Investigating the role of nicotinic acetylcholine receptors in menthol's effects in mice.

The use of menthol in tobacco products has been linked to an increased likelihood of developing nicotine dependence. The widespread use of menthol can be attributed to its unique sensory characteristics; however, emerging evidence suggests that menthol also alters sensitivity to nicotine through modulation of nicotinic acetylcholine receptors (nAChRs). Nicotinic subunits, such as ß2 and α5, are of interest due to their implications in nicotine reward, reinforcement, intake regulation, and aversion. This study, therefore, examined the in vivo relevance of ß2 and α5 nicotinic subunits on the pharmacological and behavioral effects of menthol. Data suggests that the α5 nicotinic subunit modulates menthol intake in mice. Overall, deletion or a reduction in function of the α5 subunit lessened aversion to menthol. α5 KO mice and mice possessing the humanized α5 SNP, a variant that confers a nicotine dependence phenotype in humans, demonstrated increased menthol intake compared to their WT counterparts and in a sex-related fashion for α5 SNP mice. We further reported that the modulatory effects of the α5 subunit do not extend to other aversive tastants like quinine, suggesting that deficits in α5* nAChR signaling may not abolish general sensitivity to the aversive effects of other noxious chemicals. Further probing into the role of α5 in other pharmacological properties of menthol revealed that the α5 subunit does not modulate the antinociceptive properties of menthol in mice and suggests that the in vivo differences observed are likely not due to the direct effects of menthol on α5-containing nAChRs in vitro.

Testing Association of Previously Implicated Gene Sets and Gene-Networks in Nicotine Exposed Mouse Models with Human Smoking Phenotypes.

INTRODUCTION: Smoking behaviors are partly heritable, yet the genetic and environmental mechanisms underlying smoking phenotypes are not fully understood. Developmental nicotine exposure (DNE) is a significant risk factor for smoking and leads to gene expression changes in mouse models; however, it is unknown whether the same genes whose expression is impacted by DNE are also those underlying smoking genetic liability. We examined whether genes whose expression in D1-type striatal medium spiny neurons due to DNE in the mouse are also associated with human smoking behaviors. METHODS: Specifically, we assessed whether human orthologs of mouse-identified genes, either individually or as a set, were genetically associated with five human smoking traits using MAGMA and S-LDSC while implementing a novel expression-based gene-SNP annotation methodology. RESULTS: We found no strong evidence that these genes sets were more strongly associated with smoking behaviors than the rest of the genome, but ten of these individual genes were significantly associated with three of the five human smoking traits examined (p < 2.5e-6). Three of these genes have not been reported previously and were discovered only when implementing the expression-based annotation. CONCLUSIONS: These results suggest the genes whose expression is impacted by DNE in mice are largely distinct from those contributing to smoking genetic liability in humans. However, examining a single mouse neuronal cell type may be too fine a resolution for comparison, suggesting that experimental manipulation of nicotine consumption, reward, or withdrawal in mice may better capture genes related to the complex genetics of human tobacco use. IMPLICATIONS: Genes whose expression is impacted by DNE in mouse D1-type striatal medium spiny neurons were not found to be, as a whole, more strongly associated with human smoking behaviors than the rest of the genome, though ten individual mouse-identified genes were associated with human smoking traits. This suggests little overlap between the genetic mechanisms impacted by DNE and those influencing heritable liability to smoking phenotypes in humans. Further research is warranted to characterize how developmental nicotine exposure paradigms in mice can be translated to understand nicotine use in humans and their heritable effects on smoking.

Mutation of the α5 nicotinic acetylcholine receptor subunit increases ethanol and nicotine consumption in adolescence and impacts adult drug consumption.

