EGL-3 and EGL-21 are required to trigger nocifensive response of Caenorhabditis elegans to noxious heat
Introduction
Caenorhabditis elegans (C. elegans) is a remarkable animal model system for functional genomics pertinent to mammalian and human biology and diseases (Komuniecki et al., 2012; Avila et al., 2012). C. elegans genome sequencing was finalized in 1998 and it is now comprehensively annotated and publicly available (The C. elegans Sequencing Consortium, 1998; the C. elegans genome sequencing project, 1999). This is an important benefit for proteomic investigations particularly in the context of comprehensive biochemical and signaling network studies (Tullet, 2014; Boucher and Jenna, 2013). Interestingly, adult C. elegans consists of 959 cells including 302 neurons, which make this model attractive to study neuronal communication at the physiological and molecular levels (Wittenburg and Baumeister, 1999). C. elegans is particularly useful for the study of nociception as it exhibits a well-defined and reproducible nocifensive behavior, involving a reversal and change in direction away from the noxious stimulus (Wittenburg and Baumeister, 1999; Carr and Zachariou, 2014). Following the genome sequencing of C. elegans, it was determined that specific genes encode transient receptor potential (TRP) ion channel proteins with significant sequence similarities to mammalian TRP channels including the transient receptor potential cation channel subfamily V member 1 (TRPV1) (Kahn-Kirby and Bargmann, 2006). Specifically, five TRP subfamilies including TRPV analogs (e.g. OSM-9 and OCR-1-4) were characterized (Xiao and Xu, 2011). Furthermore, it has been recently established that C. elegans TRP channels are associated with behavioral and physiological processes, including sensory transduction of thermal and chemical information (Glauser et al., 2011; Venkatachalam et al., 2014). Many C. elegans TRP channels share similar activation and regulatory mechanisms with their mammal counterparts. Interestingly, it was revealed that the thermal avoidance response of C. elegans is amplified when animals are exposed to capsaicin a well-known agonist of the TRPV1 (Wittenburg and Baumeister, 1999; Tobin et al., 2002). Furthermore, the TRPV1 can be activated by other physical and chemical stimuli including noxious heat, low pH, divalent cations and animal toxins (Yang and Zheng, 2017). The activation of the TRPV1 triggers the release of several neuropeptides, including substance P and calcitonin gene-related peptide central to synaptic and nociceptive transmission in mammals (Jara-Oseguera et al., 2008; Gazzieri et al., 2007).
Neuronal molecular communication in C. elegans was recently investigated and exploratory immunochemical analyses exposed the presence of numerous neuropeptides in C. elegans (Mills et al., 2012). Additionally, a comprehensive analysis of the genome sequence revealed several pro-neuropeptide genes, encoding a series of bioactive neuropeptides and specific neuropeptide receptors playing a central role in synaptic transmission (Li, 2005; Hu et al., 2011; Choi et al., 2013). Neuropeptides are involved in the modulation of essentially all behaviors including locomotion, mechanosensation, thermosensation and chemosensation (Komuniecki et al., 2012; Biron et al., 2008). Neuropeptides act as neuromodulators and as fast neurotransmitters. The broad existence of these neuropeptides in nematodes suggests a fundamental role of neuropeptidergic signaling in C. elegans but the molecular pathways and networks are poorly understood. Moreover, C. elegans uses classical neurotransmitter systems like acetylcholine (Ach), glutamate, γ-amunobutyric acid (GABA), serotonin, dopamine and octopamine found in mammals (Barclay et al., 2012). Several neuropeptide receptors were identified and were shown to play an important role in signal transduction. It has been recently demonstrated that neuropeptide receptor 1 (NPR-1) play a central role in locomotion and thermal avoidance. NPR-1 and its ligands FLP-18 and FLP-21 play an important role in the regulation of locomotion (Choi et al., 2013). Additionally, data suggested that TRPV channels (i.e. OSM-9 and OCR-2) and the FLP-21/ NPR-1 neuropeptide signaling pathway are essential for the nocifensive response associated to heat avoidance in C. elegans (Glauser et al., 2011). Therefore, the key proteases (e.g. ELG-3 and EGL-21) involved in the processing of pro-neuropeptides into neuropeptides can potentially regulate nociceptive behavior in C. elegans.
