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The new high selective mAChRs M3 inhibitors with IC50 in nanomolecular ranges, which can be the prototypes for effective COPD and asthma treatment drugs, were discovered with computational approaches among trifluoromethyl containing hexahydropyrimidinones/thiones. Compounds [6‐(4‐ethoxy‐3‐methoxy‐phenyl)‐4‐hydroxy‐2‐thioxo‐4‐(trifluoromethyl)hexahydropyrimidin‐5‐yl]‐phenyl‐methanone (THPT‐1) and 5‐benzoyl‐6‐(3,4‐dimethoxyphenyl)‐4‐hydroxy‐4‐(trifluoromethyl)hexahydropyrimidin‐2‐one (THPO‐4) have been proved to be a highly effective (with IC50 values of 1.62 ⋅ 10−7 M and 3.09 ⋅ 10−9 M, respectively) at the same concentrations significantly competitive inhibit the signal conduction through mAChR3 in comparison with ipratropium bromide, without significant effect on mAChR2, nicotinic cholinergic and adrenergic receptors. [ABSTRACT FROM AUTHOR]

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [3H]choline. Electrical stimulation‐evoked pronounced [3H]ACh release from cholinergic neurons. The estimated quantity of [3H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre‐synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M2‐selective antagonist AF‐DX 116, partially inhibited by M3‐selective darifenacin, and minimally by M4‐selective PD 102807. Other subtype‐selective antagonists had no effect at subtype‐selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [3H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre‐synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [3H]efflux was also enhanced by ACh uptake inhibitors (100 μM hemicholinium‐3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre‐synaptic M2/M3/M4mAChRs, AChE and post‐synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/. [ABSTRACT FROM AUTHOR]

Three-finger toxins (TFTs) are well-recognized nonenzymatic venom proteins found in snakes. However, although TFTs exhibit accelerated evolution, the drivers of this evolution remain poorly understood. The structural complexes between long-chain a-neurotoxins, a subfamily of TFTs, and their nicotinic acetylcholine receptor targets have been determined in previous research, providing an opportunity to address such questions. In the current study, we observed several previously identified positively selected sites (PSSs) and the highly variable C-terminal loop of these toxins at the toxin/receptor interface. Of interest, analysis of the molecular adaptation of the toxin-recognition regions in the corresponding receptors provided no statistical evidence for positive selection. However, these regions accumulated abundant amino acid variations in the receptors from the prey of snakes, suggesting that accelerated substitution of TFTs could be a consequence of adaptation to these variations. To the best of our knowledge, this atypical evolution, initially discovered in scorpions, is reported in snake toxins for the first time and may be applicable for the evolution of toxins from other venomous animals. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular β-amyloid protein (Aβ) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aβ caused cognition. It has high affinity with Aβ and could mediate Aβ internalization in vitro . However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aβ internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aβ is internalized by cholinergic and GABAergic neurons. The internalized Aβ were found deposits in lysosomes/endosomes and mitochondria. Aβ could form Aβ-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aβ internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aβ-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aβ. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD. [ABSTRACT FROM AUTHOR]

Acetylcholine is a neuromodulator that shapes information processing in different cortical and subcortical areas. Cell type and location specific cholinergic receptor distributions suggest that acetylcholine in macaque striate cortex should boost feed-forward driven activity, while also reducing population excitability by increasing inhibitory tone. Studies using cholinergic agonists in anesthetized primate V1 have yielded conflicting evidence for such a proposal. Here we investigated how muscarinic or nicotinic receptor blockade affect neuronal excitability and contrast response functions in awake macaque area V1. Muscarinic or nicotinic receptor blockade caused reduced activity for all contrasts tested, without affecting the contrast where neurons reach their halfmaximal response (c50). The activity reduction upon muscarinic and nicotinic blockade resulted in reduced neuronal contrast sensitivity, as assessed through neurometric functions. In the majority of cells receptor blockade was best described by a response gain model (a multiplicative scaling of responses), indicating that ACh is involved in signal enhancement, not saliency filtering in macaque V1. [ABSTRACT FROM AUTHOR]

α-Conotoxins inhibit nicotinic acetylcholine receptors (nAChRs) and are used as probes to study cholinergic pathways in vertebrates. Model organisms, such as Drosophila melanogaster, express nAChRs in their CNS that are suitable to investigate the neuropharmacology of α-conotoxins in vivo . Here we report the paired nanoinjection of native α-conotoxin PIA and two novel α-conotoxins, PIC and PIC[O7], from the injected venom of Conus purpurascens and electrophysiological recordings of their effects on the giant fiber system (GFS) of D. melanogaster and heterologously expressed nAChRs in Xenopus oocytes. α-PIA caused disruption of the function of giant fiber dorsal longitudinal muscle (GF-DLM) pathway by inhibiting the Dα7 nAChR a homolog to the vertebrate α7 nAChR, whereas PIC and PIC[O7] did not. PIC and PIC[O7] reversibly inhibited ACh-evoked currents mediated by vertebrate rodent (r)α1β1δγ, rα1β1δε and human (h)α3β2, but not hα7 nAChR subtypes expressed in Xenopus oocytes with the following selectivity: rα1β1δε > rα1β1δγ ≈ hα3β2 >> hα7. Our study emphasizes the importance of loop size and α-conotoxin sequence specificity for receptor binding. These studies can be used for the evaluation of the neuropharmacology of novel α-conotoxins that can be utilized as molecular probes for diseases such as, Alzheimer's, Parkinson's, and cancer. This article is part of the Special Issue entitled ‘Venom-derived Peptides as Pharmacological Tools.’ [ABSTRACT FROM AUTHOR]

The effects of hypertonic solution on airway smooth muscle ( ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline ( HS) inhibited acetylcholine ( ACh)-induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells ( ASMCs), ACh induced an increase in intracellular Ca2+ that was further enhanced by 5% NaCl, indicating that the HS-induced inhibition of ASM contraction was not mediated by a decrease in cytosolic Ca2+. The Rho-associated kinase ( ROCK) inhibitor Y-27632 relaxed ACh-induced precontraction of mouse tracheal rings. However, such inhibition was not observed after the relaxation induced by 5% NaCl. Moreover, the incubation of mouse tracheal rings with 5% NaCl decreased ACh-induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1. These data indicate that HS inhibits the contraction of ASM by inhibiting Ca2+ sensitization, not by decreasing intracellular Ca2+. [ABSTRACT FROM AUTHOR]

