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特征良好的昼夜节律控制系统，并且非常适合应用遗传方法，以完整行为动物的时钟神经元为目标。除了典型的转录反馈机制外，昼夜节律振荡还依赖于去极化激活的离子膜电导。提出的目的是探索细胞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。一种工程钙缓冲蛋白专门针对转基因果蝇大脑中的时钟神经元，以破坏完整生物体中的细胞钙信号，随后测量对运动活动昼夜节律的影响，昼夜节律振荡器已知转录因子组分的细胞积累，以及细胞内钙动力学。使用这种方法的初步研究表明，时钟神经元的细胞内钙缓冲导致最高剂量时自由运行的行为和细胞节律的剂量依赖性减慢，并伴有心律失常。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的昼夜节律的遗传和细胞基础非常相似，在拟议的研究中，利用果蝇的遗传可及性可以独特地获得的信息将提供对细胞振荡器功能的一般原理的见解，这些原理与哺乳动物模型系统的基本昼夜节律研究和人类昼夜节律功能紊乱的临床研究相关。人类日常休息和活动节奏的中断——通过基因突变（如晚期睡眠阶段障碍）、疾病或环境条件（如“时差反应”或夜班工人）——对公众健康、工作场所安全和经济生产力有许多不利影响。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。拟议的研究将提供对细胞振荡器功能的一般原理的见解，这些原理与哺乳动物模型系统的基本昼夜节律研究和人类昼夜节律功能紊乱的临床研究相关。人类日常休息和活动节奏的中断——通过基因突变（如晚期睡眠阶段障碍）、疾病或环境条件（如“时差反应”或夜班工人）——对公众健康、工作场所安全和经济生产力有许多不利影响。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.