Intercellular Signaling in the Circadian Clock

描述(由申请人提供)：生物钟的缺陷与多种临床疾病有关。新出现的证据表明，神经元之间在同步和维持生物钟方面存在联系。果蝇一直是阐明时钟潜在机制的强大模型，时钟的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中心。我们最近发现了PDF的G蛋白偶联受体(PDFR)。该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。生物钟是如何驱动下游神经回路来控制睡眠和醒来等行为的？PDF在神经振荡器的耦合中起什么作用？PDF重置核心振荡器和驱动节奏行为的机制是什么？该提案的具体目的是：1.绘制PDF受体在行为和分子昼夜节律中作用的细胞底物。值得注意的是，人们对介导PDF受体在昼夜行为中作用的神经底物知之甚少。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救援、过度表达和RNAi敲除。此外，我们还将评估PDFR在大脑中的分布。2.研究PDF信号在神经昼夜节律起搏器偶联中的作用。为了分析耦合，我们将在昼夜神经网络的子集中操纵时钟的速度，并分析在存在或不存在PDF信号的情况下对相互连接的振荡器的影响。3.研究PDF重置核心生物钟和输出通路的分子机制。我们将研究失去PDFR对核心时钟以及cAMP和MAPK信号通路的分子后果。利用新的电生理学方法，我们将检测外源性PDF对起搏器和输出神经元的电特性的影响。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在昼夜行为中发挥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，包括组织特异性救援研究的简便性，在已识别的起搏器神经元亚群中操纵时钟的能力，以及检查整个动物的信号通路和核心时钟的广泛遗传资源。考虑到哺乳动物系统的保守性，这项工作应该为神经肽调节人类疾病正常和紊乱的昼夜节律的机制提供洞察力。生物钟中与公共卫生相关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讯是由神经肽介导的。我们将在一个简单的动物模型中阐明昼夜节律神经肽在同步昼夜节律时钟和交流计时信息中的作用。考虑到与人类的潜在保守性，这项工作应该为神经肽调节人类疾病正常和紊乱的昼夜节律的机制提供洞察力。

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.