nAChR Gene Research Articles

Area-wide survey and monitoring of insecticide resistance in the brown planthopper, Nilaparvata lugens (Stål), from 2020 to 2023 in China

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a notorious pest affecting Asian rice crops. The evolution of insecticide resistance in BPH has emerged as a significant challenge in effectively managing this pest. This study revealed the resistance status of BPH to nine insecticides in ten provinces and Shanghai City in China from 2020 to 2023. Monitoring results showed that the resistance of BPH to triflumezopyrim, nitenpyram, and dinotefuran increased rapidly. The average resistance ratio of BPH to triflumezopyrim increased from 2.5 to 7.1 fold, nitenpyram from 18.3 to 37.7 fold, and dinotefuran from 119.5 to 268.1 fold. All populations remained extremely high resistance to imidacloprid, thiamethoxam, and buprofezin. Most field populations of BPH maintained moderate resistance to chlorpyrifos and sulfoxaflor, and high resistance to pymetrozine by rice stem dipping method. However, considering the reproduction-inhibiting character of pymetrozine, susceptible to low resistance levels to pymetrozine were monitored by Insecticide Resistance Action Committee (IRAC) NO.005 method. This result indicated that pymetrozine might lose efficacy in the control of application generation, but it could significantly inhibit the reproduction of field populations of BPH. Additionally, we compared the expression levels of 11 nicotinic acetylcholine receptor (nAChR) genes, the targets of nAChR competitive modulators, in four field populations (FY23, YH23, LJ23, LP23) and susceptible strain. The expression level of nAChR α4 was significantly reduced in all field populations, while α1, α2, α6, and α7 were significantly reduced in some field populations. Our findings provide valuable information for resistance management strategies in N. lugens and offer new insights into the resistance mechanisms of nAChR competitive modulators.

Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters.

In the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene. We constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region. The DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors. These findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

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.

Two-step method for rapid isolation of genomic DNA and validation of R81T insecticide resistance mutation in Myzus persicae

Isolation and amplification of nucleic acid (DNA) is considered a vital and potent instrument in molecular biological research. However, its functioning outside of a laboratory setting is difficult because of complex procedures that demand expert personnel and expensive equipment in addition to the fulfillment of several additional requirements. DNA isolation from minute insects is sometimes difficult, making diagnostic and genotyping procedures problematic. Thus, the current work offers a high-throughput, cost-effective, straightforward, and faster approach for isolating DNA from the aphid Myzus persicae. Intriguingly, two-step DNA extraction process yielded a high yield of extremely pure genomic DNA and required only 10 s to complete. PCR investigation aiming at amplifying the non-synonymous R81T region on the loop D site of the nAChR gene of M. persicae was subsequently utilized to successfully validate the recovered DNA. Moreover, the proposed method was compared in terms of yield and purity with conventionally used DNA isolation methods including, phenol:chloroform, salt out, and commercially available kits. In conclusion, this newly developed method would enable researchers to quickly process many biological samples used to analyze genetic diversity, mutant screening, and large spectrum diagnosis both in laboratory and field conditions.

Expression of Chrna9 is regulated by Tbx3 in undifferentiated pluripotent stem cells

It was reported that nicotinic acetylcholine receptor (nAChR)-mediated signaling pathways affect the proliferation and differentiation of pluripotent stem cells. However, detail expression profiles of nAChR genes were unrevealed in these cells. In this study, we comprehensively investigated the gene expression of α subunit of nAChRs (Chrna) during differentiation and induction of pluripotent stem cells. Mouse embryonic stem (ES) cells expressed multiple Chrna genes (Chrna3-5, 7 and 9) in undifferentiated status. Among them, Chrna9 was markedly down-regulated upon the differentiation into mesenchymal cell lineage. In mouse tissues and cells, Chrna9 was mainly expressed in testes, ES cells and embryonal F9 teratocarcinoma stem cells. Expression of Chrna9 gene was acutely reduced during differentiation of ES and F9 cells within 24 h. In contrast, Chrna9 expression was increased in induced pluripotent stem cells established from mouse embryonic fibroblast. It was shown by the reporter assays that T element-like sequence in the promoter region of Chrna9 gene is important for its activities in ES cells. Chrna9 was markedly reduced by siRNA-mediated knockdown of Tbx3, a pluripotency-related transcription factor of the T-box gene family. These results indicate that Chrna9 is a nAChR gene that are transcriptionally regulated by Tbx3 in undifferentiated pluripotent cells.

