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![PEGCs and endocannabinoids stimulate cholesterol transport by employing parallel pathways. (a) RNAi of let-767 leads to a significant increase of dauers compared to control plates in daf-7 mutant background (empty vector; from ~20% to ~80%), but has no effect on wild type (N2) dauer formation. Kruskal-Wallis oneway analysis of variance on ranks, p < 0.001, All pairwise multiple comparison procedures (Dunn's Method), (*) indicates statistically significant difference between daf-7/empty vector and daf-7/let-767 RNAi, p < 0.05. Bars represent mean values and error bars represent standard errors. The number of independent experiments is n = 6 for N2/empty vector, and N2/let-767 RNAi, n = 27 for daf-7/empty vector, n = 33 for daf-7/let-767 RNAi, and n = 6 for daf-7/let-767 RNAi+DA. [DA] = 90 nM. (b) daf-7 on let-767 RNAi at 20 ºC forms ~80% dauers. Supplementation with either of the endocannabinoids 2-AG and AEA, or the addition of methyl-C17iso suppresses dauer formation significantly. Kruskal-Wallis one-way analysis of variance on ranks, p < 0.001. Multiple comparisons versus control group (Dunn's Method). (*) indicates statistically significant difference with empty vector, p < 0.05. Bars represent mean values from at least three independent experiments and error bars represent standard errors. [2-AG] or [AEA] = 50 µM. [methyl-C17iso] = 100 µM. (c) daf-7 let-767 RNAi worms partly bypass dauer arrest if grown in excess cholesterol (130 µM). (*) indicates statistically significant difference with control plates (13 µM cholesterol). Mann-Whitney rank sum test p = 0.013. Bars represent mean values and error bars represent standard errors. The number of independent experiments is n=26 for daf-7 let-767 RNAi at 13 µM cholesterol, and n = 4 for daf-7 let-767 RNAi at 130 µM cholesterol. (d) Inhibition of the d17iso-glucosylceramide synthesis (and hence PEGCs) by PDMP treatment leads to an increase in dauer formation in daf-7 background, that can be largely suppressed by endocannabinoids, DA and excess cholesterol, but not by methyl-C17iso. One-way analysis of variance, p < 0.001. Multiple comparisons versus control group (Holm-Sidak method). (*) indicates statistically significant difference with PDMP treatment, p < 0.001 for all conditions except for empty vector (p = 0.002). Bars represent mean values and error bars represent standard errors. The number of independent experiments is n = 3 for all conditions. [DA] = 90 nM. [Cholesterol] = 130 µM. [2-AG] or [AEA] = 50 µM. [methyl-C17iso] = 100 µM. (e) mmPEGC-C22 is partly able to suppress dauer formation of daf-7;fat-3 at 20 ºC. One-way analysis of variance, p < 0.001. Multiple comparisons versus control group (Holm-Sidak method). (*) indicates statistically significant difference with solvent control, p < 0.001 for both conditions. Bars represent mean values and error bars represent standard](publication/324704425/figure/fig3/AS:618625484455944@1524503217973/PEGCs-and-endocannabinoids-stimulate-cholesterol-transport-by-employing-parallel.png)
PEGCs and endocannabinoids stimulate cholesterol transport by employing parallel pathways. (a) RNAi of let-767 leads to a significant increase of dauers compared to control plates in daf-7 mutant background (empty vector; from ~20% to ~80%), but has no effect on wild type (N2) dauer formation. Kruskal-Wallis oneway analysis of variance on ranks, p < 0.001, All pairwise multiple comparison procedures (Dunn's Method), (*) indicates statistically significant difference between daf-7/empty vector and daf-7/let-767 RNAi, p < 0.05. Bars represent mean values and error bars represent standard errors. The number of independent experiments is n = 6 for N2/empty vector, and N2/let-767 RNAi, n = 27 for daf-7/empty vector, n = 33 for daf-7/let-767 RNAi, and n = 6 for daf-7/let-767 RNAi+DA. [DA] = 90 nM. (b) daf-7 on let-767 RNAi at 20 ºC forms ~80% dauers. Supplementation with either of the endocannabinoids 2-AG and AEA, or the addition of methyl-C17iso suppresses dauer formation significantly. Kruskal-Wallis one-way analysis of variance on ranks, p < 0.001. Multiple comparisons versus control group (Dunn's Method). (*) indicates statistically significant difference with empty vector, p < 0.05. Bars represent mean