Alcohol and nicotine are commonly used during adolescence, establishing long-lasting neuroplastic alterations that influence subsequent drug use and abuse. Drinking- and smoking-related traits have been extensively associated with variation in CHRNA5 - the gene that encodes the α5 subunit of neuronal nicotinic acetylcholine receptors (nAChRs). The single nucleotide polymorphism (SNP) rs16969968 in CHRNA5 encodes an amino acid substitution (D398N) that alters the function and pharmacokinetics of α5-containing nAChR. When expressed in rodents, this variant results in increased ethanol and nicotine operant self-administration. How disruption of α5-containing nAChRs influences adolescent ethanol and nicotine intake, and how it modulates interactions between these drugs has not been previously explored. In the present study, we examined volitional ethanol and nicotine consumption in adolescent mice (post-natal day 30-43) of both sexes with mutated (SNP) or lacking (KO) the α5 nAChR subunit. The effect of adolescent alcohol or nicotine exposure on home cage consumption of the opposite drug in adulthood and its modulation by Chrna5 mutation and sex were examined. During adolescence, we found that α5 nAChR disruption increases nicotine intake in mice of both sexes, but the effect on alcohol intake was only observed in females. The sex-specific increase in alcohol consumption in α5 SNP and KO was replicated in adulthood. The effect of adolescent alcohol or nicotine exposure on subsequent intake of the opposite drug in adulthood is modulated by sex and Chrna5 mutation. These observations suggest sex differences in the genetic architecture of alcohol dependence, and modulators of alcohol and nicotine interactions.

An innate contribution of human nicotinic receptor polymorphisms to COPD-like lesions.

Chronic Obstructive Pulmonary Disease is a generally smoking-linked major cause of morbidity and mortality. Genome-wide Association Studies identified a locus including a non-synonymous single nucleotide polymorphism in CHRNA5, rs16969968, encoding the nicotinic acetylcholine receptor α5 subunit, predisposing to both smoking and Chronic Obstructive Pulmonary Disease. Here we report that nasal polyps from rs16969968 non-smoking carriers exhibit airway epithelium remodeling and inflammation. These hallmarks of Chronic Obstructive Pulmonary Disease occur spontaneously in mice expressing human rs16969968. They are significantly amplified after exposure to porcine pancreatic elastase, an emphysema model, and to oxidative stress with a polymorphism-dependent alteration of lung function. Targeted rs16969968 expression in epithelial cells leads to airway remodeling in vivo, increased proliferation and production of pro-inflammatory cytokines through decreased calcium entry and increased adenylyl-cyclase activity. We show that rs16969968 directly contributes to Chronic Obstructive Pulmonary Disease-like lesions, sensitizing the lung to the action of oxidative stress and injury, and represents a therapeutic target.

DNA methylome perturbations: an epigenetic basis for the emergingly heritable neurodevelopmental abnormalities associated with maternal smoking and maternal nicotine exposure†.

Maternal smoking during pregnancy is associated with an ensemble of neurodevelopmental consequences in children and therefore constitutes a pressing public health concern. Adding to this burden, contemporary epidemiological and especially animal model research suggests that grandmaternal smoking is similarly associated with neurodevelopmental abnormalities in grandchildren, indicative of intergenerational transmission of the neurodevelopmental impacts of maternal smoking. Probing the mechanistic bases of neurodevelopmental anomalies in the children of maternal smokers and the intergenerational transmission thereof, emerging research intimates that epigenetic changes, namely DNA methylome perturbations, are key factors. Altogether, these findings warrant future research to fully elucidate the etiology of neurodevelopmental impairments in the children and grandchildren of maternal smokers and underscore the clear potential thereof to benefit public health by informing the development and implementation of preventative measures, prophylactics, and treatments. To this end, the present review aims to encapsulate the burgeoning evidence linking maternal smoking to intergenerational epigenetic inheritance of neurodevelopmental abnormalities, to identify the strengths and weaknesses thereof, and to highlight areas of emphasis for future human and animal model research therein.

The Intergenerational Transmission of Developmental Nicotine Exposure-Induced Neurodevelopmental Disorder-Like Phenotypes is Modulated by the Chrna5 D397N Polymorphism in Adolescent Mice.

