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Understanding the endocannabinoid system in C. elegans may offer insights into basic biological processes and potential therapeutic targets for managing pain and inflammation in human. It is well established that anandamide modulates pain perception by binding to cannabinoid and vanilloid receptors, regulating neurotransmitter release and neuronal activity. One objective of this study was to demonstrate the suitability of C. elegans as a model organism for assessing the antinociceptive properties of bioactive compounds and learning about the role of endocannabinoid system in C. elegans. The evaluation of the compound anandamide (AEA) revealed antinociceptive activity by impeding C. elegans nocifensive response to noxious heat. Proteomic and bioinformatic investigations uncovered several pathways activated by AEA. Enrichment analysis unveiled significant involvement of ion homeostasis pathways, which are crucial for maintaining neuronal function and synaptic transmission, suggesting AEA's impact on neurotransmitter release and synaptic plasticity. Additionally, pathways related to translation, protein synthesis, and mTORC1 signaling were enriched, highlighting potential mechanisms underlying AEA's antinociceptive effects. Thermal proteome profiling identified NPR-32 and NPR-19 as primary targets of AEA, along with OCR-2, Cathepsin B, Progranulin, Transthyretin, and ribosomal proteins. These findings suggest a complex interplay between AEA and various cellular processes implicated in nociceptive pathways and inflammation modulation. Further investigation into these interactions could provide valuable insights into the therapeutic potential of AEA and its targets for the management of pain-related conditions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cannabis has gained popularity in recent years as a substitute treatment for pain following the risks of typical treatments uncovered by the opioid crisis. The active ingredients frequently associated with pain-relieving effects are the phytocannabinoids Δ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD), but their effectiveness and mechanisms of action are still under research. In this study, we used Caenorhabditis elegans, an ideal model organism for the study of nociception that expresses mammal ortholog cannabinoid (NPR-19 and NPR-32) and vanilloid (OSM-9 and OCR-2) receptors. Here, we evaluated the antinociceptive activity of THC and CBD, identifying receptor targets and several metabolic pathways activated following exposure to these molecules. The thermal avoidance index was used to phenotype each tested C. elegans experimental group. The data revealed for the first time that THC and CBD decreases the nocifensive response of C. elegans to noxious heat (32-35 °C). The effect was reversed 6 h post- CBD exposure but not for THC. Further investigations using specific mutants revealed CBD and THC are targeting different systems, namely the vanilloid and cannabinoid systems, respectively. Proteomic analysis revealed differences following Reactome pathways and gene ontology biological process database enrichment analyses between CBD or THC-treated nematodes and provided insights into potential targets for future drug development., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Vanilloids, including capsaicin and eugenol, are ligands of transient receptor potential channel vanilloid subfamily member 1 (TRPV1). Prolonged treatment with vanilloids triggered the desensitization of TRPV1, leading to analgesic or antinociceptive effects. Caenorhabditis elegans (C. elegans) is a model organism expressing vanilloid receptor orthologs (e.g., OSM-9 and OCR-2) that are associated with behavioral and physiological processes, including sensory transduction. We have shown that capsaicin and eugenol hamper the nocifensive response to noxious heat in C. elegans. The objective of this study was to perform proteomics to identify proteins and pathways responsible for the induced phenotype and to identify capsaicin and eugenol targets using a thermal proteome profiling (TPP) strategy. The results indicated hierarchical differences following Reactome Pathway enrichment analyses between capsaicin- and eugenol-treated nematodes. However, both treated groups were associated mainly with signal transduction pathways, energy generation, biosynthesis and structural processes. Wnt signaling, a specific signal transduction pathway, is involved following treatment with both molecules. Wnt signaling pathway is noticeably associated with pain. The TPP results show that capsaicin and eugenol target OCR-2 but not OSM-9. Further protein-protein interaction (PPI) analyses showed other targets associated with enzymatic catalysis and calcium ion binding activity. The resulting data help to better understand the broad-spectrum pharmacological activity of vanilloids., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Myocardial