Acetylcholine Receptor Subunits Research Articles

Explorations of Agonist Selectivity for the α9* nAChR with Novel Substituted Carbamoyl/Amido/Heteroaryl Dialkylpiperazinium Salts and Their Therapeutic Implications in Pain and Inflammation.

There is an urgent need for nonopioid treatments for chronic and neuropathic pain to provide effective alternatives amid the escalating opioid crisis. This study introduces novel compounds targeting the α9 nicotinic acetylcholine receptor (nAChR) subunit, which is crucial for pain regulation, inflammation, and inner ear functions. Specifically, it identifies novel substituted carbamoyl/amido/heteroaryl dialkylpiperazinium iodides as potent agonists selective for human α9 and α9α10 over α7 nAChRs, particularly compounds 3f, 3h, and 3j. Compound 3h (GAT2711) demonstrated a 230 nM potency as a full agonist at α9 nAChRs, being 340-fold selective over α7. Compound 3c was 10-fold selective for α9α10 over α9 nAChR. Compounds 2, 3f, and 3h inhibited ATP-induced interleukin-1β release in THP-1 cells. The analgesic activity of 3h was fully retained in α7 knockout mice, suggesting that analgesic effects were potentially mediated through α9* nAChRs. Our findings provide a blueprint for developing α9*-specific therapeutics for pain.

FASTMAP-a flexible and scalable immunopeptidomics pipeline for HLA- and antigen-specific T-cell epitope mapping based on artificial antigen-presenting cells.

The study of peptide repertoires presented by major histocompatibility complex (MHC) molecules and the identification of potential T-cell epitopes contribute to a multitude of immunopeptidome-based treatment approaches. Epitope mapping is essential for the development of promising epitope-based approaches in vaccination as well as for innovative therapeutics for autoimmune diseases, infectious diseases, and cancer. It also plays a critical role in the immunogenicity assessment of protein therapeutics with regard to safety and efficacy concerns. The main challenge emerges from the highly polymorphic nature of the human leukocyte antigen (HLA) molecules leading to the requirement of a peptide mapping strategy for a single HLA allele. As many autoimmune diseases are linked to at least one specific antigen, we established FASTMAP, an innovative strategy to transiently co-transfect a single HLA allele combined with a disease-specific antigen into a human cell line. This approach allows the specific identification of HLA-bound peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using FASTMAP, we found a comparable spectrum of endogenous peptides presented by the most frequently expressed HLA alleles in the world's population compared to what has been described in literature. To ensure a reliable peptide mapping workflow, we combined the HLA alleles with well-known human model antigens like coagulation factor VIII, acetylcholine receptor subunit alpha, protein structures of the SARS-CoV-2 virus, and myelin basic protein. Using these model antigens, we have been able to identify a broad range of peptides that are in line with already published and in silico predicted T-cell epitopes of the specific HLA/model antigen combination. The transient co-expression of a single affinity-tagged MHC molecule combined with a disease-specific antigen in a human cell line in our FASTMAP pipeline provides the opportunity to identify potential T-cell epitopes/endogenously processed MHC-bound peptides in a very cost-effective, fast, and customizable system with high-throughput potential.

High-Voltage Toxin'Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes.

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae, which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.

Alpha5 nicotine acetylcholine receptor subunit promotes intrahepatic cholangiocarcinoma metastasis

