Anandamide Modulates Thermal Avoidance in Caenorhabditis elegans through Vanilloid and Cannabinoid Receptor Interplay

doi:10.21203/rs.3.rs-4151433/v1

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Anandamide Modulates Thermal Avoidance in Caenorhabditis elegans through Vanilloid and Cannabinoid Receptor Interplay

https://doi.org/10.21203/rs.3.rs-4151433/v1

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published 06 Jun, 2024

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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.

Endocannabinoids

Anandamide

Caenorhabditis elegans

nociception

pain

proteomics

vanilloid receptor

cannabinoid receptor

The endocannabinoid system is comprised of a complex network of signaling molecules and receptors [1]. It has emerged as a central player in the regulation of diverse physiological processes, ranging from pain modulation and immune response to mood regulation and neuroprotection [1–3]. At the heart of this intricate system lies anandamide (AEA), an endocannabinoid specifically linked to the modulation of mood and emotions, pain perception, appetite regulation, and immune response [4–6]. AEA is synthesized facultatively within the body, primarily in response to physiological stressors, and acts as a retrograde neurotransmitter, modulating the release of other neurotransmitters and playing a pivotal role in synaptic plasticity [2, 7]. Among its primary targets is the transient receptor potential vanilloid 1 (TRPV1), a member of the transient receptor potential (TRP) ion channel family, which is integral to anandamide's role in pain perception and temperature regulation [8]. AEA interaction with the TRPV1 is of particular interest in the context of pain management, as it holds promise as an analgesic, potentially reducing the need for opioid-based pain relief, which is often associated with adverse side effects and addiction. In addition to the TRPV1 receptor, anandamide engages with two specific G protein-coupled receptors, the cannabinoid receptor type 1 (CB1), and the cannabinoid receptor type 2 (CB2) [2, 9]. CB1 receptors are predominantly localized in the central nervous system, where they mediate the psychoactive effects of cannabinoids. Meanwhile, CB2 receptors are primarily expressed in immune cells and the peripheral nervous system, playing a crucial role in immune response regulation. However, the intricate details related to the signaling pathways mediated by anandamide through CB1 and CB2 receptors remain unclear, which impacts our understanding of how cannabinoids modulate immune function, inflammation, and various neurodegenerative conditions. Consequently, delving into the interactions between anandamide and its primary targets, the TRPV1 and cannabinoid receptors, CB1 and CB2, is fundamental for unlocking the therapeutic potential of the endocannabinoid system.

While the endocannabinoid system is well-studied in vertebrates, its existence and function in simpler organisms such as the nematode Caenorhabditis elegans (C. elegans) have only recently come to light. C. elegans offers a unique opportunity to explore the evolutionary conservation and functional aspects of the endocannabinoid system in a genetically tractable model organism. The identification of endocannabinoid molecules (i.e. AEA and 2-Arachidonoylglycerol)[10] and the presence of orthologous receptors (i.e. NRP-19 and NPR32)[11] in C. elegans have opened new avenues for studying the endocannabinoid system in a simplified biological system. Additionally, C. elegans expresses several genes encoding TRP ion channels, including TRPV channels OSM-9 and OCR-2, which are essential to mediate heat avoidance behavior and are co-expressed in sensory neurons [12–15]. The ability to perceive and respond to noxious stimuli is a fundamental survival mechanism. While the molecular and neural underpinnings of nociception have been extensively explored in mammals, its evolutionarily conserved aspects, and the involvement of the endocannabinoid system in nociception remain a topic of investigation. In this context, C. elegans has emerged as a valuable model organism for studying nociception. C. elegans exhibits clear behavioral responses to noxious stimuli, which can be leveraged to study questions about the involvement of the endocannabinoid system in nociceptive pathways [7, 16–20]. Moreover, C. elegans-based assays, coupled with its ability to mimic key aspects of mammalian biology, make it a compelling choice for the early stages of drug discovery particularly for the development of novel analgesic strategies as we have previously demonstrated for molecules targeting the vanilloid[21–25] and cannabinoid systems [16].

Herein, we hypothesized that AEA interacts with vanilloid (e.g., OSM9, OCR-2) and cannabinoid (e.g. NPR-19, NPR-32) receptors in C. elegans, hampering the nocifensive response to noxious heat. Therefore, the objective of this study was to shed light on the effect of AEA on heat avoidance and identify relevant molecular targets and pathways in C. elegans.

2.1 Chemicals and reagents

All the chemicals and reagents were obtained from Fisher Scientific (Fair Lawn, NJ, USA) or Millipore Sigma (St. Louis, MO, USA). Capsaicin (Cap) was purchased from Toronto Research Chemicals (North York, ON, CAN) and Δ⁹-tetrahydrocannabinol (THC) was purchased from Cerilliant (Round Rock, TX, USA). Anandamide (AEA) was purchased from Cayman Chemical (Ann Arbor, MI, USA).

