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In vertebrates, thyrostimulin is a highly conserved glycoprotein hormone that, besides thyroid stimulating hormone (TSH), is a potent ligand of the TSH receptor. Thyrostimulin is considered the most ancestral glycoprotein hormone and orthologs of its subunits, GPA2 and GPB5, are widely conserved across vertebrate and invertebrate animals. Unlike TSH, however, the functions of the thyrostimulin neuroendocrine system remain largely unexplored. Here, we identify a functional thyrostimulin-like signaling system inCaenorhabditis elegans. We show that orthologs of GPA2 and GPB5, together with thyrotropin-releasing hormone (TRH) related neuropeptides, constitute a neuroendocrine pathway that promotes growth inC. elegans. GPA2/GPB5 signaling is required for normal body size and acts through activation of the glycoprotein hormone receptor ortholog FSHR-1.C. elegansGPA2 and GPB5 increase cAMP signaling by FSHR-1in vitro. Both subunits are expressed in enteric neurons and promote growth by signaling to their receptor in glial cells and the intestine. Impaired GPA2/GPB5 signaling causes bloating of the intestinal lumen. In addition, mutants lacking thyrostimulin-like signaling are defective in defecation behavior. Our study suggests that the thyrostimulin GPA2/GPB5 pathway is an ancient enteric neuroendocrine system that regulates intestinal function in ecdysozoans, and may ancestrally have been involved in the control of organismal growth.

Neuropeptides regulate animal physiology and behavior, making them widely studied targets of functional genetics research. While the field often relies on differential -omics approaches to build hypotheses, no such method exists for neuropeptidomics. It would nonetheless be valuable for studying behaviors suspected to be regulated by neuropeptides, especially when little information is otherwise available. This includes nictation, a phoretic strategy of Caenorhabditis elegans dauers that parallels host-finding strategies of infective juveniles of many pathogenic nematodes. We here developed a targeted peptidomics method for the model organism C. elegans and show that 161 quantified neuropeptides are more abundant in its dauer stage compared with L3 juveniles. Many of these have orthologs in the commercially relevant pathogenic nematode Steinernema carpocapsae, in whose infective juveniles, we identified 126 neuropeptides in total. Through further behavioral genetics experiments, we identify flp-7 and flp-11 as novel regulators of nictation. Our work advances knowledge on the genetics of nictation behavior and adds comparative neuropeptidomics as a tool to functional genetics workflows. ispartof: Molecular & Cellular Proteomics vol:22 issue:2 ispartof: location:United States status: published

SummaryNeuropeptides are ancient, widespread signaling molecules that underpin almost all brain functions. They constitute a broad ligand-receptor network, mainly by binding to G protein-coupled receptors (GPCRs). However, the organization of the peptidergic network and roles of many neuropeptides remain elusive, as our insight into neuropeptide-receptor interactions is limited and many peptide GPCRs in animal models and humans are still orphan receptors. Here we report a genome-wide neuropeptide-GPCR interaction map inC. elegans. By reverse pharmacology screening of over 55,384 possible interactions, we identify 461 cognate peptide-GPCR couples that uncover a broad signaling network with specific and complex combinatorial interactions encoded across and within single peptidergic genes. These interactions provide insights into neuropeptide functions and evolution. Combining our dataset with phylogenetic analysis supports peptide-receptor co-evolution and conservation of at least 14 bilaterian peptidergic systems inC. elegans. This resource lays a foundation for system-wide analysis of the peptidergic network.HighlightsSystem-wide reverse pharmacology deorphanizes 68C. eleganspeptide GPCRsDiscovery of 461 peptide-GPCR pairs and additional ligands for characterized GPCRsNeuropeptides show specific and complex combinatorial receptor interactionsPeptide-GPCR pairs support long-range conservation and expansion of peptide systems

