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起着进化上保守和重要的作用。无脊椎动物遗传学研究表明，EGFR信号是睡眠所必需的。然而，对脊椎动物的研究一直没有定论。为了解决这个问题，我发现对于白天活动的脊椎动物斑马鱼来说，EGFR信号对于促进睡眠是必要的，也是充分的。此外，我在蠕虫和斑马鱼中发现了EGFR调节睡眠的效应器。然而，关于EGFR如何在分子和神经元水平上调节睡眠，哪些因素调节EGFR驱动的睡眠，以及在什么背景下EGFR是睡眠所必需的，仍然存在几个基本问题。这项提案的目标试图回答这些特定的问题。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、遗传和药物保护， 与通常用于睡眠研究的夜间活动啮齿动物不同，斑马鱼和人类一样是白天活动的。斑马鱼幼体的光学透明性和相对简单的脊椎动物大脑有助于对完整动物的神经元功能进行非侵入性研究。斑马鱼幼体的体积很小，可以进行大规模的睡眠/清醒行为分析，产生统计上可靠的数据，并能够快速测试行为的许多遗传扰动。我们将利用这些有利的功能来进一步探索EGFR介导的睡眠调节。在特定的目标1中，我们将检验这样一个假设，即表达EGFR的神经元对于促进睡眠是必要的和充分的。我们将使用高通量光遗传学和化学遗传学分析来无创地刺激、抑制和消融自由行为的斑马鱼幼体中的这些神经元。在特定的目标2中，我将测试斑马鱼细胞应激诱导的睡眠需要EGFR信号和EGFR表达的神经元的假设，通过 利用光遗传和化学遗传方法刺激、抑制和消融它们。在具体目标3中，我们将验证EGFR信号通过激活EGFR信号诱导脑脊液睡眠促进因子的分泌来促进睡眠的假说，然后提取脑脊液并用质谱仪鉴定潜在的促睡眠脑脊液因子。在R00阶段，我将对脑脊液候选睡眠促进因子进行功能增强和功能丧失的遗传学研究，以确定它们是否对促进斑马鱼睡眠是必要的和足够的。这些实验可能揭示EGFR调节睡眠的新的遗传和神经机制。研究这些问题很重要，因为突变的EGFR信号成分与几种人类疾病有关，包括静息活动节律紊乱。通过研究这一鲜为人知的睡眠调节途径并确定新的机制，该项目可能最终导致睡眠障碍的新疗法。由于睡眠异常与包括抑郁症、精神分裂症和自闭症在内的几种神经疾病有关，在某些情况下可能是因果关系，因此该项目可能还会改进一些神经疾病的治疗方法。

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.