2.2 C. elegans strains

The N2 (Bristol) isolate of C. elegans was used as a reference strain. The mutant strains used were npr-19 (RB1668), npr-32 (RB1938), ocr-2 (CX4544), and osm-9 (CX10). N2 (Bristol) and other strains were purchased from the Caenorhabditis Genetics Center (CGC), University of Minnesota (Minneapolis, MN, USA). The strains were maintained and manipulated under standard conditions as previously described [26, 27]. The nematodes were grown and maintained on nematode growth medium (NGM) agar at 22°C in a Thermo Scientific Heratherm refrigerated incubator (Fair Lawn, NJ, USA). Analyses were performed at room temperature (∼22°C) unless otherwise noted.

2.3 C. elegans pharmacological manipulations

Cap was dissolved in Type 1 Ultrapure Water at a concentration of 25 µM. The solution was warmed for a brief period, vortexed, and sonicated for several minutes to completely dissolve the compound. THC (i.e. 1 mg/mL) and AEA (i.e. 50 mg/mL) solutions were respectively diluted in Type 1 Ultrapure Water at a concentration of 25 µM. Further dilutions of AEA 25 µM solution to concentrations of 10 µM, 5 µM and 1µM in Type 1 Ultrapure Water were performed by serial dilution. Nematodes were isolated and washed according to the protocol described by Margie et al. [27]. After 72 h of feeding and growing on 92 × 16 mm Petri dishes with NGM, the nematodes were removed from the food and exposed to Cap, THC, or AEA solutions. A 8 mL aliquot of a Cap, THC, or AEA solution was aliquoted to produce a small 2–3 mm solution film (the solution was partly absorbed by the NGM); consequently, the nematodes were swimming in solution. Nematodes were treated with Cap, THC, or AEA for 60 min, isolated, and washed thoroughly before behavioral experiments. To test for remanent (persistent) effects, after exposure to AEA, the nematodes were isolated, carefully washed, and deposited on NGM free of AEA for 6 h before behavioral experiments (i.e., 6 h latency).

2.4 Thermal avoidance assays

The behavioral analysis method for the evaluation of thermal avoidance was developed based on the previously described four-quadrant strategy [27]. We have extensively used this method in previously published studies [22–24, 28]. Briefly, experiments were performed on 92 × 16 mm Petri dishes divided into four quadrants. The nematodes tested were not fed during the experiments. A middle circle delimited (1 cm in diameter) an area where C. elegans was not considered. The Petri dishes were divided into quadrants: two stimulus areas (A and D) and two control areas (B and C). Sodium azide (0.5 M) was used to paralyze the nematodes in all quadrants. Noxious heat was created with an electronically heated metal tip (0.8 mm in diameter) producing a radial temperature gradient (32–35°C on NGM agar 2 mm from the tip measured with an infrared thermometer). The stimulus temperature was based on previous experiments [29]. Nematodes were isolated and washed according to the protocol described by Margie et al.[27]. Then, the nematodes (typically 100 to 300 young adult nematodes) were placed at the center of a marked Petri dish, and after 30 min, the plates were placed at 4°C for at least 1 h, and the number of nematodes per quadrant was counted. Nematodes that did not cross the inner circle were excluded. The thermal avoidance index (TI) was calculated using Eq. 1.

$$TI= \frac{\left[\right(A+D) - (B+C\left)\right]}{(A+B+C+D)}$$

The technical details are presented in Supplementary Figure S1. Both TI and animal avoidance percentages were used to analyze nocifensive responses to noxious heat in each tested C. elegans experimental group.

2.5 Thermal proteome profiling to identify targets

Thermal profiling for proteomics (TPP) employing a variety of ligand concentrations (TPP-CCR) enables comprehensive exploration of drug-protein interactions [30]. Specifically, it facilitates the investigation of target engagement. The underlying concept of TPP is that upon interaction with a ligand, proteins may undergo thermal stabilization, resulting in increased apparent solubility under thermal stress conditions. Nematodes were gathered in a liquid medium, centrifuged at 1,000 × g for 10 minutes, isolated, and thoroughly washed. Subsequently, the nematodes were suspended in 8 M urea/100 mM TRIS–HCl buffer (pH 8) supplemented with cOmpleteTM protease inhibitor cocktail (Roche), and aliquots were transferred into reinforced 1.5 mL homogenizer tubes containing 50 mg glass beads (500 µm). The samples were homogenized using a Bead Mill Homogenizer (Fisherbrand) with 3 bursts of 60 seconds at a speed of 5 m/s. Following homogenization, the samples were centrifuged at 12,000 × g for 10 minutes. Protein concentration in each homogenate was determined via a Bradford assay. Two hundred micrograms of protein from the lysate were subjected to a range of Anandamid concentrations (0, 10, 25, 50, and 100 µM) and subsequently heated at 60°C for 5 minutes using a heated block. Samples were then centrifuged at 12,000 × g for 10 minutes, and 100 µL aliquots of the supernatant (i.e., soluble fraction) were transferred into 1.5 mL microtubes. Reduction, alkylation, and digestion procedures were carried out as outlined in the section 2.6. Each condition was replicated three times independently (n = 3).

