic CaV2.1 Ca2+ channels

PubMed Central

Nanou, Evanthia; Yan, Jin; Whitehead, Nicholas P.; Kim, Min Jeong; Froehner, Stanley C.; Scheuer, Todd; Catterall, William A.

2016-01-01

Facilitation and inactivation of P/Q-type calcium (Ca2+) currents through the regulation of voltage-gated Ca2+ (CaV) 2.1 channels by Ca2+ sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express CaV2.1 channels. To examine the modulation of endogenous CaV2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of CaV2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively CaV2.1 channels for Ca2+ entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10–30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50–100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of CaV2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of CaV2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo. PMID:26755585

Histologic consequences of inferior oblique anastomosis to denervated lateral rectus muscle.

PubMed

Christiansen, S; Madhat, M; Baker, R S

1987-01-01

Secondary muscular neurotization has been proposed as a means of restoring contractility to paretic extraocular muscle. We studied this technique by anastomosing healthy inferior oblique muscle to lateral rectus muscle that had been denervated either orbitally or intracranially in 20 dogs. Nerve and muscle fiber growth from the inferior oblique to the lateral rectus was demonstrated but no new neuromuscular junctions were formed. Regeneration of the lesioned sixth nerve occurred frequently and may explain the restoration of function claimed after this procedure.

Myasthenic syndromes due to defects in COL13A1 and in the N-linked glycosylation pathway.

PubMed

Beeson, David; Cossins, Judith; Rodriguez-Cruz, Pedro; Maxwell, Susan; Liu, Wei-Wei; Palace, Jacqueline

2018-02-01

The congenital myasthenic syndromes (CMS) are hereditary disorders of neuromuscular transmission. The number of cases recognized, at around 1:100,000 in the United Kingdom, is increasing with improved diagnosis. The advent of next-generation sequencing has facilitated the discovery of many genes that harbor CMS-associated mutations. An emerging group of CMS, characterized by a limb-girdle pattern of muscle weakness, is caused by mutations in genes that encode proteins involved in the initial steps of the N-linked glycosylation pathway, which is surprising, since this pathway is found in all mammalian cells. However, mutations in these genes may also give rise to multisystem disorders (congenital disorders of glycosylation) or muscle disorders where the myasthenic symptoms constitute only one component within a wider phenotypic spectrum. We also report a CMS due to mutations in COL13A1, which encodes an extracellular matrix protein that is concentrated at the neuromuscular junction and highlights a role for these extracellular matrix proteins in maintaining synaptic stability that is independent of the AGRN/MuSK clustering pathway. Knowledge about the neuromuscular synapse and the different proteins involved in maintaining its structure as well as function enables us to tailor treatments to the underlying pathogenic mechanisms. © 2018 New York Academy of Sciences.

Caspase 6 has a protective role in SOD1(G93A) transgenic mice.

PubMed

Hogg, Marion C; Mitchem, Mollie R; König, Hans-Georg; Prehn, Jochen H M

2016-06-01

In amyotrophic lateral sclerosis (ALS), it has been suggested that the process of neurodegeneration starts at the neuromuscular junction and is propagated back along axons towards motor neurons. Caspase-dependent pathways are well established as a cause of motor neuron death, and recent work in other disease models indicated a role for caspase 6 in axonal degeneration. Therefore we hypothesised that caspase 6 may be involved in motor neuron death in ALS. To investigate the role of caspase 6 in ALS we profiled protein levels of caspase-6 throughout disease progression in the ALS mouse model SOD1(G93A); this did not reveal differences in caspase 6 levels during disease. To investigate the role of caspase 6 further we generated a colony with SOD1(G93A) transgenic mice lacking caspase 6. Analysis of the transgenic SOD1(G93A); Casp6(-/-) revealed an exacerbated phenotype with motor dysfunction occurring earlier and a significantly shortened lifespan when compared to transgenic SOD1(G93A); Casp6(+/+) mice. Immunofluorescence analysis of the neuromuscular junction revealed no obvious difference between caspase 6(+/+) and caspase 6(-/-) in non-transgenic mice, while the SOD1(G93A) transgenic mice showed severe degeneration compared to non-transgenic mice in both genotypes. Our data indicate that caspase-6 does not exacerbate ALS pathogenesis, but may have a protective role. Copyright © 2016 Elsevier B.V. All rights reserved.

Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A

NASA Technical Reports Server (NTRS)

Suzuki, Kazuhiro; Grinnell, Alan D.; Kidokoro, Yoshiaki

2002-01-01

The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.

Experiment K-7-18: Effects of Spaceflight in the Muscle Adductor Longus of Rats Flown in the Soviet Biosatellite Cosmos 2044. Part 1; A Study Employing Neural Cell Adhesion Molecules (N-CAM) Immunocytochemistry and Conventional Morphological Techniques (Light and Electron Microscopy)

NASA Technical Reports Server (NTRS)

Daunton, N. G.; DAmelio, F.; Wu, L.; Ilyina-Kakueva, E. I.; Krasnov, I. B.; Hyde, T. M.; Sigworth, S. K.

1994-01-01

The effects of spaceflight upon the 'slow' muscle adductor longus was examined in rats flown in the Soviet Biosatellite COSMOS 2044. Three groups - synchronous, vivarium and basal served as controls. The techniques employed included standard methods for light microscopy, N-CAM immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy, contraction bands and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leucocytes and mononuclear cells. N-CAM immunoreactivity was seen (N-CAM-IR) on the myofiber surface, satellite cells and in regenerating myofibers reminiscent of myotubes. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments that displayed varied distributive patterns. The principal electron microscopic changes of the neuromuscular junctions consisted of a decrease or absence of synaptic vesicles, degeneration of axon terminals, increased number of microtubules, vacant axonal spaces and axonal sprouting. The present observations indicate that major alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

Adaptation of Slow Myofibers: The Effect of Sustained BDNF Treatment of Extraocular Muscles in Infant Nonhuman Primates

PubMed Central

Willoughby, Christy L.; Fleuriet, Jérome; Walton, Mark M.; Mustari, Michael J.; McLoon, Linda K.

2015-01-01

Purpose. We evaluated promising new treatment options for strabismus. Neurotrophic factors have emerged as a potential treatment for oculomotor disorders because of diverse roles in signaling to muscles and motor neurons. Unilateral treatment with sustained release brain-derived neurotrophic factor (BDNF) to a single lateral rectus muscle in infant monkeys was performed to test the hypothesis that strabismus would develop in correlation with extraocular muscle (EOM) changes during the critical period for development of binocularity. Methods. The lateral rectus muscles of one eye in two infant macaques were treated with sustained delivery of BDNF for 3 months. Eye alignment was assessed using standard photographic methods. Muscle specimens were analyzed to examine the effects of BDNF on the density, morphology, and size of neuromuscular junctions, as well as myofiber size. Counts were compared to age-matched controls. Results. No change in eye alignment occurred with BDNF treatment. Compared to control muscle, neuromuscular junctions on myofibers expressing slow myosins had a larger area. Myofibers expressing slow myosin had larger diameters, and the percentage of myofibers expressing slow myosins increased in the proximal end of the muscle. Expression of BDNF was examined in control EOM, and observed to have strongest immunoreactivity outside the endplate zone. Conclusions. We hypothesize that the oculomotor system adapted to sustained BDNF treatment to preserve normal alignment. Our results suggest that BDNF treatment preferentially altered myofibers expressing slow myosins. This implicates BDNF signaling as influencing the slow twitch properties of EOM. PMID:26030102

Adaptation of slow myofibers: the effect of sustained BDNF treatment of extraocular muscles in infant nonhuman primates.

PubMed

Willoughby, Christy L; Fleuriet, Jérome; Walton, Mark M; Mustari, Michael J; McLoon, Linda K

2015-06-01

We evaluated promising new treatment options for strabismus. Neurotrophic factors have emerged as a potential treatment for oculomotor disorders because of diverse roles in signaling to muscles and motor neurons. Unilateral treatment with sustained release brain-derived neurotrophic factor (BDNF) to a single lateral rectus muscle in infant monkeys was performed to test the hypothesis that strabismus would develop in correlation with extraocular muscle (EOM) changes during the critical period for development of binocularity. The lateral rectus muscles of one eye in two infant macaques were treated with sustained delivery of BDNF for 3 months. Eye alignment was assessed using standard photographic methods. Muscle specimens were analyzed to examine the effects of BDNF on the density, morphology, and size of neuromuscular junctions, as well as myofiber size. Counts were compared to age-matched controls. No change in eye alignment occurred with BDNF treatment. Compared to control muscle, neuromuscular junctions on myofibers expressing slow myosins had a larger area. Myofibers expressing slow myosin had larger diameters, and the percentage of myofibers expressing slow myosins increased in the proximal end of the muscle. Expression of BDNF was examined in control EOM, and observed to have strongest immunoreactivity outside the endplate zone. We hypothesize that the oculomotor system adapted to sustained BDNF treatment to preserve normal alignment. Our results suggest that BDNF treatment preferentially altered myofibers expressing slow myosins. This implicates BDNF signaling as influencing the slow twitch properties of EOM.

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

PubMed Central

Nishimune, Hiroshi

2013-01-01

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses. PMID:22135013

Shared Resistance to Aging and ALS in Neuromuscular Junctions of Specific Muscles

PubMed Central

Valdez, Gregorio; Tapia, Juan C.; Lichtman, Jeff W.; Fox, Michael A.; Sanes, Joshua R.

2012-01-01

Normal aging and neurodegenerative diseases both lead to structural and functional alterations in synapses. Comparison of synapses that are generally similar but respond differently to insults could provide the basis for discovering mechanisms that underlie susceptibility or resistance to damage. Here, we analyzed skeletal neuromuscular junctions (NMJs) in 16 mouse muscles to seek such differences. We find that muscles respond in one of three ways to aging. In some, including most limb and trunk muscles, age-related alterations to NMJs are progressive and extensive during the second postnatal year. NMJs in other muscles, such as extraocular muscles, are strikingly resistant to change. A third set of muscles, including several muscles of facial expression and the external anal sphinter, succumb to aging but not until the third postnatal year. We asked whether susceptible and resistant muscles differed in rostrocaudal or proximodistal position, source of innervation, motor unit size, or fiber type composition. Of these factors, muscle innervation by brainstem motor neurons correlated best with resistance to age-related decline. Finally, we compared synaptic alterations in normally aging muscles to those in a mouse model of amyotrophic lateral sclerosis (ALS). Patterns of resistance and susceptibility were strikingly correlated in the two conditions. Moreover, damage to NMJs in aged muscles correlated with altered expression and distribution of CRMP4a and TDP-43, which are both altered in motor neurons affected by ALS. Together, these results reveal novel structural, regional and molecular parallels between aging and ALS. PMID:22485182

Restoration of Motor Defects Caused by Loss of Drosophila TDP-43 by Expression of the Voltage-Gated Calcium Channel, Cacophony, in Central Neurons.

PubMed

Lembke, Kayly M; Scudder, Charles; Morton, David B

2017-09-27

Defects in the RNA-binding protein, TDP-43, are known to cause a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar dementia. A variety of experimental systems have shown that neurons are sensitive to TDP-43 expression levels, yet the specific functional defects resulting from TDP-43 dysregulation have not been well described. Using the Drosophila TDP-43 ortholog TBPH, we previously showed that TBPH-null animals display locomotion defects as third instar larvae. Furthermore, loss of TBPH caused a reduction in cacophony , a Type II voltage-gated calcium channel, expression and that genetically restoring cacophony in motor neurons in TBPH mutant animals was sufficient to rescue the locomotion defects. In the present study, we examined the relative contributions of neuromuscular junction physiology and the motor program to the locomotion defects and identified subsets of neurons that require cacophony expression to rescue the defects. At the neuromuscular junction, we showed mEPP amplitudes and frequency require TBPH. Cacophony expression in motor neurons rescued mEPP frequency but not mEPP amplitude. We also showed that TBPH mutants displayed reduced motor neuron bursting and coordination during crawling and restoring cacophony selectively in two pairs of cells located in the brain, the AVM001b/2b neurons, also rescued the locomotion and motor defects, but not the defects in neuromuscular junction physiology. These results suggest that the behavioral defects associated with loss of TBPH throughout the nervous system can be associated with defects in a small number of genes in a limited number of central neurons, rather than peripheral defects. SIGNIFICANCE STATEMENT TDP-43 dysfunction is a common feature in neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar dementia, and Alzheimer's disease. Loss- and gain-of-function models have shown that neurons are sensitive to TDP-43 expression levels, but the specific defects caused by TDP-43 loss of function have not been described in detail. A Drosophila loss-of-function model displays pronounced locomotion defects that can be reversed by restoring the expression levels of a voltage-gated calcium channel, cacophony. We show these defects can be rescued by expression of cacophony in motor neurons and by expression in two pairs of neurons in the brain. These data suggest that loss of TDP-43 can disrupt the central circuitry of the CNS, opening up identification of alternative therapeutic targets for TDP-43 proteinopathies. Copyright © 2017 the authors 0270-6474/17/379486-12$15.00/0.

The long journey of botulinum neurotoxins into the synapse.

PubMed

Rummel, Andreas

2015-12-01

Botulinum neurotoxins (BoNT) cause the disease botulism, a flaccid paralysis of the muscle. They are also very effective, widely used medicines applied locally in sub-nanogram quantities. BoNTs are released together with several non-toxic, associated proteins as progenitor toxin complexes (PCT) by Clostridium botulinum to become highly potent oral poisons ingested via contaminated food. They block the neurotransmission in susceptible animals and humans already in nanogram quantities due to their specific ability to enter motoneurons and to cleave only selected neuronal proteins involved in neuroexocytosis. BoNTs have developed a sophisticated strategy to passage the gastrointestinal tract and to be absorbed in the intestine of the host to finally attack neurons. A non-toxic non-hemagglutinin (NTNHA) forms a binary complex with BoNT to protect it from gastrointestinal degradation. This binary M-PTC is one component of the bi-modular 14-subunit ∼760 kDa large progenitor toxin complex. The other component is the structurally and functionally independent dodecameric hemagglutinin (HA) complex which facilitates the absorption on the intestinal epithelium by glycan binding. Subsequent to its transcytosis the HA complex disrupts the tight junction of the intestinal barrier from the basolateral side by binding to E-cadherin. Now, the L-PTC can also enter the circulation by paracellular routes in much larger quantities. From here, the dissociated BoNTs reach the neuromuscular junction and accumulate via interaction with polysialo gangliosides, complex glycolipids, on motoneurons at the neuromuscular junction. Subsequently, additional specific binding to luminal segments of synaptic vesicles proteins like SV2 and synaptotagmin leads to their uptake. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had exploited before to enter their target cells, via specific cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which constitute the core components of the cellular membrane fusion machinery. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single-neuron labeling with inducible cre-mediated knockout in transgenic mice

PubMed Central

Young, Paul; Qiu, Li; Wang, Dongqing; Zhao, Shengli; Gross, James; Feng, Guoping

2011-01-01

To facilitate functional analysis of neuronal connectivity in a mammalian nervous system tightly packed with billions of cells, we developed a new technique that allows inducible genetic manipulations within fluorescently labeled single neurons in mice. We term this technique SLICK for Single-neuron Labeling with Inducible Cre-mediated Knockout. SLICK is achieved by co-expressing a drug-inducible form of cre recombinase and a fluorescent protein within the same small subsets of neurons. Thus, SLICK combines the powerful cre recombinase system for conditional genetic manipulation and the fluorescent labeling of single neurons for imaging. We demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we apply SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals. More broadly, these studies demonstrate a cre-LoxP compatible system for dissecting gene functions in single identifiable neurons. PMID:18454144

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.

PubMed

Tourovskaia, Anna; Kosar, T Fettah; Folch, Albert

2006-03-15

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.

Experiences of living with myasthenia gravis: a qualitative study with Taiwanese people.

PubMed

Chen, Yu Tai; Shih, Fu Jin; Hayter, Mark; Hou, Chang Chiu; Yeh, Jiann Horng

2013-04-01

Myasthenia gravis (MG) is an auto-immune, neuromuscular disorder, which presents with symptoms of fluctuating muscle fatigue because of a dysfunction of the neuromuscular junction. This study explores the illness experience of patients with MG, their experiences of illness, its challenges, and their coping and support strategies. In-depth interviews were undertaken with nine participants with MG (six for a generalized type of MG, three for ocular type). Data were subjected to inductive content and thematic analysis. Four themes emerged from MG patients with associated subthemes. They were "perceptions of MG," "challenges of MG, "social support," and "adapting and adjusting to MG." The study reveals the way in which individuals respond to and cope with their diagnosis. The importance of social and peer support is a key factor as well as the development of psychological strategies to live with MG. The recognition that there was a need to recognize the role of Western medicine in controlling their disease was also an important finding.

Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice

PubMed Central

Kwon, Deborah Y.; Motley, William W.; Fischbeck, Kenneth H.; Burnett, Barrington G.

2011-01-01

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by reduced levels of the survival motor neuron (SMN) protein. Here we show that the proteasome inhibitor, bortezomib, increases SMN in cultured cells and in peripheral tissues of SMA model mice. Bortezomib-treated animals had improved motor function, which was associated with reduced spinal cord and muscle pathology and improved neuromuscular junction size, but no change in survival. Combining bortezomib with the histone deacetylase inhibitor trichostatin A (TSA) resulted in a synergistic increase in SMN protein levels in mouse tissue and extended survival of SMA mice more than TSA alone. Our results demonstrate that a combined regimen of drugs that decrease SMN protein degradation and increase SMN gene transcription synergistically increases SMN levels and improves the lifespan of SMA model mice. Moreover, this study indicates that while increasing SMN levels in the central nervous system may help extend survival, peripheral tissues can also be targeted to improve the SMA disease phenotype. PMID:21693563

Synaptic activity-related classical protein kinase C isoform localization in the adult rat neuromuscular synapse.

PubMed

Besalduch, Núria; Tomàs, Marta; Santafé, Manel M; Garcia, Neus; Tomàs, Josep; Lanuza, Maria Angel

2010-01-10

Protein kinase C (PKC) is essential for signal transduction in a variety of cells, including neurons and myocytes, and is involved in both acetylcholine release and muscle fiber contraction. Here, we demonstrate that the increases in synaptic activity by nerve stimulation couple PKC to transmitter release in the rat neuromuscular junction and increase the level of alpha, betaI, and betaII isoforms in the membrane when muscle contraction follows the stimulation. The phosphorylation activity of these classical PKCs also increases. It seems that the muscle has to contract in order to maintain or increase classical PKCs in the membrane. We use immunohistochemistry to show that PKCalpha and PKCbetaI were located in the nerve terminals, whereas PKCalpha and PKCbetaII were located in the postsynaptic and the Schwann cells. Stimulation and contraction do not change these cellular distributions, but our results show that the localization of classical PKC isoforms in the membrane is affected by synaptic activity.

Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons.

PubMed

Herzog, E; Landry, M; Buhler, E; Bouali-Benazzouz, R; Legay, C; Henderson, C E; Nagy, F; Dreyfus, P; Giros, B; El Mestikawy, S

2004-10-01

Mammalian spinal motoneurons are cholinergic neurons that have long been suspected to use also glutamate as a neurotransmitter. We report that VGLUT1 and VGLUT2, two subtypes of vesicular glutamate transporters, are expressed in rat spinal motoneurons. Both proteins are present in somato-dendritic compartments as well as in axon terminals in primary cultures of immunopurified motoneurons and sections of spinal cord from adult rat. However, VGLUT1 and VGLUT2 are not found at neuromuscular junctions of skeletal muscles. After intracellular injection of biocytin in motoneurons, VGLUT2 is observed in anterogradely labelled terminals contacting Renshaw inhibitory interneurons. These VGLUT2- and VGLUT1-positive terminals do not express VAChT, the vesicular acetylcholine transporter. Overall, our study establishes for the first time that (i) mammalian spinal motoneurons express vesicular glutamate transporters, (ii) these motoneurons have the potential to release glutamate (in addition to acetylcholine) at terminals contacting Renshaw cells, and finally (iii) the VGLUTs are not present at neuromuscular synapses of skeletal muscles.

Lambert-Eaton myasthenic syndrome: the 60th anniversary of Eaton and Lambert's pioneering article.

PubMed

Lorenzoni, Paulo José; Kay, Cláudia Suemi Kamoi; Werneck, Lineu Cesar; Scola, Rosana Herminia

2018-02-01

This historical review describes the contribution of Drs. Lee M. Eaton and Edward H. Lambert to the diagnosis of myasthenic syndrome on the 60th anniversary of their pioneering article (JAMA 1957) on the disease. There are important landmarks in their article on a disorder of the neuromuscular junction associated with thoracic neoplasm and the electrophysiological criteria for Lambert-Eaton myasthenic syndrome (LEMS). After 60 years, the main electrophysiological criteria described in Eaton and Lambert's pioneering article are still currently useful in the diagnosis of LEMS.

Autoimmune synaptopathies.

PubMed

Crisp, Sarah J; Kullmann, Dimitri M; Vincent, Angela

2016-02-01

Autoantibodies targeting proteins at the neuromuscular junction are known to cause several distinct myasthenic syndromes. Recently, autoantibodies targeting neurotransmitter receptors and associated proteins have also emerged as a cause of severe, but potentially treatable, diseases of the CNS. Here, we review the clinical evidence as well as in vitro and in vivo experimental evidence that autoantibodies account for myasthenic syndromes and autoimmune disorders of the CNS by disrupting the functional or structural integrity of synapses. Studying neurological and psychiatric diseases of autoimmune origin may provide new insights into the cellular and circuit mechanisms underlying a broad range of CNS disorders.

Electrophysiological and Ultrastructural Characterization of Neuromuscular Junctions in Diaphragm Muscle of Acetylcholinesterase Knockout Mice

DTIC Science & Technology

2008-04-01

conditioned fresh air. The mice were on a 12-hr light/dark full-spectrum lighting cycle . AChE +/+ and AChE -/- mice were 58.7 ± 2.9 and 51.6 ± 3.2 days old...diaphragms were excised and pinned at resting length in Sylgard (Dow Chemical Co., Midland, MI) coated dishes containing oxygenated Krebs -Ringer solution of...measurements Hemidiaphragms with attached phrenic nerves were mounted in tissue baths at 37o C and immersed in an oxygenated Krebs -Ringer solution. To

Hexamethonium produces both twitch and tetanic depression without fade in common African toad (Bufo regularis).