Recent studies show that the frequencies of circulating follicullar helper T (cTfh) cells are significantly higher in myasthenia gravis (MG) patients compared with healthy controls (HC). And, they are positively correlated with levels of serum anti-acetylcholine receptor antibody (anti-AchR Ab). It is unclear whether cTfh cell subset frequencies are altered and what role they play in MG patients. In order to clarify this, we examined the frequencies of cTfh cell counterparts, their subsets, and circulating plasmablasts in MG patients by flow cytometry. We determined the concentrations of serum anti-AChR Ab by enzyme-linked immunosorbent assay (ELISA). We assayed the function of cTfh cell subsets by flow cytometry and real-time polymerase chain reaction (RT-PCR). We found higher frequencies of cTfh cell counterparts, cTfh-Th17 cells, and plasmablasts in MG patients compared with HC. The frequencies of cTfh cell counterparts and cTfh-Th17 cells were positively correlated with the frequencies of plasmablasts and the concentrations of anti-AChR Ab in MG patients. Functional assays showed that activated cTfh-Th17 cells highly expressed key molecular features of Tfh cells including ICOS, PD-1, and IL-21. Results indicate that, just like cTfh cell counterparts, cTfh-Th17 cells may play a role in the immunopathogenesis and the production of anti-AChR Ab of MG. [ABSTRACT FROM AUTHOR]

An array of cytokines influences the pathogenesis of early onset myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG). Patients with MG, in particular those with more severe weakness, have elevations of the pro-inflammatory cytokine IL-17 in the blood. We assessed the role of IL-17A in autoimmunity by inducing EAMG in mice with knockout of IL-17 and found a reduction of EAMG severity, but not a complete ablation of disease. The IL-17 ko mice had no evidence of weakness, low levels of acetylcholine receptor antibodies, and retention of acetylcholine receptor at the neuromuscular junction. Splenic germinal center size was reduced in EAMG IL-17 ko mice along with elevations of Foxp3 and BCL-6 gene expression, suggesting a shift away from pro-inflammatory signals. The results emphasize the importance of IL-17 in EAMG development and that IL-17 independent pathways drive the autoimmune reaction. [ABSTRACT FROM AUTHOR]

Background: Chronic graft-versus-host disease (GVHD) appears several months following allogenic hematopoietic stem cell transplantation (HSCT) and is clinically analogous to autoimmune disorder. Polymyositis is a common neuromuscular disorder in chronic GVHD, but myasthenia gravis (MG) is extremely rare. Hence, its pathophysiology and treatment have not been elucidated.Case Presentation: A 63-year-old man with a history of chronic GVHD presented with ptosis, dropped head, and dyspnea on exertion, which had worsened over the previous several months. He showed progressive decrement of compound muscle action potential in the deltoid muscle evoked by 3-Hz repetitive nerve stimulation, a positive edrophonium test, and elevated levels of serum anti-acetylcholine receptor antibodies, which suggested a diagnosis of generalized MG. No thymoma was found. Flow cytometric analysis revealed a remarkable depletion of peripheral Tregs (CD4+CD25highFOXP3+ cells, 0.24% of the total lymphocytes). Administration of prednisolone and tacrolimus was insufficient to alleviate his symptoms; however, the use of rituximab successfully improved his condition.Conclusions: Myasthenic symptoms appeared in the process of tapering prednisolone for the treatment of chronic GVHD, supporting the diagnosis of MG associated with chronic GVHD. The present case proposes a possibility that reduction of Tregs might contribute to the pathogenesis of MG underlying chronic GVHD. Immunotherapy with rituximab is beneficial for treatment of refractory MG and GVHD. [ABSTRACT FROM AUTHOR]

Experiments on random-bred albino mice showed that M1 muscarinic acetylcholine receptor agonist (TBPB) and α7n-acetylcholine receptor agonist (GTS-21) significantly reduced mortality of mice with experimental sepsis (intraperitoneally administration of E. coli) in 4 and 24 h after modeling by reducing blood concentration of proinflammatory cytokines TNF-α, IL-1β, and IL-6. Combined treatment with TBPB and GTS-21 determined their additive effect. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease, characterized by the loss of memory, multiple cognitive impairments and changes in the personality and behavior. Several decades of intense research have revealed that multiple cellular changes are involved in disease process, including synaptic damage, mitochondrial abnormalities and inflammatory responses, in addition to formation and accumulation of amyloid-β (Aβ) and phosphorylated tau. Although tremendous progress has been made in understanding the impact of neurotransmitters in the progression and pathogenesis of AD, we still do not have a drug molecule associated with neurotransmitter(s) that can delay disease process in elderly individuals and/or restore cognitive functions in AD patients. The purpose of our article is to assess the latest developments in neurotransmitters research using cell and mouse models of AD. We also updated the current status of clinical trials using neurotransmitters' agonists/antagonists in AD. [ABSTRACT FROM AUTHOR]

This retrospective study evaluated the efficiency and tolerance of rituximab in the management of resistant myasthenia gravis (MG). All patients who received rituximab for the treatment of MG between 2004 and 2015 at Pitié-Salpétrière University Hospital (Paris, France) were included. The efficacy of rituximab was evaluated every 6 months by the myasthenic muscle score (MMS), the Myasthenia Gravis Foundation of America – Clinical Classification (MGFA-CC), the MGFA Therapy Status and the Postintervention Status (PIS). All rituximab-related side effects were noted. Twenty-eight patients were included: 21 with anti-acetylcholine receptor antibodies, 3 with anti-muscle-specific tyrosine kinase antibodies and 4 seronegatives. The mean age at day 1 of RTX was 50.6 ± 12.0 years. Patients previously received 1–4 immunosuppressants. The mean follow-up was 27.2 months (range: 6–60 months). The mean total dose of rituximab was 4.8 ± 2.5 g. The initial median MMS (58.8 points) improved significantly at M6 (74.5 ± 15.0 points; p < 0.0001) and remained stable thereafter: at M12: 75.9 ± 14.0 points (p = 0.00014), at M36: 72.5 ± 13.1 points (p = 0.0013). Among 16 patients with initial severe symptoms (MGFA-CC class IV), 14 improved. The PIS showed efficacy in about 50% of patients: at M6, 12/28 (43%) patients were considered improved. This benefit remained stable thereafter: at M12: 12/24, at M24: 7/17, at M36: 6/12. One patient developed a delayed progressive multifocal leukoencephalopathy. Based on the PIS, rituximab may be efficient in 50% of patients with MG resistant to immunosuppressants. [ABSTRACT FROM AUTHOR]