Different peripheral expression patterns of the nicotinic acetylcholine receptor in dementia with Lewy bodies and Alzheimer’s disease

BackgroundThe diffuse distribution of nicotinic cholinergic receptors (nAChRs) in both brain and peripheral immune cells points out their involvement in several pathological conditions. Indeed, the deregulated function of the nAChR was previously correlated with cognitive decline and neuropsychiatric symptoms in Alzheimer’s disease (AD) and Dementia with Lewy bodies (DLB).The evaluation in peripheral immune cells of nAChR subtypes, which could reflect their expression in brain regions, is a prominent investigation area.ObjectivesThis study aims to evaluate the expression levels of both the nAChR subunits and the main known inflammatory cytokines in peripheral blood mononuclear cells (PBMCs) of patients with DLB and AD to better characterize their involvement in these two diseases.ResultsHigher gene expression levels of TNFα, IL6 and IL1β were observed in DLB and AD patients in comparison with healthy controls (HC). In our cohort, a reduction of nAChRα4, nAChRβ2 and nAChRβ4 was detected in both DLB and AD with respect to HC. Considering nAChR gene expressions in DLB and AD, significant differences were observed for nAChRα3, nAChRα4, nAChRβ2 and nAChRβ4 between the two groups. Moreover, the acetylcholine esterase (AChE) gene expression was significantly higher in DLB than in AD. Correlation analysis points out the relation between different nAChR subtype expressions in DLB (nAChRβ2 vs nAChRα3; nAChRα4 vs nAChRα3) and AD (nAChRα4 vs nAChRα3; nAChRα4 vs nAChRβ4; nAChRα7 vs nAChRα3; nAChRα7 vs nAChRα4).ConclusionsDifferent gene expressions of both pro-inflammatory cytokines and nAChR subtypes may represent a peripheral link between inflammation and neurodegeneration. Inflammatory cytokines and different nAChRs should be valid and accurate peripheral markers for the clinical diagnosis of DLB and AD. However, although nAChRs show a great biological role in the regulation of inflammation, no significant correlation was detected between nAChR subtypes and the examined cytokines in our cohort of patients.

Polymorphisms of nicotinic acetylcholine receptors in Alzheimer’s disease: a systematic review and data analysis

ObjectiveThis study was conducted to accomplish a better insight into the impact of single nucleotide polymorphisms (SNPs) of nicotinic acetylcholine receptors (nAChR) at the risk of Alzheimer’s disease (AD) and their possible pathogenicity.MethodsWe carried out a systemic review of accessible studies. The case–control studies were assessed by an electronic search of international and local databases to identify relevant studies on SNPs relating to nAChR genes in AD. Two reviewers evaluated the inclusion/exclusion criteria, summarized, and analyzed the extracted data. We used odds ratios (ORs) with 95% confidence intervals (CIs) for reporting our data. Online databases were checked for possible pathogenicity of statistically significant SNPs. Also, online databases, including NCBI, NIH, ClinVar, RegulomeDB, and Ensemble, were used to analyze and identify structure and function, DNA features, and flank sequencing in SNPs.ResultsAmong all collected SNPs, rs4779978 and rs1827294 on CHRNA7, rs1044394 on CHRNA4, and rs1127314 on CHRNB2 showed statistically significant between AD cases and controls.ConclusionsSome SNPs from the reviewed reports show evidence supporting their possible involvement in AD pathology. However, more comprehensive studies are necessary to identify the exact correlation and their role on the pathogenicity of disease.

