Intercellular Signaling in the Circadian Clock

描述（由申请人提供）：生物钟缺陷与多种临床疾病有关。新出现的证据表明，神经元之间的通信在同步和维持生物钟。果蝇是阐明生物钟基本机制的有力模型，其中许多方面与人类高度保守。在果蝇中，神经肽色素分散因子（PDF）是同步神经起搏器和调节神经输出的核心。我们最近发现了G蛋白偶联受体PDF（PDFR）。这种受体的鉴定为解决与昼夜节律起搏器功能相关的核心问题提供了机会。生物钟如何驱动下游神经回路来控制行为，如睡眠和觉醒？PDF在神经振荡器耦合中的作用是什么？PDF重置核心振荡器并驱动节律行为的机制是什么？该提案的具体目标是：1。在行为和分子昼夜节律中绘制PDF受体功能的细胞底物。值得注意的是，很少有人知道的神经基板介导PDF受体的昼夜行为。为了解决这个问题，我们将在昼夜节律和潜在的下游神经回路中使用组织特异性PDFR拯救、过表达和RNAi敲低。此外，我们将评估PDFR在大脑中的分布。2.研究PDF信号在神经昼夜节律起搏器耦合中的作用。为了分析耦合，我们将操纵昼夜节律神经网络子集中的时钟速度，并分析在存在或不存在PDF信号的情况下相互连接的振荡器的后果。3.研究PDF重置核心生物钟和输出通路的分子机制。我们将研究PDFR丢失对核心时钟以及cAMP和MAPK信号通路的分子后果。使用新的电生理方法，我们将研究外源性PDF对起搏和输出神经元电特性的影响。我们将分析PDF/PDFR，核心时钟，cAMP/MAPK和膜兴奋性突变体之间的遗传相互作用。这些研究应该阐明PDFR在昼夜节律行为中的作用所必需的分子和神经回路。他们还利用了果蝇系统的独特优势，包括组织特异性拯救研究的便利性，在已识别的起搏神经元子集中操纵时钟的能力，以及广泛的遗传资源来检查整个动物中的信号通路和核心时钟。考虑到哺乳动物系统的保守性，这项工作应该为神经肽介导人类疾病中正常和破坏的昼夜节律的机制提供见解。公众健康相关性生物钟的缺陷与多种临床疾病有关。神经起搏器与其下游靶点之间的通信由神经肽介导。我们将在一个简单的动物模型中阐明昼夜神经肽在同步生物钟和传递时间信息中的作用。考虑到与人类的潜在保护，这项工作应该提供深入了解神经肽介导人类疾病中正常和破坏昼夜节律的机制。

DESCRIPTION (provided by applicant): Defects in circadian clocks have been implicated in a variety of clinical disorders. Emerging evidence implicates communication between neurons in synchronizing and sustaining circadian clocks. The fruit fly Drosophila has been a powerful model to elucidate the underlying mechanisms of clocks, many aspects of which are highly conserved with humans. In the fruit fly, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is central to synchronizing neural pacemakers and regulating neural outputs. We have recently identified the G-protein coupled receptor for PDF (PDFR). The identification of this receptor affords an opportunity to address central questions related to circadian pacemaker function. How do circadian clocks drive downstream neural circuits to control behavior, such as sleep and wake? What is the role of PDF in coupling of neural oscillators? What are the mechanisms by which PDF resets core oscillators and drives rhythmic behaviors? The specific aims of the proposal are: 1. To map the cellular substrates of PDF receptor function in behavioral and molecular circadian rhythms. Remarkably little is known about the neural substrates that mediate PDF receptor action in circadian behavior. To address this issue, we will use tissue-specific PDFR rescue, overexpression, and RNAi knockdown in circadian and potential downstream neural circuits. In addition, we will assess the distribution of PDFR in the brain. 2. To examine the role of PDF signaling in coupling of neural circadian pacemakers. To assay coupling, we will manipulate the speed of the clock in subsets of the circadian neural network and assay the consequences on interconnected oscillators in the presence or absence of PDF signaling. 3. To examine the molecular mechanisms by which PDF resets the core circadian clock and output pathways. We will examine the molecular consequences of loss of PDFR on the core clock as well as cAMP and MAPK signaling pathways. Using novel electrophysiological approaches, we will examine the effects of exogenous PDF on electrical properties of pacemaker and output neurons. We will analyze genetic interactions between PDF/PDFR, core clock, cAMP/MAPK, and membrane excitability mutants. These studies should elucidate the molecular and neural circuitry essential for PDFR action in circadian behavior. They also exploit the unique advantages of the Drosophila system, including the ease of tissue- specific rescue studies, the ability to manipulate the clock in identified subsets of pacemaker neurons, and the extensive genetic resources to examine signaling pathways and the core clock in the whole animal. Given the conservation with mammalian systems, this work should provide insights into the mechanisms by which neuropeptides mediate normal and disrupted circadian rhythms in human disease. PUBLIC HEALTH RELEVANCE Defects in circadian clocks have been implicated in a variety of clinical disorders. Communication between neural pacemakers and to their downstream targets is mediated by neuropeptides. We will elucidate the role of a circadian neuropeptide in synchronizing circadian clocks and communicating timing information in a simple animal model. Given the potential conservation with humans, this work should provide insight into the mechanisms by which neuropeptides mediate normal and disrupted circadian rhythms in human disease.