azyx-1 is a new gene that overlaps with zyxin and affects its translation in C. elegans, impacting muscular integrity and locomotion.

Overlapping genes are widely prevalent; however, their expression and consequences are poorly understood. Here, we describe and functionally characterize a novel zyx-1 overlapping gene, azyx-1, with distinct regulatory functions in Caenorhabditis elegans. We observed conservation of alternative open reading frames (ORFs) overlapping the 5' region of zyxin family members in several animal species, and find shared sites of azyx-1 and zyxin proteoform expression in C. elegans. In line with a standard ribosome scanning model, our results support cis regulation of zyx-1 long isoform(s) by upstream initiating azyx-1a. Moreover, we report on a rare observation of trans regulation of zyx-1 by azyx-1, with evidence of increased ZYX-1 upon azyx-1 overexpression. Our results suggest a dual role for azyx-1 in influencing zyx-1 proteoform heterogeneity and highlight its impact on C. elegans muscular integrity and locomotion.

Mass Spectrometry-Driven Discovery of Neuropeptides Mediating Nictation Behavior of Nematodes.

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.

Signaling via the FLP-14/FRPR-19 neuropeptide pathway sustains nociceptive response to repeated noxious stimuli in C. elegans.

In order to thrive in constantly changing environments, animals must adaptively respond to threatening events. Noxious stimuli are not only processed according to their absolute intensity, but also to their context. Adaptation processes can cause animals to habituate at different rates and degrees in response to permanent or repeated stimuli. Here, we used a forward genetic approach in Caenorhabditis elegans to identify a neuropeptidergic pathway, essential to prevent fast habituation and maintain robust withdrawal responses to repeated noxious stimuli. This pathway involves the FRPR-19A and FRPR-19B G-protein coupled receptor isoforms produced from the frpr-19 gene by alternative splicing. Loss or overexpression of each or both isoforms can impair withdrawal responses caused by the optogenetic activation of the polymodal FLP nociceptor neuron. Furthermore, we identified FLP-8 and FLP-14 as FRPR-19 ligands in vitro. flp-14, but not flp-8, was essential to promote withdrawal response and is part of the same genetic pathway as frpr-19 in vivo. Expression and cell-specific rescue analyses suggest that FRPR-19 acts both in the FLP nociceptive neurons and downstream interneurons, whereas FLP-14 acts from interneurons. Importantly, genetic impairment of the FLP-14/FRPR-19 pathway accelerated the habituation to repeated FLP-specific optogenetic activation, as well as to repeated noxious heat and harsh touch stimuli. Collectively, our data suggest that well-adjusted neuromodulation via the FLP-14/FRPR-19 pathway contributes to promote nociceptive signals in C. elegans and counteracts habituation processes that otherwise tend to rapidly reduce aversive responses to repeated noxious stimuli.

NPY/NPF-Related Neuropeptide FLP-34 Signals from Serotonergic Neurons to Modulate Aversive Olfactory Learning in Caenorhabditis elegans.

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.

Antagonism between neuropeptides and monoamines in a distributed circuit for pathogen avoidance.

Pathogenic infection elicits behaviors that promote recovery and survival of the host. After exposure to the pathogenic bacterium Pseudomonas aeruginosa PA14, the nematode Caenorhabditis elegans modifies its sensory preferences to avoid the pathogen. Here, we identify antagonistic neuromodulators that shape this acquired avoidance behavior. Using an unbiased cell-directed neuropeptide screen, we show that AVK neurons upregulate and release RF/RYamide FLP-1 neuropeptides during infection to drive pathogen avoidance. Manipulations that increase or decrease AVK activity accelerate or delay pathogen avoidance, respectively, implicating AVK in the dynamics of avoidance behavior. FLP-1 neuropeptides drive pathogen avoidance through the G protein-coupled receptor DMSR-7, as well as other receptors. DMSR-7 in turn acts in multiple neurons, including tyraminergic/octopaminergic neurons that receive convergent avoidance signals from the cytokine DAF-7/transforming growth factor ß. Neuromodulators shape pathogen avoidance through multiple mechanisms and targets, in agreement with the distributed neuromodulatory connectome of C. elegans.

Ancestral glycoprotein hormone-receptor pathway controls growth in C. elegans.

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 in Caenorhabditis elegans. We show that orthologs of GPA2 and GPB5, together with thyrotropin-releasing hormone (TRH) related neuropeptides, constitute a neuroendocrine pathway that promotes growth in C. elegans. GPA2/GPB5 signaling is required for normal body size and acts through activation of the glycoprotein hormone receptor ortholog FSHR-1. C. elegans GPA2 and GPB5 increase cAMP signaling by FSHR-1 in 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 show an increased defecation cycle period. 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.

System-wide mapping of peptide-GPCR interactions in C. elegans.

Neuropeptides and peptide hormones 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 peptides remain elusive, as our insight into peptide-receptor interactions is limited and many peptide GPCRs are still orphan receptors. Here we report a genome-wide peptide-GPCR interaction map in Caenorhabditis 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 peptide functions and evolution. Combining our dataset with phylogenetic analysis supports peptide-receptor co-evolution and conservation of at least 14 bilaterian peptidergic systems in C. elegans. This resource lays a foundation for system-wide analysis of the peptidergic network.

Ascaris suum Informs Extrasynaptic Volume Transmission in Nematodes.

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.

Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone.

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.

Neuromedin U signaling regulates retrieval of learned salt avoidance in a C. elegans gustatory circuit.

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.

Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer's disease mouse models.

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 Aß 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.

ß-arrestin 2 regulates Aß generation and γ-secretase activity in Alzheimer's disease.

ß-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 ß-arrestin 2 expression is elevated in two independent cohorts of individuals with Alzheimer's disease. Overexpression of ß-arrestin 2 leads to an increase in amyloid-ß (Aß) peptide generation, whereas genetic silencing of Arrb2 (encoding ß-arrestin 2) reduces generation of Aß 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 Aß(40) and Aß(42). Two GPCRs implicated previously in Alzheimer's disease (GPR3 and the ß(2)-adrenergic receptor) mediate their effects on Aß generation through interaction with ß-arrestin 2. ß-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 ß-arrestin 2 as a new therapeutic target for reducing amyloid pathology and GPCR dysfunction in Alzheimer's disease.