2.6 Proteomic analysis

Cultured nematodes (with or without AEA exposure) were collected in liquid medium and centrifuged at 1,000 ×g for 10 min, after which they were collected and washed thoroughly. Nematodes were resuspended in 8 M urea 100 mM TRIS-HCl buffer (pH 8) containing cOmplete™ protease inhibitor cocktail (Roche Diagnostic Canada, Laval, QC, Canada). The suspension was aliquoted into reinforced 1.5 mL homogenizer tubes containing 25 mg of 500 µm glass beads. The samples were homogenized using a Bead Mill Homogenizer (Fisher Scientific) with five bursts of 60 s each at a speed of 5 m/s, followed by centrifugation at 12,000 × g for 10 min. The protein concentration in each homogenate was determined using the Bradford assay. Protein (100 µg) was extracted using ice-cold acetone precipitation (1:5, v/v). The protein pellet was dissolved in 100 µL of 50 mM TRIS-HCl buffer (pH 8), and the solution was mixed with a Disruptor Genie at maximum speed (2,800 rpm) for 15 min and sonicated to improve the protein dissolution yield. The proteins were denatured by heating at 120°C for 10 min using a heated reaction block. The solution was allowed to cool for 15 min. Proteins were reduced using 20 mM dithiothreitol (DTT) at 60°C for 1h. Proteins were then alkylated with 40 mM iodoacetamide (IAA) in the dark at room temperature for 30 min. Proteomic-grade trypsin (5 µg) was added to this mixture and incubated at 37°C for 24 h. Protein digestion was quenched by adding 10 µL (∼10% v/v) of a 1% trifluoroacetic acid (TFA) solution. The samples were centrifuged at 12,000 × g for 10 min and 100 µL of the supernatant was transferred to injection vials for analysis.

A label-free precursor ion-based MS¹ quantification workflow was used. A nano-flow Thermo Scientific Vanquish Neo UHPLC system (San Jose, CA, USA) was set up for pre-concentration mode using a Thermo Scientific PepMap Neo 5 µm C18 300 µm × 5 mm Trap Cartridge in back-flush configuration. Rapid sample loading (20 µL/min flow rate) was performed on a trap cartridge. Tryptic peptides were separated using a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in an 80% acetonitrile and 20% water mixture (B) using a 120-minute gradient from 5–50% B at a flow rate of 300 nL/min. A Thermo Scientific PepMap Neo C18 2 µm × 75 µm × 500 mm nano column was connected to the system and coupled to a Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer, with a Thermo Nanospray Flex ion source. The Nanospray electrode was set to 2200 V, and the ion transfer tube temperature was set to 200°C. Mass spectrometry (MS) was performed in the positive ion mode operating in the TOP-12 data-dependent acquisition (DDA) mode. A DDA cycle entailed one MS¹ survey scan (m/z 375–1200) acquired at 70,000 resolution (FWHM) and precursor ions meeting the user-defined criteria for monoisotopic precursor intensity (dynamic acquisition of MS²-based TOP-12 most intense ions with a minimum 1×10⁴ intensity threshold) were isolated using the quadrupole (2.0 Da isolation width), activated by HCD (28 NCE) and fragment ions was detected in the ORBITRAP at a resolution of 17,500 (FWHM). Data were processed using Thermo Proteome Discoverer (version 3.0) in conjunction with SEQUEST-HT using default settings unless otherwise specified. Peptides and proteins were identified using SEQUEST-HT based on the reference proteome extracted from UniProt (C. elegans taxon identifier 6239) as FASTA sequences. Parameters were set as follows: MS¹ tolerance of 10 ppm, MS² mass tolerance of 0.02 Da for Orbitrap detection, enzyme specificity was set as trypsin with two missed cleavages allowed, cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation was set as a variable modification. The minimum peptide length was set to six amino acids. Data was further analyzed using Percolator [31]. Proteins were identified using a 1% false discovery rate (FDR) threshold. For protein quantification and comparative analysis, we used the peak integration feature in Proteome Discoverer 3.0. For each identified protein, the average ion intensity of unique peptides was used to determine the protein abundance.

