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: 8913108
• 负责人: Henry A. Lester
• 金额: $ 40.58万
• 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/8913108
• 关键词: Agonist; American Psychiatric Association; Animals; Binding; Biological Assay; Biological Markers; Biological Models; Brain; Brain region; Catabolism; Cell membrane; Cotinine; Data; Dependence; Diagnostic and Statistical Manual of Mental Disorders; Disease; Engineering; Event; Exposure to; Flavoring; Fluorescence; Future; Head; Health; Human; Immunoblotting; Immunofluorescence Immunologic; Individual; Knock-in Mouse; Label; Lead; Measures; Menthol; Mus; Muscle; Neurons; Nicotine; Nicotine Dependence; Nicotinic Receptors; Nomenclature; Pathway interactions; Positioning Attribute; Procedures; Production; Proteins; Research; Resolution; Staging; 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; interest; nicotinic receptor beta2; receptor; research study; 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.

描述（由申请人提供）：该项目对烟草产品研究中心的几个部分做出了回应。尼古丁依赖性（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，其基于所有其他与烟草相关的疾病的基础。建立一种在动物中建立尼古丁依赖性的生物标志物，然后利用该生物标志物以获取有关Menthol行为的信息，将为 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志物必须位于大脑区域，单个细胞类型以及轴突与轴突隔室的水平。在维持接触尼古丁期间，必须发展生物标志物。此外，该生物标志物应立即被利用用于薄荷醇的研究。选择的生物标志物将包括检测β2烟碱乙酰胆碱受体（NACHR）亚基的总水平（细胞内加质膜）。鼠标大脑是一个完全合适的模型系统。该方法开发，然后开始利用，这是一种生产水平的测定，用于确定小鼠脑中β2* NACHR的上调。维持接触尼古丁会产生β2* NACHR的上调，并且认为这种上调是尼古丁依赖性的某些早期阶段所必需的和足够的。 AIM 1开发了一种相当有效，定量但解剖学低分辨率的方法 一种免疫印迹技术（“ Western印迹”），用于量化小鼠大脑各个区域中β2亚基的量。该方法将导致有价值的数据。 AIM 2开发了一种高分辨率的半定量方法，该方法识别出上调发生的神经元细胞类型，以及在上调上调节的亚细胞区域（轴突与somatendritic）。 AIM 2采用了最近开发的beta2-GFP敲击小鼠菌株。 AIM 2的大多数使用直接荧光用于GFP标记的beta2亚基；免疫荧光用于增强灵敏度。 AIM 2不在进一步工作的生产途径上，但是AIM 2的数据是理解和验证Beta2生物标志物所需的最低限度。 AIM 3使用这些工具来检验一个重要的假设：即使在静脉内交付时，薄荷醇也会上调NACHR。基本的假设是，除了可能通过气道上皮细胞吸收尼古丁的吸收或可能降低尼古丁分解代谢外，薄荷醇通过事件在神经元水平上通过事件增加了烟草依赖性。该项目的总体结果将是一种生产级别的程序，可用于评估Beta2亚基生物标志物中的水平，这是关键实验变量的函数，该函数感兴趣烟草产品中心。此外，目标3将使用beta2生物标志物开始回答重要的问题，而Menthol在哪里？ FDA需要此关键信息才能评估 /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，例如可替宁和烟草烟雾中的其他化合物。 beta2生物标志物测定也将适合于工程具有辅助蛋白改变的小鼠。因此，该项目以高影响力，高意义和高度适当的方法对RFA做出反应。