values from at least three independent experiments and error bars represent standard errors. [2-AG] or [AEA] = 50 µM. [methyl-C17iso] = 100 µM. (c) daf-7 let-767 RNAi worms partly bypass dauer arrest if grown in excess cholesterol (130 µM). (*) indicates statistically significant difference with control plates (13 µM cholesterol). Mann-Whitney rank sum test p = 0.013. Bars represent mean values and error bars represent standard errors. The number of independent experiments is n=26 for daf-7 let-767 RNAi at 13 µM cholesterol, and n = 4 for daf-7 let-767 RNAi at 130 µM cholesterol. (d) Inhibition of the d17iso-glucosylceramide synthesis (and hence PEGCs) by PDMP treatment leads to an increase in dauer formation in daf-7 background, that can be largely suppressed by endocannabinoids, DA and excess cholesterol, but not by methyl-C17iso. One-way analysis of variance, p < 0.001. Multiple comparisons versus control group (Holm-Sidak method). (*) indicates statistically significant difference with PDMP treatment, p < 0.001 for all conditions except for empty vector (p = 0.002). Bars represent mean values and error bars represent standard errors. The number of independent experiments is n = 3 for all conditions. [DA] = 90 nM. [Cholesterol] = 130 µM. [2-AG] or [AEA] = 50 µM. [methyl-C17iso] = 100 µM. (e) mmPEGC-C22 is partly able to suppress dauer formation of daf-7;fat-3 at 20 ºC. One-way analysis of variance, p < 0.001. Multiple comparisons versus control group (Holm-Sidak method). (*) indicates statistically significant difference with solvent control, p < 0.001 for both conditions. Bars represent mean values and error bars represent standard
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Proper cholesterol transport is crucial for the functionality of cells. In C. elegans, certain cholesterol derivatives called dafachronic acids (DAs) govern the entry into diapause. In their absence, worms form a developmentally arrested dauer larva. Thus, cholesterol transport to appropriate places for DA biosynthesis warrants the reproductive gro...
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... the elongation of long-chain and branched-chain fatty acids. RNAi of LET-767 in wild type worms leads to multiple developmental phenotypes in the first generation and early larval arrest in the second generation (L1-L2) 22 . Indeed, RNAi against LET-767 in daf-7 mutants grown at 20 °C significantly increases the Daf-c pheno- type of the strain (Fig. 4a). GC-MS analysis of the relative fatty acid profiles of control and let-767 RNAi animals confirmed that RNAi treatment leads to a strong decrease in the levels of C15iso and C17iso mmBCFAs, but not of PUFAs (Supplementary Table 3). Accordingly, as seen in Fig. 4b, supplementation with C17iso mmBCFA methyl esters rescued the let-767 ...
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... mutants grown at 20 °C significantly increases the Daf-c pheno- type of the strain (Fig. 4a). GC-MS analysis of the relative fatty acid profiles of control and let-767 RNAi animals confirmed that RNAi treatment leads to a strong decrease in the levels of C15iso and C17iso mmBCFAs, but not of PUFAs (Supplementary Table 3). Accordingly, as seen in Fig. 4b, supplementation with C17iso mmBCFA methyl esters rescued the let-767 RNAi-induced dauer arrest of daf-7 worms. Astoundingly, both 2-AG and AEA also rescued the dauer formation observed in daf-7 animals subjected to let-767 RNAi (Fig. 4b). To exclude the possibility that endocannabinoids act by elevating levels of mmBCFAs, i.e. by ...
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... in the levels of C15iso and C17iso mmBCFAs, but not of PUFAs (Supplementary Table 3). Accordingly, as seen in Fig. 4b, supplementation with C17iso mmBCFA methyl esters rescued the let-767 RNAi-induced dauer arrest of daf-7 worms. Astoundingly, both 2-AG and AEA also rescued the dauer formation observed in daf-7 animals subjected to let-767 RNAi (Fig. 4b). To exclude the possibility that endocannabinoids act by elevating levels of mmBCFAs, i.e. by inducing (a) not yet identified alter- native 3-ketoacyl-CoA reductase(s), we performed GC-MS analysis of mmBCFA in daf-7 let-767 RNAi worms in the presence of 2-AG (Supplementary Table 4). This experiment clearly shows that endocannabinoid ...