Dexmedetomidine attenuates lipopolysaccharide-induced inflammation through macrophageal IL-10 expression following α7 nAchR activation

Dexmedetomidine, a highly selective α2-adrenoceptor agonist, has been recently reported to alleviate systemic inflammatory response induced by lipopolysaccharide (LPS), in addition to its sedative, analgesic, bradycardic and hypotensive properties. This study aimed to illustrate the molecular mechanisms underlying dexmedetomidine-induced anti-inflammation. In the LPS-pretreated mice, subcutaneous injection of dexmedetomidine reduced the spleen weight as well as serum and spleen expression of proinflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β, and increased serum and spleen expression of IL-10, a known anti-inflammatory cytokine. In addition, dexmedetomidine-attenuated proinflammatory cytokine reduction was entirely inhibited by selective α7 nicotinic acetylcholine receptor (nAChR) antagonist methyllycaconitine but not α2-adrenoceptor antagonist yohimbine. Dexmedetomidine also increased macrophageal IL-10 expression in the presence and absence of LPS, which was also attenuated by methyllycaconitine but not yohimbine. Furthermore, the stimulatory effect of dexmedetomidine on the expression of IL-10 was also reduced by the α7 nAChR gene silencer siRNA/α7 nAChR. Lastly, pretreatment with the IL-10 neutralizing antibody reversed dexmedetomidine-supressed expression of proinflammatory cytokines. Our findings illustrate that dexmedetomidine-induced anti-inflammation is through macrophageal expression of IL-10 following activation of α7 nAchRs but not α2-adrenoceptors.

Several nAChRs gene variants are associated with phenotypes of heroin addiction in Chinese Han population

Heroin addiction is a chronic and complex brain disease. Nicotinic acetylcholine receptors (nAChRs) have been shown as major control points in many of the neurological and physiological disorders involved in heroin addiction. In the present study, thirty-three SNPs across nine nAChR genes were selected and probed for their associations with heroin addiction phenotypes in 801 unrelated northwestern Chinese Han patients. We found that rs2565055 in CHRNA2 gene was associated with daily dose of methadone treatment, and rs2672215, rs2672216 and rs2741865 in CHRNA10 gene were associated with the duration of the transition from first use to dependence (DTFUD). Cox multivariable regression analysis revealed that rs3743075, rs6495309 in CHRNA3, rs2304297 in CHRNA6, and rs1948 in CHRNB4 were associated with sexual desire in patients with heroin addiction. These findings were further supported by the identification of a haplotype block spanning CHRNA5, CHRNA3, and CHRNB4 that is correlated with changes in sexual desire after long-term heroin use. Our findings highlight associations between polymorphisms in nAChRs genes and the phenotypes of heroin addiction in the Chinese Han population. We suggest several nAChRs subunits as potential novel targets for the treatment of heroin addiction.

Loss of α-9 Nicotinic Acetylcholine Receptor Subunit Predominantly Results in Impaired Postural Stability Rather Than Gaze Stability.

The functional role of the mammalian efferent vestibular system (EVS) is not fully understood. One proposal is that the mammalian EVS plays a role in the long-term calibration of central vestibular pathways, for example during development. Here to test this possibility, we studied vestibular function in mice lacking a functional α9 subunit of the nicotinic acetylcholine receptor (nAChR) gene family, which mediates efferent activation of the vestibular periphery. We focused on an α9 (−/−) model with a deletion in exons 1 and 2. First, we quantified gaze stability by testing vestibulo-ocular reflex (VOR, 0.2–3 Hz) responses of both α9 (−/−) mouse models in dark and light conditions. VOR gains and phases were comparable for both α9 (−/−) mutants and wild-type controls. Second, we confirmed the lack of an effect from the α9 (−/−) mutation on central visuo-motor pathways/eye movement pathways via analyses of the optokinetic reflex (OKR) and quick phases of the VOR. We found no differences between α9 (−/−) mutants and wild-type controls. Third and finally, we investigated postural abilities during instrumented rotarod and balance beam tasks. Head movements were quantified using a 6D microelectromechanical systems (MEMS) module fixed to the mouse’s head. Compared to wild-type controls, we found head movements were strikingly altered in α9 (−/−) mice, most notably in the pitch axis. We confirmed these later results in another α9 (−/−) model, with a deletion in the exon 4 region. Overall, we conclude that the absence of the α9 subunit of nAChRs predominately results in an impairment of posture rather than gaze.

