Nicotinic Acetylcholine Receptor Subunit Genes Research Articles

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.

Dual nicotinic acetylcholine receptor subunit gene knockouts reveal limits to functional redundancy

The nicotinic acetylcholine receptor (nAChR) subunit gene family consists of ten members in Drosophila melanogaster. The mature nAChR is a pentamer assembled from these subunits. Despite recent advances in the in vitro expression of some receptor subunit combinations (nAChR subtypes), the in vivo combinations and stoichiometry of these subtypes remains poorly defined. In addition, there are many potential nAChR signalling roles for different subtypes in insect behaviour, development and physiology. Prior work has shown that nAChR subunit mutants can display altered sleep and mating behaviour, disrupted hormone signalling and reduced locomotion, climbing ability and longevity. Teasing out the specific receptor subunits that are involved in these different functions is potentially made more difficult given that the structural similarity between members of gene families often means that there is a degree of functional redundancy. In order to circumvent this, we created a dual knockout strain for the Dα1 and Dβ2 nAChR subunit genes and examined four traits including insecticide resistance. These subunits had been previously implicated in the response to a neonicotinoid insecticide, imidacloprid. The use of the dual knockout revealed that Dα1 and Dβ2 subunits are involved in signalling that leads to the inflation of wings following adult emergence from the pupal case. The Dβ1 subunit had previously been implicated as a contributor to this function. The lack of a phenotype or low penetrance of the phenotype in the Dα1 and Dβ2 single mutants compared to the dual knockout suggests that these subunits are, to some extent, functionally redundant. We also observed stronger reductions in climbing ability and longevity in the dual knockout. Our findings demonstrate that a dual knockout approach to examining members of the nAChR subunit gene family may increase the power of genetic approaches linking individual subunits and combinations thereof to particular biological functions. This approach will be valuable as the nAChRs are so widely expressed in the insect brain that they are likely to have many functions that hereto remain undetected.

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.

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.

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.

The Power of Drosophila melanogaster for Modeling Neonicotinoid Effects on Pollinators and Identifying Novel Mechanisms.

Neonicotinoids are the most widely used insecticides in the world and are implicated in the widespread population declines of insects including pollinators. Neonicotinoids target nicotinic acetylcholine receptors which are expressed throughout the insect central nervous system, causing a wide range of sub-lethal effects on non-target insects. Here, we review the potential of the fruit fly Drosophila melanogaster to model the sub-lethal effects of neonicotinoids on pollinators, by utilizing its well-established assays that allow rapid identification and mechanistic characterization of these effects. We compare studies on the effects of neonicotinoids on lethality, reproduction, locomotion, immunity, learning, circadian rhythms and sleep in D. melanogaster and a range of pollinators. We also highlight how the genetic tools available in D. melanogaster, such as GAL4/UAS targeted transgene expression system combined with RNAi lines to any gene in the genome including the different nicotinic acetylcholine receptor subunit genes, are set to elucidate the mechanisms that underlie the sub-lethal effects of these common pesticides. We argue that studying pollinators and D. melanogaster in tandem allows rapid elucidation of mechanisms of action, which translate well from D. melanogaster to pollinators. We focus on the recent identification of novel and important sublethal effects of neonicotinoids on circadian rhythms and sleep. The comparison of effects between D. melanogaster and pollinators and the use of genetic tools to identify mechanisms make a powerful partnership for the future discovery and testing of more specific insecticides.

Inhibiting the proteasome reduces molecular and biological impacts of the natural product insecticide, spinosad.

Insecticide targets are often identified by mutations that confer resistance, but the intricacies of insecticide binding and downstream processes leading to insect death often remain obscure. Mutations in α6-like nicotinic acetylcholine receptor subunit genes have been associated with high levels of resistance to spinosad in many insect species, including Drosophila melanogaster. Here, we aimed to expand our understanding of the effects of the natural product insecticide spinosad on its protein target, the α6 subunit, using genetic tools available in D. melanogaster. Functional, fluorescently tagged Dα6 subunits (Dα6YFP ) were developed to allow observation of the protein in vivo. Larvae expressing Dα6YFP were exposed to a sub-lethal concentration of spinosyn A (0.025 ppm) for 6 days, leading to a 64% reduction in fluorescence relative to unexposed larvae. Direct application of high doses of spinosyn A to dissected larval brains resulted in a visible 38.25% decrease in Dα6YFP within 20 min, indicating that degradation of the Dα6 protein occurred in response to spinosyn A exposure. Chemical inhibition of the proteasome system using the multiple myeloma treatment drug, PS-341 reduced loss of Dα6YFP in response to spinosyn A at the 20-min time point to 6.35%. In addition, in vivo administration of PS-341 prior to spinosad exposure reduced the effect of spinosad on larval activity. Based on these data, we propose that exposure to spinosad leads to degradation of the α6-like target protein, a potentially novel element in the mode of action of spinosyns that may contribute to their toxicity towards insects. © 2021 Society of Chemical Industry.

