Calcium Signaling in Circadian Clock Neurons

描述(申请人提供)：这项建议的长期目标是阐明细胞内钙信号在昼夜节律时钟神经元中的作用。实验将在果蝇--黑腹果蝇身上进行，果蝇拥有一个功能复杂、解剖学特征良好的昼夜节律控制系统，它独特地服从于以完整行为动物的时钟神经元为目标的遗传方法的应用。除了典型的转录反馈机制外，昼夜振荡还依赖于去极化激活的离子膜电导。提出的目的是探索一种假设，即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。一种经过工程的钙缓冲蛋白专门针对转基因果蝇大脑中的神经元提供时钟，以扰乱完整活体中的细胞钙信号，随后测量运动活动对昼夜节律的影响、昼夜节律振荡器已知转录因子成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自由奔跑的行为和细胞节律的剂量依赖性减慢，并在最高剂量下发生心律失常。建议的目标将确定相关钙信号的亚细胞位置，它们的时间动态，它们的干扰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在遗传和细胞基础上有很大的相似性，在拟议的研究中利用果蝇的遗传可及性获得的唯一信息将为深入了解细胞振荡器功能的一般原理提供见解，这些原理与在哺乳动物模型系统中进行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。人类日常休息和活动节奏的中断--通过基因突变(如晚期睡眠相障碍)、疾病或环境条件(如“时差”或夜班工人)--对公共健康、工作场所安全和经济生产力有许多不利后果。了解这些节律的细胞机制是开发药物和其他治疗方法改善它们的关键。这些拟议的研究将深入了解细胞振荡器功能的一般原理，这些原理既与在哺乳动物模型系统中进行的基本昼夜节律研究有关，也与人类昼夜节律功能障碍的临床研究相关。人类日常休息和活动节奏的中断--通过基因突变(如晚期睡眠相障碍)、疾病或环境条件(如“时差”或夜班工人)--对公共健康、工作场所安全和经济生产力有许多不利后果。了解这些节律的细胞机制是开发药物和其他治疗方法改善它们的关键。

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.