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In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.

Rationale The cholinergic system has long been linked to cognitive processes. Two main classes of acetylcholine (ACh) receptors exist in the human brain, namely muscarinic and nicotinic receptors, of which several subtypes occur. Objectives This review seeks to provide an overview of previous findings on the influence of cholinergic receptor manipulations on cognition in animals and humans, with particular emphasis on the role of selected cholinergic receptor subtypes. Furthermore, the involvement of these receptor subtypes in the regulation of emotion and brain electrical activity as measured by electroencephalography (EEG) shall be addressed since these domains are considered to be important modulators of cognitive functioning. Results In regard to cognition, the muscarinic receptor subtypes have been implicated mainly in memory functions, but have also been linked to attentional processes. The nicotinic α7 receptor subtype is involved in working memory, whereas the α4β2* subtype has been linked to tests of attention. Both muscarinic and nicotinic cholinergic mechanisms play a role in modulating brain electrical activity. Nicotinic receptors have been strongly associated with the modulation of depression and anxiety. Conclusions Cholinergic receptor manipulations have an effect on cognition, emotion, and brain electrical activity as measured by EEG. Changes in cognition can result from direct cholinergic receptor manipulation or from cholinergically induced changes in vigilance or affective state.

The Drosophila melanogaster heart is a popular model in which to study cardiac physiology and development. Progress has been made in understanding the role of endogenous compounds in regulating cardiac function in this model. It is well characterized that common neurotransmitters act on many peripheral and non-neuronal tissues as they flow through the hemolymph of insects. Many of these neuromodulators, including acetylcholine (ACh), have been shown to act directly on the D. melanogaster larval heart. ACh is a primary neurotransmitter in the central nervous system (CNS) of vertebrates and at the neuromuscular junctions on skeletal and cardiac tissue. In insects, ACh is the primary excitatory neurotransmitter of sensory neurons and is also prominent in the CNS. A full understanding regarding the regulation of the Drosophila cardiac physiology by the cholinergic system remains poorly understood. Here we use semi-intact D. melanogaster larvae to study the pharmacological profile of cholinergic receptor subtypes, nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), in modulating heart rate (HR). Cholinergic receptor agonists, nicotine and muscarine both increase HR, while nAChR agonist clothianidin exhibits no significant effect when exposed to an open preparation at concentrations as low as 100 nM. In addition, both nAChR and mAChR antagonists increase HR as well but also display capabilities of blocking agonist actions. These results provide evidence that both of these receptor subtypes display functional significance in regulating the larval heart’s pacemaker activity.

In vivo , synaptic receptor densities were maintained over minutes by a rapid exchange with nonsynaptic receptor pools and over hours through turnover. These changes and receptor dynamics may represent the initial phases of synaptic efficacy modulation before eventual structural modification involving spine growth or retraction. Synaptic plasticity underlies the adaptability of the mammalian brain, but has been difficult to study in living animals. Here we imaged the synapses between pre- and postganglionic neurons in the mouse submandibular ganglion in vivo , focusing on the mechanisms that maintain and regulate neurotransmitter receptor density at postsynaptic sites. Normally, synaptic receptor densities were maintained by rapid exchange of receptors with nonsynaptic regions (over minutes) and by continual turnover of cell surface receptors (over hours). However, after ganglion cell axons were crushed, synaptic receptors showed greater lateral mobility and there was a precipitous decline in insertion. These changes led to near-complete loss of synaptic receptors and synaptic depression. Disappearance of postsynaptic spines and presynaptic terminals followed this acute synaptic depression. Therefore, neurotransmitter receptor dynamism associated with rapid changes in synaptic efficacy precedes long-lasting structural changes in synaptic connectivity.

Here we review recent studies on the mode of action of the cholinergic anthelmintics (levamisole, pyrantel etc.). We also include material from studies on the free living nematode Caenorhabditis elegans. The initial notion that these drugs act on a single receptor population, while attractive, has proven to be an oversimplification. In both free living and parasitic nematodes there are multiple types of nicotinic acetylcholine receptor (nAChR) on the somatic musculature. Each type has different (sometimes subtly so) pharmacological properties. The implications of these findings are: (1) combinations of anthelmintic that preferentially activate a broad range of nAChR types would be predicted to be more effective; (2) in resistant isolates of parasite where a subtype has been lost, other cholinergic anthelmintics may remain effective. Not only are there multiple types of nAChR, but relatively recent research has shown these receptors can be modulated; it is possible to increase the response of a parasite to a fixed concentration of drug by altering the receptor properties (e.g. phosphorylation state). These findings offer a potential means of increasing efficacy of existing compounds as an alternative to the costly and time consuming development of new anthelmintic agents.