Neurotransmitter-initiated signaling pathway were reported to play an important role in regulating the malignant phenotype of tumor cells. Cancer cells could exhibit a “neural addiction” property and build up local nerve networks to achieve an enhanced neurotransmitter-initiated signaling through nerve growth factor-mediated axonogenesis. Targeting the dysregulated nervous systems might represent a novel strategy for cancer treatment. However, whether intrahepatic cholangiocarcinoma (ICC) could build its own nerve networks and the role of neurotransmitters in the progression ICC remains largely unknown. Immunofluorescence staining and Enzyme-linked immunosorbent assay suggested that ICC cells and the infiltrated nerves could generate a tumor microenvironment rich in acetylcholine that promotes ICC metastasis by inducing epithelial-mesenchymal transition (EMT). Acetylcholine promoted ICC metastasis through interacting with its receptor, alpha 5 nicotine acetylcholine receptor subunits (CHRNA5). Furthermore, acetylcholine/CHRNA5 axis activated GSK3β/β-catenin signaling pathway partially through the influx of Ca2+-mediated activation of Ca/calmodulin-dependent protein kinases (CAMKII). In addition, acetylcholine signaling activation also expanded nerve infiltration through increasing the expression of Brain-Derived Neurotrophic Factor (BDNF), which formed a feedforward acetylcholine-BDNF axis to promote ICC progression. KN93, a small-molecule inhibitor of CAMKII, significantly inhibited the migration and enhanced the sensitivity to gemcitabine of ICC cells. Above all, Acetylcholine/CHRNA5 axis increased the expression of β-catenin to promote the metastasis and resistance to gemcitabine of ICC via CAMKII/GSK3β signaling, and the CAMKII inhibitor KN93 may be an effective therapeutic strategy for combating ICC metastasis.

Using RNA interference targeting a nicotinic acetylcholine receptor subunit to counteract insecticide accommodation mechanisms: example of the β1 subunit in the imidacloprid-accommodated American cockroach, Periplaneta americana.

Insecticide accommodation and resistance are limiting factors to the much-needed increase in agricultural production. Various physiological and cellular modifications, such as the changes of insecticide molecular targets, have been linked to these events. Thus, a previous study demonstrated that the imidacloprid accommodation set up by the cockroach Periplaneta americana after an exposure to a sublethal dose of this insecticide involves functional alterations of two nicotinic acetylcholine receptor (nAChR) subtypes. As RNA interference (RNAi) is one of the most promising strategies for controlling pest insects, we evaluated, in this study, the use of RNAi that targets the β1 nAChR subunit to counteract the imidacloprid accommodation phenomenon in cockroaches. Interestingly, we showed that ingestion of dsRNA-β1 increased the sensitivity to imidacloprid of accommodated cockroaches. Thus, we have demonstrated for the first time that RNAi that targets an nAChR subunit can counteract the accommodation mechanism to insecticide targeting nAChRs set up by an insect.

Analysis of insecticide resistance and de novo transcriptome assembly of resistance associated genes in the European grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae).

The European grapevine moth Lobesia botrana (Denis & Shiffermüller 1776) is an economically important pest of the vine-growing areas worldwide. Chemical insecticides have been used for its control; however, its resistance status is largely unknown in many regions. We monitored the susceptibility of several L. botrana populations from Greece and Turkey. In addition, based on RNAseq transcriptome analysis, we identified and phylogenetically classify the cytochrome P450 genes of L. botrana, as well as analysed target site sequences and looked for the presence of known resistance mutations. Resistance against chlorantraniliprole, alpha-cypermethrin, spinetoram, etofenprox, and acetamiprid was very low (below 2.5-fold in all cases, compared to a reference strain from Greece) in all populations from Greece that were included in the study. However, resistance against indoxacarb (4-30-fold), spinosad (5-59-fold), and deltamethrin (18-30 fold) was detected in the L. botrana populations from Turkey, compared to a reference population from Turkey. De novo transcriptome assembly and manual annotation, and subsequent PCR-based analysis of insecticide target sequences (i.e. voltage-gated sodium channel - VGSC: target of pyrethroids and oxadiazines; nicotinic acetylcholine receptor subunit a6 - nAChR_α6: target of spinosad; ryanodine receptor - RyR: target of diamides; glutamate-gated chloride channel - GluCl: target of avermectins and; acetylcholinesterase - AChE: target of organophosphates) showed the absence of known resistance mutations in all specimens from both countries. Finally, the L. botrana CYPome (116 genes) was manually analysed and phylogenetically characterised, to provide resources for future studies that will aim the analysis of metabolic resistance.

Histone Methyltransferase G9a Plays an Essential Role on Nicotine Preference in Zebrafish.