Constructing and Tuning Excitatory Cholinergic Synapses: The Multifaceted Functions of Nicotinic Acetylcholine Receptors in Drosophila Neural Development and Physiology.

Nicotinic acetylcholine receptors (nAchRs) are widely distributed within the nervous system across most animal species. Besides their well-established roles in mammalian neuromuscular junctions, studies using invertebrate models have also proven fruitful in revealing the function of nAchRs in the central nervous system. During the earlier years, both in vitro and animal studies had helped clarify the basic molecular features of the members of the Drosophila nAchR gene family and illustrated their utility as targets for insecticides. Later, increasingly sophisticated techniques have illuminated how nAchRs mediate excitatory neurotransmission in the Drosophila brain and play an integral part in neural development and synaptic plasticity, as well as cognitive processes such as learning and memory. This review is intended to provide an updated survey of Drosophila nAchR subunits, focusing on their molecular diversity and unique contributions to physiology and plasticity of the fly neural circuitry. We will also highlight promising new avenues for nAchR research that will likely contribute to better understanding of central cholinergic neurotransmission in both Drosophila and other organisms.

Association Study of Polymorphisms in Neuronal Nicotinic Acetylcholine Receptor Subunit Genes With Schizophrenia in the Han Chinese Population.

ObjectiveTo investigate the relation between nicotinic acetylcholine receptor subunit (nAChR) genes and schizophrenia, and the relation between tag single nucleotide polymorphism (rs1317286, rs1044396, rs6494212, rs16969968, and rs684513) and schizophrenia in Han Chinese people. MethodsThe protein-protein interaction (PPI) network among nAChR protein and 350 proteins encoded by schizophrenia-related susceptibility genes was constructed through the String database to explore whether nAChR genes were associated with schizophrenia in these known databases. Then, five single nucleotide polymorphisms (SNPs) of CHRNA3 (rs1317286), CHRNA4 (rs1044396), CHRNA7 (rs6494212), and CHRNA5 (rs16969968, rs684513) were analyzed in a sample of 1,035 schizophrenic patients and 816 healthy controls. The interaction between the markers was analyzed using multifactor dimensionality reduction (MDR) software. Power analysis was performed using the Quanto program. ResultsThere are no significant differences in genotype or allele distribution were identified between the patients and controls (p>0.05). The haplotypes constructed by four markers rs1317286, rs6494212, rs16969968, and rs684513 were not associated with schizophrenia either. However, a significant association between models made of rs1317286, rs1044396, rs6494212, and rs684513 and schizophrenia was revealed in interaction analysis (p<0.05). ConclusionThe nAChR protein may have effects on the development of schizophrenia through the interaction with proteins encoded by schizophrenia-related susceptibility genes, but no relation was found between selected polymorphisms and schizophrenia in the collected Han Chinese people. However, interaction analysis suggested four-SNP model has an important effect on schizophrenia.