2.7 Bioinformatics

The abundance ratio (log₂): (experimental group)/(N2 control), abundance ratio p-value: (experimental group)/(N2 control), and accession columns were extracted from the datasets generated using Proteome Discoverer 3.0. Adjusted p-value was determined using the Benjamini-Hochberg method. Proteins with a single high-scoring peptide hit were not excluded based on recommendations [32]. Volcano plots were generated using all identified and quantified proteins with log₂ ratios and p-values. Proteins with a p-value of > 0.05 were not used for further analysis. Additionally, only proteins with an absolute log₂ ratio ≥ 1.0 were used for bioinformatics analysis. Gene Ontology (GO) and Reactome enrichment analyses were performed using Metascape [33], in which all differentially expressed proteins (DEPs) were used (absolute log₂ ratio ≥ 1.0; p-value ≤ 0.05). Further pathway analysis was performed using ClueGO[34] and Cytoscape,[35] using the Reactome pathway databases. For TPP experiments, proteins with a p-value of > 0.01 (i.e. 1% FDR) were excluded. Protein − protein interaction (PPI) network analyses using AEA targets listed in Table 1 was performed using STRING[36] and Markov cluster (MCL) algorithm [37].

Table 1

Summary of AEA proteins target identified using thermal proteome profiling method using compound concentration range mode (TTP-CCR).
Uniprot ID	Gene	Description	Fold change (100µM AEA)	Adjusted p-value
G5EGB1	lec-2	Galectin	45.375	0.000002
P43508	cpr-4	Cathepsin B-like cysteine proteinase 4	18.799	0.000014
Q17754	Tin-9.1	Mitochondrial import inner membrane translocase subunit Tim9	10.428	0.000002
Q9U2A8	rpl-43	Large ribosomal subunit protein eL43	10.378	0.000001
Q9U362	pgrn-1	Progranulin homolog	8.983	0.000127
Q19749	dlat-1	Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial	7.994	0.000160
O17891	F55B11.2	DUF19 domain-containing protein	6.646	< 0.000001
O44145	perm-2	PERMeable eggshell	5.933	< 0.000001
O62053	ule-4	UPF0375 protein ule-4	5.871	0.000003
P52814	rpl-39	Large ribosomal subunit protein eL39	5.757	0.000413
O16309	fkb-3	peptidylprolyl isomerase	4.948	< 0.000001
Q17849	hsp-25	SHSP domain-containing protein	4.380	0.000003
H2KZV8	mlp-1	LIM zinc-binding domain-containing	4.356	0.000215
Q1XFY9	rps-24	Small ribosomal subunit protein eS24	4.216	0.000019
O44144	perm-4	PERMeable eggshell 4	4.095	0.000125
Q9XVA9	F01D5.5	ShKT domain-containing protein	4.055	< 0.000001
P49632	ubq-2	Ubiquitin-ribosomal protein eL40 fusion; might be involved	3.882	0.000004
P37165	ubl-1	Ubiquitin-like protein 1-ribosomal protein eS31 fusion protein	3.750	0.000001
Q19837	spp-18	Saposin B-type domain-containing protein	3.550	0.000003
Q86G91	flp-33	FMRF-Like Peptide	3.438	0.010361
G5EBJ8	Y59E9AR.1	Major sperm protein	3.285	0.002672
P34460	eef-1B.1	Probable elongation factor 1-beta/1-delta 1	3.199	0.000157
O44512	isp-1	Cytochrome b-c1 complex subunit Rieske, mitochondrial	3.063	0.000001
Q9XXL3	ssp-31	Sperm-specific class P protein 31	2.974	< 0.000001
Q17561	aly-1	Chromatin target of PRMT1 protein C-terminal domain-containing protein	2.926	0.000088
Q95QA6	pat-12	Protein pat-12	2.913	0.000152
O62388	W01D2.1	Ribosomal protein L37	2.595	0.000056
Q86FL8	spp-5	Saposin B-type domain-containing protein	2.501	0.002523
P91020	C07D8.6	NADP-dependent oxidoreductase domain-containing protein	2.489	0.000030
O45713	rbm-3.2	RRM domain-containing protein	2.447	0.000011
Q22288	ttr-15	Transthyretin-like protein 15	2.385	0.000017
P91914	rpl-27	Large ribosomal subunit protein eL27	2.322	0.000081
Q20206	rps-11	Small ribosomal subunit protein uS17	2.298	0.000006
P91913	rla-1	Large ribosomal subunit protein P1	2.272	0.000732
P48166	rpl-36.A	Large ribosomal subunit protein eL42	2.268	0.002545
Q21268	K07C11.7	metallophosphoesterase domain containing	2.259	0.000267
Q9TYS3	ftn-2	Ferritin;	2.220	0.000129
O45181	nucl-1	RRM domain-containing protein	2.149	0.000004
Q95XX2	clec-85	C-type lectin domain-containing protein	2.112	0.000007
P49196	rps-12	Small ribosomal subunit protein eS12	2.028	< 0.000001
O61880	F59B1.2	Secreted protein	2.013	0.000044
P55853	smo-1	Small ubiquitin-related modifier	2.000	< 0.000001
Q17594	npr-19	G-protein coupled receptors family 1 profile domain-containing protein	10.880	0.008462
O62403	npr-32	G-protein coupled receptors family 1 profile domain-containing protein	107.444	0.007707
G5EBV8	osm-9	Ion transport domain-containing protein	1.671	< 0.000001
O61220	ocr-2	Ion transport domain-containing protein	2.055	0.009336
False discovery rates for multiple comparisons were performed using the Benjamini-Hochberg method.