Neural circuit synaptic connectivities (the connectome) provide the anatomical foundation for our understanding of nematode nervous system function. However, other nonsynaptic routes of communication are known in invertebrates including extrasynaptic volume transmission (EVT), which enables short- and/or long-range communication in the absence of synaptic connections. Although EVT has been highlighted as a facet of Caenorhabditis elegans neurosignaling, no experimental evidence identifies body cavity fluid (pseudocoelomic fluid; PCF) as a vehicle for either neuropeptide or biogenic amine transmission. In the parasitic nematode Ascaris suum, FMRFamide-like peptides encoded on flp-18 potently stimulate female reproductive organs but are expressed in cells that are anatomically distant from the reproductive organ, with no known synaptic connections to this tissue. Here we investigate nonsynaptic neuropeptide signaling in nematodes mediated by the body cavity fluid. Our data show that (i) A. suum PCF (As-PCF) contains a catalog of neuropeptides including FMRFamide-like peptides and neuropeptide-like proteins, (ii) the A. suum FMRFamide-like peptide As-FLP-18A dominates the As-PCF peptidome, (iii) As-PCF potently modulates nematode reproductive muscle function ex vivo, mirroring the effects of synthetic FLP-18 peptides, (iv) As-PCF activates the C. elegans FLP-18 receptors NPR-4 and -5, (v) As-PCF alters C. elegans behavior, and (vi) FLP-18 and FLP-18 receptors display pan-phylum distribution in nematodes. This study provides the first direct experimental evidence to support an extrasynaptic volume route for neuropeptide transmission in nematodes. These data indicate nonsynaptic signaling within the nematode functional connectome and are particularly pertinent to receptor deorphanization approaches underpinning drug discovery programs for nematode pathogens. ispartof: Acs Chemical Neuroscience vol:12 issue:17 pages:3176-3188 ispartof: location:United States status: published

SUMMARYPathogenic infection elicits sickness behaviors that promote recovery and survival of the host. For example, following infection with the pathogenic bacterium Pseudomonas aeruginosa PA14, the nematode Caenorhabditis elegans modifies its sensory preferences to avoid the pathogen. Here we identify antagonistic neuromodulatory circuits that shape this sickness behavior. Using an unbiased cell-directed neuropeptide screen, we show that AVK neurons upregulate and release FMRFamide-like FLP-1 neuropeptides during infection to drive pathogen avoidance. Manipulations that increase or decrease AVK signaling accelerate or delay pathogen avoidance, respectively, implicating AVK in the dynamics of sickness behavior. FLP-1 neuropeptides act via the G-protein-coupled receptor DMSR-7 in RIM/RIC neurons to reduce tyraminergic/octopaminergic signaling that opposes pathogen avoidance. RIM/RIC neurons relay parallel signals from neuropeptides and the cytokine TGF-β that represent internal and external regulators of pathogen avoidance. Our results demonstrate that antagonism between neuromodulatory systems results in slow, graded transitions between alternative behavioral states.

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., Reilly et al identify a neuropeptide-neuropeptide receptor (NP/NPR) module that is active in male but not in hermaphrodite C. elegans. Using a neuropeptide rescue paradigm, they demonstrate that receptor expression within pre-motor neurons mediates coordination of male-specific and core locomotor circuitries.

Neural circuit synaptic connectivities (the connectome) provide the anatomical foundation for our understanding of nematode nervous system function. However, other non-synaptic routes of communication are known in invertebrates including extrasynaptic volume transmission (EVT), which enables short- and/or long-range communication in the absence of synaptic connections. Although EVT has been highlighted as a facet of Caenorhabditis elegans neurosignaling, no experimental evidence identifies body cavity fluid (pseudocoelomic fluid; PCF) as a vehicle for either neuropeptide or biogenic amine transmission. In the parasitic nematode Ascaris suum FMRFamide-like peptides encoded on flp-18 potently stimulate female reproductive organs but are only expressed in cells that are anatomically distant from the reproductive organ, with no known synaptic connections to this tissue. Here we report a new non-synaptic mode of signaling in nematodes mediated by neuropeptides within the PCF. Our data show that: (i) A. suum PCF (As-PCF) contains a catalogue of neuropeptides including FMRFamide-like peptides and neuropeptide-like proteins; (ii) the A. suum FMRFamide-like peptide As-FLP-18A dominates the As-PCF peptidome; (iii) As-PCF potently modulates nematode reproductive muscle function ex vivo, mirroring the effects of synthetic FLP-18 peptides; (iv) As-PCF activates the C. elegans FLP-18 receptors NPR-4 and -5; (v) As-PCF alters C. elegans behavior and, (vi) FLP-18 and FLP-18 receptors display pan-phylum distribution in nematodes. Here we provide the first direct experimental evidence that supports an extrasynaptic volume route for neuropeptide transmission in nematodes. These data demonstrate non-synaptic signaling within the nematode functional connectome and are pertinent to receptor deorphanisation approaches underpinning drug discovery programs for nematode pathogens.

