Nicotinic Acetylcholine Receptor Alpha Subunit Research Articles

Imidacloprid effects on acetylcholinesterase and nicotinic acetylcholine receptor in Apis mellifera. Experimental and molecular modeling approaches

Although the neonicotinoid insecticides have good selectivity towards insects rather than vertebrates, they have severe effects on honeybee production and pollination activities. Therefore, the effects of imidacloprid (IMI), the most used neonicotinoid, on the two main bioreceptors, acetylcholinesterase (AChE) and nicotinic acetylcholine receptor alpha subunit (nAChRα1) of honeybees were examined to identify their roles in honeybee toxicity and possible binding sites which assist in selecting and designing neonicotinoids. In vivo, IMI showed a high inhibitory effect on AChE (IC50 5.63 mg/L); however, the effect was much lower in vitro experiment (IC50 719 mg/L). This result induced us to examine the IMI effect on AChE gene expression which revealed that the AChE-2 gene expression was severely affected by IMI explaining the observed high enzyme inhibition. In addition, although toxicity increased by increasing exposure to IMI (LC50 2.9 mg/L after 4h and 0.75 mg/L after 48h), AChE was not elevated (IC50 5.63 and 5.52 mg/L respectively). Besides, Despite resuming most enzyme activity (77% during 2 h and 84.14% after 4 h), a high mortality level was observed with LC50 2.9 mg/L. These results reinforced that the observed high toxicity is a multifactor process. Accordingly, Molecular modeling and docking of IMI into honeybee AChE and nAChRα1were also performed to examine their possible interactions and identify the important binding sites. Results models indicated that the first two binding sites in AChE were found in the esteratic subunit in the active site explaining the observed in vitro inhibition. In nAChRα1, four of the highest five free energy binding sites are located in the large TM3-TM4 loop and one in the extracellular loops. Consequently, the present work revealed that IMI toxicity is attributed to various factors including direct interaction with both AChE and nAChRα1 as well as downregulating AChE-2 gene expression.

Biomarker development for neonicotinoid exposure in soil under interaction with the synergist piperonyl butoxide in Folsomia candida

Pesticide toxicity is typically assessed by exposing model organisms to individual compounds and measuring effects on survival and reproduction. These tests are time-consuming, labor-intensive, and do not accurately capture the effect of pesticide mixtures. Moreover, it is unfeasible to screen the nearly infinite combinations of mixtures for synergistic effects on model organisms. Therefore, reliable molecular indicators of pesticide exposure have to be identified, i.e., biomarkers. These biomarkers can form the basis of rapid and economical screening procedures to assess the toxicity of pesticides even under synergistic interaction with other pollutants. In this study, we screened the expression patterns of eight genes for suitability as a biomarker for neonicotinoid exposure in the soil ecotoxicological model Folsomia candida (springtails). Springtails were exposed to the neonicotinoids imidacloprid and thiacloprid either alone or with various levels of piperonyl butoxide (PBO), which inhibits cytochrome P450 enzymes (CYPs): a common point of synergistic interaction between neonicotinoid and other pesticides. First, we confirmed PBO as a potency enhancer for neonicotinoid toxicity to springtail fecundity, and then used it as a tool to confirm biomarker robustness. We identified two genes that are reliably indicative for neonicotinoid exposure even under metabolic inhibition of CYPs by PBO, nicotinic acetylcholine receptor–subunit alpha 1 (nAchR) and sodium-coupled monocarboxylate transporter (SMCT). These results can form the basis for developing high-throughput screening procedures for neonicotinoid exposure in varying mixture compositions.

Low doses of the organic insecticide spinosad trigger lysosomal defects, elevated ROS, lipid dysregulation, and neurodegeneration in flies.

Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit alpha 6 (nAChRα6), reducing the cholinergic response. We show that the nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.

Mechanosensation: Alpha-7 nAChR transduces sound signals in earless C. elegans

How do organisms without specialized auditory systems perceive and transduce sound? In this issue of Neuron, Iliff etal. (2021) investigate the functional mechanism of airborne sound sensation in Caenorhabditis elegans and highlight the crucial role of alpha-7 nicotinic acetylcholine receptor subunits in mechanotransduction.

Nicotinic Acetylcholine Receptor Subunit α7 Mediates Cigarette Smoke-Induced PD-L1 Expression in Human Bronchial Epithelial Cells.