Psychostimulants regulate behavioral responses in zebrafish via epigenetic mechanisms. We have previously shown that DNA methylation and histone deacetylase (HDAC) inhibition abolish nicotine-induced conditioned place preference (CPP) but little is known about the role of histone methylation in addictive-like behaviors. To assess the influence of histone methylation on nicotine-CPP, zebrafish were treated with a histone (H3) lysine-9 (K9) dimethyltransferase G9a/GLP inhibitor, BIX-01294 (BIX), which was administered before conditioning sessions. We observed a dual effect of the inhibitor BIX: at high doses inhibited while at low doses potentiated nicotine reward. Transcriptional expression of α6 and α7 subunits of the nicotinic acetylcholine receptor and of G9a, DNA methyl transferase-3, and HDAC-1 was upregulated in zebrafish with positive scores for nicotine-CPP. Changes in relative levels of these mRNA molecules reflected the effects of BIX on nicotine reward. BIX treatment per sé did not affect transcriptional levels of epigenetic enzymes that regulate trimethylation or demethylation of H3. BIX reduced H3K9me2 protein levels in a dose-dependent manner in key structures of the reward pathway. Thus, our findings indicated that different doses of BIX differentially affect nicotine CPP via strong or weak inhibition of G9a/GLP activity. Additionally, we found that the lysine demethylase inhibitor daminozide abolished nicotine-CPP and drug seeking. Our data demonstrate that H3 methylation catalyzed by G9a/GLP is involved in nicotine-CPP induction. Dimethylation of K9 at H3 is an important epigenetic modification that should be considered as a potential therapeutic target to treat nicotine reward and perhaps other drug addictions.

A comparative study of the performance of E. coli and K. phaffii for expressing α-cobratoxin

Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His6-tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs.

CHRNA9 as a New Prognostic Marker and Potential Therapeutic Target in Glioma.

Background: The nicotinic acetylcholine receptor (nAChR) subunit alpha-9 (CHRNA9) is a unique cholinergic receptor, which is involved in tumor proliferation, apoptosis, metastasis and chemotherapy resistance. However, the correlation between the expression level of CHRNA9 in glioma and the clinical features and prognosis of glioma patients has not been clarified. The aim of this study was to verify the expression level of CHRNA9 in glioma and its effect on prognosis by bioinformatics methods. Methods: The RNA-seq data of glioma and normal samples were obtained from the TCGA and GTEx databases. Bioinformatics methods were utilized to analyze the differential expression of CHRNA9 between tumor samples and normal samples. The potential association between CHRNA9 and the clinicopathological features of glioma patients was also investigated. The Kaplan-Meier method and Cox regression were utilized to analyze the relationship between CHRNA9 expression level and survival time and prognostic value of glioma patients. Enrichment analysis was applied to predict gene function and signaling pathways associated with CHRNA9. Experimental verification was performed using tumor tissues and paracancerous tissues from glioma patients. Results: The results of bioinformatics analysis showed that the expression of CHRNA9 was increased in glioma tissues, correlating with poor prognosis and reduced patient survival time. Enrichment analysis suggested that CHRNA9 may interact with the JAK/STAT pathway. CHRNA9 was also found to be abnormally expressed in various other tumors and associated with the expression levels of numerous immune checkpoints in glioma. The findings from the analysis of clinical samples revealed that the expression levels of both mRNA and protein of CHRNA9 in glioma tissues were higher than those in paracancerous tissues. Similarly, the mRNA expression levels of STAT3, IL-6, and TNF-α, which are crucial factors in the STAT3 pathway, were elevated in glioma tissues compared to paracancerous tissues. Conclusion: CHRNA9 is a potential prognostic marker and immunotherapy target for glioma, with its mechanism of action potentially linked to the STAT3 pathway.

Nicotine Abuse and Neurodegeneration: Novel Pharmacogenetic Targets to Aid Quitting and Reduce the Risk of Dementia.