Nicotine Patch Improved Autosomal Dominant Sleep-Related Hypermotor Epilepsy

Autosomal dominant sleep-related hypermotor epilepsy (ADSHE) is a genetic focal epilepsy associated with mutations in the nicotinic acetylcholine receptor. Mutations in two major nicotinic acetylcholine receptor subunit genes, CHRNA4 and CHRNB2, are commonly associated with ADSHE.1Saberi B. Tehrani B.S. Pore-forming M2 domains of CHRNA4 and CHRNB2 mutations in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) phenotype.Am J Biomed Sci Res. 2019; 1: 222-223Crossref Google Scholar In up to one-third of patients with ADSHE, seizures are refractory to antiseizure medications. Here, we report complete seizure freedom with transdermal nicotine in two pediatric patients from the same family with medically refractory ADSHE. Both patients, who are siblings, presented with exclusively nocturnal focal seizures in childhood, at ages seven years (Patient 1) and 4.5 years (Patient 2). Elelctroencephalographies (EEGs) demonstrated focal electroclinical seizures with hypermotor features and interictal epileptiform discharges in sleep (Fig A and B). Genetic testing revealed a heterozygous missense mutation in CHRNA4 (c.851C>T P.Ser284Leu), which has been described as a common pathogenic gain-of-function mutation in ADSHE. Patient 2 was also diagnosed with an autism spectrum disorder. Both patients had a high nightly seizure burden with minimal improvement on numerous antiseizure medications. A trial of low-dose (7 mg/24 hour) nicotine patch was initiated at ages 16 years (Patient 1) and 14 years (Patient 2). Clinical seizures stopped in both the first night the patch was applied. Follow-up overnight EEGs confirmed seizure freedom and showed normalization or near-normalization of their interictal EEGs (Fig C). Both patients have experienced sustained seizure freedom for over 18 months with nicotine patch 7 mg/24 hour in addition to oxcarbazepine. Transdermal nicotine has been reported to provide marked seizure improvement in a subset of patients with ADSHE.2Fox J. Thodeson D.M. Dolce A.M. Nicotine: a targeted therapy for epilepsy due to nAChR gene variants.J Child Neurol. 2020; 36: 371-377Crossref PubMed Scopus (3) Google Scholar,3Lossius K. de Saint Martin A. Myren-Svelstad S. et al.Remarkable effect of transdermal nicotine in children with CHRNA4-related autosomal dominant sleep-related hypermotor epilepsy.Epilepsy Behav. 2020; 105: 106944Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar The siblings reported here have the same CHRNA4 mutation as a 19-year-old patient who had considerable seizure improvement, although not complete resolution, with transdermal nicotine at a higher dose of 14 mg/24 hour.2Fox J. Thodeson D.M. Dolce A.M. Nicotine: a targeted therapy for epilepsy due to nAChR gene variants.J Child Neurol. 2020; 36: 371-377Crossref PubMed Scopus (3) Google Scholar Lossius et al. described striking improvement in seizures and neuropsychological test performance in three six- to 10-year-old children with a different CHRNA4 mutation (c.839C>T P.Ser280Phe) who were treated with 3.5 to 7 mg/24 hour nicotine.3Lossius K. de Saint Martin A. Myren-Svelstad S. et al.Remarkable effect of transdermal nicotine in children with CHRNA4-related autosomal dominant sleep-related hypermotor epilepsy.Epilepsy Behav. 2020; 105: 106944Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar With different patient characteristics, age as well as specific mutation might contribute to the efficacy of nicotine patches in controlling seizure activity. Christensen et al. discussed how nicotine increases neuronal firing of cholinergic cells in younger animals, which in turn affects the action potential shape and afterhyperpolarization.4Christensen M.H. Ishibashi M. Nielsen M.L. Leonard C.S. Kohlmeier K.A. Age-related changes in nicotine response of cholinergic and non-cholinergic laterodorsal tegmental neurons: implications for the heightened adolescent susceptibility to nicotine addiction.Neuropharmacology. 2014; 85: 263-283Crossref PubMed Scopus (15) Google Scholar Nicotine has been found to cause age-dependent changes in gene expression in adolescent female rat brains.5Polesskaya O.O. Fryxell K.J. Merchant A.D. et al.Nicotine causes age-dependent changes in gene expression in the adolescent female rat brain.Neurotoxicol Teratol. 2007; 29: 126-140Crossref PubMed Scopus (33) Google Scholar In adolescence, GABAergic signaling in the prefrontal cortex, an important brain region associated with ADSHE, is still developing.6Caballero A. Tseng K.Y. GABAergic function as a limiting factor for prefrontal maturation during adolescence.Trends Neurosci. 2016; 39: 441-448Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Therefore, age might be an important factor when determining whether nicotine patch treatment may be beneficial for patients with ADSHE, with regard to both seizure improvement and neuropsychologic outcomes. A limitation of our report is that our patients are from the same family and therefore may possess unique characteristics that influenced their dramatic response to transdermal nicotine. This possibility might prevent generalization of our results to all patients with same mutation in CHRNA4. Nevertheless, the two siblings reported here add to the small number of pediatric reports documenting the successful use of nicotine patches in ADSHE. Future research is needed to establish whether the nicotine patch could become an adjunct or first-line treatment in a subset of pediatric patients with ADSHE. The authors wish to thank all the patients, family members, and staff members who participated in the study.