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... GC-MS analysis of mmBCFA in daf-7 let-767 RNAi worms in the presence of 2-AG (Supplementary Table 4). This experiment clearly shows that endocannabinoid treatment does not increase mmBCFA. In consistency with impaired cholesterol trafficking in daf-7 let-767 (RNAi), dauer formation was partly inhibited by high cholesterol levels in the medium (Fig. ...
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... second approach to deplete PEGCs was to treat daf-7 with PDMP, an inhibitor of the final glucosylation step in the synthesis of GlcCer (Supplementary Figure 6). We found that DA, high cholesterol and endocannabi- noids rescue daf-7 dauers generated in the presence of PDMP (Fig. 4d) 10 . However, as expected, PDMP treatment could not be rescued by addition of mmBCFA C17iso methyl ester because PDMP inhibits a later step of the synthesis of PEGCs. Thus, PEGCs deficiency could be rescued by supplementation with ...
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... Supplementation with either of the endocannabinoids 2-AG and AEA, or the addition of methyl-C17iso suppresses dauer formation significantly. Kruskal-Wallis one-way analysis of variance on ranks, p < 0.001. Based on our data, we propose a tentative scheme of the interaction between endocannabinoids and PEGC in the control of cholesterol transport (Fig. 4f). Multiple reactions produce the precursors diacylglycerol (DAG), N-acyl-phosphatidylethanolamine (NAPE), and GlcCer required for the synthesis of 2-AG, AEA and PEGCs, respectively. The conversions of these precursors to the active compounds are carried out by not yet identified enzymes in C. elegans. 2-AG and AEA are probably produced ...
Citations
... Another emerging role for ECS is its involvement in food intake (Aguilera Vasquez & Nielsen, 2022;Galles et al., 2018). Different lengths of food deprivation differently affect snails' behavior and learning and memory abilities (Ito et al., 2015;Kagan et al., 2022Kagan et al., , 2023Ito et al., 2017;Rivi, Benatti, Actis et al., 2022;Totani et al., 2019). ...
The endocannabinoid system (ECS) plays an important role in neuroprotection, neuroplasticity, energy balance, modulation of stress, and inflammatory responses, acting as a critical link between the brain and the body's peripheral regions, while also offering promising potential for novel therapeutic strategies. Unfortunately, in humans, pharmacological inhibitors of different ECS enzymes have led to mixed results in both preclinical and clinical studies. As the ECS has been highly conserved throughout the eukaryotic lineage, the use of invertebrate model organisms like the pond snail Lymnaea stagnalis may provide a flexible tool to unravel unexplored functions of the ECS at the cellular, synaptic, and behavioral levels. In this study, starting from the available genome and transcriptome of L. stagnalis, we first identified putative transcripts of all ECS enzymes containing an open reading frame. Each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate organisms. Sequences were confirmed by qualitative PCR and sequencing. Then, we investigated the transcriptional effects induced by different stress conditions (i.e., bacterial LPS injection, predator scent, food deprivation, and acute heat shock) on the expression levels of the enzymes of the ECS in Lymnaea's central ring ganglia. Our results suggest that in Lymnaea as in rodents, the ECS is involved in mediating inflammatory and anxiety‐like responses, promoting energy balance, and responding to acute stressors. To our knowledge, this study offers the most comprehensive analysis so far of the ECS in an invertebrate model organism. image
... Experiments conducted by Galles et al. (2018) indicated that endocannabinoids can promote cholesterol recycling in a Caenorhabditis elegans mutant for the ncr1 and ncr2 genes, which are orthologous to the human Niemann Pick C disease gene npc1. Similar results were obtained by Bartoll et al. (2020), demonstrated that inhibition of fatty acid hydrolase (FAAH), involved in endocannabinoid breakdown, reduces sphingomyelin and cholesterol accumulation in NPC models. ...
