Acetylcholine Receptor Function Research Articles

B-094 Assessment of a Live Cell-based Flow Cytometry Assay for MuSK-IgG4 Testing in Myasthenia Gravis

Abstract Background Myasthenia gravis (MG) is caused by disrupting the function of postsynaptic acetylcholine receptors (AChRs) at the neuromuscular junction. 85% of MG patients have autoantibodies targeting AChRs, while 6% of MG patients have autoantibodies targeting the muscle-specific receptor tyrosine kinase (MuSK). MuSK is located on the muscle membrane and is activated by the low-density lipoprotein receptor-related protein 4 (LRP4) upon the binding with ligand Agrin. Activated MuSK undergoes autophosphorylation and stimulates downstream signaling to cluster AChRs, which is important for the formation and maintenance of neuromuscular synapses. IgG4 antibodies that bind MuSK are thought to directly disrupt the ability of MuSK to cluster AChR. The gold standard methodology for MuSK antibody testing is radioimmunoprecipitation assay (RIA) using anti-human IgG secondary antibodies. Performing RIA methods in the clinical laboratory is a burden due to radioactive biohazard concerns and the analytical limitations of these assays. This study is to assess the analytical and clinical performance of a live cell-based flow cytometry assay as a potential replacement for the MuSK RIA. Methods HEK293T cells were transiently transfected with a vector co-expressing human MuSK protein and GFP. Patient sera were incubated with a mix of HEK293 cells (GFP-positive cells that express MuSK protein on the cell surface and GFP-negative cells that do no express MuSK) followed by incubation with anti-human IgG4 secondary antibody conjugated with alexa fluor 647 (AF647). An IgG binding index (IBI) was calculated for each sample as the ratio of the median AF647 fluorescence intensity (MFI) of the MuSK-expressing cells (GFP+) to the MFI of the non-MuSK expressing cells (GFP-). An arbitrary positive IBI cut-off of 2.0 was selected. A preliminary assessment of assay imprecision was performed for the flow cytometry assay, as well as a method comparison study to compare this assay to RIA utilizing a cohort of 130 (34 positive and 96 negative) residual samples previously tested by the gold-standard assay. 133 disease control samples were also tested to assess the clinical specificity of the flow cytometry-based assay. Results To assess the imprecision, eight samples with varying IBIs were tested with 5 replicates in each assay over five days. The coefficient of variation (CV) for intra-assay replicates ranged from 1% to 7% for all samples, while CV for inter-assay replicates ranged from 2% to 15%. Based on the 130 samples previously tested by RIA, the Positive Percentage Agreement (PPA) between these two assays was 76.5% and the negative Percentage Agreement (NPA) was 100%. Of the discordant samples (n=8), clinical information was available for only 1 sample (positive by RIA, negative by flow). This patient had a final non-MG diagnosis (ALS). All control samples were negative on the flow cytometry-based assay. Conclusions The live cell-based flow cytometry MuSK IgG4 assay demonstrated encouraging preliminary analytical performance that warrants further development and validation.

Therapeutic Potential of 1-(2-Chlorophenyl)-6,7-dimethoxy-3-methyl-3,4-dihydroisoquinoline.

The synthesized compound 1-(2-chlorophenyl) 6-7-dimethoxy-3-methyl-3,4-dihydroisoquinoline (DIQ) was investigated as a biological agent. Its potential to affect muscle contractility was predicted through in silico PASS analysis. Based on the in silico analysis, its capabilities were experimentally investigated. The study aimed to investigate the effects of DIQ on the ex vivo spontaneous contractile activity (CA) of smooth muscle (SM) tissue. DIQ was observed to reduce the strength of Ca2+-dependent contractions in SM preparations (SMP), possibly by increasing cytosolic Ca2+ levels through the activation of a voltage-gated L-type Ca2+ channel. DIQ potently affected calcium currents by modulating the function of muscarinic acetylcholine receptors (mAChRs) and 5-hydroxytryptamine (5-HT) receptors at a concentration of 50 μM. Immunohistochemical tests showed a 47% reduction in 5-HT2A and 5-HT2B receptor activity in SM cells and neurons in the myenteric plexus (MP), further confirming the effects of DIQ. Furthermore, a significant inhibition of neuronal activity was observed when the compound was co-administered with 5-HT to SM tissues. The conducted experiments confirm the ability of the isoquinoline analog to act as a physiologically active molecule to control muscle contractility and related physiological processes.

The human-specific nicotinic receptor subunit CHRFAM7A reduces α7 receptor function in human induced pluripotent stem cells-derived and transgenic mouse neurons.

