Jerry A. Stitzel

Variants in Nicotinic Receptors and Risk for Nicotine Dependence

OBJECTIVE A recent study provisionally identified numerous genetic variants as risk factors for the transition from smoking to the development of nicotine dependence, including an amino acid change in the alpha5 nicotinic cholinergic receptor (CHRNA5). The purpose of this study was to replicate these findings in an independent data set and more thoroughly investigate the role of genetic variation in the cluster of physically linked nicotinic receptors, CHRNA5-CHRNA3-CHRNB4, and the risk of smoking. METHOD Individuals from 219 European American families (N=2,284) were genotyped across this gene cluster to test the genetic association with smoking. The frequency of the amino acid variant (rs16969968) was studied in 995 individuals from diverse ethnic populations. In vitro studies were performed to directly test whether the amino acid variant in the CHRNA5 influences receptor function. RESULTS A genetic variant marking an amino acid change showed association with the smoking phenotype (p=0.007). This variant is within a highly conserved region across nonhuman species, but its frequency varied across human populations (0% in African populations to 37% in European populations). Furthermore, functional studies demonstrated that the risk allele decreased response to a nicotine agonist. A second independent finding was seen at rs578776 (p=0.003), and the functional significance of this association remains unknown. CONCLUSIONS This study confirms that at least two independent variants in this nicotinic receptor gene cluster contribute to the development of habitual smoking in some populations, and it underscores the importance of multiple genetic variants contributing to the development of common diseases in various populations.

Multiple distinct risk loci for nicotine dependence identified by dense coverage of the complete family of nicotinic receptor subunit (CHRN) genes

Tobacco smoking continues to be a leading cause of preventable death. Recent research has underscored the important role of specific cholinergic nicotinic receptor subunit (CHRN) genes in risk for nicotine dependence and smoking. To detect and characterize the influence of genetic variation on vulnerability to nicotine dependence, we analyzed 226 SNPs covering the complete family of 16 CHRN genes, which encode the nicotinic acetylcholine receptor (nAChR) subunits, in a sample of 1,050 nicotine‐dependent cases and 879 non‐dependent controls of European descent. This expanded SNP coverage has extended and refined the findings of our previous large‐scale genome‐wide association and candidate gene study. After correcting for the multiple tests across this gene family, we found significant association for two distinct loci in the CHRNA5–CHRNA3–CHRNB4 gene cluster, one locus in the CHRNB3–CHRNA6 gene cluster, and a fourth, novel locus in the CHRND–CHRNG gene cluster. The two distinct loci in CHRNA5–CHRNA3–CHRNB4 are represented by the non‐synonymous SNP rs16969968 in CHRNA5 and by rs578776 in CHRNA3, respectively, and joint analyses show that the associations at these two SNPs are statistically independent. Nominally significant single‐SNP association was detected in CHRNA4 and CHRNB1. In summary, this is the most comprehensive study of the CHRN genes for involvement with nicotine dependence to date. Our analysis reveals significant evidence for at least four distinct loci in the nicotinic receptor subunit genes that each influence the transition from smoking to nicotine dependence and may inform the development of improved smoking cessation treatments and prevention initiatives.

Genetic influences on nicotine responses.

Male mice from 19 inbred strains were tested for the effects of nicotine on six responses: respiratory rate, acoustic startle response, Y-maze crosses, Y-maze rears, heart rate and body temperature. Dose-response curves were constructed for each strain on each test in a multitest battery. Results indicated that the responses were strongly influenced by the genotype of the animal. Comparison of the results from the six tests measured in this study and the results previously reported for nicotine-induced seizures in these same strains indicated that the responses could be grouped into two major classes: a set characterized by Y-maze crosses, Y-maze rears and body temperature and a set characterized by seizure sensitivity and seizure latency. Responses observed for respiratory rate and startle response shared characteristics with both of these sets, while nicotine effect on heart rate was fairly unique. The results have identified strains of mice which are differentially sensitive to the effects of nicotine.

Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5

Nicotine dependence risk and lung cancer risk are associated with variants in a region of chromosome 15 encompassing genes encoding the nicotinic receptor subunits CHRNA5, CHRNA3 and CHRNB4. To identify potential biological mechanisms that underlie this risk, we tested for cis-acting eQTLs for CHRNA5, CHRNA3 and CHRNB4 in human brain. Using gene expression and disease association studies, we provide evidence that both nicotine-dependence risk and lung cancer risk are influenced by functional variation in CHRNA5. We demonstrated that the risk allele of rs16969968 primarily occurs on the low mRNA expression allele of CHRNA5. The non-risk allele at rs16969968 occurs on both high and low expression alleles tagged by rs588765 within CHRNA5. When the non-risk allele occurs on the background of low mRNA expression of CHRNA5, the risk for nicotine dependence and lung cancer is significantly lower compared to those with the higher mRNA expression. Together, these variants identify three levels of risk associated with CHRNA5. We conclude that there are at least two distinct mechanisms conferring risk for nicotine dependence and lung cancer: altered receptor function caused by a D398N amino acid variant in CHRNA5 (rs16969968) and variability in CHRNA5 mRNA expression.

A Risk Allele for Nicotine Dependence in CHRNA5 Is a Protective Allele for Cocaine Dependence

BACKGROUND A nonsynonymous coding polymorphism, rs16969968, of the CHRNA5 gene that encodes the alpha-5 subunit of the nicotinic acetylcholine receptor (nAChR) has been found to be associated with nicotine dependence. The goal of this study was to examine the association of this variant with cocaine dependence. METHODS Genetic association analysis was performed in two independent samples of unrelated case and control subjects: 1) 504 European Americans participating in the Family Study on Cocaine Dependence (FSCD) and 2) 814 European Americans participating in the Collaborative Study on the Genetics of Alcoholism (COGA). RESULTS In the FSCD, there was a significant association between the CHRNA5 variant and cocaine dependence (odds ratio = .67 per allele, p = .0045, assuming an additive genetic model), but in the reverse direction compared with that previously observed for nicotine dependence. In multivariate analyses that controlled for the effects of nicotine dependence, both the protective effect for cocaine dependence and the previously documented risk effect for nicotine dependence were statistically significant. The protective effect for cocaine dependence was replicated in the COGA sample. In COGA, effect sizes for habitual smoking, a proxy phenotype for nicotine dependence, were consistent with those observed in FSCD. CONCLUSIONS The minor (A) allele of rs16969968, relative to the major G allele, appears to be both a risk factor for nicotine dependence and a protective factor for cocaine dependence. The biological plausibility of such a bidirectional association stems from the involvement of nAChRs with both excitatory and inhibitory modulation of dopamine-mediated reward pathways.

An autoradiographic analysis of cholinergic receptors in mouse brain

Autoradiographic techniques were used to localize cholinergic receptors in the central nervous system of female DBA mice. Nicotinic receptors were identified using [3H]-L-nicotine and alpha-[125I]-bungarotoxin (BTX); [3H]-quinuclidinyl benzilate (QNB) was used to examine muscarinic receptor binding. There was little overlap between the regional distribution of binding sites for these ligands. Nicotine binding was highest in thalamic nuclei, the superior colliculus and the interpeduncular nucleus. For BTX binding, high density receptor populations were identified in the hippocampus, caudate putamen, colliculi (superior and inferior) and various nuclei in the hypothalamus and hindbrain. Muscarinic receptors were distributed more uniformly than nicotinic receptors; the colliculi, hippocampus and cerebral cortex had the highest level of QNB binding. Species differences between rats and mice in terms of cholinergic receptor binding are discussed.

The Incentive Salience of Alcohol: Translating the Effects of Genetic Variant in CNR1

CONTEXT The gene that codes for cannabinoid receptor 1 (CNR1) represents an important target for investigations designed to elucidate individual differences in the etiology of alcohol dependence. OBJECTIVE To achieve a better understanding of the role of the CNR1 gene in the etiology and treatment of alcohol dependence. DESIGN The present investigation spans multiple levels of analysis, including receptor binding in postmortem brain tissue, neuroimaging, human laboratory models, and analyses of treatment outcome data. RESULTS Findings indicate that the C allele of rs2023239 is associated with greater CB1 binding in the prefrontal cortex, greater alcohol cue-elicited brain activation in the midbrain and prefrontal cortex, greater subjective reward when consuming alcohol, and more positive outcomes after treatment with a medication that targets the mesocorticolimbic neurocircuitry. In addition, there were strong correlations between cue-elicited brain activation and alcohol consumption measures in individuals with the C allele. CONCLUSION Individuals with the C allele may be more susceptible to changes in the mesocorticolimbic neurocircuitry that is involved in the attribution of incentive salience after repeated exposure to alcohol.

