Fig 4 - uploaded by Matthew D Nelson
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FRPR-4 is partially required for the feeding quiescence response to heat shock. Reducing frpr-4 function by mutation (A) or by RNA interference (B) impairs the feeding quiescence response to a 30-minute 35°C heat shock (Protocol 1 (See methods); Student’s t-test, average of 10 trials, N≥20 worms per trial, *p < .05, **p < .005). (C) A fosmid containing the wild-type frpr-4 gene restores in frpr-4(ok2376) mutants the feeding quiescence response to a 30-minute 35°C heat shock (Protocol 1 (See methods)) but not at other temperatures (Student’s t-test, average of 4 trials for each temperature, N≥20 worms per trial, **p < .005.) (D) frpr-4(ok2376) animals suppress the feeding quiescence in response to a 30-minute 33°C heat shock but not at the other temperatures tested (Student’s t-test, average of 3 trials, N≥20 worms per trial, **P < .005). (E) frpr-4(ok2376) worms display normal locomotion quiescence in response to heat stress at all temperatures tested (Average of 2 trials, 12 worms per trial).
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Neuropeptides signal through G-protein coupled receptors (GPCRs) to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regul...
Citations
... Overexpression of npr-38 disrupts sleep Overexpression of GPCRs can induce gain-of-function phenotypes. 46,47 We predicted that npr-38 overexpression would heighten stress-induced sleep and DTS. We made transgenic, multi-copy overexpression lines. ...
Although essential and conserved, sleep is not without its challenges that must be overcome; most notably, it renders animals vulnerable to threats in the environment. Infection and injury increase sleep demand, which dampens sensory responsiveness to stimuli, including those responsible for the initial insult. Stress-induced sleep in Caenorhabditis elegans occurs in response to cellular damage following noxious exposures the animals attempted to avoid. Here, we describe a G-protein-coupled receptor (GPCR) encoded by npr-38, which is required for stress-related responses including avoidance, sleep, and arousal. Overexpression of npr-38 shortens the avoidance phase and causes animals to initiate movement quiescence and arouse early. npr-38 functions in the ADL sensory neurons, which express neuropeptides encoded by nlp-50, also required for movement quiescence. npr-38 regulates arousal by acting on the DVA and RIS interneurons. Our work demonstrates that this single GPCR regulates multiple aspects of the stress response by functioning in sensory and sleep interneurons.
... Heat stress causes the release of LIN-3/EGF, which acts on its receptor LET-23/EGFR present on the ALA neuron, triggering the release of FLP-13 neuropeptide that is required for feeding and locomotory quiescent behavior (Nelson et al., 2014). The FLP-13 neuropeptide released from the ALA neuron as a result of stress acts on the receptor FRPR-4 on the DVA neuron to modulate the posture of the animals during quiescence (Nelson et al., 2015). The transcription factor LIM-6 controls both peptidergic and GABAergic function in the RIS neuron. ...
One of the reasons that most multicellular animals survive and thrive is because of the adaptable and plastic nature of their nervous systems. For an organism to survive, it is essential for the animal to respond and adapt to environmental changes. This is achieved by sensing external cues and translating them into behaviors through changes in synaptic activity. The nervous system plays a crucial role in constantly evaluating environmental cues and allowing for behavioral plasticity in the organism. Multiple neurotransmitters and neuropeptides have been implicated as key players for integrating sensory information to produce the desired output. Because of its simple nervous system and well-established neuronal connectome, C. elegans acts as an excellent model to understand the mechanisms underlying behavioral plasticity. Here, we critically review how neuropeptides modulate a wide range of behaviors by allowing for changes in neuronal and synaptic signaling. This review will have a specific focus on feeding, mating, sleep, addiction, learning and locomotory behaviors in C. elegans. With a view to understand evolutionary relationships, we explore the functions and associated pathophysiology of C. elegans neuropeptides that are conserved across different phyla. Further, we discuss the mechanisms of neuropeptidergic signaling and how these signals are regulated in different behaviors. Finally, we attempt to provide insight into developing potential therapeutics for neuropeptide-related disorders.