Acetylcholine receptors at innervated neuromuscular junctions are very stable, with half-lives reported to be 6 to 13 days. Their turnover is described as a first-order process, implying a single population of receptors. In this study, two subpopulations of acetylcholine receptors at normally innervated junctions have been identified. One has a rapid turnover rate with a half-life of 18.7 hours, similar to that of extrajunctional receptors, and the other has a slow turnover rate with a half-life of 12.4 days. The rapidly turned over subpopulation represents approximately 20 percent of the total junctional receptors. This finding may account for the discrepancies in previous reports of turnover rates and may explain the rapid reversibility in vivo of agents that ``irreversibly'' block acetylcholine receptors. This finding also implies that the synthesis rate of junctional acetylcholine receptors may be higher than previous estimates. The rapidly turned-over subpopulation may represent receptors that were newly inserted into the neuromuscular junction and that were not yet stabilized by an influence of the motor nerve.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a 'model hopping' approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

Myasthenia gravis is an autoimmune disease that is characterised by muscle weakness and fatigue, is B-cell mediated, and is associated with antibodies directed against the acetylcholine receptor, muscle-specific kinase (MUSK), lipoprotein-related protein 4 (LRP4), or agrin in the postsynaptic membrane at the neuromuscular junction. Patients with myasthenia gravis should be classified into subgroups to help with therapeutic decisions and prognosis. Subgroups based on serum antibodies and clinical features include early-onset, late-onset, thymoma, MUSK, LRP4, antibody-negative, and ocular forms of myasthenia gravis. Agrin-associated myasthenia gravis might emerge as a new entity. The prognosis is good with optimum symptomatic, immunosuppressive, and supportive treatment. Pyridostigmine is the preferred symptomatic treatment, and for patients who do not adequately respond to symptomatic therapy, corticosteroids, azathioprine, and thymectomy are first-line immunosuppressive treatments. Additional immunomodulatory drugs are emerging, but therapeutic decisions are hampered by the scarcity of controlled studies. Long-term drug treatment is essential for most patients and must be tailored to the particular form of myasthenia gravis.

Abstract Background Myasthenia gravis (MG) is categorized into several subgroups, including seronegative MG. Seronegative human patients are well documented, but seronegative dogs remain clinically uncharacterized and their prevalence unknown. Objectives This study aims to evaluate the clinical presentation, diagnosis, treatment, and outcome of canine MG subgroups. Animals One hundred sixty-seven owner-owned dogs diagnosed with MG from three referral centers. Methods Retrospective case series. We classified myasthenic dogs into subgroups, adhering to human guidelines. Results We classified 167 dogs into four subgroups: acetylcholine receptor (AChR) antibody-positive generalized (49.7%, n = 83/167), focal (19.2%, n = 32/167) and thymoma-associated MG (9%, n = 15/167) and seronegative MG (22.2%, n = 37/167). Dogs with thymoma-associated MG were older (median 102 months; Interquartile Range (IQR) 96–120; p < 0.001) and seronegative dogs were younger (median 30 months; IQR 11.5–66; p = 0.017), compared to the generalized subgroup (median 67 months; IQR 36–96). Seronegative dogs presented less frequently with megaesophagus, compared to the generalized subgroup (63.8% vs. 85.7%; Odds Ratio 3.4; 95% confidence intervals (C.I.) 1.4–8.9; p = 0.025). Myasthenic dogs' survival time was significantly reduced when thymoma (Hazard Ratio (H.R.) 3.7; 95% C.I. 1.4–9.9; p = 0.028) or esophageal weakness (H.R. 3.8; 95% C.I. 2.0–7.0; p < 0.001) was present. Conversely, a higher likelihood of remission was achieved when esophageal weakness was absent (H.R. 3.8; 95% C.I. 1.4–10.0; p = 0.007). Conclusion and Clinical Importance Dogs with seronegative MG are more common than previously reported. Myasthenic subgroups differ in presentation and outcome, with esophageal weakness key to survival and remission. Diagnostic tests for seronegative dogs and effective treatments for esophageal weakness in myasthenic dogs are urgently needed.

ObjectiveTo investigate the association between changes in anti-acetylcholine receptor antibody (AChR Ab) levels induced by immunosuppressive treatment and myasthenia gravis (MG) prognosis at 1-year post-treatment in patients with MG.MethodsWe included 53 consecutive AChR Ab-positive patients with MG whose AChR Ab levels were remeasured within 100 days of initiating immunosuppressive treatment (median remeasuring time post-treatment: 71 (55–84) days). The AChR Ab level reduction rate (RR-AChRAb, %/day) adjusted for the time between treatment initiation, and AChR Ab level remeasurement was calculated as follows: (pretreatment–post-treatment AChR Ab level)/pretreatment AChR Ab level/days between therapy initiation and AChR Ab level remeasurement ×100. Participants were divided into two groups based on the cut-off value of RR-AChR Ab, determined using receiver operating characteristic analyses for achieving minimal manifestation (MM) or better status at 1-year postimmunosuppressive treatment. The Myasthenia Gravis Foundation of America postintervention status and MG activity of daily living (MG-ADL) score at 1-year post-treatment were compared between the two groups.ResultsThe RR-AChRAb cut-off value was 0.64%/day. The high RR-AChRAb group had a higher ratio of MM or better status (90% vs 65%, p=0.03) and lower MG-ADL score (median; 1 vs 2, p=0.04) than the low RR-AChRAb group. Kaplan-Meier analyses showed the early MM achievement in the high RR-AChRAb group (p=0.002, log-rank test).ConclusionsHigh RR-AChRAb is associated with a favourable outcome at 1-year post-treatment. AChR Ab remeasurement within 100 days of therapy may be useful for predicting AChR Ab-positive MG outcomes at 1-year post-treatment.