α3* Nicotinic Acetylcholine Receptors in the Habenula-Interpeduncular Nucleus Circuit Regulate Nicotine Intake.

Allelic variation in CHRNA3, the gene encoding the α3 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to tobacco dependence and smoking-related diseases, but little is known about the role for α3-containing (α3*) nAChRs in regulating the addiction-related behavioral or physiological actions of nicotine. α3* nAChRs are densely expressed by medial habenula (mHb) neurons, which project almost exclusively to the interpeduncular nucleus (IPn) and are known to regulate nicotine avoidance behaviors. We found that Chrna3tm1.1Hwrt hypomorphic mice, which express constitutively low levels of α3* nAChRs, self-administer greater quantities of nicotine (0.4 mg kg-1 per infusion) than their wild-type littermates. Microinfusion of a lentivirus vector to express a short-hairpin RNA into the mHb or IPn to knock-down Chrna3 transcripts markedly increased nicotine self-administration behavior in rats (0.01-0.18 mg kg-1 per infusion). Using whole-cell recordings, we found that the α3β4* nAChR-selective antagonist α-conotoxin AuIB almost completely abolished nicotine-evoked currents in mHb neurons. By contrast, the α3β2* nAChR-selective antagonist α-conotoxin MII only partially attenuated these currents. Finally, micro-infusion of α-conotoxin AuIB (10 μm) but not α-conotoxin MII (10 μm) into the IPn in rats increased nicotine self-administration behavior. Together, these data suggest that α3β4* nAChRs regulate the stimulatory effects of nicotine on the mHb-IPn circuit and thereby regulate nicotine avoidance behaviors. These findings provide mechanistic insights into how CHRNA3 risk alleles can increase the risk of tobacco dependence and smoking-related diseases in human smokers.SIGNIFICANCE STATEMENT Allelic variation in CHRNA3, which encodes the α3 nicotinic acetylcholine receptor (nAChR) subunit gene, increases risk of tobacco dependence but underlying mechanisms are unclear. We report that Chrna3 hypomorphic mice consume greater quantities of nicotine than wild-type mice and that knock-down of Chrna3 gene transcripts in the habenula or interpeduncular nucleus (IPn) increases nicotine intake in rats. α-Conotoxin AuIB, a potent antagonist of the α3β4 nAChR subtype, reduced the stimulatory effects of nicotine on habenular neurons, and its infusion into the IPn increased nicotine intake in rats. These data suggest that α3β4 nAChRs in the habenula-IPn circuit regulate the motivational properties of nicotine.

Dissecting the genetic overlap of smoking behaviors, lung cancer, and chronic obstructive pulmonary disease: A focus on nicotinic receptors and nicotine metabolizing enzyme.

Smoking is a major contributor to lung cancer and chronic obstructive pulmonary disease (COPD). Two of the strongest genetic associations of smoking-related phenotypes are the chromosomal regions 15q25.1, encompassing the nicotinic acetylcholine receptor subunit genes CHRNA5-CHRNA3-CHRNB4, and 19q13.2, encompassing the nicotine metabolizing gene CYP2A6. In this study, we examined genetic relations between cigarettes smoked per day, smoking cessation, lung cancer, and COPD. Data consisted of genome-wide association study summary results. Genetic correlations were estimated using linkage disequilibrium score regression software. For each pair of outcomes, z-score-z-score (ZZ) plots were generated. Overall, heavier smoking and decreased smoking cessation showed positive genetic associations with increased lung cancer and COPD risk. The chromosomal region 19q13.2, however, showed a different correlational pattern. For example, the effect allele-C of the sentinel SNP (rs56113850) within CYP2A6 was associated with an increased risk of heavier smoking (z-score = 19.2; p = 1.10 × 10-81 ), lung cancer (z-score = 8.91; p = 5.02 × 10-19 ), and COPD (z-score = 4.04; p = 5.40 × 10-5 ). Surprisingly, this allele-C (rs56113850) was associated with increased smoking cessation (z-score = -8.17; p = 2.52 × 10-26 ). This inverse relationship highlights the need for additional investigation to determine how CYP2A6 variation could increase smoking cessation while also increasing the risk of lung cancer and COPD likely through increased cigarettes smoked per day.

