Jerry A. Stitzel - US grants

DESCRIPTION (Provided by applicant): It is the long-term objective of this proposal to identify inbred mouse strains that express functionally distinct isoforms of the neuronal nicotinic receptor subunits alpha3, alpha4, alpha6, alpha7, and beta2. These subunits were chosen based on their potential roles in mediating the dependence-causing actions of nicotine. Identification of functionally distinct nicotinic receptor isoforms will be initiated by mutation analysis of the exons of the genes that encode these receptor subunits. Approximately forty inbred mouse strains of distinct genetic origins will be evaluated in the mutation screen. Once the mutation screen identifies nicotinic receptor subunit isoforms in mice with amnino acid differences, the appropriate cDNAs will be generated. Subsequently, the variant nicotinic receptor subunit isoforms will be evaluated in vitro for their pharmacological and functional properties. These experiments will lead to the establishment of a catalogue of mouse strains with known variations in nicotinic receptor subtype functional properties. This catalogue of mouse strains will serve as an important additional resource to evaluate the role of the various nicotinic receptor subunits in modulating the dependence-causing actions of nicotine. It has been estimated that 25 percent of all premature deaths in the United States are due to tobacco use. Despite the widespread knowledge of the health risks of smoking, approximately 25 percent of adults in the United States continue to smoke. Among smokers, nearly 80 percent say that they would like to quit although less than 5 percent successfully do so. It is well established that nicotine is the major dependence-causing agent in tobacco and that the initial actions of nicotine in the brain are mediated by nicotinic acetyicholine receptors. However, which nicotinic receptor subtypes mediate the dependence-causing actions of nicotine is poorly understood. To date, only modest success, at best, has been achieved in resolving this issue. The mouse strain nicotinic receptor "function" catalogue that will result from the studies outlined in this proposal will add a valuable resource for addressing this issue. By elucidating which nicotinic receptor subtypes are critical for the establishment of nicotine dependence, more effective pharmacological strategies for smoking cessation may be developed.

[unreadable] DESCRIPTION (provided by applicant): In the United States, the use of tobacco products is responsible for over 450,000 premature deaths per year (about 20% of all premature deaths) and accounts for around $100 billion dollars per year in health care costs. Despite these facts, approximately 25% of adults in the United States continue to smoke. Typically, however, even heavy smokers can abstain from smoking during the normal sleep cycle and wake no more frequently during the night than do non-smokers. This observation suggests that there may be circadian patterns in which craving for nicotine is reduced at night. In support of this possibility, recent studies have demonstrated that sensitivity to drugs of abuse is generally reduced at night as well as altered by mutations in genes that regulate the circadian clock. In addition, melatonin, a hormone whose synthesis peaks at night, decreases craving in abstinent smokers and reduces sensitivity to the rewarding properties of some drugs of abuse in rodents. Despite this information, very little research has been done to assess circadian variations in sensitivity to nicotine. In the experiments outlined in this proposal, we will perform exploratory studies in mice in order to define the circadian pattern of sensitivity to the acute effects of nicotine. We also will examine whether any identified variation in sensitivity to nicotine is associated with circadian variations in melatonin synthesis, nicotinic receptor expression and/or nicotine metabolism. Finally, we will determine whether melatonin signaling through melatonin receptors is required for circadian variations in nicotine sensitivity, nicotinic receptor expression and nicotine metabolism. These studies will identify time points, mouse strains and potential mechanisms that will guide future studies to understand the mechanisms that regulate altered sensitivity to nicotine over the course of the circadian cycle. Such knowledge may provide novel insights into the neurobiology of nicotine addiction. to public health: Understanding the role of daily variations in nicotine sensitivity and the potential role of melatonin on regulating these daily variations in nicotine sensitivity may provide insight into why smokers typically can sleep through the night without the need for a cigarette. This knowledge would be extremely useful for the development of better methods to treat nicotine addiction. [unreadable] [unreadable] [unreadable]