... Previous studies have linked the entirety of the gene to a receptor based on binding studies and full transgenic rescue 29,65,66 . Here, we employ a rescue-by-feeding assay, following the design of RNAi feeding protocols, to rescue individual peptides 37 . ...
... Here, we employ a rescue-by-feeding assay, following the design of RNAi feeding protocols, to rescue individual peptides 37 . While "feeding" of peptides through soaking is a valid approach, there are many constraints on such approaches, the most prominent being the ability to acquire purified peptides 29,65 . Using our rescue-byfeeding approach, we provide access to the peptide to the worms directly through their food source. ...
... FRPR-16, however, is more closely related to the fly FMRFamide Receptor 67 . Interestingly, while some C. elegans FRPR receptors function as FLP receptors 19,64,65 , at least one receptor within the same evolutionary clade (DAF-37) acts as a chemosensor for pheromones 68 . The evolutionary distance between NPR-10 and FRPR-16 suggests that these two receptors have undergone convergent evolution. ...
Dioecious species are a hallmark of the animal kingdom, with opposing sexes responding differently to identical sensory cues. Here, we study the response of C. elegans to the small-molecule pheromone, ascr#8, which elicits opposing behavioral valences in each sex. We identify a novel neuropeptide-neuropeptide receptor (NP/NPR) module that is active in males, but not in hermaphrodites. Using a novel paradigm of neuropeptide rescue that we established, we leverage bacterial expression of individual peptides to rescue the sex-specific response to ascr#8. Concurrent biochemical studies confirmed individual FLP-3 peptides differentially activate two divergent receptors, NPR-10 and FRPR-16. Interestingly, the two of the peptides that rescued behavior in our feeding paradigm are related through a conserved threonine, suggesting that a specific NP/NPR combination sets a male state, driving the correct behavioral valence of the ascr#8 response. Receptor expression within pre-motor neurons reveals novel coordination of male-specific and core locomotory circuitries.
... The C. elegans genome encodes three classes of neuropeptides: FMRFamide-like peptides (FLP), insulin-like peptides (INS), and non-FLP/insulin neuropeptide-like peptides (NLP) [22][23][24] , encoding over 300 individual neuropeptides through 131 genes that modulate the functional connectome 9,25,26 . The complexity of the neuropeptide genome, combined with extrasynaptic neuropeptides signaling makes elucidating the role of individual peptides difficult, as canonical studies often rely on null mutations and transgenic rescues [27][28][29][30] . As such, these studies are often incomplete, as full gene rescue restores complete preproproteins and makes discrimination of discrete peptide function impossible 9,31 . ...
Animals constantly respond to changes in their environment and internal states via neuromodulation. Neuropeptide genes modulate neural circuits by encoding either multiple copies of the same neuropeptide or different neuropeptides. This architectural complexity makes it difficult to determine the function of discrete and active neuropeptides. Here, we present a novel genetic tool that facilitates functional analysis of individual peptides. We engineered Escherichia coli bacteria to express active peptides and fed loss-of-function Caenorhabditis elegans to rescue gene activity. Using this approach, we rescued the activity of different neuropeptide genes with varying lengths and functions: trh-1, ins-6, and pdf-1. While some peptides are functionally redundant, others exhibited unique and previously uncharacterized functions. The mechanism of peptide uptake is reminiscent of RNA interference, suggesting convergent mechanisms of gene regulation in organisms. Our rescue-by-feeding paradigm provides a high-throughput screening strategy to elucidate the functional landscape of neuropeptide genes regulating different behavioral and physiological processes.
... 46 After heat stress exposure, frpr-4 mutation suppressed feeding quiescence. 31 TKR-1 overexpression could confer a resistance to UV irradiation and heat stress. 28 In neuronal cells, we found that the GPCRs of NPR-9, NPR-12, DCAR-1, and GTR-1 functioned upstream of JNK MAPK signaling to control PS-NPs toxicity (Figure 2 and Table S4). ...