Key points Phasic release of acetylcholine (ACh) in the neocortex facilitates attentional processes., Acting at a single metabotropic receptor subtype, ACh exerts two opposing actions in cortical pyramidal neurons: transient inhibition and longer-lasting excitation., Cholinergic inhibitory responses depend on calcium release from intracellular calcium stores, and run down rapidly at resting membrane potentials when calcium stores become depleted., We demonstrate that cholinergic excitation promotes calcium entry at subthreshold membrane potentials to rapidly refill calcium stores, thereby maintaining the fidelity of inhibitory cholinergic signalling., We propose a 'unifying hypothesis' for M1 receptor signalling whereby inhibitory and excitatory responses to ACh in pyramidal neurons represent complementary mechanisms governing rapid calcium cycling between the endoplasmic reticulum, the cytosol and the extracellular space., Abstract Gq-coupled M1-type muscarinic acetylcholine (ACh) receptors (mAChRs) mediate two distinct electrophysiological responses in cortical pyramidal neurons: transient inhibition driven by calcium-dependent small conductance potassium ('SK') channels, and longer-lasting and voltage-dependent excitation involving non-specific cation channels. Here we examine the interaction of these two cholinergic responses with respect to their contributions to intracellular calcium dynamics, testing the 'unifying hypothesis' that rundown of inhibitory SK responses at resting membrane potentials (RMPs) reflects depletion of intracellular calcium stores, while mAChR-driven excitation acts to refill those stores by promoting voltage-dependent entry of extracellular calcium. We report that fidelity of cholinergic SK responses requires the continued presence of extracellular calcium. Inhibitory responses that diminished after repetitive ACh application at RMPs were immediately rescued by pairing mAChR stimulation with subthreshold depolarization (∼10 mV from RMPs) initiated with variable delay (up to 500 ms) after ACh application, but not by subthreshold depolarization preceding mAChR stimulation. Further, rescued SK responses were time-locked to ACh application, rather than to the timing of subsequent depolarizing steps, suggesting that cholinergic signal transduction itself is not voltage-sensitive, but that depolarization facilitates rapid cycling of extracellular calcium through the endoplasmic reticulum to activate SK channels. Consistent with this prediction, rescue of SK responses by subthreshold depolarization required the presence of extracellular calcium. Our results demonstrate that, in addition to gating calcium release from intracellular stores, mAChR activation facilitates voltage-dependent refilling of calcium stores, thereby maintaining the ongoing fidelity of SK-mediated inhibition in response to phasic release of ACh. [ABSTRACT FROM AUTHOR]

Fishes of north-temperate latitudes exhibit marked seasonal changes in electrical excitability of the heart partly as an outcome of temperature-dependent changes in the density of major K ion currents: delayed rectifiers (I, I) and background inward rectifier (I). In the arctic teleost, navaga cod ( Eleginus navaga), I and I are strongly up-regulated in winter. The current study tests the hypothesis that the ligand-gated K current, the acetylcholine-activated inward rectifier, I, is also modified by seasonal acclimatization in atrial myocytes of navaga. In sinoatrial preparations of the summer-acclimatized (SA) navaga, 10 M carbamylcholine chloride (CCh) caused slowing of heart rate, shortening of atrial action potential (AP) duration and a drastic reduction of AP amplitude, eventually resulting in inexcitability. In winter-acclimatized (WA) atria CCh slowed HR and reduced AP duration, but reduction of AP amplitude was modest and never resulted in inexcitability. The difference in cholinergic response between SA and WA navaga is explained by seasonal changes in I density. The peak density of I, induced by 10 M CCh, at the common experimental temperature (+6 °C) was 0.97 ± 0.28 pA/pF in SA navaga but only 0.183 ± 0.013 pA/pF in WA navaga (a 5.3-fold difference, P < 0.05). At acclimatization temperatures of the fish I density was 2.8 ± 0.50 (at +12 °C) and 0.11 ± 0.06 pA/pF (at +3 °C) (a 26-fold difference, P < 0.05) for SA and WA navaga, respectively. Thus, acclimatization to summer induces a drastic up-regulation of the atrial I, which effectively shortens atrial AP. The reverse temperature compensation of the atrial I may be advantageous in summer under variable water temperatures and oxygen concentrations by reducing workload of the heart. [ABSTRACT FROM AUTHOR]

Background and purpose A single, oral dose of 3 mg/day tacrolimus, approved for myasthenia gravis ( MG) treatment in Japan, was shown to reduce steroid dose and anti-acetylcholine receptor ( AChR) antibody titers as well as to improve MG symptoms. However, no studies have investigated the association between tacrolimus concentration and its clinical efficacy in MG. In this study, we aimed to determine the optimal tacrolimus concentration for MG treatment. Methods The trough tacrolimus concentration in 51 patients with MG (positive for anti- AChR antibody, n = 48; negative for anti- AChR and anti-muscle-specific tyrosine kinase antibodies, n = 3) who received 3 mg/day tacrolimus for more than 1 year was measured using a chemiluminescent enzyme immunoassay. The clinical characteristics of patients with MG as well as the dose of prednisolone used before and after tacrolimus treatment were evaluated retrospectively. Results The median trough tacrolimus concentration was 5.4 (range, 2.9-7.6) ng/mL, which was correlated with 'minimal manifestation or better status' ( P = 0.0190, r = 0.3273) and the reduction in anti- AChR antibody 1 year after tacrolimus initiation ( P = 0.0170, r = 0.3465). When the cut-off value for tacrolimus was defined as 4.8 ng/mL using a receiver operating characteristic curve, patients with adequate tacrolimus concentration (≥4.8 ng/mL) showed more reduction in anti- AChR antibody titers and more improvement in MG-related activities in daily life scores. More patients with adequate tacrolimus concentration achieved 'minimal manifestation or better status' compared with those with low tacrolimus concentration. Conclusions An adequate tacrolimus concentration is required for better MG prognosis. [ABSTRACT FROM AUTHOR]

Developmental nicotine exposure (DNE) is associated with increased risk of cardiorespiratory, intellectual, and behavioral abnormalities in neonates, and is a risk factor for apnea of prematurity, altered arousal responses and Sudden Infant Death Syndrome. Alterations in nicotinic acetylcholine receptor signaling (nAChRs) after DNE lead to changes in excitatory neurotransmission in neural networks that control breathing, including a heightened excitatory response to AMPA microinjection into the hypoglossal motor nucleus. Here, we report on experiments designed to probe possible postsynaptic and presynaptic mechanisms that may underlie this plasticity. Pregnant dams were exposed to nicotine or saline via an osmotic mini-pump implanted on the 5th day of gestation. We used whole-cell patch clamp electrophysiology to record from hypoglossal motoneurons (XIIMNs) in thick medullary slices from neonatal rat pups ( N = 26 control and 24 DNE cells). To enable the translation of our findings to breathing-related consequences of DNE, we only studied XIIMNs that were receiving rhythmic excitatory drive from the respiratory central pattern generator. Tetrodotoxin was used to isolate XIIMNs from presynaptic input, and their postsynaptic responses to bath application of l -glutamic acid (glutamate) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were studied under voltage clamp. DNE had no influence on inward current magnitude evoked by either glutamate or AMPA. However, in cells from DNE animals, bath application of AMPA was associated with a right shift in the amplitude distribution ( P = 0.0004), but no change in the inter-event interval distribution of miniature excitatory postsynaptic currents (mEPSCs). DNE had no influence on mEPSC amplitude or frequency evoked by glutamate application, or under (unstimulated) baseline conditions. Thus, in the presence of AMPA, DNE is associated with a small but significant increase in quantal size, but no change in the probability of glutamate release. [ABSTRACT FROM AUTHOR]

The article discusses myasthenia gravis, an autoimmune disease, in which antibodies bind to acetylcholine receptors and induce weakness of skeletal muscles that include eye muscles with diplopia and ptosis. It offers information on the diagnostic tests used in detecting the disease including neurophysiological test, ice-pack test and supplementary antibody test. It also highlights updated treatment algorithms and individualized therapy based on biomarkers.