Simple SummaryLung cancer is strongly associated with tobacco smoking. Nicotine in tobacco smoke can be transformed into a carcinogen after in vivo metabolism. However, the mechanism of how nicotine metabolites modulate a pro-tumorigenic microenvironment remains largely unknown. We have explored the effects of nicotine stimulation on nicotinic acetylcholine receptor subunit alpha 7 (nAChRα7)-mediated PD-L1 expression in human bronchial epithelial cells (HBECs). PD-L1 is an important protein contributing to cancer immune escape and is currently the key biomarker for lung cancer immunotherapy. Here, we found that the nicotine metabolite NNK can induce PD-L1 expression in HBECs by acting through the nicotine receptor nAChRα7. This study has a direct impact on the understanding of smoking-related changes in bronchial epithelial cells and potential immune escape in smoking-related carcinogenesis and has immuno-therapeutic implications via PD-L1 in lung cancer. Further studies are warranted to examine the upstream inhibition of PD-L1-related pathways, which could be mediated through nicotinic acetylcholine receptors in smokers, and may ultimately aid in the prevention and treatment of lung cancer.Tobacco smoking is the top risk factor for lung cancer development. Nicotine in cigarettes can induce addiction, and its derivatives become potent carcinogens after metabolic activation and activate oncogenic signaling in lung epithelial cells through their expressed nicotinic acetylcholine receptors (nAChRs). However, the effects of smoking on the tumor immune microenvironment are under investigation. In the current study, we investigated whether nicotine activation of nicotinic acetylcholine receptor subunit α7 (nAChRα7, CHRNA7) would induce PD-L1 expression in lung epithelial cells. The expression levels of nAChRα7 and PD-L1 in eight human bronchial epithelial cell (HBEC) lines were measured after treatment with cigarette smoke extract (CSE) or nicotine derivatives. The results showed that PD-L1 expression levels increased in HBECs after exposure to CSE or nicotine derivatives. This induction of PD-L1 expression could be diminished by treatment with CHRNA7 small-interfering RNA, and the relevant signaling was mediated via STAT3 phosphorylation and NRF2 expression. In summary, this study demonstrated that the well-known nicotine derivative-activated nAChRα7 could induce STAT3/NRF2 pathways and subsequently promote PD-L1 expression in normal lung epithelial cells. This information provides mechanistic insight into cigarette smoke-induced immune evasion in lung epithelial cells.

Altered neuronal physiology, development, and function associated with a common chromosome 15 duplication involving CHRNA7

BackgroundCopy number variants (CNVs) linked to genes involved in nervous system development or function are often associated with neuropsychiatric disease. While CNVs involving deletions generally cause severe and highly penetrant patient phenotypes, CNVs leading to duplications tend instead to exhibit widely variable and less penetrant phenotypic expressivity among affected individuals. CNVs located on chromosome 15q13.3 affecting the alpha-7 nicotinic acetylcholine receptor subunit (CHRNA7) gene contribute to multiple neuropsychiatric disorders with highly variable penetrance. However, the basis of such differential penetrance remains uncharacterized. Here, we generated induced pluripotent stem cell (iPSC) models from first-degree relatives with a 15q13.3 duplication and analyzed their cellular phenotypes to uncover a basis for the dissimilar phenotypic expressivity.ResultsThe first-degree relatives studied included a boy with autism and emotional dysregulation (the affected proband-AP) and his clinically unaffected mother (UM), with comparison to unrelated control models lacking this duplication. Potential contributors to neuropsychiatric impairment were modeled in iPSC-derived cortical excitatory and inhibitory neurons. The AP-derived model uniquely exhibited disruptions of cellular physiology and neurodevelopment not observed in either the UM or unrelated controls. These included enhanced neural progenitor proliferation but impaired neuronal differentiation, maturation, and migration, and increased endoplasmic reticulum (ER) stress. Both the neuronal migration deficit and elevated ER stress could be selectively rescued by different pharmacologic agents. Neuronal gene expression was also dysregulated in the AP, including reduced expression of genes related to behavior, psychological disorders, neuritogenesis, neuronal migration, and Wnt, axonal guidance, and GABA receptor signaling. The UM model instead exhibited upregulated expression of genes in many of these same pathways, suggesting that molecular compensation could have contributed to the lack of neurodevelopmental phenotypes in this model. However, both AP- and UM-derived neurons exhibited shared alterations of neuronal function, including increased action potential firing and elevated cholinergic activity, consistent with increased homomeric CHRNA7 channel activity.ConclusionsThese data define both diagnosis-associated cellular phenotypes and shared functional anomalies related to CHRNA7 duplication that may contribute to variable phenotypic penetrance in individuals with 15q13.3 duplication. The capacity for pharmacological agents to rescue some neurodevelopmental anomalies associated with diagnosis suggests avenues for intervention for carriers of this duplication and other CNVs that cause related disorders.