In this study, the association of expressional alterations in neuronal G protein-coupled receptors (GPCRs) with induction of protective response to polystyrene nanoparticles (PS-NPs) was investigated in Caenorhabditis elegans. On the basis of both phenotypic analysis and expression levels, the alterations in expressions of NPR-1, NPR-4, NPR-8, NPR-9, NPR-12, DCAR-1, GTR-1, DOP-2, SER-4, and DAF-37 in neuronal cells mediated the protective response to PS-NPs exposure. In neuronal cells, NPR-9, NPR-12, DCAR-1, and GTR-1 controlled the PS-NPs toxicity by activating or inhibiting JNK-1/JNK MAPK signaling. Neuronal NPR-8, NPR-9, DCAR-1, DOP-2, and DAF-37 controlled the PS-NPs toxicity by activating or inhibiting MPK-1/ERK MAPK signaling. Neuronal NPR-4, NPR-8, NPR-9, NPR-12, GTR-1, DOP-2, and DAF-37 controlled the PS-NPs toxicity by activating or inhibiting DBL-1/TGF-β signaling. Neuronal NPR-1, NPR-4, NPR-12, and GTR-1 controlled the PS-NPs toxicity by activating or inhibiting DAF-7/TGF-β signaling. Our data provides an important neuronal basis for induction of protective response to PS-NPs in C. elegans.
... In contrast to its relatively minor roles in DTS, NLP-14 is more important during SIS, where the removal of all or subsets of peptides causes strong defects in movement and defecation quiescence. Numerous neuropeptides regulate movement quiescence, including FLP-11, secreted from the RIS (Konietzka et al., 2020) and FLP-13, FLP-24, and NLP-8 (Nath et al., 2016;Nelson et al., 2015), released from ALA. These molecules signal through many GPCRs (Iannacone et al., 2017;Nelson et al., 2015), reducing cAMP/PKA signaling in different cells (Cianciulli et al., 2019). ...
... Numerous neuropeptides regulate movement quiescence, including FLP-11, secreted from the RIS (Konietzka et al., 2020) and FLP-13, FLP-24, and NLP-8 (Nath et al., 2016;Nelson et al., 2015), released from ALA. These molecules signal through many GPCRs (Iannacone et al., 2017;Nelson et al., 2015), reducing cAMP/PKA signaling in different cells (Cianciulli et al., 2019). The orcokinins can be added to this expanding list of somnogenic neuropeptides. ...
Orcokinin neuropeptides are conserved among ecdysozoans, but their functions are incompletely understood. Here, we report a role for orcokinin neuropeptides in the regulation of sleep in the nematode Caenorhabditis elegans. The C. elegans orcokinin peptides, which are encoded by the nlp-14 and nlp-15 genes, are necessary and sufficient for quiescent behaviors during developmentally timed sleep (DTS) as well as during stress-induced sleep (SIS). The five orcokinin neuropeptides encoded by nlp-14 have distinct but overlapping functions in the regulation of movement and defecation quiescence during SIS. We suggest that orcokinins may regulate behavioral components of sleep-like states in nematodes and other ecdysozoans.
... For instance, the mechanosensory role described in FMRFamide-like peptides family in several nematodes were particularly interesting [16,[21][22][23][24][25][26] and this peptide family is also present in M. incognita [27]. The GPCRs associated to these peptides, such as the C. elegans flp-32 receptor [28] (uniprot G5EEB1) and FRPR-4 receptor [29] (uniprot A0A131MCZ4), have been shown to be involved in the regulation of sexual and locomotion behaviour of nematodes and are therefore interesting targets for inhibiting nematodes parasitism on plants. In this respect, NPR-1 receptor from C. elegans [30,31] (UniProt Q18534) and NPR-4 receptor from C. elegans and Brugia malayi [32] (Uniprot A0A078BS36 and A0A0J9XSQ0 respectively) were interesting targets for our purpose because the proteins were annotated as neuropeptide receptors. ...
