CAREER: Neuropeptidergic and cAMP-mediated regulation of stress-induced sleep in C. elegans

睡眠对生命至关重要，在所有经过仔细研究的动物身上都观察到了睡眠。令人惊讶的是，睡眠的功能仍然是自然界最大的谜团之一。在生病期间，免疫系统会加强睡眠。因此，一种功能可能涉及资源的重新分配，允许在受伤或感染期间发生损伤后进行细胞修复。调节睡眠的基因正在变得更好地被理解；有趣的是，这些基因在从线虫等无脊椎动物到人类的远亲动物中都是保守的。通过在相对简单的动物中识别新的睡眠基因及其在细胞通路中的相互作用，人们可能会对更复杂的动物(如人类)的睡眠调节和功能有深入的了解。该项目使用线虫秀丽线虫的遗传和行为方法的组合，包括遗传操作、神经活动的实时监测和动物视频分析，在细胞和生物水平上操纵和表征睡眠。值得注意的是，这项研究中使用的基因发现方法将被纳入大学生理学教学实验室和费城公立学校的科学课程，为专注于遗传学、全基因组测序和行为的学生提供独特的STEM体验。线虫在暴露于可能损害他们细胞的环境应激源后表现出睡眠，这种行为被称为应激诱导睡眠(SIS)。SIS的拟议功能被定义为一段行为静止期，专门用于在受到侮辱或伤害后发生的细胞修复。在302个细胞的线虫神经系统中，SIS主要由单一的中间神经元ALA诱导。ALA释放神经肽，这些神经肽导致不同方面的行为平静。最近，由NLP-14基因编码的神经肽被发现是SIS的中枢调节因子，很可能是从ALA释放出来的。NLP-14肽对SIS既是必要的，也是充分的。这些促进睡眠的多肽可能通过促进细胞内环磷酸腺苷(CAMP)的减少发挥作用，这些细胞在清醒时含有高水平的cAMP。这个项目将阐明ALA和NLP-14多肽与下游电路的联系。这将通过使用活体生物传感器测量睡眠动物的cAMP水平来进行检验。此外，新的下游细胞将通过激活红光激活的腺苷环化酶，IlaC22，在细胞组中被识别。它将研究腺苷一磷酸激活激酶的激活机制(S)，它是一种代谢主调节因子，将细胞资源从生长转移到修复，它在SIS的下游发挥作用。此外，该项目还将确定NLP-14多肽在一个由三部分组成的过程中的特定作用：(I)使用CRISPR-CAS9去除单个多肽，(Ii)在新的转基因菌株中过度表达单个多肽，以及(Iii)识别NLP-14受体的正向遗传抑制筛选(S)。基因筛查将被纳入高中外展实验室练习，以加强费城社区的STEM教育。这将通过实地考察和建立名为WormCam.的网络广播行为平台来实现。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Sleep is essential for life and has been observed in all animals closely studied. Surprisingly, the function of sleep remains one of nature's greatest mysteries. During sickness, sleep is enhanced by the immune system. Thus, one function may involve the redistribution of resources allowing for cellular repair following damage that occurs during injury or infection. The genes that regulate sleep are becoming better understood; interestingly, these genes are conserved among distantly related animals from invertebrates, like nematodes, to humans. By identifying novel sleep genes and their interactions in cellular pathways in relatively simple animals, one may gain insights into sleep regulation and function in more complex animals, like humans. This project uses a combination of genetic and behavioral approaches in the nematode Caenorhabditis elegans, including genetic manipulations, live monitoring of neural activity and animal video analyses, to manipulate and characterize sleep at the cellular and organismal level. Notably, the gene discovery approaches used in this study will be incorporated into college physiology teaching labs and Philadelphia public school science classes, to provide unique STEM experiences for students focusing on genetics, whole-genome sequencing and behavior.C. elegans display sleep following exposure to environmental stressors that may damage their cells, a behavior called stress-induced sleep (SIS). The proposed function of SIS is defined as a period of behavioral quiescence dedicated for cellular repairs that occur following insult or injury. Out of the 302-celled C. elegans nervous system, SIS is largely induced by a single interneuron, the ALA. The ALA releases neuropeptides, which induce different aspects of behavioral quiescence. Recently, neuropeptides encoded by the gene nlp-14 were identified to be central regulators of SIS and are likely thought to be released from the ALA. NLP-14 peptides are both necessary and sufficient for SIS. These sleep-promoting peptides may function by facilitating the reduction of intracellular cyclic adenosine monophosphate (cAMP) in a subset of cells that contain high levels of cAMP during times of wakefulness. This project will elucidate the connections of the ALA and NLP-14 peptides to downstream circuitry. This will be examined via the measurement of cAMP levels in sleeping animals using an in vivo biosensor. Additionally, novel downstream cells will be identified through the activation of a red-light activated adenylyl cyclase, IlaC22, in groups of cells. It will investigate the mechanism(s) of activation of adenosine monophosphate-activated kinase (AMPK), a metabolic master regulator that diverts cellular resources from growth to repair, which functions downstream of SIS. Also, this project will determine specific roles for the NLP-14 peptides in a three-part process: (i) the removal of individual peptides using CRISPR-Cas9, (ii) over expression of individual peptides in new transgenic strains, and (iii) a forward genetic suppressor screen to identify NLP-14 receptor(s). The genetic screen will be incorporated into high school outreach laboratory exercises to enhance STEM education in the Philadelphia community. This will be accomplished through field trips and the establishment of a webcast behavioral platform called WormCam.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.