We investigated the impact of the human-specific gene CHRFAM7A on the function of α7 nicotinic acetylcholine receptors (α7 nAChRs) in two different types of neurons: human-induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. dupα7, the gene product of CHRFAM7A, which lacks a major part of the extracellular N-terminal ligand-binding domain, co-assembles with α7, the gene product of CHRNA7. We assessed the receptor function in hiPSC-derived cortical and SCG neurons with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists PNU282987 and choline with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously, we demonstrate that CHRFAM7A has a major impact on nicotinic α7 nAChRs by reducing calcium transients in response to all three agonists.

Advances in Understanding and Managing Myasthenia Gravis: Current Trends and Future Directions.

Myasthenia gravis (MG) is an autoimmune illness characterized by autoantibodies against the acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), and an increasing number of extra postsynaptic proteins. Pathogenic autoantibodies reduce the number of functional AChRs in the neuromuscular junction's (NMJ)muscle end plate. The cause of the autoimmune response is unknown, but thymic abnormalities and immune regulatory deficiencies are significant. The disease's incidence is likely influenced by genetic predisposition, with sex hormones and exercise playing a role. MGcan affect any age, race, or gender and can be caused by any stressor, with infections being the most frequent cause. Treatment focuses on airway support and the triggering incident. MG is a rare autoimmune disease causing fatigue-inducing weakness in the axial, respiratory, leg, and bulbar muscles. Initially affecting the eyes, most MGpatients experience at least one worsening symptom during their illness. The disease is mainly caused by antibodies against the AChR, dependence on the immune system within cells, and engagement of the complement system. The complement system plays a significant role in MG, and complement inhibition can both prevent the onset and slow its development. Ocular MG affects around 15% of people, with most patients having blocking antibodies against the cholinergic receptor. There may be correlations between thymoma and other autoimmune conditions, especially thyroid illness. Treatment and management for MGinvolve removing autoantibodies from circulation or blocking effector mechanisms using techniques such as complement inhibition, plasmapheresis, and B-cell elimination.

The role of α7 nicotinic acetylcholine receptors in post-acute sequelae of covid-19

Post-Acute Sequelae of COVID-19 or Long COVID becomes evident some weeks to months following acute COVID-19. Symptoms include cognitive impairment and varying degrees of memory loss with no definitive etiologies or efficacious therapies forthcoming even after four years of the SARS-Cov2 pandemic virus. The aim of this review is to demonstrate the important role of α7 nicotinic acetylcholine receptors in both acute COVID-19 and Long COVID. Evidence presented implicates immune mechanisms stimulated by SARS-Cov-2 S-protein fragment 674–685 that possesses homology with α7-specific ligands. Cognitive dysfunctions observed in Long COVID patients may be derived from anti-idiotypic α7-specific antibodies stimulated by (674−685)-specific antibodies. Therapeutic interventions capable of neutralizing these antibodies and restoring full functions of α7 nicotinic acetylcholine receptors appear to be of paramount importance in post-acute sequelae of COVID-19.

Extreme Paralysis Following Rocuronium Administration in a Myasthenia Gravis Patient: A Case Report.

The use of paralytics during rapid sequence intubation (RSI) in patients with myasthenia gravis (MG) remains a controversial topic in emergency medicine. Due to fewer functional acetylcholine receptors, these patients can be both sensitive and resistant to different types of neuromuscular blocking agents (NMBA). Their atypical sensitivity to non-depolarizing NMBAs such as rocuronium can increase both the duration and depth of paralysis after its use at typical RSI doses. However, the extent of rocuronium's prolonged duration of effect in patients with MG has yet to be quantified in an emergency department setting. We describe a case wherein a full RSI dose of 1.2 milligrams per kilogram of rocuronium led to a prolonged 232-minute duration of paralysis in a patient with MG. This sustained paralysis was suspected but only confirmed after the patient received the reversal agent sugammadex. Once administered, an acute change in neurologic function was seen, and the patient was emergently taken to the operating room for neurosurgical intervention. When intubating patients with MG, many emergency physicians are aware that using paralytics during RSI provides several challenges. If not properly dose-reduced, rocuronium may exert its paralytic effects for up to four hours in patients with MG. This unique case highlights the importance of personalizing care for this patient population before, during, and after RSI.

Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology

The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (− 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.Graphical abstract

Modelling organophosphate intoxication in C. elegans highlights nicotinic acetylcholine receptor determinants that mitigate poisoning.