Not Goanna Get Me: Mutations in the Savannah Monitor Lizard (Varanus exanthematicus) Nicotinic Acetylcholine Receptor Confer Reduced Susceptibility to SympatricCobra Venoms.

Antagonistic coevolutionary relationships provide intense selection pressure which drive changes in the genotype. Predator-prey interactions have caused some venomous snakes and their predators/prey to evolve α-neurotoxin resistance through changes at the orthosteric site of nicotinic acetylcholine receptors. The presence of negatively charged amino acids at orthosteric site positions 191 and 195 is the ancestral state. These negatively charged amino acids have exerted a selection pressure for snake venom α-neurotoxins to evolve with strong positive charges on their molecular surface, with the opposite-charge attraction facilitating the binding by the neurotoxins. We aimed to test the effects of a series of mutations whereby one or both negatively charged amino acids are replaced by uncharged residues to ascertain if this was a novel form of reduced venom susceptibility in the varanid species. Using a biolayer interferometry assay, we tested the relative binding of α-neurotoxin-rich snake venoms against the orthosteric sites of V. giganteus (Perentie) and V. komodoensis (Komodo dragon), which both possess the negatively charged aspartic acid at position 191; V. mertensi (Merten's water monitor), which alsohas aspartic acid at position 195; and Varanus exanthematicus (savannah monitor), which lacks negatively charged amino acids at both positions 191 and 195. The orthosteric sites of these species are otherwise identical. In order to complete the structure-function relationship examination, we also tested a mutant version with the negatively charged aspartic acid at both positions 191 and 195. It was demonstrated that the presence of a negatively charged amino acid at either position 191 or 195 is crucial for the successful binding of snake venom α-neurotoxins, with V. giganteus, V. komodoensis and V. mertensi all strongly bound. The mutant version containing a negatively charged amino acid at both positions was bound equipotently to the native forms of V. giganteus, V. komodoensis and V. mertensi. Thus, the presence of a negatively charged amino acid at both positions does not increase binding affinity. In contrast, Varanus exanthematicus, lacking a negatively charged amino acid at either position, displayed dramatically less sensitivity to neurotoxins compared with the other species. V. exanthematicus is distinguished from the other species examined in this study by being a small, terrestrial, slow-moving species living sympatrically with a high density of large cobra species that have neurotoxin-rich venoms. Thus, this vulnerable prey item seems to haveevolved a novel form of reduced susceptibility to snake venom neurotoxins under a strong selection pressures from these neurotoxic predators. These results therefore contribute to the body of knowledge of predator/prey chemical arm races while providing novel insights into the structure-activity relationships of the orthosteric site of the nicotinic acetylcholine receptor alpha-subunit.

CHRNA5 belongs to the secondary estrogen signaling network exhibiting prognostic significance in breast cancer.

Cholinergic signals can be important modulators of cellular signaling in cancer. We recently have shown that knockdown of nicotinic acetylcholine receptor subunit alpha 5, CHRNA5, diminishes the proliferative potential of breast cancer cells. However, modulation of CHRNA5 expression in the context of estrogen signaling and its prognostic implications in breast cancer remained unexplored. Meta-analyses of large breast cancer microarray cohorts were used to evaluate the association of CHRNA5 expression with estrogen (E2) treatment, estrogen receptor (ER) status and patient prognosis. The results were validated through RT-qPCR analyses of multiple E2 treated cell lines, CHRNA5 depleted MCF7 cells and across a breast cancer patient cDNA panel. We also calculated a predicted secondary (PS) score representing direct/indirect induction of gene expression by E2 based on a public dataset (GSE8597). Co-expression analysis was performed using a weighted gene co-expression network analysis (WGCNA) pipeline. Multiple other publicly available datasets such as CCLE, COSMIC and TCGA were also analyzed. Herein we found that CHRNA5 expression was induced by E2 in a dose- and time-dependent manner in breast cancer cell lines. ER- breast tumors exhibited higher CHRNA5 expression levels than ER+ tumors. Independent meta-analysis for survival outcome revealed that higher CHRNA5 expression was associated with a worse prognosis in untreated breast cancer patients. Furthermore, CHRNA5 and its co-expressed gene network emerged as secondarily induced targets of E2 stimulation. These targets were largely downregulated by exposure to CHRNA5 siRNA in MCF7 cells while the response of primary ESR1 targets was dependent on the direction ofthe PS-score. Moreover, primary and secondary target genes were uncoupled and clustered distinctly based on multiple public datasets. Our findings strongly associate increased expression of CHRNA5 and its co-expression network with secondary E2 signaling and a worse prognosis in breast cancer.

