Mechanisms of Sleep Regulation by EGFR-Mediated Neural and Molecular Circuits

一些证据表明，表皮生长因子受体（EGFR）信号可能在调节睡眠中发挥进化保守和重要的作用。无脊椎动物遗传学研究表明，EGFR信号是睡眠所必需的。然而，对脊椎动物的研究还没有定论。为了解决这个问题，我发现EGFR信号传导对于促进斑马鱼（一种昼行性脊椎动物）的睡眠是必要的，也是足够的。此外，我确定了蠕虫和斑马鱼中EGFR调节睡眠的效应子。然而，关于EGFR如何在分子和神经元水平上调节睡眠，什么因素介导EGFR驱动的睡眠，以及在什么情况下EGFR是睡眠所必需的，仍然存在一些基本问题。本提案的目的是要回答这些具体问题。斑马鱼非常适合解决这些问题，因为它们表现出哺乳动物睡眠的行为，解剖，遗传和药理学保护， 而且与通常用于睡眠研究的夜行性啮齿动物不同，斑马鱼像人类一样是昼行性动物。斑马鱼幼鱼的光学透明度和相对简单的脊椎动物脑有助于在完整动物中进行神经元功能的非侵入性研究。斑马鱼幼体的小尺寸允许大规模的睡眠/觉醒行为测定，其产生统计学上稳健的数据，并能够快速测试许多遗传扰动对行为的影响。我们将利用这些有利的功能，进一步探讨EGFR介导的睡眠调节。在具体目标1中，我们将测试EGFR表达神经元对于促进睡眠是必要且充分的假设。我们将使用高通量的光遗传学和化学遗传学检测来刺激，抑制和消融这些神经元在自由行为的斑马鱼幼虫非侵入性。在具体目标2中，我将通过以下方法检验斑马鱼细胞应激诱导的睡眠需要EGFR信号传导和EGFR表达神经元的假设： 使用光遗传学和化学遗传学方法刺激、抑制和消除它们。在具体目标3中，我们将检验EGFR信号传导通过激活EGFR信号传导诱导CSF睡眠促进因子分泌来促进睡眠的假设，然后提取CSF并使用质谱法表征潜在的睡眠促进CSF因子。在R 00阶段，我将进行CSF候选睡眠促进因子的功能获得和功能丧失遗传学研究，以确定它们是否是促进斑马鱼睡眠所必需和足够的。这些实验可能揭示EGFR调节睡眠的新的遗传和神经机制。调查这些问题是重要的，因为突变的EGFR信号传导成分与几种人类疾病有关，包括休息-活动节律紊乱。通过研究这种知之甚少的睡眠调节途径并确定新的机制，该项目最终可能导致睡眠障碍的新疗法。由于睡眠异常与几种神经系统疾病有关，包括抑郁症，精神分裂症和自闭症，并且在某些情况下可能是因果关系，因此该项目还可能导致改善某些神经系统疾病的治疗方法。

Several lines of evidence suggest that epidermal growth factor receptor (EGFR) signaling may play an evolutionarily conserved and important role in regulating sleep. Invertebrate genetic studies have demonstrated that EGFR signaling is required for sleep. Vertebrate studies, however, have been inconclusive. To address this, I found that EGFR signaling is both necessary and sufficient to promote sleep in zebrafish, a diurnal vertebrate. Furthermore, I identified effectors of EGFR-regulated sleep in worms and zebrafish. However, several fundamental questions remain regarding how EGFR regulates sleep at the molecular and neuronal levels, what factors mediate EGFR-driven sleep, and in what contexts EGFR is required for sleep. The objectives of this proposal seek to answer these particular questions. Zebrafish are well suited to address these questions because they exhibit behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep, and unlike nocturnal rodents that are commonly used for sleep research, zebrafish are diurnal like humans. The optical transparency and relatively simple vertebrate brain of zebrafish larvae facilitate noninvasive studies of neuronal function in intact animals. The small size of zebrafish larvae allows for large-scale sleep/wake behavioral assays that generate statistically robust data and enable rapid testing of many genetic perturbations on behavior. We will exploit these advantageous features to further explore the EGFR-mediated sleep regulation. In Specific Aim 1, we will test the hypothesis that egfr-expressing neurons are necessary and sufficient to promote sleep. We will use high-throughput optogenetic and chemical-genetic assays to stimulate, inhibit and ablate these neurons noninvasively in freely behaving zebrafish larvae. In Specific Aim 2, I will test the hypothesis that cellular stress-induced sleep in zebrafish requires EGFR signaling and egfr-expressing neurons by stimulating, inhibiting and ablating them using optogenetic and chemogenetic approaches. In Specific Aim 3, we will test the hypothesis that EGFR signaling promotes sleep by inducing the secretion of CSF sleep promoting factors by activating EGFR signaling, then extracting CSF and characterizing potential sleep promoting CSF factors using mass spectrometry. During the R00 phase, I will perform gain- and loss-of-function genetic studies of CSF candidate sleep-promoting factors to determine whether they are necessary and sufficient to promote sleep in zebrafish. These experiments may reveal novel genetic and neurological mechanisms through which EGFR regulates sleep. Investigating these questions is important because mutated EGFR signaling components are implicated in several human disorders, including disrupted rest-activity rhythms. By investigating this poorly understood sleep regulatory pathway and identifying new mechanism, this project may eventually lead to new therapies for sleep disorders. Because abnormal sleep is associated with several neurological disorders, including depression, schizophrenia and autism, and may be causal in some cases, this project may also lead to improved therapies for some neurological disorders.