Genetic Influences Over Nicotine Withdrawal

描述(申请人提供)：根据世界卫生组织的数据，世界上每分钟大约售出1000万支香烟，每8秒就有人死于烟草使用(世卫组织，2002年)。尼古丁是烟草的主要成瘾成分，与尼古丁戒断相关的情感和躯体症状导致戒烟困难。虽然目前的戒烟疗法是有帮助的，但没有一种方法能声称有非常高的成功率。因此，很有必要更好地了解尼古丁戒断背后的行为和生物学因素。吸烟的持续性受遗传因素的影响，最近发现尼古丁依赖与CHRNA5-CHRNA3-CHRNB4 nAChR基因簇的单核苷酸多态(SNP)之间的相关性。这种应用建立在这样的观察基础上，即缺乏含有？5的烟碱型乙酰胆碱受体(？5*nAChRs)可以消除躯体的戒断迹象，并减少焦虑样反应。内侧缰核(MHB)和脚间核(IPN)表达nAChRs。实验室最近的研究表明，在MHB或IPN中注射nAChR拮抗剂甲戊胺足以促使尼古丁处理的小鼠出现戒断症状。我们将首先询问引起D398N氨基酸变化的rs16969968 SNP是否可以改变MHB和IPN中表达的？5*nAChR亚型的生物物理和药理学特性。膜片钳实验将在包含D398或N398？5亚单位变体的nAChRs上的异源表达系统中进行。其次，为了研究？5*nAChRs在MHB和IPN中的直接作用，我们将利用慢病毒载体在？5基因缺失小鼠的MHB和IPN中表达？5基因变异体，或在野生型小鼠的MHB和IPN中下调内源性？5的水平。来自这些小鼠的MHB和IPN切片将用于膜片钳实验，以确定基因操作对尼古丁诱导的尖峰放电频率增加的影响。在其他实验中，注射慢病毒的小鼠将暴露在饮用水中的尼古丁中六周，以研究甲基淀粉沉淀物的戒断。这些研究将确定？5*nAChRs的表达是否对尼古丁戒断的表现是必要和充分的，以及rs16969968 15 SNP产生的潜在分子变化是否在体内转化为不同的戒断症状。 公共卫生相关性：我们的研究将利用基因工程小鼠和新的基因技术，并将结合行为分析、分子生物学和药理学来研究已识别的单核苷酸多态对尼古丁戒断表现的影响。这些研究可能会导致个性化戒烟疗法的发展。

DESCRIPTION (provided by applicant): According to the World Health Organization, about 10 million cigarettes are sold every minute in the world and every eight seconds someone dies from tobacco use (WHO, 2002). Nicotine is the main addictive component of tobacco, and the affective and somatic symptoms associated with nicotine withdrawal contribute to the difficulty in quitting tobacco use. While current therapies for smoking cessation are helpful, none can claim a very high rate of success. Therefore, there is a great need for a better understanding of the behavioral and biological factors underlying nicotine withdrawal. The persistence of cigarette smoking is influenced by genetic factors as highlighted by the recently found correlation between nicotine dependence and single nucleotide polymorphism (SNP) in the CHRNA5-CHRNA3-CHRNB4 nAChR gene cluster. This application builds on the observation that lack of ?5-containing nicotinic acetylcholine receptors (?5* nAChRs) abolishes somatic signs of withdrawal and reduces anxiety-like responses. ?5* nAChRs are expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies from the lab have shown that injection of the nAChR antagonist, mecamylamine, in either the MHb or IPN is sufficient to precipitate withdrawal signs in nicotine-treated mice. We will first ask whether the rs16969968 SNP, which causes a D398N aminoacid change, can alter the biophysical and pharmacological properties of the ?5* nAChR subtypes expressed in MHb and IPN. Patch clamp experiments will be conducted in heterologous expression systems on nAChRs comprising the D398 or N398 ?5 subunit variants. Second, to investigate the direct role of ?5* nAChRs in MHb and IPN, lentiviral vectors will be used to either express ?5 gene variants in the MHb and IPN of ?5 null mice or to knock down the levels of endogenous ?5 in the MHb and IPN of wild type mice. MHb and IPN slices from those mice will be used in patch clamping experiments to determine the effect of the genetic manipulations on nicotine-induced increases in spike firing frequency. In other experiments, lentivirus-injected mice will be exposed to nicotine in the drinking water for six weeks to study mecamylamine-precipitated withdrawal. The studies will determine whether expression of ?5* nAChRs is necessary and sufficient for the manifestation of nicotine withdrawal and whether the potential molecular changes produced by the rs16969968 15 SNP translate into different withdrawal symptoms in vivo. PUBLIC HEALTH RELEVANCE: Our studies will take advantage of genetically engineered mice and novel genetic techniques, and will combine behavioral analysis, molecular biology, and pharmacology to study the influence of an identified single nucleotide polymorphism on the manifestations of nicotine withdrawal. The studies could lead to the development of personalized smoking cessation therapies.