Validation of gene editing efficiency with CRISPR-Cas9 system directly in rat zygotes using electroporation mediated delivery and embryo culture

Successful use of the CRISPR-Cas9 system for gene manipulation relies on identifying effective and efficient guide RNA sequences (gRNAs). When the goal is to create transgenic animal/rodent models by knocking-in desired sequences using homology-directed repair (HDR), selecting effective guides becomes even more critical to minimize developmental time and resources. Currently, validation experiments for gRNAs for generating rat models are carried out using immortalized rat cells. However, there are several limitations with using such cell lines, including ploidy of the genome, non-predictive transfection efficiency, and the ability to identify gene modifications efficiently within diverse cell populations. Since embryos are authentic representatives of live animals compared to cell lines, validating CRISPR guides for their nuclease activity in freshly isolated embryos will provide greater accuracy of in vivo gene editing efficiency. In contrast to microinjections, delivery by electroporation is a more accessible method that can be simple and does not require special skills and equipment. We demonstrate an accessible workflow to either delete or edit target genes in vivo in rats using the efficient editing of a human mutation in alpha7 nicotinic acetylcholine receptor subunit (CHRNA7) ortholog using electroporation as a delivery method for CRISPR-Cas9 ribonucleoprotein complexes in rat embryos.•Upon identifying CRISPR targets at the desired genetic alteration site, we designed homologydriven repair (HDR) templates for effective and easy identification of gene editing by Restriction Fragment Length Polymorphism (RFLP).•Cultured rat embryos can be genotyped to assess CRISPR activity as seen by either presence of indels resulting from NHEJ or knock-in of repair template resulting from homology driven repair.•Heteroduplex mobility assay (HMA) and Restriction Fragment Length Polymorphism (RFLP) of PCR products can be performed reliably and reproducibly at a low-cost.

Targeting the cholinergic anti-inflammatory pathway with vagus nerve stimulation in patients with Covid-19?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), at the origin of the worldwide COVID-19 pandemic, is characterized by a dramatic cytokine storm in some critical patients with COVID-19. This storm is due to the release of high levels of pro-inflammatory cytokines such as interleukin (IL)-1 β, IL-6, tumor necrosis factor (TNF), and chemokines by respiratory epithelial and dendritic cells, and macrophages. We hypothesize that this cytokine storm and the worsening of patients’ health status can be dampened or even prevented by specifically targeting the vagal-driven cholinergic anti-inflammatory pathway (CAP). The CAP is a concept that involves an anti-inflammatory effect of vagal efferents by the release of acetylcholine (ACh). Nicotinic acetylcholine receptor alpha7 subunit (α7nAChRs) is required for ACh inhibition of macrophage-TNF release and cytokine modulation. Hence, targeting the α7nAChRs through vagus nerve stimulation (VNS) could be of interest in the management of patients with SARS-CoV-2 infection. Indeed, through the wide innervation of the organism by the vagus nerve, especially the lungs and gastrointestinal tract, VNS appears as a serious candidate for a few side effect treatment that could dampen or prevent the cytokine storm observed in COVID-19 patients with severe symptoms. Finally, a continuous vagal tone monitoring in patients with COVID-19 could be used as a predictive marker of COVID-19 illness course but also as a predictive marker of response to COVID-19 treatment such as VNS or others.