Organophosphate intoxication via acetylcholinesterase inhibition executes neurotoxicity via hyper stimulation of acetylcholine receptors. Here, we use the organophosphate paraoxon-ethyl to treat C. elegans and use its impact on pharyngeal pumping as a bio-assay to model poisoning through these neurotoxins. This assay provides a tractable measure of acetylcholine receptor mediated contraction of body wall muscle. Investigation of the time dependence of organophosphate treatment and the genetic determinants of the drug-induced inhibition of pumping highlight mitigating modulation of the effects of paraoxon-ethyl. We identified mutants that reduce acetylcholine receptor function protect against the consequence of intoxication by organophosphates. Data suggests that reorganization of cholinergic signalling is associated with organophosphate poisoning. This reinforces the under investigated potential of using therapeutic approaches which target a modulation of nicotinic acetylcholine receptor function to treat the poisoning effects of this important class of neurotoxins.

Muscle-type nicotinic acetylcholine receptor structure, function, and pharmacology.

Nicotinic acetylcholine receptors mediate fast chemical transmission throughout the central and peripheral nervous systems. The nicotinic receptor subtype found on the motor endplate at the neuromuscular junction has been an important model system for understanding receptor structure, function, signaling, and pharmacology. The nicotinic receptor found in the electric organs of fish, including the Torpedo ray, is homologous to the human muscle-type nicotinic receptor and serves as an abundant natural source for biochemical and structural studies. Here we leverage the Torpedo nicotinic receptor in structural biology and electrophysiological studies to understand how clinically relevant drug classes regulate receptor conformation and activity. The findings reveal how different types of functional antagonists stabilize distinct non-conducting states to inhibit neuromuscular transmission.

Acetylcholine Receptors in Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are well known for their regenerative potential. Even though the ability of MSCs to proliferate and differentiate has been studied extensively, there remains much to learn about the signaling mechanisms and pathways that control proliferation and influence the differentiation phenotype. In recent years, there has been growing evidence for the utility of non-neuronal cholinergic signaling systems and that acetylcholine (ACh) plays an important ubiquitous role in cell-to-cell communication. Indeed, cholinergic signaling is hypothesized to occur in stem cells and ACh synthesis, as well as in ACh receptor (AChR) expression, has been identified in several stem cell populations, including MSCs. Furthermore, AChRs have been found to influence MSC regenerative potential. In humans, there are two major classes of AChRs, muscarinic AChRs and nicotinic AChRs, with each class possessing several subtypes or subunits. In this review, the expression and function of AChRs in different types of MSC are summarized with the aim of highlighting how AChRs play a pivotal role in regulating MSC regenerative function.

B cell-specific mAb–siRNA conjugates improve experimental myasthenia

Myasthenia gravis (MG) is a debilitating autoimmune disease characterized by muscle fatigue and weakness caused by autoantibody- and complement-mediated damage to the neuromuscular junction. This study sought to compare the efficacy of unique sets of monoclonal antibody–siRNA conjugates, individually (mono) or in combination (duo), against the crucial receptors predominantly or solely expressed on two subsets of B cells—plasma B cells and their precursor (transitional mature B) cells in a mouse model of MG. At the optimized doses, the conjugates, likely due to the combined activities of mAb and siRNA, substantially decreased the expression levels of CD268 (B cell-activating factor receptor) in mature B cells and CD269 (B-cell maturation antigen) in plasma cells concomitantly with reducing the levels of acetylcholine receptor (AChR)-specific autoantibodies. PEGylation, but not pretreatment with an antibody against type 1 interferon receptor, further improved duoconjugate-induced reduction in the autoantibody levels. Our results show that the duoconjugate treatment significantly improved the clinical symptoms of MG, consistent with the preservation of bungarotoxin-bound functional AChRs. In the future, developing similar target-specific combination molecules can potentially turn into a new and effective therapeutic approach for MG.

GluR2Q and GluR2R AMPA Subunits are not Targets of lypd2 Interaction.

A large family of prototoxin-like molecules endogenous to mammals, Ly6 proteins have been implicated in the regulation of cell signaling processes across multiple species. Previous work has shown that certain members of the Ly6 family are expressed in the brain and target nicotinic acetylcholine receptor and potassium channel function. Structural similarities between Ly6 proteins and alpha-neurotoxins suggest the possibility of additional ionotropic receptor targets. Here, we investigated the possibility of lypd2 as a novel regulator of AMPA receptor (AMPAR) function. In particular, we focused on potential interactions with the Q/R isoforms of the GluR2 subunit, which have profound impacts on AMPAR permeability to calcium during neuronal stimulation. We find that although lypd2 and GluR2 share overlapping expression patterns in the mouse hippocampus, there was no interaction between lypd2 and either GluR2Q or GluR2R isoform. These results underscore the importance of continuing to investigate novel targets for Ly6 interaction and regulation.

