Beta2 nicotine receptor subunits: biomarkers for dependence

Beta2 尼古丁受体亚基：依赖的生物标志物

• 批准号: 9328036
• 负责人: Henry A. Lester
• 金额: $ 45.71万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9328036
• 关键词: Agonist; American Psychiatric Association; Anatomy; Animals; Binding; Biological Assay; Biological Markers; Biological Models; Brain; Brain region; Catabolism; Cell membrane; Cotinine; DSM-V; Data; Dependence; Disease; Engineering; Event; Exposure to; Flavoring; Fluorescence; Future; Health; Human; Immunofluorescence Immunologic; Individual; Intravenous; Knock-in Mouse; Label; Measures; Menthol; Mus; Muscle; Neurons; Nicotine; Nicotine Dependence; Nicotinic Receptors; Nomenclature; Pathway interactions; Positioning Attribute; Procedures; Production; Proteins; Research; Resolution; Techniques; Testing; Tobacco; Tobacco Control Research; Tobacco Dependence; Tobacco Use Disorder; Tobacco smoke; Tobacco use; Up-Regulation; Validation; Western Blotting; Withdrawal; Work; absorption; addiction; airway epithelium; cell type; experimental study; interest; nicotinic receptor beta2; public health relevance; tool

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.

项目描述（由申请人提供）：本项目响应烟草制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和烟草戒断，292.0）是与烟草相关的疾病，是所有其他烟草相关疾病的基础。在动物中建立尼古丁依赖的生物标志物，然后利用这种生物标志物获得关于薄荷醇作用的信息，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组分相关的生物标志物必须定位于脑区域、个体细胞类型和轴突与体树突隔室的水平。生物标志物必须在持续暴露于尼古丁期间形成。此外，这种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β 2烟碱乙酰胆碱受体（nAChR）亚单位（细胞内加质膜）的总水平。小鼠脑是一个完全合适的模型系统。该方法开发，然后开始利用，生产水平的测定，以确定在小鼠大脑中的β 2 * nAChRs的上调。持续暴露于尼古丁会导致β 2 * nAChR上调，并且假设这种上调对于尼古丁依赖的某些早期阶段来说是必要且足够的。Aim 1开发了一种相当有效的，定量的，但在解剖学上分辨率较低的方法， 免疫印迹技术（“western印迹”），用于定量小鼠脑不同区域中β 2亚基的量。这种方法将产生有价值的数据。目的2开发了一种更高分辨率的半定量方法，用于识别上调发生的神经元细胞类型，以及上调发生的亚细胞区域（轴突与体树突）。目标2采用最近开发的β 2-GFP基因敲入小鼠品系。大多数Aim 2使用GFP标记的β 2亚基的直接荧光;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β 2生物标志物所需的最低限度。目标3使用这些工具来测试一个重要的假设：即使在静脉注射时，薄荷脑也会上调nAChR。潜在的假设是，除了可能增加气道上皮对尼古丁的吸收或可能降低尼古丁催化剂外，薄荷醇还通过神经元水平的事件增加烟草依赖。该项目的总体结果将是一个生产水平的程序，可用于评估β 2亚基生物标志物的水平，作为烟草产品中心感兴趣的关键实验变量的函数。此外，目标3将使用β 2生物标志物来开始回答重要的问题，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，包括调味剂，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。β 2生物标志物测定也将适用于经工程改造以具有辅助蛋白改变的小鼠。因此，本项目以高影响、高意义和高度适当的方法响应RFA。