Cholinergic Receptor Nicotinic Alpha Research Articles

Repurposing dextromethorphan and metformin for treating nicotine-induced cancer by directly targeting CHRNA7 to inhibit JAK2/STAT3/SOX2 signaling

Smoking is one of the most impactful lifestyle-related risk factors in many cancer types including esophageal squamous cell carcinoma (ESCC). As the major component of tobacco and e-cigarettes, nicotine is not only responsible for addiction to smoking but also a carcinogen. Here we report that nicotine enhances ESCC cancer malignancy and tumor-initiating capacity by interacting with cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) and subsequently activating the JAK2/STAT3 signaling pathway. We found that aberrant CHRNA7 expression can serve as an independent prognostic factor for ESCC patients. In multiple ESCC mouse models, dextromethorphan and metformin synergistically repressed nicotine-enhanced cancer-initiating cells (CIC) properties and inhibited ESCC progression. Mechanistically, dextromethorphan non-competitively inhibited nicotine binding to CHRNA7 while metformin downregulated CHRNA7 expression by antagonizing nicotine-induced promoter DNA hypomethylation of CHRNA7. Since dextromethorphan and metformin are two safe FDA-approved drugs with minimal undesirable side-effects, the combination of these drugs has a high potential as either a preventive and/or a therapeutic strategy against nicotine-promoted ESCC and perhaps other nicotine-sensitive cancer types as well.

Chr15q25 genetic variant (rs16969968) independently confers risk of lung cancer, COPD and smoking intensity in a prospective study of high-risk smokers

ImportanceWhile cholinergic receptor nicotinic alpha 5 (CHRNA5) variants have been linked to lung cancer, chronic obstructive pulmonary disease (COPD) and smoking addiction in case–controls studies, their corelationship is not well...

CHRNA1 promotes the pathogenesis of primary focal hyperhidrosis

The aim of the study was to elucidate the involvement of cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) in the pathogenesis of primary focal hyperhidrosis (PFH). The hyperhidrosis mouse model was constructed using pilocarpine injection. The expression levels of CHRNA1 in sweat gland tissues of PFH patients and hyperhidrosis mice were compared using Western blots and quantitative real-time PCR (qRT-PCR) analyses. Sweat secretion in hyperhidrosis mice treated with small-interfering RNA (siRNA) targeting CHRNA1 (si-CHRNA1) or non-specific siRNA were compared. Sweat secretory granules in the sweat gland cells of hyperhidrosis mice were examined using transmission electron microscopy. The serum level of acetylcholine was measured using enzyme-linked immunosorbent assay, while markers associated with PFH, including Aquaporin 5 (AQP5) and Calcium Voltage-Gated Channel Subunit Alpha1 C (CACNA1C), were assessed using immunohistochemical assay and Western blots. Brain-derived neurotrophic factor (BDNF) and Neuregulin 1 (NRG-1) in sympathetic ganglia axons of hyperhidrosis mice were quantified using Western blots. CHRNA1 up-regulation is a characteristic of the sweat glands of PFH patients and Hyperhidrosis mice. Silencing CHRNA1 decreased sweat secretion and the number of sweat secretory granules of hyperhidrosis mice. Serum acetylcholine, as well as AQP5 and CACNA1C expression in the sweat glands, was reduced by siCHRNA1. BDNF1 and NRG-1 levels in the sympathetic ganglia axons were also attenuated by siCHRNA1 treatment. CHRNA1 up-regulation is a potential biomarker of PFH and downregulating CHRNA1 could alleviate the symptoms of PFH through inactivating the sympathetic system.

The Positive Allosteric Modulation of alpha7-Nicotinic Cholinergic Receptors by GAT107 Increases Bacterial Lung Clearance in Hyperoxic Mice by Decreasing Oxidative Stress in Macrophages.