Bierut and colleagues recently demonstrated that a polymorphism in the gene that encodes the alphas subunit, CHRNA5, is associated with nicotine dependence in human subjects. In addition, our group has shown that alphaS-containing nAChRs are involved in modulating nAChR function and also demonstrated that a polymorphism in the gene that encodes the mouse alphas subunit, ChrnaS, is associated with sensitivity to the high dose effects of nicotine. Nonetheless, the role of the nicotinic acetylcholine receptor (nAChR) alphas subunit in modulating the drug addiction process and brain function is poorly understood. In accordance with the overall goals of the COGEND Program Project, which are the identification of genes, environmental features, and biological mechanisms that predispose or protect individuals from the onset and persistence of nicotine dependence, we plan to conduct a series of experiments using three mouse genetic models of ChrnaS to address the basic mechanism(s) through which ChmaS/alphaS might contribute to nicotine addiction. The three mouse models we will utilize include!) Mice in which ChrnaS has been deleted (ChrnaS KO mice);2) Mice in which naturally-occurring allelic variants of ChmaS have been exchanged between two inbred mouse strains (C3.D2Chma5 and D2.C3Chma5);and 3) Mice in which the human nonsynonymous SNP has been introduced (ChmaS D398N Kl). With these mouse models, we will 1) define the brain regions and neurotransmitter systems whose function is modulated by alphas containing nAChRs;and 2) determine the role of ChrnaS in regulating behaviors that can be modeled in mice that are thought to be components of the addiction process, including drug reinforcement, drug aversion, tolerance development and withdrawal. Because the influence of ChrnaS on drug abuse related phenotypes could occur in adolescence and/or adulthood, both age groups will be assessed for nAChR function and behavior. Relevance to public health: Genes are known to play a significant part in determining whether an individual will become a smoker. In this proposal, the influence of a genetic difference in a specific gene called ChrnaS will be studied in mice to determine how this gene might affect how an individual responds to the addictive substance in tobacco, nicotine. This study should provide information that will improve our understanding of how genes influence the use of nicotine-containing products.

DESCRIPTION (provided by applicant): Smoking is responsible for approximately one in five premature deaths each year in the USA making it, without question, the single most preventable cause of premature death. In addition, lung cancer, which is predominantly caused by smoking, is the leading cause of cancer deaths in the USA. Recently, a series of 9 papers all reported that single nucleotide polymorphisms (SNPs) within the nicotinic receptor gene cluster CHRNA5-CHRNA3-CHRNB4 are associated with various smoking-related behaviors including nicotine dependence, level of smoking, age of initiation and subjective effects of smoking. Moreover, four studies also reported that variants in this same gene cluster are associated with risk for lung cancer. Whether the association between lung cancer and this gene cluster is indirect though the association with smoking or represents an independent finding remains to be determined. Regardless, due to the multiple replications of the association findings, studies to identify the polymorphism or polymorphisms in this region that influences risk for the single most preventable cause of premature death and the most common cause of cancer death are highly warranted. Therefore, in response to RFA-DA-09-003, which based upon the executive summary "focuses solely on functional characterization of gene variants which are strongly suggested to be associated with common, complex human diseases identified through candidate gene, GWAS, and other approaches", we propose a series of experiments to evaluate the function of an exceptionally strong candidate SNP in this region, rs16969968. This SNP is a non-synonymous SNP in the nicotinic receptor 15 subunit that leads to an asparagine for aspartic acid substitution at amino acid position 398. We previously have shown that this SNP affect the function of 142215 nicotinic receptors in vitro. In the studies outlined in this application, we will utilize a knockin mouse model in which the "at risk" asparagine codon has replaced the protective aspartic acid codon in Chrna5 to address the functional relevance of this polymorphism with regards to brain function and lung cancer susceptibility. We also will utilize in vitro experiments to determine whether the CHRNA5 D398N polymorphism affects the function of a subtype of nicotinic receptor (132415) that are expressed in the peripheral nervous system as well as in non-neuronal cells, including bronchial epithelial cells and lung cancer cell lines. PUBLIC HEALTH RELEVANCE: This project will test whether a specific mutation that is associated with risk for nicotine dependence and lung cancer in humans affects brain function and lung cancer susceptibility in a mouse model. Results will lead to a better appreciation of the genetics of these diseases and hopefully provide insight that may lead to more effective treatments for these conditions.