Modulation of "Protective" Nicotine Perception and Use Profile by Flavorants: Preliminary Findings in E-cigarettes.

Characterizing flavors are widely available in e-cigarettes and motivate initiation and continued use. Flavors may enhance appeal and facilitate development of addiction to tobacco products through modulation of tobacco products' reinforcing or aversive actions. Palatable flavors (eg, fruit) may increase appeal through primary reinforcing properties. Menthol's cooling and anesthetic effects may increase appeal by counteracting nicotine's aversive effects. Genetics provide a method for modeling individual differences in sensitivity to nicotine's effects. A common polymorphism, rs16969968, encoded in the α5 nicotinic acetylcholine receptor subunit gene (CHRNA5), is a well-recognized marker for smoking risk and reduces sensitivity to nicotine aversiveness. This pilot study tested how flavors impacted e-cigarette appeal and self-administration. In a single testing day, cigarette smokers (N = 32; 94% menthol-smokers) self-administered e-cigarettes containing e-liquids differing in nicotine level (0 mg/mL, 24 mg/mL) and flavor (unflavored, menthol, fruit-flavored) within directed and ad libitum e-cigarette paradigms. Subjective drug effects, number of puffs, rs16969968 genotype, plasma nicotine, and menthol glucuronide levels were collected. Menthol partially ameliorated nicotine aversiveness; fruit did not. In nicotine's absence, fruit flavor increased self-reported preference and ad libitum use relative to menthol-containing or unflavored e-liquids. Individuals with high-smoking-risk rs16969968 genotype (N = 7) reported greater craving alleviation following directed administration of nicotine-containing e-liquids, showed a trend rating nicotine-containing e-liquids as less harsh, and self-administered more nicotine during ad libitum compared to individuals with low-smoking-risk genotype (N = 23). While menthol countered aversiveness of nicotine-containing e-liquids, fruit flavor increased appeal of nicotine-free e-liquids. These preliminary findings suggest menthol and fruit flavor increase e-cigarettes' appeal through distinct mechanisms. This study provides a detailed characterization of the effects of flavors (unflavored, menthol, fruit), nicotine (0 mg/mL, 24 mg/mL) and their interactions on the subjective drug effects and ad libitum self-administration of e-cigarettes. Genetics were used to assess these effects in higher-smoking-risk (diminished sensitivity to nicotine aversiveness) and lower-risk groups. Findings could inform impact of regulation of flavors or nicotine in e-cigarettes, and their impacts on vulnerable sub-populations.

Exploring the role of low-frequency and rare exonic variants in alcohol and tobacco use

BackgroundAlcohol and tobacco use are heritable phenotypes. However, only a small number of common genetic variants have been identified, and common variants account for a modest proportion of the heritability. Therefore, this study aims to investigate the role of low-frequency and rare variants in alcohol and tobacco use. MethodsWe meta-analyzed ExomeChip association results from eight discovery cohorts and included 12,466 subjects and 7432 smokers in the analysis of alcohol consumption and tobacco use, respectively. The ExomeChip interrogates low-frequency and rare exonic variants, and in addition a small pool of common variants. We investigated top variants in an independent sample in which ICD-9 diagnoses of “alcoholism” (N = 25,508) and “tobacco use disorder” (N = 27,068) had been assessed. In addition to the single variant analysis, we performed gene-based, polygenic risk score (PRS), and pathway analyses. ResultsThe meta-analysis did not yield exome-wide significant results. When we jointly analyzed our top results with the independent sample, no low-frequency or rare variants reached significance for alcohol consumption or tobacco use. However, two common variants that were present on the ExomeChip, rs16969968 (p = 2.39 × 10−7) and rs8034191 (p = 6.31 × 10−7) located in CHRNA5 and AGPHD1 at 15q25.1, showed evidence for association with tobacco use. DiscussionLow-frequency and rare exonic variants with large effects do not play a major role in alcohol and tobacco use, nor does the aggregate effect of ExomeChip variants. However, our results confirmed the role of the CHRNA5-CHRNA3-CHRNB4 cluster of nicotinic acetylcholine receptor subunit genes in tobacco use.

