Analysis of EGFR-dependent sleep in C. elegans

描述(申请人提供)：睡眠障碍损害认知能力，慢性睡眠不足与心脏代谢疾病的风险增加有关，包括肥胖和高血压。尽管睡眠具有基本的重要性，但它的功能仍然存在争议，执行睡眠的分子机制也才刚刚开始阐明。睡眠被认为是由保守的遗传机制调节的，在过去的十年里，睡眠领域已经扩展到非哺乳动物生物，允许应用新的遗传方法。我们已经在线虫体内开发了一种可诱导睡眠的系统，我们正在使用该系统强大的分子遗传学工具来发现睡眠调节的新成分。我们发现，线虫表皮生长因子(EGF)同源物LIN-3的强制表达在任何阶段都会诱导一种睡眠状态。通过分析对这一效应的抵抗力，我们已经确定了EGF诱导睡眠的几个效应器，包括EGF受体(let-23/EGFR)和与UNC-103同源的ERG(ether-a-go-go-go)，UNC-103是一种电压门控钾通道。我们的目标是确定在睡眠中抑制活动所需的UNC-103活动所在的神经元。我们将研究响应EGF表达的UNC-103水平和定位的潜在变化，以建立睡眠信号及其对神经元兴奋性的下游影响之间的联系。我们的研究有望阐明哺乳动物的睡眠调节，因为EGF配体在哺乳动物中的应用具有保守的安眠作用。我们还将研究线虫睡眠行为的时间，这种行为是随着蜕皮而不是昼夜周期而有节奏地发生的。我们将研究LIN-3在蜕皮周期中的振荡表达，以将行为与发育时间线索联系起来。这一分析与哺乳动物的生物钟对睡眠的调节有关，因为蜕皮周期是由昼夜节律基因per的线虫同源基因调节的。最后，我们将确定睡眠调节的其他效应器。我们已经发现，单个神经元ALA中的EGFR激活触发了EGF诱导的睡眠。我们假设这个神经元释放一个信号，进而触发整个神经网络的睡眠。我们将通过基于RNAi的功能基因组学来鉴定这个信号，并使用分子遗传学方法来表征它的功能。

DESCRIPTION (provided by applicant): Disordered sleep impairs cognitive performance, and chronic sleep loss is associated with increased risk of cardiometabolic diseases including obesity and hypertension. Despite the fundamental importance of sleep, its function remains controversial and the molecular mechanisms by which it is executed are only beginning to be elucidated. Sleep is recognized to be regulated by conserved genetic mechanisms, and in the past decade the sleep field has expanded to non- mammalian organisms, allowing the application of novel genetic approaches. We have developed an inducible-sleep system in the nematode C. elegans, and we are using the powerful molecular- genetic tools of this system to discover novel components of sleep regulation. We have found that forced expression of the C. elegans Epidermal Growth Factor (EGF) homolog LIN-3 induces a sleep-like state at any stage. By assaying for resistance to this effect we have identified several effectors of EGF-induced sleep, including the EGF receptor (LET-23/EGFR) and the ERG (ether-a- go-go related gene) homolog UNC-103, a voltage-gated potassium channel. We aim to determine the neurons in which UNC-103 activity is required for the inhibition of activity during sleep. We will examine potential changes in UNC-103 levels and localization in response to EGF expression, to make a connection between sleep signals and their downstream effects on neuronal excitability. Our studies are expected to shed light on mammalian sleep regulation, as EGF ligand administration in mammals has a conserved soporific effect. We will also investigate the timing of C. elegans sleep behavior, which occurs rhythmically with molting rather than with circadian periodicity. We will examine LIN-3 expression for oscillation with the molting cycle, to connect behavior with developmental timing cues. This analysis is relevant to the regulation of sleep by biological clocks in mammals, as the molting cycle is regulated by the C. elegans homolog of the circadian gene PER. Lastly, we will identify additional effectors of sleep regulation. We have found that EGFR activation within a single neuron, ALA, triggers EGF-induced sleep. We hypothesize that this neuron releases a signal that in turn triggers sleep across the neuronal network. We will identify this signal through RNAi-based functional genomics, and characterize its function using molecular-genetic approaches.