Maternal tobacco smoking during pregnancy constitutes developmental nicotine exposure (DNE) and is associated with nicotine dependence and neurodevelopmental disorders in both children and grandchildren as well as animal models thereof. Genetic variants such as the CHRNA5 single nucleotide polymorphism (SNP) rs16969968, which leads to an aspartic acid to asparagine substitution at amino acid position 398 (D398N) in the alpha-5 nicotinic acetylcholine receptor subunit, can also confer risk for nicotine dependence and neurodevelopmental disorders in the absence of DNE. However, the degrees to which, the consequences of maternal smoking on offspring outcomes are influenced by genetic variants and interactions therewith are not well understood. Addressing this void in the literature, the present study utilizes a DNE mouse model engineered to possess the equivalent of the human D398N SNP in CHRNA5 (D397N SNP in mice) to assess how the N397 risk allele impacts the induction and intergenerational transmission of a range of neurodevelopmental disorder-related behavioral phenotypes in first- and second-generation DNE offspring. Results reveal that offspring possessing the N397 variant in the absence of DNE as well as DNE offspring and grand offspring possessing theD397 variant exhibit analogous neurodevelopmental disorder-like phenotypes including hyperactivity, risk-taking behaviors, aberrant rhythmicity of activity, and enhanced nicotine consumption. DNE amplified these behavioral anomalies in first-generation N397 progeny, but the severity of DNE-evoked behavioral perturbations did not significantly differ between first-generation D397 and N397 DNE mice for any measure. Remarkably, the behavioral profiles of second-generation N397 DNE progeny closely resembled DNE-naive D397 mice, suggesting that the N397 variant may protect against the intergenerational transmission of DNE-induced neurodevelopmental disorder-like behaviors.

Developmental nicotine exposure engenders intergenerational downregulation and aberrant posttranslational modification of cardinal epigenetic factors in the frontal cortices, striata, and hippocampi of adolescent mice.

BACKGROUND: Maternal smoking of traditional or electronic cigarettes during pregnancy, which constitutes developmental nicotine exposure (DNE), heightens the risk of neurodevelopmental disorders including ADHD, autism, and schizophrenia in children. Modeling the intergenerationally transmissible impacts of smoking during pregnancy, we previously demonstrated that both the first- and second-generation adolescent offspring of nicotine-exposed female mice exhibit enhanced nicotine preference, hyperactivity and risk-taking behaviors, aberrant rhythmicity of home cage activity, nicotinic acetylcholine receptor and dopamine transporter dysfunction, impaired furin-mediated proBDNF proteolysis, hypocorticosteronemia-related glucocorticoid receptor hypoactivity, and global DNA hypomethylation in the frontal cortices and striata. This ensemble of multigenerational DNE-induced behavioral, neuropharmacological, neurotrophic, neuroendocrine, and DNA methylomic anomalies recapitulates the pathosymptomatology of neurodevelopmental disorders such as ADHD, autism, and schizophrenia. Further probing the epigenetic bases of DNE-induced multigenerational phenotypic aberrations, the present study examined the expression and phosphorylation of key epigenetic factors via an array of immunoblot experiments. RESULTS: Data indicate that DNE confers intergenerational deficits in corticostriatal DNA methyltransferase 3A (DNMT3A) expression accompanied by downregulation of methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) in the frontal cortices and hippocampi, while the expression of ten-eleven translocase methylcytosine dioxygenase 2 (TET2) is unaltered. Moreover, DNE evokes multigenerational abnormalities in HDAC2 (Ser394) but not MeCP2 (Ser421) phosphorylation in the frontal cortices, striata, and hippocampi. CONCLUSIONS: In light of the extensive gene regulatory roles of DNMT3A, MeCP2, and HDAC2, the findings of this study that DNE elicits downregulation and aberrant posttranslational modification of these factors in both first- and second-generation DNE mice suggest that epigenetic perturbations may constitute a mechanistic hub for the intergenerational transmission of DNE-induced neurodevelopmental disorder-like phenotypes.

The Role of A Priori-Identified Addiction and Smoking Gene Sets in Smoking Behaviors.

INTRODUCTION: Smoking is a leading cause of death, and genetic variation contributes to smoking behaviors. Identifying genes and sets of genes that contribute to risk for addiction is necessary to prioritize targets for functional characterization and for personalized medicine. METHODS: We performed a gene set-based association and heritable enrichment study of two addiction-related gene sets, those on the Smokescreen Genotyping Array and the nicotinic acetylcholine receptors, using the largest available GWAS summary statistics. We assessed smoking initiation, cigarettes per day, smoking cessation, and age of smoking initiation. RESULTS: Individual genes within each gene set were significantly associated with smoking behaviors. Both sets of genes were significantly associated with cigarettes per day, smoking initiation, and smoking cessation. Age of initiation was only associated with the Smokescreen gene set. Although both sets of genes were enriched for trait heritability, each accounts for only a small proportion of the single nucleotide polymorphism-based heritability (2%-12%). CONCLUSIONS: These two gene sets are associated with smoking behaviors, but collectively account for a limited amount of the genetic and phenotypic variation of these complex traits, consistent with high polygenicity. IMPLICATIONS: We evaluated evidence for the association and heritable contribution of expert-curated and bioinformatically identified sets of genes related to smoking. Although they impact smoking behaviors, these specifically targeted genes do not account for much of the heritability in smoking and will be of limited use for predictive purposes. Advanced genome-wide approaches and integration of other 'omics data will be needed to fully account for the genetic variation in smoking phenotypes.