DESCRIPTION (provided by applicant): Human genetic studies have identified the cluster of nicotinic receptor genes (CHRNA5/A3/B4) on chromosome 15 as strong candidates for association with drug abuse-related behaviors, including smoking, alcohol phenotypes, and cocaine dependence. These genes have also been associated with lung cancer, but it is remains unclear what aspects of this association may be mediated through smoking behavior. In addition, we have found evidence that the intergenic region between the CHRNA3 and CHRNB4 genes is associated with more general measures of disinhibitory behavior, including conduct disorder, which is a known risk factor for early initiation of drugs and later problems. There is evidence from molecular, pharmacological, and animal studies that transcriptional regulation of these genes is likely to be co-regulated and complex, and that the 13 and 24 subunits might be good targets for development of smoking cessation drugs. We have initiated studies to assess the putative functional differences of alleles for SNPs that have been implicated in our human genetic studies. Our results provide evidence that at least two SNPs in this region may lead to differences in gene expression, using luciferase-gene assays in immortalized cell culture lines and in primary mouse neuronal embryonic cell cultures. The goals of this application are to extend these findings in several ways. First, we will use these assays to evaluate how the DNA sequence surrounding these SNPs may affect their ability to differentially drive gene expression ("promoter bashing"). Second, we will examine the effects of these SNPs in different cell types, including a variety of immortalized neuronal cells, lung cancer cells, and primary mouse neuronal embryonic cells (gene x cell type interaction). Third, we will challenge the cells with nicotine to assess whether this drug modifies gene expression patterns (gene x environment interaction). Fourth, we will determine whether allele-specific changes in gene expression using the luciferase assays lead to corresponding differences in protein subunit and receptor expression. Finally, we will assess function of nicotinic receptors whose subunit constituents were generated using our in vitro system containing different alleles for individual SNPs and haplotypes of SNPs. We expect the methods developed and applied to these genes to be easily applied to future studies of other nicotinic receptor genes that may also be associated with drug abuse-related behaviors. PUBLIC HEALTH RELEVANCE: Results from this project will facilitate a better understanding of how naturally occurring variations in the CHRNA3 and CHRNB4 genes might contribute to the underlying molecular mechanisms responsible for individual differences in behaviors relevant to drug use and abuse. Such knowledge should lead to the development of improved prevention and treatment of individuals who suffer from these disorders.

DESCRIPTION (provided by applicant):.Although many smokers attempt to quit each year, few are successful. The real world success rate for smoking abstinence at one year following a cessation attempt is an abysmal 4%. One of the reasons why smokers relapse is the cognitive impairment produced by nicotine withdrawal. In fact, this nicotine withdrawal symptom is predictive of relapse. Another phenomenon of chronic nicotine exposure is the up-regulation of ?2 nAChRs in brain. However, the role of this up-regulation in nicotine dependence is not clear. In animal models, nicotine withdrawal also produces cognitive impairment as well as up-regulation of ?2* nAChRs. Recent data have suggested that withdrawal-induced learning deficits in mice are dependent upon the up-regulation of ?2* nAChRs in the hippocampus. The mechanisms for nAChR up-regulation are not fully elucidated and hypotheses vary considerably and depend upon the in vitro system used for the mechanistic studies. Importantly, in brain, up-regulation is not uniform: it occurs in some brain regions but not others. Therefore, whatever the mechanism of up-regulation, it must be cell-type specific. It is known that modifications of serine residues in the large intracellular loop of the ?nAChR subunit are phosphorylated by PKA and PKC and modulate the expression and function of ?2 nAChR. Since phosphorylation can be cell-type specific, it is plausible that differential phosphorylation f the ?2* nAChR contributes to the cell-type specific up- regulation. Consistent with this possibility, at least one phosphorylation appears to be important for up- regulation of ?2 nAChRs in vitro. Our preliminary data also indicate that the kinase CDK5 alters ?2 nAChR expression and a putative CDK5 phosphorylation site in the ?subunit significantly affects function of ?2 nAChRs expressed in HEK293T cells. This site has not previously been implicated in ?2* expression or function. In this proposal, we will assess the role of the three putative serine phosphorylation sites in the intracellular loop of the ?nAChR-subunit for effects on cellular distribution, up-regulation, function and withdrawal-induced learning deficits. These experiments will completed by re-expressing wild type and phosphorylation site mutant ?subunits in primary cultured neurons derived from ?knockout mice (Specific Aim 1) and in the hippocampus of ?knockout mice (Specific Aim 2).