Protective effect of alpha-linoleic acid on Aβ-induced oxidative stress, neuroinflammation, and memory impairment by alteration of α7 nAChR and NMDAR gene expression in the hippocampus of rats

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects many older people around the world. Numerous studies are underway to evaluate the protective effects of natural products in AD. Alpha-linoleic acid (ALA) is an essential unsaturated fatty acid that exhibits neuroprotective outcomes in rat models of ischemic stroke and Parkinson's disease. This research aimed to investigate the effect of ALA on oxidative stress, neuroinflammation, neuronal death, and memory deficit induced by amyloid-beta (Aβ) peptide. After intrahippocampal injection of Aβ1−42, rats received ALA (150 μg/kg, subcutaneously) for 14 consecutive days. ALA decreased the levels of malondialdehyde and nitric oxide, enhanced glutathione content, and increased the activity of catalase in the hippocampus of the rat model of AD. It also reduced the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, nuclear factor-kappa B, and N-methyl-d-aspartate receptor subunits NR2A and NR2B mRNAs in the hippocampus, prevented the neuronal loss in the CA1 region, and enhanced the expression of α7 nicotinic acetylcholine receptor. In addition, ALA allowed Aβ1−42-injected rats to spend less time and distance to reach the hidden platform in the Morris water maze test and to swim longer in the target quadrant. We concluded that ALA reduces the biochemical, molecular, histological, and behavioral changes caused by Aβ1−42 and it may be an effective option for treating AD.

Characterization and expression analysis of tandemly duplicated nicotinic acetylcholine receptors in pearl oysters after stimulation of pathogen-related molecular patterns.

Nicotinic acetylcholine receptors (nAChRs) are a class of ligand-gated ion channels that participate in signal transduction and are reported to play an important role in the immunomodulation of vertebrates and invertebrates. Previous studies have shown that the nAChRs in mollusks have undergone large-scale expansion after tandem repeats and retrotransposition, with the most expansion observed in bivalves. This study characterized the sequence of a tandem repeat nAChR unique to several bivalve mollusks and investigated its functions in Pinctada fucata martensii. Firstly, phylogenetic analysis revealed that the tandem arrays of nAChRs existed before bivalve differentiation and m ost tandem-replicated nAChR genes have a conserved genomic structure and domain combination. In present study, five tandemly duplicated nAChR genes were cloned from P. f. martensii and designated as PmnAChR-1 to PmnAChR-5. qRT-PCR analysis revealed that five PmnAChRs were specifically expressed in adult gills. In addition, after PAMP stimulation, the expression of PmnAChRs in hemocytes of P. f. martensii were strongly induced but exhibited different responses to different stimuli. PmnAChR-1, PmnAChR-4, and PmnAChR-5 exhibited strong and wide responsiveness to lipopolysaccharide (LPS) stimulation but had no response to peptidoglycan (PGN) stimulation. PmnAChR-2 expression was notably upregulated at 6h after PGN challenge but had no response to LPS stimulation. Polyinosinic-polycytidylic acid challenge upregulated nearly all PmnAChRs, except for PmnAChR-5. Furthermore, Pm-miR-873-3p, Pm-miR-4577, Pm-miR-103a-3p, and Pm-miR-6753-3p were identified as the regulatory miRNA of PmnAChR-1, PmnAChR-3, PmnAChR-4, and PmnAChR-5, respectively. These findings suggested that these tandem arrays of nAChRs are unique to bivalves, and the tandem duplication of nAChR genes may be involved in the immune regulation process after pathogen stimulation.

