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

职业：神经肽能和 cAMP 介导的线虫应激诱导睡眠调节

• 批准号: 1845020
• 负责人: Matthew Nelson
• 金额: $ 75万
• 依托单位: St Joseph's University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1845020&HistoricalAwards=false
• 关键词: CAREER; Neuropeptidergic; cAMP; mediated; regulation

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.

睡眠对于生命至关重要，并且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。生病期间，免疫系统可以增强睡眠。因此，一种功能可能涉及资源的重新分配，以允许在损伤或感染过程中发生损伤后进行细胞修复。人们对调节睡眠的基因有了更深入的了解。有趣的是，这些基因在从无脊椎动物（如线虫）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。该项目结合了秀丽隐杆线虫的遗传和行为方法，包括遗传操作、神经活动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其细胞的环境压力源后会表现出睡眠，这种行为称为压力诱导睡眠（SIS）。 SIS 的拟议功能被定义为专门用于损伤或损伤后发生的细胞修复的行为静止期。在 302 细胞线虫神经系统中，SIS 很大程度上是由单个中间神经元 ALA 诱导的。 ALA 释放神经肽，诱导不同方面的行为静止。 最近，由 nlp-14 基因编码的神经肽被确定为 SIS 的中央调节因子，并且可能被认为是从 ALA 中释放的。 NLP-14 肽对于 SIS 来说既是必要的也是充分的。这些促进睡眠的肽可能通过促进在清醒时含有高水平 cAMP 的细胞亚群中细胞内环磷酸腺苷 (cAMP) 的减少而发挥作用。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的 cAMP 水平来检查。此外，新的下游细胞将通过细胞组中红光激活的腺苷酸环化酶 IlaC22 的激活来识别。它将研究单磷酸腺苷激活激酶 (AMPK) 的激活机制，AMPK 是一种代谢主调节因子，可将细胞资源从生长转移到修复，在 SIS 下游发挥作用。此外，该项目将确定 NLP-14 肽在三部分过程中的具体作用：(i) 使用 CRISPR-Cas9 去除单个肽，(ii) 在新转基因菌株中过度表达单个肽，以及 (iii) ）用于鉴定 NLP-14 受体的正向遗传抑制筛选。基因筛查将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完成。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力优点和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

An AMPK biosensor for Caenorhabditis elegans. microPublication Biology

用于秀丽隐杆线虫的 AMPK 生物传感器。

• DOI: 10.17912/micropub.biology.000596.
• 发表时间: 2022-07
• 期刊: microPublication biology
• 作者: Carman L;Schuck RJ;Li E;Nelson MD
• 通讯作者: Nelson MD

The neuropeptide receptor npr-38 regulates avoidance and stress-induced sleep in Caenorhabditis elegans

神经肽受体 npr-38 调节秀丽隐杆线虫的回避和压力诱导的睡眠

• DOI: 10.1016/j.cub.2023.06.042
• 发表时间: 2023-07
• 期刊: Current Biology
• 影响因子: 9.2
• 作者: Le, Emily;McCarthy, Teagan;Honer, Madison;Curtin, Caroline E.;Fingerut, Jonathan;Nelson, Matthew D.
• 通讯作者: Nelson, Matthew D.

RPamide neuropeptides NLP-22 and NLP-2 act through GnRH-like receptors to promote sleep and wakefulness in C. elegans

RPamide 神经肽 NLP-22 和 NLP-2 通过 GnRH 样受体发挥作用，促进线虫的睡眠和觉醒

• DOI: 10.1038/s41598-020-66536-2
• 发表时间: 2020-06-18
• 期刊: Scientific Reports
• 影响因子: 4.6
• 作者: Petrus Van der Auwera;L. Frooninckx;Kristen Buscemi;Ryan T Vance;Jan Watteyne;Olivier Mirabeau;L. Temmerman;Wouter De Haes;Luca Fancsalszky;A. Gottschalk;D. Raizen;M. Nelson;L. Schoofs;Isabel Beets
• 通讯作者: Isabel Beets

Orcokinin neuropeptides regulate sleep in Caenorhabditis elegans

Orcokinin 神经肽调节秀丽隐杆线虫的睡眠

• DOI: 10.1080/01677063.2020.1830084
• 发表时间: 2020-09
• 期刊: Journal of Neurogenetics
• 影响因子: 1.9
• 作者: Honer M;Buscemi K;Barrett N;Riazati N;Orlando G;Nelson MD
• 通讯作者: Nelson MD