2.8 Statistical analysis

Behavioral data were analyzed using the non-parametric Kruskal–Wallis test, followed by the post-hoc Dunn test for multiple comparisons. Significance was set a priori at p ≤ 0.05. Statistical analyses were performed using GraphPad PRISM (version 10.2.0).

In recent studies, we demonstrated that C. elegans can serve as a valuable model organism for assessing the antinociceptive properties of bioactive compounds. Utilizing proteomics techniques, researchers have gained insights into valuable aspects such as differential expression and protein-protein interactions, facilitating the investigation of underlying biological pathways [16, 21–25]. In this study, our initial objective was to ascertain whether there existed any behavioral bias in C. elegans using our experimental setup. To test this, we conducted an evaluation of the mobility and bias of both wild-type (N2) and specific mutant nematodes, with and without exposure to AEA. The experiments were conducted at room temperature (≈ 22˚C), maintaining consistency across all quadrants. As illustrated in Fig. 1, our findings indicate the absence of any quadrant selection bias among all experimental groups of C. elegans, regardless of exposure to AEA (10 µM). The data reveal a uniform dispersion of nematodes throughout the Petri dish 30 minutes after initial placement at the center. Furthermore, exposure to AEA for sixty minutes did not induce significant alterations in nematode mobility or behavioral biases.

3.1 Evaluation of antinociceptive activity of AEA

We investigated the antinociceptive activity of AEA in this study in an attempt to elucidate the role of endocannabinoids in the nociceptive behavior of C. elegans. In Fig. 2, our data revealed that AEA (≥ 10 µM) hampers the nocifensive response of C. elegans to noxious heat following a 1-hour exposure period. AEA plays a crucial role in modulating pain perception in mammals, which is likely related to its ability to bind to vanilloid and cannabinoid receptors. AEA actions involve regulating neuronal activity and synaptic transmission pathways involved in pain signaling, contributing to the overall management of pain sensation. The results obtained highlights AEA play a significant role in modulating nociceptive behavior in C. elegans. This is coherent with AEA acting as a pain modulator in mammals.

To elucidate potential targets of AEA, we conducted experiments using specific C. elegans mutants (ocr-2, osm-9, npr-19, and npr-32). All tested mutants exhibited reduced sensitivity to noxious heat compared to the WT (N2) control, albeit remaining responsive. To identify targets, mutants were exposed to 10 µM of AEA for 60 minutes prior to conducting heat avoidance experiments. Results depicted in Fig. 3 indicate quantifiable antinociceptive effects of AEA in all mutants. While the data suggest AEA evoke antinociceptive effect on the ocr-2 mutant, thorough inspection does not support this assertion, precluding a conclusive determination of AEA targeting OCR-2 specifically. However, results from mutant exposure to AEA hint at redundancy in receptor involvement and potential compensatory mechanisms. Notably, all single mutants tested retained sensitivity to heat, albeit reduced compared to WT, indicating potential redundancy in receptor function. Indeed, double mutants (e.g., ocr-2/osm-9) have exhibited insensitivity to noxious heat [38]. It's worth noting inconclusive outcomes about targets were also obtained when testing vanilloids in previous studies [23, 25]. In C. elegans, thermal nociceptive neurons express heat-sensitive TRPV channels, primarily OCR-2 and OSM-9, with more supporting data for OCR-2 as the receptor channel targeted by vanilloids [21, 22].