Cholinergic receptor nicotinic alpha 5 subunit polymorphisms are associated with smoking cessation success in women

BackgroundThe identification of variants in the nicotinic acetylcholine receptor (nAChR) subunit genes associated with smoking phenotypes are increasingly important for prevention and treatment of nicotine dependence. In the context of personalized medicine, the aims of this study were to evaluate whether cholinergic receptor nicotinic alpha 2 (CHRNA2), cholinergic receptor nicotinic alpha 3 (CHRNA3), cholinergic receptor nicotinic alpha 5 (CHRNA5) and cholinergic receptor nicotinic beta 3 (CHRNB3) polymorphisms were associated with nicotine dependence severity, and to investigate possible pharmacogenetics markers of smoking cessation treatment.MethodsThis study cohort enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores (Issa score) were analyzed for nicotine dependence. CHRNA2 (rs2472553), CHRNA3 (rs1051730), CHRNA5 (rs16969968 and rs2036527) and CHRNB3 (rs6474413) polymorphisms were genotyped by high resolution melting analysis.ResultsFemales with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527 polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively, compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P = 0.03, n = 389; P = 0.01, n = 391). The GA or AA genotypes for the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR = 1.63; 95% CI = 1.04 to 2.54; P = 0.03 and OR = 1.59, 95% CI = 1.02 to 2.48; P = 0.04; respectively). We did not find association of these polymorphisms with nicotine dependence related scores. Polymorphisms in the CHRNA2, CHRNA3 and CHRNB3 genes were not associated with the phenotypes studied.ConclusionCHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These findings might contribute to advances in personalized medicine.

Smoking Interacts With CHRNA5, a Nicotinic Acetylcholine Receptor Subunit Gene, to Influence the Risk of IBD-Related Surgery.

BackgroundInflammatory bowel disease (IBD) is a chronic luminal disease with genetic and environmental factors affecting phenotype. This study evaluated the relationship between CHRNA5, a nicotinic receptor subunit gene, and smoking in predicting IBD-related surgery as well as the relationship between CHRNA5 and nicotine dependence.MethodsParticipants completed a smoking questionnaire and were genotyped for CHRNA5 rs16969968. Demographic and clinical data were obtained from medical records. Wilcoxon, ANOVA, Chi square, and Fisher’s exact tests were used for comparisons. Logistic regression was used to evaluate the effect of clinical and genetic predictors on surgery, stratified by disease subtype given paradoxical effects of smoking. Kaplan-Meier curves were used to examine the effect of smoking and genotype on time to surgery. (Significance: P < 0.05 for main effects; P < 0.2 for interaction terms)Results400 (65.8%) patients had Crohn’s disease (CD) and 208 (34.2%) had ulcerative colitis (UC). 298 (49%) underwent an IBD-related surgery. There was a trend towards significance between rs16969968 and smoking behavior (smoking status [P = 0.05], nicotine dependence [AA > AG > GG; P = 0.08]). Smoking and genotype were not independently associated with surgery in UC or CD. However, interaction between rs16969968 and smoking in predicting surgery was observed for both UC (OR = 2.72; P = 0.05) and CD (OR = 2.88; P = 0.1). CHRNA5 genotype, but not smoking, predicted time to surgery in patients with UC (P = 0.007) but not in patients with CD. The interaction between smoking and genotype was not significantly associated with time to surgery in UC or CD.ConclusionsThe CHRNA5 rs16969968 A variant interacts with smoking to influence IBD-related surgery.

α5 nAChR modulation of the prefrontal cortex makes attention resilient

A loss-of-function polymorphism in the α5 nicotinic acetylcholine receptor (nAChR) subunit gene has been linked to both drug abuse and schizophrenia. The α5 nAChR subunit is strategically positioned in the prefrontal cortex (PFC), where a loss-of-function in this subunit may contribute to cognitive disruptions in both disorders. However, the specific contribution of α5 to PFC-dependent cognitive functions has yet to be illustrated. In the present studies, we used RNA interference to knockdown the α5 nAChR subunit in the PFC of adult rats. We provide evidence that through its contribution to cholinergic modulation of cholinergic modulation of neurons in the PFC, the α5 nAChR plays a specific role in the recovery of attention task performance following distraction. Our combined data reveal the potent ability of this subunit to modulate the PFC and cognitive functions controlled by this brain region that are impaired in disease.