PubMed

Ajibola, E S; Adebayo, A O; Thomas, F C; Rahman, S A; Gbadebo, A M; Odunbaku, T A

2009-12-01

The study was designed to investigate the nature of the cholinoceptors at the sciatic nerve-gastrocnemius muscle junction of the common African toad (Bufo regularis). Using myographic technique, the twitch properties of the sciatic-gastrocnemius muscle preparation of the common African toad was studied. Both the twitch height and peak tetanic height were measured as a percentage of control. Hexamethonium at a concentration of 0.1 mM significantly [P<0.05] reduced the mean twitch height from 2.62 cm to 1.0 cm and mean peak tetanic height from 5.38 cm to 4.32 cm. Hexamethonium, however does not produce tetanic fade at the same concentration. We hypothesized that the cholinoceptors of the neuromuscular junction of the common African toad (Bufo regularis) resemble the developing synapse of African clawed toad (Xenopus laevis) and may contain muscarinic M1 autoreceptors at the pre juntional membrane.

Activity-dependent degeneration of axotomized neuromuscular synapses in WldS mice

PubMed Central

Brown, R.; Hynes-Allen, A.; Swan, A.J.; Dissanayake, K.N.; Gillingwater, T.H.; Ribchester, R.R.

2015-01-01

Activity and disuse of synapses are thought to influence progression of several neurodegenerative diseases in which synaptic degeneration is an early sign. Here we tested whether stimulation or disuse renders neuromuscular synapses more or less vulnerable to degeneration, using axotomy as a robust trigger. We took advantage of the slow synaptic degeneration phenotype of axotomized neuromuscular junctions in flexor digitorum brevis (FDB) and deep lumbrical (DL) muscles of Wallerian degeneration-Slow (WldS) mutant mice. First, we maintained ex vivo FDB and DL nerve-muscle explants at 32 °C for up to 48 h. About 90% of fibers from WldS mice remained innervated, compared with about 36% in wild-type muscles at the 24-h checkpoint. Periodic high-frequency nerve stimulation (100 Hz: 1 s/100 s) reduced synaptic protection in WldS preparations by about 50%. This effect was abolished in reduced Ca2+ solutions. Next, we assayed FDB and DL innervation after 7 days of complete tetrodotoxin (TTX)-block of sciatic nerve conduction in vivo, followed by tibial nerve axotomy. Five days later, only about 9% of motor endplates remained innervated in the paralyzed muscles, compared with about 50% in 5 day-axotomized muscles from saline-control-treated WldS mice with no conditioning nerve block. Finally, we gave mice access to running wheels for up to 4 weeks prior to axotomy. Surprisingly, exercising WldS mice ad libitum for 4 weeks increased about twofold the amount of subsequent axotomy-induced synaptic degeneration. Together, the data suggest that vulnerability of mature neuromuscular synapses to axotomy, a potent neurodegenerative trigger, may be enhanced bimodally, either by disuse or by hyperactivity. PMID:25617654

Antivenom for Neuromuscular Paralysis Resulting From Snake Envenoming

PubMed Central

Silva, Anjana; Hodgson, Wayne C.; Isbister, Geoffrey K.

2017-01-01

Antivenom therapy is currently the standard practice for treating neuromuscular dysfunction in snake envenoming. We reviewed the clinical and experimental evidence-base for the efficacy and effectiveness of antivenom in snakebite neurotoxicity. The main site of snake neurotoxins is the neuromuscular junction, and the majority are either: (1) pre-synaptic neurotoxins irreversibly damaging the presynaptic terminal; or (2) post-synaptic neurotoxins that bind to the nicotinic acetylcholine receptor. Pre-clinical tests of antivenom efficacy for neurotoxicity include rodent lethality tests, which are problematic, and in vitro pharmacological tests such as nerve-muscle preparation studies, that appear to provide more clinically meaningful information. We searched MEDLINE (from 1946) and EMBASE (from 1947) until March 2017 for clinical studies. The search yielded no randomised placebo-controlled trials of antivenom for neuromuscular dysfunction. There were several randomised and non-randomised comparative trials that compared two or more doses of the same or different antivenom, and numerous cohort studies and case reports. The majority of studies available had deficiencies including poor case definition, poor study design, small sample size or no objective measures of paralysis. A number of studies demonstrated the efficacy of antivenom in human envenoming by clearing circulating venom. Studies of snakes with primarily pre-synaptic neurotoxins, such as kraits (Bungarus spp.) and taipans (Oxyuranus spp.) suggest that antivenom does not reverse established neurotoxicity, but early administration may be associated with decreased severity or prevent neurotoxicity. Small studies of snakes with mainly post-synaptic neurotoxins, including some cobra species (Naja spp.), provide preliminary evidence that neurotoxicity may be reversed with antivenom, but placebo controlled studies with objective outcome measures are required to confirm this. PMID:28422078

MAPK3 at the Autism-Linked Human 16p11.2 Locus Influences Precise Synaptic Target Selection at Drosophila Larval Neuromuscular Junctions.

PubMed

Park, Sang Mee; Park, Hae Ryoun; Lee, Ji Hye

2017-02-01

Proper synaptic function in neural circuits requires precise pairings between correct pre- and post-synaptic partners. Errors in this process may underlie development of neuropsychiatric disorders, such as autism spectrum disorder (ASD). Development of ASD can be influenced by genetic factors, including copy number variations (CNVs). In this study, we focused on a CNV occurring at the 16p11.2 locus in the human genome and investigated potential defects in synaptic connectivity caused by reduced activities of genes located in this region at Drosophila larval neuromuscular junctions, a well-established model synapse with stereotypic synaptic structures. A mutation of rolled , a Drosophila homolog of human mitogen-activated protein kinase 3 ( MAPK3 ) at the 16p11.2 locus, caused ectopic innervation of axonal branches and their abnormal defasciculation. The specificity of these phenotypes was confirmed by expression of wild-type rolled in the mutant background. Albeit to a lesser extent, we also observed ectopic innervation patterns in mutants defective in Cdk2, Gα q , and Gp93, all of which were expected to interact with Rolled MAPK3. A further genetic analysis in double heterozygous combinations revealed a synergistic interaction between rolled and Gp93 . In addition, results from RT-qPCR analyses indicated consistently reduced rolled mRNA levels in Cdk2 , Gα q , and Gp93 mutants. Taken together, these data suggest a central role of MAPK3 in regulating the precise targeting of presynaptic axons to proper postsynaptic targets, a critical step that may be altered significantly in ASD.

Effect of temperature on endplate potential rundown and recovery in rat diaphragm.

PubMed

Moyer, M; van Lunteren, E

2001-05-01

The amplitude of neuromuscular junction end-plate potentials (EPPs) decreases quickly within a train but recovers nearly completely from train to train during intermittent stimulation. Rundown has been shown to be dependent not only on the rate of transmitter release but also on the rate of replenishment of the depleted neurotransmitter at the site of release. Two groups of processes have been proposed for synaptic vesicle recycling, both of which involve multiple energy-requiring steps and enzymatic reactions and which therefore would be expected to be very temperature-sensitive. The present study tested the hypothesis that low temperature therefore increases the rate of EPP amplitude rundown. Studies were performed in vitro on rat diaphragm and used micro-conotoxin to allow normal-sized EPPs to be recorded from intact fibers. EPP amplitude rundown during intermittent stimulation at 20 and 50 Hz (duty cycle 333 ms) was greater at 20 degrees C than it was at 37 degrees C. Initially, temperature affected only intra-train rundown but, over longer periods of stimulation, both intra- and inter-train rundown were significantly accelerated by cold temperature. Cumulative EPP amplitudes were calculated by successively adding the amplitudes of each EPP during the stimulation period to provide an estimate of total neurotransmitter release in the neuromuscular junction. The cumulative EPP amplitude was significantly lower at 20 degrees C than it was at 37 degrees C during both 20 and 50 Hz stimulation. These data indicate that the mechanism involved in EPP amplitude rundown and recovery is temperature-sensitive, with a greater decrement in EPP amplitude at cold than at warm temperatures.

Induced formation and maturation of acetylcholine receptor clusters in a defined 3D bio-artificial muscle.

PubMed

Wang, Lin; Shansky, Janet; Vandenburgh, Herman

2013-12-01

Dysfunction of the neuromuscular junction is involved in a wide range of muscular diseases. The development of neuromuscular junction through which skeletal muscle is innervated requires the functional modulation of acetylcholine receptor (AchR) clustering on myofibers. However, studies on AchR clustering in vitro are mostly done on monolayer muscle cell culture, which lacks a three-dimensional (3D) structure, a prominent limitation of the two-dimensional (2D) system. To enable a better understanding on the structure-function correlation underlying skeletal muscle innervation, a muscle system with a well-defined geometry mimicking the in vivo muscular setting is needed. Here, we report a 3D bio-artificial muscle (BAM) bioengineered from green fluorescent protein-transduced C3H murine myoblasts as a novel in vitro tissue-based model for muscle innervation studies. Our cell biological and molecular analysis showed that this BAM is structurally similar to in vivo muscle tissue and can reach the perinatal differentiation stage, higher than does 2D culture. Effective clustering and morphological maturation of AchRs on BAMs induced by agrin and laminin indicate the functional activity and plasticity of this BAM system toward innervation. Taken together, our results show that the BAM provides a favorable 3D environment that at least partially recapitulates real physiological skeletal muscle with regard to innervation. With a convenience of fabrication and manipulation, this 3D in vitro system offers a novel model for studying mechanisms underlying skeletal muscle innervation and testing therapeutic strategies for relevant nervous and muscular diseases.

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis.

PubMed

Tüzün, Erdem; Scott, Benjamin G; Goluszko, Elzbieta; Higgs, Stephen; Christadoss, Premkumar

2003-10-01

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.

Ubiquitin–Synaptobrevin Fusion Protein Causes Degeneration of Presynaptic Motor Terminals in Mice

PubMed Central

Liu, Yun; Li, Hongqiao; Sugiura, Yoshie; Han, Weiping; Gallardo, Gilbert; Khvotchev, Mikhail; Zhang, Yinan; Kavalali, Ege T.; Südhof, Thomas C.

2015-01-01

Protein aggregates containing ubiquitin (Ub) are commonly observed in neurodegenerative disorders, implicating the involvement of the ubiquitin proteasome system (UPS) in their pathogenesis. Here, we aimed to generate a mouse model for monitoring UPS function using a green fluorescent protein (GFP)-based substrate that carries a “noncleavable” N-terminal ubiquitin moiety (UbG76V). We engineered transgenic mice expressing a fusion protein, consisting of the following: (1) UbG76V, GFP, and a synaptic vesicle protein synaptobrevin-2 (UbG76V-GFP-Syb2); (2) GFP-Syb2; or (3) UbG76V-GFP-Syntaxin1, all under the control of a neuron-specific Thy-1 promoter. As expected, UbG76V-GFP-Syb2, GFP-Syb2, and UbG76V-GFP-Sytaxin1 were highly expressed in neurons, such as motoneurons and motor nerve terminals of the neuromuscular junction (NMJ). Surprisingly, UbG76V-GFP-Syb2 mice developed progressive adult-onset degeneration of motor nerve terminals, whereas GFP-Syb2 and UbG76V-GFP-Syntaxin1 mice were normal. The degeneration of nerve terminals in UbG76V-GFP-Syb2 mice was preceded by a progressive impairment of synaptic transmission at the NMJs. Biochemical analyses demonstrated that UbG76V-GFP-Syb2 interacted with SNAP-25 and Syntaxin1, the SNARE partners of synaptobrevin. Ultrastructural analyses revealed a marked reduction in synaptic vesicle density, accompanying an accumulation of tubulovesicular structures at presynaptic nerve terminals. These morphological defects were largely restricted to motor nerve terminals, as the ultrastructure of motoneuron somata appeared to be normal at the stages when synaptic nerve terminals degenerated. Furthermore, synaptic vesicle endocytosis and membrane trafficking were impaired in UbG76V-GFP-Syb2 mice. These findings indicate that UbG76V-GFP-Syb2 may compete with endogenous synaptobrevin, acting as a gain-of-function mutation that impedes SNARE function, resulting in the depletion of synaptic vesicles and degeneration of the nerve terminals. SIGNIFICANCE STATEMENT Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (UbG76V-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration. PMID:26290230

An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

PubMed

Karakesisoglou, I; Yang, Y; Fuchs, E

2000-04-03

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.

Ocular myasthenia gravis: A review

PubMed Central

Nair, Akshay Gopinathan; Patil-Chhablani, Preeti; Venkatramani, Devendra V; Gandhi, Rashmin Anilkumar

2014-01-01

Myasthenia gravis (MG) is a disease that affects the neuro-muscular junction resulting in classical symptoms of variable muscle weakness and fatigability. It is called the great masquerader owing to its varied clinical presentations. Very often, a patient of MG may present to the ophthalmologist given that a large proportion of patients with systemic myasthenia have ocular involvement either at presentation or during the later course of the disease. The treatment of ocular MG involves both the neurologist and ophthalmologist. Thus, the aim of this review was to highlight the current diagnosis, investigations, and treatment of ocular MG. PMID:25449931

Communication Breakdown: The Impact of Ageing on Synapse Structure

PubMed Central

Petralia, Ronald S.; Mattson, Mark P.; Yao, Pamela J.

2014-01-01

Impaired synaptic plasticity is implicated in the functional decline of the nervous system associated with ageing. Understanding the structure of ageing synapses is essential to understanding the functions of these synapses and their role in the ageing nervous system. In this review, we summarize studies on ageing synapses in vertebrates and invertebrates, focusing on changes in morphology and ultrastructure. We cover different parts of the nervous system, including the brain, the retina, the cochlea, and the neuromuscular junction. The morphological characteristics of aged synapses could shed light on the underlying molecular changes and their functional consequences. PMID:24495392

Ineffectiveness of 3,4-diaminopyridine as a therapy for type C botulism.

USGS Publications Warehouse

Siegel, L.S.; Price, J.I.

1987-01-01

Clostridium botulinum neurotoxins inhibit acetylcholine release at neuromuscular junctions. Agents stimulating neurotransmitter efflux, such as 3,4-diaminopyridine (3,4-DAP), could be useful for botulism therapy. Treatment with 3,4-DAP (8 mg/kg hourly, beginning 3 hr after toxin injection) failed to increase the survival times of mice receiving 10, 20 or 40 LD50 type C, but did prolong the survival of those receiving 20 LD50 type A. This difference in 3,4-DAP efficacy may reflect variations in the molecular mechanism of action of types A and C botulinum neurotoxins.

Isaacs' syndrome in a patient with dermatomyositis: case report and review of the literature.

PubMed

Lertnawapan, Ratchaya; Kulkantrakorn, Kongkiat

2017-08-01

This is a case report of Isaacs' syndrome in dermatomyositis. The patient presented with proximal muscle weakness, rash, elevated muscle enzyme, myopathic electromyograph and typical muscle biopsy. Ultimately he developed typical symptoms of Isaacs' syndrome which is an autoimmune channelopathy from voltage gated potassium channel antibody (anti-VGKC) leading to dysfunction of axonal discharge at neuromuscular junctions. It shares some similar characteristics with dermatomyositis such as autoimmunity, its association with malignancy and the response to treatment. © 2016 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.

PubMed Central

Uchitel, O D; Protti, D A; Sanchez, V; Cherksey, B D; Sugimori, M; Llinás, R

1992-01-01

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel. Images PMID:1348859

P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.

PubMed

Uchitel, O D; Protti, D A; Sanchez, V; Cherksey, B D; Sugimori, M; Llinás, R

1992-04-15

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.

Effect of pyridostigmine on in vivo and in vitro respiratory muscle of mdx mice.

PubMed

Amancio, Gabriela de Cássia Sousa; Grabe-Guimarães, Andrea; Haikel, Dridi; Moreau, Johan; Barcellos, Neila Marcia Silva; Lacampagne, Alain; Matecki, Stefan; Cazorla, Olivier

2017-09-01

The current work was conducted to verify the contribution of neuromuscular transmission defects at the neuromuscular junction to Duchenne Muscular Dystrophy disease progression and respiratory dysfunction. We tested pyridostigmine and pyridostigmine encapsulated in liposomes (liposomal PYR), an acetylcholinesterase inhibitor to improve muscular contraction on respiratory muscle function in mdx mice at different ages. We evaluated in vivo with the whole-body plethysmography, the ventilatory response to hypercapnia, and measured in vitro diaphragm strength in each group. Compared to C57BL10 mice, only 17 and 22 month-old mdx presented blunted ventilatory response, under normocapnia and hypercapnia. Free pyridostigmine (1mg/kg) was toxic to mdx mice, unlike liposomal PYR, which did not show any side effect, confirming that the encapsulation in liposomes is effective in reducing the toxic effects of this drug. Treatment with liposomal PYR, either acute or chronic, did not show any beneficial effect on respiratory function of this DMD experimental model. The encapsulation in liposomes is effective to abolish toxic effects of drugs. Copyright © 2017. Published by Elsevier B.V.

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

PubMed Central

Chen, Justin L.; Qian, Hongwei; Liu, Yingying; Bernardo, Bianca C.; Beyer, Claudia; Watt, Kevin I.; Thomson, Rachel E.; Connor, Timothy; Turner, Bradley J.; McMullen, Julie R.; Larsson, Lars; McGee, Sean L.; Harrison, Craig A.

2013-01-01

Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders. PMID:24145169

A kinetic model for chemical neurotransmission

NASA Astrophysics Data System (ADS)

Ramirez-Santiago, Guillermo; Martinez-Valencia, Alejandro; Fernandez de Miguel, Francisco

Recent experimental observations in presynaptic terminals at the neuromuscular junction indicate that there are stereotyped patterns of cooperativeness in the fusion of adjacent vesicles. That is, a vesicle in hemifusion process appears on the side of a fused vesicle and which is followed by another vesicle in a priming state while the next one is in a docking state. In this talk we present a kinetic model for this morphological pattern in which each vesicle state previous to the exocytosis is represented by a kinetic state. This chain states kinetic model can be analyzed by means of a Master equation whose solution is simulated with the stochastic Gillespie algorithm. With this approach we have reproduced the responses to the basal release in the absence of stimulation evoked by the electrical activity and the phenomena of facilitation and depression of neuromuscular synapses. This model offers new perspectives to understand the underlying phenomena in chemical neurotransmission based on molecular interactions that result in the cooperativity between vesicles during neurotransmitter release. DGAPA Grants IN118410 and IN200914 and Conacyt Grant 130031.

Presynaptic Disorders: Lambert-Eaton Myasthenic Syndrome and Botulism.

PubMed

Gable, Karissa L; Massey, Janice M

2015-08-01

Lambert-Eaton myasthenic syndrome (LEMS) and botulism are acquired presynaptic nerve terminal disorders of the neuromuscular junction. Lambert-Eaton myasthenic syndrome is an idiopathic or paraneoplastic autoimmune syndrome in which autoantibodies of the P/Q-type voltage-gated calcium channel play a role in decreasing the release of acetylcholine, resulting in clinical symptoms of skeletal muscle weakness, diminished reflexes, and autonomic symptoms. Paraneoplastic LEMS is most often associated with small cell lung cancer. Diagnosis is confirmed by positive serologic testing and electrophysiological studies, which display characteristic features of low compound muscle action potentials, a decrement at 3Hz repetitive nerve stimulation, and facilitation with exercise or high-frequency repetitive stimulation. Treatment involves cancer monitoring and treatment, 3,4-diaminopyridine, immunosuppressive medications, and acetylcholinesterase inhibitors. Botulism is another presynaptic disorder of neuromuscular transmission. Clinical features classically involve cranial and bulbar palsies followed by descending weakness of the limbs, respiratory failure, and autonomic dysfunction. Electrodiagnostic testing is important in the evaluation and diagnosis. Treatment is supportive, and administration of antitoxin is beneficial in selected cases. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

The effect of potassium on exocytosis of transmitter at the frog neuromuscular junction.

PubMed Central

Ceccarelli, B; Fesce, R; Grohovaz, F; Haimann, C

1988-01-01

1. Electrophysiology and morphology have been combined to investigate the time course of the exocytosis of quanta of neurotransmitter induced by elevated concentrations of K+ at the frog neuromuscular junction. 2. Replicas of freeze-fractured resting nerve terminals fixed in the presence of 20 mM-K+ showed images of fusion of synaptic vesicles with the presynaptic axolemma which were closely associated with the active zones. After 1 min in 20 nM-K+ fusions appeared also outside the active zones, and by 5 min they became uniformly distributed over the presynaptic membrane. 3. The average total density of fusions was not significantly different at the various times examined since it decreased at the active zones while it increased over the rest of the membrane. 4. Resting terminals fixed in 20 mM-K+ released 33,000-45,000 quanta after the addition of fixative; terminals stimulated by 20 mM-K+ for 1-5 min released 50,000-100,000 quanta during fixation. The fixative potentiated K+-induced transmitter release. 5. Fusions were uniformly distributed in terminals pre-incubated for 5 min in 20 mM-K+ without added Ca2+, stimulated by adding Ca2+ for 30 s, and then fixed. Conversely, after 5 min stimulation in hypertonic Ringer solution fusions remained predominantly located near the active zones. A similar distribution was observed after 15 min stimulation by a lower concentration of K+ (15 mM). 6. At all concentrations of K+ tested (10, 15, 20, 25 mM) miniature end-plate potential (MEPP) rate attained a steady-state value within 10-15 min. Values from a single junction were generally lower at higher concentrations of K+, which indicates partial inactivation of the secretion-recycling process. 7. The data indicate that K+ initially activates exocytosis at the active zones. Subsequently, ectopic exocytosis is activated while sites at the active zones appear to undergo partial inactivation. These phenomena are not related to the intensity or to the amount of previous secretion. Images Fig. 1 Fig. 2 Fig. 3 Fig. 8 Fig. 10 PMID:2902217

Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

PubMed

Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

2016-01-15

After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis. Copyright © 2015 Elsevier B.V. All rights reserved.