Supplemental oxygen therapy with supraphysiological concentrations of oxygen (hyperoxia; >21% O2) is a life-saving intervention for patients experiencing respiratory distress. However, prolonged exposure to hyperoxia can compromise bacterial clearance processes, due to oxidative stress-mediated impairment of macrophages, contributing to the increased susceptibility to pulmonary infections. This study reports that the activation of the α7 nicotinic acetylcholine receptor (α7nAChR) with the delete allosteric agonistic-positive allosteric modulator, GAT107, decreases the bacterial burden in mouse lungs by improving hyperoxia-induced lung redox imbalance. The incubation of RAW 264.7 cells with GAT107 (3.3 µM) rescues hyperoxia-compromised phagocytic functions in cultured macrophages, RAW 264.7 cells, and primary bone marrow-derived macrophages. Similarly, GAT107 (3.3 µM) also attenuated oxidative stress in hyperoxia-exposed macrophages, which prevents oxidation and hyper-polymerization of phagosome filamentous actin (F-actin) from oxidation. Furthermore, GAT107 (3.3 µM) increases the (1) activity of superoxide dismutase 1; (2) activation of Nrf2 and (3) the expression of heme oxygenase-1 (HO-1) in macrophages exposed to hyperoxia. Overall, these data suggest that the novel α7nAChR compound, GAT107, could be used to improve host defense functions in patients, such as those with COVID-19, who are exposed to prolonged periods of hyperoxia.

Nicotinic acetylcholine receptors α7 and α9 modifies tobacco smoke risk for multiple sclerosis

Introduction: Tobacco smoke exposure is an established risk factor for multiple sclerosis (MS), yet how it confers risk is not known. Evidence from observational studies suggests nicotine may be a protective component. Animal studies further support this hypothesis, demonstrating nicotine’s protective effect in MS is mediated by the presence and absence of α7 and α9 nicotinic acetylcholine receptors (nAChRs), respectively. Objective: To determine if variation in the genes encoding α7 and α9 nAChRs (cholinergic receptor nicotinic alpha 7 (CHRNA7) and alpha 9 (CHRNA9)) will modify MS risk conferred by tobacco smoking. Methods: A multi-stage gene-environment (G×E) framework was utilized, including a case–control analysis (286 cases, 176 controls) with haplotype- and gene-based analyses, followed by an extension case-only (1053 cases) analysis for overlapping variants. Results: The results suggest that CHRNA7 and CHRNA9 modifies MS risk conferred by tobacco smoke, where risk among smokers was increased in carriers of the minor CHRNA9 haplotype and in non-carriers the minor CHRNA7 haplotype. The findings are consistent with the pharmacology of these receptors and animal studies of MS. Conclusion: This study implicates novel processes in MS initiation and demonstrate the need for further G×E studies to advancing our understanding of the missing heritability of MS.

Genetic Variant in CHRNA5 and Response to Varenicline and Combination Nicotine Replacement in a Randomized Placebo-Controlled Trial.

It is unclear if genetic variants affect smoking cessation treatment response. This study tested whether variants in the cholinergic receptor nicotinic alpha 5 subunit (CHRNA5)predict response to smoking cessation medication by directly comparing the two most effective smoking cessation pharmacotherapies. In this genotype-stratified randomized, double-blind, placebo-controlled clinical trial (May 2015-August 2019 in St Louis, Missouri), smokers were randomized by genotype in blocks of six (1:1:1 ratio) to three conditions: 12weeks of placebo (n=273), combination nicotine patch and lozenge (combination nicotine replacement therapy, cNRT, n=275), or varenicline (n=274). All participants received counseling and were followed for 12months. The primary end point was biochemically verified 7-day point prevalence abstinence at the end of treatment (EOT, week 12). Trial registration and eligibility criteria are on clinicaltrials.gov (https://clinicaltrials.gov/) (NCT02351167). We conducted the genetic analyses separately for 516 European ancestry (EA) smokers and 306 non-EA smokers (including 270 African American smokers). In African American smokers, there was a genotype-by-treatment interaction for EOT abstinence (χ2 =10.7, degrees of freedom=2. P=0.0049): specifically, cNRT was more effective in smokers with rs16969968 GG genotype than was placebo, while varenicline was more effective in smokers of GA/AA genotypes. In EA ancestry smokers, there was no significant genotype-by-treatment interaction. In the whole sample, although both were effective at EOT, only varenicline, and not cNRT, was significantly effective relative to placebo at 6-month follow-up. Importantly, this study suggests that genetic information can further enhance smoking cessation treatment effectiveness.