... Therefore, the findings presented in this study enable us to suggest a scenario, in which the deficits observed in cholesterol trafficking in aged astrocytes could be intensified during inflammatory processes in the elderly brain. Galles et al. (2018) showed that endocannabinoids are able to mobilize cholesterol in C. elegans mutant for nrc-1 and nrc-2 (orthologous for npc-1 and npc-2, respectively (Guix et al., 2021). If this hypothesis holds true, cannabinoids could not only aid in the redistribution and release of cholesterol from aged astrocytes but also facilitate its redistribution within aged neurons, promoting its exit from the lysosome and enabling it to reach various intracellular membranes to ensure proper neuronal function. ...
Cholesterol is crucial for the proper functioning of eukaryotic cells, especially neurons, which rely on cholesterol to maintain their complex structure and facilitate synaptic transmission. However, brain cells are isolated from peripheral cholesterol by the blood–brain barrier and mature neurons primarily uptake the cholesterol synthesized by astrocytes for proper function. This study aimed to investigate the effect of aging on cholesterol trafficking in astrocytes and its delivery to neurons. We found that aged astrocytes accumulated high levels of cholesterol in the lysosomal compartment, and this cholesterol buildup can be attributed to the simultaneous occurrence of two events: decreased levels of the ABCA1 transporter, which impairs ApoE-cholesterol export from astrocytes, and reduced expression of NPC1, which hinders cholesterol release from lysosomes. We show that these two events are accompanied by increased microR-33 in aged astrocytes, which targets ABCA1 and NPC1. In addition, we demonstrate that the microR-33 increase is triggered by oxidative stress, one of the hallmarks of aging. By coculture experiments, we show that cholesterol accumulation in astrocytes impairs the cholesterol delivery from astrocytes to neurons. Remarkably, we found that this altered transport of cholesterol could be alleviated through treatment with endocannabinoids as well as cannabidiol or CBD. Finally, according to data demonstrating that aged astrocytes develop an A1 phenotype, we found that cholesterol buildup is also observed in reactive C3+ astrocytes. Given that reduced neuronal cholesterol affects synaptic plasticity, the ability of cannabinoids to restore cholesterol transport from aged astrocytes to neurons holds significant implications in aging and inflammation.
... 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][17][18][19][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][22][23][24][25] and cannabinoid systems [16]. ...
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.
... The endocannabinoids also work through other receptors such as OCTR-1 (α 2A -adrenergic-like octopamine receptor) and SER-4 (5-HT 1A -like serotonin receptor) to influence nociception and locomotion [87]. Studies found that 2-AG acted as an endogenous modulator of TRPV signal transduction, influencing sterol mobilisation via the IGF-1 signalling pathway [88] and stimulating cholesterol trafficking [89] in nematodes. AEA has a bidirectional effect on feeding in C. elegans, which is mediated by NPR-19 [90]. ...
There is a significant global upsurge in the number and proportion of older persons in the population. With this comes an increasing prevalence of age-related conditions which pose a major challenge to healthcare systems. The development of anti-ageing treatments may help meet this challenge by targeting the ageing process which is a common denominator to many health problems. Cannabis-like compounds (cannabinoids) are reported to improve quality of life and general well-being in human trials, and there is increasing preclinical research highlighting that they have anti-ageing activity. Moreover, preclinical evidence suggests that endogenous cannabinoids regulate ageing processes. Here, we review the anti-ageing effects of the cannabinoids in various model systems, including the most extensively studied nematode model, Caenorhabditis elegans . These studies highlight that the cannabinoids lengthen healthspan and lifespan, with emerging evidence that they may also hinder the development of cellular senescence. The non-psychoactive cannabinoid cannabidiol (CBD) shows particular promise, with mechanistic studies demonstrating it may work through autophagy induction and activation of antioxidative systems. Furthermore, CBD improves healthspan parameters such as diminishing age-related behavioural dysfunction in models of both healthy and accelerated ageing. Translation into mammalian systems provides an important next step. Moreover, looking beyond CBD, future studies could probe the multitude of other cannabis constituents for their anti-ageing activity.
... 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][17][18][19][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][22][23][24][25] and cannabinoid systems [16]. ...
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.
... In addition, the endocannabinoid system in C. elegans exhibits several similarities with that in mammals. C. elegans has several endocannabinoids structurally related to cannabinoid receptor ligands in mammals including anandamide and 2-arachidonoylglycerol [33]. Moreover, recent studies have identified receptor components of the C. elegans cannabinoid system with orthologs in mammals, NPR-19 (a CB1/2 ortholog) and NPR-32 (a GPR18/55 ortholog) [34][35][36]. ...