ELECTRON MICROSCOPIC LOCALIZATION OF ACETYLCHOLINESTERASE AND NONSPECIFIC CHOLINESTERASE AT THE NEUROMUSCULAR JUNCTION BY THE GOLD-THIOCHOLINE AND GOLD-THIOLACETIC ACID METHODS

PubMed Central

Davis, Richard; Koelle, George B.

1967-01-01

By means of the gold-thiocholine (AuThCh) and gold-thiolacetic acid (AuThAc) methods, it has been demonstrated electron microscopically that acetylcholinesterase (AChE) is located at the prejunctional axoplasmic membrane and the postjunctional sarcoplasmic membrane, including the full lengths of its invaginations, at the motor end plate of mouse intercostal muscle. Nonspecific cholinesterase (ChE) is present in relatively low concentrations at the same sites, and in greater concentrations in the teloglial Schwann sheath cells. Significant amounts of reaction product appeared in the junctional cleft only after prolonged incubation with both methods. The identification of AChE and ChE was confirmed by the use of appropriate concentrations of several selective inhibitors. In confirmation of previous studies by light microscopy, the AuThCh method is more specific for AChE and ChE, whereas the AuThAc method allows greater accuracy of localization. PMID:6033530

The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling.

PubMed

Ryan, Kerrianne; Lu, Zhiyuan; Meinertzhagen, Ian A

2016-12-06

Left-right asymmetries in brains are usually minor or cryptic. We report brain asymmetries in the tiny, dorsal tubular nervous system of the ascidian tadpole larva, Ciona intestinalis . Chordate in body plan and development, the larva provides an outstanding example of brain asymmetry. Although early neural development is well studied, detailed cellular organization of the swimming larva's CNS remains unreported. Using serial-section EM we document the synaptic connectome of the larva's 177 CNS neurons. These formed 6618 synapses including 1772 neuromuscular junctions, augmented by 1206 gap junctions. Neurons are unipolar with at most a single dendrite, and few synapses. Some synapses are unpolarised, others form reciprocal or serial motifs; 922 were polyadic. Axo-axonal synapses predominate. Most neurons have ciliary organelles, and many features lack structural specialization. Despite equal cell numbers on both sides, neuron identities and pathways differ left/right. Brain vesicle asymmetries include a right ocellus and left coronet cells.

Modeling study of mecamylamine block of muscle type acetylcholine receptors.

PubMed

Ostroumov, Konstantin; Shaikhutdinova, Asya; Skorinkin, Andrey

2008-04-01

The blocking action of mecamylamine on different types of nicotinic acetylcholine receptors (nAChRs) has been extensively studied and used as a tool to characterize the nAChRs from different synapses. However, mechanism of mecamylamine action was not fully explored for all types of nAChRs. In the present study, we provide brief description of the mecamylamine action on muscle nAChRs expressed at the frog neuromuscular junction. In this preparation mecamylamine block of nAChRs was accompanied by a use-dependent block relief induced by membrane depolarization combined with the activation of nAChRs by endogenous agonist acetylcholine (ACh). Further, three kinetic models of possible mecamylamine interaction with nAChRs were analyzed including simple open channel block, symmetrical trapping block and asymmetrical trapping block. This analysis suggested that mecamylamine action could be described on the basis of trapping mechanism, when the antagonist remained inside the channel even in the absence of bound agonist. Such receptors with trapped mecamylamine inside were predicted to have a closing rate constant about three times faster than resting one and a fast voltage-dependent unblocking rate constant. Specific experimental conditions and morphological organization of the neuromuscular synapses were considered to simulate time course of the mecamylamine block development. Thus, likewise for the neuronal nAChRs, the trapping mechanism determined the action of mecamylamine on synaptic neuromuscular currents evoked by the endogenous agonist acetylcholine (ACh), however specific morphological organization of the synaptic transmission delayed time development of the currents block.

Venomous snake bites, scorpions, and spiders.

PubMed

Kularatne, S A M; Senanayake, Nimal

2014-01-01

Neurologic dysfunction due to natural neurotoxins is an important, but neglected, public health hazard in many parts of the world, particularly in the tropics. These toxins are produced by or found among a variety of live forms that include venomous snakes, arthropods such as scorpions, spiders, centipedes, stinging insects (Hymenoptera), ticks, certain poisonous fish, shellfish, crabs, cone shells, skin secretions of dart-poison frogs, and bacterial poisons such as botulinum toxin. These toxins commonly act on neuromuscular transmission at the neuromuscular junction where acetylcholine is the neurotransmitter, but in certain situations the toxins interfere with neurotransmitters such as GABA, noradrenaline, adrenaline, dopamine, and γ-aminobutyrate. Of the toxins, α-toxins and κ-toxins (e.g., Chinese krait, Bungarus multicinctus) act on the postsynaptic membrane, blocking the receptors, whilst β-toxin (e.g., common krait, B. caeruleus) acts on the presynaptic membrane, causing impairment of acetylcholine release. Conversely, dendrotoxins of the African mamba enhance acetylcholine release. The toxins of scorpions and spiders commonly interfere with voltage-gated ion channels. Clinically, the cardinal manifestation is muscle paralysis. In severe cases respiratory paralysis could be fatal. Effective antivenoms are the mainstay of treatment of envenoming, but their lack of availability is the major concern in the regions of the globe where they are desperately needed. Interestingly, some toxins have proved to be valuable pharmaceutical agents, while some others are widely exploited to study neuromuscular physiology and pathology. © 2014 Elsevier B.V. All rights reserved.

Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS

PubMed Central

Rudnick, Noam D.; Griffey, Christopher J.; Guarnieri, Paolo; Gerbino, Valeria; Wang, Xueyong; Piersaint, Jason A.; Tapia, Juan Carlos; Rich, Mark M.; Maniatis, Tom

2017-01-01

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non–cell-autonomous manner to promote disease progression. PMID:28904095

Muscarinic Ca2+ responses resistant to muscarinic antagonists at perisynaptic Schwann cells of the frog neuromuscular junction.

PubMed Central

Robitaille, R; Jahromi, B S; Charlton, M P

1997-01-01

1. Acetylcholine causes a rise of intracellular Ca2+ in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction. The signalling pathway was characterized using the fluorescent Ca2+ indicator fluo-3 and fluorescence microscopy. 2. Nicotinic antagonists had no effect on Ca2+ responses evoked by ACh and no Ca2+ responses were evoked with the nicotinic agonist nicotine. The muscarinic agonists muscarine and oxotremorine-M induced Ca2+ signals in PSCs. 3. Ca2+ responses remained unchanged when extracellular Ca2+ was removed, indicating that they are due to the release of Ca2+ from internal stores. Incubation with pertussis toxin did not alter the Ca2+ signals induced by muscarine, but did block depression of transmitter release induced by adenosine and prevented Ca2+ responses in PSCs induced by adenosine. 4. The general muscarinic antagonists atropine, quinuclidinyl benzilate and N-methyl-scopolamine failed to block Ca2+ responses to muscarinic agonists. Atropine (at 20,000-fold excess concentration) also failed to reduce the proportion of cells responding to a threshold muscarine concentration sufficient to cause responses in less than 50% of cells. Only the allosteric, non-specific blocker, gallamine (1-10 microM) was effective in blocking muscarine-induced Ca2+ responses. 5. In preparations denervated 7 days prior to experiments, low concentrations of atropine reversibly and completely blocked Ca2+ responses to muscarine. 6. The lack of blockade by general muscarinic antagonists in innervated, in situ preparations suggests that muscarinic Ca2+ responses at PSCs are not mediated by any of the five known muscarinic receptors or that post-translational modification prevented antagonist binding. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 PMID:9365908

Facilitation through Buffer Saturation: Constraints on Endogenous Buffering Properties

PubMed Central

Matveev, Victor; Zucker, Robert S.; Sherman, Arthur

2004-01-01

Synaptic facilitation (SF) is a ubiquitous form of short-term plasticity, regulating synaptic dynamics on fast timescales. Although SF is known to depend on the presynaptic accumulation of Ca2+, its precise mechanism is still under debate. Recently it has been shown that at certain central synapses SF results at least in part from the progressive saturation of an endogenous Ca2+ buffer (Blatow et al., 2003), as proposed by Klingauf and Neher (1997). Using computer simulations, we study the magnitude of SF that can be achieved by a buffer saturation mechanism (BSM), and explore its dependence on the endogenous buffering properties. We find that a high SF magnitude can be obtained either by a global saturation of a highly mobile buffer in the entire presynaptic terminal, or a local saturation of a completely immobilized buffer. A characteristic feature of BSM in both cases is that SF magnitude depends nonmonotonically on the buffer concentration. In agreement with results of Blatow et al. (2003), we find that SF grows with increasing distance from the Ca2+ channel cluster, and increases with increasing external Ca2+, [Ca2+]ext, for small levels of [Ca2+]ext. We compare our modeling results with the experimental properties of SF at the crayfish neuromuscular junction, and find that the saturation of an endogenous mobile buffer can explain the observed SF magnitude and its supralinear accumulation time course. However, we show that the BSM predicts slowing of the SF decay rate in the presence of exogenous Ca2+ buffers, contrary to experimental observations at the crayfish neuromuscular junction. Further modeling and data are required to resolve this aspect of the BSM. PMID:15111389

How the nerves reached the muscle: Bernard Katz, Stephen W. Kuffler, and John C. Eccles-Certain implications of exile for the development of twentieth-century neurophysiology.

PubMed

Stahnisch, Frank W

2017-01-01

This article explores the work by Bernard Katz (1911-2003), Stephen W. Kuffler (1913-1980), and John C. Eccles (1903-1997) on the nerve-muscle junction as a milestone in twentieth-century neurophysiology with wider scientific implications. The historical question is approached from two perspectives: (a) an investigation of twentieth-century solutions to a longer physiological dispute and (b) an examination of a new kind of laboratory and academic cooperation. From this vantage point, the work pursued in Sydney by Sir John Carew Eccles' team on the neuromuscular junction is particularly valuable, since it contributed a central functional element to modern physiological understanding regarding the function and structure of the human and animal nervous system. The reflex model of neuromuscular action had already been advanced by neuroanatomists such as Georg Prochaska (1749-1820) in Bohemia since the eighteenth century. It became a major component of neurophysiological theories during the nineteenth century, based on the law associated with the names of François Magendie (1783-1855) in France and Charles Bell (1774-1842) in Britain regarding the functional differences of the sensory and motor spinal nerves. Yet, it was not until the beginning of the twentieth century that both the histological and the neurophysiological understanding of the nerve-muscle connection became entirely understood and the chemical versus electrical transmission further elicited as the mechanisms of inhibition. John C. Eccles, Bernard Katz, and Stephen W. Kuffler helped to provide some of the missing links for modern neurophysiology. The current article explores several of their scientific contributions and investigates how the context of forced migration contributed to these interactions in contingently new ways.

Presynaptic and postsynaptic effects of the venom of the Australian tiger snake at the neuromuscular junction

PubMed Central

Datyner, M. E.; Gage, P. W.

1973-01-01

1. Crude venom (TSV) from the Australian tiger snake (Notechis scutatus scutatus) has both presynaptic and postsynaptic effects at the neuromuscular junctions of toads. 2. TSV (50 μg/ml) rapidly blocked indirectly elicited muscle twitches without affecting the compound action potential in the sciatic nerve or twitches elicited by direct stimulation. 3. Low concentrations of the venom (1-10 μg/ml) reduced the amplitude of miniature endplate potentials (m.e.p.ps) and inhibited the depolarization of muscle fibres normally caused by carbachol. It was concluded that a fraction of the venom binds to acetylcholine receptors. 4. The frequency of m.e.p.ps was at first increased by TSV at a concentration of 1 μg/ml. Occasional, high frequency `bursts' of m.e.p.ps were recorded in some preparations. The mean frequency of m.e.p.ps appeared to fall after several hours in the venom. 5. The quantal content of endplate potentials (e.p.ps) was reduced by the venom. With low concentrations (1 μg/ml), an initial increase in quantal content was often seen. When the quantal content was markedly depressed there was no parallel reduction in the amplitude of nerve terminal spikes recorded extracellularly, though a later fall in size and slowing of time course was often seen. 6. There was evidence that TSV eventually changed the normal Poisson characteristics of the spontaneous release of quanta and this may be correlated with electronmicroscopic changes in nerve terminals. 7. Tiger snake antivenene counteracted the postsynaptic, but not the presynaptic effects of TSV when they had developed. PMID:4367126

NLP-12 engages different UNC-13 proteins to potentiate tonic and evoked release.

PubMed

Hu, Zhitao; Vashlishan-Murray, Amy B; Kaplan, Joshua M

2015-01-21

A neuropeptide (NLP-12) and its receptor (CKR-2) potentiate tonic and evoked ACh release at Caenorhabditis elegans neuromuscular junctions. Increased evoked release is mediated by a presynaptic pathway (egl-30 Gαq and egl-8 PLCβ) that produces DAG, and by DAG binding to short and long UNC-13 proteins. Potentiation of tonic ACh release persists in mutants deficient for egl-30 Gαq and egl-8 PLCβ and requires DAG binding to UNC-13L (but not UNC-13S). Thus, NLP-12 adjusts tonic and evoked release by distinct mechanisms. Copyright © 2015 the authors 0270-6474/15/351038-05$15.00/0.

Muscle contractility decrement and correlated morphology during the pathogenesis of streptozotocin-diabetic mice.

PubMed

Fahim, M A; el-Sabban, F; Davidson, N

1998-06-01

Peripheral neuropathy of both motor and sensory nerves has been well documented in diabetes mellitus, but the evidence for physiological and correlated morphological changes during the pathogenesis of myopathy is scarce. In the present report, we have chosen the dorsiflexor muscle of adult male mice as a model for studying in situ muscle contraction and neuromuscular ultrastructure during the pathogenesis of streptozotocin-induced diabetes. Thirty mice (30 g bodyweight) were injected once i.p. with streptozotocin solution (200 mg/Kg) to induce experimental diabetes mellitus. Comparative analyses of in situ muscle isometric contractile characteristics were studied (at 1 Hz, 5 Hz and 30 Hz nerve stimulation) in urethane-anesthetized (2 mg/g, i.p.) control and diabetic mice at three time points, 2 weeks, 4 weeks, and 8 weeks postinjection. Synaptic delay was also recorded in diabetic and age-matched control mice. There was a significant increase in synaptic delay in both 4-week and 8-week diabetic mice compared with control mice (8.9 +/- 1.2 msec and 7.6 +/- 0.6 msec, respectively, compared with 6.1 +/- 0.5 msec). At all three stimulation frequencies, diabetes did not affect muscle contractile speed but significantly reduced the twitch tension after 8 weeks, with no changes at 2 weeks or 4 weeks. The recorded single-twitch tension values were 2.6 +/- 0.3 g, 2.1 +/- 0.6 g, 2.2 +/- 0.7 g, and 1.2 +/- 0.1 g for control, 2 weeks, 4 weeks, and 8 weeks, respectively. At 30 Hz, the recorded tension values were 4.6 +/- 1.6 g, 3.1 +/- 1.2 g, 3.1 +/- 1.1 g, and 2.1 +/- 1.0 g for control, 2 weeks, 4 weeks, and 8 weeks, respectively. Ultrastructural changes in neuromuscular junctions were similar to those that have been described in disuse and aging. These changes were observed after 8 weeks and included serve loss of synaptic vesicles, electron-dense bodies, and myelin-like figures as well as degeneration of mitochondria. The results reveal that streptozotocin-induced diabetes affects presynaptically the neuromuscular junction as well as muscle itself. Actions at both sites may contribute to the functional alterations seen in muscle contractile properties and may play a role in the pathogenesis of diabetic neuromyopathy.

Agrin mutations lead to a congenital myasthenic syndrome with distal muscle weakness and atrophy.

PubMed

Nicole, Sophie; Chaouch, Amina; Torbergsen, Torberg; Bauché, Stéphanie; de Bruyckere, Elodie; Fontenille, Marie-Joséphine; Horn, Morten A; van Ghelue, Marijke; Løseth, Sissel; Issop, Yasmin; Cox, Daniel; Müller, Juliane S; Evangelista, Teresinha; Stålberg, Erik; Ioos, Christine; Barois, Annie; Brochier, Guy; Sternberg, Damien; Fournier, Emmanuel; Hantaï, Daniel; Abicht, Angela; Dusl, Marina; Laval, Steven H; Griffin, Helen; Eymard, Bruno; Lochmüller, Hanns

2014-09-01

Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of rare diseases resulting from impaired neuromuscular transmission. Their clinical hallmark is fatigable muscle weakness associated with a decremental muscle response to repetitive nerve stimulation and frequently related to postsynaptic defects. Distal myopathies form another clinically and genetically heterogeneous group of primary muscle disorders where weakness and atrophy are restricted to distal muscles, at least initially. In both congenital myasthenic syndromes and distal myopathies, a significant number of patients remain genetically undiagnosed. Here, we report five patients from three unrelated families with a strikingly homogenous clinical entity combining congenital myasthenia with distal muscle weakness and atrophy reminiscent of a distal myopathy. MRI and neurophysiological studies were compatible with mild myopathy restricted to distal limb muscles, but decrement (up to 72%) in response to 3 Hz repetitive nerve stimulation pointed towards a neuromuscular transmission defect. Post-exercise increment (up to 285%) was observed in the distal limb muscles in all cases suggesting presynaptic congenital myasthenic syndrome. Immunofluorescence and ultrastructural analyses of muscle end-plate regions showed synaptic remodelling with denervation-reinnervation events. We performed whole-exome sequencing in two kinships and Sanger sequencing in one isolated case and identified five new recessive mutations in the gene encoding agrin. This synaptic proteoglycan with critical function at the neuromuscular junction was previously found mutated in more typical forms of congenital myasthenic syndrome. In our patients, we found two missense mutations residing in the N-terminal agrin domain, which reduced acetylcholine receptors clustering activity of agrin in vitro. Our findings expand the spectrum of congenital myasthenic syndromes due to agrin mutations and show an unexpected correlation between the mutated gene and the associated phenotype. This provides a good rationale for examining patients with apparent distal myopathy for a neuromuscular transmission disorder and agrin mutations. © The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The aging neuromuscular system and motor performance

PubMed Central

Keenan, Kevin G.

2016-01-01

Age-related changes in the basic functional unit of the neuromuscular system, the motor unit, and its neural inputs have a profound effect on motor function, especially among the expanding number of old (older than ∼60 yr) and very old (older than ∼80 yr) adults. This review presents evidence that age-related changes in motor unit morphology and properties lead to impaired motor performance that includes 1) reduced maximal strength and power, slower contractile velocity, and increased fatigability; and 2) increased variability during and between motor tasks, including decreased force steadiness and increased variability of contraction velocity and torque over repeat contractions. The age-related increase in variability of motor performance with aging appears to involve reduced and more variable synaptic inputs that drive motor neuron activation, fewer and larger motor units, less stable neuromuscular junctions, lower and more variable motor unit action potential discharge rates, and smaller and slower skeletal muscle fibers that coexpress different myosin heavy chain isoforms in the muscle of older adults. Physical activity may modify motor unit properties and function in old men and women, although the effects on variability of motor performance are largely unknown. Many studies are of cross-sectional design, so there is a tremendous opportunity to perform high-impact and longitudinal studies along the continuum of aging that determine 1) the influence and cause of the increased variability with aging on functional performance tasks, and 2) whether lifestyle factors such as physical exercise can minimize this age-related variability in motor performance in the rapidly expanding numbers of very old adults. PMID:27516536

The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling

PubMed Central

Ryan, Kerrianne; Lu, Zhiyuan; Meinertzhagen, Ian A

2016-01-01

Left-right asymmetries in brains are usually minor or cryptic. We report brain asymmetries in the tiny, dorsal tubular nervous system of the ascidian tadpole larva, Ciona intestinalis. Chordate in body plan and development, the larva provides an outstanding example of brain asymmetry. Although early neural development is well studied, detailed cellular organization of the swimming larva’s CNS remains unreported. Using serial-section EM we document the synaptic connectome of the larva’s 177 CNS neurons. These formed 6618 synapses including 1772 neuromuscular junctions, augmented by 1206 gap junctions. Neurons are unipolar with at most a single dendrite, and few synapses. Some synapses are unpolarised, others form reciprocal or serial motifs; 922 were polyadic. Axo-axonal synapses predominate. Most neurons have ciliary organelles, and many features lack structural specialization. Despite equal cell numbers on both sides, neuron identities and pathways differ left/right. Brain vesicle asymmetries include a right ocellus and left coronet cells. DOI: http://dx.doi.org/10.7554/eLife.16962.001 PMID:27921996

Seronegative myasthenia gravis associated with malignant thymoma.

PubMed

Richards, Jason; Howard, James F

2017-05-01

Myasthenia gravis (MG) is generally caused by antibodies directed against the neuromuscular junction, including antibodies against the postsynaptic nicotinic acetylcholine receptor (AChR). Pathologic abnormalities of the thymus gland, including thymoma, are associated with MG. We report a 56-year-old woman who presented with double vision. Single fiber EMG confirmed myasthenia gravis. AChR, striational muscle and MuSK antibodies were absent in the serum. Chest CT demonstrated a malignant thymoma. We report the first case of seronegative myasthenia gravis associated with malignant thymoma. The case challenges the conventional wisdom that all patients with thymoma associated MG test positive for antibodies against AChR. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.

2010-10-19

Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Gyanendra; Agarwal, Rakhi; Swaminathan, Subramanyam

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. In addition, the structure–activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of themmore » show low micromolar IC 50 values.« less

[Applications of botulinum toxin in Neurology].

PubMed

Garcia-Ruiz, Pedro J

2013-07-07

At present, botulinum toxin (BT) is one of the most fundamental available drugs in Neurology, only comparable with levodopa. BT is currently used in those entities characterized by excessive muscle contraction, including dystonia and spasticity. In addition, BT has been used to control pain associated with increased muscle contraction in dystonia and spasticity, but also is useful to control chronic pain not associated with muscle contraction, such as chronic daily headache. Finally, BT is useful in sialorrhoea and bruxism. The mechanism of action is complex, mainly acting on terminal neuromuscular junction, but also exhibiting analgesic properties, probably through inhibition of pain neurotransmitters release. Copyright © 2012 Elsevier España, S.L. All rights reserved.