Neuropsychiatric factor and polymorphism gene in internet addiction

Internet addiction (IA) has been described as ‘excessive or poorly controlled preoccupation, urges, or behaviors regarding computer use and internet access that lead to impairment or distress. Excessive internet user has emerged as a leading cause of behavioral and developmental problems in adolescents. There are several factors that influence the occurrence of internet addiction. The risk factors can be categorized by a social factor, psychiatric factor and biological factor. Some studies have found varied results. Molecular and functional imaging techniques have been increasingly used for the analysis of neurobiological changes and neurochemical correlates of IA. MRI (Magnetic Resonance Imaging) studies demonstrate that structural changes in the frontal cortex are associated with functional abnormalities in Internet-addicted subjects. Recent research has implicated the role of the striatal dopaminergic system in the behavioral maladaptation associated with IA. It provided evidence for a molecular genetic link between serotonergic and dopaminergic neurotransmission and Internet addiction like dopamine polymorphism the DRD2/ANKKI Taq Ia and COMT Val158metpolymorphism and the gene coding for the nicotinic acetylcholine receptor subunit alpha 4 (CHRNA4). As both the genetic and personality traits are known to play a role in other addiction it is likely that same mechanism (biopsychosocial) underlie Internet addiction.

Involvement of the Cholinergic Parameters and Glial Cells in Learning Delay Induced by Glutaric Acid: Protection by N-Acetylcysteine.

Dysfunction of basal ganglia neurons is a characteristic of glutaric acidemia type I (GA-I), an autosomal recessive inherited neurometabolic disease characterized by deficiency of glutaryl-CoA dehydrogenase (GCDH) and accumulation of glutaric acid (GA). The affected patients present clinical manifestations such as motor dysfunction and memory impairment followed by extensive striatal neurodegeneration. Knowing that there is relevant striatal dysfunction in GA-I, the purpose of the present study was to verify the performance of young rats chronically injected with GA in working and procedural memory test, and whether N-acetylcysteine (NAC) would protect against impairment induced by GA. Rat pups were injected with GA (5μmolg body weight-1, subcutaneously; twice per day; from the 5th to the 28th day of life) and were supplemented with NAC (150mg/kg/day; intragastric gavage; for the same period). We found that GA injection caused delay procedural learning; increase of cytokine concentration, oxidative markers, and caspase levels; decrease of antioxidant defenses; and alteration of acetylcholinesterase (AChE) activity. Interestingly, we found an increase in glial cell immunoreactivity and decrease in the immunoreactivity of nuclear factor-erythroid 2-related factor 2 (Nrf2), nicotinic acetylcholine receptor subunit alpha 7 (α7nAChR), and neuronal nuclei (NeuN) in the striatum. Indeed, NAC administration improved the cognitive performance, ROS production, neuroinflammation, and caspase activation induced by GA. NAC did not prevent neuronal death, however protected against alterations induced by GA on Iba-1 and GFAP immunoreactivities and AChE activity. Then, this study suggests possible therapeutic strategies that could help in GA-I treatment and the importance of the striatum in the learning tasks.

Nicotinic Acetylcholine Receptor Alpha7 Subunit Mediates Vagus Nerve Stimulation-Induced Neuroprotection in Acute Permanent Cerebral Ischemia by a7nAchR/JAK2 Pathway.

BackgroundThe role of nicotinic acetylcholine receptor alpha7 subunit (a7nAchR) in the treatment of acute cerebral ischemia by VNS has not been thoroughly clarified to date. Therefore, this study aimed to investigate the specific role of a7nAchR and explore whether this process is involved in the mechanisms of VNS-induced neuroprotection in rats undergoing permanent middle cerebral artery occlusion (PMCAO) surgery.Material/MethodsRats received a7nAChR antagonist (A) or antagonist placebo injection for control (AC), followed by PMCAO and VNS treatment, whereas the a7nAChR agonist (P) was utilized singly without VNS treatment but only with PMCAO pretreatment. The rats were randomly divided into 6 groups: sham PMCAO, PMCAO, PMCAO+VNS, PMCAO+VNS+A, PMCAO+VNS+AC, and PMCAO+P. Neurological function and cerebral infarct volume were measured to evaluate the level of brain injury at 24 h after PMCAO or PMCAO-sham. Moreover, the related proteins levels of a7nAChR, p-JAK2, and p-STAT3 in the ischemic penumbra were assessed by Western blot analysis.ResultsRats pretreated with VNS had significantly improved neurological function and reduced cerebral infarct volume after PMCAO injury (p<0.05). In addition, VNS enhanced the levels of a7nAchR, p-JAK2, and p-STAT3 in the ischemic penumbra (p<0.05). However, inhibition of a7nAchR not only attenuated the beneficial neuroprotective effects induced by VNS, but also decreased levels of p-JAK2 and p-STAT3. Strikingly, pharmacological activation of a7nAchR can partially substitute for VNS-induced beneficial neurological protection.ConclusionsThese results suggest that a7nAchR is a pivotal mediator of VNS-induced neuroprotective effects on PMCAO injury, which may be related to suppressed inflammation via activation of the a7nAchR/JAK2 anti-inflammatory pathway.