Is There a Genetic Basis for Information Search Propensity? A Genotyping Experiment

In this work we explore the question of whether there is a genetic basis for people’s different propensities to search for information. Specifically, we test whether the rs1044396 polymorphism—one variation in the cholinergic receptor nicotinic alpha 4 (

Transcriptomic profiling of the enteric nervous system (ENS) in the pig colon: regional heterogeneity and implication in physiological functions

The ENS modulates a variety of physiological functions in the gastrointestinal tract. However, little is known about the regional transcriptomic heterogeneity in the ENS. The pig is a relevant experimental model due to its similarity to the human in nutrition, physiology and metabolic process, particularly in the ENS. This study aims to characterize the transcriptome profile of enteric ganglia and regional differences in the pig colon. Full thickness tissue samples of the proximal, transverse and distal colon (pC, tC, dC) were collected from adult Yucatan minipigs (n=10). We captured 25–40 ganglia/pig using Leica LMD6000 laser microdissection system from inner submucosal and myenteric plexus (ISP, MP), which were processed for RNA sequencing on Hiseq 3000. Pathway enrichment analysis was exploited to identify the biological pathways that were statistically enriched in a series of differentially expressed gene (DEG) lists. The discordance in biological pathways was assessed between two plexi and among the segments. Although both tC and dC displayed the pathways related to the muscular contraction (MC), the regulatory gene networks were different. In the ISP, the top‐ranking ADORA1 (adenosine A1 receptor) interacted with SSTR1 (somatostatin receptor 1) in the tC and ADORA1 or VIP in the dC contributed to MC regulation. While in the MP, we found interaction of top‐ranking ADRA2C (alpha‐2C‐adrenergic receptor) and ADRA1A in the tC and network among ADRA2C, ADRA1A, EDNRA (endothelin receptor type A) and EDNRB in the dC. The pC showed pathway prominence in both MC and inflammatory defense. The regulatory genes involved in inflammation were highly ranked in the DEG lists. Among them, complement C3‐C3aR network in the ISP regulated the inflammatory activities, while in the MP it involved TRPV1‐TRPM4 interaction. A similarity of the pathway popularity stood out in pC‐MP compared to tC‐ or dC‐MP. TRPV1 was highly ranked in pC‐MP rather than in tC‐ or dC‐MP. In addition, ADORA2A and ADORA2B (pC vs tC) and ACHE (acetylcholinesterase) (pC vs dC) in pC‐MP pointed to the anti‐inflammatory effects. When comparing with pC‐MP, major categories of the pathway enrichment were the same between tC‐ and dC‐MP, with CaSR (calcium‐sensing receptor) signaling mediating the neuronal calcium regulation. Surprisingly, the variance in the ISP among segments was strikingly smaller, which was also validated by weighted gene coexpression network analysis. The topmost ranking CHRNA6 (cholinergic receptor nicotinic alpha 6 subunit) and CHRNB3 were referred to as mediation of cholinergic neurotransmission in pC‐ISP (vs dC‐ISP), the component of the renin‐angiotensin system as maintenance of electrolyte homeostasis in tC‐ISP (vs pC‐ISP), and NOS1, P2RY1 (purinergic receptor P2Y1) and NPY2R (neuropeptide Y receptor Y2) as the modulation of colonic motility in dC‐ISP (vs pC‐ISP). Collectively, these data present the transcriptomic profile with distinct patterns in different colonic segments, suggesting that genetic diversity is required for the ENS in orchestrating physiological functions in the colon including regulation of inflammation, ion transport and motility.Support or Funding Information(NIH) SPARC OT2OD023899‐01 grant (P‐QY, YT)

Double blind, two dose, randomized, placebo-controlled, cross-over clinical trial of the positive allosteric modulator at the alpha7 nicotinic cholinergic receptor AVL-3288 in schizophrenia patients.