Nicotine dependence has deleterious neurological impacts. Previous studies found an association between cigarette smoking and accelerating age-related thinning of the brain's cortex and subsequent cognitive decline. Smoking is considered the third most common risk factor for dementia, which prompted the inclusion of smoking cessation in dementia prevention strategies. Traditional pharmacologic options for smoking cessation include nicotine transdermal patches, bupropion and varenicline. However, based on smokers' genetic makeup, pharmacogenetics can be used to develop novel therapies to replace these traditional approaches. Genetic variability of cytochrome P450 2A6 has a major impact on smokers' behavior and their response to quitting therapies. Gene polymorphism in nicotinic acetylcholine receptor subunits also has a great influence on the ability to quit smoking. In addition, polymorphism of certain nicotinic acetylcholine receptors was found to affect the risk of dementia and the impact of tobacco smoking on the development of Alzheimer's disease. Nicotine dependence involves the activation of pleasure response through the stimulation of dopamine release. Central dopamine receptors, catechol-o-methyltransferase and the dopamine transporter protein, regulate synaptic dopamine levels. The genes of these molecules are potential targets for novel smoking cessation drugs. Pharmacogenetic studies of smoking cessation also investigated other molecules, such as ANKK1 and dopamine-beta-hydroxylase (DBH). In this perspective article, we aim to highlight the promising role of pharmacogenetics in the development of effective drugs for smoking cessation, which can increase the success rate of smoking quitting plans and ultimately reduce the incidence of neurodegeneration and dementia.

Nicotinic acetylcholine receptor signaling maintains epithelial barrier integrity.

Disruption of epithelial barriers is a common disease manifestation in chronic degenerative diseases of the airways, lung, and intestine. Extensive human genetic studies have identified risk loci in such diseases, including in chronic obstructive pulmonary disease (COPD) and inflammatory bowel diseases. The genes associated with these loci have not fully been determined, and functional characterization of such genes requires extensive studies in model organisms. Here, we report the results of a screen in Drosophila melanogaster that allowed for rapid identification, validation, and prioritization of COPD risk genes that were selected based on risk loci identified in human genome-wide association studies (GWAS). Using intestinal barrier dysfunction in flies as a readout, our results validate the impact of candidate gene perturbations on epithelial barrier function in 56% of the cases, resulting in a prioritized target gene list. We further report the functional characterization in flies of one family of these genes, encoding for nicotinic acetylcholine receptor (nAchR) subunits. We find that nAchR signaling in enterocytes of the fly gut promotes epithelial barrier function and epithelial homeostasis by regulating the production of the peritrophic matrix. Our findings identify COPD-associated genes critical for epithelial barrier maintenance, and provide insight into the role of epithelial nAchR signaling for homeostasis.

Hepatocyte CHRNA4 mediates the MASH-promotive effects of immune cell-produced acetylcholine and smoking exposure in mice and humans

Metabolic dysfunction-associated steatohepatitis (MASH) is a leading risk factor for liver cirrhosis and hepatocellular carcinoma. Here, we report that CHRNA4, a subunit of nicotinic acetylcholine receptors (nAChRs), is an accelerator of MASH progression. CHRNA4 also mediates the MASH-promotive effects induced by smoking. Chrna4 was expressed specifically in hepatocytes and exhibited increased levels in mice and patients with MASH. Elevated CHRNA4 levels were positively correlated with MASH severity. We further revealed that during MASH development, acetylcholine released from immune cells or nicotine derived from smoking functioned as an agonist to activate hepatocyte-intrinsic CHRNA4, inducing calcium influx and activation of inflammatory signaling. The communication between immune cells and hepatocytes via the acetylcholine-CHRNA4 axis led to the production of a variety of cytokines, eliciting inflammation in liver and promoting the pathogenesis of MASH. Genetic and pharmacological inhibition of CHRNA4 protected mice from diet-induced MASH. Targeting CHRNA4 might be a promising strategy for MASH therapeutics.

Immunodetection of Truncated Forms of the α6 Subunit of the nAChR in the Brain of Spinosad Resistant Ceratitis capitata Phenotypes.