Developmental nicotine exposure elicits multigenerational disequilibria in proBDNF proteolysis and glucocorticoid signaling in the frontal cortices, striata, and hippocampi of adolescent mice.

Maternal smoking of conventional or vapor cigarettes during pregnancy, a form of developmental nicotine exposure (DNE), enhances the risk of neurodevelopmental disorders such as ADHD, autism, and schizophrenia in children. Modeling the multigenerational effects of smoking during pregnancy and nursing in the first- (F1) and second- (F2) generation adolescent offspring of oral nicotine-treated female C57BL/6J mice, we have previously reported that DNE precipitates intergenerational transmission of nicotine preference, hyperactivity and impulsivity-like behaviors, altered rhythmicity of home cage activity, corticostriatal nicotinic acetylcholine receptor and dopamine transporter dysfunction, and corticostriatal global DNA methylome deficits. In aggregate, these DNE-evoked behavioral, neuropharmacological, and epigenomic anomalies mirror fundamental etiological aspects of neurodevelopmental disorders including ADHD, autism, and schizophrenia. Expanding this line of research, the current study profiled the multigenerational neurotrophic and neuroendocrine consequences of DNE. Results reveal impaired proBDNF proteolysis as indicated by proBDNF-BDNF imbalance, downregulation of the proBDNF processing enzyme furin, atypical glucocorticoid receptor (GR) activity as implied by decreased relative nuclear GR localization, and deficient basal plasma corticosterone (CORT) levels in adolescent DNE offspring and grandoffspring. Collectively, these data recapitulate the BDNF deficits and HPA axis dysregulation characteristic of neurodevelopmental disorders such as ADHD, autism, and schizophrenia as well as the children of maternal smokers. Notably, as BDNF is a quintessential mediator of neurodevelopment, our prior findings of multigenerational DNE-induced behavioral and neuropharmacological abnormalities may stem from neurodevelopmental insults conferred by the proBDNF-BDNF imbalance detected in DNE mice. Similarly, our findings of multigenerational GR hypoactivity may contribute to the increased risk-taking behaviors and aberrant circadian rhythmicity of home cage activity that we previously documented in first- and second-generation DNE mice.

Developmental nicotine exposure precipitates multigenerational maternal transmission of nicotine preference and ADHD-like behavioral, rhythmometric, neuropharmacological, and epigenetic anomalies in adolescent mice.

Maternal smoking during pregnancy, a form of developmental nicotine exposure (DNE), is associated with increased nicotine use and neurodevelopmental disorders such as ADHD in children. Here, we characterize the behavioral, rhythmometric, neuropharmacological, and epigenetic consequences of DNE in the F1 (first) and F2 (second) generation adolescent offspring of mice exposed to nicotine prior to and throughout breeding. We assessed the effects of passive oral methylphenidate (MPH) administration and voluntary nicotine consumption on home cage activity rhythms and activity and risk-taking behaviors in the open field. Results imply a multigenerational predisposition to nicotine consumption in DNE mice and demonstrate ADHD-like diurnal and nocturnal hyperactivity and anomalies in the rhythmicity of home cage activity that are reversibly rescued by MPH and modulated by voluntary nicotine consumption. DNE mice are hyperactive in the open field and display increased risk-taking behaviors that are normalized by MPH. Pharmacological characterization of nicotinic and dopaminergic systems in striatum and frontal cortex reveals altered expression and dysfunction of nicotinic acetylcholine receptors (nAChRs), hypersensitivity to nicotine-induced nAChR-mediated dopamine release, and impaired dopamine transporter (DAT) function in DNE mice. Global DNA methylation assays indicate DNA methylome deficits in striatum and frontal cortex of DNE mice. Collectively, our data demonstrate that DNE enhances nicotine preference, elicits hyperactivity and risk-taking behaviors, perturbs the rhythmicity of activity, alters nAChR expression and function, impairs DAT function, and causes DNA hypomethylation in striatum and frontal cortex of both first and second-generation adolescent offspring. These findings recapitulate multiple domains of ADHD symptomatology.