Despite their theoretical rationale, nicotinic alpha-7 acetylcholine (nα7) receptor agonists, have largely failed to demonstrate efficacy in placebo-controlled trials in schizophrenia. AVL-3288 is a nα7 positive allosteric modulator (PAM), which is only active in the presence of the endogenous ligand (acetylcholine), and thustheoretically less likely to cause receptor desensitization. We evaluated the efficacy of AVL-3288 in a Phase 1b, randomized, double-blind, placebo-controlled, triple cross-over study. Twenty-four non-smoking, medicated, outpatients with schizophrenia or schizoaffective disorder and a Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) ≥62 were randomized. Each subject received 5 days of AVL-3288 (10, 30 mg) and placeboacross three separate treatment weeks. The primary outcome measurewas the RBANS total scale score, with auditory P50 evoked potential suppression the key target engagement biomarker. Secondary outcome measures include task-based fMRI (RISE task), mismatch negativity, the Scale for the Assessment of Negative Symptoms of Schizophrenia (SANS) and the Brief Psychiatric Rating Scale (BPRS). Twenty-four subjects were randomized and treated without any clinically significant treatment emergent adverse effects. Baseline RBANS (82 ± 17) and BPRS (41 ± 13) scores were consistent with moderate impairment. Primary outcomes were negative, with non-significant worsening for both active groups vs. placebo in the P50 and minimal between group changes on the RBANS. In conclusion, the results did not indicate efficacy of the compound, consistent with most prior results for the nα7 target.

Maternal high fat diet consumption reduces liver alpha7 nicotinic cholinergic receptor expression and impairs insulin signalling in the offspring

The activation of nicotinic acetylcholine receptor α7 subunit (α7nAChR) has been associated to anti-inflammatory response in macrophages. High-fat diet (HFD) consumption during pregnancy and lactation impairs the cholinergic anti-inflammatory pathway in liver and white adipose tissue of offspring. In order to evaluate the relationship between damage in the cholinergic anti-inflammatory pathway and insulin resistance (IR) development, the liver of offspring of obese dams was investigated. Additionally, the capacity of α7nAChR activation to reduce IR induced by saturated fatty acid was investigated in hepatoma cell line. Initially, female mice were subjected to either standard chow (SC) or HFD during pregnancy and lactation period. After weaning, only male offspring from HFD dams (HFD-O) and SC dams (SC-O) were fed with the SC diet. Hepatic α7nAChR expression was downregulated, and hepatic TNF-α, IL-1β, and pIKK level, but not pJNK, were elevated in the HFD-O compared to SC-O mice. Besides, hepatic expression of TNF-α in response to lipopolysaccharide (LPS) was higher in HFD-O than SC-O mice. Insulin-stimulated phosphorylation of the AKT was lower in HFD-O compared to SC-O. Additionally, insulin-stimulated phosphorylation of the AKT in KOα7Alb-Cre mice fed HFD was lower than WT mice fed HFD. In hepatoma cell line, palmitate increased IL-6 and TNF-α expressions and pJNK level. These effects were accompanied by reduced capacity of insulin to stimulate AKT phosphorylation. PNU or nicotine reduced cytokine expression and JNK activation, but improved insulin resistance induced by palmitate. Our results suggest that maternal obesity impairs hepatic α7nAChR expression and AKT phosphorylation in the offspring. In vitro studies suggest that α7nAChR activation has potential to reduce deleterious effect of saturated fatty acids on insulin signalling.