Background: Congenital myasthenic syndromes (CMSs) occur as a result of genetic mutations that cause aberrations in structure and/or function of proteins involved in neuromuscular transmission. Acetylcholine receptor epsilon (ε) subunit (CHRNE) gene mutations account for about 30-50 % of genetically diagnosed cases. We report a rare CHRNE gene mutation in a South Asian female with CMS. Case presentation: A 17-year-old Maldivian female presented with bilateral partial ptosis, fatigable proximal muscle weakness and slurring of speech noted since the age of 2 years. She could not run, had difficulty negotiating stairs and rising from a seated position, and fatigues when speaking at length. Her birth and past medical histories were otherwise unremarkable. There is no parental consanguinity or family history of muscle disorders. On examination, she had a BMI of 18 kg/m2, bilateral fatigable partial ptosis, complete external ophthalmoplegia and fatigable proximal muscle weakness (MRC grade 4/5). Apart from spinal scoliosis the rest of the examination was normal. Haematological and biochemical investigations including serum lactate level and thyroid functions were normal. Acetylcholine receptor antibodies and muscle specific kinase antibodies were not detected in serum. Repetitive nerve stimulation showed marked decrement (>30 %) in nerve-muscle pairs in the face and forearm. Her DNA sequencing revealed a c.183-187dupCTCAC mutation in CHRNE. She remained functionally independent on pyridostigmine treatment. Conclusions: This case describes a rare mutation of the CHRNE gene in CMS and highlights the relevance of genetic diagnosis in CMS. It further adds to map the occurrence of such mutations in Asian populations. [ABSTRACT FROM AUTHOR]

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/ oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·μm-2 human muscarinic M1 receptor identified a ~75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/ oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior. [ABSTRACT FROM AUTHOR]

Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulateβ2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation ofα4* nAChRs, complementing that of chronic nicotine alone, which upregulatesα4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescentnAChRsubunits, exposure to 500 nMmenthol alone also increasednAChRnumber and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunitcontaining nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway. [ABSTRACT FROM AUTHOR]

The author discusses the roles of factors from perivascular tissue in regulation of vascular tone. He states the generation of systemic circulation through sympathetic innervation, circulating hormones and adaptation to transmural pressure and recognition of perivascular tissue (PVT) to regulate tone development through the release of soluble factors, and augmentation of acetylcholine-induced nitric oxide (NO) by endothelium-dependent relaxations.

In recent years, the discovery of new autoantigens and the use of sensitive assays have expanded the clinical spectrum of myasthenia gravis (MG). In particular, antibodies binding to clustered acetylcholine receptors and to the low-density lipoprotein receptor-related protein 4 have not only bridged a significant gap in diagnosis but also have relevant clinical implications. MG management includes different therapeutic options, from symptomatic agents as the only therapy in mildly affected cases to combined long-term immunosuppression and thymectomy in patients with severe disabling disease. MG biological diversity can influence the response to therapies and should be taken into account when planning treatment. Biologic agents are promising, though their use is currently limited to patients with refractory disease. [ABSTRACT FROM PUBLISHER]

Alpha 7 subunits of nicotinic acetylcholine receptors (nAChRs) are expressed in microglia and astrocyte and are involved in the suppression of neuroinflammation. Over the past decade, many reports showbeneficial effects of nicotine. Here we review the function of nAChR in glial cells and how nicotine attenuates neuronal death induced by activated microglia. One is Ca2+-dependent action. Nicotine causes transienct increase in intracellular Ca2+ in phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)-dependent manner, and negatively modulates LPS-induced release of pro-inflammatory cytokines. The seconed is adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent action. Nicotine may attenuate NOX and subsequently inhibiting acidosis, therefore inhibit activation of H+ channel in activated microglia. The inhibitory effect of nicotine on H+ current is due to the activation of α7 nAChR. On the other hand, nicotine does not affect increased expression of H+ channel, HVCN1, in activated microglia. Especially the phagocytic NOX (NOX2) is the best studied ROS-generating system and is mainly expressed in microglia in the CNS. Since a role of NOX2 in Alzheimer's and cerebrovascular disease and other neurological diseases relating to smoking is implicated, inhibiting NOX2 and consecutive acidosis and activation of H+ channel in microglia may have a therapeutic potential in neuroinflammatory diseases. [ABSTRACT FROM AUTHOR]

The author reflects on the mechanism of modal gating in adult mouse endplate acetylcholine receptor (AChR) channels in reference to research paper by A. Auerbach and colleagues. Topics discussed include requirement of presynaptic release of acetylcholine by the neuromuscular transmission in mammals and its impact on skeletal muscles, several nebulous mechanisms for stable modal gating, and mutation-induced structural changes for binding pocket results in modal gating in AChRs.

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability ("modes"). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5--β5' linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity. [ABSTRACT FROM AUTHOR]

Genetic and functional studies have revealed that both common and rare variants of several nicotinic acetylcholine receptor subunits are associated with nicotine dependence (ND). In this study, we identified variants in 30 candidate genes including nicotinic receptors in 200 sib pairs selected from the Mid-South Tobacco Family population with equal numbers of African Americans (AAs) and European Americans (EAs). We selected 135 of the rare and common variants and genotyped them in the Mid-South Tobacco Case-Control (MSTCC) population, which consists of 3088 AAs and 1430 EAs. None of the genotyped common variants showed significant association with smoking status (smokers vs non-smokers), Fagerström Test for ND scores or indexed cigarettes per day after Bonferroni correction. Rare variants in NRXN1, CHRNA9, CHRNA2, NTRK2, GABBR2, GRIN3A, DNM1, NRXN2, NRXN3 and ARRB2 were significantly associated with smoking status in the MSTCC AA sample, with weighted sum statistic (WSS) P-values ranging from 2.42 × 10−3 to 1.31 × 10−4 after 106 phenotype rearrangements. We also observed a significant excess of rare nonsynonymous variants exclusive to EA smokers in NRXN1, CHRNA9, TAS2R38, GRIN3A, DBH, ANKK1/DRD2, NRXN3 and CDH13 with WSS P-values between 3.5 × 10−5 and 1 × 10−6. Variants rs142807401 (A432T) and rs139982841 (A452V) in CHRNA9 and variants V132L, V389L, rs34755188 (R480H) and rs75981117 (N549S) in GRIN3A are of particular interest because they are found in both the AA and EA samples. A significant aggregate contribution of rare and common coding variants in CHRNA9 to the risk for ND (SKAT-C: P=0.0012) was detected by applying the combined sum test in MSTCC EAs. Together, our results indicate that rare variants alone or combined with common variants in a subset of 30 biological candidate genes contribute substantially to the risk of ND. [ABSTRACT FROM AUTHOR]