? DESCRIPTION (provided by applicant): Genetics clearly contributes to individual risk for nicotine dependence in humans and variations in nicotine sensitivity in experimental animals. In mice, several behavioral and physiological responses to nicotine have been demonstrated to be influenced by genetics. However, none of the specific genes that contribute to the genetic influence on nicotine sensitivity in mice have been identified. These genes remain an untapped source of information that almost certainly will improve our understanding of what drives individual differences in nicotine sensitivity. For example, the recent discovery that variants in human CHRNA5 are associated with risk for nicotine dependence in humans led to follow-up studies in rodents which not only helped to identify a specific neuronal pathway critical for controlling the level of nicotine consumption, but also demonstrated that this gene impacts individual differences in nicotine self-administration not by increased sensitivity to the reinforcng effects of nicotine at low doses but rather a lack of the loss of reinforcement at aversive doses. This is just one of many examples where the identification of a gene that contributes to individual variability in a phenotypic measure can lead to significant insights into the underlying biology of the measure. We previously have mapped chromosomal regions that harbor genes or genes that contribute to individual differences in oral nicotine intake in mice and it is the goal f this project to follow up this initial finding to identify the genes that contribute to variation i nicotine intake. For this, we will again map chromosomal regions that impact nicotine intake but this time in a panel of mice that will allow us to define with much greater precision the regions i the genome that harbor genes that impact nicotine intake. We will follow this up with selective breeding to produce lines of mice that differ in nicotine intake. The selection process should produce mouse lines that are enriched for alleles that are involved in increasing or decreasing nicotine intake. Finally, we will perform whole genome sequencing on the selected lines. We will use these sequencing data to establish whether the chromosomal regions identified through mapping are enriched through the selection process and to identify all variants within the region. We also will analyze the sequence data for other potential chromosomal regions that have been enriched through selection for nicotine consumption. This multi-tiered approach should allow us to substantially narrow the search for variants that influence nicotine intake and potentially lead to the identification of causal variants (functional variants that contribute to individual variabiity in nicotine consumption).