Miscellanea Dependent generalized functional linear models.

This paper considers testing for no effect of functional covariates on response variables in multivariate regression. We use generalized estimating equations to determine the underlying parameters and establish their joint asymptotic normality. This is then used to test the significance of the effect of predictors on the vector of response variables. Simulations demonstrate the importance of considering existing correlation structures in the data. To explore the effect of treating genetic data as a function, we perform a simulation study using gene sequencing data and find that the performance of our test is comparable to that of another popular method used in sequencing studies. We present simulations to explore the behaviour of our test under varying sample size, cluster size and dimension of the parameter to be estimated, and an application where we are able to confirm known associations between nicotine dependence and neuronal nicotinic acetylcholine receptor subunit genes.

Expression patterns for nicotinic acetylcholine receptor subunit genes in smoking-related lung cancers.

Cigarette smoking is associated with increased risk for all histologic types of lung cancer, but why the strength of this association is stronger for squamous cell carcinoma than adenocarcinoma of the lung (SQC-L, ADC-L) is not fully understood. Because nicotine and tobacco-specific nitrosamines contribute to carcinogenesis by activating nicotinic acetylcholine receptors (nAChRs) on lung tumors and epithelial cells, we investigated whether differential expression of nAChR subtypes in these tumors could explain their different association with smoking. Expression of nAChR subunit genes in paired tumor and non-tumor lung specimens from 40 SQC-L and 38 ADC-L patients was analyzed by quantitative PCR. Compared to normal lung, both tumors share: i) transcriptional dysregulation of CHRNA3/CHRNA5/CHRNB4 (α3, α5, β4 subunits) at the chromosomal locus that predisposes to lung cancer; and ii) decreased expression of CHRFAM7A (dupα7 subunit); this last subunit negatively modulates α7-nAChR activity in oocytes. In contrast, CHRNA7 (α7 subunit) expression was increased in SQC-L, particularly in smokers and non-survivors, while CHRNA4 (α4 subunit) expression was decreased in ADC-L. Thus, over-representation of cancer-stimulating α7-nAChR in SQC-L, also potentiated by smoking, and under-representation of cancer-inhibiting α4β2-nAChR in ADC-L could explain the different tobacco influences on the tumorigenic process in each cancer type.

Short-term nicotine exposure induces long-lasting modulation of gustatory plasticity in Caenorhabditis elegans

Nicotine administration induces many effects on animal behavior. In wild-type Caenorhabditis elegans, gustatory plasticity results in reduced chemotaxis toward NaCl of otherwise attractive concentrations after pre-exposure to 100mM NaCl in the absence of food. However, acute nicotine administration during a 15min pre-exposure period inhibits gustatory plasticity, whereas chronic nicotine administration during worm development facilitates the plasticity. To investigate the relationship between the duration of nicotine administration and its effects, we exposed worms to nicotine for various periods during development. The modulatory effect of nicotine on gustatory plasticity was gradually switched from inhibition to facilitation with increased duration of nicotine administration. Moreover, inhibition of plasticity was sustained after relatively short-term chronic administration, with effects lasting for 45h after the removal of nicotine. Similar to the acute inhibitory effect after 15min nicotine pre-exposure, the inhibitory effect after short-term chronic administration was dependent on the nicotinic acetylcholine receptor subunit genes lev-1 and unc-29, and genes involved in serotonin biosynthesis bas-1 and tph-1. The impaired inhibition in bas-1 and tph-1mutants was recovered by exogenous serotonin, demonstrating that serotonin plays an important role in the long-lasting inhibitory effects of short-term chronic nicotine exposure.

Expression profile of nicotinic acetylcholine receptor subunits in the brain of HIV-1 transgenic rats given chronic nicotine treatment.

Abuse of addictive substances, including cigarettes, is much greater in HIV-1-infected individuals than in the general population and challenges the efficiency of highly active anti-retroviral therapy (HAART). The HIV-1 transgenic (HIV-1Tg) rat, an animal model used to study drug addiction in HIV-1-infected patients on HAART, displays abnormal neurobehavioral responses to addictive substances. Given that the cholinergic system plays an essential part in the central reward circuitry, we evaluated the expression profile of nine nicotinic acetylcholine receptor (nAChR) subunit genes in the central nervous system (CNS) of HIV-1Tg rats. We found that nAChR subunits were differentially expressed in various brain regions in HIV-1Tg rats compared to F344 control rats, with more subunits altered in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of the HIV-1Tg rats than in other brain regions. We also found that chronic nicotine treatment (0.4mg/kg/day) decreased the mRNA expression of nAChR subunits α6, β3, and β4 in the VTA of HIV-1Tg rats, whereas expression of α4 and α6 subunits in the NAc increased. No such changes were observed in F344 rats. Together, our data suggest that HIV-1 proteins alter the expression of nAChRs, which may contribute to the vulnerability to cigarette smoking addiction in HIV-1 patients.