infraction (MI) is the principal risk factor for the onset of heart failure (HF). Investigations regarding the physiopathology of MI progression to HF have revealed the concerted engagement of other tissues, such as the autonomic nervous system and the medulla oblongata (MO), giving rise to systemic effects, important in the regulation of heart function. Cardiac sympathetic afferent denervation following application of resiniferatoxin (RTX) attenuates cardiac remodelling and restores cardiac function following MI. While the physiological responses are well documented in numerous species, the underlying molecular responses during the initiation and progression from MI to HF remains unclear. We obtained multi-tissue time course proteomics with a murine model of HF induced by MI in conjunction with RTX application. We isolated tissue sections from the left ventricle (LV), MO, cervical spinal cord and cervical vagal nerves at four time points over a 12-week study. Bioinformatic analyses consistently revealed a high statistical enrichment for metabolic pathways in all tissues and treatments, implicating a central role of mitochondria in the tissue-cellular response to both MI and RTX. In fact, the additional functional pathways found to be enriched in these tissues, involving the cytoskeleton, vesicles and signal transduction, could be downstream of responses initiated by mitochondria due to changes in neuronal pulse frequency after a shock such as MI or the modification of such frequency communication from the heart to the brain after RTX application. Development of future experiments, based on our proteomic results, should enable the dissection of more precise mechanisms whereby metabolic changes in neuronal and cardiac tissues can effectively ameliorate the negative physiological effects of MI via RTX application., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Resiniferatoxin (RTX) is a metabolite extracted from Euphorbia resinifera. RTX is a potent capsaicin analog with specific biological activities resulting from its agonist activity with the transient receptor potential channel vanilloid subfamily member 1 (TRPV1). RTX has been examined as a pain reliever, and more recently, investigated for its ability to desensitize cardiac sensory fibers expressing TRPV1 to improve chronic heart failure (CHF) outcomes using validated animal models. Caenorhabditis elegans (C. elegans) expresses orthologs of vanilloid receptors activated by capsaicin, producing antinociceptive effects. Thus, we used C. elegans to characterize the antinociceptive properties and performed proteomic profiling to uncover specific signaling networks. After exposure to RTX, wild-type (N2) and mutant C. elegans were placed on petri dishes divided into quadrants for heat stimulation. The thermal avoidance index was used to phenotype each tested C. elegans experimental group. The data revealed for the first time that RTX can hamper the nocifensive response of C. elegans to noxious heat (32 - 35 °C). The effect was reversed 6 h after RTX exposure. Additionally, we identified the RTX target, the C. elegans transient receptor potential channel OCR-3. The proteomics and pathway enrichment analysis results suggest that Wnt signaling is triggered by the agonistic effects of RTX on C. elegans vanilloid receptors., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Eugenol, a known vanilloid, was frequently used in dentistry as a local analgesic in addition, antibacterial and neuroprotective effects were also reported. Eugenol, capsaicin and many vanilloids are interacting with the transient receptor potential vanilloid 1 (TRPV1) in mammals and the TRPV1 is activated by noxious heat. The pharmacological manipulation of the TRPV1 has been shown to have therapeutic value. Caenorhabditis elegans (C. elegans) express TRPV orthologs (e.g. OCR-2, OSM-9) and it is a commonly used animal model system to study nociception as it displays a well-defined and reproducible nocifensive behavior. After exposure to vanilloid solutions, C. elegans wild type (N2) and mutants were placed on petri dishes divided in quadrants for heat stimulation. Thermal avoidance index was used to phenotype each tested C. elegans experimental groups. The results showed that eugenol, vanillin and zingerone can hamper nocifensive response of C. elegans to noxious heat (32-35 °C) following a sustained exposition. Also, the effect was reversed 6 h post exposition. Furthermore, eugenol and vanillin did not target specifically the OCR-2 or OSM-9 but zingerone did specifically target the OCR-2 similarly to capsaicin. Further structural and physicochemical analyses were performed. Key parameters for quantitative structure-property relationships (QSPR), quantitative structure-activity relationships (QSAR) and frontier orbital analyses suggest similarities and dissimilarities amongst the tested vanilloids and capsaicin in accordance with the relative anti-nociceptive effects observed.

