Beta2 nicotine receptor subunits: biomarkers for dependence

描述(由申请者提供)：该项目响应烟草产品研究计划中心的几个部分。尼古丁依赖(烟草使用障碍，DSM-5,305.1，烟草戒断，292.0)是一种烟草相关障碍，是所有其他烟草相关疾病的基础。建立动物尼古丁依赖的生物标记物，然后利用这个生物标记物来了解薄荷醇的作用，将对以下方面做出重要贡献 烟草控制研究。与人类尼古丁依赖的不同成分相关的生物标记物必须定位于大脑区域、单个细胞类型以及轴突与躯体树突之间的水平。生物标记物必须在持续接触尼古丁的过程中形成。此外，这一生物标志物应立即用于薄荷醇的研究。选择的生物标志物将包括检测β2烟碱型乙酰胆碱受体(NAChR)亚单位(细胞内+质膜)的总水平。小鼠大脑是一个完全合适的模型系统。该方法开发并开始开发一种生产水平的分析方法，用于确定小鼠大脑中Beta2*nAChRs的上调。持续接触尼古丁会导致β2*nAChRs上调，这种上调被认为对尼古丁依赖的早期阶段既是必要的，也是充分的。目标1开发了一种相当有效的、定量的、但在解剖学上分辨率低的方法，细化 一种免疫印迹技术(“蛋白质印迹”)，用于量化小鼠大脑不同区域中的Beta2亚单位的数量。这种方法将带来有价值的数据。目的2开发一种更高分辨率、半定量的方法，识别发生上调的神经细胞类型，以及发生上调的亚细胞区域(轴突与躯体树突状细胞)。AIM 2使用了最近开发的一种Beta2-GFP敲入小鼠。大多数AIM 2使用GFP标记的Beta2亚单位的直接荧光；免疫荧光用于提高灵敏度。目标2不在进一步工作的生产途径上，但目标2的数据是理解和验证Beta2生物标志物所必需的最低限度。AIM 3使用这些工具来检验一个重要的假设：薄荷醇即使在静脉注射时也会上调nAChRs。基本的假设是，薄荷醇通过神经元水平的事件增加烟草依赖，此外可能增加呼吸道上皮对尼古丁的吸收或可能减少尼古丁分解代谢。该项目的总体结果将是一个生产级程序，可用于根据烟草产品中心感兴趣的关键实验变量来评估Beta2亚单位生物标记物的水平。此外，目标3将使用Beta2生物标记物来开始回答这个重要的问题，薄荷醇在哪里起作用？FDA需要这些关键信息来评估/测量薄荷醇。未来的项目可以测试其他变量，包括调味料、其他可能的成瘾成分，如可替宁，以及烟草烟雾中的其他化合物。Beta2生物标记物检测也将适用于经过改造而具有辅助蛋白变化的小鼠。因此，本项目对RFA的回应具有很高的影响力、很高的意义、非常合适的方法。

DESCRIPTION (provided by applicant): This project responds to several sections in Center for Tobacco Products Research Plan. Nicotine dependence (Tobacco Use Disorder, DSM-5, 305.1, and Tobacco Withdrawal, 292.0) is the tobacco-related disorder that underlies all other tobacco-related diseases. Establishing a biomarker for nicotine dependence in animals, and then exploiting this biomarker for information on menthol's actions, will contribute importantly to tobacco control research. A biomarker relevant to distinct components of human nicotine dependence must be localized at the level of brain region, of individual cell types, and of axonal vs. somatodendritic compartments. The biomarker must develop during maintained exposure to nicotine. Furthermore, this biomarker should immediately be exploited for studies on menthol. The biomarker chosen will consist of detecting the total level of beta2 nicotinic acetylcholine receptor (nAChR) subunits (intracellular plus plasma membrane). Mouse brain is an entirely appropriate model system. The approach develops, and then begins to exploit, a production-level assay for determining the upregulation of beta2* nAChRs in mouse brain. Maintained exposure to nicotine produces upregulation of beta2* nAChRs, and such upregulation is hypothesized to be both necessary and sufficient for some early stages of nicotine dependence. Aim 1 develops a rather efficient, quantitative, but anatomically low-resolution approach, refining an immunoblot technique ("western blots") for quantifying the amount of beta2 subunits in various regions of mouse brain. The approach will lead to valuable data. Aim 2 develops a higher-resolution, semiquantitative approach that identifies the neuronal cell types in which upregulation occurs, as well as the subcellular regions (axonal vs. somatodendritic) in which upregulation occurs. Aim 2 employs a recently developed strain of beta2-GFP knock-in mice. Most of Aim 2 uses direct fluorescence for the GFP-labeled beta2 subunit; immunofluorescence is used to enhance sensitivity. Aim 2 is not on the production pathway for further work, but the data of Aim 2 are the minimum necessary for understanding and validating the beta2 biomarker. Aim 3 uses these tools to test an important hypothesis: that menthol upregulates nAChRs even when delivered intravenously. The underlying assumption is that menthol increases tobacco dependence via events at the level of neurons, in addition to possible increased absorption of nicotine through airway epithelium or possible decreased nicotine catabolism. The overall results of this project will be a production-level procedure that can be used to assess levels in the beta2 subunit biomarker as a function of key experimental variables that interest the Center for Tobacco Products. Furthermore Aim 3 will use the beta2 biomarker to begin answering the important question, where does menthol act? FDA requires this key information in order to evaluate / measure menthol. Future projects can test additional variables including flavorings, additional possible addictive components such as cotinine, and other compounds in tobacco smoke. The beta2 biomarker assay will also be appropriate for mice engineered to possess alterations in accessory proteins. Therefore this project responds to the RFA with high impact, high significance, and a highly appropriate approach.