The mesolimbic dopamine (DA) system is known to play a role in cue-mediated reward-seeking for natural rewards and drugs of abuse. Specifically, cholinergic and glutamatergic receptors in the ventral tegmental area (VTA) have been shown to regulate cue-induced drug-seeking. However, the potential role of these VTA receptors in regulating cue-induced reward seeking for natural rewards is unknown. Here, we examined whether blockade of VTA acetylcholine receptors (AChRs) and N-methyl- d -aspartate receptors (NMDARs) would alter cue-induced sucrose seeking in male Sprague-Dawley rats. Subjects underwent 10 days of sucrose self-administration training (fixed ratio 1 schedule) followed by 7 days of forced abstinence. On withdrawal day 7, rats received bilateral VTA infusion of vehicle, the muscarinic AChR antagonist scopolamine (2.4 or 24 μg/side), the nicotinic AChR antagonist mecamylamine (3 or 30 μg/side), or the NMDAR antagonist AP-5 (0.1 or 1 μg/side) immediately prior to examination of cue-induced sucrose-seeking. Scopolamine infusion led to robust attenuation, but did not completely block, sucrose-seeking behavior. In contrast, VTA administration of mecamylamine or AP-5 did not alter cue-induced sucrose-seeking. Together, the data suggest that VTA muscarinic AChRs, but not nicotinic AChRs nor NMDARs, facilitate the ability of food-associated cues to drive seeking behavior for a food reward. [ABSTRACT FROM AUTHOR]

Nicotinic acetylcholine receptors (nAChRs) play an important role in the modulation of many cognitive functions but their role in integrated network activity remains unclear. This is at least partly because of the complexity of the cholinergic circuitry and the difficulty in comparing results from in vivo studies obtained under diverse experimental conditions and types of anesthetics. Hence the role of nAChRs in the synchronization of cortical activity during slow-wave sleep is still controversial, with some studies showing they are involved in ACh-dependent EEG desynchronization, and others suggesting that this effect is mediated exclusively by muscarinic receptors. Here we use an in vitro model of endogenous network activity, in the form of recurring self-maintained depolarized states (Up states), which allows us to examine the role of high-affinity nAChRs on network dynamics in a simpler form of the cortical microcircuit. We find that mice lacking nAChRs containing the β2-subunit (β2-nAChRs) have longer and more frequent Up states, and that this difference is eliminated when β2-nAChRs in wild-type mice are blocked. We further show that endogenously released ACh can modulate Up/Down states through the activation of both β2- and α7-containing nAChRs, but through distinct mechanisms: α7-nAChRs affect only the termination of spontaneous Up states, while β2-nAChRs also regulate their generation. Finally we provide evidence that the effects of β2-subunit-containing, but not α7-subunit-containing nAChRs, are mediated through GABAB receptors. To our knowledge this is the first study documenting direct nicotinic modulation of Up/Down state activity. [ABSTRACT FROM AUTHOR]

We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two wellresolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent. [ABSTRACT FROM AUTHOR]

Resistin is a pro-inflammatory cytokine involved in the pathogenesis of autoimmune diseases. To investigate serum resistin levels in patients with myasthenia gravis (MG) and determine if there are associations between resistin levels and disease severity, we measured serum resistin levels in 102 patients with anti-acetylcholine receptor antibody-positive MG (AChR-MG). We further analyzed associations between serum resistin levels and clinical variables in patients with MG. Our findings demonstrate that serum resistin levels are elevated in patients with AChR-generalized MG and AChR-MG with thymoma and are correlated with disease severity. Resistin has potential as a useful serum biomarker for inflammation in AChR-MG. [ABSTRACT FROM AUTHOR]

In the current research work, rivastigmine (RV)-loaded in situ gelling nanostructured lipid carriers (NLCs) were developed for nose to brain delivery. NLCs were fabricated by ethanol injection method using glyceryl monosterate, Capmul MCM C8, Lecithin and Tween 80. NLCs showed average particle size of 123.2 ± 2.3 nm with entrapment efficiency of 68.34 ± 3.4%. DSC, XRD and IR studies showed complete amorphization and incorporation of the drug into nanoparticles. NLCs were incorporated into an in situ gelling system using 0.8% gellan gum and 15% Lutrol F 127. RV in situ gel showed excellent elasticity, rheology, mucoadhesion and adhesiveness to facilitate its adhesion to the upper nasal mucosa. NLC-based in situ gel showed a 2-fold increase in nasal permeation of the drug over plain RV solution, in situ gelling NLCs showed a 3-fold increase in enzyme inhibition efficacy. [ABSTRACT FROM AUTHOR]

Purpose: The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. The syntheses and C-11 labeling of two potent enantiopure VAChT inhibitors are reported here. Procedures: Two VAChT inhibitors, (±)- 2 and (±)- 6, were successfully synthesized. A chiral HPLC column was used to resolve the enantiomers from each corresponding racemic mixture for in vitro characterization. The radiosyntheses of (−)-[C] 2 and (−)-[C] 6 from the corresponding desmethyl phenol precursor was accomplished using [C]methyl iodide or [C]methyl triflate, respectively. Results: The synthesis of (−)-[C] 2 was accomplished with 40-50 % radiochemical yield (decay-corrected), SA > 480 GBq/μmol (EOB), and radiochemical purity >99 %. Synthesis of (−)-[C] 6 was accomplished with 5-10 % yield, SA > 140 GBq/μmol (EOB), and radiochemical purity >97 %. The radiosynthesis and dose formulation of each tracer was completed in 55-60 min. Conclusions: Two potent enantiopure VAChT ligands were synthesized and C-labeled with good radiochemical yield and specific activity. [ABSTRACT FROM AUTHOR]