Depletion of miR-380 mitigates human bronchial epithelial cells injury to improve chronic obstructive pulmonary disease through targeting CHRNA4

BackgroundChronic obstructive pulmonary disease (COPD) not only causes respiratory damage, but also affects circulatory function, which can be life-threatening in severe cases. Therefore, it is very essential to reveal the molecular mechanism of its pathogenesis. MethodsHuman Bronchial Epithelial cells were exposed to 20% cigarette smoke extract (CSE) condition to simulate the cells in COPD patients. GEO database was applied to analyze the expression of miR-380 in COPD patients. The expression level of miR-380 in CSE model was determined with qRT-PCR. Cells proliferation, apoptosis, and inflammation response-related factors were detected with cell counting kit 8, flow cytometry, and Western blot, respectively. A correlation between miR-380 and Cholinergic Receptor Nicotinic Alpha 4 subunit (CHRNA4) was predicted with bioinformatics software, and confirmed by dual luciferase assay. Rescue assay was applied to explore further relationship between miR-380 and CHRNA4. ResultsmiR-380 showed a tendency of high expression in COPD patients and CSE models. Overexpression of miR-380 promoted the inhibitory effect of cells proliferation, and promotion effects of cells apoptosis and inflammation response, which were caused by CSE. CHRNA4, which was lower expressed in COPD patients, was affirmed as a target of miR-380 and negatively modulated by miR-380. Rescue assay indicated that exhausting of CHRNA4 attenuated the moderating effects of miR-380 inhibitor on cells damage induced by CSE. ConclusionsDepletion of miR-380 alleviated cells damage caused by CSE through targeting CHRNA4, suggesting that miR-380/CHRNA4 may serve as novel therapeutic targets for COPD treatment.

Genes Involved in Neurodevelopment, Neuroplasticity and Major Depression: No Association for CACNA1C, CHRNA7 and MAPK1.

ObjectiveGenetics factors are likely to play a role in the risk, clinical presentation and treatment outcome in major depressive disorder (MDD). In this study, we investigated the role of three candidate genes for MDD; calcium voltage-gated channel subunit alpha1 C (CACNA1C ), cholinergic receptor nicotinic alpha 7 subunit (CHRNA7 ), and mitogen-activated protein kinase 1 (MAPK1).MethodsTwo-hundred forty-two MDD patients and 326 healthy controls of Korean ancestry served as samples for the analyses. Thirty-nine single nucleotide polymorphisms (SNPs) within CACNA1C, CHRNA7, and MAPK1 genes were genotyped and subsequently tested for association with MDD (primary analysis) and other clinical features (symptoms’ severity, age of onset, history of suicide attempt, treatment outcome) (secondary analyses). Single SNPs, haplotypes and epistatic analyses were performed.ResultsSingle SNPs were not associated with disease risk and clinical features. However, a combination of alleles (haplotype) within MAPK1 was found associated with MDD-status. Secondary analyses detected a possible involvement of CACNA1C haplotype in resistance to antidepressant treatment.ConclusionThese data suggest a role for MAPK1 and CACNA1C in MDD risk and treatment resistance, respectively. However, since many limitations characterize the analysis, the results must be considered with great caution and verified.

Increased Levels of Kynurenic Acid in the Cerebrospinal Fluid in Patients with Hydrocephalus.

Normal pressure hydrocephalus (NPH) is a potentially reversible neurological syndrome commonly characterized by gait disturbance, urinary incontinence, and dementia. Hydrocephalus e-vacuo (He-v) is also characterized by the occurrence of dementia but does not show gait disturbance or urinary incontinence and has no evident cerebrospinal fluid (CSF) pressure elevation. Kynurenic acid (KYNA), an endogenous metabolite of the L-kynurenine (L-KYN) pathway of L-tryptophan (L-TRP) degradation, is an antagonist of glutamate N-methyl-D-aspartic acid and alpha-7 nicotinic cholinergic receptors that have been linked to dementia. We investigated KYNA, L-KYN, and L-TRP levels in human CSF and serum during the aging process in 30 healthy control individuals. In addition, clinical parameters and L-TRP metabolites in CSF and serum were evaluated in four patients with NPH and five with He-v. KYNA, L-KYN, and L-TRP levels in CSF and serum were determined using highperformance liquid chromatography. Healthy controls showed a significant decrease in serum albumin with age. Compared with their corresponding controls and unlike patients with He-v, patients with NPH (age ≤ 50 years) had significant increases in CSF protein (241%, p < 0.001), CSF albumin (246%, p < 0.001), CSF IgG (328%, p < 0.001), and CSF:serum IgG (321%, p < 0.001) and CSF:serum albumin (257%, p < 0.001) ratios. Controls had significant increases in KYNA, L-KYN, and L-TRP levels in the CSF with advancing age but not in the serum. Compared with the corresponding controls, KYNA levels were significantly increased in the CSF of patients with NPH (141%, p < 0.05) and He-v (225%; p < 0.01). Additionally, the serum levels of KYNA were increased in patients with NPH and He-v to 161% and 156% of controls, respectively (both p < 0.01). The serum levels of L-KYN and L-TRP were significantly reduced in patients with He-v but not in patients with NPH. C-reactive protein, as a marker of inflammation, was significantly increased in the serum of patients with He-v but not in patients with NPH, compared with the corresponding controls. The aging process is related to elevated CSF levels of KYNA, L-KYN, and L-TRP levels. There are significant differences in clinical parameters between the two forms of hydrocephalus and these differences might have diagnostic utility. The occurrence of dementia in patients with either form of hydrocephalus might be at least partly related to elevated KYNA levels in the CNS and/or periphery.