In mammals, several neuropeptides are synthesized by the action of pro-protein convertases (PCs) and endopeptidases during the axonal transport (Hook et al., 2008; Saidi et al., 2016; Ruiz and Beaudry, 2016; Saidi and Beaudry, 2017; Ben Salem et al., 2018). Recently, it was revealed that C.elegans egl-3 gene encodes a protein (i.e. EGL-3) with 57% sequence homology compared to mammalian pro-protein convertase type 2 (PC2) (Kass et al., 2001). EGL-3 is a serine endoprotease which cleaves pro-proteins at paired basic amino acids as shown in Fig. 1 and is an ortholog of the human PC2. EGL-3 is an essential enzyme involved in the maturation of pro-neuropeptides to active neuropeptides in C. elegans (Hook, 2008; Li and Kim, 2008). Analogously to human, C. elegans egl-21 gene encodes a protein (i.e. EGL-21) that is an ortholog of the human carboxypeptidase E and this enzyme is broadly expressed in several neurons. EGL-21 is essential for removing basic residues from the C-terminal following EGL-3 pro-neuropeptide processing (Jacob and Kaplan, 2003). Thus, we believe that egl-3 and egl-21 mutant animals will lack mature neuropeptides and consequently synaptic chemical communication will be impaired. Changes in nocifensive behavior are orchestrated by specific altered activity during synaptic communication in C. elegans.
The objectives of the study were to characterize nocifensive responses of wild type (N2 strain), egl-3 and elg-21 mutants as well as specific neuropeptide mutant nematodes (flp-21 and flp-18) and neuropeptide receptor npr-1 following exposition to noxious heat (i.e. 33–35 °C). Chemotaxis results are shown in supplementary figures. Relative quantification of FLP-21 and FLP-18 related neuropeptides were performed using high performance liquid chromatography coupled to a hybrid Orbitrap high-resolution mass spectrometer.
Section snippets
Chemicals and reagents
All chemicals and reagents were obtained from Fisher Scientific (Fair Lawn, NJ, USA) or MilliporeSigma (St-Louis, MO, USA). For mass spectrometry analysis, formic acid, water (HPLC-MS Optima grade), acetonitrile (HPLC-MS Optima grade), trifluoroacetic acid (TFA), were purchased from Fisher Scientific.
C. elegans strains
The N2 (Bristol) isolate of C. elegans was used as a reference strain. Mutant strains used in this work included: egl-21 (KP2018); egl-21 (MT1206); egl-3 (MT1541); egl-3 (VC461); flp-18 (AX1410);
C. elegans thermal avoidance behavior
Thermal avoidance assays are widely used as a model to study how sensory information is integrated to alter nematode behavior. Noxious temperatures (>30 °C) provoke a temperature avoidance response in C. elegans that can be quantified using a standard thermal avoidance assay. Comprehensive studies have suggested that AFD neurons are the main thermosensors in C. elegans (Mori and Ohshima, 1995; Koutarou et al., 2004). Also, FLP neurons located in the head and PHC neurons in the tail act as
Conclusion
The thermal avoidance behavior of egl-3 and egl-21 mutant C. elegans was significantly hampered compared to WT(N2) C. elegans. Moreover, flp-18, flp-21 and npr-1 mutant C. elegans displayed a similar phenotype. EGL-3 pro-protein convertase and EGL-21 carboxypeptidase E are essential enzymes for the maturation of pro-neuropeptides to active neuropeptides in C. elegans. The ability to avoid noxious heat is strongly associated with the neuropeptide receptor gene npr-1. FLP-18/FLP-21/NPR-1
Acknowledgements
This project was funded by the National Sciences and Engineering Research Council of Canada (F. Beaudry discovery grant no. RGPIN-2015-05071). The mass spectrometry analyses were performed using an infrastructure and operation funded by the Canadian Foundation for Innovation (CFI) and the Fonds de Recherche du Québec (FRQ), Government of Quebec (F. Beaudry CFI John R. Evans Leaders grant no. 36706). A PhD scholarship was awarded to J. Ben Salem with a grant obtained from Fondation de France (
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