Purpose: The vesicular acetylcholine transporter (VAChT) is a specific biomarker for imaging presynaptic cholinergic neurons. Herein, two potent and selective C-labeled VAChT inhibitors were evaluated in rodents and nonhuman primates for imaging VAChT in vivo. Procedures: For both (−)-[C] 2 and (−)-[C] 6, biodistribution, autoradiography, and metabolism studies were performed in male Sprague Dawley rats. Positron emission tomography (PET) brain studies with (−)-[C] 2 were performed in adult male cynomolgus macaques; 2 h dynamic data was acquired, and the regions of interest were drawn by co-registration of the PET images with the MRI. Results: The resolved enantiomers (−)- 2 and (−)- 6 were very potent and selective for VAChT in vitro ( K < 5 nM for VAChT with >35-fold selectivity for VAChT vs. σ receptors); both radioligands, (−)-[C] 2 and (−)-[C] 6, demonstrated high accumulation in the VAChT-enriched striatum of rats. (−)-[C] 2 had a higher striatum to cerebellum ratio of 2.4-fold at 60 min; at 30 min, striatal uptake reached 0.550 ± 0.086 %ID/g. Uptake was also specific and selective; following pretreatment with (±)- 2, striatal uptake of (−)-[C] 2 in rats at 30 min decreased by 50 %, while pretreatment with a potent sigma ligand had no significant effect on striatal uptake in rats. In addition, (−)-[C] 2 displayed favorable in vivo stability in rat blood and brain. PET studies of (−)-[C] 2 in nonhuman primates indicate that it readily crosses the blood-brain barrier (BBB) and provides clear visualization of the striatum; striatal uptake reaches the maximum at 60 min, at which time the target to nontarget ratio reached ~2-fold. Conclusions: The radioligand (−)-[C] 2 has high potential to be a suitable PET radioligand for imaging VAChT in the brain of living subjects. [ABSTRACT FROM AUTHOR]

The thymus plays a primary role in early-onset Myasthenia Gravis (MG) mediated by anti-acetylcholine receptor (AChR) antibodies. As we recently showed an inflammatory and anti-viral signature in MG thymuses, we investigated in detail the contribution of interferon (IFN)-I and IFN-III subtypes in thymic changes associated with MG. We showed that IFN-I and IFN-III subtypes, but especially IFN-β, induced specifically α-AChR expression in thymic epithelial cells (TECs). We also demonstrated that IFN-β increased TEC death and the uptake of TEC proteins by dendritic cells. In parallel, we showed that IFN-β increased the expression of the chemokines CXCL13 and CCL21 by TECs and lymphatic endothelial cells, respectively. These two chemokines are involved in germinal center (GC) development and overexpressed in MG thymus with follicular hyperplasia. We also demonstrated that the B-cell activating factor (BAFF), which favors autoreactive B-cells, was overexpressed by TECs in MG thymus and was also induced by IFN-β in TEC cultures. Some of IFN-β effects were down-regulated when cell cultures were treated with glucocorticoids, a treatment widely used in MG patients that decreases the number of thymic GCs. Similar changes were observed in vivo. The injections of Poly(I:C) to C57BL/6 mice triggered a thymic overexpression of IFN-β and IFN-α2 associated with increased expressions of CXCL13, CCL21, BAFF, and favored the recruitment of B cells. These changes were not observed in the thymus of IFN-I receptor KO mice injected with Poly(I:C), even if IFN-β and IFN-α2 were overexpressed. Altogether, these results demonstrate that IFN-β could play a central role in thymic events leading to MG by triggering the overexpression of α-AChR probably leading to thymic DC autosensitization, the abnormal recruitment of peripheral cells and GC formation. [ABSTRACT FROM AUTHOR]

Myasthenia gravis (MG) is an autoimmune disease mainly mediated by anti-acetylcholine receptor (AChR) antibodies. In the late onset, a thymoma, tumor of the thymus, is quite frequent. However, the events leading to thymoma and MG are not understood. As thymoma-associated MG (MG-T) patients also display anti-interferon type I (IFN-I) neutralizing antibodies, we investigated if MG-T could be associated with an anti-viral signature. RT-PCR analyses demonstrated huge increases of IFN-I subtypes, IFN-α2, -α8, -ω and -β, in thymoma-associated MG but not in thymomas without MG or in control thymuses. Next, we investigated if dsRNA signaling pathway involvement could be observed in MG-T, as recently observed in early-onset MG. We observed an abnormal regulation of dsRNA-sensing molecules with an increase of toll-like receptor 3 (TLR3), and a decrease of protein kinase R (PKR) and dsRNA helicases (RIG-I and MDA5) in thymoma from MG patients. We also detected a decreased expression of p53, the tumor suppressor that is known to be down-regulated by dsRNA. Altogether, these results strongly suggest that MG-T could be linked to a viral infection. As p16 (CDKN2A), a marker of HPV infections, was up-regulated in MG-T, we thus screened DNA from thymomas for human papillomavirus (HPV) by real-time PCR using HPV consensus SPF10 primers. RT-PCR results were negative for all samples tested. We confirmed the absence of HPV DNA detection by end point PCR using FAP primers to amplify a larger panel of HPV genotypes. Our data clearly demonstrate INF-I overexpression together with the activation of innate immunity pathways in thymoma-associated MG suggesting that MG might develop after a pathogen infection. We were not able to relate thymoma to HPV infections and the implication of other pathogens is discussed. [ABSTRACT FROM AUTHOR]

The nicotinic acetylcholine receptor (nAChR) and the acetylcholine binding protein (AChBP) are pentameric oligomers in which binding sites for nicotinic agonists and competitive antagonists are found at selected subunit interfaces. The nAChR spontaneously exists in multiple conformations associated with its activation and de-sensitization steps, and conformations are selectively stabilized by binding of agonists and antagonists. In the nAChR, agonist binding and the associated conformational changes accompanying activation and desensitization are cooperative. AChBP, which lacks the transmembrane spanning and cytoplasmic domains, serves as a homology model of the extracellular domain of the nAChRs. We identified unique cooperative binding behavior of a number of 4,6-disubstituted 2-aminopyrimidines to Lymnaea AChBP, with different molecular variants exhibiting positive, nH > 1.0, and negative cooperativity, nH < 1.0. Therefore, for a distinctive set of ligands, the extracellular domain of a nAChR surrogate suffices to accommodate cooperative interactions. X-ray crystal structures of AChBP complexes with examples of each allowed the identification of structural features in the ligands that confer differences in cooperative behavior. Both sets of molecules bind at the agonist-antagonist site, as expected from their competition with epi-batidine. An analysis of AChBP quaternary structure shows that cooperative ligand binding is associated with a blooming or flare conformation, a structural change not observed with the classical, noncooperative, nicotinic ligands. Positively and negatively cooperative ligands exhibited unique features in the detailed binding determinants and poses of the complexes. [ABSTRACT FROM AUTHOR]