? DESCRIPTION (provided by applicant): Despite warnings of the teratogenic effects of tobacco use during pregnancy, estimates show that between 15 and 30% of all pregnant women in the US smoke. This leads to an alarming 700,000 children each year that are exposed to tobacco constituents in utero. Among the many reported negative outcomes of in utero nicotine exposure is a 3-5 fold elevated risk for the development of nicotine dependence. Rodent studies have supported the findings that developmental nicotine exposure increases negative outcomes, including increased risk for nicotine consumption. The mechanism(s) underlying the increased rate of smoking among in utero exposed children have not been elucidated. However, the women most prone to continue smoking during pregnancy are those at elevated genetic risk for nicotine dependence. Therefore, the elevated risk of nicotine dependence in their offspring could be due to inherited risk factors, in utero exposure, or both. Our laboratory recently has developed a mouse model that carries a human genetic variation in a gene named CHRNA5. Importantly for this application, women who have the risk version of this gene are less likely to stop smoking during pregnancy. Preliminary data from the mouse model indicate that mice with the risk version of the gene and exposed to nicotine in utero will consume the highest levels of nicotine suggesting an additive effect of genetic risk and environment. Strikingly and unexpectedly, however, offspring with the non-risk variant that were developmentally exposed to nicotine exhibited a dramatic reduction in nicotine intake during adolescence as compared to their genotype matched controls. This novel finding indicates that the direction of effect of developmental nicotine exposure on subsequent risk for nicotine intake is genotype dependent and challenges the notion that prenatal nicotine exposure alone is a risk factor for the development of nicotine dependence. To better appreciate how genotype and developmental nicotine exposure interact to dramatically alter outcomes, this exploratory project proposes two specific aims. Because alterations in nicotine consumption can be through altered reward or/and aversion to nicotine, we will determine the impact of nicotine exposure and genotype on these motivated behaviors. We also will assess whether the interaction between genotype and nicotine exposure to alter nicotine intake is due to the genotype of the offspring. In addition, we will assess the effect of genotype and prenatal nicotine exposure on the functional properties and structure of layer VI prefrontal cortical pyramidal cells that project to select brain areas relevant to reward and aversion. Understanding how genotype modifies the effect of developmental nicotine exposure on nicotine reward and brain circuits that modulate these behaviors will provide unique insight into the behavioral and neurophysiological mechanisms through which prenatal nicotine exposure alters risk for nicotine dependence.

PROJECT SUMMARY/ABSTRACT Less than 1 in 10 individuals who attempt to quit smoking remain abstinent for 1 year. This poor quit rate is driven, in part, by the fact that currently available drugs used to aid in smoking cessation are only moderately effective, at best. Thus, there is a great need to develop novel drugs that are more effective for smoking cessation. It is the goal of this project to use a novel genetic strategy to identify new biological targets for the potential development of novel smoking cessation drugs. The genetic strategy is based upon identifying modifier genes that alter nicotine responses in mice that have a null mutation in Chrna5, the gene that codes for the nicotinic receptor ?5 subunit. In effect, modifier genes are genes that contribute to physiological and/or molecular processes that are important for the behavior of interest but that generally go undetected in the absence of a perturbation in the gene that they modify. Because variants in Chrna5 alter risk for nicotine dependence in humans and studies in rodents clearly demonstrate that Chrna5 is critical for many nicotine- related behaviors, we believe that identifying genes that modify the effect of Chrna5 deletion on nicotine behaviors will uncover new genes relevant to nicotine dependence that may serve as novel targets for novel smoking cessation pharmacotherapies. Importantly, the behaviors that we plan to screen for modifiers are not only dependent upon Chrna5, but also dependent uponthe medial habenula-IPN pathway, a neural pathway that is thought to play a critical role in nicotine dependence. To identify genetic modifiers of the effect of Chrna5 deletion on nicotine behaviors, we propose 3 aims. In specific aim 1, we will breed the Chrna5 null mutation onto each of the B6-ChrA/J chromosome substitution strains (CSS) and identify chromosomes that harbor modifier genes for the effect of Chrna5 deletion on three nicotine behaviors, oral nicotine intake, somatic signs of nicotine withdrawal, and nicotine conditioned place preference. For specific aim 2, we will fine map those chromosomes that harbor modifier genes using sequential congenic strains. Typically, 3 generations of congenic strains starting from a CSS strain provides mapping resolution equivalent to that of any high resolution mapping population. Finally, in specific aim 3, we will use RNA-seq to identify genes whose expression is altered by the identified modifier genes. Importantly, we will use a state of the art genetic strategy that will allow us to examine gene expression in a neural cell population that is highly relevant to the behaviors: Chrna5 expressing cells of the interpeduncular nucleus. By combining the results of this aim with modifier loci identified through aims 1 and 2, we expect to narrow the list of potential candidate modifier genes and identify pathways specifically impacted by the modifier genes. In short, we believe that this strategy will lead to the identification of previously unknown genes and/or genetic pathways that contribute to the physiological and/or molecular processes important for the response to nicotine. These genes and/or pathways may serve as novel targets for the development of new pharmacotherapies to aid in smoking cessation.