Root-knot nematodes (RKN), from the Meloidogyne genus, have a worldwide distribution and cause severe economic damage to many life-sustaining crops. Because of their lack of specificity and danger to the environment, most chemical nematicides have been banned from use. Thus, there is a great need for new and safe compounds to control RKN. Such research involves identifying beforehand the nematode proteins essential to the invasion. Since G protein-coupled receptors GPCRs are the target of a large number of drugs, we have focused our research on the identification of putative nematode GPCRs such as those capable of controlling the movement of the parasite towards (or within) its host. A datamining procedure applied to the genome of Meloidogyne incognita allowed us to identify a GPCR, belonging to the neuropeptide GPCR family that can serve as a target to carry out a virtual screening campaign. We reconstructed a 3D model of this receptor by homology modeling and validated it through extensive molecular dynamics simulations. This model was used for large scale molecular dockings which produced a filtered limited set of putative antagonists for this GPCR. Preliminary experiments using these selected molecules allowed the identification of an active compound, namely C260-2124, from the ChemDiv provider, which can serve as a starting point for further investigations.
... Neuropeptides regulate sleep across phylogeny by signaling through GPCRs (TROJANOWSKI AND RAIZEN 2016); during SIS, the ALA neuron releases neuropeptides that act on unknown target cells to initiate sleep NATH et al. 2016), which may occur by inhibiting cAMP/PKA. We proposed that cells expressing the gene twk-16, including the DVA interneuron (SALKOFF et al. 2001), were potential downstream targets of ALA signaling, based upon the expression pattern of the sleep-promoting neuropeptide receptor, FRPR-4 ( NELSON et al. 2015). To test this hypothesis, we generated transgenic lines expressing IlaC22 from the twk-16 promoter. ...
Sleep is evolutionarily conserved, thus studying simple invertebrates such as Caenorhabditis elegans can provide mechanistic insight into sleep with single cell resolution. A conserved pathway regulating sleep across phylogeny involves cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that functions in neurons by activating protein kinase A. C. elegans sleep in response to cellular stress caused by environmental insults [stress-induced sleep (SIS)], a model for studying sleep during sickness. SIS is controlled by simple neural circuitry, thus allowing for cellular dissection of cAMP signaling during sleep. We employed a red-light activated adenylyl cyclase, IlaC22, to identify cells involved in SIS regulation. We found that pan-neuronal activation of IlaC22 disrupts SIS through mechanisms independent of the cAMP response element binding protein. Activating IlaC22 in the single DVA interneuron, the paired RIF interneurons, and in the CEPsh glia identified these cells as wake-promoting. Using a cAMP biosensor, epac1-camps, we found that cAMP is decreased in the RIF and DVA interneurons by neuropeptidergic signaling from the ALA neuron. Ectopic overexpression of sleep-promoting neuropeptides coded by flp-13 and flp-24, released from the ALA, reduced cAMP in the DVA and RIFs, respectively. Overexpression of the wake-promoting neuropeptides coded by pdf-1 increased cAMP levels in the RIFs. Using a combination of optogenetic manipulation and in vivo imaging of cAMP we have identified wake-promoting neurons downstream of the neuropeptidergic output of the ALA. Our data suggest that sleep- and wake-promoting neuropeptides signal to reduce and heighten cAMP levels during sleep, respectively.
... In C. elegans, the ALA neuron, the neuropeptide FLP-13, and the G-protein-coupled receptor FRPR-4 have been shown to be critical in stress-induced quiescence, with their absences leading to suppression of quiescence and overexpression leading to increased quiescence after cellular stress (Nelson et al. 2014, Nelson et al. 2015. Therefore, with RAGE expressed pan-neuronally, it was hypothesized that the RAGE-related detrimental pathways will lead to a generalized neurodegeneration that results in suppression of quiescence post-heat stressor, as shown previously in the nematodes lacking the ALA neuron or FLP-13 (Nelson et al. 2014(Nelson et al. , 2015. ...
... In C. elegans, the ALA neuron, the neuropeptide FLP-13, and the G-protein-coupled receptor FRPR-4 have been shown to be critical in stress-induced quiescence, with their absences leading to suppression of quiescence and overexpression leading to increased quiescence after cellular stress (Nelson et al. 2014, Nelson et al. 2015. Therefore, with RAGE expressed pan-neuronally, it was hypothesized that the RAGE-related detrimental pathways will lead to a generalized neurodegeneration that results in suppression of quiescence post-heat stressor, as shown previously in the nematodes lacking the ALA neuron or FLP-13 (Nelson et al. 2014(Nelson et al. , 2015. ...
