Title: Novel gonadotropin-releasing hormone, tachykinin and neuromedin U neuropeptide signaling systems in Caenorhabditis elegans
Other Titles: Identificatie van gonadotropine-releasing hormoon, tachykinine en neuromedine U neuropeptide signaalsystemen in Caenorhabditis elegans
Authors: Frooninckx, Lotte
Issue Date: 27-Apr-2015
Abstract: To survive organisms need to be capable to sense environmental cues and respond properly in order to maintain internal homeostasis. The nervous and endocrine systems ensure that the coordination and integration of internal and external stimuli works properly. Neuropeptides are small signaling molecules that play an important role as neurotransmitters, neurohormones or neuromodulators in these processes. Neuropeptides generally signal through G protein-coupled receptors (GPCRs). The identification of their corresponding GPCRs is an important step in elucidating the function of neuropeptides. In this study, we aim to investigate neuropeptidergic signaling in the roundworm Caenorhabditis elegans. This transparent model organism is amenable to genetic manipulation and has a relatively simple and defined nervous system, allowing us to investigate how neuropeptides guide behavior and physiological processes by analyzing the neural circuits they act on.
In the first place we performed an in silico prediction of the repertoire of neuropeptide GPCRs encoded in the genome of C. elegans using the MEME/MAST tool. Here,nbsp;common motif sequences of previously deorphanized neuropeptide GPCRs were used to scan the entire C. elegans genome for the presence of these motifs. Doing so, 129 hypothetical C. elegans neuropeptide GPCRs were manually annotated. From this list three groups of orphan receptors related to gonadotropin-releasing hormone/adipokinetic hormone (GnRH/AKH), tachykinin or neuromedin U/pyrokinin (NMU/PK) signaling systems were chosen for ligand identificationnbsp;a combined in silico and library-based reverse pharmacology approach. A total ofnbsp;receptors (GNRR-3, TKR-1, TKR-2 and NMUR-1)nbsp;coupled to their activating ligands in an in vitro assay. For the functional analysis we focused on the GnRH/AKH and tachykinin signaling systems.
NLP-22, one of the neuropeptides that activated the GnRH/AKH-like receptor GNRR-3 in our in vitro assay, was recently shown to induce sleep-like behavior in C. elegans. Like sleep in vertebrates, this behavior is actively regulated by neural circuits and engages multiple neurochemical systems, including neuropeptides. Genetic overexpression of gnrr-3 indicates that this receptor also modulates sleep-like behavior. However, in contrast to NLP-22, this receptor seems to inhibit sleep. Besides NLP-22, GNRR-3nbsp;also activated by NLP-2 peptides in our in vitro assay. Like gnrr-3, overexpression of nlp-2 inhibits sleep. This effect isnbsp;when nlp-2 is overexpressed in gnrr-3 deletion mutants,nbsp;that NLP-2 peptides interact with GNRR-3 in vivo as well. Sleep is generally characterized by a decline in locomotion and feeding, and reduced responsiveness to arousing stimuli. NLP-2/GNRR-3 signaling seems to specifically inhibit the reduction of locomotion during sleep, whereas thenbsp;sleep characteristics are unperturbed. nlp-2 transcript levels are cyclic and in phase with the transcription of lin-42, which encodes the PERIOD homolog that regulates the timing of sleep-like behavior in C. elegans. In vivo localization of nlp-2 shows thatnbsp;neuropeptide is expressed in the sensory AWA neurons, suggesting thatnbsp;could inhibit sleep in response tonbsp;cues which are sensed by AWA.
Tachykinins are a multifunctional family of neuropeptides that occur in both vertebrates and invertebrates. In this study we demonstrate that tachykinins are conserved in nematodes as well. The genome of C. elegans encodes two tachykinin receptors, TKR-1 and TKR-2. Bothnbsp;are activated in vitro bynbsp;in silico predicted C. elegans tachykinin neuropeptides. In vivo localization of the tachykinin receptors and the tachykinin peptides shows that they are expressed in the nervous system. Based on the identified expression pattern, their role in navigation, chemotaxis, nociception and adaptation were investigated. However, we did not observe an effect of impaired tachykinin signaling on the performed assays.
In summary, four novel C. elegans neuropeptidenbsp;systems were identified. Our results indicate that the NLP-2/GNRR-3 system modulates sleep behavior and the neuronal expression pattern of the tachykinin system suggests a modulatory role in nociception. The powerful genetic tools and high-throughput phenotyping assays endow this model organism with unique advantages to investigate the cellular and molecular function of the identified signaling systems.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Animal Physiology and Neurobiology Section - miscellaneous

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