3.2 Proteomic and bioinformatic investigations

Anandamide showed antinociceptive activity in C. elegans but data derived from specific mutants may suggest compensatory effects from the vanilloid and cannabinoid system, respectively. The vanilloid and cannabinoid systems are closely related to each other [39]. Thus, we used mass spectrometry-based proteomics and network biology to decipher the pathways activated by AEA. Label-free proteomics was performed on C. elegans exposed to AEA (25 µM) for 1 h. The volcano plots in Fig. 4 show several DEPs (≥ 2-fold) in response to the AEA treatment. Tables S1 (supplementary file) contain the log₂ fold changes and a p-values for all DEPs identified. Enrichment analysis of Reactome pathways and Gene Ontology Biological Process database in Fig. 5 revealed that the upregulated DEPs are connected to pathways and biological processes strongly associated with the activation of the vanilloid system. Ion homeostasis (Reactome identifier R-CEL-5578775) is an essential pathway for maintaining the electrical properties of neurons, enabling them to generate and transmit signals, communicate with other neurons, and undergo adaptive changes in response to stimuli. Dysfunction in ion homeostasis can lead to neuronal excitability. Transient receptor potential vanilloid channels are versatile ion channels that play an important role in ion homeostasis, particularly through its ability to mediate the influx of calcium ions. It is well described that AEA can bind to and activate mammalian TRPV1 channels [40, 41]. The activation of the TRPV1 by AEA or other ligands triggers calcium influx and depolarization of nociceptive neurons. However, prolonged TRPV1 activation results in desensitization, wherein the channel becomes less responsive to subsequent stimuli. This desensitization led to a reduction in pain perception in mammals. The activation of vanilloid receptors by AEA is part of the broader role of endocannabinoids in modulating neuronal activity and neurotransmitter release. This is also coherent with biological processes such as monoatomic cation homeostasis (Gene Ontology identifier GO:0055080) and programmed cell death (Gene Ontology identifier GO:0012501). The maintenance of ion homeostasis, including monoatomic cation homeostasis, plays a crucial role in determining cell fate, and disruptions in these processes can contribute to programmed cell death [42]. In mammals, the increase in intracellular calcium, as a result of TRPV1 activation, can activate apoptotic pathways [43, 44]. It’s important to note that while there is evidence supporting a role for TRPV1 in cell death, the precise mechanisms, and conditions under which C. elegans vanilloid receptors contributes to apoptotic pathways remain to be defined. Interestingly, Resiniferatoxin-induced TRPV1 activation can lead to cell death but our recent study in C. elegans has not shown nematode apoptosis following Resiniferatoxin exposure [45]. The effects can vary depending on factors such as concentration and duration of exposure. Therefore, further studies are needed to fully understand the intricacies between C. elegans vanilloid receptors and apoptosis. Collectively, the strong enrichment of Ion homeostasis (Reactome identifier R-CEL-5578775), monoatomic cation homeostasis (Gene Ontology identifier GO:0055080) and programmed cell death (Gene Ontology identifier GO:0012501) suggest that AEA bind and activate C. elegans vanilloid receptors (e.g. OSM-9, OCR-2).

Other strongly enriched pathways are Translation (Reactome identifier R-CEL-72766) and its child term Mitochondrial translation elongation (Reactome identifier R-CEL-5389840). Vanilloid receptor activation can lead to the influx of calcium ions (Ca²⁺) into the cell. This calcium influx is a key event in the modulation of neurotransmitter synthesis and release as it has been shown for neuropeptides. Elevated intracellular calcium levels activate various intracellular signaling pathways including calcium-dependent enzymes and transcription factors. In mammals, TRPV1-mediated calcium influx can activate transcription factors, such as CREB (cAMP response element-binding protein) leading to the upregulation of genes encoding proneuropeptides or proteins involved in proneuropeptide biosynthesis. Neuropeptides act as neurotransmitters and are central to trigger nocifensive response in C. elegans [28, 46]. Consequently, the upregulation of translation associated pathways is intimately linked to the activation of the vanilloid receptors. Other pathways including protein localization to membrane (Gene Ontology identifier GO:0072657), cellular component assembly (Gene Ontology identifier GO:0022607), membrane organization (Gene Ontology identifier GO:0061024), and vesicle organization (Gene Ontology identifier GO:0016050) are also related to translation and protein synthesis, protein transport and exocytosis. ATP synthesis is vital for anabolism reaction. This could explain the significant enrichment of the biological process proton motive force-driven ATP synthesis (Gene Ontology identifier GO:0015986). Activation of vanilloid receptors can also trigger an influx of calcium ions resulting in a change in state or activity of neurons. This is coherent with the significant enrichment of the biological process response to abiotic stimulus (Gene Ontology identifier GO:0009628). The mechanistic target of rapamycin complex 1 (mTORC1) is a key cellular signaling complex that plays a central role in regulating various cellular processes, including protein synthesis, cell growth, metabolism, and autophagy. Another interesting enriched pathway is Amino acids regulate mTORC1 (Reactome identifier R-CEL-9639288). In mammals, mTORC1 has been shown to play a role in regulating synaptic plasticity, which is crucial for the processing of pain signals in the central nervous system [47, 48]. mTORC1 has been implicated in the molecular mechanisms underlying neuronal sensitization, contributing to the amplification of pain signals [49, 50]. Calcium influx through TRPV1, especially in the context of sensory neurons, impact mTORC1 signaling [51]. Studies suggested that targeting mTORC1 may provide a new avenue for developing therapeutic interventions to alleviate chronic pain [52]. It is a noteworthy observation that exposure to anandamide trigger mTORC1 associated pathway in C. elegans.

