MAPK signaling: gates, oscillators and circadian timing

项目摘要/摘要 实际上，人类生理和行为的每一个方面都受到固有的24小时(昼夜节律)的调节。 计时过程。在这个时钟计时系统的中心是视交叉上核。 下丘脑。SCN时钟的一个关键特征是对MAPK的严格、实时、依赖的调节 (p44/42丝裂原活化蛋白激酶)途径。这种现象的两个例子是《每日新闻》 MAPK通路激活状态的振荡和光的时钟门控调节 通路的响应性。重要的是，时钟产生的、时间分隔的MAPK调节 信号似乎在SCN的计时和夹带中起着核心作用。此外，MAPK的日常门禁 信令可能是所有振荡器种群的基本设计原则，因此，MAPK节奏 可能会对一系列生理过程产生深远的影响。考虑到这些影响， 令人惊讶的是，我们对细胞机制和突触电路的了解仍然相对较少 对MAPK活性进行昼夜节律控制。在这里，我们假设MAPK的昼夜调节 信号是SCN细胞振荡器固有的(细胞自主)特征，这种MAPK节奏是一个关键 生物钟调节基本和复杂生理状态的机械积木。为了测试 在这一假设下，我们提出了以下一套实验目标。在目标1中，我们将识别细胞和 引起MAPK途径节律性调节的SCN的网络特性。为此， 我们将，A)确定MAPK节律是否是细胞自主的，或者它们是否源于 细胞间SCN网络，以及B)确定产生MAPK活性的细胞内信号事件 节奏。在目标2中，我们建议描述基于分子、细胞和系统的机制，通过这些机制 SCN时钟选通光诱导的MAPK通路激活。为了解决这一在很大程度上未被探索的现象， 我们将，A)确定分子门何时和如何打开，以及B)，测试细胞质ERK 支架蛋白PEA-15是MAPK信号的主要昼夜节律门。值得注意的是，我们最近 确认PEA-15是SCN中MAPK信号的调制器，以及它动态调节ERK的能力 信号转导使其成为MAPK信号门控的一个有吸引力的候选者。在目标3中，我们建议采用 选择性靶向方法在SCN核心区和壳区内转基因干扰MAPK信号 为了解决MAPK信号在A)昼夜节律的产生和B)携带 生物钟。此外，有条件的PEA-15 KO和点突变PEA-15转基因小鼠株系将 用于测试PEA-15磷酸化导致ERK快速解离的模型，我们假设这是一种 启动光致相移的关键步骤。总而言之，这些数据将提供基本的新 对MAPK信号和生物钟之间的关系的洞察，并指出了 其中，时钟门控MAPK信号的失调可能导致中枢神经系统的紊乱。

Project Summary/Abstract Virtually every aspect of human physiology and behavior is modulated by an inherent 24 hour (circadian) timing process. At the center of this clock timing system is the suprachiasmatic nucleus (SCN) of the hypothalamus. A key feature of the SCN clock is the tight, time-of-day, dependent regulation of the MAPK (p44/42 mitogen-activated protein kinase) pathway. Two examples of this phenomenon are the daily oscillations in the activation state of the MAPK pathway, and the clock-gated regulation of the photic responsiveness of the pathway. Importantly, the clock-generated, temporally-delimited, regulation of MAPK signaling appears to play a central role in SCN timing and entrainment. Further, the daily gating of MAPK signaling may be an underlying design principal of all oscillator populations, and as such, MAPK rhythms could have profound and far-reaching effects on a range of physiological processes. Given these implications, it is surprising that we still know relatively little about the cellular mechanisms and synaptic circuits that confer circadian control over MAPK activity. Here, we hypothesize that the circadian regulation of MAPK signaling is an inherent (cell autonomous) feature of SCN cellular oscillators and that this MAPK rhythm is a key mechanistic building-block by which the circadian clock modulates both basic and complex physiological states. To test this hypothesis, we propose the following set of experimental goals. In Aim 1, we will identify the cellular and network properties of the SCN that give rise to the rhythmic regulation of the MAPK pathway. To this end, we will, A) Determine whether MAPK rhythms are cell autonomous or whether they result from an intercellular SCN network, and B) Determine the intracellular signaling events that generate MAPK activity rhythms. In Aim 2 we propose to characterize the molecular, cellular and systems-based mechanisms by which the SCN clock gates light-evoked MAPK pathway activation. To address this largely unexplored phenomenon, we will, A) determine when and how the molecular gate opens, and B), test whether the cytoplasmic ERK scaffold protein PEA-15 serves as the principal circadian gate on MAPK signaling. Of note, we recently identified PEA-15 as a modulator of MAPK signaling in the SCN, and its capacity to dynamically regulate ERK signaling makes it an attractive candidate for the gating of MAPK signaling. In Aim 3 we propose to employ a selective targeting approach to transgenically disrupt MAPK signaling within the SCN core and shell regions to address the roles of MAPK signaling in A) the generation of circadian rhythms, and B) the entrainment of the circadian clock. Further, conditional PEA-15 KO and point mutant PEA-15 transgenic mouse lines will be used to test a model in which PEA-15 phosphorylation leads to rapid ERK dissociation, which we posit to be a key step in the initiation of light-evoked phase-shifting. Together, these data will provide fundamental new insights into the relationship between MAPK signaling and the circadian clock, and point to potential ways in which the dysregulation of clock-gated MAPK signaling could contribute to disorders of the CNS.