A calcium/calcineurin signaling cascade regulates neuronal cannabinoid receptors

描述（由申请人提供）：本项目将测试一种Ca2+和钙调素依赖性磷酸酶，钙调神经磷酸酶，调节神经元中1型大麻素受体（CB 1）的转录的假设。CB1是哺乳动物脑中植物和内源性大麻素的主要靶点。CB1激动剂正在研究作为急性和慢性神经元变性的治疗，而拮抗剂似乎有希望治疗尼古丁、酒精、可卡因、海洛因、吗啡成瘾以及肥胖症。因此，有强烈的兴趣，在确定的生理，治疗和病理作用的CB1信号。目前，我们对大麻素的行为和药理作用以及CB1的结构、功能和信号传导有了大量的了解。然而，我们对调控转录CB1的因子几乎一无所知。我的实验室已经证明，去极化和Ca2+依赖的信号级联调节CB1 mRNA和蛋白质的表达在原代培养的颗粒神经元从啮齿动物小脑皮质。我们的初步数据表明钙调神经磷酸酶（CaN）在这一过程中的主要作用。我们提出两个具体目标。AIM 1.目的：验证钙离子依赖性激活CaN抑制大鼠小脑皮质颗粒神经元CB1 mRNA转录的假说。我们将明确确定CaN及其下游效应，NFAT，在CB1转录的生物化学，免疫化学，分子生物学和显微镜技术相结合的作用。AIM 2.为了验证CaN依赖性抑制CB1基因是通过包含多个NFAT反应位点的启动子区和位于5'非翻译区的假设。公共卫生相关性：大麻是青少年滥用最多的非法药物，长期消费使年轻滥用者容易染上更严重的毒瘾。大麻素受体（CB1）拮抗剂正在测试作为治疗各种药物成瘾，包括尼古丁，酒精，可卡因和鸦片。因此，有强烈的兴趣，在确定的生理，治疗和病理作用的CB1信号。该项目将测试的假设，钙离子和钙调素依赖性磷酸酶，钙调神经磷酸酶，调节1型大麻素受体（CB1）在培养的小脑神经元的转录。我们在培养的神经元中的结果将作为未来在动物中进行功能研究的基础。

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.