MECHANISMS OF CIRCADIAN CLOCK OUTPUT

描述（由申请人提供）：最近对昼夜节律分子振荡器的定义已经允许识别昼夜节律计时的关键起搏器神经元。这些信息现在提供了重新审视有关行为昼夜节律调节的细胞基础的基本问题的可能性。我们对昼夜节律起搏器神经元将信息从生物钟传递到大脑和身体的分子和信号机制感兴趣。我们研究的一个主要焦点是起搏器神经元的传递信号。遗传证据表明，神经肽PDF（色素分散因子）是果蝇的主要昼夜节律递质。我们的工作假设是PDF是起搏器网络中一个重要的同步代理。在之前的研究周期中，我们研究了PDF受体（PDF- r）的表达位置，我们开发了实时成像方法来研究其在体内的激活，并筛选了参与其信号通路的激酶和磷酸酶。这些结果为我们现在提出的一系列研究奠定了基础——分析PDF-R信号转导机制及其对神经元活动的影响。PDF-R是B类肽GPCR -该类别包括哺乳动物PACAP和VIP肽的受体。我们将追求与PDF受体信号传导相关的两个具体目标。首先，我们将分析PDF-R信号小体的组成，从鉴定相关的腺苷酸环化酶开始。不同的起搏器的信号小体是不同的，它们的分子定义将极大地促进我们对昼夜生理学的理解。其次，我们将确定PDF-R信号及其失活是如何由内化控制的，通过内吞作用-初步的遗传证据表明，这是适当的PDF-R信号传导的关键步骤。最后，在Aim 3中，我们将利用新的成像技术（称为OCPI）快速实时测量所有150个果蝇大脑起搏器的神经元活动-每分钟一次，长达24小时。当对PDF进行遗传和药理学操作时，这将使我们能够将PDF- r信号数据与正常的起搏器生理学联系起来。生物计时对于许多体内平衡生理事件是必不可少的。对这些机制的挑战（来自轮班工作时间表或季节变化）会导致严重到足以包括临床疾病的破坏。我们的研究集中在生物钟输出的进化保守分子机制上，并将确定更好的治疗干预措施来纠正这种情况。

DESCRIPTION (provided by applicant): The recent definition of the circadian molecular oscillator has permitted identification of the critical pacemaker neurons underlying circadian timekeeping. That information now presents the possibility to re- examine fundamental questions regarding the cellular basis for circadian regulation of behavior. We are interested in the molecules and signaling mechanisms by which circadian pacemaker neurons transmit information from the clock to the brain and body. A major focus of our studies concerns transmitter signaling by pacemaker neurons. Genetic evidence indicates the neuropeptide PDF (Pigment Dispersing Factor) is a principal circadian transmitter in Drosophila. Our working hypothesis is that PDF is an important synchronizing agent within the pacemaker network. In the previous research cycle, we studied where the receptor for PDF (PDF-R) is expressed, we developed realtime imaging methods to study its activation in vivo, and screened for kinases and phosphatases that participate in its signaling pathways. Those outcomes helped establish a foundation for a cycle of studies we now propose - to analyze PDF-R signal transduction mechanisms and consequences on neuronal activity. PDF-R is a Class B peptide GPCR - that category includes receptors for the mammalian PACAP and VIP peptides. We will pursue two specific aims related to PDF receptor signaling. First, we will analyze the composition of the PDF-R signalosomes, starting with the identification of associated adenylate cyclases. The signalosomes are different for different pacemakers - their molecular definition will greatly advance our understanding of circadian physiology. Second, we will determine how PDF-R signaling and its inactivation are controlled by internalization, via endocytotsis - preliminary genetic evidence suggests this is a key step in proper PDF-R signaling. Finally, in Aim 3, we will exploit novel imaging technology (called OCPI) for fast, realtime measures of neuronal activity in all 150 Drosophila brain pacemakers - once per minute, for up to 24 hours. When performed with genetic and pharmacological manipulations of PDF, this will permit us to relate PDF-R signaling data to normal pacemaker physiology. Biological timekeeping is essential for numerous homeostatic physiological events. Challenges to these mechanisms (from shift-work schedules or seasonal change) cause disruptions severe enough to include clinical conditions. Our studies focus on evolutionarily-conserved molecular mechanisms of circadian clock output and will identify leads for better therapeutic interventions to redress such conditions.