Aversive learning is fundamental for animals to increase chances of survival. In addition to classical neurotransmitters, neuropeptides have emerged to modulate such complex behaviors. Among them, neuropeptide Y (NPY) is well known to promote aversive memory acquisition in mammals. Here we identify an NPY/neuropeptide F (NPF)-related neuropeptide system in Caenorhabditis elegans and show that this FLP-34/NPR-11 system is required for learning negative associations, a process that is reminiscent of NPY signaling in mammals. The Caenorhabditis elegans NPY/NPF ortholog FLP-34 displays conserved structural hallmarks of bilaterian-wide NPY/NPF neuropeptides. We show that it is required for aversive olfactory learning after pairing diacetyl with the absence of food, but not for appetitive olfactory learning in response to butanone. To mediate diacetyl learning and thus integrate the aversive food context with the diacetyl odor, FLP-34 is released from serotonergic neurons and signals through its evolutionarily conserved NPY/NPF GPCR, NPR-11, in downstream AIA interneurons. NPR-11 activation in the AIA integration center results in avoidance of a previously attractive stimulus. This study opens perspectives for a deeper understanding of stress conditions in which aversive learning results in excessive avoidance.SIGNIFICANCE STATEMENT Aversive learning evolved early in evolution to promote avoidance of dangerous and stressful situations. In addition to classical neurotransmitters, neuropeptides are emerging as modulators of complex behaviors, including learning and memory. Here, we identified the evolutionary ortholog of neuropeptide Y/neuropeptide F in the nematode Caenorhabditis elegans, and we discovered that it is required for olfactory aversive learning. In addition, we elucidated the neural circuit underlying this avoidance behavior, and we discovered a novel coordinated action of Caenorhabditis elegans neuropeptide Y/neuropeptide F and serotonin that could aid in our understanding of the molecular mechanisms underlying stress disorders in which excessive avoidance results in maladaptive behaviors. ispartof: Journal Of Neuroscience vol:40 issue:31 pages:6018-6034 ispartof: location:United States nrpages: 17 status: published

Aversive learning is fundamental for animals to increase chances of survival. In addition to classical neurotransmitters, neuropeptides have emerged to modulate such complex behaviors. Among them, neuropeptide Y (NPY) is well known to promote aversive memory acquisition in mammals. Here we identify an NPY/neuropeptide F (NPF)-related neuropeptide system in Caenorhabditis elegans and show that this FLP-34/NPR-11 system is required for learning negative associations, a process that is reminiscent of NPY signaling in mammals. The Caenorhabditis elegans NPY/NPF ortholog FLP-34 displays conserved structural hallmarks of bilaterian-wide NPY/NPF neuropeptides. We show that it is required for aversive olfactory learning after pairing diacetyl with the absence of food, but not for appetitive olfactory learning in response to butanone. To mediate diacetyl learning and thus integrate the aversive food context with the diacetyl odor, FLP-34 is released from serotonergic neurons and signals through its evolutionarily conserved NPY/NPF GPCR, NPR-11, in downstream AIA interneurons. NPR-11 activation in the AIA integration center results in avoidance of a previously attractive stimulus. This study opens perspectives for a deeper understanding of stress conditions in which aversive learning results in excessive avoidance. SIGNIFICANCE STATEMENT Aversive learning evolved early in evolution to promote avoidance of dangerous and stressful situations. In addition to classical neurotransmitters, neuropeptides are emerging as modulators of complex behaviors, including learning and memory. Here, we identified the evolutionary ortholog of neuropeptide Y/neuropeptide F in the nematode Caenorhabditis elegans, and we discovered that it is required for olfactory aversive learning. In addition, we elucidated the neural circuit underlying this avoidance behavior, and we discovered a novel coordinated action of Caenorhabditis elegans neuropeptide Y/neuropeptide F and serotonin that could aid in our understanding of the molecular mechanisms underlying stress disorders in which excessive avoidance results in maladaptive behaviors.

Learning and memory are regulated by neuromodulatory pathways, but the contribution and temporal requirement of most neuromodulators in a learning circuit are unknown. Here we identify the evolutionarily conserved neuromedin U (NMU) neuropeptide family as a regulator of C. elegans gustatory aversive learning. The NMU homolog CAPA-1 and its receptor NMUR-1 are required for the retrieval of learned salt avoidance. Gustatory aversive learning requires the release of CAPA-1 neuropeptides from sensory ASG neurons that respond to salt stimuli in an experience-dependent manner. Optogenetic silencing of CAPA-1 neurons blocks the expression, but not the acquisition, of learned salt avoidance. CAPA-1 signals through NMUR-1 in AFD sensory neurons to modulate two navigational strategies for salt chemotaxis. Aversive conditioning thus recruits NMU signaling to modulate locomotor programs for expressing learned avoidance behavior. Because NMU signaling is conserved across bilaterian animals, our findings incite further research into its function in other learning circuits., Learning and memory are regulated by neuropeptides. Here, the authors show that the neuropeptide CAPA-1 and its receptor NMUR-1 are required to retrieve learned salt avoidance in C. elegans. CAPA-1/NMUR-1 activation in AFD sensory neurons modulates locomotor programs to express learned avoidance.