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer.

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is an important susceptibility locus for nicotine addiction and lung cancer. Depletion of CHRNA5 has been associated with reduced cell viability, increased apoptosis and alterations in cellular motility in different cancers yet not in breast cancer. Herein we first showed the expression of CHRNA5 was variable and positively correlated with the fraction of total genomic alterations in breast cancer cell lines and tumors indicating its potential role in DNA damage response (DDR). Next, we demonstrated that silencing of CHRNA5 expression in MCF7 breast cancer cell line by RNAi affected expression of genes involved in cytoskeleton, TP53 signaling, DNA synthesis and repair, cell cycle, and apoptosis. The transcription profile of CHRNA5 depleted MCF7 cells showed a significant positive correlation with that of A549 lung cancer cell line while exhibiting a negative association with the CHRNA5 co-expression profile obtained from Cancer Cell Line Encylopedia (CCLE). Moreover, it exhibited high similarities with published MCF7 expression profiles obtained from exposure to TP53 inducer nutlin-3a and topoisomerase inhibitors. We then demonstrated that CHRNA5 siRNA treatment reduced cell viability and DNA synthesis indicating G1 arrest while it significantly increased apoptotic sub-G1 cell population. Accordingly, we observed lower levels of phosphorylated RB (Ser807/811) and an increased BAX/BCL2 ratio in RNAi treated MCF7 cells. We also showed that CHRNA5 RNAi transcriptome correlated negatively with DDR relevant gene expression profile in breast cancer gene expression datasets while the coexposure to topoisomerase inhibitors in the presence of CHRNA5 RNAi enhanced chemosensitivity potentially due to reduced DDR. CHRNA5 RNAi consistently lowered total CHEK1 mRNA and protein levels as well as phosphorylated CHEK1 (Ser345) in MCF7 cells. We also detected a significant positive correlation between the expression levels of CHRNA5 and CHEK1 in CCLE, TCGA and METABRIC breast cancer datasets. Our study suggests CHRNA5 RNAi is associated with cell cycle inhibition, apoptosis as well as reduced DDR and increased drug sensitivity in breast cancer yet future studies are warranted since dose- and cell line-specific differences exist in response to CHRNA5 depletion. Gene expression microarray data can be accessed from GEO database under the accession number GSE89333.

Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1

Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry-based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n= 15) and performed immunohistochemical analyses. Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre miceexposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.

An immune-beige adipocyte communication via nicotinic acetylcholine receptor signaling.