FGF–2 is required to prevent astrogliosis in the facial nucleus after facial nerve injury and mechanical stimulation of denervated vibrissal muscles

PubMed Central

Hizay, Arzu; Seitz, Mark; Grosheva, Maria; Sinis, Nektarios; Kaya, Yasemin; Bendella, Habib; Sarikcioglu, Levent; Dunlop, Sarah A.; Angelov, Doychin N.

2016-01-01

Abstract Recently, we have shown that manual stimulation of paralyzed vibrissal muscles after facial-facial anastomosis reduced the poly-innervation of neuromuscular junctions and restored vibrissal whisking. Using gene knock outs, we found a differential dependence of manual stimulation effects on growth factors. Thus, insulin-like growth factor-1 and brain-derived neurotrophic factor are required to underpin manual stimulation-mediated improvements, whereas FGF-2 is not. The lack of dependence on FGF-2 in mediating these peripheral effects prompted us to look centrally, i.e. within the facial nucleus where increased astrogliosis after facial-facial anastomosis follows "synaptic stripping". We measured the intensity of Cy3-fluorescence after immunostaining for glial fibrillary acidic protein (GFAP) as an indirect indicator of synaptic coverage of axotomized neurons in the facial nucleus of mice lacking FGF-2 (FGF-2-/- mice). There was no difference in GFAP-Cy3-fluorescence (pixel number, gray value range 17–103) between intact wildtype mice (2.12± 0.37×107) and their intact FGF-2-/- counterparts (2.12± 0.27×107) nor after facial-facial anastomosis +handling (wildtype: 4.06± 0.32×107; FGF-2-/-: 4.39±0.17×107). However, after facial-facial anastomosis, GFAP-Cy3-fluorescence remained elevated in FGF-2-/--animals (4.54±0.12×107), whereas manual stimulation reduced the intensity of GFAP-immunofluorescence in wild type mice to values that were not significantly different from intact mice (2.63± 0.39×10 ). We conclude that FGF-2 is not required to underpin the beneficial effects of manual stimulation at the neuro-muscular junction, but it is required to minimize astrogliosis in the brainstem and, by implication, restore synaptic coverage of recovering facial motoneurons. PMID:28276669

Mild aerobic training with blood flow restriction increases the hypertrophy index and MuSK in both slow and fast muscles of old rats: Role of PGC-1α.

PubMed

Bahreinipour, Mohammad-Ali; Joukar, Siyavash; Hovanloo, Fariborz; Najafipour, Hamid; Naderi, Vida; Rajiamirhasani, Alireza; Esmaeili-Mahani, Saeed

2018-06-01

Existing evidence emphasize the role of mitochondrial dysfunction in sarcopenia which is revealed as loss of skeletal muscle mass and neuromuscular junction remodeling. We assessed the effect of low-intensity aerobic training along with blood flow restriction on muscle hypertrophy index, muscle-specific kinase (MuSK), a pivotal protein of the neuromuscular junction and Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) in aged male rats. Animals groups were control (CTL), sham (Sh), leg blood flow restriction (BFR), exercise (Ex), sham + exercise (Sh + Ex), and BFR plus exercise (BFR + Ex) groups. The exercise groups were trained with low intensity exercise for 10 weeks. 48 h after the last training session, animals were sacrificed under anesthesia. Soleus and EDL muscles were isolated, hypertrophy index was estimated and MuSK and PGC-1α were measured by western blot method. Hypertrophy index enhanced in soleus and Extensor digitorum longus (EDL) muscles of BFR + Ex group (P < 0.01 versus CTL and Sh groups, and P < 0.001 versus other groups). The MuSK protein of soleus and EDL muscles increased in BFR + Ex group (P < 0.01 and P < 0.001, respectively) in comparison with CTL and Sh groups. In BFR + Ex group, the PGC-1α protein increased in both soleus and EDL (P < 0.001 compared to other groups). Also the PGC-1α of soleus muscle was higher in Ex and Sh + Ex groups versus CTL and Sh groups (P < 0.05). Findings suggest that low endurance exercise plus BFR improves the MuSK and hypertrophy index of both slow and fast muscles of elderly rats probably through the rise of PGC-1α expression. Copyright © 2018. Published by Elsevier Inc.

Congenital myasthenic syndromes due to mutations in ALG2 and ALG14.

PubMed

Cossins, Judith; Belaya, Katsiaryna; Hicks, Debbie; Salih, Mustafa A; Finlayson, Sarah; Carboni, Nicola; Liu, Wei Wei; Maxwell, Susan; Zoltowska, Katarzyna; Farsani, Golara Torabi; Laval, Steven; Seidhamed, Mohammed Zain; Donnelly, Peter; Bentley, David; McGowan, Simon J; Müller, Juliane; Palace, Jacqueline; Lochmüller, Hanns; Beeson, David

2013-03-01

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Neuromuscular diseases: Diagnosis and management.

PubMed

Mary, P; Servais, L; Vialle, R

2018-02-01

Neuromuscular diseases (NMDs) affect the peripheral nervous system, which includes the motor neurons and sensory neurons; the muscle itself; or the neuromuscular junction. Thus, the term NMDs encompasses a vast array of different syndromes. Some of these syndromes are of direct relevance to paediatric orthopaedic surgeons, either because the presenting manifestation is a functional sign (e.g., toe-walking) or deformity (e.g., pes cavus or scoliosis) suggesting a need for orthopaedic attention or because orthopaedic abnormalities requiring treatment develop during the course of a known NMD. The main NMDs relevant to the orthopaedic surgeon are infantile spinal muscular atrophy (a motor neuron disease), peripheral neuropathies (chiefly, Charcot-Marie-Tooth disease), congenital muscular dystrophies, progressive muscular dystrophies, and Steinert myotonic dystrophy (or myotonic dystrophy type 1). Muscle weakness is a symptom shared by all these conditions. The paediatric orthopaedic surgeon must be familiar, not only with the musculoskeletal system, but also with many other domains (particularly respiratory and cardiac function and nutrition) that may interfere with the treatment and require preoperative management. Good knowledge of the natural history of each NMD is essential to ensure optimal timing of the therapeutic interventions, which must be performed under the best possible conditions in these usually frail patients. Timing is particularly crucial for the treatment of spinal deformities due to paraspinal muscle hypotonia during growth: depending on the disease and natural history, the treatment may involve non-operative methods or growing rods, followed by spinal fusion. A multidisciplinary approach is always required. Finally, the survival gains achieved in recent years increasingly require attention to preparing for adult life, to orthopaedic problems requiring treatment before the patient leaves the paediatric environment, and to the transition towards the adult healthcare system. Copyright © 2017. Published by Elsevier Masson SAS.

Action of Micrurus dumerilii carinicauda coral snake venom on the mammalian neuromuscular junction.

PubMed

Serafim, Francine G; Reali, Marielga; Cruz-Höfling, Maria Alice; Fontana, Marcos D

2002-02-01

The venoms of coral snakes (mainly Micrurus species) have pre- and/or postsynaptic actions, but only a few of these have been studied in detail. We have investigated the effects of Micrurus dumerilii carinicauda coral snake venom on neurotransmission in rat isolated phrenic nerve-diaphragm muscle and chick biventer cervicis preparations stimulated directly or indirectly. M. d. carinicauda venom (5 or 10 microg/ml) produced neuromuscular blockade in rat (85-90% in 291.8+/-7.3 min and 108.3+/-13.8, respectively; n=5) and avian (95.0+/-2.0 min; 5 microg/ml, n=5) preparations. Neostigmine (5.8 microM) and 3,4-diaminopyridine (230 microM) partially reversed the venom-induced neuromuscular blockade in rat nerve-muscle preparations. In neither preparation did the venom depress the twitch response elicited by direct muscle stimulation. The contractures induced by acetylcholine in chick preparations were inhibited by the venom (95-100%; n=4; p<0.05). In rat preparations, the venom produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps control frequency=69.3+/-5.0/min and control amplitude=0.4+/-0.2 mV) until these were abolished. Neostigmine (5.8 microM) and 3,4-diaminopyridine (230 microM) partially antagonized this blockade of m.e.p.ps. The resting membrane potential was not altered with the venom (10 microg/ml). M. d. carinicauda venom produced dose-dependent morphological changes in indirectly stimulated mammal preparations. Twenty-five per cent of muscle fibers were affected by a venom concentration of 5 microg/ml, whilst 60.7% were damaged by 10 microg of venom/ml. In biventer cervicis preparations, the morphological changes were slower in onset and were generally characterized by undulating fibers and, to a lesser extent, by zones of disintegrating myofibrils. A venom concentration of 5 microg/ml damaged 52.2% of the fibers. These findings indicate that M. d. carinicauda venom has neurotoxic and myotoxic effects and that the neuromuscular blockade involves mainly a postsynaptic action.

Molecular Mechanisms of Treadmill Therapy on Neuromuscular Atrophy Induced via Botulinum Toxin A

PubMed Central

Tsai, Sen-Wei; Chen, Hsiao-Ling

2013-01-01

Botulinum toxin A (BoNT-A) is a bacterial zinc-dependent endopeptidase that acts specifically on neuromuscular junctions. BoNT-A blocks the release of acetylcholine, thereby decreasing the ability of a spastic muscle to generate forceful contraction, which results in a temporal local weakness and the atrophy of targeted muscles. BoNT-A-induced temporal muscle weakness has been used to manage skeletal muscle spasticity, such as poststroke spasticity, cerebral palsy, and cervical dystonia. However, the combined effect of treadmill exercise and BoNT-A treatment is not well understood. We previously demonstrated that for rats, following BoNT-A injection in the gastrocnemius muscle, treadmill running improved the recovery of the sciatic functional index (SFI), muscle contraction strength, and compound muscle action potential (CMAP) amplitude and area. Treadmill training had no influence on gastrocnemius mass that received BoNT-A injection, but it improved the maximal contraction force of the gastrocnemius, and upregulation of GAP-43, IGF-1, Myo-D, Myf-5, myogenin, and acetylcholine receptor (AChR) subunits α and β was found following treadmill training. Taken together, these results suggest that the upregulation of genes associated with neurite and AChR regeneration following treadmill training may contribute to enhanced gastrocnemius strength recovery following BoNT-A injection. PMID:24327926

Deletion of Pofut1 in Mouse Skeletal Myofibers Induces Muscle Aging-Related Phenotypes in cis and in trans

PubMed Central

Zygmunt, Deborah A.; Singhal, Neha; Kim, Mi-Lyang; Cramer, Megan L.; Crowe, Kelly E.; Xu, Rui; Jia, Ying; Adair, Jessica; Martinez-Pena y Valenzuela, Isabel; Akaaboune, Mohammed; White, Peter; Janssen, Paulus M.

2017-01-01

ABSTRACT Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein O-fucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on α dystroglycan glycosylation. PMID:28265002

Botulinum Toxin for Rhinitis.

PubMed

Ozcan, Cengiz; Ismi, Onur

2016-08-01

Rhinitis is a common clinical entity. Besides nasal obstruction, itching, and sneezing, one of the most important symptoms of rhinitis is nasal hypersecretion produced by nasal glands and exudate from the nasal vascular bed. Allergic rhinitis is an IgE-mediated inflammatory reaction of nasal mucosa after exposure to environmental allergens. Idiopathic rhinitis describes rhinitis symptoms that occur after non-allergic, noninfectious irritants. Specific allergen avoidance, topical nasal decongestants, nasal corticosteroids, immunotherapy, and sinonasal surgery are the main treatment options. Because the current treatment modalities are not enough for reducing rhinorrhea in some patients, novel treatment options are required to solve this problem. Botulinum toxin is an exotoxin generated by Clostridium botulinum. It disturbs the signal transmission at the neuromuscular and neuroglandular junction by inhibiting the acetylcholine release from the presynaptic nerve terminal. It has been widely used in neuromuscular, hypersecretory, and autonomic nerve system disorders. There have been a lot of published articles concerning the effect of this toxin on rhinitis symptoms. Based on the results of these reports, intranasal botulinum toxin A administration appears to be a safe and effective treatment method for decreasing rhinitis symptoms in rhinitis patients with a long-lasting effect. Botulinum toxin type A will be a good treatment option for the chronic rhinitis patients who are resistant to other treatment methods.

Neuromuscular plasticity in the locust after permanent removal of an excitatory motoneuron of the extensor tibiae muscle.

PubMed

Büschges, A; Djokaj, S; Bässler, D; Bässler, U; Rathmayer, W

2000-01-01

The capacity of the larval insect nervous system to compensate for the permanent loss of one of the two excitatory motoneurons innervating a leg muscle was investigated in the locust (Locusta migratoria). In the fourth instar, the fast extensor tibiae (FETi) motoneuron in the mesothoracic ganglion was permanently removed by photoinactivation with a helium-cadmium laser. Subsequently, the animals were allowed to develop into adulthood. When experimental animals were tested as adults after final ecdysis, fast-contracting fibers in the most proximal region of the corresponding extensor muscle, which are normally predominantly innervated by FETi only, uniformly responded to activity of the slow extensor tibiae (SETi) neuron. In adult operated animals, single pulses to SETi elicited large junctional responses in the fibers which resulted in twitch contractions of these fibers similar to the responses to FETi activity in control animals. The total number of muscle fibers, their properties as histochemically determined contractional types (fast and slow), and their distribution were not affected by photoinactivation of FETi. Possible mechanisms enabling the larval neuromuscular system to compensate for the loss of FETi through functionally similar innervation by a different motoneuron, i.e. SETi, are discussed. Copyright 2000 John Wiley & Sons, Inc.

Nematode cholinergic pharmacology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segerberg, M.A.

1989-01-01

Nematode acetylcholine (ACh) receptors were characterized using both biochemical and electrophysiological techniques, including: (1) receptor binding studies in crude homogenates of the free-living nematode Caenorhabditis elegans and the parasitic nematode Ascaris lumbricoides with the high-affinity probe ({sup 3}H)N-methylscopolamine (({sup 3}H)NMS) which binds to muscarinic receptors in many vertebrate and invertebrate tissues (2) measurement of depolarization and contraction induced by a variety of cholinergic agents, including N-methylscopolamine (NMS), in an innervated dorsal muscle strip preparation of Ascaris; (3) examination of the antagonistic actions of d-tubocurarine (dTC) and NMS at dorsal neuromuscular junction; (4) measurement of input resistance changes in Ascaris commissuralmore » motorneurons induced by ACh, dTC, NMS, pilocarpine and other cholinergic drugs.« less

Early onset bilateral juvenile myasthenia gravis masquerading as simple congenital ptosis.

PubMed

Alam, Md Shahid; Devi Nivean, Pratheeba

2017-01-01

Myasthenia gravis is an autoimmune disorder affecting the neuromuscular junction. Ocular myasthenia gravis presents as ptosis with extraocular motility restriction and is prone to be misdiagnosed as third nerve palsy or congenital or aponeurotic ptosis. Juvenile ocular myasthenia gravis in very young children is difficult to diagnose and can be easily labeled as a case of congenital ptosis, the more so when the condition is bilateral. We present a case of a two-year-old child who presented with bilateral ptosis and was diagnosed as a case of simple congenital ptosis elsewhere with the advice to undergo tarsofrontalis sling surgery. The child was diagnosed with juvenile myasthenia gravis on thorough history, examination, and systemic evaluation and was started on anti-myasthenic treatment.

Redox factor-1 in muscle biopsies of patients with inclusion-body myositis.

PubMed

Broccolini, A; Engel, W K; Alvarez, R B; Askanas, V

2000-06-16

To determine whether redox factor-1 (Ref-1) participates in the pathogenesis of inclusion-body myositis (IBM), we immunolocalized Ref-1 in muscle biopsies of IBM patients by light- and electron-microscopy. Approximately 70-80% of the IBM vacuolated muscle fibers had focal inclusions strongly immunoreactive for Ref-1. By immunoelectronmicroscopy, Ref-1 was localized to paired-helical filaments, 6-10 nm amyloid-like fibrils and amorphous material. Virtually all regenerating and necrotic muscle fibers in various muscle biopsies had diffusely strong Ref-1 immunoreactivity. At all neuromuscular junctions, postsynaptically there was strong Ref-1 immunoreactivity. Our study suggests that Ref-1 plays a role in IBM pathogenesis, and in other pathologic and normal processes of human muscle.

Anesthetic Management of a Patient With Antimuscle-Specific Kinase Antibody-Positive Myasthenia Gravis Undergoing an Open Cholecystectomy: A Case Report.

PubMed

Akatsu, Masahiko; Ikegami, Yukihiro; Tase, Choichiro; Nishikawa, Koichi

2017-03-15

Myasthenia gravis (MG) is an autoimmune disease characterized by the production of antibodies against the acetylcholine receptor, muscle-specific kinase (MuSK), or other proteins at the neuromuscular junction. MG with antibodies against MuSK (MuSK-MG) has been described recently. Here, we report the first case of anesthetic management of a patient with MuSK-MG undergoing an open cholecystectomy. In our case, propofol and remifentanil-based anesthesia were used for successful management without using muscle relaxants. Patients with MuSK-MG have predominantly ocular, bulbar, and respiratory symptoms that may increase the risk of aspiration. Anesthesiologists need to pay attention to perioperative respiratory failure and respiratory crisis.

Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome.

PubMed

Andersen, Søren M; Ling, Chang-Chun; Zhang, Ping; Townson, Kate; Willison, Hugh J; Bundle, David R

2004-04-21

Guillain-Barré syndrome is a postinfectious, autoimmune neuropathy resulting in neuromuscular paralysis. Auto-antibodies, often induced by bacterial infection, bind to human gangliosides possessing monosialoside and diasialoside epitopes and impair the function of nerve junctions, where these ganglioside structures are highly enriched. Truncated gangliosides representive of GD3, GQ1b and GM2 epitopes have been synthesized as methyl glycosides and as a glycosides of an eleven carbon tether. The synthetic oligosaccharide ligands are structural mimics of these highly complex ganglioside epitopes and via their ability to neutralize or remove auto-antibodies have the potential for therapy, either as soluble blocking ligands administered systemically, or as immuno-affinity ligands for use as extracorporeal immunoadsorbents.

The immunoglobulin-like domains 1 and 2 of the protein tyrosine phosphatase LAR adopt an unusual horseshoe-like conformation

PubMed Central

Biersmith, Bridget H.; Hammel, Michal; Geisbrecht, Erika R.; Bouyain, Samuel

2011-01-01

Neurogenesis depends on exquisitely regulated interactions between macromolecules on the cell surface and in the extracellular matrix. In particular, interactions between proteoglycans and members of the type IIa subgroup of receptor protein tyrosine phosphatases underlie critical developmental processes such as the formation of synapses at the neuromuscular junction and the migration of axons to their appropriate targets. We report here the crystal structures of the first and second immunoglobulin-like domains of the Drosophila type IIa receptor Dlar and its mouse homologue LAR. These two domains adopt an unusual antiparallel arrangement that has not been previously observed in tandem repeats of immunoglobulin-like domains and that is presumably conserved in all type IIa receptor protein tyrosine phosphatases. PMID:21402080

Graphene quantum dots for ultrasensitive detection of acetylcholinesterase and its inhibitors

NASA Astrophysics Data System (ADS)

Li, Nan; Wang, Xuewan; Chen, Jie; Sun, Lei; Chen, Peng

2015-09-01

Graphene quantum dots (GQDs) are emerging zero-dimensional materials promising a wide spectrum of novel applications including development of optical sensors. Herein, a GQD-based fluorometric sensor is devised to detect acetylcholinesterase (AChE, a critical enzyme in central nervous system and neuromuscular junctions) with an ultralow detection limit (0.58 pM with S/N of 5.0), using a photoluminescence ‘turn-off’ mechanism. This simple ‘mix-and-detect’ platform can also be employed to sense a variety of compounds that can directly or indirectly inhibit the enzymatic activities of AChE, such as nerve gases, pesticides, and therapeutic drugs. As the proof-of-concept demonstrations, we show the sensitive detection of paraoxon (a pesticide), tacrine (a drug to treat Alzheimer’s disease), and dopamine (an important neurotransmitter).

Glutamatergic postsynaptic block by Pamphobeteus spider venoms in crayfish.

PubMed

Araque, A; Ferreira, W; Lucas, S; Buño, W

1992-01-31

The effects of toxins from venom glands of two south american spiders (Pamphobeteus platyomma and P. soracabae) on glutamatergic excitatory synaptic transmission were studied in the neuromuscular junction of the opener muscle of crayfish. The toxins selectively and reversibly blocked both excitatory postsynaptic currents and potentials in a dose-dependent manner. They also reversibly abolished glutamate-induced postsynaptic membrane depolarization. They had no effect on resting postsynaptic membrane conductance nor on postsynaptic voltage-gated currents. The synaptic facilitation and the frequency of miniature postsynaptic potentials were unaffected by the toxins, indicating that presynaptic events were not modified. Picrotoxin, a selective antagonist of the gamma-aminobutyric acid (GABA)A receptor, did not modify toxin effects. We conclude that both toxins specifically block the postsynaptic glutamate receptor-channel complex.

[Descending paralysis caused by wound botulism. A case report].

PubMed

Hiersemenzel, L P; Jermann, M; Waespe, W

2000-02-01

We report on the history and clinical findings of an injecting drug abuser in the Canton of Zurich who presented with multiple deep abscesses in the arms and legs. A diagnosis of wound botulism was made based on his clinical presentation with a rapidly progressing descending paralysis starting at the cranial nerves, a neuromuscular junction disorder on neurophysiologic testing, and normal findings on lumbar puncture. Several cases of wound botulism have occurred in i.v. drug abuse in Switzerland since 1997. We suspect subcutaneous injections of contaminated heroin containing Clostridium spores as sites of entry. Wound botulism caused by Clostridium botulinum is a rare cause of rapidly progressing, generalized, flaccid paralysis and should be considered in patients with a history of i.v. drug abuse presenting with descending paralysis.

Neurexin and Neuroligin Mediate Retrograde Synaptic Inhibition in C. elegans

PubMed Central

Hu, Zhitao; Hom, Sabrina; Kudze, Tambudzai; Tong, Xia-Jing; Choi, Seungwon; Aramuni, Gayane; Zhang, Weiqi; Kaplan, Joshua M.