The effects of oral nicotine administration and abstinence on sleep in male C57BL/6J mice.

BACKGROUND: Sleep disturbances are common in smoking cessation attempts and are predictive of relapse. Despite this knowledge, there is no established animal model to study the effect of nicotine abstinence on sleep and EEG parameters. OBJECTIVES: The present study was conducted to characterize sleep and wakefulness in male C57BL/6J mice during periods of oral nicotine administration and abstinence. METHODS: Male C57BL/6J mice were implanted with EEG/EMG recording devices. EEG/EMG data were recorded continuously for a period of 4 weeks. At the beginning of week 2, 200 µg/ml of nicotine was added to the 0.2% saccharin vehicle drinking solution. Following a 2-week period of oral nicotine administration, abstinence was initiated by excluding the nicotine from the 0.2% saccharin vehicle drinking solution. EEG/EMG were analyzed at pre-nicotine baseline, during nicotine administration, and on days 1, 2, and 5 of abstinence from nicotine. RESULTS: Oral nicotine administration decreased total sleep time during the active phase, consistent with the stimulant actions of nicotine. In contrast, NREM sleep quantity was increased during the active phase on nicotine abstinence day 1 and REM sleep was decreased during days 2 and 5 of abstinence. Further, sleep fragmentation was increased during the inactive phase on all days of abstinence. Oral nicotine administration and abstinence from nicotine also altered EEG relative power frequencies during the inactive and active phase. CONCLUSIONS: Both oral nicotine administration and abstinence lead to sleep disturbances in mice. Similarities between this model and human reports on the effect of nicotine/nicotine withdrawal on sleep support its utility in examining the molecular mechanisms that modulate the relationship between sleep, nicotine, and nicotine abstinence/withdrawal.

A novel genetic marker of decreased inflammation and improved survival after acute myocardial infarction.

The CHRNA5 gene encodes a neurotransmitter receptor subunit involved in multiple processes, including cholinergic autonomic nerve activity and inflammation. Common variants in CHRNA5 have been linked with atherosclerotic cardiovascular disease. Association of variation in CHRNA5 and specific haplotypes with cardiovascular outcomes has not been described. The aim of this study was to examine the association of CHRNA5 haplotypes with gene expression and mortality among patients with acute myocardial infarction (AMI) and explore potential mechanisms of this association. Patients (N = 2054) hospitalized with AMI were genotyped for two common variants in CHRNA5. Proportional hazard models were used to estimate independent association of CHRNA5 haplotype with 1-year mortality. Both individual variants were associated with mortality (p = 0.0096 and 0.0004, respectively) and were in tight LD (D' = 0.99). One haplotype, HAP3, was associated with decreased mortality one year after AMI (adjusted HR = 0.42, 95% CI 0.26, 0.68; p = 0.0004). This association was validated in an independent cohort (N = 637) of post-MI patients (adjusted HR = 0.23, 95% CI 0.07, 0.79; p = 0.019). Differences in CHRNA5 expression by haplotype were investigated in human heart samples (n = 28). Compared with non-carriers, HAP3 carriers had threefold lower cardiac CHRNA5 mRNA expression (p = 0.023). Circulating levels of the inflammatory marker hsCRP were significantly lower in HAP3 carriers versus non-carriers (3.43 ± 4.2 versus 3.91 ± 5.1; p = 0.0379). Activation of the inflammasome, an important inflammatory complex involved in cardiovascular disease that is necessary for release of the pro-inflammatory cytokine IL-1 ß, was assessed in bone marrow-derived macrophages (BMDM) from CHRNA5 knockout mice and wild-type controls. In BMDM from CHRNA5 knockout mice, IL-1ß secretion was reduced by 50% compared to wild-type controls (p = 0.004). Therefore, a common haplotype of CHRNA5 that results in reduced cardiac expression of CHRNA5 and attenuated macrophage inflammasome activation is associated with lower mortality after AMI. These results implicate CHRNA5 and the cholinergic anti-inflammatory pathway in survival following AMI.