Hedgehog-interacting protein acts in the habenula to regulate nicotine intake

Hedgehog-interacting protein (HHIP) sequesters Hedgehog ligands to repress Smoothened (SMO)-mediated recruitment of the GLI family of transcription factors. Allelic variation in HHIP confers risk of chronic obstructive pulmonary disease and other smoking-related lung diseases, but underlying mechanisms are unclear. Using single-cell and cell-type-specific translational profiling, we show that HHIP expression is highly enriched in medial habenula (MHb) neurons, particularly MHb cholinergic neurons that regulate aversive behavioral responses to nicotine. HHIP deficiency dysregulated the expression of genes involved in cholinergic signaling in the MHb and disrupted the function of nicotinic acetylcholine receptors (nAChRs) through a PTCH-1/cholesterol-dependent mechanism. Further, CRISPR/Cas9-mediated genomic cleavage of the Hhip gene in MHb neurons enhanced the motivational properties of nicotine in mice. These findings suggest that HHIP influences vulnerability to smoking-related lung diseases in part by regulating the actions of nicotine on habenular aversion circuits.

The transforming growth factor beta ligand TIG-2 modulates the function of neuromuscular junction and muscle energy metabolism in Caenorhabditis elegans.

Deciphering the physiological function of TGF-β (the transforming growth factor beta) family ligands is import for understanding the role of TGF-β in animals' development and aging. Here, we investigate the function of TIG-2, one of the ligands in Caenorhabditis elegans TGF-β family, in animals' behavioral modulation. Our results show that a loss-of-function mutation in tig-2 gene result in slower locomotion speed in the early adulthood and an increased density of cholinergic synapses, but a decreased neurotransmitter release at neuromuscular junctions (NMJs). Further tissue-specific rescue results reveal that neuronal and intestinal TIG-2 are essential for the formation of cholinergic synapses at NMJs. Interestingly, tig-2(ok3416) mutant is characterized with reduced muscle mitochondria content and adenosine triphosphate (ATP) production, although the function of muscle acetylcholine receptors and the morphology muscle fibers in the mutant are comparable to that in wild-type animals. Our result suggests that TIG-2 from different neuron and intestine regulates worm locomotion by modulating synaptogenesis and neurotransmission at NMJs, as well as energy metabolism in postsynaptic muscle cells.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR.Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Systemic injection of nicotinic acetylcholine receptor antagonist mecamylamine affects licking, eyelid size, and locomotor and autonomic activities but not temporal prediction in male mice

Nicotinic acetylcholine receptors are thought to be associated with a wide range of phenomena, such as movement, learning, memory, attention, and addiction. However, the causal relationship between nicotinic receptor activity and behavior remains unclear. Contrary to the studies that examined the functions of muscarinic acetylcholine receptors, the role of the nicotinic acetylcholine receptors on behavior has not been examined as extensively. Here, we examined the effects of intraperitoneal injection of mecamylamine, a nicotinic acetylcholine receptor antagonist, on the performance of male mice in a head-fixed temporal conditioning task and a free-moving open-field task. The head-fixed experimental setup allowed us to record and precisely quantify the licking response while the mice performed the behavioral task with no external cues. In addition, by combining the utility of the head-fixed experimental design with computer vision analysis based on deep learning algorithms, we succeeded in quantifying the eyelid size of awake mice. In the temporal conditioning task, we delivered a 10% sucrose solution every 10 s using a blunt-tipped needle placed within the licking distance of the mice. After the training, the mice showed increased anticipatory licking toward the timing of sucrose delivery, suggesting that the mice could predict the timing of the reward. Systemic injection of mecamylamine decreased licking behavior and caused eye closure but had no effect on learned conditioned predictive behavior in the head-fixed temporal conditioning task. In addition, the injection of mecamylamine decreased spontaneous locomotor activity in a dose-dependent manner in the free-moving open-field task. The results in the open-field experiments further revealed that the effect of mecamylamine on fecal output and urination, suggesting the effects on autonomic activities. Our achievement of successful eyelid size recording has potential as a useful approach in initial screening for drug discovery. Our study paves a way forward to understanding the role of nicotinic acetylcholine receptors on learning and behavior.