The α6 subunit of the nicotinic acetylcholine receptor (nAChR) has been proposed as the target for spinosad in insects. Point mutations that result in premature stop codons in the α6 gene of Ceratitis capitata flies have been previously associated with spinosad resistance, but it is unknown if these transcripts are translated and if so, what is the location of the putative truncated proteins. In this work, we produced a specific antibody against C. capitata α6 (Ccα6) and validated it by ELISA, Western blotting and immunofluorescence assays in brain tissues. The antibody detects both wild-type and truncated forms of Ccα6 in vivo, and the protein is located in the cell membrane of the brain of wild-type spinosad sensitive flies. On the contrary, the shortened transcripts present in resistant flies generate putative truncated proteins that, for the most part, fail to reach their final destination in the membrane of the cells and remain in the cytoplasm. The differences observed in the locations of wild-type and truncated α6 proteins are proposed to determine the susceptibility or resistance to spinosad.

Use of double-stranded RNA targeting β2 divergent nicotinic acetylcholine receptor subunit to control pea aphid Acyrthosiphon pisum at larval and adult stages.

In recent years, the use of the RNA interference technology (RNAi) has emerged as one of the new strategies for species-specific control of insect pests. Its specificity depends on the distinctiveness of the target gene sequence for a given species. In this work, we assessed in the pea aphid Acyrthosiphon pisum (A. pisum) the use of a double-stranded RNA (dsRNA) that targets the β2 divergent nicotinic acetylcholine receptor (nAChR) subunit (dsRNA-β2), which shares low sequence identity with other subunits, to control populations of this pest at different developmental stages. Because nAChRs are targeted by neonicotinoid insecticides such as imidacloprid, we also assessed the effect of dsRNA-β2 coupled to this insecticide on aphid survival. Finally, because the effect of a control agent on beneficial insect must be considered before any use of new pest management strategies, the acute toxicity of dsRNA-β2 combined with imidacloprid was evaluated on honeybee Apis mellifera. In this work, we demonstrated that dsRNA-β2 alone has an insecticidal effect on aphid larvae and adults. Moreover, dsRNA-β2 and imidacloprid effects on aphid larvae and adults were additive, meaning that dsRNA-β2 did not alter the efficacy of imidacloprid on these two developmental stages. Also, no obvious acute toxicity on Apis mellifera was reported. Using RNAi that targets β2 divergent nAChR subunit is effective alone or combined with imidacloprid to control A. pisum at larval and adult stages. Because no obvious Apis mellifera mortality has been reported, this RNAi-based pest management strategy should be considered to control insect pest. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The knockout of the nicotinic acetylcholine receptor subunit gene α1 (nAChR α1) through CRISPR/Cas9 technology exposes its involvement in the resistance of Spodoptera exigua to insecticides

Insect nicotinic acetylcholine receptors (nAChRs) are the directed targets of many insecticides. However, there have been no reports on the molecular characterization of the nAChR gene family or the causal association between nAChR α1 and resistance to insecticides in S. exigua, which is a significant agricultural pest. In this study, we identified a total of 9 candidate nAChR subunits in S. exigua, namely nAChR α1-α7 and nAChR β1-β2. For functional validation roles of Seα1 in insecticide resistance of S. exigua, we introduced a ∼ 1041-bp deletion of the Seα1 gene in a homozygous mutant strain (Seα1-KO) by CRISPR/Cas9 genome editing system, resulting in a premature truncation of the Seα1 protein and the subsequent loss of functional transmembrane (TM) 3 and TM4 elements. Compared with WH-S strain (wild-type strain), the Seα1-KO strain exhibited 2.62-folds resistant to trifluoropyrimidine, 8.3-folds resistant to dimehypo, and 5.28-folds resistant to dinotefuran, but no significant change in susceptibility to emamectin benzoate, spinetoram, lambda-cyhalothrin, permethrin and chlorpyrifos. Thus, this study has laid a solid foundation for investigating the role of nAChRs in S. exigua, and provides evidence for the crucial involvement of the α1 subunit in the mechanism of trifluoropyrimidine, dimehypo, and dinotefuran in S. exigua. Moreover, it provides a reference for the value of Seα1 subunit and its homologues in other species as insecticide targets.