Beige adipocytes have been recently shown to regulate energy dissipation when activated, and help organisms defend against hypothermia and obesity. Prior reports indicate beige-like adipocytes exist in adult humans and may present novel opportunities to curb the global epidemic in obesity and metabolic illnesses. In an effort to identify unique features of activated beige adipocytes, we uncovered that the cholinergic receptor nicotinic alpha 2 subunit (Chrna2) is induced in subcutaneous fat during the activation of these cells, and that acetylcholine-producing immune cells within this tissue regulate this signaling pathway via paracrine mechanisms. CHRNA2 functions selectively in uncoupling protein 1 (Ucp1)+ beige adipocytes, increasing thermogenesis through a cAMP and PKA pathway. Furthermore, this signaling via CHRNA2 is conserved and present in human subcutaneous adipocytes. Inactivation of Chrna2 in mice compromises the cold-induced thermogenic response selectively in subcutaneous fat and exacerbates high-fat diet-induced obesity and associated metabolic disorders, indicating that even partial loss of beige fat regulation in vivo leads to detrimental consequences. Our results reveal a beige-selective immune-adipose interaction mediated through CHRNA2 and identify a novel function of nicotinic acetylcholine receptors (nAChRs) in energy metabolism. These findings may lead to identification of therapeutic targets to counteract human obesity.

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.

20.3 OPEN CHROMATIN ANALYSES INFORM FUNCTIONAL NONCODING GWAS VARIANTS IN HIPSC MODEL OF MENTAL DISORDERS

BackgroundNeuropsychiatric disorders, including schizophrenia (SZ), afflict a significant fraction of the population. Recent genome-wide association studies (GWAS) under the framework of the Psychiatric Genomics Consortium (PGC), along with large-scale sequencing efforts, have identified a plethora of disease risk loci with common and/or rare risk variants. Translating these exciting genomic findings into causation and disease biology offers the promise of developing more tailored therapies in psychiatry. However, understanding the disease biology underlying most GWAS findings remains challenging: (1) The paucity of disease-relevant biological materials for assaying molecular and cellular phenotypes associated with risk loci; (2) Most disease variants lie within poorly-annotated noncoding parts of the genome for which functional interpretation is challenging; and (3) Each locus often contains many genes/variants equivalently associated with the disease due to linkage disequilibrium, and it is difficult to identify which are the likely causal gene/variant. Human neurons derived from induced pluripotent stem cells (iPSCs), both monolayer cultures (2D model) and the emerging brain organoids (3D model), provide a promising alternative to human brains for recapitulating cellular phenotypes relevant to psychiatric disorders. CRISPR/Cas9 editing further strengthens the utility of these models by enabling the generation of isogenic lines with essentially the same genetic background on which allelic effects of a risk variant can be directly compared, thus increasing the sensitivity to detect typically small effects of a GWAS variant.MethodsTo functionally assess the relevance of noncoding sequences in neuropsychiatric disorders, we hypothesized that disease-relevant noncoding sequences likely overlap with cell-specific open chromatin regions (OCRs). We have carried out a genome-wide OCR profiling of excitatory neuronal differentiation from human iPSCs using an Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq).ResultsWe found that OCRs in neurons were enriched SZ risk variants in neural OCRs and can help prioritize putatively functional SZ risk variants that may impact OCRs and consequently, cellular development. At a leading SZ-risk locus flanking MIR137, we further examined the functional effects of a prioritized common GWAS SNP rs1198588 in CRISPR/Cas9-edited hiPSCs, and found that SZ-risk allele of rs1198588 altered MIR137 expression, OCR dynamics and dendrite arborization/synapse maturation. To systematically identify such disease risk variants that may affect OCR, we further carried out a proof-of-concept analysis of allele-specific open chromatin (ASoC) of in hIPSC-derived neurons. We found that Heterozygous SNPs showing ASoC are more prevalent in neurons than in hiPSCs. Out of the 12 schizophrenia GWAS-implicated SNPs that we found in neuronal OCRs of this single individual, two SNPs showed ASoC and are thus putatively functional: one lies within the 5’-UTR of CHRNA5 (cholinergic receptor, nicotinic, alpha 5) and the other is in the promoter region of VPS45, a Sec1 family gene involved in synaptic transmission. We are currently in the process of replicating the observed landscape of ASoC in iPSC-derived neurons from a larger sample pool.DiscussionOur study suggests that OCR profiling in a human iPSC model of neuron differentiation can predict functional noncoding sequences that regulate neurodevelopment.

Effects of repeated long-term psychosocial stress and acute cannabinoid exposure on mouse corticostriatal circuitries: Implications for neuropsychiatric disorders.

