Calcium Signaling in Circadian Clock Neurons

描述（由申请人提供）：本提案的长期目标是阐明细胞内钙信号在生物钟神经元中的作用。实验将在果蝇（Drosophila melanogaster）中进行，果蝇具有功能复杂且解剖学上充分表征的昼夜节律控制系统，并且独特地适合于应用靶向完整行为动物中的时钟神经元的遗传方法。除了典型的转录反馈机制，昼夜节律振荡还依赖于去极化激活的离子膜电导。所提出的目的是探索由膜去极化触发的细胞内钙信号是细胞昼夜节律振荡器的核心组成部分的假设。工程化钙缓冲蛋白特异性靶向转基因果蝇脑中的时钟神经元，以破坏完整活生物体中的细胞钙信号，然后测量对运动活动的昼夜节律、昼夜振荡器的已知转录因子组分的细胞积累和细胞内钙动力学的影响。使用这种方法的初步研究表明，时钟神经元中的细胞内钙缓冲导致自由运行的行为和细胞节律的剂量依赖性减慢，在最高剂量下具有抑制性。提出的目标将确定相关钙信号的亚细胞位置，它们的时间动态，它们的破坏对细胞节律的详细影响，以及它们转导所需的下游钙敏感信号通路。由于果蝇和哺乳动物昼夜节律的遗传和细胞基础非常相似，因此在拟议的研究中利用果蝇的遗传可及性可以获得独特的信息，这些信息将深入了解细胞振荡器功能的一般原理，这些原理与哺乳动物模型系统中的基本昼夜节律研究和人类昼夜节律功能障碍的临床研究都相关。通过基因突变（如晚期睡眠期障碍）、疾病或环境条件（如时差反应或夜班工人）破坏人类的日常休息和活动节奏，对公共卫生、工作场所安全和经济生产力产生了许多不利影响。了解这些节律的细胞机制是开发药物和其他治疗方法的关键。拟议的研究将提供洞察细胞振荡器功能的一般原则，这是相关的基础昼夜节律的研究在哺乳动物模型系统和人类的昼夜节律功能紊乱的临床研究。通过基因突变（如晚期睡眠期障碍）、疾病或环境条件（如时差反应或夜班工人）破坏人类的日常休息和活动节奏，对公共卫生、工作场所安全和经济生产力产生了许多不利影响。了解这些节律的细胞机制是开发药物和其他治疗方法的关键。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to elucidate the roles of intracellular calcium signals in circadian clock neurons. Experiments will be carried out in the fruit fly, Drosophila melanogaster, which has a functionally sophisticated and anatomically well- characterized circadian control system and is uniquely amenable to application of genetic methods that target clock neurons in the intact behaving animal. In addition to canonical transcriptional feedback mechanisms, circadian oscillation also relies upon depolarization-activated ionic membrane conductances. The proposed aims explore the hypothesis that intracellular calcium signals triggered by membrane depolarization are a core component of the cellular circadian oscillator. An engineered calcium buffer protein is specifically targeted to clock neurons in the brains of transgenic flies to disrupt cellular calcium signals in the intact living organism, followed by measurement of effects on circadian rhythms of locomotor activity, cellular accumulation of known transcription factor components of the circadian oscillator, and intracellular calcium dynamics. Preliminary studies using this approach indicate that intracellular calcium buffering in clock neurons leads to dose-dependent slowing of free-running behavioral and cellular rhythms with arrhythmicity at the highest dose. The proposed aims will identify the subcellular location of the relevant calcium signals, their temporal dynamics, the detailed effects their disruption has on cellular rhythms, and the downstream calcium-sensitive signaling pathways required for their transduction. Because of the great similarity in the genetic and cellular bases for circadian rhythmicity in flies and mammals, the information that can uniquely be obtained exploiting the genetic accessibility of Drosophila in the proposed studies will provide insight into general principles of cellular oscillator function that are relevant both to the basic circadian research done in mammalian model systems and to clinical research on human disorders of circadian function. Disruption of daily rhythms of rest and activity in human beings--through genetic mutation (as in advanced sleep phase disorder), disease, or environmental conditions (as in "jet lag" or for night shift workers)--has many adverse consequences for public health, workplace safety, and economic productivity. Understanding the cellular mechanisms of these rhythms is key to developing drugs and other treatments for their amelioration. The proposed studies will provide insight into general principles of cellular oscillator function that are relevant both to the basic circadian research done in mammalian model systems and to clinical research on human disorders of circadian function. Disruption of daily rhythms of rest and activity in human beings--through genetic mutation (as in advanced sleep phase disorder), disease, or environmental conditions (as in "jet lag" or for night shift workers)--has many adverse consequences for public health, workplace safety, and economic productivity. Understanding the cellular mechanisms of these rhythms is key to developing drugs and other treatments for their amelioration.