2013-01-01

The synaptic adhesion molecules Neurexin and Neuroligin alter the development and function of synapses and are linked to autism in humans. We find that C. elegans Neurexin (NRX-1) and Neuroligin (NLG-1) mediate a retrograde synaptic signal that inhibits neurotransmitter release at neuromuscular junctions. Retrograde signaling was induced in mutants lacking a muscle microRNA (miR-1) and was blocked in mutants lacking NLG-1 or NRX-1. Release was rapid and abbreviated when the retrograde signal was on whereas release was slow and prolonged when retrograde signaling was blocked. The retrograde signal adjusted release kinetics by inhibiting exocytosis of synaptic vesicles (SVs) that are distal to the site of calcium entry. Inhibition of release was mediated by increased pre-synaptic levels of Tomosyn, an inhibitor of SV fusion. PMID:22859820

The Arabidopsis thaliana ortholog of a purported maize cholinesterase gene encodes a GDSL-lipase

PubMed Central

Muralidharan, Mrinalini; Buss, Kristina; Larrimore, Katherine E.; Segerson, Nicholas A.; Kannan, Latha

2013-01-01

Acetylcholinesterase is an enzyme that is intimately associated with regulation of synaptic transmission in the cholinergic nervous system and in neuromuscular junctions of animals. However the presence of cholinesterase activity has been described also in non-metazoan organisms such as slime molds, fungi and plants. More recently, a gene purportedly encoding for acetylcholinesterase was cloned from maize. We have cloned the Arabidopsis thaliana homolog of the Zea mays gene, At3g26430, and studied its biochemical properties. Our results indicate that the protein encoded by the gene exhibited lipase activity with preference to long chain substrates but did not hydrolyze choline esters. The At3g26430 protein belongs to the SGNH clan of serine hydrolases, and more specifically to the GDS(L) lipase family. PMID:23430565

Electromyographic studies of motor control in humans.

PubMed

Shahani, B T; Wierzbicka, M M

1987-11-01

Electromyography and electroneurography have proved to be useful in investigation and understanding of a variety of neurologic disorders. In most laboratories, however, these electrodiagnostic techniques have been used to help in the diagnosis of diseases that affect the peripheral nerves, neuromuscular junctions, or skeletal muscle fibers. Although major advances in electronic and computer technology have made it possible to study, quantitate, and document reflex activity in intact human subjects, most neurologists still rely on gross clinical observations and most electromyographers continue to use conventional techniques of EMG and nerve conduction studies to differentiate "myopathy" from "neuropathy." This article is a review of some of the electromyographic techniques that have been used in the authors' laboratory for the study of normal and abnormal motor control in man and the treatment of patients with disorders of motor control.

Small molecule non-peptide inhibitors of botulinum neurotoxin serotype E: Structure-activity relationship and a pharmacophore model.

PubMed

Kumar, Gyanendra; Agarwal, Rakhi; Swaminathan, Subramanyam

2016-09-15

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. The structure-activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of them show low micromolar IC50 values. Copyright © 2016 Elsevier Ltd. All rights reserved.

Distribution of serine/threonine kinase SAD-B in mouse peripheral nerve synapse.

PubMed

Hagiwara, Akari; Harada, Kenu; Hida, Yamato; Kitajima, Isao; Ohtsuka, Toshihisa

2011-05-11

The serine/threonine kinase SAD regulates neural functions such as axon/dendrite polarization and neurotransmitter release. In the vertebrate central nervous system, SAD-B, a homolog of Caenorhabditis elegans SAD-1, is associated with synaptic vesicles and the active zone cytomatrix in nerve terminals. However, the distribution of SAD-B in the peripheral nervous system remains elusive. Here, we show that SAD-B is specifically localized to neuromuscular junctions. Although the active zone protein bassoon showed a punctated signal indicating its localization to motor end plates, SAD-B shows relatively diffuse localization indicating its association with both the active zone and synaptic vesicles. Therefore, SAD kinase may regulate neurotransmitter release from motor end plates in a similar manner to its regulation of neurotransmitter release in the central nervous system.

Small molecule non-peptide inhibitors of botulinum neurotoxin serotype E: Structure–activity relationship and a pharmacophore model

DOE PAGES

Kumar, Gyanendra; Agarwal, Rakhi; Swaminathan, Subramanyam

2016-06-18

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. In addition, the structure–activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of themmore » show low micromolar IC 50 values.« less

Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

PubMed Central

Hazell, Gareth; Shabanpoor, Fazel; Saleh, Amer F.; Bowerman, Melissa; Meijboom, Katharina E.; Zhou, Haiyan; Muntoni, Francesco; Talbot, Kevin; Gait, Michael J.; Wood, Matthew J. A.

2016-01-01

The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA. PMID:27621445

Nur77 coordinately regulates expression of genes linked to glucose metabolism in skeletal muscle.

PubMed

Chao, Lily C; Zhang, Zidong; Pei, Liming; Saito, Tsugumichi; Tontonoz, Peter; Pilch, Paul F

2007-09-01

Innervation is important for normal metabolism in skeletal muscle, including insulin-sensitive glucose uptake. However, the transcription factors that transduce signals from the neuromuscular junction to the nucleus and affect changes in metabolic gene expression are not well defined. We demonstrate here that the orphan nuclear receptor Nur77 is a regulator of gene expression linked to glucose utilization in muscle. In vivo, Nur77 is preferentially expressed in glycolytic compared with oxidative muscle and is responsive to beta-adrenergic stimulation. Denervation of rat muscle compromises expression of Nur77 in parallel with that of numerous genes linked to glucose metabolism, including glucose transporter 4 and genes involved in glycolysis, glycogenolysis, and the glycerophosphate shuttle. Ectopic expression of Nur77, either in rat muscle or in C2C12 muscle cells, induces expression of a highly overlapping set of genes, including glucose transporter 4, muscle phosphofructokinase, and glycogen phosphorylase. Furthermore, selective knockdown of Nur77 in rat muscle by small hairpin RNA or genetic deletion of Nur77 in mice reduces the expression of a battery of genes involved in skeletal muscle glucose utilization in vivo. Finally, we show that Nur77 binds the promoter regions of multiple genes involved in glucose metabolism in muscle. These results identify Nur77 as a potential mediator of neuromuscular signaling in the control of metabolic gene expression.

Nur77 coordinately regulates expression of genes linked to glucose metabolism in skeletal muscle

PubMed Central

Chao, Lily C.; Zhang, Zidong; Pei, Liming; Saito, Tsugumichi; Tontonoz, Peter; Pilch, Paul F.

2008-01-01

Innervation is important for normal metabolism in skeletal muscle, including insulin-sensitive glucose uptake. However, the transcription factors that transduce signals from the neuromuscular junction to the nucleus and affect changes in metabolic gene expression are not well defined. We demonstrate here that the orphan nuclear receptor Nur77 is a regulator of gene expression linked to glucose utilization in muscle. In vivo, Nur77 is preferentially expressed in glycolytic compared to oxidative muscle and is responsive to β-adrenergic stimulation. Denervation of rat muscle compromises expression of Nur77 in parallel with that of numerous genes linked to glucose metabolism, including GLUT4 and genes involved in glycolysis, glycogenolysis, and the glycerophosphate shuttle. Ectopic expression of Nur77, either in rat muscle or in C2C12 muscle cells, induces expression of a highly overlapping set of genes, including GLUT4, muscle phosphofructokinase, and glycogen phosphorylase. Furthermore, selective knockdown of Nur77 in rat muscle by shRNA or genetic deletion of Nur77 in mice reduces the expression of a battery of genes involved in skeletal muscle glucose utilization in vivo. Finally, we show that Nur77 binds the promoter regions of multiple innervation-dependent genes in muscle. These results identify Nur77 as a potential mediator of neuromuscular signaling in the control of metabolic gene expression. PMID:17550977

Combined Guillain-Barré syndrome and myasthenia gravis.

PubMed

Hsieh, Meng-Ying; Chan, Oi-Wa; Lin, Jainn-Jim; Lin, Kuang-Lin; Hsia, Shao-Hsuan; Wang, Huei-Shyong; Chiu, Cheng-Hsun

2013-10-01

Guillain-Barré syndrome and myasthenia gravis both lead to muscle weakness but the two combined is uncommon. Detection of these entities can help identify forms of autoimmune neuromuscular diseases that may respond to immunotherapy. This report sought to characterize the clinical features of these two entities when combined. This report is of a case of combined Guillain-Barré syndrome and myasthenia gravis. The clinical features were analyzed and correlated to those published in English literature from 1960 to 2012. Ten reports and 12 cases, including the present case, were reviewed. There were 12 patients (4 women and 8 men), aged 17 to 84 years, with combined Guillain-Barré syndrome and myasthenia gravis. Four had post-infectious Guillain-Barré syndrome followed by the development of myasthenia gravis concurrently or concomitantly within one month. All cases had symptoms of ptosis and areflexia. The other common presentations were limb weakness, oculobulbar weakness, and respiratory involvement. Functional outcome was mentioned in 10 patients and seven had good outcome (Hughes scale ≤ 2). Detection of ptosis with or without ophthalmoplegia, distribution of limb weakness, and reflex can help in recognizing combined Guillain-Barré syndrome and myasthenia gravis. The early recognition of this combination of peripheral nervous and neuro-muscular junction inflammation is important for initial treatment and prognosis. Copyright © 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

PubMed

Mayer, U; Saher, G; Fässler, R; Bornemann, A; Echtermeyer, F; von der Mark, H; Miosge, N; Pöschl, E; von der Mark, K

1997-11-01

Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction

PubMed Central

Ghazanfari, Nazanin; Morsch, Marco; Reddel, Stephen W; Liang, Simon X; Phillips, William D

2014-01-01

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. PMID:24860174

Rab3-GAP controls the progression of synaptic homeostasis at a late stage of vesicle release.

PubMed

Müller, Martin; Pym, Edward C G; Tong, Amy; Davis, Graeme W

2011-02-24

Homeostatic signaling systems stabilize neural function through the modulation of neurotransmitter receptor abundance, ion channel density, and presynaptic neurotransmitter release. Molecular mechanisms that drive these changes are being unveiled. In theory, molecular mechanisms may also exist to oppose the induction or expression of homeostatic plasticity, but these mechanisms have yet to be explored. In an ongoing electrophysiology-based genetic screen, we have tested 162 new mutations for genes involved in homeostatic signaling at the Drosophila NMJ. This screen identified a mutation in the rab3-GAP gene. We show that Rab3-GAP is necessary for the induction and expression of synaptic homeostasis. We then provide evidence that Rab3-GAP relieves an opposing influence on homeostasis that is catalyzed by Rab3 and which is independent of any change in NMJ anatomy. These data define roles for Rab3-GAP and Rab3 in synaptic homeostasis and uncover a mechanism, acting at a late stage of vesicle release, that opposes the progression of homeostatic plasticity. Copyright © 2011 Elsevier Inc. All rights reserved.

Number of junctional acetylcholine receptors: control by neural and muscular influences in the rat.

PubMed

Andreose, J S; Fumagalli, G; Lømo, T

1995-03-01

1. The number of acetylcholine receptors (AChRs) per neuromuscular junction in soleus muscles of adult rats was estimated from counts of 125I-alpha-bungarotoxin binding sites. The muscles were either denervated, denervated and electrically stimulated, paralysed by botulinum toxin (BoTX), or paralysed by tetrodotoxin (TTX). 2. After denervation, the number of junctional AChRs was normal after 18 days and then fell to 54 and 35% of normal after 33 and 57 days, respectively. 3. Direct high frequency muscle stimulation (100 Hz) maintained a normal number of junctional AChRs for at least 2 months when the stimulation started on the day of denervation. When the stimulation was started progressively later, the effect of the stimulation on AChR number disappeared within about a week. The disappearance was gradual and appeared to affect all the muscle fibres equally. 4. Stimulation at 100 Hz, starting on the day of denervation and stopping after 18 days, did not prevent the endplates from losing AChRs during the subsequent 15 days without stimulation. Thus 100 Hz stimulation and innervation are not equivalent in their effects on junctional AChR number. 5. Direct low frequency muscle stimulation from the day of denervation did not maintain a normal number of junctional AChRs, as the number of AChRs fell to 70 and 62% of normal after 33 days of stimulation at 20 and 10 Hz, respectively. 6. Endplates paralysed by BoTX or TTX for 33 days lost about as many junctional AChRs (54 and 55%) as endplates denervated for 33 days (46%). Direct stimulation at 100 Hz during the last 15 days of BoTX treatment reduced but did not prevent this AChR loss (36% loss at 33 days). 7. The results show that when motor nerve terminals in rat soleus muscles are removed by axotomy, they leave a 'trace' which, in conjunction with appropriate muscle stimulation, can maintain a normal number of AChRs in the postsynaptic region. In non-stimulated muscles the trace responsible for this maintenance disappears within about a week. In stimulated muscles it persists for at least 2 months. From indirect evidence it appears that the trace is a factor, or the postsynaptic effect of a factor, released by impulse activity in the nerve, and that its degradation after denervation is accelerated by the acute effects of nerve degeneration.

Comparison of Whole Body SOD1 Knockout with Muscle-Specific SOD1 Knockout Mice Reveals a Role for Nerve Redox Signaling in Regulation of Degenerative Pathways in Skeletal Muscle.

PubMed

Sakellariou, Giorgos K; McDonagh, Brian; Porter, Helen; Giakoumaki, Ifigeneia I; Earl, Kate E; Nye, Gareth A; Vasilaki, Aphrodite; Brooks, Susan V; Richardson, Arlan; Van Remmen, Holly; McArdle, Anne; Jackson, Malcolm J

2018-02-01

Lack of Cu,Zn-superoxide dismutase (CuZnSOD) in homozygous knockout mice (Sod1 -/- ) leads to accelerated age-related muscle loss and weakness, but specific deletion of CuZnSOD in skeletal muscle (mSod1KO mice) or neurons (nSod1KO mice) resulted in only mild muscle functional deficits and failed to recapitulate the loss of mass and function observed in Sod1 -/- mice. To dissect any underlying cross-talk between motor neurons and skeletal muscle in the degeneration in Sod1 -/- mice, we characterized neuromuscular changes in the Sod1 -/- model compared with mSod1KO mice and examined degenerative molecular mechanisms and pathways in peripheral nerve and skeletal muscle. In contrast to mSod1KO mice, myofiber atrophy in Sod1 -/- mice was associated with increased muscle oxidative damage, neuromuscular junction degeneration, denervation, nerve demyelination, and upregulation of proteins involved in maintenance of myelin sheaths. Proteomic analyses confirmed increased proteasomal activity and adaptive stress responses in muscle of Sod1 -/- mice that were absent in mSod1KO mice. Peripheral nerve from neither Sod1 -/- nor mSod1KO mice showed increased oxidative damage or molecular responses to increased oxidation compared with wild type mice. Differential cysteine (Cys) labeling revealed a specific redox shift in the catalytic Cys residue of peroxiredoxin 6 (Cys47) in the peripheral nerve from Sod1 -/- mice. Innovation and Conclusion: These findings demonstrate that neuromuscular integrity, redox mechanisms, and pathways are differentially altered in nerve and muscle of Sod1 -/- and mSod1KO mice. Results support the concept that impaired redox signaling, rather than oxidative damage, in peripheral nerve plays a key role in muscle loss in Sod1 -/- mice and potentially sarcopenia during aging. Antioxid. Redox Signal. 28, 275-295.

Kv1.1 knock-in ataxic mice exhibit spontaneous myokymic activity exacerbated by fatigue, ischemia and low temperature

PubMed Central

Brunetti, Orazio; Imbrici, Paola; Botti, Fabio Massimo; Pettorossi, Vito Enrico; D'Adamo, Maria Cristina; Valentino, Mario; Zammit, Christian; Mora, Marina; Gibertini, Sara; Di Giovanni, Giuseppe; Muscat, Richard; Pessia, Mauro

2012-01-01

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K+ channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1V408A/+). Here, we investigated the neuromuscular transmission of Kv1.1V408A/+ ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve–muscle from Kv1.1+/+ and Kv1.1V408A/+ mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca2 + signals that occurred abnormally only in preparations dissected from Kv1.1V408A/+ mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca2 + homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K+ channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult. PMID:22609489

A Randomized Controlled Trial of an Online Relapse Prevention Program for Adolescents in Substance Abuse Treatment

ERIC Educational Resources Information Center

Trudeau, Kimberlee J.; Black, Ryan A.; Kamon, Jody L.; Sussman, Steve

2017-01-01

Background: An Internet-based relapse prevention supplement to adolescent substance abuse treatment programming is a promising modality to reinforce treatment gains and enhance recovery; however, an evidence base is lacking. Objective: To assess the efficacy of the online Navigating my Journey (NmJ) program. Methods: 129 adolescent-aged…

A Generalist Looks Back

NASA Astrophysics Data System (ADS)

Salpeter, Edwin E.

I fled with my parents from Hitler's Austria to Australia and studied physics at Sydney University. I obtained my Ph.D. in quantum electrodynamics with Rudolf Peierls at Birmingham University and came to Cornell to work with Hans Bethe. I have stayed at Cornell ever since, and I have essentially had only a single job in my whole life, but have switched fields quite often. I worked in nuclear astrophysics and in late-stellar evolution, estimated the Initial Mass Function for star formation and the metal enrichment of the interstellar medium. I suggested black hole accretion as the energy source for quasars, worked on molecule formation on dust grain surfaces, and was involved in 21-cm studies of gas clouds and disk galaxies. I collaborated with my wife on the neurobiology of the neuromuscular junction and with one of my daughters on the epidemiology of tuberculosis.

Structural and biophysical characterisation of agrin laminin G3 domain constructs.

PubMed

Tidow, Henning; Mattle, Daniel; Nissen, Poul

2011-01-01

Agrin mediates accumulation of acetylcholine receptors (AChRs) at the developing neuromuscular junction, but has also been implicated as a regulator of central nervous system (CNS) synapses. A C-terminal region of agrin (Ag-C20) binds to the α3 subunit of the sodium-potassium ATPase (NKA) in CNS neurons suggesting that α3NKA is a neuronal agrin receptor, whereas a shorter agrin fragment (Ag-C15) was shown to act as a competitive antagonist. Here, we show that the agrin C22 construct, which represents the naturally occurring neurotrypsin cleavage product, constitutes a well-folded, stable domain, while the deletion of 48 residues that correspond to strands β1-β4 of the agrin laminin G3 domain imposed by the agrin C15 construct leads to a misfolded protein.

Recurrent excitation between motoneurones propagates across segments and is purely glutamatergic

PubMed Central

Bhumbra, Gardave S.

2018-01-01

Spinal motoneurones (Mns) constitute the final output for the execution of motor tasks. In addition to innervating muscles, Mns project excitatory collateral connections to Renshaw cells (RCs) and other Mns, but the latter have received little attention. We show that Mns receive strong synaptic input from other Mns throughout development and into maturity, with fast-type Mns systematically receiving greater recurrent excitation than slow-type Mns. Optical recordings show that activation of Mns in one spinal segment can propagate to adjacent segments even in the presence of intact recurrent inhibition. While it is known that transmission at the neuromuscular junction is purely cholinergic and RCs are excited through both acetylcholine and glutamate receptors, here we show that neurotransmission between Mns is purely glutamatergic, indicating that synaptic transmission systems are differentiated at different postsynaptic targets of Mns. PMID:29538375

Botulism in children: a diagnostic dilemma in developing countries.

PubMed

Khan, Muhammad Rehan; Maheshwari, Prem Kumar; Ibrahim, Shahnaz Hamid; Haque, Anwarul

2013-06-01

Botulism is a well-known disease of the neuromuscular junction. It is a rare but curable cause of paralysis in paediatric population. In addition to classical clinical signs and symptoms, the diagnosis of botulism requires laboratory confirmation of intoxication by various biological tests. These include demonstration of botulinum toxin in serum or isolation of the Clostridium botulinum from stool/gastric aspirates. However, it is not always possible to confirm intoxication due to unavailability of technical facilities, especially in resource limited countries like Pakistan. Under these circumstances, electrophysiological studies serve as an excellent diagnostic tool. These studies can provide quick diagnosis of botulism so that early administration of botulism immunoglobulin, if available, can reduce morbidity, mortality and length of stay in hospital. We report a case of botulism from Pakistan diagnosed on the basis of electrophysiological studies.

Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.

PubMed

Albin, Stephanie D; Davis, Graeme W

2004-08-04

Here, we show that postsynaptic p21-activated kinase (Pak) signaling diverges into two genetically separable pathways at the Drosophila neuromuscular junction. One pathway controls glutamate receptor abundance. Pak signaling within this pathway is specified by a required interaction with the adaptor protein Dreadlocks (Dock). We demonstrate that Dock is localized to the synapse via an Src homology 2-mediated protein interaction. Dock is not necessary for Pak localization but is necessary to restrict Pak signaling to control glutamate receptor abundance. A second genetically separable function of Pak kinase signaling controls muscle membrane specialization through the regulation of synaptic Discs-large. In this pathway, Dock is dispensable. We present a model in which divergent Pak signaling is able to coordinate two different features of postsynaptic maturation, receptor abundance, and muscle membrane specialization.

Respiratory chain deficiency in aged spinal motor neurons☆

PubMed Central

Rygiel, Karolina A.; Grady, John P.; Turnbull, Doug M.