... As RAGE-related pathways are associated with increased cellular stress via syntheses of toxic metabolites and reactive oxygen species, it is possible that the increased quiescence seen after heat shock is due to upregulation of FLP-13 and FRPR-4 in the RAGE-expressing nematodes (Singh et al. 2001). A similar phenomenon has been discussed previously, when the overexpression of FLP-13 and FRPR-4 independently leads to increased quiescence post-heat stressor (Nelson et al. 2014(Nelson et al. , 2015. ...
The receptor for advanced glycation products (RAGE) is a cell surface, multi-ligand receptor belonging to the immunoglobulin superfamily; this receptor is implicated in a variety of maladies, via inflammatory pathways and induction of oxidative stress. Currently, RAGE is being studied using a limited number of mammalian in vivo, and some complementary in vitro, models. Here, we present a Caenorhabditis elegans model for the study of RAGE-related pathology: a transgenic strain, expressing RAGE in all neurons, was generated and subsequently tested behaviorally, developmentally, and morphologically. In addition to RAGE expression being associated with a significantly shorter lifespan, the following behavioral observations were made when RAGE-expressing worms were compared to the wild type: RAGE-expressing worms showed an impaired dopaminergic system, evaluated by measuring the fluorescent signal of GFP tagging; these worms exhibited decreased locomotion—both general and following ethanol exposure—as measured by counting body bends in adult worms; RAGE expression was also associated with impaired recovery of quiescence and pharyngeal pumping secondary to heat shock, as a significantly smaller fraction of RAGE-expressing worms engaged in these behaviors in the 2 h immediately following the heat shock. Finally, significant developmental differences were also found between the two strains: RAGE expression leads to a significantly smaller fraction of hatched eggs 24 h after laying and also to a significantly slower developmental speed overall. As evidence for the role of RAGE in a variety of neuropathologies accumulates, the use of this novel and expedient model should facilitate the elucidation of relevant underlying mechanisms and also the development of efficient therapeutic strategies.
... Therefore, we expected these transgenes to cause overexpression of SEB-3/NLP-49. Overexpression of neuropeptide precursor genes or neuropeptide receptors has previously been shown to cause gain-of-function phenotypes opposite to those seen in the respective null mutants (see, for example, [33][34][35][36]). Our data indicated that both neuropeptide and receptor rescue strains showed similar locomotion changes compared with wild-type, and that these changes were generally in the opposite direction to those seen for nlp-49 and seb-3 mutants (figure 2a). ...
Neuropeptide signalling has been implicated in a wide variety of biological processes in diverse organisms, from invertebrates to humans. The Caenorhabditis elegans genome has at least 154 neuropeptide precursor genes, encoding over 300 bioactive peptides. These neuromodulators are thought to largely signal beyond ‘wired’ chemical/electrical synapse connections, therefore creating a ‘wireless’ network for neuronal communication. Here, we investigated how behavioural states are affected by neuropeptide signalling through the G protein-coupled receptor SEB-3, which belongs to a bilaterian family of orphan secretin receptors. Using reverse pharmacology, we identified the neuropeptide NLP-49 as a ligand of this evolutionarily conserved neuropeptide receptor. Our findings demonstrate novel roles for NLP-49 and SEB-3 in locomotion, arousal and egg-laying. Specifically, high-content analysis of locomotor behaviour indicates that seb-3 and nlp-49 deletion mutants cause remarkably similar abnormalities in movement dynamics, which are reversed by overexpression of wild-type transgenes. Overexpression of NLP-49 in AVK interneurons leads to heightened locomotor arousal, an effect that is dependent on seb-3. Finally, seb-3 and nlp-49 mutants also show constitutive egg-laying in liquid medium and alter the temporal pattern of egg-laying in similar ways. Together, these results provide in vivo evidence that NLP-49 peptides act through SEB-3 to modulate behaviour, and highlight the importance of neuropeptide signalling in the control of behavioural states.
This article is part of a discussion meeting issue ‘Connectome to behaviour: modelling C. elegans at cellular resolution’.
