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.