Nicotine Impairs Macrophage Control of Mycobacterium tuberculosis.

Pure nicotine impairs macrophage killing of Mycobacterium tuberculosis (MTB), but it is not known whether the nicotine component in cigarette smoke (CS) plays a role. Moreover, the mechanisms by which nicotine impairs macrophage immunity against MTB have not been explored. To neutralize the effects of nicotine in CS extract, we used a competitive inhibitor to the nicotinic acetylcholine receptor (nAChR)-mecamylamine-as well as macrophages derived from mice with genetic disruption of specific subunits of nAChR. We also determined whether nicotine impaired macrophage autophagy and whether nicotine-exposed T regulatory cells (Tregs) could subvert macrophage anti-MTB immunity. Mecamylamine reduced the CS extract increase in MTB burden by 43%. CS extract increase in MTB was also significantly attenuated in macrophages from mice with genetic disruption of either the α7, ß2, or ß4 subunit of nAChR. Nicotine inhibited autophagosome formation in MTB-infected THP-1 cells and primary murine alveolar macrophages, as well as increased the intracellular MTB burden. Nicotine increased migration of THP-1 cells, consistent with the increased number of macrophages found in the lungs of smokers. Nicotine induced Tregs to produce transforming growth factor-ß. Naive mouse macrophages co-cultured with nicotine-exposed Tregs had significantly greater numbers of viable MTB recovered with increased IL-10 production and urea production, but no difference in secreted nitric oxide as compared with macrophages cocultured with unexposed Tregs. We conclude that nicotine in CS plays an important role in subverting macrophage control of MTB infection.

Nicotine reverses hypofrontality in animal models of addiction and schizophrenia.

The prefrontal cortex (PFC) underlies higher cognitive processes that are modulated by nicotinic acetylcholine receptor (nAChR) activation by cholinergic inputs. PFC spontaneous default activity is altered in neuropsychiatric disorders, including schizophrenia-a disorder that can be accompanied by heavy smoking. Recently, genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) in the human CHRNA5 gene, encoding the α5 nAChR subunit, that increase the risks for both smoking and schizophrenia. Mice with altered nAChR gene function exhibit PFC-dependent behavioral deficits, but it is unknown how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying behavior. Here we show that mice expressing a human α5 SNP exhibit neurocognitive behavioral deficits in social interaction and sensorimotor gating tasks. Two-photon calcium imaging in awake mouse models showed that nicotine can differentially influence PFC pyramidal cell activity by nAChR modulation of layer II/III hierarchical inhibitory circuits. In α5-SNP-expressing and α5-knockout mice, lower activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased somatostatin (SOM) interneuron inhibitory drive over layer II/III pyramidal neurons. The decreased activity observed in α5-SNP-expressing mice resembles the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction. Chronic nicotine administration reversed this hypofrontality, suggesting that administration of nicotine may represent a therapeutic strategy for the treatment of schizophrenia, and a physiological basis for the tendency of patients with schizophrenia to self-medicate by smoking.

The ß3 subunit of the nicotinic acetylcholine receptor: Modulation of gene expression and nicotine consumption.

Genetic factors explain approximately half of the variance in smoking behaviors, but the molecular mechanism by which genetic variation influences behavior is poorly understood. SNPs in the putative promoter region of CHRNB3, the gene that encodes the ß3 subunit of the nicotinic acetylcholine receptor (nAChR), have been repeatedly associated with nicotine behaviors. In this work we sought to identify putative function of three SNPs in the promoter region of CHRNB3 on in vitro gene expression. Additionally, we used ß3 null mutant mice as a model of reduced gene expression to assess the effects on nicotine behaviors. The effect of rs13277254, rs6474413, and rs4950 on reporter gene expression was examined using a luciferase reporter assay. A major and minor parent haplotype served as the background on which alleles at the three SNPs were flipped onto different backgrounds (e.g. minor allele on major haplotype background). Constructs were tested in three human cell lines: BE(2)-C, SH-SY5Y and HEK293T. In all cell types the major haplotype led to greater reporter gene expression compared to the minor haplotype, and results indicate that this effect is driven by rs6474413. Moreover, mice lacking the ß3 subunit showed reduced voluntary nicotine consumption compared that of wildtype animals. These data provide evidence that the protective genetic variant at rs6474413 identified in human genetic studies reduces gene expression and that decreased ß3 gene expression in mice reduces nicotine intake. This work contributes to our understanding of the molecular mechanisms that contribute to the human genetic associations of tobacco behaviors.