Acetylcholine-Stimulated [3H]GABA Release from Mouse Brain Synaptosomes is Modulated by α4β2 and α4α5β2 Nicotinic Receptor Subtypes

Nicotinic acetylcholine receptor (nAChR) agonists stimulate the release of GABA from GABAergic nerve terminals, but the nAChR subtypes that mediate this effect have not been elucidated. The studies reported here used synaptosomes derived from the cortex, hippocampus, striatum, and thalamus of wild-type and α4-, α5-, α7-, β2-, and β4-null mutant mice to identify nAChR subtypes involved in acetylcholine (ACh)-evoked GABA release. Null mutation of genes encoding the α4 or β2 subunits resulted in complete loss of ACh-stimulated [3H]GABA release in all four brain regions. In contrast, α5 gene deletion exerted a small but significant decrease in maximal ACh-evoked [3H]GABA release in hippocampus and striatum, with a more profound effect in cortex. Acetylcholine-stimulated [3H]GABA release from thalamic synaptosomes was not significantly affected by α5 gene deletion. No effect was detected in the four brain regions examined in α7- or β4-null mutant mice. Further analysis of ACh-evoked [3H]GABA release revealed biphasic concentration-response relationships in the four brain regions examined from all wild-type animals and in α5 null mutant mice. Moreover, a selective reduction in the maximum response of the high-affinity component was apparent in α5-null mutant mice. The results demonstrate that α4β2-type nAChRs are critical for ACh-stimulated [3H]GABA release from all four brain regions examined. In addition, the results suggest that α5-containing receptors on GABAergic nerve terminals comprise a fraction of the high ACh-sensitivity component of the concentration-response curve and contribute directly to the ability of nicotinic agonists to evoke GABA release in these regions.

Influence of kinetics of nicotine administration on tolerance development and receptor levels.

Nicotine was administered intravenously to DBA mice through cannulae implanted in the jugular veins. Five groups of animals were treated: a control group which received saline and four nicotine treatment groups. All of the nicotine treatment groups received a dose of 4.0 mg/kg/hr. The first group received continuous infusion, the second group received 1 mg/kg pulses four times an hour, the third group received 2 mg/kg pulses twice an hour, and the fourth group received 4 mg/kg pulses once an hour. After a 10-day treatment period, the animals were tested for tolerance to an acute intraperitoneal administration of nicotine. Tolerance was measured using a test battery composed of the following tests: respiratory rate, acoustic startle response, Y-maze crosses and rears, heart rate, and body temperature. Mice from each of the four nicotine treatment groups were tolerant to the acute effect of nicotine, but the extent of tolerance varied among the groups as follows: continuous infusion less than 1 mg/kg pulses four times/hr less than 2 mg/kg pulses twice/hr less than 4 mg/kg pulse once/hr. Chronic nicotine infusion resulted in significant increases in the binding of L-[3H]nicotine in all six brain regions assayed and in significant increases in the binding of alpha-[125I]bungarotoxin binding in cerebral cortex and hippocampus. All increases in binding resulted from increases in Bmax for these ligands. In contrast to the effects observed for tolerance development, the increases in [3H]nicotine binding were not significantly affected by the kinetics of nicotine infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional characterization of mouse α4β2 nicotinic acetylcholine receptors stably expressed in HEK293T cells

Mouse α4β2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mmα4β2, was assessed using an aequorin‐based luminescence method that measures agonist‐evoked changes in intracellular calcium. Agonist‐elicited changes in intracellular calcium were due primarily to direct entry of calcium through the α4β2 channel, although release of calcium from intracellular stores contributed ˜ 28% of the agonist‐evoked response. Agonist pharmacologies were very similar between the mmα4β2 cells and most cell lines that stably express human α4β2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro‐β‐erythroidine (DHβE), the predominant α4β2 nAchR expressed in the mmα4β2 cells exhibits a pharmacology that most resembles the DHβE‐sensitive component of 86Rb+ efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mmα4β2 nAchR was found to be atypically sensitive to blockade by the presumed α7‐selective antagonist methyllycaconitine (MLA), exhibiting an IC50 value of 31 ± 0.1 nm. Similar IC50 values have been reported for the MLA inhibition of nicotine‐stimulated dopamine release, a response that is mediated by β2‐subunit‐containing nAchRs and not α7‐subunit‐containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for α7‐containing nAchRs as previously thought.