β-arrestins are associated with numerous aspects of G protein-coupled receptor (GPCR) signaling and regulation and accordingly influence diverse physiological and pathophysiological processes. Here we report that β-arrestin 2 expression is elevated in two independent cohorts of individuals with Alzheimer's disease. Overexpression of β-arrestin 2 leads to an increase in amyloid-β (Aβ) peptide generation, whereas genetic silencing of Arrb2 (encoding β-arrestin 2) reduces generation of Aβ in cell cultures and in Arrb2(-/-) mice. Moreover, in a transgenic mouse model of Alzheimer's disease, genetic deletion of Arrb2 leads to a reduction in the production of Aβ(40) and Aβ(42). Two GPCRs implicated previously in Alzheimer's disease (GPR3 and the β(2)-adrenergic receptor) mediate their effects on Aβ generation through interaction with β-arrestin 2. β-arrestin 2 physically associates with the Aph-1a subunit of the γ-secretase complex and redistributes the complex toward detergent-resistant membranes, increasing the catalytic activity of the complex. Collectively, these studies identify β-arrestin 2 as a new therapeutic target for reducing amyloid pathology and GPCR dysfunction in Alzheimer's disease.

The orphan G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer's disease (AD). However, given the vast resources required to develop and evaluate any new therapy for AD and the multiple failures involved in translational research, demonstration of the pathophysiological relevance of research findings in multiple disease-relevant models is necessary before initiating costly drug development programs. We evaluated the physiological consequences of loss of Gpr3 in four AD transgenic mouse models, including two that contain the humanized murine Aβ sequence and express similar amyloid precursor protein (APP) levels as wild-type mice, thereby reducing potential artificial phenotypes. Our findings reveal that genetic deletion of Gpr3 reduced amyloid pathology in all of the AD mouse models and alleviated cognitive deficits in APP/PS1 mice. Additional three-dimensional visualization and analysis of the amyloid plaque burden provided accurate information on the amyloid load, distribution, and volume in the structurally intact adult mouse brain. Analysis of 10 different regions in healthy human postmortem brain tissue indicated that GPR3 expression was stable during aging. However, two cohorts of human AD postmortem brain tissue samples showed a correlation between elevated GPR3 and AD progression. Collectively, these studies provide evidence that GPR3 mediates the amyloidogenic proteolysis of APP in four AD transgenic mouse models as well as the physiological processing of APP in wild-type mice, suggesting that GPR3 may be a potential therapeutic target for AD drug development.

Deficits in several neurotransmitter systems are characteristic features of the brains of AD patients. The majority of neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs) or 7-transmembrane receptors (7TMRs). It has recently been appreciated that a small family of multifunctional GPCR regulatory known as the β-arrestins which play an almost universal role in facilitating traditional GPCR desensitization, are also capable of initiating distinct signals in their own right, conveying receptor subtype-specific signaling events. These signals are often both spatially and temporally distinct, and result in unique cellular and physiological or pathophysiological consequences. As mediators of GPCR desensitization, trafficking and cell signaling, the β-arrestins provide a putative basis to understand GPCR dysfunction in AD. Here, we report that β-arrestin 2 levels are elevated in two independent cohorts of patients with AD. Genetic deletion of Arrb2 (β-arrestin 2) reduces accumulation of the amyloid-β (Aβ) peptide in an AD mouse model. Consistent with these observations, endogenous murine Aβ generation is also reduced in Arrb2-/- mice. Elucidation of the mechanism of the β-arrestin 2-mediated effect on Aβ levels indicates that recruitment of β-arrestin 2 to two GPCRs implicated in the pathogenesis of AD, GPR3 and the β2-adrenergic receptor (β2-AR), is required for the promotion of Aβ release. Collectively, these studies identify β-arrestin 2 as a novel avenue for targeting amyloid pathology and GPCR dysfunction in AD.