2014-01-01

Sarcopenia, muscle wasting, and strength decline with age, is an important cause of loss of mobility in the elderly individuals. The underlying mechanisms are uncertain but likely to involve defects of motor nerve, neuromuscular junction, and muscle. Loss of motor neurons with age and subsequent denervation of skeletal muscle has been recognized as one of the contributing factors. This study investigated aspects of mitochondrial biology in spinal motor neurons from elderly subjects. We found that protein components of complex I of mitochondrial respiratory chain were reduced or absent in a proportion of aged motor neurons–a phenomenon not observed in fetal tissue. Further investigation showed that complex I-deficient cells had reduced mitochondrial DNA content and smaller soma size. We propose that mitochondrial dysfunction in these motor neurons could lead to the cell loss and ultimately denervation of muscle fibers. PMID:24684792

Synaptic sprouting increases the uptake capacities of motoneurons in amyotrophic lateral sclerosis mice

PubMed Central

Millecamps, Stéphanie; Nicolle, Delphine; Ceballos-Picot, Irène; Mallet, Jacques; Barkats, Martine

2001-01-01

Using adenoviruses encoding reporter genes as retrograde tracers, we assessed the capacity of motoneurons to take up and retrogradely transport adenoviral particles injected into the muscles of transgenic mice expressing the G93A human superoxide dismutase mutation, a model of amyotrophic lateral sclerosis. Surprisingly, transgene expression in the motoneurons was significantly higher in symptomatic mice than in control or presymptomatic mice. Using botulinum toxin to induce nerve sprouting at neuromuscular junctions, we showed that the unexpectedly high level of motoneurons retrograde transduction results, at least in part, from newly acquired uptake properties of the sprouts. These findings demonstrate the remarkable uptake properties of amyotrophic lateral sclerosis motoneurons in response to denervation and the rationale of using intramuscular injections of adenoviruses to overexpress therapeutic proteins in motor neuron diseases. PMID:11404466

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

PubMed

Ghazanfari, Nazanin; Morsch, Marco; Reddel, Stephen W; Liang, Simon X; Phillips, William D

2014-07-01

Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

Zebrafish CaV2.1 Calcium Channels Are Tailored for Fast Synchronous Neuromuscular Transmission

PubMed Central

Naranjo, David; Wen, Hua; Brehm, Paul

2015-01-01

The CaV2.2 (N-type) and CaV2.1 (P/Q-type) voltage-dependent calcium channels are prevalent throughout the nervous system where they mediate synaptic transmission, but the basis for the selective presence at individual synapses still remains an open question. The CaV2.1 channels have been proposed to respond more effectively to brief action potentials (APs), an idea supported by computational modeling. However, the side-by-side comparison of CaV2.1 and CaV2.2 kinetics in intact neurons failed to reveal differences. As an alternative means for direct functional comparison we expressed zebrafish CaV2.1 and CaV2.2 α-subunits, along with their accessory subunits, in HEK293 cells. HEK cells lack calcium currents, thereby circumventing the need for pharmacological inhibition of mixed calcium channel isoforms present in neurons. HEK cells also have a simplified morphology compared to neurons, which improves voltage control. Our measurements revealed faster kinetics and shallower voltage-dependence of activation and deactivation for CaV2.1. Additionally, recordings of calcium current in response to a command waveform based on the motorneuron AP show, directly, more effective activation of CaV2.1. Analysis of calcium currents associated with the AP waveform indicate an approximately fourfold greater open probability (PO) for CaV2.1. The efficient activation of CaV2.1 channels during APs may contribute to the highly reliable transmission at zebrafish neuromuscular junctions. PMID:25650925

Role of membrane cholesterol in spontaneous exocytosis at frog neuromuscular synapses: reactive oxygen species–calcium interplay

PubMed Central

Petrov, Alexey M; Yakovleva, Anastasiya A; Zefirov, Andrey L

2014-01-01

Using electrophysiological and optical techniques, we studied the mechanisms by which cholesterol depletion stimulates spontaneous transmitter release by exocytosis at the frog neuromuscular junction. We found that methyl-β-cyclodextrin (MCD, 10 mm)-mediated exhaustion of cholesterol resulted in the enhancement of reactive oxygen species (ROS) production, which was prevented by the antioxidant N-acetyl cysteine (NAC) and the NADPH oxidase inhibitor apocynin. An increase in ROS levels occurred both extra- and intracellularly, and it was associated with lipid peroxidation in synaptic regions. Cholesterol depletion provoked a rise in the intracellular Ca2+ concentration, which was diminished by NAC and transient receptor potential vanilloid (TRPV) channel blockers (ruthenium red and capsazepine). By contrast, the MCD-induced rise in [Ca2+]i remained unaffected if Ca2+ release from endoplasmic stores was blocked by TMB8 (8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride). The effects of cholesterol depletion on spontaneous release and exocytosis were significantly reduced by the antioxidant, intracellular Ca2+ chelation with BAPTA-AM and blockers of TRPV channels. Bath application of the calcineurin antagonist cyclosporine A blocked MCD-induced enhancement of spontaneous release/exocytosis, whereas okadaic acid, an inhibitor of phosphatases PP1 and PP2A, had no effect. Thus, our findings indicate that enhancement of spontaneous exocytosis induced by cholesterol depletion may depend on ROS generation, leading to an influx of Ca2+ via TRPV channels and, subsequently, activation of calcineurin. PMID:25326454

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development.

PubMed

Lu, Cecilia S; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David

2014-09-26

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins.

MicroRNA-8 promotes robust motor axon targeting by coordinate regulation of cell adhesion molecules during synapse development

PubMed Central

Lu, Cecilia S.; Zhai, Bo; Mauss, Alex; Landgraf, Matthias; Gygi, Stephen; Van Vactor, David

2014-01-01

Neuronal connectivity and specificity rely upon precise coordinated deployment of multiple cell-surface and secreted molecules. MicroRNAs have tremendous potential for shaping neural circuitry by fine-tuning the spatio-temporal expression of key synaptic effector molecules. The highly conserved microRNA miR-8 is required during late stages of neuromuscular synapse development in Drosophila. However, its role in initial synapse formation was previously unknown. Detailed analysis of synaptogenesis in this system now reveals that miR-8 is required at the earliest stages of muscle target contact by RP3 motor axons. We find that the localization of multiple synaptic cell adhesion molecules (CAMs) is dependent on the expression of miR-8, suggesting that miR-8 regulates the initial assembly of synaptic sites. Using stable isotope labelling in vivo and comparative mass spectrometry, we find that miR-8 is required for normal expression of multiple proteins, including the CAMs Fasciclin III (FasIII) and Neuroglian (Nrg). Genetic analysis suggests that Nrg and FasIII collaborate downstream of miR-8 to promote accurate target recognition. Unlike the function of miR-8 at mature larval neuromuscular junctions, at the embryonic stage we find that miR-8 controls key effectors on both sides of the synapse. MiR-8 controls multiple stages of synapse formation through the coordinate regulation of both pre- and postsynaptic cell adhesion proteins. PMID:25135978

Diagnostic yield of electromyography in children with myopathic disorders.

PubMed

Ghosh, Partha S; Sorenson, Eric J

2014-08-01

Interpretation of pediatric electromyography interpretation in myopathic disorders is technically challenging. We assessed our electromyographic experience with respect to sensitivity and specificity in pediatric myopathy. We did a retrospective chart review of patients ≤18 years between 2009 and 2013. Two hundred twenty-four electromyographic studies were reviewed with the following referral diagnoses: myopathy, muscle weakness, neuromuscular disorders, myositis, myalgia, myoglobinuria, myasthenia, myotonia, cramps, periodic paralysis, hypotonia, and developmental delay. Only children who had an electromyography and muscle biopsy were included for analysis. Patients with neurogenic electromyography and neuromuscular junction disorders were excluded. Myopathic electromyography was defined as short duration, low amplitude, polyphasic motor unit potentials with rapid recruitment. Seventy-two patients were included (age range, 6 months-18 years). The following observations were made: group A: myopathic electromyography or pathognomonic of muscle disease and biopsy or genetically confirmed myopathy (32 cases); group B: myopathic electromyography but biopsy normal or nondiagnostic (12 cases); group C: normal electromyography but biopsy or genetically confirmed myopathy (three cases, all with metabolic myopathy); and group D: electromyography normal and biopsy normal or nondiagnostic (25 cases). The most common diagnoses were congenital myopathy (seven cases), metabolic myopathy (six cases), muscular dystrophy (six cases), genetically confirmed myopathy (five cases), myopathy, undefined (five cases), and inflammatory myopathy (four cases). Pediatric electromyography was 91% sensitive and 67% specific in myopathic disorders. The metabolic myopathies were commonly missed by electromyography. Copyright © 2014 Elsevier Inc. All rights reserved.

Association of myasthenia gravis and Behçet's disease: A case report.

PubMed

Kisabay, Aysin; Sari, Ummu Serpil; Boyaci, Recep; Batum, Melike; Yilmaz, Hikmet; Selcuki, Deniz

2016-01-01

Myasthenia gravis is a disease of neuromuscular junction due to auto-immune destruction of the acetylcholine receptors. Behçet's disease, on the other hand, is a multisystemic vascular-inflammatory disease. Both conditions are not common in the general population although their association has not been reported in the literature. We wanted to present our patient who developed clinical course of myasthenia gravis following discontinuation of medications due to complications of corticosteroid for Behçet's disease. It was observed that clinical findings of myasthenia gravis recovered following restarting steroid treatment and he did not experience attacks of both conditions. Although Myasthenia gravis and Behçet's disease are distinct entities clinically as well as in terms of pathogenesis, they share common physiopathological features and their treatment is based on their common features. Copyright © 2016 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Guidelines for pre-clinical assessment of the acetylcholine receptor-specific passive transfer myasthenia gravis model - recommendations for methods and experimental designs

PubMed Central

Kusner, Linda L.; Losen, Mario; Vincent, Angela; Lindstrom, Jon; Tzartos, Socrates; Lazaridis, Konstantinos; Martinez-Martinez, Pilar

2015-01-01

Antibodies against the muscle acetylcholine receptor (AChR) are the most common cause of myasthenia gravis (MG). Passive transfer of AChR antibodies from MG patients into animals reproduces key features of human disease, including antigenic modulation of the AChR, complement-mediated damage of the neuromuscular junction, and muscle weakness. Similarly, AChR antibodies generated by active immunization in experimental autoimmune MG models can subsequently be passively transferred to other animals and induce weakness. The passive transfer model is useful to test therapeutic strategies aimed at the effector mechanism of the autoantibodies. Here we summarize published and unpublished experience using the AChR passive transfer MG model in mice, rats and rhesus monkeys, and give recommendations for the design of preclinical studies in order to facilitate translation of positive and negative results to improve MG therapies. PMID:25743217

Animal models of myasthenia gravis: utility and limitations

PubMed Central

Mantegazza, Renato; Cordiglieri, Chiara; Consonni, Alessandra; Baggi, Fulvio

2016-01-01

Myasthenia gravis (MG) is a chronic autoimmune disease caused by the immune attack of the neuromuscular junction. Antibodies directed against the acetylcholine receptor (AChR) induce receptor degradation, complement cascade activation, and postsynaptic membrane destruction, resulting in functional reduction in AChR availability. Besides anti-AChR antibodies, other autoantibodies are known to play pathogenic roles in MG. The experimental autoimmune MG (EAMG) models have been of great help over the years in understanding the pathophysiological role of specific autoantibodies and T helper lymphocytes and in suggesting new therapies for prevention and modulation of the ongoing disease. EAMG can be induced in mice and rats of susceptible strains that show clinical symptoms mimicking the human disease. EAMG models are helpful for studying both the muscle and the immune compartments to evaluate new treatment perspectives. In this review, we concentrate on recent findings on EAMG models, focusing on their utility and limitations. PMID:27019601

Acetylcholinesterase activity in soleus muscle intrafusal and extrafusal fibres in tail suspended rats.

PubMed

Tang, Bin; Fan, Xiao-li; Wu, Su-di

2002-10-01

Objective. To explore the mechanisms involved in muscle atrophy and conversion of the fiber types induced by simulated weightlessness. Method. Weightlessness was simulated by tail suspension of female rats. Intrafusal and extrafusal fibers of soleus muscles in the rat were examined histochemically for their activity of acetylcholinesterase (AChE) and succinic dehydrogenase (SDH) in 7 d, 14 d, 21 d tail-suspended groups and control groups. Result. Staining for succinic dehydrogenase showed that simulated weightlessness caused obvious atrophy and change in fiber type composition in soleus muscle, with decrease of the proportion of type I fiber and increase of type II fiber. Acetylcholinesterase activities of intrafusal and extrafusal fibers were both decreased significantly after 21 d tail suspension. Conclusion. Simulated weightlessness could induce decrease of AChE activity in neuromuscular junctions, which might be linked with decrease in motor neuron activity.

Presynaptic neurones may contribute a unique glycoprotein to the extracellular matrix at the synapse

NASA Astrophysics Data System (ADS)

Caroni, Pico; Carlson, Steven S.; Schweitzer, Erik; Kelly, Regis B.

1985-04-01

As the extracellular matrix at the original site of a neuromuscular junction seems to play a major part in the specificity of synaptic regeneration1-5, considerable attention has been paid to unique molecules localized to this region6-11. Here we describe an extracellular matrix glycoprotein of the elasmobranch electric organ that is localized near the nerve endings. By immunological criteria, it is synthesized in the cell bodies, transported down the axons and is related to a glycoprotein in the synaptic vesicles of the neurones that innervate the electric organ. It is apparently specific for these neurones, as it cannot be detected elsewhere in the nervous system of the fish. Therefore, neurones seem to contribute unique extracellular matrix glycoproteins to the synaptic region. Synaptic vesicles could be involved in transporting these glycoproteins to or from the nerve terminal surface.

Plakins in striated muscle.

PubMed

Boyer, Justin G; Bernstein, Marija A; Boudreau-Larivière, Céline

2010-03-01

Striated muscle cells contain numerous architectural proteins that contribute to the function of muscle as generators of mechanical force. Among these proteins are crosslinkers belonging to the plakin family, namely plectin, microtubule-actin crosslinking factor (ACF7/MACF1), bullous pemphigoid antigen 1 (Bpag1/dystonin), and desmoplakin. These plakin family members, in particular plectin and Bpag1/dystonin, exist as several isoforms. The domain organization of these plakin variants dictates their subcellular location and the proteins with which they interact. Several studies suggest that plakins exert unique functions within various compartments of the muscle cell including the sarcolemma, the sarcomere, both neuromuscular and myotendinous junctions in skeletal muscle, and the intercalated discs in cardiac muscle. Plakins may also regulate the cellular placement and function of specific organelles, notably the nucleus, mitochondria, Golgi apparatus, and sarcoplasmic reticulum. Here we review and summarize our current knowledge of the function of plakins in striated muscle cells.

Differential effects of myostatin deficiency on motor and sensory axons.

PubMed

Jones, Maria R; Villalón, Eric; Northcutt, Adam J; Calcutt, Nigel A; Garcia, Michael L

2017-12-01

Deletion of myostatin in mice (MSTN -/- ) alters structural properties of peripheral axons. However, properties like axon diameter and myelin thickness were analyzed in mixed nerves, so it is unclear whether loss of myostatin affects motor, sensory, or both types of axons. Using the MSTN -/- mouse model, we analyzed the effects of increasing the number of muscle fibers on axon diameter, myelin thickness, and internode length in motor and sensory axons. Axon diameter and myelin thickness were increased in motor axons of MSTN -/- mice without affecting internode length or axon number. The number of sensory axons was increased without affecting their structural properties. These results suggest that motor and sensory axons establish structural properties by independent mechanisms. Moreover, in motor axons, instructive cues from the neuromuscular junction may play a role in co-regulating axon diameter and myelin thickness, whereas internode length is established independently. Muscle Nerve 56: E100-E107, 2017. © 2017 Wiley Periodicals, Inc.

ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function

PubMed Central

Sharma, Aarti; Lyashchenko, Alexander K.; Lu, Lei; Nasrabady, Sara Ebrahimi; Elmaleh, Margot; Mendelsohn, Monica; Nemes, Adriana; Tapia, Juan Carlos; Mentis, George Z.; Shneider, Neil A.

2016-01-01

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations. PMID:26842965

MicroRNAs and the Evolution of Insect Metamorphosis.

PubMed

Belles, Xavier

2017-01-31

MicroRNAs (miRNAs) are involved in the regulation of a number of processes associated with metamorphosis, either in the less modified hemimetabolan mode or in the more modified holometabolan mode. The miR-100/let-7/miR-125 cluster has been studied extensively, especially in relation to wing morphogenesis in both hemimetabolan and holometabolan species. Other miRNAs also participate in wing morphogenesis, as well as in programmed cell and tissue death, neuromaturation, neuromuscular junction formation, and neuron cell fate determination, typically during the pupal stage of holometabolan species. A special case is the control of miR-2 over Kr-h1 transcripts, which determines adult morphogenesis in the hemimetabolan metamorphosis. This is an elegant example of how a single miRNA can control an entire process by acting on a crucial mediator; however, this is a quite exceptional mechanism that was apparently lost during the transition from hemimetaboly to holometaboly.

Neuronal Lipid Metabolism: Multiple Pathways Driving Functional Outcomes in Health and Disease

PubMed Central

Tracey, Timothy J.; Steyn, Frederik J.; Wolvetang, Ernst J.; Ngo, Shyuan T.

2018-01-01

Lipids are a fundamental class of organic molecules implicated in a wide range of biological processes related to their structural diversity, and based on this can be broadly classified into five categories; fatty acids, triacylglycerols (TAGs), phospholipids, sterol lipids and sphingolipids. Different lipid classes play major roles in neuronal cell populations; they can be used as energy substrates, act as building blocks for cellular structural machinery, serve as bioactive molecules, or a combination of each. In amyotrophic lateral sclerosis (ALS), dysfunctions in lipid metabolism and function have been identified as potential drivers of pathogenesis. In particular, aberrant lipid metabolism is proposed to underlie denervation of neuromuscular junctions, mitochondrial dysfunction, excitotoxicity, impaired neuronal transport, cytoskeletal defects, inflammation and reduced neurotransmitter release. Here we review current knowledge of the roles of lipid metabolism and function in the CNS and discuss how modulating these pathways may offer novel therapeutic options for treating ALS. PMID:29410613

Curare: the South American arrow poison.

PubMed

Lee, M R

2005-02-01

The history of curare is both curious and convoluted. A product of South American culture it emerged in the sixteenth century from the mists of antiquity at the same time as quinine, coca, and chocolate. Like quinine, at first came the extract but no plant, and later the plant but no chemical compound. It took more than 300 years and the efforts of many explorers and scientists to resolve the problem. These included Condamine, Humboldt, Brodie, Waterton, Bernard, Dale, Walker, and King. Finally, the pure compound d-tubocurarine was isolated from the liana Chondrodendron and synthesised. Its specific physiological action was blockade of the effect of acetylcholine at the neuro-muscular junction. Such a paralytic poison could be used to kill oneself or others. The bizarre plot to kill the Prime Minister, Lloyd George, during the First World War is described. Fortunately this nefarious plan was thwarted by the Secret Service!

A SMN-Dependent U12 Splicing Event Essential for Motor Circuit Function

PubMed Central

Lotti, Francesco; Imlach, Wendy L.; Saieva, Luciano; Beck, Erin S.; Hao, Le T.; Li, Darrick K.; Jiao, Wei; Mentis, George Z.; Beattie, Christine E.; McCabe, Brian D.; Pellizzoni, Livio

2012-01-01

SUMMARY Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a novel protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA. PMID:23063131

Boosted Regeneration and Reduced Denervated Muscle Atrophy by NeuroHeal in a Pre-clinical Model of Lumbar Root Avulsion with Delayed Reimplantation.

PubMed

Romeo-Guitart, David; Forés, Joaquim; Navarro, Xavier; Casas, Caty

2017-09-20

The "gold standard" treatment of patients with spinal root injuries consists of delayed surgical reconnection of nerves. The sooner, the better, but problems such as injury-induced motor neuronal death and muscle atrophy due to long-term denervation mean that normal movement is not restored. Herein we describe a preclinical model of root avulsion with delayed reimplantation of lumbar roots that was used to establish a new adjuvant pharmacological treatment. Chronic treatment (up to 6 months) with NeuroHeal, a new combination drug therapy identified using a systems biology approach, exerted long-lasting neuroprotection, reduced gliosis and matrix proteoglycan content, accelerated nerve regeneration by activating the AKT pathway, promoted the formation of functional neuromuscular junctions, and reduced denervation-induced muscular atrophy. Thus, NeuroHeal is a promising treatment for spinal nerve root injuries and axonal regeneration after trauma.

A network of autism linked genes stabilizes two pools of synaptic GABAA receptors

PubMed Central

Tong, Xia-Jing; Hu, Zhitao; Liu, Yu; Anderson, Dorian; Kaplan, Joshua M

2015-01-01

Changing receptor abundance at synapses is an important mechanism for regulating synaptic strength. Synapses contain two pools of receptors, immobilized and diffusing receptors, both of which are confined to post-synaptic elements. Here we show that immobile and diffusing GABAA receptors are stabilized by distinct synaptic scaffolds at C. elegans neuromuscular junctions. Immobilized GABAA receptors are stabilized by binding to FRM-3/EPB4.1 and LIN-2A/CASK. Diffusing GABAA receptors are stabilized by the synaptic adhesion molecules Neurexin and Neuroligin. Inhibitory post-synaptic currents are eliminated in double mutants lacking both scaffolds. Neurexin, Neuroligin, and CASK mutations are all linked to Autism Spectrum Disorders (ASD). Our results suggest that these mutations may directly alter inhibitory transmission, which could contribute to the developmental and cognitive deficits observed in ASD. DOI: http://dx.doi.org/10.7554/eLife.09648.001 PMID:26575289

Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation

PubMed Central

Li, Xiling; Goel, Pragya; Chen, Catherine; Angajala, Varun; Chen, Xun

2018-01-01

Postsynaptic compartments can be specifically modulated during various forms of synaptic plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic homeostatic plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular junction, where a retrograde enhancement of presynaptic neurotransmitter release compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP induction and expression, we have developed a genetic manipulation to reduce postsynaptic receptor expression at one of the two muscles innervated by a single motor neuron. We find that PHP can be induced and expressed at a subset of synapses, over both acute and chronic time scales, without influencing transmission at adjacent release sites. Further, homeostatic modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and expression mechanisms are locally transmitted and restricted to specific synaptic compartments. PMID:29620520

Critical analysis of the use of onabotulinumtoxinA (botulinum toxin type A) in migraine.

PubMed

Robertson, Carrie E; Garza, Ivan

2012-01-01

OnabotulinumtoxinA, a neurotoxin, has been studied in numerous trials as a novel preventive therapy for migraine headache. The data would support that it may be effective at reducing headache days in patients suffering from chronic migraine (≥15 headache days/month, with eight or more of those migraine headache days). The mechanism by which onabotulinumtoxinA exerts its effects on migraine is not yet understood. It is known to inhibit acetylcholine release at the neuromuscular junction, but this probably does not explain the observed antinociceptive properties noted in preclinical and clinical trials. This review will discuss the known mechanisms of action of botulinum toxin type A, and will review the available randomized, placebo-controlled trials that have looked at its efficacy as a migraine preventative. We also describe the onabotulinumtoxinA injection sites used at our institution.