A multiancestry study identifies novel genetic associations with CHRNA5 methylation in human brain and risk of nicotine dependence.

Nicotine dependence is influenced by chromosome 15q25.1 single nucleotide polymorphisms (SNPs), including the missense SNP rs16969968 that alters function of the α5 nicotinic acetylcholine receptor (CHRNA5) and noncoding SNPs that regulate CHRNA5 mRNA expression. We tested for cis-methylation quantitative trait loci (cis-meQTLs) using SNP genotypes and DNA methylation levels measured across the IREB2-HYKK-PSMA4-CHRNA5-CHRNA3-CHRNB4 genes on chromosome 15q25.1 in the BrainCloud and Brain QTL cohorts [total N = 175 European-Americans and 65 African-Americans (AAs)]. We identified eight SNPs that were significantly associated with CHRNA5 methylation in prefrontal cortex: P ranging from 6.0 × 10(-10) to 5.6 × 10(-5). These SNP-methylation associations were also significant in frontal cortex, temporal cortex and pons: P ranging from 4.8 × 10(-12) to 3.4 × 10(-3). Of the eight cis-meQTL SNPs, only the intronic CHRNB4 SNP rs11636753 was associated with CHRNA5 methylation independently of the known SNP effects in prefrontal cortex, and it was the most significantly associated SNP with nicotine dependence across five independent cohorts (total N = 7858 European ancestry and 3238 AA participants): P = 6.7 × 10(-4), odds ratio (OR) [95% confidence interval (CI)] = 1.11 (1.05-1.18). The rs11636753 major allele (G) was associated with lower CHRNA5 DNA methylation, lower CHRNA5 mRNA expression and increased nicotine dependence risk. Haplotype analyses showed that rs11636753-G and the functional rs16969968-A alleles together increased risk of nicotine dependence more than each variant alone: P = 3.1 × 10(-12), OR (95% CI) = 1.32 (1.22-1.43). Our findings identify a novel regulatory SNP association with nicotine dependence and connect, for the first time, previously observed differences in CHRNA5 mRNA expression and nicotine dependence risk to underlying DNA methylation differences.

Crucial role of nicotinic α5 subunit variants for Ca2+ fluxes in ventral midbrain neurons.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit modulate nicotine consumption, and the human CHRNA5 rs16969968 polymorphism, causing the replacement of the aspartic acid residue at position 398 with an asparagine (α5DN), has recently been associated with increased use of tobacco and higher incidence of lung cancer. We show that in ventral midbrain neurons, the α5 subunit is essential for heteromeric nAChR-induced intracellular-free Ca(2+) concentration elevations and that in α5(-/-) mice, a class of large-amplitude nicotine-evoked currents is lost. Furthermore, the expression of the α5DN subunit is not able to restore nicotinic responses, indicating a loss of function by this subunit in native neurons. To understand how α5DN impairs heteromeric nAChR functions, we coexpressed α4, α5, or α5DN subunits with a dimeric concatemer (ß2α4) in a heterologous system, to obtain nAChRs with fixed stoichiometry. Both α5(ß2α4)2 and α5DN(ß2α4)2 nAChRs yielded similar levels of functional expression and Ca(2+) permeability, measured as fractional Ca(2+) currents (8.2 ± 0.7% and 8.0 ± 1.9%, respectively), 2-fold higher than α4(ß2α4)2. Our results indicate that the loss of function of nicotinic responses observed in α5DN-expressing ventral midbrain neurons is neither due to an intrinsic inability of this subunit to form functional nAChRs nor to an altered Ca(2+) permeability but likely to intracellular modulation.

Melatonin administration alters nicotine preference consumption via signaling through high-affinity melatonin receptors.