Airway mucus hypersecretion is a significant clinical and pathological feature of chronic inflammatory airway diseases. Its clinical presentations include recurrent coughing and phlegm. Airway mucus is closely associated with the occurrence, development and prognosis of chronic inflammatory airway diseases and critically affects the lung function, quality of life, hospitalization rate and mortality of patients with chronic inflammatory airway diseases. Therefore, expectorant therapies targeting the potential mechanisms of mucus hypersecretion have been the focus of numerous studies. Conventional expectorants are mainly mucoactive medicines, including nausea-stimulating expectorants, mucolytics, mucokinetics, and proteases and nucleases. In addition, certain traditional Chinese herbal medicines and non-mucoactive agents, including muscarinic acetylcholine receptor antagonists, corticosteroids, leukotriene receptor antagonists and macrolide antibiotics, have also shown expectorant effects. Several novel medicines for expectorant therapy have emerged, including cholesterol-lowering statins, epidermal growth factor receptor tyrosine kinase inhibitors, phosphodiesterase-4 inhibitors, stanozolol, surfactants, flavonoids, tachykinin receptor antagonists, protease inhibitors, cytokine antagonists and purinergic agonists. With the increasing number of multidisciplinary studies, the effectiveness of expectorant therapy for the treatment of chronic inflammatory airway diseases has been confirmed. Therefore, the development of novel expectorants and the standardization of expectorant therapy are the direction and focus of future studies, thus benefiting patients who have a chronic inflammatory airway disease. [ABSTRACT FROM AUTHOR]

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ. [ABSTRACT FROM AUTHOR]

Objective: To observe the effect of pregnenolone (PREG) intervention on the cholinergic system function and the synaptic protein 1 (SYP1) expression in different brain regions of aged rats. Method: Twenty-four-month-old male Sprague Dawley rats intraperitoneally injected every other day for one month were divided into blank control group, solvent control group, PREG (0.5 mg/kg) intervention group and PREG (2.0 mg/kg) intervention group. The rats were sacrificed 2 d after the intervention and the corresponding regions of brain tissue were separated and cryopreserved. Western blot analysis was used to detect the expression level of choline acetyltransferase (ChAT), SYP1, serum PREG and the activity of ChAT and acetylcholinesterase (AChE) in different brain regions. In addition, the semiquantitative changes in the expression level of ChAT and SYP1 in frontal lobe and hippocampus were tested by immunohistochemistry. Result: Western blot and immunohistochemistry analysis showed that PREG (2.0 mg/kg) administration led to a significant increase of ChAT and SYP1 expressions in frontal lobe, temporal lobe, and hippocampus regions ( p < 0.05). The result of enzyme-linked immunosorbent assay showed that PREG (2.0 mg/kg) administration significantly increased ChAT activity and serum PREG levels and caused a decrease in AChE activity ( p < 0.05); while PREG (0.5 mg/kg) only elevated levels of serum PREG. Conclusion: PREG significantly improved the synaptic plasticity of memory-related brain areas of aged rats, significantly increased brain cholinergic activity and thus helps to improve learning and memory in aged rats. [ABSTRACT FROM AUTHOR]

Abstract: Botulinum neurotoxin A (BoNT/A) induces muscle paralysis by enzymatically cleaving the presynaptic SNARE protein SNAP-25, which results in lasting inhibition of acetylcholine release at the neuromuscular junction. A rapid and sensitive in vitro assay for BoNT/A is required to replace the mouse lethality assay (LD50) in current use. We have developed a fully automated sensor to assay the endoprotease activity of BoNT/A. We produced monoclonal antibodies (mAbs) that recognize SNAP-25 neo-epitopes specifically generated by BoNT/A action. Recombinant SNAP-25 was coupled to the sensor surface of a surface plasmon resonance (SPR) system and samples containing BoNT/A were injected over the substrate sensor. Online substrate cleavage was monitored by measuring binding of mAb10F12 to a SNAP-25 neo-epitope. The SNAP-25-chip assay was toxin serotype-specific and detected 55fM BoNT/A (1 LD50/ml) in 5min and 0.4fM (0.01 LD50/ml) in 5h. Time-course and dose-response curves were linear, yielding a limit of quantification of 0.03 LD50/ml. This label-free method is 100 times more sensitive than the mouse assay, potentially providing rapid read-out of small amounts of toxin for environmental surveillance and the quality control of pharmaceutical preparations. [Copyright &y& Elsevier]

Highlights: [•] Arecoline inhibits the formation of agrin-induced AChR clusters in C2C12 myotubes. [•] Arecoline destabilizes agrin-induced and spontaneous AChR clusters in C2C12 myotubes. [•] Arecoline inhibits the expression of myogenin in C2C12 myotubes. [ABSTRACT FROM AUTHOR]

Oscillations in the coordinated firing of brain neurons have been proposed to play important roles in perception, cognition, attention, learning, navigation, and sensory-motor control. The network theta rhythm has been associated with properties of spatial navigation, as has the firing of entorhinal grid cells and hippocampal place cells. Two recent studies reduced the theta rhythm by inactivating the medial septum (MS) and demonstrated a correlated reduction in the characteristic hexagonal spatial firing patterns of grid cells. These results, along with properties of intrinsic membrane potential oscillations (MPOs) in slice preparations of medial entorhinal cortex (MEC), have been interpreted to support oscillatory interference models of grid cell firing. The current article shows that an alternative self-organizing map (SOM) model of grid cells can explain these data about intrinsic and network oscillations without invoking oscillatory interference. In particular, the adverse effects of MS inactivation on grid cells can be understood in terms of how the concomitant reduction in cholinergic inputs may increase the conductances of leak potassium (K+) and slow and medium after-hyperpolarization (sAHP and mAHP) channels. This alternative model can also explain data that are problematic for oscillatory interference models, including how knockout of the HCN1 gene in mice, which flattens the dorsoventral gradient in MPO frequency and resonance frequency, does not affect the development of the grid cell dorsoventral gradient of spatial scales, and how hexagonal grid firing fields in bats can occur even in the absence of theta band modulation. These results demonstrate how models of grid cell self-organization can provide new insights into the relationship between brain learning and oscillatory dynamics. [ABSTRACT FROM AUTHOR]

Abstract: Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875 – fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms. [Copyright &y& Elsevier]