A Drosophila model for developmental nicotine exposure.

Despite the known health risks of tobacco smoking, many people including pregnant women continue smoking. The effects of developmental nicotine exposure are known, but the underlying mechanisms are not well understood. Drosophila melanogaster is a model organism that can be used for uncovering genetic and molecular mechanisms for drugs of abuse. Here I show that Drosophila can be a model to elucidate the mechanisms for nicotine’s effects on a developing organism. Drosophila reared on nicotine food display developmental and behavioral effects similar to those in mammals including decreased survival and weight, increased developmental time, and decreased sensitivity to acute nicotine and ethanol. The Drosophila nicotinic acetylcholine receptor subunit alpha 7 (Dα7) mediates some of these effects. A novel role for Dα7 on ethanol sedation in Drosophila is also shown. Future research taking advantage of the genetic and molecular tools for Drosophila will allow additional discovery of the mechanisms behind the effects of nicotine during development.

Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample.

Variation in genes coding for nicotinic acetylcholine receptor (nAChR) subunits affect cognitive processes and may contribute to the genetic architecture of neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs) in the CHRNA4 gene that codes for the alpha4 subunit of alpha4/beta2-containing receptors have previously been implicated in aspects of (mostly visual) attention and smoking-related behavioral measures. Here we investigated the effects of six synonymous but functional CHRNA4 exon 5 SNPs on the N100 event-related potential (ERP), an electrophysiological endophenotype elicited by a standard auditory oddball. A total of N = 1,705 subjects randomly selected from the general population were studied with electroencephalography (EEG) as part of the German Multicenter Study on nicotine addiction. Two of the six variants, rs1044396 and neighboring rs1044397, were significantly associated with N100 amplitude. This effect was pronounced in females where we also observed an effect on reaction time. Sequencing of the complete exon 5 region in the population sample excluded the existence of additional/functional variants that may be responsible for the observed effects. This is the first large-scale population-based study investigation the effects of CHRNA4 SNPs on brain activity measures related to stimulus processing and attention. Our results provide further evidence that common synonymous CHRNA4 exon 5 SNPs affect cognitive processes and suggest that they also play a role in the auditory system. As N100 amplitude reduction is considered a schizophrenia-related endophenotype the SNPs studied here may also be associated with schizophrenia outcome measures.

Variants near CHRNA3/5 and APOE have age- and sex-related effects on human lifespan.

Lifespan is a trait of enormous personal interest. Research into the biological basis of human lifespan, however, is hampered by the long time to death. Using a novel approach of regressing (272,081) parental lifespans beyond age 40 years on participant genotype in a new large data set (UK Biobank), we here show that common variants near the apolipoprotein E and nicotinic acetylcholine receptor subunit alpha 5 genes are associated with lifespan. The effects are strongly sex and age dependent, with APOE ɛ4 differentially influencing maternal lifespan (P=4.2 × 10−15, effect −1.24 years of maternal life per imputed risk allele in parent; sex difference, P=0.011), and a locus near CHRNA3/5 differentially affecting paternal lifespan (P=4.8 × 10−11, effect −0.86 years per allele; sex difference P=0.075). Rare homozygous carriers of the risk alleles at both loci are predicted to have 3.3–3.7 years shorter lives.