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 Δ⁹-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.
... Experiments conducted by Galles et al. [21] indicated that endocannabinoids can promote cholesterol recycling in a C. elegans mutant for the ncr1 and ncr2 genes, which are orthologous to the human Niemann Pick C disease gene npc1. Similar results were obtained by Bartoll et al. [22] , who demonstrated that inhibition of Fatty Acid Hydrolase (FAAH), involved in endocannabinoid breakdown, reduces sphingomyelin and cholesterol accumulation in NPC models. ...
... Finally, taking into account previous results from Galles et al. [21] , where it was shown that feeding C. elegans with AEA or 2-AG is able to inhibit dauer formation caused by impaired cholesterol trafficking in Niemann-Pick C1 mutants, we demonstrated that the lysosomal cholesterol accumulation in aged astrocytes can be alleviated by the treatment not only with the endocannabinoids AEA and 2-AG but also with the phytocannabinoid CBD. Additionally, we showed that AEA treatment is able to improve the neuronal uptake of cholesterol from aged astrocytes. ...
Cholesterol is crucial for the proper functioning of eukaryotic cells, especially neurons, which rely on cholesterol to maintain their complex structure and facilitate synaptic transmission. However, brain cells are isolated from peripheral cholesterol by the blood-brain barrier and mature neurons primarily uptake the cholesterol synthesized by astrocytes for proper function. This study aimed to investigate the effect of aging on cholesterol trafficking in astrocytes and its delivery to neurons.
Using in vitro and in vivo models of aging, we found that aged astrocytes accumulated high levels of cholesterol in the lysosomal compartment, and this cholesterol buildup can be attributed to the simultaneous occurrence of two events: decreased levels of the ABCA1 transporter which impairs ApoE-cholesterol export from astrocytes, and reduced expression of NPC1, which hinders cholesterol release from lysosomes. We show that these two events are accompanied by increased microR33 in aged astrocytes, which is known to downregulate ABCA1 and NPC1. In addition, we demonstrate that the microR33 increase is triggered by oxidative stress, one of the hallmarks of aging.
By co-culture experiments we also show that aging in vitro impairs the cholesterol delivery from astrocytes to neurons. Remarkably, we found that this altered transport of cholesterol could be alleviated through treatment with endocannabinoids as well as cannabidiol or CBD. Given that reduced neuronal cholesterol affects synaptic plasticity, the ability of cannabinoids to restore cholesterol transport from aged astrocytes to neurons holds significant implications in the field of aging.
... By comparison, there have been fewer dissections of the specific mechanisms controlling developmental rate in C. elegans. Many such investigations have focused on pathways controlling developmental progression in the absence of food or certain nutrients, revealing critical roles for IIS signaling, TORC1, lipid metabolism, the one-carbon cycle, and micronutrients in dauer formation and larval arrest (Baugh and Sternberg, 2006;Galles et al., 2018;Long et al., 2002;Watson et al., 2014;Watts et al., 2018). However, our understanding of the role of TORC2 signaling in development remains largely incomplete, confined to the DCC, a gut-neuronal axis linking TORC2 to TGF-β signaling, and CDC-42 induced neuronal protrusions (Alan et al., 2013;O'Donnell et al., 2018;Webster et al., 2013). ...
Both Hedgehog (Hh) signaling and target of rapamycin complex 2 (TORC2) are central, evolutionarily conserved pathways that regulate development and metabolism. In C. elegans , loss of essential TORC2 component RICTOR ( rict-1 ) causes delayed development, shortened lifespan, reduced brood, small size, and increased fat. Here we report that knockdown of Hedgehog-related morphogen grd-1 and its Patched-related receptor ptr-11 rescues delayed development in TORC2 loss of function mutants, indicating an unexpected role for grd-1 / ptr-11 in slowing developmental rate downstream of nutrient sensing pathways. Further, we implicate chronic stress transcription factor pqm-1 as a key transcriptional effector of grd-1 / ptr-11 in slowing whole-organism growth. We propose that the TORC2/ grd-1 / ptr-11 / pqm-1 signaling relay acts as a critical executor of growth to slow development when C. elegans encounters unfavorable growth conditions.