Impaired learning and memory performance is often found in aging as an early sign of dementia. It is associated with neuronal loss and reduced functioning of cholinergic networks. Here we present evidence that the neurokinin3 receptors (NK3-R) and their influence on acetylcholine (ACh) release may represent a crucial mechanism that underlies age-related deficits in learning and memory. Repeated pharmacological stimulation of NK3-R in aged rats was found to improve learning in the water maze and in object-place recognition. This treatment also enhanced in vivo acetylcholinergic activity in the frontal cortex, hippocampus, and amygdala but reduced NK3-R mRNA expression in the hippocampus. Furthermore, NK3-R agonism incurred a significantly higher increase in ACh levels in aged animals that showed superior learning than in those that were most deficient in learning. Our findings suggest that the induced activation of ACh, rather than basal ACh activity, is associated with superior learning in the aged. To test whether natural variation in NK3-R function also determines learning and memory performance in aged humans, we investigated 209 elderly patients with cognitive impairments. We found that of the 15 analyzed single single-nucleotide ploymorphism (SNPs) of the NK3-R-coding gene, TACR3, the rs2765 SNP predicted the degree of impairment of learning and memory in these patients. This relationship could be partially explained by a reduced right hippocampus volume in a subsample of 111 tested dementia patients. These data indicate the NK3-R as an important target to predict and improve learning and memory performance in the aged organism. [ABSTRACT FROM AUTHOR]

Abstract: Anatoxin-a is one of the common and major cyanobacterial neurotoxins acting as a powerful agonist at nicotinic acetylcholine receptors (nAChR). In recent years, the toxin has become the focus of public attention, due to the mass development of cyanobacteria (cyanobacterial blooms) in freshwater bodies triggered by eutrophication and climate change. Anatoxin-a is suspected to have a distinct toxic mechanism depending on physiological and nervous systems in exposed organisms. The numerous researches have been actively conducted with respect to the toxic effects of anatoxin-a on mammals; however, little research has aimed at its possible effects on aquatic plants, wherein well-structured nervous system is absent with the lack of various components of the acetylcholine mechanism. In this study, submerged macrophyte Ceratophyllum demersum (C. demersum) was adopted to examine the effects of anatoxin-a on morphological (growth), physiological (photosynthetic pigment contents) and biochemical (hydrogen peroxide level, biotransformation and antioxidative enzymes) responses in the aquatic plant at environmentally relevant concentrations (0.005, 0.05, 0.5, 5 and 50 μg/L). The significant elevation of antioxidative enzymes in parallel with increased formation of hydrogen peroxide appeared from 0.5 μg/L of anatoxin-a. In the measurement of photosynthetic pigments, the decrease in chlorophyll a content was detected at 5 and 50 μg/L, whereas the increase in carotenoids/total chlorophyll was observed from 0.05 μg/L. Accordingly, the alteration in growth was manifested in the presence of 5 and 50 μg/L of anatoxin-a. The results clearly indicate that anatoxin-a can disrupt homeostasis of C. demersum through induction of oxidative stress; furthermore this aquatic plant possesses effective defense mechanisms to cope with low concentrations of anatoxin-a. [Copyright &y& Elsevier]

The objectives of the study are to study the clinical features of myasthenia gravis in southern China. A retrospective study was carried out on all patients who were diagnosed with myasthenia gravis at the First Affiliated Hospital of Sun Yat-sen University during 1987-2009. Of the 2,154 myasthenia gravis patients, the gender ratio (male:female) was 1:1.15. The median age at onset was 18 years. There was a single peak distribution of age at onset, and 44.8 % were children (≤14 years) at first onset. 1,766 patients (82.0 %) only had ocular symptoms at onset. 1,451 patients (67.4 %) were classified as Osserman grade I. 250 unselected patients received anti-acetylcholine receptor antibodies test, in which only 51.2 % were positive. Computed tomography scan/magnetic resonance Imaging of chest were done in 1,354 patients, of which 899 patients (66.4 %) had thymic hyperplasia and 201(14.8 %) had thymoma. There were 150 patients (7.0 %) with myasthenia gravis combined with other autoimmune diseases, in which hyperthyroidism was most common (84 %). 189 (8.8 %) patients experienced 267 episodes of crisis. The rate of family myasthenia gravis was 1.6 % (35/2,154). In conclusion, the clinical features and demography of myasthenia gravis patients in this study are significantly different from prior studies on other regions and ethnic groups. [ABSTRACT FROM AUTHOR]

Anti-acetylcholine receptor antibodies (anti-AChR-Ab) are responsible for the failure of neuromuscular junction in myasthenia gravis (MG). Some anti-AChR-Ab-seronegative MG patients have anti-muscle-specific tyrosine kinase antibodies (anti-MuSk-Ab). Here, the anti-AChR-Ab was tested in 250 MG outpatients from Southern China. While anti-MuSk-Ab was tested in 66 patients who had no anti-AChR-Ab in blood serum, but none of them was positive. The antibodies were measured by a radioimmunoprecipitation assay. The frequency of anti-AChR-Ab was 51.2 %. The percentage of anti-AChR-Ab in ocular type was lower than generalized type (44.9 vs. 66.2 %, P = 0.002). Seronegative MG was characterized by a lower percentage of thymoma than seropositive patients ( P = 0.013). It seemed to be less severe in seronegative MG than seropositive MG in these 250 patients. In ocular type, seronegative MG mainly manifesting blepharoptosis but seldom diplopia or eyeball fixation related to ocular movement disability ( P = 0.016). While in generalized type, seronegative MG was characterized by a lower percentage of bulbar muscle involvements than seropositive patients ( P = 0.005). Logistic regression analysis revealed that bulbar weakness was affected by the existence of anti-AChR antibodies (OR = 3.524, P = 0.015). Besides, seronegative MG tended to be characterized by a lower percentage of neck extensor involvement, but this did not reach significance. The percentage of anti-AChR antibodies was much lower than other countries. Seronegative MG has characteristic clinical features that are different from features of the remaining seropositive MG. This emphasises the predictive value of anti-AChR antibodies analysis in MG patients. [ABSTRACT FROM AUTHOR]

Abstract: Kynurenic acid (KYNA), a major tryptophan metabolite, is a glutamate receptor antagonist, which is also reported to inhibit α7 nicotinic acetylcholine receptors (α7nAChRs). Due to variations in experimental approaches, controversy has arisen regarding the ability of KYNA to directly influence α7nAChR function. Here we summarize current concepts of KYNA neurobiology and review evidence pertaining to the proposed role of KYNA as an endogenous modulator of α7nAChRs and synaptic transmission. As dysfunction of α7nAChRs plays a major role in the pathophysiology of central nervous system disorders, elucidation of KYNA''s action on this receptor subtype has significant therapeutic implications. [Copyright &y& Elsevier]

The synaptic adhesion molecules neurexin and neuroligin alter the development and function of synapses and are linked to autism in humans. Here, we found that Caenorhabditis elegans neurexin (NRX-1) and neuroligin (NLG-1) mediated a retrograde synaptic signal that inhibited 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 presynaptic levels of tomosyn, an inhibitor of SV fusion. [ABSTRACT FROM AUTHOR]