Expression of nicotinic acetylcholine receptor subunits alpha 4, alpha 7 and beta 2 in human internal mammary arteries of non-smokers and smokers

Nicotinic acetylcholine receptors (nAChR) are widely expressed in non-neuronal tissue, but data about their expression in vascular tissue are rare. To study the expression of nAChR α4, α7 and β2 in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in human arteries of smokers and non-smokers, arteriae thoracicae internae dissected for coronary artery bypass grafting were analysed immunhistochemically and by polymerase chain reaction (PCR). ECs of the tunica intima and the vasa vasorum as well as the VSMCs of the tunica media showed clear staining for nAChR α4, α7 and β2, without significant differences between non-smokers and smokers in all vascular layers. Further verification of nAChR α4, α7 and β2 expression of whole-tissue homogenates using PCR analysis showed no differences in the subtype expression between non-smokers and smokers. This provides an important basis for further investigations using positron electron tomography (PET) tracers for imaging of nAChRs in vascular health and disease.

HnRNP L and hnRNP LL antagonistically modulate PTB-mediated splicing suppression of CHRNA1 pre-mRNA

CHRNA1 gene, encoding the muscle nicotinic acetylcholine receptor alpha subunit, harbors an inframe exon P3A. Inclusion of exon P3A disables assembly of the acetylcholine receptor subunits. A single nucleotide mutation in exon P3A identified in congenital myasthenic syndrome causes exclusive inclusion of exon P3A. The mutation gains a de novo binding affinity for a splicing enhancing RNA-binding protein, hnRNP LL, and displaces binding of a splicing suppressing RNA-binding protein, hnRNP L. The hnRNP L binds to another splicing repressor PTB through the proline-rich region and promotes PTB binding to the polypyrimidine tract upstream of exon P3A, whereas hnRNP LL lacking the proline-rich region cannot bind to PTB. Interaction of hnRNP L with PTB inhibits association of U2AF65 and U1 snRNP with the upstream and downstream of P3A, respectively, which causes a defect in exon P3A definition. HnRNP L and hnRNP LL thus antagonistically modulate PTB-mediated splicing suppression of exon P3A.

A novel prophylactic effect of furosemide treatment on autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)

The transgenic rat strain S284L-TG harbors the S284L mutant of the neuronal nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4), which is responsible for human autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). S284L-TG rats have epileptic seizure phenotypes during slow-wave sleep, similar to those in NFLE. We previously demonstrated that γ-aminobutyric acid (GABA)ergic action of these rats was suppressed before the onset of ADNFLE seizures, and that glutamate release in the epileptic focus lesion was increased at the onset of epilepsy. Here, mRNA analysis revealed that Cl(-)-accumulating Na-K-2Cl cotransporter 1 (NKCC1) levels were increased and Cl(-)-extruding K-Cl cotransporter 1 and 2 (KCC1 and KCC2) levels were decreased at the onset of ADNFLE seizures in S284L-TG rat frontal cortexes, which perturbed the GABAergic inhibitory system. The reversal potentials (EGABA) of GABAA receptor-mediated currents in cortical layer V pyramidal neurons of S284L-TG rats also changed their polarity from hyperpolarization to depolarization, and S284L-TG miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs), significantly increased in both amplitude and frequency. Administration of 25mg/kg/day furosemide before, but not after, the onset of interictal discharges prevented idiopathic epileptic activity, reversed the depolarizing shift of EGABA and increased mEPSC amplitude to normal levels. These data indicate that early treatment with an agent that normalizes pathogenesis has a prophylactic effect on epilepsy. We propose a strategy for prophylactic medication against idiopathic epilepsy through the suppression of epileptogenesis and/or ictogenesis.

The Role of the CHRNA4 Gene in Internet Addiction

Recent studies from Asia provided first evidence for a molecular genetic link between serotonergic and dopaminergic neurotransmission and Internet addiction. The present report offers data on a new candidate gene in the investigation of Internet addiction-the gene coding for the nicotinic acetylcholine receptor subunit alpha 4 (CHRNA4). A case-control study was carried out. The participants were recruited from a large gene data bank, including people from the general population and from a university setting. A total of 132 participants with problematic Internet use and 132 age- and sex-matched controls participated in the study. Participants provided DNA samples and filled in the Internet Addiction Test Questionnaire. The T- variant (CC genotype) of the rs1044396 polymorphism on the CHRNA4 gene occurred significantly more frequently in the case group. Further analyses revealed that this effect was driven by females. Combined with the findings from other studies, the present data point in the direction that rs1044396 exerts pleiotropic effects on a vast range of behaviors, including cognition, emotion, and addiction.