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

睡眠对生命至关重要，在所有仔细研究的动物中都观察到了这一点。 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，免疫系统会增强睡眠。因此，一种功能可能涉及资源的重新分配，允许在损伤或感染期间发生的损伤后进行细胞修复。人们对调节睡眠的基因越来越了解;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，人们可以深入了解更复杂的动物（如人类）的睡眠调节和功能。该项目在线虫线虫中使用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，以在细胞和有机体水平上操纵和表征睡眠。 值得注意的是，本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学的学生提供独特的STEM体验。秀丽线虫在暴露于可能损害其细胞的环境压力源后显示睡眠，这种行为称为压力诱导睡眠（SIS）。SIS的拟议功能被定义为一段时间的行为静止期，专门用于损伤或损伤后发生的细胞修复。在302个细胞的C.在elegans神经系统中，SIS主要由单个中间神经元ALA诱导。ALA释放神经肽，诱导行为静止的不同方面。 最近，由基因nlp-14编码的神经肽被鉴定为SIS的中枢调节因子，并且可能被认为是从ALA释放的。NLP-14肽对于SIS是必要的和足够的。这些促进睡眠的肽可以通过促进在清醒期间含有高水平cAMP的细胞亚群中细胞内环磷酸腺苷（cAMP）的减少来起作用。该项目将阐明ALA和NLP-14肽与下游电路的连接。这将通过使用体内生物传感器测量睡眠动物中的cAMP水平来检查。此外，新的下游细胞将通过在细胞群中激活红光激活的腺苷酸环化酶IlaC 22来鉴定。它将研究腺苷一磷酸激活激酶（AMPK）的激活机制，AMPK是一种代谢主调节剂，将细胞资源从生长转移到修复，其功能在SIS下游。此外，该项目将确定NLP-14肽在三部分过程中的特定作用：（i）使用CRISPR-Cas9去除单个肽，（ii）在新的转基因菌株中过度表达单个肽，以及（iii）正向遗传抑制筛选以识别NLP-14受体。遗传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。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值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.