SummaryIntroductionVulnerability to psychiatric manifestations is achieved by the influence of genetic and environment including stress and cannabis consumption. Here, we used a psychosocial stress model based on resident‐intruder confrontations to study the brain corticostriatal‐function, since deregulation of corticostriatal circuitries has been reported in many psychiatric disorders. CB 1 receptors are widely expressed in the central nervous system and particularly, in both cortex and striatum brain structures.Aims and methodsThe investigation presented here is addressed to assess the impact of repeated stress following acute cannabinoid exposure on behavior and corticostriatal brain physiology by assessing mice behavior, the concentration of endocannabinoid and endocannabinoid‐like molecules and changes in the transcriptome.ResultsStressed animals urinated frequently; showed exacerbated scratching activity, lower striatal N‐arachidonylethanolamine (AEA) levels and higher cortical expression of cholinergic receptor nicotinic alpha 6. The cannabinoid agonist WIN55212.2 diminished locomotor activity while the inverse agonist increased the distance travelled in the center of the open field. Upon CB 1 activation, N‐oleoylethanolamide and N‐palmitoylethanolamide, two AEA congeners that do not interact directly with cannabinoid receptors, were enhanced in the striatum. The co‐administration with both cannabinoids induced an up‐regulation of striatal FK506 binding protein 5. The inverse agonist in controls reversed the effects of WIN55212.2 on motor activity. When Rimonabant was injected under stress, the cortical levels of 2‐arachidonoylglycerol were maximum. The agonist and the antagonist influenced the cortical expression of cholinergic receptor nicotinic alpha 6 and serotonin transporter neurotransmitter type 4 in opposite directions, while their co‐administration tended to produce a null effect under stress.ConclusionsThe endocannabinoid system had a direct effect on serotoninergic neurotransmission and glucocorticoid signaling. Cholinergic receptor nicotinic alpha‐6 was shown to be deregulated in response to stress and following synthetic cannabinoid drugs thus could confer vulnerability to cannabis addiction and psychosis. Targeting the receptors of endocannabinoids and endocannabinoid‐like mediators might be a valuable option for treating stress‐related neuropsychiatric symptoms.

Targeted high-throughput sequencing of candidate genes for chronic obstructive pulmonary disease.

BackgroundReduced lung function in patients with chronic obstructive pulmonary disease (COPD) is likely due to both environmental and genetic factors. We report here a targeted high-throughput DNA sequencing approach to identify new and previously known genetic variants in a set of candidate genes for COPD.MethodsExons in 22 genes implicated in lung development as well as 61 genes and 10 genomic regions previously associated with COPD were sequenced using individual DNA samples from 68 cases with moderate or severe COPD and 66 controls matched for age, gender and smoking. Cases and controls were selected from the Obstructive Lung Disease in Northern Sweden (OLIN) studies.ResultsIn total, 37 genetic variants showed association with COPD (p < 0.05, uncorrected). Several variants previously discovered to be associated with COPD from genetic genome-wide analysis studies were replicated using our sample. Two high-risk variants were followed-up for functional characterization in a large eQTL mapping study of 1,111 human lung specimens. The C allele of a synonymous variant, rs8040868, predicting a p.(S45=) in the gene for cholinergic receptor nicotinic alpha 3 (CHRNA3) was associated with COPD (p = 8.8 x 10−3). This association remained (p = 0.003 and OR = 1.4, 95 % CI 1.1-1.7) when analysing all available cases and controls in OLIN (n = 1,534). The rs8040868 variant is in linkage disequilibrium with rs16969968 previously associated with COPD and altered expression of the CHRNA5 gene. A follow-up analysis for detection of expression quantitative trait loci revealed that rs8040868-C was found to be significantly associated with a decreased expression of the nearby gene cholinergic receptor, nicotinic, alpha 5 (CHRNA5) in lung tissue.ConclusionOur data replicate previous result suggesting CHRNA5 as a candidate gene for COPD and rs8040868 as a risk variant for the development of COPD in the Swedish population.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-016-0309-y) contains supplementary material, which is available to authorized users.