Katleen Peymen

The FMRFamide-Like Peptide Family in Nematodes

In the three decades since the FMRFamide peptide was isolated from the mollusk Macrocallista nimbosa, structurally similar peptides sharing a C-terminal RFamide motif have been identified across the animal kingdom. FMRFamide-like peptides (FLPs) represent the largest known family of neuropeptides in invertebrates. In the phylum Nematoda, at least 32 flp-genes are classified, making the FLP system of nematodes unusually complex. The diversity of the nematode FLP complement is most extensively mapped in Caenorhabditis elegans, where over 70 FLPs have been predicted. FLPs have shown to be expressed in the majority of the 302 C. elegans neurons including interneurons, sensory neurons, and motor neurons. The vast expression of FLPs is reflected in the broad functional repertoire of nematode FLP signaling, including neuroendocrine and neuromodulatory effects on locomotory activity, reproduction, feeding, and behavior. In contrast to the many identified nematode FLPs, only few peptides have been assigned a receptor and there is the need to clarify the pathway components and working mechanisms of the FLP signaling network. Here, we review the diversity, distribution, and functions of FLPs in nematodes.

Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans.

Significance The hypothalamic neuropeptide TRH (thyrotropin-releasing hormone) is one of the major endocrine factors that regulate vertebrate physiology. For decades the general assumption has been that TRH neuropeptides are not present in protostomes, at least not in ecdysozoans, despite the presence of TRH receptor orthologs in these phyla. Here we identify a TRH-related neuropeptide–receptor pathway in the nematode Caenorhabditis elegans. TRH-like neuropeptides activate the C. elegans TRH receptor ortholog in cell-culture cells. Using RNAi and CRISPR/Cas9 reverse genetics, we discovered that TRH-related signaling in the pharyngeal system promotes C. elegans growth. Our study provides evidence of a functional TRH neuropeptide–receptor pathway in invertebrates, suggesting that TRH signaling had evolved in a bilaterian ancestor more than 700 million years ago. In vertebrates thyrotropin-releasing hormone (TRH) is a highly conserved neuropeptide that exerts the hormonal control of thyroid-stimulating hormone (TSH) levels as well as neuromodulatory functions. However, a functional equivalent in protostomian animals remains unknown, although TRH receptors are conserved in proto- and deuterostomians. Here we identify a TRH-like neuropeptide precursor in Caenorhabditis elegans that belongs to a bilaterian family of TRH precursors. Using CRISPR/Cas9 and RNAi reverse genetics, we show that TRH-like neuropeptides, through the activation of their receptor TRHR-1, promote growth in C. elegans. TRH-like peptides from pharyngeal motor neurons are required for normal body size, and knockdown of their receptor in pharyngeal muscle cells reduces growth. Mutants deficient for TRH signaling have no defects in pharyngeal pumping or isthmus peristalsis rates, but their growth defect depends on the bacterial diet. In addition to the decrease in growth, trh-1 mutants have a reduced number of offspring. Our study suggests that TRH is an evolutionarily ancient neuropeptide, having its origin before the divergence of protostomes and deuterostomes, and may ancestrally have been involved in the control of postembryonic growth and reproduction.

Molecular characterization of a short neuropeptide F signaling system in the tsetse fly, Glossina morsitans morsitans

Neuropeptides of the short neuropeptide F (sNPF) family are widespread among arthropods and found in every sequenced insect genome so far. Functional studies have mainly focused on the regulatory role of sNPF in feeding behavior, although this neuropeptide family has pleiotropic effects including in the control of locomotion, osmotic homeostasis, sleep, learning and memory. Here, we set out to characterize and determine possible roles of sNPF signaling in the haematophagous tsetse fly Glossina morsitans morsitans, a vector of African Trypanosoma parasites causing human and animal African trypanosomiasis. We cloned the G. m. morsitans cDNA sequences of an sNPF-like receptor (Glomo-sNPFR) and precursor protein encoding four Glomo-sNPF neuropeptides. All four Glomo-sNPF peptides concentration-dependently activated Glomo-sNPFR in a cell-based calcium mobilization assay, with EC50 values in the nanomolar range. Gene expression profiles in adult female tsetse flies indicate that the Glomo-sNPF system is mainly restricted to the nervous system. Glomo-snpfr transcripts were also detected in the hindgut of adult females. In contrast to the Drosophila sNPF system, tsetse larvae lack expression of Glomo-snpf and Glomo-snpfr genes. While Glomo-snpf transcript levels are upregulated in pupae, the onset of Glomo-snpfr expression is delayed to adulthood. Expression profiles in adult tissues are similar to those in other insects suggesting that the tsetse sNPF system may have similar functions such as a regulatory role in feeding behavior, together with a possible involvement of sNPFR signaling in osmotic homeostasis. Our molecular data will enable further investigations into the functions of sNPF signaling in tsetse flies.

Characterization of G protein-coupled receptors by a fluorescence-based calcium mobilization assay.

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16 construct is co-transfected. Following ligand binding, activation of the Gα16 subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.

Characterization of a neuropeptide F receptor in the tsetse fly, Glossina morsitans morsitans

Neuropeptides related to mammalian neuropeptide Y (NPY) and insect neuropeptide F (NPF) are conserved throughout Metazoa and intimately involved in a wide range of biological processes. In insects NPF is involved in regulating feeding, learning, stress and reproductive behavior. Here we identified and characterized an NPF receptor of the tsetse fly, Glossina morsitans morsitans, the sole transmitter of Trypanosoma parasites causing sleeping sickness. We isolated cDNA sequences encoding tsetse NPF (Glomo-NPF) and its receptor (Glomo-NPFR), and examined their spatial and temporal expression patterns using quantitative PCR. In tsetse flies, npfr transcripts are expressed throughout development and most abundantly in the central nervous system, whereas low expression is found in the flight muscles and posterior midgut. Expression of npf, by contrast, shows low transcript levels during development but is strongly expressed in the posterior midgut and brain of adult flies. Expression of Glomo-npf and its receptor in the brain and digestive system suggests that NPF may have conserved neuromodulatory or hormonal functions in tsetse flies, such as in the regulation of feeding behavior. Cell-based activity studies of the Glomo-NPFR showed that Glomo-NPF activates the receptor up to nanomolar concentrations. The molecular data of Glomo-NPF and Glomo-NPFR paves the way for further investigation of its functions in tsetse flies.

Corrigendum: the FMRFamide-like peptide family in nematodes

[This corrects the article on p. 90 in vol. 5, PMID: 24982652.].