Capsaicin is the most abundant pungent molecule identified in red chili peppers, and it is widely used for food flavoring, in pepper spray for self-defense devices and recently in ointments for the relief of neuropathic pain. Capsaicin and several other related vanilloid compounds are secondary plant metabolites. Capsaicin is a selective agonist of the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). After exposition to vanilloid solution, Caenorhabditis elegans wild type (N2) and mutants were placed on petri dishes divided in quadrants for heat stimulation. Thermal avoidance index was used to phenotype each tested C. elegans experimental groups. The data revealed for the first-time that capsaicin can impede nocifensive response of C. elegans to noxious heat (32-35 °C) following a sustained exposition. The effect was reversed 6 h post capsaicin exposition. Additionally, we identified the capsaicin target, the C. elegans transient receptor potential channel OCR-2 and not OSM-9. Further experiments also undoubtedly revealed anti-nociceptive effect for capsaicin analogues, including olvanil, gingerol, shogaol and curcumin.

In mammals, glutamate is an important excitatory neurotransmitter. Glutamate and glutamate receptors are found in areas specifically involved in pain sensation, transmission and transduction such as peripheral nervous system, spinal cord and brain. In C. elegans, several studies have suggested glutamate pathways are associated with withdrawal responses to mechanical stimuli and to chemical repellents. However, few evidences demonstrate that glutamate pathways are important to mediate nocifensive response to noxious heat. The thermal avoidance behavior of C. elegans was studied and results illustrated that mutants of glutamate receptors (glr-1, glr-2, nmr-1, nmr-2) behaviors was not affected. However, results revealed that all strains of eat-4 mutants, C. elegans vesicular glutamate transporters, displayed defective thermal avoidance behaviors. Due to the interplay between the glutamate and the FLP-18/FLP-21/NPR-1 pathways, we analyzed the effectors FLP-18 and FLP-21 at the protein level, we did not observe biologically significant differences compared to N2 (WT) strain (fold-change < 2) except for the IK602 strain. The data presented in this manuscript reveals that glutamate signaling pathways are essential to elicit a nocifensive response to noxious heat in C. elegans.

Neuropeptides are derived from large and inactive proteins which require endoproteolytic processing for the biosynthesis of the bioactive peptides. The maturation of pro-neuropeptide to neuropeptide is believed to be performed by ortholog pro-protein convertase EGL-3 in Caenorhabditis elegans (C. elegans). Furthermore, ortholog of Cathepsin L, CPL-1 are found in C. elegans and can potentially cleave paired basic amino acids at the N-terminal suggesting the presence of both pathways. The objective of this study was to decipher the role of EGL-3 in the proteolysis of FMRF amide-related peptides (FLPs) or neuropeptide-like proteins (NLPs) using synthetic surrogate peptides based on a universal enzymatic cleavage pattern published by Schechter and Berger and used widely in enzymology. The results show evidence that proteolysis controls FLP-21 and NLP-8 related neuropeptide levels in C. elegans. Surrogate peptides were degraded rapidly when exposed to C. elegans S9 fractions leading to the formation of specific peptide fragments related to EGL-3 and CPL-1 pathway. The results suggest that CPL-1 pathway does not compensate for the loss of the EGL-3 pathway. Proteolysis of pro-neuropeptides associated to FLP-21 and NLP-8 in elg-3 mutants are severely hampered leading to a lack of mature bioactive neuropeptides.

Substance P (SP) play a central role in nociceptive transmission and it is an agonist of the Neurokinin-1 receptor located in the lamina I of the spinal cord. SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons. Proprotein convertases (PCs) are extensively expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous protachykinins has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis surrogate protachykinins (i.e. Tachykinin 20-68 and Tachykinin 58-78) using cellular fractions of spinal cords from wild type (WT), PC1(-/+) and PC2(-/+) animals and mass spectrometry. Full-length Tachykinin 20-68 and Tachykinin 58-78 was incubated for 30 min in WT, PC1(-/+) and PC2(-/+) mouse spinal cord S9 fractions and specific C-terminal peptide fragments were identified and quantified by mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and Tachykinin 58-71, an important SP precursor, with over 50 % reduction of the rate of formation in mutant PC1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein.

Vanilloid receptors have a central role in the processing of nociceptive stimuli. TRPV1 null mice showed significant decrease in response to heat noxious stimuli. However, thermal sensitivity is still present suggesting that the TRPV1 is not an exclusive transducer of thermal stimuli. Additionally, tachykinin peptides play a central role in pain processing and expression levels may also contribute in modifying the pain threshold. The LC-MS/MS analysis revealed that SP and NKA were significantly down-regulated in TRPV1(-/-) in spinal cord and brain tissues. In spinal cord, SP concentrations were 23.4 % lower (p < 0.0049) and NKA concentrations were 22.0 % lower (p < 0.0022) in TRPV1 null mice. Additionally, brain SP concentrations were 26.9 % lower (p < 0.0260) and brain NKA concentrations were 31.9 % lower (p < 0.0063) in TRPV1 null mice. These results clearly demonstrate that TPRV1 null mice exhibit lower SP and NKA concentrations in the central nervous system. The deficit of thermal responses may also be related to the down-regulations of SP and NKA.