The expression of nicotinic acetylcholine receptor subunits and their associations with local immune cells and prognosis in oral squamous cell carcinoma.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that may be responsible for cancer cell proliferation, epithelial-mesenchymal transition (EMT), and immune regulation. However, little is known about the associations of different nAChR subunits with tumor microenvironment in oral squamous cell carcinoma (OSCC). We retrospectively reviewed pathology samples from 75 OSCC patients by immunohistochemistry. In addition, a cohort of 307 OSCC patients in The Cancer Genome Atlas was analyzed. Subunit α1 was specific to peri-OSCC skeletal muscle. Increased α1 was associated with increased CD44 (cancer stem cells), increased CD3 and 8 (T cells), increased CD56 and 16 (natural killer cells), a decreased T stage, and an increased N stage. Increased α3 was associated with increased CD56 and 16. Increased α5 was associated with decreased CD3, 8, and 56, a decreased T stage, an increased N stage, worse survival, and decreased epithelial features. Increased α7 was associated with increased CD3, 8, 56, and 16, decreased tumor/peritumor ratios of CD3, 8, and 56 immune cells, and increased epithelial features. Increased local immune cells were associated with a better prognosis. α5 is the only subunit associated with decreased local immune cells and worse survival, while α1, α3, and α7 are associated with increased local immune cells in OSCC. α5 and α7 are correlated with different EMT states to be mesenchymal-like and epithelial-like OSCC, respectively. Protein expression data of the nAChR subunits, complementary to gene expression data, could provide meaningful information regarding the EMT status of OSCC associated with immune responses and prognosis.

Cooling Down Inflammation in the Cardiovascular System via the Nicotinic Acetylcholine Receptor.

Inflammation is a major player in many cardiovascular diseases including hypertension, atherosclerosis, myocardial infarction, and heart failure. In many individuals, these conditions coexist and mutually exacerbate each other's progression. The pathophysiology of these diseases entails the active involvement of both innate and adaptive immune cells. Immune cells that possess the α7 subunit of the nicotinic acetylcholine receptor (α7-nAChR) on their surface, have the potential to be targeted through both pharmacological and electrical stimulation of the cholinergic system. The cholinergic system regulates the inflammatory response to various stressors in different organ systems via systematically suppressing spleen-derived monocytes and chemokines, and locally improving immune cell function. Research on the cardiovascular system has demonstrated the potential for atheroma plaque stabilization and regression as favorable outcomes. Smaller infarct size and reduced fibrosis have been associated with improved cardiac function and a decrease in adverse cardiac remodeling. Furthermore, enhanced electrical stability of the myocardium can lead to a reduction in the incidence of ventricular tachyarrhythmia. Additionally, improving mitochondrial dysfunction and decreasing oxidative stress can result in less myocardial tissue damage caused by reperfusion injury. Restoring baroreflex activity and reduction in renal damage can promote blood pressure regulation and help counteract hypertension. Thus, the present review highlights the potential of nicotinic acetylcholine receptor activation as a natural approach to alleviate the adverse consequences of inflammation in the cardiovascular system.

Expression pattern of nicotinic acetylcholine receptor subunit transcripts in neurons and astrocytes in the ventral tegmental area and locus coeruleus.

Acetylcholine is the endogenous agonist for the neuronal nicotinic acetylcholine receptor (nAChR) system, which is involved in attention, memory, affective behaviours and substance use disorders. Brain nAChRs are highly diverse with 11 different subunits that can form multiple receptor subtypes, each with distinct receptor and pharmacological properties. Different neuronal cell types can also express different nAChR subtypes, resulting in highly complex cholinergic signalling. Identifying which nAChR subunit transcripts are expressed in cell types can provide an indication of which nAChR combinations are possible and which receptor subtypes may be most pharmacologically relevant to target. In addition to differences in expression across cell types, nAChRs also undergo changes in expression levels from adolescence to adulthood. In this study, we used fluorescent in situ hybridization to identify and quantify the expression of α4, α5, α6, β2 and β3 nAChR subunit transcripts in dopaminergic, GABAergic, glutamatergic and noradrenergic neurons and astrocytes in the ventral tegmental area (VTA) and locus coeruleus (LC) in adult and adolescent, male and female C57BL/6J mice. There were distinct differences in the pattern of nAChR subunit transcript expression between the two brain regions. LC noradrenergic neurons had high prevalence of α6, β2 and β3 expression, with very low expression of α4, suggesting the α6(non-α4)β2β3 receptor as a main subtype in these neurons. VTA astrocytes from adult mice showed greater prevalence of α5, α6, β2 and β3 transcript compared with adolescent mice. These data highlight the complex nAChR expression patterns across brain region and cell type.