In mammals, the endocannabinoid system is involved in maintaining immune homeostasis, and endocannabinoids can influence the activity of immune cells. CB2 receptors, in particular, are expressed on immune cells, and their activation has been associated with anti-inflammatory effects [53]. AEA can activate CB2 receptors on immune cells, leading to the modulation of cytokine production and other immune responses [54]. There is some evidence to suggest that AEA may interact with TLR4, and their crosstalk could have implications for immune regulation and inflammatory responses [55–57]. Following C. elegans exposition to AEA, enrichment analysis of down-regulated DEPs revealed that the pathway Toll Like Receptor 4 (TLR4) Cascade (Reactome identifier R-CEL-166016) was significantly enriched. Activation of TLR4 initiates signaling cascades that lead to the release of pro-inflammatory cytokines and chemokines and it has been linked to the sensitization of nociceptive pathways [58]. AEA has shown to activate CB2 receptors leading to anti-inflammatory and immunomodulatory effects in mammals [54]. In C. elegans, this effect is most likely triggered by the interaction of AEA with NPR-19 or NPR-32, which are orthologs of cannabinoid receptors. Moreover, the activation of cannabinoid receptors leads to a reduction in cAMP levels. The inhibition of cAMP by cannabinoid receptors is associated with the modulation of neurotransmitter release, which explains the enrichment of the biological process adenylate cyclase-modulating G protein-coupled receptor signaling pathway (Gene Ontology identifier GO:0007188). Another interesting significantly enriched biological process is the positive regulation of the nervous system development (Gene Ontology identifier GO:0051962), which is related to the modulation of chemical synaptic transmission (Gene Ontology identifier GO:0050804). The positive regulation of synaptogenesis includes processes that promote the establishment of functional chemical synapses, and they are useful to trigger responses to noxious stimuli. Following AEA treatment, synaptogenesis is negatively modulated in C. elegans contributing to the antinociceptive effect observed. Conversely, the biological process translational initiation (Gene Ontology identifier GO:0006413) could be related to the regulation of neuropeptide synthesis. Neuropeptides are key chemical synapse players and function as neurotransmitters or neuromodulators. Therefore, AEA treatment likely inhibits neurotransmitters involved in typical pain response.

3.3 Thermal proteome profiling for unbiased identification of AEA targets

To identify the principal targets of AEA, we employed a TPP approach with a ligand concentration range (TTP-CCR) [30, 59]. The choice of a 60˚C temperature challenge for the TPP-CCR experiments was based on the determination of the protein’s melting temperature (Tm) established in a previous study [21]. As shown in Fig. 6A, we detected 757 proteins in the soluble fraction following the thermal challenge at 60˚C. Further analysis of this dataset, allowed us to identify 46 targets with fold-change ≥ 2 and adjusted p-value ≤ 0.01 as shown in Fig. 6B. As exhibited in the heat map, protein concentrations in the soluble fraction increases significantly with ligand concentration. Remarkably, in C. elegans, cannabinoid receptor orthologs NPR-32 and, to a lesser degree, NPR-19 emerge as primary targets of AEA. Moreover, vanilloid receptor ortholog OCR-2 is also targeted by AEA but with most likely less affinity based on the increase in solubility observed following AEA exposure. Interestingly, OCR-2 was the primary target of capsaicin, a well known ligand of the mammalian TRPV1 [21, 22].