Summary statement
Developmental rate in C. elegans is dramatically slowed in animals deficient in nutrient-sensitive target of rapamycin complex 2 signaling and slowing is effected by increased activity of a previously uncharacterized Hh-r/Ptr signaling relay.
... It was recently discovered that the C. elegans glycolipids phosphoethanolamine glucosylceramides (PEGCs) stimulate the growth of worms by as yet unknown mechanism under conditions of cholesterol scarcity [14]. We also recently reported that the best studied endocannabinoids (eCBs), 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamine (AEA), lipid messengers that elicit a plethora of biological functions in mammals, enhance traffic of cholesterol in C. elegans [15]. We found that these eCBs stimulate worm growth under conditions of cholesterol scarcity and reverse the developmental arrest of ncr-2; ncr-1 mutants [15]. ...
... We also recently reported that the best studied endocannabinoids (eCBs), 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamine (AEA), lipid messengers that elicit a plethora of biological functions in mammals, enhance traffic of cholesterol in C. elegans [15]. We found that these eCBs stimulate worm growth under conditions of cholesterol scarcity and reverse the developmental arrest of ncr-2; ncr-1 mutants [15]. ncr-1 and ncr-2 encode proteins with homology to the human Niemann-Pick type C (NP-C) disease gene (NPC-1) and are involved in intracellular cholesterol trafficking in C. elegans [16]. ...
... ocr-2 encodes a channel of the TRPV subfamily that functions in C. elegans olfaction, nociception and osmosensation [24]. We found that, similar to C. elegans fat-3 and fat-4 mutants, which are deficient in PUFAs and display aberrant cholesterol mobilization [15], ocr-2 animals displayed a high incidence of arrested dauer-like larvae in the first generation without cholesterol (Fig 3A and 3B). While 2-AG partially suppresses dauer formation of fat mutants [15], 2-AG was unable to rescue the arrest of ocr-2 animals starved from cholesterol. ...
The nematode Caenorhabditis elegans requires exogenous cholesterol to survive and its depletion leads to early developmental arrest. Thus, tight regulation of cholesterol storage and distribution within the organism is critical. Previously, we demonstrated that the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) plays a key role in C. elegans since it modulates sterol mobilization. However, the mechanism remains unknown. Here we show that mutations in the ocr-2 and osm-9 genes, coding for transient receptors potential V (TRPV) ion channels, dramatically reduce the effect of 2-AG in cholesterol mobilization. Through genetic analysis in combination with the rescue of larval arrest induced by sterol starvation, we found that the insulin/IGF-1signaling (IIS) pathway and UNC-31/CAPS, a calcium-activated regulator of neural dense-core vesicles release, are essential for 2-AG-mediated stimulation of cholesterol mobilization. These findings indicate that 2-AG-dependent cholesterol trafficking requires the release of insulin peptides and signaling through the DAF-2 insulin receptor. These results suggest that 2-AG acts as an endogenous modulator of TRPV signal transduction to control intracellular sterol trafficking through modulation of the IGF-1 signaling pathway.
... Elimination of the two homologues of mammalian NPC1, whose mutation causes Niemann-Pick type C (OMIM 257220), caused impaired development of C. elegans that could be rescued by specific glycosphingolipids [202] and endocannabinoids [203]. Modula-tion of the levels of proteins associated with the Endosomal Sorting Complex Required for Transport (ESCRT), which is a cellular mechanism that contributes to membrane remodelling and repair [204], almost fully supressed lysosomal defects and cell death in a C. elegans model for mucolipidosis type IV (OMIM 253000) [205]. ...
The lack of available treatments and fatal outcome in most lysosomal storage disorders (LSDs) have spurred research on pathological mechanisms and novel therapies in recent years. In this effort, experimental methodology in cellular and animal models have been developed, with aims to address major challenges in many LSDs such as patient-to-patient variability and brain condition. These techniques and models have advanced knowledge not only of LSDs but also for other lysosomal disorders and have provided fundamental insights into the biological roles of lysosomes. They can also serve to assess the efficacy of classical therapies and modern drug delivery systems. Here, we summarize the techniques and models used in LSD research, which include both established and recently developed in vitro methods, with general utility or specifically addressing lysosomal features. We also review animal models of LSDs together with cutting-edge technology that may reduce the need for animals in the study of these devastating diseases.