The interaction of nicotinic acetylcholine receptor subunits Ldα3, Ldα8 and Ldβ1 with neonicotinoids in Colorado potato beetle, Leptinotarsa decemlineata

The Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), is an extremely destructive notifiable quarantine pest. Over the last two decades, neonicotinoid insecticides, particularly thiamethoxam and imidacloprid, have been used to control it in Xinjiang, and local field populations have developed different levels of resistance in consequence. However, the contributions of nicotinic acetylcholine receptors (nAChRs) to neonicotinoid resistance are currently poorly understood in CPB. Previous studies have shown that nAChRα1, α3, α8 and β1 are major target subunits for neonicotinoids in some model and important agricultural insects including nAChRα1 subunit of L. decemlineata (Ldα1). In this study, the expression levels of Ldα3, Ldα8 and Ldβ1 following 72 h of treatments with median lethal doses of thiamethoxam and imidacloprid were compared using real-time quantitative PCR. These genes were then individually and simultaneously knocked down with Ldα1 by RNA interference (RNAi) using a double-stranded RNA (dsRNA) feeding method for six days to explore their roles in CPB susceptibility to imidacloprid and thiamethoxam. The results showed that the expressions of Ldα3, Ldα8 and Ldβ1 were significantly decreased by 36.99–74.89% after thiamethoxam and imidacloprid treatments, compared with the control. The significant downregulation of the target genes resulting from RNAi significantly reduced the mortality of adults exposed to thiamethoxam and imidacloprid by 34.53% –56.44% and 28.78%–43.93%, respectively. Furthermore, the adult survival rates were not affected by every dsRNA-feeding treatment, while the body weight of the test adults significantly deceased after four and six days of individual gene RNAi. This study showed that Ldα3, Ldα8 and Ldβ1 are down-regulated by thiamethoxam and imidacloprid and play important roles in the tolerance of CPB to neonicotinoids.

Involvement of CHRNA6 in the Immune Response in Lung Squamous Cell Carcinoma and its Potential as a Drug Target for the Disease.

Lung squamous cell carcinoma (LUSC) is a subtype of lung cancer with a poor prognosis and limited treatment options. Previous studies show that some components of the cholinergic pathway may play important roles in the tumorigenesis of lung cancer, including LUSC. The purpose of this study is to investigate the involvement of cholinergic genes in immune infiltration in LUSC, and identify the key genes in the pathway and analyze their potential as targets for LUSC treatment and novel drugs. We first screened the cholinergic genes associated with immune infiltration in LUSC based on transcriptomic samples and explored the correlation between the key genes and immune infiltrating cells and immune pathways. Then, we assessed the effect of immunotherapeutic response in the high and low-expression groups of key genes in vitro. And finally, we screened potential drugs for the treatment of LUSC. We found that the expression of CHRNA6, the gene encoding the α6 subunit of nicotinic acetylcholine receptors (nAChR), was significantly correlated with the proportion of immune infiltrating cells in LUSC, and the high expression level of the gene was associated with poor prognosis of the disease. Also, the proportion of Tregs, M1 macrophages, and resting mast cells was correlated with the expression of CHRNA6. In addition, LUSC patients with higher CHRNA6 expression levels had better immunotherapy responses. Furthermore, we found that the drugs, i.e., adavosertib, varbulin and pyrazoloacridine, had a strong affinity with CHRNA6, with adavosertib binding most stably with the protein. CHRNA6 may be associated with immune infiltration in LUSC and affects patient prognosis and immunotherapeutic response by regulating immune cells and immune pathways. In addition, adavosertib may be a potential drug for the treatment of LUSC.