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin.

PubMed

Wang, Chien-Hsiang; Huang, Yi-Chun; Chen, Pei-Yi; Cheng, Ying-Ju; Kao, Hsiu-Hua; Pi, Haiwei; Chien, Cheng-Ting

2017-05-10

Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. Here, we show that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth.

ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function.

PubMed

Sharma, Aarti; Lyashchenko, Alexander K; Lu, Lei; Nasrabady, Sara Ebrahimi; Elmaleh, Margot; Mendelsohn, Monica; Nemes, Adriana; Tapia, Juan Carlos; Mentis, George Z; Shneider, Neil A

2016-02-04

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), including some of the most aggressive, juvenile-onset forms of the disease. FUS loss-of-function and toxic gain-of-function mechanisms have been proposed to explain how mutant FUS leads to motor neuron degeneration, but neither has been firmly established in the pathogenesis of ALS. Here we characterize a series of transgenic FUS mouse lines that manifest progressive, mutant-dependent motor neuron degeneration preceded by early, structural and functional abnormalities at the neuromuscular junction. A novel, conditional FUS knockout mutant reveals that postnatal elimination of FUS has no effect on motor neuron survival or function. Moreover, endogenous FUS does not contribute to the onset of the ALS phenotype induced by mutant FUS. These findings demonstrate that FUS-dependent motor degeneration is not due to loss of FUS function, but to the gain of toxic properties conferred by ALS mutations.

Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97.

PubMed

Bachmann, André; Timmer, Marco; Sierralta, Jimena; Pietrini, Grazia; Gundelfinger, Eckart D; Knust, Elisabeth; Thomas, Ulrich

2004-04-15

Stardust (Sdt) and Discs-Large (Dlg) are membrane-associated guanylate kinases (MAGUKs) involved in the organization of supramolecular protein complexes at distinct epithelial membrane compartments in Drosophila. Loss of either Sdt or Dlg affects epithelial development with severe effects on apico-basal polarity. Moreover, Dlg is required for the structural and functional integrity of synaptic junctions. Recent biochemical and cell culture studies have revealed that various mammalian MAGUKs can interact with mLin-7/Veli/MALS, a small PDZ-domain protein. To substantiate these findings for their in vivo significance with regard to Sdt- and Dlg-based protein complexes, we analyzed the subcellular distribution of Drosophila Lin-7 (DLin-7) and performed genetic and biochemical assays to characterize its interaction with either of the two MAGUKs. In epithelia, Sdt mediates the recruitment of DLin-7 to the subapical region, while at larval neuromuscular junctions, a particular isoform of Dlg, Dlg-S97, is required for postsynaptic localization of DLin-7. Ectopic expression of Dlg-S97 in epithelia, however, was not sufficient to induce a redistribution of DLin-7. These results imply that the recruitment of DLin-7 to MAGUK-based protein complexes is defined by cell-type specific mechanisms and that DLin-7 acts downstream of Sdt in epithelia and downstream of Dlg at synapses.

Selective depolarization of the muscle membrane in frog nerve-muscle preparations by a chromatographically purified extract of the dinoflagellate Ostreopsis lenticularis

PubMed Central

Meunier, Frédéric A; Mercado, José A; Molgó, Jordi; Tosteson, Thomas R; Escalona de Motta, Gladys

1997-01-01

The actions of a chromatographically identified extract of the marine dinoflagellate Ostreopsis lenticularis, named ostreotoxin-3 (OTX-3), were studied on frog isolated neuromuscular preparations. OTX-3 (1–10 μg ml−1) applied to cutaneous pectoris nerve-muscle preparations depolarized skeletal muscle fibres and caused spontaneous contractions. The depolarization was neither reversed by prolonged washing nor by (+)-tubocurarine. OTX-3 decreased the amplitude of miniature end plate potentials (m.e.p.ps) but did not affect their frequency. Extracellular recording of compound action potentials revealed that OTX-3 affected neither excitability nor conduction along intramuscular nerve branches. End-plate potentials (e.p.ps) elicited by nerve stimulation were reduced in amplitude by OTX-3 and even showed reversed polarity in junctions deeply depolarized by the toxin. Membrane depolarization induced by OTX-3 was decreased about 70% in muscles pretreated for 30 min with 10 μM tetrodotoxin. In contrast, muscles pretreated with 5 μM μ-conotoxin GIIIA were completely insensitive to OTX-3-induced depolarization. OTX-3 did not affect e.p.p. amplitude and the quantal content of e.p.ps in junctions in which muscle depolarization was abolished by μ-conotoxin GIIIA. OTX-3 is a novel type of sodium-channel activating toxin that discriminates between nerve and skeletal muscle membranes. PMID:9249261

Influence of phospholipasic inhibition on neuromuscular activity of Bothrops fonsecai snake venom.

PubMed

Schezaro-Ramos, Raphael; Collaço, Rita de Cássia O; Randazzo-Moura, Priscila; Rocha, Thalita; Cogo, José Carlos; Rodrigues-Simioni, Léa

2017-05-01

Bothrops fonsecai (B. fonsecai), a pitviper endemic to southeastern Brazil, has a venom mainly composed by snake venom phospholipases (PLA 2 ) and metalloproteases, compounds that could interfere with neuromuscular junction in vitro. In this work, we investigated the role of PLA 2 in the myotoxicity and neuromuscular blockade caused by B. fonsecai venom using different procedures frequently associated with PLA 2 activity inhibition: 24 °C bath temperature, Ca 2+ - Sr 2+ replacement and chemical modification with p-bromophenacyl bromide (p-BPB). Mice extensor digitorum longus preparations (EDL) were incubated with usual or modified Tyrode solution (prepared with Ca 2+ or Sr 2+ respectively) at 24 °C or 37 °C (as controls) and in addition of B. fonsecai venom (100 μg/mL) alone or after its incubation with buffer (24 h, 23 °C) on the absence (alkylation control) and presence of p-BPB; all muscle were processed for histological analysis. The PLA 2 , proteolytic and amidolytic activities under the same conditions (24 °C or 37 °C, Ca 2+ - Sr 2+ replacement, absence or presence p-BPB) were also assessed. The B. fonsecai venom caused total neuromuscular blockade after 100 min of incubation, in Ca 2+ Tyrode solution at 37 °C (usual conditions); on Sr 2+ Tyrode solution (37 °C) the twitch height were 31.7 ± 7.4% of basal, and at 24 °C (Ca 2+ Tyrode solution) were 53.6 ± 7.0% of basal. The alkylation of PLA 2 with p-BPB promoted a great blockade decrease at 100 min of incubation (88.7 ± 5.7% of basal), but it was also observed on alkylation control preparations (66.2 ± 6.6%). The venom produced 50% of blockade at 40.5 ± 5.9 min, in Ca 2+ Tyrode solution at 37 °C. The protocols delayed the time for 50% blockade: 105.7 ± 7.1 min (at 24 °C, in Ca 2+ Tyrode solution) and 71.1 ± 9.0 min (at 37 °C, in Sr 2+ Tyrode solution). Regarding p-BPB incubation and alkylation control preparations, 50% of blockade was not reached during the 120 min of venom incubation. Regarding to enzymatic activities, the 24 °C protocol reduced not only PLA 2 (to 62.3%) but also proteolytic (52.3%) and amidolytic (73.4%) activities, as well as observed on p-BPB alkylation protocol which markedly inhibited all enzymes (<10%). The alkylation control promoted the same proteolytic and amidolytic inhibition but no reduction of PLA 2 activity; Ca 2+ - Sr 2+ replacement reduced only the PLA 2 activity (to 15.3%). We observed a strict relation between the inhibition of PLA 2 activity and the myotoxicity. On the other hand, this relation was not observed with neuromuscular blockade, suggesting that blockade and muscle damage may not be strictly related. It suggests that the neuromuscular blockade may be induced by non-catalytic PLA 2 or other venom components, such as metalloproteinases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endogenous purines modulate K+ -evoked ACh secretion at the mouse neuromuscular junction.

PubMed

Guarracino, Juan F; Cinalli, Alejandro R; Veggetti, Mariela I; Losavio, Adriana S

2018-06-01

At the mouse neuromuscular junction, adenosine triphosphate (ATP) is co-released with the neurotransmitter acetylcholine (ACh), and once in the synaptic cleft, it is hydrolyzed to adenosine. Both ATP/adenosine diphosphate (ADP) and adenosine modulate ACh secretion by activating presynaptic P2Y 13 and A 1 , A 2A , and A 3 receptors, respectively. To elucidate the action of endogenous purines on K + -dependent ACh release, we studied the effect of purinergic receptor antagonists on miniature end-plate potential (MEPP) frequency in phrenic diaphragm preparations. At 10 mM K + , the P2Y 13 antagonist N-[2-(methylthio)ethyl]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with (dichloromethylene)bis[phosphonic acid], tetrasodium salt (AR-C69931MX) increased asynchronous ACh secretion while the A 1 , A 3 , and A 2A antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(±)-dihydropyridine-3,5-, dicarboxylate (MRS-1191), and 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261) did not modify neurosecretion. The inhibition of equilibrative adenosine transporters by S-(p-nitrobenzyl)-6-thioinosine provoked a reduction of 10 mM K + -evoked ACh release, suggesting that the adenosine generated from ATP is being removed from the synaptic space by the transporters. At 15 and 20 mM K + , endogenous ATP/ADP and adenosine bind to inhibitory P2Y 13 and A 1 and A 3 receptors since AR-C69931MX, DPCPX, and MRS-1191 increased MEPP frequency. Similar results were obtained when the generation of adenosine was prevented by using the ecto-5'-nucleotidase inhibitor α,β-methyleneadenosine 5'-diphosphate sodium salt. SCH-58261 only reduced neurosecretion at 20 mM K + , suggesting that more adenosine is needed to activate excitatory A 2A receptors. At high K + concentration, the equilibrative transporters appear to be saturated allowing the accumulation of adenosine in the synaptic cleft. In conclusion, when motor nerve terminals are depolarized by increasing K + concentrations, the ATP/ADP and adenosine endogenously generated are able to modulate ACh secretion by sequential activation of different purinergic receptors. © 2018 Wiley Periodicals, Inc.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria Angel; Cilleros, Victor; Tomàs, Josep Maria

2016-01-01

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes. © 2017 S. Karger AG, Basel.

Kv1.1 knock-in ataxic mice exhibit spontaneous myokymic activity exacerbated by fatigue, ischemia and low temperature.

PubMed

Brunetti, Orazio; Imbrici, Paola; Botti, Fabio Massimo; Pettorossi, Vito Enrico; D'Adamo, Maria Cristina; Valentino, Mario; Zammit, Christian; Mora, Marina; Gibertini, Sara; Di Giovanni, Giuseppe; Muscat, Richard; Pessia, Mauro

2012-09-01

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K(+) channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1(V408A/+)). Here, we investigated the neuromuscular transmission of Kv1.1(V408A/+) ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve-muscle from Kv1.1(+/+) and Kv1.1(V408A/+) mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca(2+) signals that occurred abnormally only in preparations dissected from Kv1.1(V408A/+) mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca(2+) homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K(+) channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult. Copyright © 2012 Elsevier Inc. All rights reserved.

A case series of re-establishment of neuromuscular block with rocuronium after sugammadex reversal.

PubMed

Iwasaki, Hajime; Sasakawa, Tomoki; Takahoko, Kenichi; Takagi, Shunichi; Nakatsuka, Hideki; Suzuki, Takahiro; Iwasaki, Hiroshi

2016-06-01

We report the use of rocuronium to re-establish neuromuscular block after reversal with sugammadex. The aim of this study was to investigate the relationship between the dose of rocuronium needed to re-establish neuromuscular block and the time interval between sugammadex administration and re-administration of rocuronium. Patients who required re-establishment of neuromuscular block within 12 h after the reversal of rocuronium-induced neuromuscular block with sugammadex were included. After inducing general anesthesia and placing the neuromuscular monitor, the protocol to re-establish neuromuscular block was as follows. An initial rocuronium dose of 0.6 mg/kg was followed by additional 0.3 mg/kg doses every 2 min until train-of-four responses were abolished. A total of 11 patients were enrolled in this study. Intervals between sugammadex and second rocuronium were 12-465 min. Total dose of rocuronium needed to re-establish neuromuscular block was 0.6-1.2 mg/kg. 0.6 mg/kg rocuronium re-established neuromuscular block in all patients who received initial sugammadex more than 3 h previously. However, when the interval between sugammadex and second rocuronium was less than 2 h, more than 0.6 mg/kg rocuronium was necessary to re-establish neuromuscular block.

Drosophila Importin-α2 Is Involved in Synapse, Axon and Muscle Development

PubMed Central

Mosca, Timothy J.; Schwarz, Thomas L.

2010-01-01

Nuclear import is required for communication between the cytoplasm and the nucleus and to enact lasting changes in gene transcription following stimuli. Binding to an Importin-α molecule in the cytoplasm is often required to mediate nuclear entry of a signaling protein. As multiple isoforms of Importin-α exist, some may be responsible for the entry of distinct cargoes rather than general nuclear import. Indeed, in neuronal systems, Importin-α isoforms can mediate very specific processes such as axonal tiling and communication of an injury signal. To study nuclear import during development, we examined the expression and function of Importin-α2 in Drosophila melanogaster. We found that Importin-α2 was expressed in the nervous system where it was required for normal active zone density at the NMJ and axonal commissure formation in the central nervous system. Other aspects of synaptic morphology at the NMJ and the localization of other synaptic markers appeared normal in importin-α2 mutants. Importin-α2 also functioned in development of the body wall musculature. Mutants in importin-α2 exhibited errors in muscle patterning and organization that could be alleviated by restoring muscle expression of Importin-α2. Thus, Importin-α2 is needed for some processes in the development of both the nervous system and the larval musculature. PMID:21151903

21 CFR 882.5810 - External functional neuromuscular stimulator.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false External functional neuromuscular stimulator. 882.5810 Section 882.5810 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... External functional neuromuscular stimulator. (a) Identification. An external functional neuromuscular...

21 CFR 882.5810 - External functional neuromuscular stimulator.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false External functional neuromuscular stimulator. 882.5810 Section 882.5810 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... External functional neuromuscular stimulator. (a) Identification. An external functional neuromuscular...

Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody

PubMed Central

Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

2013-01-01

In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft. PMID:23440963

Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody.

PubMed

Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

2013-05-15

In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft.

Adaptability of the Immature Ocular Motor Control System: Unilateral IGF-1 Medial Rectus Treatment.

PubMed

Willoughby, Christy L; Fleuriet, Jérome; Walton, Mark M; Mustari, Michael J; McLoon, Linda K

2015-06-01

Unilateral treatment with sustained release IGF-1 to one medial rectus muscle in infant monkeys was performed to test the hypothesis that strabismus would develop as a result of changes in extraocular muscles during the critical period of development of binocularity. Sustained release IGF-1 pellets were implanted unilaterally on one medial rectus muscle in normal infant monkeys during the first 2 weeks of life. Eye position was monitored using standard photographic methods. After 3 months of treatment, myofiber and neuromuscular size, myosin composition, and innervation density were quantified in all rectus muscles and compared to those in age-matched controls. Sustained unilateral IGF-1 treatments resulted in strabismus for all treated subjects; 3 of the 4 subjects had a clinically significant strabismus of more than 10°. Both the treated medial rectus and the untreated ipsilateral antagonist lateral rectus muscles had significantly larger myofibers. No adaptation in myofiber size occurred in the contralateral functionally yoked lateral rectus or in myosin composition, neuromuscular junction size, or nerve density. Sustained unilateral IGF-1 treatment to extraocular muscles during the sensitive period of development of orthotropic eye alignment and binocularity was sufficient to disturb ocular motor development, resulting in strabismus in infant monkeys. This could be due to altering fusion of gaze during the early sensitive period. Serial measurements of eye alignment suggested the IGF-1-treated infants received insufficient coordinated binocular experience, preventing the establishment of normal eye alignment. Our results uniquely suggest that abnormal signaling by the extraocular muscles may be a cause of strabismus.

Adaptability of the Immature Ocular Motor Control System: Unilateral IGF-1 Medial Rectus Treatment

PubMed Central

Willoughby, Christy L.; Fleuriet, Jérome; Walton, Mark M.; Mustari, Michael J.; McLoon, Linda K.

2015-01-01

Purpose. Unilateral treatment with sustained release IGF-1 to one medial rectus muscle in infant monkeys was performed to test the hypothesis that strabismus would develop as a result of changes in extraocular muscles during the critical period of development of binocularity. Methods. Sustained release IGF-1 pellets were implanted unilaterally on one medial rectus muscle in normal infant monkeys during the first 2 weeks of life. Eye position was monitored using standard photographic methods. After 3 months of treatment, myofiber and neuromuscular size, myosin composition, and innervation density were quantified in all rectus muscles and compared to those in age-matched controls. Results. Sustained unilateral IGF-1 treatments resulted in strabismus for all treated subjects; 3 of the 4 subjects had a clinically significant strabismus of more than 10°. Both the treated medial rectus and the untreated ipsilateral antagonist lateral rectus muscles had significantly larger myofibers. No adaptation in myofiber size occurred in the contralateral functionally yoked lateral rectus or in myosin composition, neuromuscular junction size, or nerve density. Conclusions. Sustained unilateral IGF-1 treatment to extraocular muscles during the sensitive period of development of orthotropic eye alignment and binocularity was sufficient to disturb ocular motor development, resulting in strabismus in infant monkeys. This could be due to altering fusion of gaze during the early sensitive period. Serial measurements of eye alignment suggested the IGF-1-treated infants received insufficient coordinated binocular experience, preventing the establishment of normal eye alignment. Our results uniquely suggest that abnormal signaling by the extraocular muscles may be a cause of strabismus. PMID:26030103

Deciphering the role of a coleopteran steering muscle via free flight stimulation.

PubMed

Sato, Hirotaka; Vo Doan, Tat Thang; Kolev, Svetoslav; Huynh, Ngoc Anh; Zhang, Chao; Massey, Travis L; van Kleef, Joshua; Ikeda, Kazuo; Abbeel, Pieter; Maharbiz, Michel M

2015-03-16

Testing hypotheses of neuromuscular function during locomotion ideally requires the ability to record cellular responses and to stimulate the cells being investigated to observe downstream behaviors [1]. The inability to stimulate in free flight has been a long-standing hurdle for insect flight studies. The miniaturization of computation and communication technologies has delivered ultra-small, radio-enabled neuromuscular recorders and stimulators for untethered insects [2-8]. Published stimulation targets include the areas in brain potentially responsible for pattern generation in locomotion [5], the nerve chord for abdominal flexion [9], antennal muscles [2, 10], and the flight muscles (or their excitatory junctions) [7, 11-13]. However, neither fine nor graded control of turning has been demonstrated in free flight, and responses to the stimulation vary widely [2, 5, 7, 9]. Technological limitations have precluded hypotheses of function validation requiring exogenous stimulation during flight. We investigated the role of a muscle involved in wing articulation during flight in a coleopteran. We set out to identify muscles whose stimulation produced a graded turning in free flight, a feat that would enable fine steering control not previously demonstrated. We anticipated that gradation might arise either as a function of the phase of muscle firing relative to the wing stroke (as in the classic fly b1 muscle [14, 15] or the dorsal longitudinal and ventral muscles of moth [16]), or due to regulated tonic control, in which phase-independent summation of twitch responses produces varying amounts of force delivered to the wing linkages [15, 17, 18]. Copyright © 2015 Elsevier Ltd. All rights reserved.

Synaptic defects associated with s-inclusion body myositis are prevented by copper.

PubMed

Aldunate, R; Minniti, A N; Rebolledo, D; Inestrosa, N C

2012-08-01

Sporadic-inclusion body myositis (s-IBM) is the most common skeletal muscle disorder to afflict the elderly, and is clinically characterized by skeletal muscle degeneration. Its progressive course leads to muscle weakness and wasting, resulting in severe disability. The exact pathogenesis of this disease is unknown and no effective treatment has yet been found. An intriguing aspect of s-IBM is that it shares several molecular abnormalities with Alzheimer's disease, including the accumulation of amyloid-β-peptide (Aβ). Both disorders affect homeostasis of the cytotoxic fragment Aβ(1-42) during aging, but they are clinically distinct diseases. The use of animals that mimic some characteristics of a disease has become important in the search to elucidate the molecular mechanisms underlying the pathogenesis. With the aim of analyzing Aβ-induced pathology and evaluating the consequences of modulating Aβ aggregation, we used Caenorhabditis elegans that express the Aβ human peptide in muscle cells as a model of s-IBM. Previous studies indicate that copper treatment increases the number and size of amyloid deposits in muscle cells, and is able to ameliorate the motility impairments in Aβ transgenic C. elegans. Our recent studies show that neuromuscular synaptic transmission is defective in animals that express the Aβ-peptide and suggest a specific defect at the nicotine acetylcholine receptors level. Biochemical analyses show that copper treatment increases the number of amyloid deposits but decreases Aβ-oligomers. Copper treatment improves motility, synaptic structure and function. Our results suggest that Aβ-oligomers are the toxic Aβ species that trigger neuromuscular junction dysfunction.

21 CFR 882.5860 - Implanted neuromuscular stimulator.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Implanted neuromuscular stimulator. 882.5860... neuromuscular stimulator. (a) Identification. An implanted neuromuscular stimulator is a device that provides electrical stimulation to a patient's peroneal or femoral nerve to cause muscles in the leg to contract, thus...

Neuromuscular adaptations predict functional disability independently of clinical pain and psychological factors in patients with chronic non-specific low back pain.

