A calcium/calcineurin signaling cascade regulates neuronal cannabinoid receptors

描述(申请人提供)：这个项目将测试一种钙离子和钙调蛋白依赖的磷酸酶，钙调神经磷酸酶，调节神经元中1型大麻素受体(CB1)转录的假设。CB1是哺乳动物大脑中植物和内源性大麻素的主要靶标。CB1激动剂正在被研究用于治疗急性和慢性神经元变性，而拮抗剂似乎有望治疗尼古丁、酒精、可卡因、海洛因、吗啡成瘾以及肥胖。因此，人们对确定CB1信号的生理、治疗和病理效应非常感兴趣。目前，我们对大麻素的行为和药理作用，以及CB1的结构、功能和信号转导有了很大的了解。然而，我们对调控转录CB1的因素几乎一无所知。我的实验室已经证明，在原代培养的啮齿动物小脑皮质颗粒神经元中，去极化和钙依赖的信号级联调节CB1mRNA和蛋白的表达。我们的初步数据表明，钙调神经磷酸酶(CaN)在这一过程中发挥了主要作用。我们提出了两个具体目标。目的1.验证钙依赖激活CaN抑制啮齿动物小脑皮质颗粒神经元CB1mRNA转录的假说。我们将结合生化、免疫化学、分子生物学和显微技术，最终确定CaN及其下游效应因子NFAT在CB1转录中的作用。目的2.验证CB1基因的CaN依赖的抑制是通过包含多个NFAT反应位点的启动子区域和位于5‘非翻译区的假说。与公共卫生有关：大麻是青少年中滥用最多的非法药物，长期消费使青少年吸毒者更容易上瘾。大麻素受体(CB1)拮抗剂正在接受测试，用于治疗各种药物成瘾，包括尼古丁、酒精、可卡因和阿片类药物。因此，人们对确定CB1信号的生理、治疗和病理效应非常感兴趣。这个项目将测试一种钙离子和钙调蛋白依赖的磷酸酶，钙调神经磷酸酶，调节来自小脑的培养神经元中1型大麻受体(CB1)的转录的假设。我们在培养神经元上的结果将作为未来动物功能研究的基础。

DESCRIPTION (provided by applicant): This project will test the hypothesis that a Ca2+ and calmodulin-dependent phosphatase, calcineurin, regulates the transcription of type 1 cannabinoid receptors (CB1) in neurons. CB1 is the primary target for plant and endogenous cannabinoids in mammalian brain. CB1 agonists are being studied as treatments for acute and chronic neuronal degeneration, whereas antagonists appear promising to treat addictions to nicotine, alcohol, cocaine, heroin, morphine, as well as obesity. Thus there is intense interest in defining the physiological, therapeutic and pathological effects of CB1 signaling. Currently, we understand a great deal about the behavioral and pharmacological effects of cannabinoids, and CB1 structure, function and signaling. However, we know almost nothing about factors regulating the transcription CB1. My laboratory has demonstrated that a depolarization and Ca2+-dependent signaling cascade regulates expression of CB1 mRNA and protein in primary cultures of granule neurons from rodent cerebellar cortex. Our preliminary data point to primary role for calcineurin (CaN) in this process. We propose 2 specific aims. AIM 1. To test the hypothesis that Ca2+-dependent activation of CaN represses the transcription of CB1 mRNA in granule neurons from rodent cerebellar cortex. We will definitively determine the role of CaN and its downstream effector, NFAT, in CB1 transcription using a combination of biochemical, immunochemical, molecular biological and microscopic techniques. AIM 2. To test the hypothesis that CaN-dependent repression of the CB1 gene is via a promoter region containing multiple NFAT responsive sites and lying in the 5' untranslated region. PUBLIC HEALTH RELEVANCE: Cannabis is the most abused illegal drug in adolescents and chronic consumption predisposes young abusers to more serious addictions. Cannabinoid receptor (CB1) antagonists are being tested as treatments for a variety of drug addictions, including nicotine, alcohol, cocaine, and opiates. Thus, there is intense interest in defining the physiological, therapeutic and pathological effects of CB1 signaling. This project will test the hypothesis that a Ca2+ and calmodulin-dependent phosphatase, calcineurin, regulates the transcription of type 1 cannabinoid receptors (CB1) in cultured neurons from the cerebellum. Our results in cultured neurons will serve as a basis for future functional studies in animals.