Role of nicotinic acetylcholine receptor subunits in the mode of action of neonicotinoid, sulfoximine and spinosyn insecticides in Drosophila melanogaster

Insecticides remain valuable tools for the control of insect pests that significantly impact human health and agriculture. A deeper understanding of insecticide targets is important in maintaining this control over pests. Our study systematically investigates the nicotinic acetylcholine receptor (nAChR) gene family, in order to identify the receptor subunits critical to the insect response to insecticides from three distinct chemical classes (neonicotinoids, spinosyns and sulfoximines). Applying the CRISPR/Cas9 gene editing technology in D. melanogaster, we were able to generate and maintain homozygous mutants for eight nAChR subunit genes. A ninth gene (Dβ1) was investigated using somatic CRISPR in neural cells to overcome the low viability of the homozygous germline knockout mutant. These findings highlight the specificity of the spinosyn class insecticide, spinosad, to receptors containing the Dα6 subunit. By way of contrast, neonicotinoids are likely to target multiple receptor subtypes, beyond those receptor subunit combinations previously identified. Significant differences in the impacts of specific nAChR subunit deletions on the resistance level of flies to neonicotinoids imidacloprid and nitenpyram indicate that the receptor subtypes they target do not completely overlap. While an R81T mutation in β1 subunits has revealed residues co-ordinating binding of sulfoximines and neonicotinoids differ, the resistance profiles of a deletion of Dβ1 examined here provide new insights into the mode of action of sulfoxaflor (sulfoximine) and identify Dβ1 as a key component of nAChRs targeted by both these insecticide classes. A comparison of resistance phenotypes found in this study to resistance reported in insect pests reveals a strong conservation of subunit targets across many different insect species and that mutations have been identified in most of the receptor subunits that our findings would predict to have the potential to confer resistance.

Identification of the Aedes aegypti nAChR gene family and molecular target of spinosad.

Spinosad is an insecticide with unique mode of action (MOA) of disrupting nicotinic acetylcholine receptor and is efficacious against many insect species. Mutations in the nicotinic acetylcholine receptor (nAChR) α6 subunit have been identified that are associated with levels of spinosad resistance, but the molecular characterization of the nAChR gene family and a causative association between nAChR α6 and resistance to spinosad in Aedes aegypti, a primary vector of many arboviruses, have not yet been reported. In this study, we identified 10 candidate nAChR subunits in Ae. Aegypti, nAChRα1-α9 and nAChRβ1, showing similarly orthologous relationships with Anopheles gambiae. With the application of the CRISPR/Cas9 genome editing system, we introduced a 32-bp deletion at the 5' end of the Aaeα6 (Ae. aegypti nAChR α6) gene in a homozygous mutant strain (Aaeα6-KO). The mutation produced two successive pre-mature stop codons, resulting in loss of function in the target receptor. The Aaeα6-KO mutant strain exhibited a 320-fold level of resistance to spinosad compared with wildtype. A recessive mode of inheritance for spinosad resistance was found in the Aaeα6-KO strain. CRISPR/Cas9 introduced truncated Aaeα6 receptor in Ae. aegypti resulted in an increased level of resistance to spinosad, suggesting that the conserved nAChR α6 subunit is the target for spinosad insecticide. © 2020 Society of Chemical Industry.

First report of voltage‐gated sodium channel M918V and molecular diagnostics of nicotinic acetylcholine receptor R81T in the cotton aphid