Another top target is LEC-2 (G5EGB1). This protein is an ortholog of mammalian galectins. Galectins, a class of proteins implicated in various biological processes, including pain modulation, exhibit potential roles in pain perception and processing [60, 61]. Research suggests galectins may influence pain pathways, offering insights into novel therapeutic targets[62] for managing pain conditions through modulation of galectin activity or expression levels. Investigating the potential crosstalk between AEA signaling and galectin function could unveil novel insights into pain modulation mechanisms and therapeutic avenues. Another interesting top target is CPR-4 (P43508), a Cathepsin B-like cysteine proteinase. Cathepsin B, a lysosomal protease, has been implicated in various pain conditions due to its involvement in neuronal inflammation and neuropathic pain mechanisms [63]. Cathepsin B's role extends beyond pain to include inflammation modulation, as it participates in cytokine processing and activation, implicating it as a potential target for anti-inflammatory interventions [64, 65]. Cathepsin B may interact with endocannabinoids like AEA, potentially influencing their metabolism and signaling pathways. Another top target is PGRN-1 (Q9U362), a C. elegans progranulin ortholog. Progranulin, a multifunctional growth factor, exhibits potential implications in pain modulation, with studies linking its deficiency to heightened pain sensitivity [66, 67]. Investigations into progranulin's mechanisms suggest its involvement in neuroinflammation[68] and nociceptive signaling pathways, highlighting its promising role as a therapeutic target for managing pain disorders. Progranulin might intersect with anandamide for its regulatory role in various processes associated with pain and inflammation. Research exploring the potential interplay between progranulin and anandamide signaling pathways could provide novel insights into their mutual regulatory mechanisms and associated physiological implications. Additionally. we were also intrigued by the presence of TTR-15 (Q22288), a Transthyretin-like protein in the list of AEA targets. Transthyretin, a carrier protein primarily transporting thyroid hormones and retinol-binding proteins, may play a role in pain and inflammation modulation. Intriguingly, we have showed a link between transthyretin and pain [69, 70]. However, there isn't a well-established association between transthyretin and anandamide or the endocannabinoid system. Additionally, as disclosed in Table 1, several small and large ribosomal proteins were identified as top targets. Protein-protein interaction network analyses performed with STRING (V.12.0) (Fig. 7) revealed there is an important cluster regrouping several proteins from GTP hydrolysis and joining of the 60S ribosomal subunit (Reactome identifier R-CEL-72706) including specifically small and large ribosomal proteins, essential to protein synthesis. This metabolic pathway may have an impact on nociceptive and inflammation pathways through its involvement in cellular signaling cascades [71–73]. Moreover, small and large ribosomal proteins play important roles in cytokine/chemokine production [74, 75]. Exploring the potential interplay between AEA and small and large ribosomal proteins could reveal novel regulatory mechanisms.

This study has shown, for the first-time, that AEA is implicated in modulating nocifensive response to noxious heat in C. elegans following a controlled and prolonged exposition to low doses of the compound. Additionally, our data identified that AEA targeted vanilloid and cannabinoid receptors, but functional redundancy between OCR-2/OSM-9 and NPR-19/NPR-32 seems to be at play, precluding the identification of specific receptors. This was further confirmed following Reactome pathway and Gene Ontology Biological Process enrichment analyses. Additionally, AEA was found to exert modulatory effects on the immune system, which agrees with the known significant role of the immune system in the perception and modulation of pain in mammals. Inflammatory responses, orchestrated by immune cells such as macrophages, T cells, and cytokines, contribute to the sensitization of peripheral nerves and the transmission of pain signals to the central nervous system. Additionally, immune cells release signaling molecules that directly activate pain receptors or sensitize neurons, amplifying pain sensations. Conversely, immune-modulating therapies can alleviate pain by reducing inflammation and immune cell activation. Understanding the intricate interplay between the immune system, pain pathways and AEA may lead the development of effective treatments for pain-related disorders.

Conflict of interest

The authors declare no conflict of interest.

Funding information

This project was funded by the National Sciences and Engineering Research Council of Canada (F. Beaudry discovery grant no. RGPIN-2020-05228). Laboratory equipment was funded by the Canadian Foundation for Innovation (CFI) and the Fonds de Recherche du Québec (FRQ), the Government of Quebec (F. Beaudry CFI John R. Evans Leaders grant no. 36706 and 42043). F. Beaudry is the holder of the Canada Research Chair in metrology of bioactive molecule and target discovery (grant no. CRC-2021-00160). This research was undertaken, partly, thanks to funding from the Canada Research Chairs Program.

Author Contribution

M.A. and F.B. conceived and designed this study. M.A., J.B.S., J.D.C. conducted the experiments. M.A., J.B.S., J.D.C. and F.B. conducted data analyses and wrote the manuscript. All the authors have read and approved the final manuscript.

Acknowledgements

The Université de Montréal partially provided financial support to M. Abdollahi and J. Ben Salem. A Ph.D. scholarship was awarded to J. Ben Salem from the Fonds de recherche du Québec – Santé (FRQS).

Data Availability

The data that support the findings from this study are available from the corresponding author upon reasonable request.

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Anandamide Modulates Thermal Avoidance in Caenorhabditis elegans through Vanilloid and Cannabinoid Receptor Interplay

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Chemicals and reagents

2.2 C. elegans strains

2.3 C. elegans pharmacological manipulations

2.4 Thermal avoidance assays

2.5 Thermal proteome profiling to identify targets

2.6 Proteomic analysis

2.7 Bioinformatics

2.8 Statistical analysis

3. Results and discussion

3.1 Evaluation of antinociceptive activity of AEA

3.2 Proteomic and bioinformatic investigations

3.3 Thermal proteome profiling for unbiased identification of AEA targets

4. Conclusion

Declarations

Conflict of interest

Funding information

Author Contribution

Acknowledgements

Data Availability

References

Additional Declarations

Supplementary Files

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