MicroRNA Regulation of Nicotinic Acetylcholine Receptor Expression and Function

描述（由申请人提供）：烟草使用每年在全世界造成500万人死亡，是西方世界可预防死亡的主要原因。尼古丁是烟草中令人上瘾的成分，它结合并激活了一个配体门控离子通道家族，即尼古丁乙酰胆碱受体（nAChRs）。多巴胺能（DAergic）中边缘奖赏通路中受体的激活被认为是成瘾开始的基础。了解尼古丁对nAChR影响的困难之一在于存在多种nAChR亚型，每种亚型都表现出独特的电生理特性和对尼古丁的不同亲和力。已经鉴定出十二个不同的神经元nAChR亚基（12-110和22-24）。五个亚基共同组装形成受体，每个通道的亚基组成决定其药理学和生物物理特性。慢性尼古丁暴露改变了nAChR亚型的表达，这可能导致尼古丁依赖；然而，调控这些变化的潜在机制尚不清楚。越来越多的证据表明，尼古丁和香烟烟雾会改变21-24个核苷酸长的小调控分子的表达，这些分子被称为microrna （miRNAs）。据预测，mirna可以调节超过60%的人类基因组和大多数哺乳动物蛋白质编码基因，通常通过结合靶mrna的3'-非翻译区（3'- utr）的互补位点并引导它们进入RNA诱导的沉默复合体（RISC）。据报道，一种miRNA miR-1在秀丽隐杆线虫中靶向nAChR，导致nAChR特性的改变。我们对哺乳动物14和22 nAChR基因进行了生物信息学分析，发现它们含有几种已知mirna的预测结合位点。本研究将探讨尼古丁诱导的miRNA表达变化通过改变nAChR亚型的表达介导中脑边缘通路（奖励通路）功能变化的假设。为了解决这个问题，我将结合分子生物学、电生理学和药理学。在AIM 1中，我将检查mirna是否调节nAChR亚基14和22。在AIM 2中，我将研究尼古丁对在DAergic中脑边缘通路中起作用的mirna表达水平的影响。在AIM 3中，我将测试mirna是否必要和/或足以引起尼古丁诱导的中脑能神经元nAChR功能特性的变化。最终，更好地了解控制尼古丁依赖相关行为的分子过程将导致戒烟新疗法的发展。

DESCRIPTION (provided by applicant): Tobacco use causes five million deaths worldwide annually and is the leading cause of preventable mortality in the Western world. Nicotine is the addictive component of tobacco that binds to and activates a family of ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs). Activation of the receptors in the dopaminergic (DAergic) mesolimbic reward pathway is thought to underlie the initiation of addiction. One of the difficulties in understanding nicotine's effect on nAChRs arises from the existence of multiple nAChR subtypes, each exhibiting unique electrophysiological properties and varying affinities for nicotine. Twelve distinct neuronal nAChR subunits have been identified (12-110 and 22-24). Five subunits co-assemble to form receptors with the subunit composition of each channel determining its pharmacological and biophysical properties. Chronic nicotine exposure alters the expression of nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear. A growing body of evidence indicates that nicotine and cigarette smoke alters the expression of small 21-24 nucleotide long regulatory molecules, referred to as microRNAs (miRNAs). miRNAs are predicted to regulate over 60% of the human genome and the majority of all mammalian protein coding genes, typically by binding to complementary sites in the 3'-untranslated regions (3'-UTRs) of target mRNAs and guiding them to an RNA- induced silencing complex (RISC). A miRNA, miR-1, has been reported to target nAChRs in C. elegans, leading to changes in nAChR properties. We carried out bioinformatics analysis of mammalian 14 and 22 nAChR genes and found that they contain predicted binding sites for several known miRNAs. This proposal will investigate the hypothesis that nicotine-induced changes in miRNA expression mediate functional changes in the mesolimbic pathway (the reward pathway) by altering the expression of nAChR subtypes. To address this, I will use a combination of molecular biology, electrophysiology, and pharmacology. In AIM 1 I will examine if miRNAs regulate nAChR subunits 14 and 22. In AIM 2 I will look at nicotine's effect on the expression levels of miRNAs that function within the DAergic mesolimbic pathway. In AIM 3 I will test if miRNAs are necessary and/or sufficient to cause nicotine-induced changes in nAChR functional properties of DAergic neurons from the midbrain. Ultimately, gaining a better understanding of the molecular processes that act to control nicotine dependence-associated behaviors will lead to the development of novel therapies for smoking cessation. PUBLIC HEALTH RELEVANCE: Tobacco use is the second leading cause of death in the world and the single largest preventable cause of disease and premature mortality. Nicotine, the primary addictive component of tobacco, is among the most addictive substances of abuse. The goal of this project is to gain a better understanding of the molecular processes that act to control expression of the neuronal receptors for nicotine. We anticipate that insights gained from this project will help lead to the development of therapies for smoking cessation.