RATIONALE: While it is known that tobacco use varies across the 24-h day, the time-of-day effects are poorly understood. Findings from several previous studies indicate a potential role for melatonin in these time-of-day effects; however, the specific underlying mechanisms have not been well characterized. Understanding of these mechanisms may lead to potential novel smoking cessation treatments. OBJECTIVE: The objective of this study is examine the role of melatonin and melatonin receptors in nicotine free-choice consumption METHODS: A two-bottle oral nicotine choice paradigm was utilized with melatonin supplementation in melatonin-deficient mice (C57BL/6J) or without melatonin supplementation in mice proficient at melatonin synthesis (C3H/Ibg) compared to melatonin-proficient mice lacking both or one of the high-affinity melatonin receptors (MT1 and MT2; double-null mutant DM, or MT1 or MT2). Preference for bitter and sweet tastants also was assessed in wild-type and MT1 and MT2 DM mice. Finally, home cage locomotor monitoring was performed to determine the effect of melatonin administration on activity patterns. RESULTS: Supplemental melatonin in drinking water significantly reduced free-choice nicotine consumption in C57BL/6J mice, which do not produce endogenous melatonin, while not altering activity patterns. Independently, genetic deletion of both MT1 and MT2 receptors in a melatonin-proficient mouse strain (C3H) resulted in significantly more nicotine consumption than controls. However, single genetic deletion of either the MT1 or MT2 receptor alone did not result in increased nicotine consumption. Deletion of MT1 and MT2 did not impact taste preference. CONCLUSIONS: This study demonstrates that nicotine consumption can be affected by exogenous or endogenous melatonin and requires at least one of the high-affinity melatonin receptors. The fact that expression of either the MT1 or MT2 melatonin receptor is sufficient to maintain lower nicotine consumption suggests functional overlap and potential mechanistic explanations.

Natural genetic variability of the neuronal nicotinic acetylcholine receptor subunit genes in mice: Consequences and confounds.

Recent human genetic studies have identified genetic variants in multiple nicotinic acetylcholine receptor (nAChR) subunit genes that are associated with risk for nicotine dependence and other smoking-related measures. Genetic variability also exists in the nAChR subunit genes in mice. Most studies on mouse nAChR subunit gene variability to date have focused on Chrna4, the gene that encodes the α4 nAChR subunit and Chrna7, the gene that encodes the α7 nAChR subunit. However, genetic variability exists for all nAChR genes in mice. In this review, we will describe what is known about nAChR subunit gene polymorphisms in mice and how it relates to variability in nAChR expression and function in brain. The relationship between nAChR genetic variability in mice and the effects of nicotine on several behavioral and physiological measures also will be discussed. In addition, an overview of the contribution of other genetic variation to nicotine sensitivity in mice will be provided. Finally, the potential for natural genetic variability to confound and/or modify the results of studies that utilize genetically engineered mice will be considered. As an example of the ability of a natural genetic variant to modify the effect of an engineered mutation, data will be presented that demonstrate that the effect of Chrna5 deletion on oral nicotine intake is dependent upon naturally occurring variant alleles of Chrna4. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

Functional characterization of SNPs in CHRNA3/B4 intergenic region associated with drug behaviors.

The cluster of human neuronal nicotinic receptor genes (CHRNA5/A3/B4) (15q25.1) has been associated with a variety of smoking and drug-related behaviors, as well as risk for lung cancer. CHRNA3/B4 intergenic single nucleotide polymorphisms (SNPs) rs1948 and rs8023462 have been associated with early initiation of alcohol and tobacco use, and rs6495309 has been associated with nicotine dependence and risk for lung cancer. An in vitro luciferase expression assay was used to determine whether these SNPs and surrounding sequences contribute to differences in gene expression using cell lines either expressing proteins characteristic of neuronal tissue or derived from lung cancers. Electrophoretic mobility shift assays (EMSAs) were performed to investigate whether nuclear proteins from these cell lines bind SNP alleles differentially. Results from expression assays were dependent on cell culture type and haplotype. EMSAs indicated that rs8023462 and rs6495309 bind nuclear proteins in an allele-specific way. Additionally, GATA transcription factors appeared to bind rs8023462 only when the minor/risk allele was present. Much work has been done to describe the rat Chrnb4/a3 intergenic region, but few studies have examined the human intergenic region effects on expression; therefore, these studies greatly aid human genetic research as it relates to observed nicotine phenotypes, lung cancer risk and potential underlying genetic mechanisms. Data from these experiments support the hypothesis that SNPs associated with human addiction-related phenotypes and lung cancer risk can affect gene expression, and are potential therapeutic targets. Additionally, this is the first evidence that rs8023462 interacts with GATA transcription factors to influence gene expression.