The M3 Muscarinic Acetylcholine Receptor Promotes Epidermal Differentiation

The M3 muscarinic acetylcholine receptor is predominantly expressed in the basal epidermal layer where it mediates the effects of the autocrine/paracrine cytotransmitter acetylcholine. Patients with the autoimmune blistering disease pemphigus develop autoantibodies to M3 muscarinic acetylcholine receptor and show alterations in keratinocyte adhesion, proliferation, and differentiation, suggesting that M3 muscarinic acetylcholine receptor controls these cellular functions. Chmr3-/- mice display altered epidermal morphology resembling that seen in patients with pemphigus vulgaris. In this study, we characterized the cellular and molecular mechanisms through which M3 muscarinic acetylcholine receptor controls epidermal structure and function. We used single-cell RNA sequencing to evaluate keratinocyte heterogeneity and identify differentially expressed genes in specific subpopulations of epidermal cells in Chmr3-/- neonatal mice. We found that Chmr3-/- mice feature abnormal epidermal morphology characterized by accumulation of nucleated basal cells, shrinkage of basal keratinocytes, and enlargement of intercellular spaces. These morphologic changes were associated with upregulation of cell proliferation genes and downregulation of genes contributing to epidermal differentiation, extracellular matrix formation, intercellular adhesion, and cell arrangement. These findings provide, to our knowledge, previously unreported insights into how acetylcholine controls epidermal differentiation and lay a groundwork for future translational studies evaluating the therapeutic potential of cholinergic drugs in dermatology.

Auditory mismatch responses are differentially sensitive to changes in muscarinic acetylcholine versus dopamine receptor function.

The auditory mismatch negativity (MMN) has been proposed as a biomarker of NMDA receptor (NMDAR) dysfunction in schizophrenia. Such dysfunction may be caused by aberrant interactions of different neuromodulators with NMDARs, which could explain clinical heterogeneity among patients. In two studies (N = 81 each), we used a double-blind placebo-controlled between-subject design to systematically test whether auditory mismatch responses under varying levels of environmental stability are sensitive to diminishing and enhancing cholinergic vs. dopaminergic function. We found a significant drug × mismatch interaction: while the muscarinic acetylcholine receptor antagonist biperiden delayed and topographically shifted mismatch responses, particularly during high stability, this effect could not be detected for amisulpride, a dopamine D2/D3 receptor antagonist. Neither galantamine nor levodopa, which elevate acetylcholine and dopamine levels, respectively, exerted significant effects on MMN. This differential MMN sensitivity to muscarinic versus dopaminergic receptor function may prove useful for developing tests that predict individual treatment responses in schizophrenia.

Expression and Function of Nicotinic Acetylcholine Receptors in Induced Regulatory T Cells

A contribution of the cholinergic system to immune cell function has been suggested, though the role of nicotine and its receptors in T cells, especially regulatory T (Treg) cells, is unclear. We herein investigated the expression and function of nicotinic acetylcholine receptors (nAChRs) in murine-induced Treg (iTreg) cells. Upon differentiation of naive BALB/c T cells into iTreg cells and other T-cell subsets, the effect of nicotine on cytokine production and proliferation of iTreg cells was examined. The expression of nAChRs and its regulatory mechanisms were comparatively analyzed among T-cell subsets. Stimulation-induced transforming growth factor-β1 (TGF-β1) production of iTreg cells was suppressed by nicotine, whereas interleukin (IL)-10 production and proliferation was not affected. α2-, α5-, α9-, and β2-nAChRs were differentially expressed in naive, Th1, Th2, Th9, Th17, and iTreg cells. Among these cell types, the α9-nAChR was particularly upregulated in iTreg cells via its gene promoter, but not through tri-methylation at the 4th lysine residue of the histone H3-dependent mechanisms. We conclude that the immunoregulatory role of Treg cells is modified by the cholinergic system, probably through the characteristic expression of nAChRs.

Expression of Concern: Wang et al., “Dissociating β-Amyloid from α7 Nicotinic Acetylcholine Receptor by a Novel Therapeutic Agent, S 24795, Normalizes α7 Nicotinic Acetylcholine and NMDA Receptor Function in Alzheimer's Disease Brain”

JNeurosci is publishing an Expression of Concern for the article, “Dissociating β-Amyloid from α7 Nicotinic Acetylcholine Receptor by a Novel Therapeutic Agent, S 24795, Normalizes α7 Nicotinic Acetylcholine and NMDA Receptor Function in Alzheimer's Disease Brain,” by Hoau-Yan Wang, Andres