One Receptor, Two Functions: The GPCR DMSR-1 Regulates Sleep Onset and Duration in C. elegans
Year: 2025
Type: dissertation
Abstract: Summary Background Many reports suggest that vulnerable populations do not get enough sleep, mainly due to a misalignment between their biological needs and societal structures. Moreover, a significant portion of the population contends with diverse clinical sleep disorders, which diminish quality of life and entail a substantial societal cost. Developing solutions for sleep disorders and tailoring public health guidelines requires a fundamental understanding of the molecular and cellular mechanisms that regulate sleep. Basic research using genetically tractable model organisms provides a powerful approach to uncover these core principles. C. elegans is a powerful model organism for sleep research. Sleep occurs throughout all steps of the C. elegans lifecycle as a response to different biological needs. Starvation in C. elegans is probably among the strongest drivers of sleep. Prolonged starvation causes L1 larvae to halt development, shifting their physiology from growth to stress resistance. In this condition, L1 arrest larvae undergo frequent wake-sleep cycles regulated by the activation of the ring interneuron RIS. However, RIS activation is necessary in all types of sleep: starvation-induced L1 and adult sleep, developmentally timed sleep, as well as the stress-induced one. While previously unknown, the target receptor for FLP-11 was recently identified in a system-wide screen; of several binding GPCRs, DMSR-1 showed the highest potency in vitro. DMSR-1 had been previously linked to stress-induced sleep regulated by the ALA-released peptide FLP-13. However, how this newly identified FLP-11/DMSR-1 pathway regulates L1 arrest sleep remained to be elucidated. Research questions and hypotheses This thesis aimed to discover and characterize the FLP-11/DMSR-1 signaling pathway controlling C. elegans sleep. This involved two primary objectives. The first was to identify the pathway's core molecular components and physiological functions. This included identifying DMSR-1 as the primary FLP-11 receptor, validating its genetic dependence on FLP-11, and testing its role in survival and protective gene expression. The second objective was to characterize the pathway's circuit-level and cellular mechanisms by mapping the dual functions of DMSR-1 to distinct cholinergic and GABAergic neurons and providing direct evidence for its autocrine inhibitory role within the RIS neuron. Methods To dissect the FLP-11 signaling pathway, I measured the sleep behavior of C. elegans by long-term imaging in agarose microfluidic devices. This technique was used to screen FLP-11 receptor mutants and to perform genetic interaction studies. To test if DMSR-1 acts downstream of RIS, I performed long-term calcium imaging of the RIS neuron and manipulated it optogenetically. To map the dual functions of DMSR-1 to neuronal subpopulations, I used a newly designed conditional allele of dmsr-1 that, when combined with cell-specific driver lines of FLP recombinase, caused the knockdown of the receptor. I then measured the sleep behavior by long-term imaging in agarose microchambers in the resulting knockdown mutants. To evaluate the role of acetylcholine in C. elegans sleep, I treated C. elegans L1 arrest larvae with aldicarb and measured sleep behavior and RIS calcium activity. To test the autocrine feedback loop on RIS, I measured the calcium activity of the neuron after specifically knocking down dmsr-1 in that same cell. Finally, I connected the behavioral role of DMSR-1 to key physiological outcomes by performing a survival assay and measuring the expression of a transcriptional reporter of the hsp-12.6 gene. Results First, I found that the G-protein coupled receptor DMSR-1 mediates L1 Arrest Sleep in C. elegans, while the other receptors that bind FLP-11 in vitro (DMSR-7, FRPR-8, and NPR-22) are not essential for sleep. Then, I showed that DMSR-1 functions in C. elegans sleep in a manner that is dependent on FLP-11 signaling and that the receptor functions downstream of the RIS neuron. Moreover, since the receptor binds, in vitro, also to the peptides FLP-25 and FLP-13, I showed that these peptides are not required for L1 arrest sleep. Finally, I showed that the dmsr-1 mutation impairs survival and protective gene expression. With cell-specific dmsr-1 knockdowns in several neuronal subpopulations, I discovered that the receptor triggers sleep by acting in cholinergic neurons and negatively regulates it in GABAergic neurons. DMSR-1 in cholinergic neurons acts downstream of RIS, where it exerts its sleep-promoting activity. Moreover, I showed that pharmacological enhancement of cholinergic signaling antagonizes sleep, suggesting that DMSR-1 activity is necessary to lower cholinergic tone and allow sleep onset. As shown for the global dmsr-1 mutant, DMSR-1's effects on sleep are linked to the physiological effects of this behavior. Therefore, the function of the receptor in cholinergic neurons is necessary to induce protective gene expression. Finally, applying RIS-specific knockdown, I mapped the negative sleep-regulation function of DMSR-1 to the sleep-active neuron RIS itself, and I showed with calcium imaging that the FLP-11/DMSR-1 pathway inhibits RIS activity in an autocrine fashion. The activity of the receptor in RIS not only limits sleep duration, but it also reduces the overexpression of protective genes like hsp-12.6. Conclusion With this PhD project, I have improved the understanding of how sleep is induced and limited in C. elegans. Specifically, I discovered and characterized the FLP-11/DMSR-1 signaling pathway essential for controlling L1 arrest sleep in C. elegans. FLP-11 through DMSR-1 has a dual function on the worm's sleep. Acting on cholinergic neurons, it has a sleep-promoting function that corresponds to the inhibition of cholinergic signaling. On the other hand, RIS activity is regulated by FLP-11 and DMSR-1 through an autocrine negative feedback loop to limit sleep duration. This dual function of DMSR-1 is not limited to a behavioral outcome, but is linked to the physiological effects of L1 arrest sleep: survival during starvation and protective gene expression.
Author: Rossi, Lorenzo
Language: English
Subfield: Cognitive Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7155661921