AbstractThe cotton aphid, Aphis gossypii Glover, is one of the most important agricultural insect pests. Pyrethroid and neonicotinoid insecticides have generally shown excellent control of A. gossypii, but many populations of this pest have developed resistance against these classes of insecticides. The success of insecticide resistance management strategies requires detailed knowledge of both phenotype and genotype of the target insect pest. In this study, we attempted to understand the molecular status of insecticide resistance in cotton aphid populations in Xinjiang Uygur Autonomous Region of China, the major cotton planting region of China. In addition to the previously reported M918L mutation, we discovered another substitution (M918V) in the voltage‐gated sodium channel (VGSC). Moreover, we developed a molecular assay that could be used to detect precisely the R81T mutation in the nicotinic acetylcholine receptor (nAChR). This survey revealed that 918L was the predominant VGSC allele with a frequency ranging from 50.0% to 56.7%. Notably, appreciable frequencies (between 10% and 40%) of the resistance 81T allele of the nAChR gene were detected in three investigated populations. The prevalent co‐occurrence of both VGSC 918L/V and nAchR 81T indicates a worrisome situation of multiple resistance to both pyrethroids and neonicotinoids.

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in Bradysia odoriphaga

Bradysia odoriphaga is a destructive insect pest, damaging more than 30 crop species. Nicotinic acetylcholine receptors (nAChRs) mediating fast excitatory transmission in the central nervous system in insects are the molecular targets of some economically important insecticides including imidacloprid, which has been widely used to control B. odoriphaga in China since 2013. However, the clear characterization about nAChRs in B. odoriphaga is still unknown. Hence, our objective is to identify and characterize the nAChR gene family in B. odoriphaga based on the transcriptome database and sequence, phylogenetic and expression profiles analysis. In this study, we cloned seven nAChR subunit genes from B. odoriphaga, including Boα1, Boα2, Boα3, Boα7, Boα8, Boβ1 and Boβ3. Sequence analysis revealed that the seven nAChR subunits of B. odoriphaga shared the typical structural features with Drosophila melanogaster nAChR α1 subunit, including an extracellular N-terminal domain containing six functional loops (loop A-F), a signature Cys-loop with two disulfide bond-forming cysteines separated by 13 amino acid residues, and four typical transmembrane helices (TM1-TM4) in the C-terminal region. Phylogenetic analysis suggested that seven nAChR subunit genes in B. odoriphaga are evolutionarily conserved among four model insects, including D. melanogaster, Bombyx mori, Apis mellifera and Tribolium castaneum. Meanwhile, nAChR α4, α5, α6 and β2 subunit genes may potentially exist in B. odoriphaga, which need further study. Furthermore, quantitative real-time PCR analysis revealed the specific expression pattern of nAChR subunits in three body parts including head, thorax and abdomen, and developmental expression pattern of nAChR subunits throughout the B. odoriphaga life cycle. These results provided necessary information for further investigating the diverse functions of nAChRs in B. odoriphaga.

Massive expansion and diversity of nicotinic acetylcholine receptors in lophotrochozoans

BackgroundNicotinic acetylcholine receptors (nAChRs) are among the oldest and most conserved transmembrane receptors involved in signal transduction. Despite the prevalence and significance of cholinergic signaling, the diversity and evolution of nAChRs are not fully understood.ResultBy comparative genomic analysis, we found massive expansions of nAChR genes in molluscs and some other lophotrochozoans. The expansion is particularly pronounced in stationary bivalve molluscs with simple nervous systems, with the number of nAChR genes ranging from 99 to 217 in five bivalves, compared with 10 to 29 in five ecdysozoans and vertebrates. The expanded molluscan nAChR genes tend to be intronless and in tandem arrays due to retroposition followed by tandem duplication. Phylogenetic analysis revealed diverse nAChR families in the common ancestor of bilaterians, which subsequently experienced lineage-specific expansions or contractions. The expanded molluscan nAChR genes are highly diverse in sequence, domain structure, temporal and spatial expression profiles, implying diversified functions. Some molluscan nAChR genes are expressed in early development before the development of the nervous system, while others are involved in immune and stress responses.ConclusionThe massive expansion and diversification of nAChR genes in bivalve molluscs may be a compensation for reduced nervous systems as part of adaptation to stationary life under dynamic environments, while in vertebrates a subset of specialized nAChRs are retained to work with advanced nervous systems. The unprecedented diversity identified in molluscs broadens our view on the evolution and function of nAChRs that are critical to animal physiology and human health.