PubMed

Dubois, Jean-Daniel; Abboud, Jacques; St-Pierre, Charles; Piché, Mathieu; Descarreaux, Martin

2014-08-01

Patients with chronic low back pain exhibit characteristics such as clinical pain, psychological symptoms and neuromuscular adaptations. The purpose of this study was to determine the independent contribution of clinical pain, psychological factors and neuromuscular adaptations to disability in patients with chronic low back pain. Clinical pain intensity, pain catastrophizing, fear-avoidance beliefs, anxiety, neuromuscular adaptations to chronic pain and neuromuscular responses to experimental pain were assessed in 52 patients with chronic low back pain. Lumbar muscle electromyographic activity was assessed during a flexion-extension task (flexion relaxation phenomenon) to assess both chronic neuromuscular adaptations and neuromuscular responses to experimental pain during the task. Multiple regressions showed that independent predictors of disability included neuromuscular adaptations to chronic pain (β=0.25, p=0.006, sr(2)=0.06), neuromuscular responses to experimental pain (β=-0.24, p=0.011, sr(2)=0.05), clinical pain intensity (β=0.28, p=0.002, sr(2)=0.08) and psychological factors (β=0.58, p<0.001, sr(2)=0.32). Together, these predictors accounted for 65% of variance in disability (R(2)=0.65 p<0.001). The current investigation revealed that neuromuscular adaptations are independent from clinical pain intensity and psychological factors, and contribute to inter-individual differences in patients' disability. This suggests that disability, in chronic low back pain patients, is determined by a combination of factors, including clinical pain, psychological factors and neuromuscular adaptations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sugammadex: A Review of Neuromuscular Blockade Reversal.

PubMed

Keating, Gillian M

2016-07-01

Sugammadex (Bridion(®)) is a modified γ-cyclodextrin that reverses the effect of the steroidal nondepolarizing neuromuscular blocking agents rocuronium and vecuronium. Intravenous sugammadex resulted in rapid, predictable recovery from moderate and deep neuromuscular blockade in patients undergoing surgery who received rocuronium or vecuronium. Recovery from moderate neuromuscular blockade was significantly faster with sugammadex 2 mg/kg than with neostigmine, and recovery from deep neuromuscular blockade was significantly faster with sugammadex 4 mg/kg than with neostigmine or spontaneous recovery. In addition, recovery from neuromuscular blockade was significantly faster when sugammadex 16 mg/kg was administered 3 min after rocuronium than when patients spontaneously recovered from succinylcholine. Sugammadex also demonstrated efficacy in various special patient populations, including patients with pulmonary disease, cardiac disease, hepatic dysfunction or myasthenia gravis and morbidly obese patients. Intravenous sugammadex was generally well tolerated. In conclusion, sugammadex is an important option for the rapid reversal of rocuronium- or vecuronium-induced neuromuscular blockade.

Molecular characterization and functional analysis of a salmon louse (Lepeophtheirus salmonis, Krøyer 1838) heme peroxidase with a potential role in extracellular matrixes.

PubMed

Øvergård, Aina-Cathrine; Eichner, Christiane; Nilsen, Frank; Dalvin, Sussie

2017-04-01

Heme peroxidases are the most abundant type of peroxidase catalyzing a H 2 O 2 -dependent oxidation of a wide variety of substrates. They are involved in numerous processes like the innate immune response, hormone and prostaglandin synthesis and crosslinking of proteins within extracellular matrixes (ECM) as well as molecules within the cuticle and chorion of arthropods and nematodes. In the present study, a Lepeophtheirus salmonis heme peroxidase (LsHPX) 1 was characterized. Amino acids in the active site of heme peroxidases were conserved, and the predicted protein sequence showed the highest similarity to genes annotated as chorion peroxidases and genes suggested to be involved in cuticle hardening or adhesion. LsHPX1 exhibited a dynamic expression during ontogenesis and during the nauplius molting cycle. Transcripts were localized to muscle cells near the muscle-tendon junction, in nerve tissue especially at neuromuscular junctions, subcuticular epithelium, subepithelial cells facing the hemolymph, exocrine glands within the subepithelial tissue and in isolated cells within the testis. Knock-down of LsHPX1 in nauplius larvae decreased the swimming activity of emerging copepodids. Histological analysis of knock-down animals revealed increased spacing between myofibers and changes in subepithelial and exocrine gland tissue. Considering these results, the potential role of LsHPX1 in crosslinking molecules of salmon louse ECMs is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

Plasticity of the transverse tubules following denervation and subsequent reinnervation in rat slow and fast muscle fibres.

PubMed

Takekura, Hiroaki; Tamaki, Hiroyuki; Nishizawa, Tomie; Kasuga, Norikatsu

2003-01-01

We have studied the effects of short term denervation followed by reinnervation on the ultrastructure of the membrane systems and on the content of and distribution of key proteins involved in calcium regulation of fast-twitch (FT) extensor digitorum longus (EDL) and slow-twitch (ST) soleus (SOL) muscle fibres. Ischiadic nerve freezing resulted in total lack of neuromuscular transmission for 3 days followed by a slow recovery, but no decline in twitch force elicited by direct stimulation. The latter measurements indicate no significant atrophy within this time frame. The membrane systems of skeletal muscle fibres were visualized using Ca92+)-K3Fe(CN)6-OsO4 techniques and observed using a high voltage electron microscope. [3H]nitrendipine binding was used to detect levels of dihydropyridine receptor (DHPR) expression. The Ca2+ pumping free sarcoplasmic reticulum domains were not affected by the denervation, but the Ca2+ release domains were dramatically increased, particularly in the FT-EDL muscle fibres. The increase is evidenced by a doubling up of the areas of contacts between SR and transverse (t-) tubules, so that in place of the normal triadic arrangement, pentadic and heptadic junctions, formed by multiple interacting layers of ST and t-tubules are seen. Frequency of pentads and heptads increases and declines in parallel to the denervation and reinnervation but with a delay. Immunofluorecence and electron microscopy observations show presence of DHPR and ryanodine receptor clusters at pentads and heptads junctions. A significant (P < 0.01) positive correlation between the level of [3H]nitrendipine binding component and the frequency pentads and heptads was observed in both the FT-EDL and ST-SOL muscle fibres indicating that overexpression of DHPRs accompanies the build up extra junctional contacts. The results indicate that denervation reversibly affects the domains of the membrane systems involved in excitation-contraction coupling.

The effects of neuromuscular training on knee joint motor control during sidecutting in female elite soccer and handball players.

PubMed

Zebis, Mette K; Bencke, Jesper; Andersen, Lars L; Døssing, Simon; Alkjaer, Tine; Magnusson, S Peter; Kjaer, Michael; Aagaard, Per

2008-07-01

The project aimed to implement neuromuscular training during a full soccer and handball league season and to experimentally analyze the neuromuscular adaptation mechanisms elicited by this training during a standardized sidecutting maneuver known to be associated with non-contact anterior cruciate ligament (ACL) injury. The players were tested before and after 1 season without implementation of the prophylactic training and subsequently before and after a full season with the implementation of prophylactic training. A total of 12 female elite soccer players and 8 female elite team handball players aged 26 +/- 3 years at the start of the study. The subjects participated in a specific neuromuscular training program previously shown to reduce non-contact ACL injury. Neuromuscular activity at the knee joint, joint angles at the hip and knee, and ground reaction forces were recorded during a sidecutting maneuver. Neuromuscular activity in the prelanding phase was obtained 10 and 50 ms before foot strike on a force plate and at 10 and 50 ms after foot strike on a force plate. Neuromuscular training markedly increased before activity and landing activity electromyography (EMG) of the semitendinosus (P < 0.05), while quadriceps EMG activity remained unchanged. Neuromuscular training increased EMG activity for the medial hamstring muscles, thereby decreasing the risk of dynamic valgus. This observed neuromuscular adaptation during sidecutting could potentially reduce the risk for non-contact ACL injury.

Case Report: Ocular Myasthenia Gravis Associated with In Vitro Fertilization Procedures.

PubMed

Yoo, Yung Ju; Han, Sang Beom; Yang, Hee Kyung; Hwang, Jeong-Min

2018-05-01

Ocular myasthenia gravis is a localized form of myasthenia gravis, which is a postsynaptic disorder of the neuromuscular junction that causes fluctuating weakness of extraocular muscles resulting from autoimmune mechanisms. In women with myasthenia, changes in sex hormone levels and administration of corticosteroids can trigger or worsen symptoms of myasthenia gravis. To describe a case of seronegative ocular myasthenia gravis whose first symptom appeared a day after in vitro fertilization procedure. A 37-year-old woman suddenly developed mild ptosis and fluctuating diplopia that worsened in the evening. Before the development of symptoms, she had undergone in vitro fertilization procedure and had taken oral steroids. Ocular motility examination revealed an intermittent exotropia in primary gaze at both distance and near. The neostigmine test confirmed her diagnosis as ocular myasthenia gravis. When taking a history for young women with sudden onset of binocular diplopia, steroids and sex hormones should be taken into account, which may trigger or exacerbate symptoms of ocular myasthenia gravis.

The neurophysiological effects of dry needling in patients with upper trapezius myofascial trigger points: study protocol of a controlled clinical trial

PubMed Central

Abbaszadeh-Amirdehi, Maryam; Ansari, Noureddin Nakhostin; Naghdi, Soofia; Olyaei, Gholamreza; Nourbakhsh, Mohammad Reza

2013-01-01

Introduction Dry needling (DN) is an effective method for the treatment of myofascial trigger points (MTrPs). There is no report on the neurophysiological effects of DN in patients with MTrPs. The aim of the present study will be to assess the immediate neurophysiological efficacy of deep DN in patients with upper trapezius MTrPs. Methods and analysis A prospective, controlled clinical trial was designed to include patients with upper trapezius MTrPs and volunteered healthy participants to receive one session of DN. The primary outcome measures are neuromuscular junction response and sympathetic skin response. The secondary outcomes are pain intensity and pressure pain threshold. Data will be collected at baseline and immediately after intervention. Ethics and dissemination This study protocol has been approved by the Research Council, School of Rehabilitation and the Ethics Committee of Tehran University of Medical Sciences. The results of the study will be disseminated in a peer-reviewed journal and presented at international congresses. PMID:23793673

Major Histocompatibility Complex I Expression by Motor Neurons and Its Implication in Amyotrophic Lateral Sclerosis

PubMed Central

Nardo, Giovanni; Trolese, Maria Chiara; Bendotti, Caterina

2016-01-01

Neuronal expression of major histocompatibility complex I (MHCI)-related molecules in adults and during CNS diseases is involved in the synaptic plasticity and axonal regeneration with mechanisms either dependent or independent of their immune functions. Motor neurons are highly responsive in triggering the expression of MHCI molecules during normal aging or following insults and diseases, and this has implications in the synaptic controls, axonal regeneration, and neuromuscular junction stability of these neurons. We recently reported that MHCI and immunoproteasome are strongly activated in spinal motor neurons and their peripheral motor axon in a mouse model of familial amyotrophic lateral sclerosis (ALS) during the course of the disease. This response was prominent in ALS mice with slower disease progression in which the axonal structure and function was better preserved than in fast-progressing mice. This review summarizes and discusses our observations in the light of knowledge about the possible role of MHCI in motor neurons providing additional insight into the pathophysiology of ALS. PMID:27379008

Properties of slow- and fast-twitch muscle fibres in a mouse model of amyotrophic lateral sclerosis.

PubMed

Atkin, Julie D; Scott, Rachel L; West, Jan M; Lopes, Elizabeth; Quah, Alvin K J; Cheema, Surindar S

2005-05-01

This investigation was undertaken to determine if there are altered histological, pathological and contractile properties in presymptomatic or endstage diseased muscle fibres from representative slow-twitch and fast-twitch muscles of SOD1 G93A mice in comparison to wildtype mice. In presymptomatic SOD1 G93A mice, there was no detectable peripheral dysfunction, providing evidence that muscle pathology is secondary to motor neuronal dysfunction. At disease endstage however, single muscle fibre contractile analysis demonstrated that fast-twitch muscle fibres and neuromuscular junctions are preferentially affected by amyotrophic lateral sclerosis-induced denervation, being unable to produce the same levels of force when activated by calcium as muscle fibres from their age-matched controls. The levels of transgenic SOD1 expression, aggregation state and activity were also examined in these muscles but there no was no preference for muscle fibre type. Hence, there is no simple correlation between SOD1 protein expression/activity, and muscle fibre type vulnerability in SOD1 G93A mice.

Application of Pyridostigmine in Pediatric Gastrointestinal Motility Disorders: A Case Series.

PubMed

Manini, Mhd Louai; Camilleri, Michael; Grothe, Rayna; Di Lorenzo, Carlo

2018-04-01

Gastrointestinal (GI) motility disorders are common in children. Treatment is challenging with limited medical and surgical options. Pyridostigmine, an acetyl cholinesterase inhibitor, increases acetylcholine at the neuromuscular junction promoting intestinal contractions. Little is known about the role and dosing of pyridostigmine in pediatric GI motility disorders. We present a case series of children with GI dysmotility managed with oral pyridostigmine. Patients' diagnoses include chronic intestinal pseudo-obstruction, gastroparesis with delayed small bowel transit, chronic constipation with failure to thrive, and prolonged ileus after pelvic surgery with chronic opioid use. Pyridostigmine was effective and safe in all cases. Pyridostigmine decreased abdominal distention, increased bowel movement frequency, and improved enteral feeding tolerance. Effective dosing ranged between 0.25-2.0 mg/kg/day. One patient experienced cramping abdominal pain while on pyridostigmine, but pain resolved after medication was discontinued. We found oral pyridostigmine to be helpful in children with different GI motility problems. Pyridostigmine should be considered in such patients when other treatment interventions have not been beneficial.

Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca2+ Channels

PubMed Central

Astorga, César; Jorquera, Ramón A.; Ramírez, Mauricio; Kohler, Andrés; López, Estefanía; Delgado, Ricardo; Córdova, Alex; Olguín, Patricio; Sierralta, Jimena

2016-01-01

The DLG-MAGUK subfamily of proteins plays a role on the recycling and clustering of glutamate receptors (GLUR) at the postsynaptic density. discs-large1 (dlg) is the only DLG-MAGUK gene in Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments we study the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. Our results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. Our results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo. PMID:27573697

Presynaptic DLG regulates synaptic function through the localization of voltage-activated Ca(2+) Channels.

PubMed

Astorga, César; Jorquera, Ramón A; Ramírez, Mauricio; Kohler, Andrés; López, Estefanía; Delgado, Ricardo; Córdova, Alex; Olguín, Patricio; Sierralta, Jimena

2016-08-30

The DLG-MAGUK subfamily of proteins plays a role on the recycling and clustering of glutamate receptors (GLUR) at the postsynaptic density. discs-large1 (dlg) is the only DLG-MAGUK gene in Drosophila and originates two main products, DLGA and DLGS97 which differ by the presence of an L27 domain. Combining electrophysiology, immunostaining and genetic manipulation at the pre and postsynaptic compartments we study the DLG contribution to the basal synaptic-function at the Drosophila larval neuromuscular junction. Our results reveal a specific function of DLGS97 in the regulation of the size of GLUR fields and their subunit composition. Strikingly the absence of any of DLG proteins at the presynaptic terminal disrupts the clustering and localization of the calcium channel DmCa1A subunit (Cacophony), decreases the action potential-evoked release probability and alters short-term plasticity. Our results show for the first time a crucial role of DLG proteins in the presynaptic function in vivo.

Uptake and intracellular fate of cholera toxin subunit b-modified mesoporous silica nanoparticle-supported lipid bilayers (aka protocells) in motoneurons.

PubMed

Gonzalez Porras, Maria A; Durfee, Paul; Giambini, Sebastian; Sieck, Gary C; Brinker, C Jeffrey; Mantilla, Carlos B

2018-04-01

Cholera toxin B (CTB) modified mesoporous silica nanoparticle supported lipid bilayers (CTB-protocells) are a promising, customizable approach for targeting therapeutic cargo to motoneurons. In the present study, the endocytic mechanism and intracellular fate of CTB-protocells in motoneurons were examined to provide information for the development of therapeutic application and cargo delivery. Pharmacological inhibitors elucidated CTB-protocells endocytosis to be dependent on the integrity of lipid rafts and macropinocytosis. Using immunofluorescence techniques, live confocal and transmission electron microscopy, CTB-protocells were primarily found in the cytosol, membrane lipid domains and Golgi. There was no difference in the amount of motoneuron activity dependent uptake of CTB-protocells in neuromuscular junctions, consistent with clathrin activation at the axon terminals during low frequency activity. In conclusion, CTB-protocells uptake is mediated principally by lipid rafts and macropinocytosis. Once internalized, CTB-protocells escape lysosomal degradation, and engage biological pathways that are not readily accessible by untargeted delivery methods. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

NASA Astrophysics Data System (ADS)

Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

2016-04-01

Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

Neurophysiological and clinical effects of dry needling in patients with upper trapezius myofascial trigger points.

PubMed

Abbaszadeh-Amirdehi, Maryam; Ansari, Noureddin Nakhostin; Naghdi, Soofia; Olyaei, Gholamreza; Nourbakhsh, Mohammad Reza

2017-01-01

Dry needling (DN) is a widely used in treatment of myofascial trigger points (MTrPs). The purpose of this pretest-posttest clinical trial was to investigate the neurophysiological and clinical effects of DN in patients with MTrPs. A sample of 20 patients (3 man, 17 women; mean age 31.7 ± 10.8) with upper trapezius MTrPs received one session of deep DN. The outcomes of neuromuscular junction response (NMJR), sympathetic skin response (SSR), pain intensity (PI) and pressure pain threshold (PPT) were measured at baseline and immediately after DN. There were significant improvements in SSR latency and amplitude, pain, and PPT after DN. The NMJR decreased and returned to normal after DN. A single session of DN to the active upper trapezius MTrP was effective in improving pain, PPT, NMJR, and SSR in patients with myofascial trigger points. Further studies are needed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Flaccid quadriplegia due to thyrotoxic myopathy.

PubMed

Couillard, Philippe; Wijdicks, Eelco F M

2014-04-01

Acute flaccid paralysis is an important clinical problem in neurological critical care. After implementing life-supporting measures, it is imperative to identify the correct diagnosis to provide timely appropriate care. Thyrotoxicosis is a recognized cause of myopathy, but rarely of quadriplegia. Here, we report a case of hyperthyroidism with severe weakness. Case report and video demonstration of clinical examination. We describe a case of a 59-year-old woman with Grave's disease who presented to the hospital with progressive shortness of breath secondary to atrial fibrillation with rapid ventricular response. Following contrast administration, she had a pulseless electrical activity arrest from which she recovered without cognitive sequelae, but with flaccid quadriplegia, facial diplegia, and hypophonia. CK was mildly elevated and electrolytes were essentially normal. Nerve conduction studies and electromyography demonstrated features supporting an acute myopathy without evidence of neuromuscular junction conduction abnormality. Normalization of thyroid hormones resulted in slow, but steady improvement over months after which she regained ambulation. Acute flaccid quadriplegia can result from thyrotoxicosis. With normalization of thyroid function, recovery can be expected.

Diffusion and binding constants for acetylcholine derived from the falling phase of miniature endplate currents.

PubMed Central

Land, B R; Harris, W V; Salpeter, E E; Salpeter, M M

1984-01-01

In previous papers we studied the rising phase of a miniature endplate current (MEPC) to derive diffusion and forward rate constants controlling acetylcholine (AcCho) in the intact neuromuscular junction. The present study derives similar values (but with smaller error ranges) for these constants by including experimental results from the falling phase of the MEPC. We find diffusion to be 4 X 10(-6) cm2 s-1, slightly slower than free diffusion, forward binding to be 3.3 X 10(7) M-1 s-1, and the distance from an average release site to the nearest exit from the cleft to be 1.6 micron. We also estimate the back reaction rates. From our values we can accurately describe the shape of MEPCs under different conditions of receptor and esterase concentration. Since we suggest that unbinding is slower than isomerization, we further predict that there should be several short "closing flickers" during the total open time for an AcCho-ligated receptor channel. PMID:6584895

Coexistence of primary biliary cirrhosis and myasthenia gravis: a case study.

PubMed

Horigome, H; Nomura, T; Saso, K; Joh, T; Ohara, H; Akita, S; Sobue, S; Mizuno, Y; Kato, Y; Itoh, M

2000-01-01

We present a case that suggests a relationship between primary biliary cirrhosis and myasthenia gravis. A 43-year-old Japanese woman was admitted to the Nagoya City University Medical School, First Department of Internal Medicine with abnormal liver function in August 1991. She had had ptosis of the right eye since 1990. She had not been treated for liver disease. Ptosis of the right eye and hepatomegaly were present. Serum laboratory examinations revealed elevated biliary enzymes and IgM levels; tests were positive for antimitochondrial antibody and antiacetylcholine antibody. Liver histology revealed chronic non-suppurative destructive cholangitis and led to a diagnosis of primary biliary cirrhosis. The tensilon test was positive. Electromyography with repetitive motor nerve stimulation revealed a neuromuscular junction defect; i.e., the primary characteristic of myasthenia gravis. The patient was diagnosed with myasthenia gravis. Although the development of myasthenia gravis has previously been reported in patients with primary biliary cirrhosis during D-penicillamine administration, this is a very rare case of the coexistence of both diseases before such treatment.

Patterns of Weakness, Classification of Motor Neuron Disease & Clinical Diagnosis of Sporadic ALS

PubMed Central

Statland, Jeffrey M.; Barohn, Richard J.; McVey, April L.; Katz, Jonathan; Dimachkie, Mazen M.

2015-01-01

Synopsis When approaching the patient with suspected motor neuron disease (MND) the pattern of weakness on exam helps distinguish MND from other diseases of peripheral nerves, the neuromuscular junction, or muscle. MND is a clinical diagnosis supported by findings on electrodiagnostic testing, in the absence of other abnormalities on neuroimaging or serological testing. MNDs exist on a spectrum: from a pure lower motor neuron; to mixed upper and lower motor neuron; to a pure upper motor neuron variant in addition to regional variants restricted to the arms, legs or bulbar region. Amyotrophic lateral sclerosis (ALS) is a progressive mixed upper and lower motor neuron disorder, most commonly sporadic (~85%), which is invariably fatal. The only FDA approved treatments for ALS are riluzole, which prolongs life by about 3 months, and dextromethorphan/quinidine which provides symptomatic relief for pseudobulbar affect (inappropriate bouts of laughter or crying). Here we describe a pattern approach to identifying motor neuron disease, and clinical features of sporadic ALS. PMID:26515618