Prokineticin 2 and Suprachiasmatic Circadian Output

描述（由申请人提供）：生理和行为在日常环境条件下的组织是一种适应，基本上发生在所有生物体内。昼夜节律由三个组成部分调节：昼夜节律起搏器、输入机制和输出机制。在哺乳动物中，驱动昼夜节律的主要起搏器位于下丘脑前部的视交叉上核（SCN）。环境的光暗循环使SCN时钟进入24小时的一天。SCN神经元的同步导致协调的昼夜节律输出，调节表达的节奏。SCN内分子钟机制的清晰视图已经出现。SCN生物钟的分子时钟由自调节转录和翻译反馈回路组成，这些反馈回路既有积极的因素，也有消极的因素。同样，光输入如何重置SCN时钟以与环境光/暗周期同步的信号通路也正在出现。与SCN起搏器时钟和输入通路的分子机制相比，我们对SCN昼夜节律起搏器发送定时信息以控制生理和行为节律的输出机制知之甚少。Prokineticin 2 （PK2）是一种富含半胱氨酸的蛋白，最近被证明是传递昼夜运动节律的SCN输出分子。PK2符合SCN生物钟真正输出分子的标准：1)PK2是分泌分子；2) PK2的转录受核心时钟基因调控，PK2 mRNA在SCN中振荡幅度较大；3) PK2的产生对光的夹带有响应；4) PK2受体在初级SCN输出靶区表达；5)当内源性PK2水平较低时，在主观夜间使用脑室内（ICV）给药可抑制夜间高强度的车轮跑步活动。我们建议进一步研究PK2信号在SCN生物钟输出机制中的作用。具体来说，PK2蛋白在细胞体、SCN神经元终末区和脑脊液中的节律将通过定量免疫细胞化学和/或放射免疫测定来研究。PK2是否是介导SCN生物钟和光掩蔽输出的共同信号将被研究。SCN的PK2节律输出如何响应光/暗周期的突变也将被研究。此外，PK2对大鼠SCN生物钟控制的运动和睡眠/觉醒节律的影响将通过急性和慢性输注PK2和PK2拮抗剂来研究。此外，PK2基因将在小鼠中被破坏，其对SCN控制的昼夜行为节律以及核心SCN昼夜循环的影响将被检查。最后，将通过遗传方法研究SCN PK2的输出途径。这些拟议的研究将帮助我们进一步了解PK2信号在调节SCN昼夜节律装置的时间信息输出中的机制，并可能对未来治疗许多昼夜节律障碍（如时差、轮班工作综合征和慢性失眠）产生重大影响

DESCRIPTION (provided by applicant): Organization of physiology and behavior with recurring daily environmental conditions is an adaptation that occurs in essentially all living organisms. Circadian rhythms are regulated by three components: the circadian pacemaker, an input mechanism and an output mechanism. In mammals, the master pacemaker driving circadian rhythms resides in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. Environmental light-dark cycles entrain the SCN clock to the 24-hr day. Synchronization of SCN neurons leads to coordinated circadian outputs that regulate expressed rhythms. A clear view of molecular clock mechanisms within the SCN has emerged. The molecular clockwork of the SCN circadian clock consists of auto-regulatory transcriptional and translational feedback loops that have both positive and negative elements. Similarly, the signal pathway of how light input resets SCN clock to synchronize with the environmental light/dark cycle is also emerging. In contrast to molecular mechanisms of SCN pacemaker clockwork and input pathway, relatively little is known about the output mechanism by which the SCN circadian pacemaker sends timing information to control physiological and behavioral rhythms. Prokineticin 2 (PK2), a cysteine-rich protein, has recently been shown as a SCN output molecule that transmits the circadian locomotor rhythm. PK2 fulfill the criteria expected for a bona fide output molecule from SCN circadian clock: 1) PK2 is a secreted molecule; 2) The transcription of PK2 is regulated by core clock genes, and PK2 mRNA oscillates in the SCN with high magnitude; 3) The production of PK2 is responsive to light entrainment; 4) Receptor for PK2 is expressed in primary SCN output target areas; and 5) Intracerebroventricular (ICV) administration of PK2 at subjective night, when endogenous PK2 levels are low, suppressed high nocturnal wheel-running activity. We propose to further investigate the role of PK2 signaling in the output mechanism of the SCN circadian clock. Specifically, the rhythms of PK2 protein in the cell bodies, terminal areas of SCN neurons and cerebral spinal fluid will be investigated by quantitative immunocytochemistry and/or radioimmunoassay. Whether PK2 is the common signal that mediates the output of SCN circadian clock and light masking will be investigated. How the PK2 rhythmic output from the SCN responds to abrupt shifts of light/dark cycle will also be investigated. Moreover, the effects of PK2 on SCN circadian clock-controlled locomotor and sleep/wake rhythms will be investigated by acute and chronic infusion of PK2 and PK2 antagonist in rats. Furthermore, the PK2 gene will be disrupted in mice and its effect on SCN-controlled circadian behavioral rhythms as well as core SCN circadian loops will be examined. Finally, the SCN PK2 output pathway will be investigated by a genetic approach. These proposed studies should help us gain further insight into the mechanism of PK2 signaling in mediating the output of timing information from the SCN circadian crock, and could have a major impact on the future treatment of a number of circadian disorders such as jet-lag, shift work syndrome, and chronic insomnia