Chromosome 15q25 (CHRNA3-CHRNB4) Variation Indirectly Impacts Lung Cancer Risk in Chinese Males.

IntroductionRecently, genome-wide association studies (GWAS) in Caucasian populations have identified an association between single nucleotide polymorphisms (SNPs) in the CHRNA5-A3-B4 nicotinic acetylcholine receptor subunit gene cluster on chromosome 15q25, lung cancer risk and smoking behaviors. However, these SNPs are rare in Asians, and there is currently no consensus on whether SNPs in CHRNA5-A3-B4 have a direct or indirect carcinogenic effect through smoking behaviors on lung cancer risk. Though some studies confirmed rs6495308 polymorphisms to be associated with smoking behaviors and lung cancer, no research was conducted in China. Using a case-control study, we decided to investigate the associations between CHRNA3 rs6495308, CHRNB4 rs11072768, smoking behaviors and lung cancer risk, as well as explore whether the two SNPs have a direct or indirect carcinogenic effect on lung cancer.MethodsA total of 1025 males were interviewed using a structured questionnaire (204 male lung cancer patients and 821 healthy men) to acquire socio-demographic status and smoking behaviors. Venous blood samples were collected to measure rs6495308 and rs11072768 gene polymorphisms. All subjects were divided into 3 groups: non-smokers, light smokers (1–15 cigarettes per day) and heavy smokers (>15 cigarettes per day).ResultsCompared to wild genotype, rs6495308 and rs11072768 variant genotypes reported smoking more cigarettes per day and a higher pack-years of smoking (P<0.05). More importantly, among smokers, both rs6495308 CT/TT and rs11072768 GT/GG had a higher risk of lung cancer compared to wild genotype without adjusting for potential confounding factors (OR = 1.36, 95%CI = 1.09–1.95; OR = 1.11, 95%CI = 1.07–1.58 respectively). Furthermore, heavy smokers with rs6495308 or rs11072768 variant genotypes have a positive interactive effect on lung cancer after adjustment for potential confounding factors (OR = 1.13, 95%CI = 1.01–3.09; OR = 1.09, 95%CI = 1.01–3.41 respectively). However, No significant associations were found between lung cancer risk and both rs6495308 and rs11072768 genotypes among non-smokers and smokers after adjusting for age, occupation, and education.ConclusionThis study confirmed both rs6495308 and rs11072768 gene polymorphisms association with smoking behaviors and had an indirect link between gene polymorphisms and lung cancer risk.

An Adolescent Substance Prevention Model Blocks the Effect of CHRNA5 Genotype on Smoking During High School.

Prevention intervention programs reduce substance use, including smoking, but not all individuals respond. We tested whether response to a substance use prevention/intervention program varies based upon a set of five markers (rs16969968, rs1948, rs578776, rs588765, and rs684513) within the cluster of nicotinic acetylcholine receptor subunit genes (CHRNA5/A3/B4). Participants (N = 424) were randomly assigned to either control condition, or a family-based intervention in grade 6 and a school-based drug preventive intervention in grade 7. Smoking in the past month was assessed in grades 9-12 using a four-point scale (0 = never smoked, 1 = smoked but not in last month, 2 = one or a few times, 3 = about once a week or more). There was a main effect of both the intervention (b = -0.24, P < .05) and genotype at rs16969968 (b = 0.14, P < .05) on high school smoking. Using dummy coding to allow for nonlinear effects, individuals with the A/A genotype smoked more often than those with G/G (b = 0.33, P < .05). A genotype × intervention effect was found with reduced smoking among those with A/A and G/A genotypes to levels similar to those with the G/G genotype (G/G vs. A/A: b = -0.67, P < .05; A/G vs. A/A: b = -0.61, P < .05; G/G vs. A/G ns). Results were nonsignificant for the other four markers. Preventive interventions can reduce the genetic risk for smoking from rs16969968.