Neuroprotective effect of erythropoietin on anesthesia-induced neurotoxicity through the modulation of autophagy in Caenorhabditis elegans.

BACKGROUND: The anti-oxidative, anti-inflammatory, and anti-apoptotic effects of erythropoietin may provide neuroprotective effects. Erythropoietin also modulates autophagy signaling that may play a role in anesthesia-induced neurotoxicity (AIN). Herein, we investigated whether AIN can be attenuated by the neuroprotective effect of erythropoietin in the Caenorhabditis elegans (C. elegans). METHODS: Synchronized worms were divided into the control, Iso, EPO, and EPO-Iso groups. The chemotaxis index (CI) was evaluated when they reached the young adult stage. The lgg-1::GFP-positive puncta per seam cell were used to determine the autophagic events. The erythropoietin-mediated pathway of autophagy was determined by measuring the genetic expression level of let-363, bec-1, atg-7, atg-5, and lgg-3. RESULTS: Increased lgg-1::GFP puncta were observed in the Iso, EPO, and EPO-Iso groups. In the Iso group, only the let-363 level decreased significantly as compared to that in the control group (P = 0.009). bec-1 (P < 0.001), atg-5 (P = 0.012), and lgg-3 (P < 0.001) were expressed significantly more in the EPO-Iso group than in the Iso groups. Repeated isoflurane exposure during development decreased the CI. Erythropoietin could restore the decreased CI by isoflurane significantly in the EPO-Iso group. CONCLUSIONS: Erythropoietin showed neuroprotective effects against AIN and modulated the autophagic pathway in C. elegans. This experimental evidence of erythropoietin-related neuroprotection against AIN may be correlated with the induced autophagic degradation process that was sufficient for handling enhanced autophagy induction in erythropoietin-treated worms.

Adaptive capacity to dietary Vitamin B12 levels is maintained by a gene-diet interaction that ensures optimal life span.

Diet regulates complex life-history traits such as longevity. For optimal lifespan, organisms employ intricate adaptive mechanisms whose molecular underpinnings are less known. We show that Caenorhabditis elegans FLR-4 kinase prevents lifespan differentials on the bacterial diet having higher Vitamin B12 levels. The flr-4 mutants are more responsive to the higher B12 levels of Escherichia coli HT115 diet, and consequently, have enhanced flux through the one-carbon cycle. Mechanistically, a higher level of B12 transcriptionally downregulates the phosphoethanolamine methyltransferase pmt-2 gene, which modulates phosphatidylcholine (PC) levels. Pmt-2 downregulation activates cytoprotective gene expression through the p38-MAPK pathway, leading to increased lifespan only in the mutant. Evidently, preventing bacterial B12 uptake or inhibiting one-carbon metabolism reverses all the above phenotypes. Conversely, supplementation of B12 to E. coli OP50 or genetically reducing PC levels in the OP50-fed mutant extends lifespan. Together, we reveal how worms maintain adaptive capacity to diets having varying micronutrient content to ensure a normal lifespan.

Regulation of monoamine levels by typical and atypical antipsychotics in Caenorhabditis elegans mutant for nuclear distribution element genes.

Mammalian nuclear distribution genes encode proteins with essential roles in neuronal migration and brain formation during embryogenesis. The implication of human nuclear distribution genes, namely nudC and NDE1 (Nuclear Distribution Element 1)/NDEL1 (Nuclear Distribution Element-Like 1), in psychiatric disorders including schizophrenia and bipolar disorder, has been recently described. The partial loss of NDEL1 expression results in neuronal migration defects, while ndel1 null knockout (KO) leads to early embryonic lethality in mice. On the other hand, loss-of-function of the orthologs of nuclear distribution element genes (nud) in Caenorhabditis elegans renders viable worms and influences behavioral endophenotypes associated with dopaminergic and serotoninergic pathways. In the present work, we evaluated the role of nud genes in monoamine levels at baseline and after the treatment with typical or atypical antipsychotics. Dopamine, serotonin and octopamine levels were significantly lower in homozygous loss-of-function mutant worms KO for nud genes compared with wild-type (WT) C. elegans at baseline. While treatment with antipsychotics determined significant differences in monoamine levels in WT, the nud KO mutant worms appear to respond differently to the treatment. According to the best of our knowledge, we are the first to report the influence of nud genes in the monoamine levels changes in response to antipsychotic drugs, ultimately placing the nuclear distribution genes family at the cornerstone of pathways involved in the modulation of monoamines in response to different classes of antipsychotic drugs.

Caffeic and Dihydrocaffeic Acids Promote Longevity and Increase Stress Resistance in Caenorhabditis elegans by Modulating Expression of Stress-Related Genes.

Caffeic and dihydrocaffeic acid are relevant microbial catabolites, being described as products from the degradation of different phenolic compounds i.e., hydroxycinnamoyl derivatives, anthocyanins or flavonols. Furthermore, caffeic acid is found both in free and esterified forms in many fruits and in high concentrations in coffee. These phenolic acids may be responsible for a part of the bioactivity associated with the intake of phenolic compounds. With the aim of progressing in the knowledge of the health effects and mechanisms of action of dietary phenolics, the model nematode Caenorhabditis elegans has been used to evaluate the influence of caffeic and dihydrocaffeic acids on lifespan and the oxidative stress resistance. The involvement of different genes and transcription factors related to longevity and stress resistance in the response to these phenolic acids has also been explored. Caffeic acid (CA, 200 µM) and dihydrocaffeic acid (DHCA, 300 µM) induced an increase in the survival rate of C. elegans under thermal stress. Both compounds also increased the mean and maximum lifespan of the nematode, compared to untreated worms. In general, treatment with these acids led to a reduction in intracellular ROS concentrations, although not always significant. Results of gene expression studies conducted by RT-qPCR showed that the favorable effects of CA and DHCA on oxidative stress and longevity involve the activation of several genes related to insulin/IGF-1 pathway, such as daf-16, daf-18, hsf-1 and sod-3, as well as a sirtuin gene (sir-2.1).

Identification of the 5-HT1A serotonin receptor as a novel therapeutic target in a C. elegans model of Machado-Joseph disease.

Machado-Joseph disease (MJD) or Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder that affects movement coordination leading to a premature death. Despite several efforts, no disease-modifying treatment is yet available for this disease. Previous studies pinpointed the modulation of serotonergic signaling, through pharmacological inhibition of the serotonin transporter SERT, as a promising therapeutic approach for MJD/SCA3. Here, we describe the 5-HT1A receptor as a novel therapeutic target in MJD, using a C. elegans model of ATXN3 proteotoxicity. Chronic and acute administration of befiradol (also known as NLX-112), a highly specific 5-HT1A agonist, rescued motor function and suppressed mutant ATXN3 aggregation. This action required the 5-HT1A receptor orthologue in the nematode, SER-4. Tandospirone, a clinically tested 5-HT1A receptor partial agonist, showed a limited impact on animals' motor dysfunction on acute administration and a broader receptor activation profile upon chronic treatment, its effect depending on 5-HT1A but also on the 5-HT6/SER-5 and 5-HT7/SER-7 receptors. Our results support high potency and specificity of befiradol for activation of 5-HT1A/SER-4 receptors and highlight the contribution of the auto- and hetero-receptor function to the therapeutic outcome in this MJD model. Our study deepens the understanding of serotonergic signaling modulation in the suppression of ATXN3 proteotoxicity and suggests that a potent and selective 5-HT1A receptor agonist such as befiradol could constitute a promising therapeutic agent for MJD.

A Dihydroflavonoid Naringin Extends the Lifespan of C. elegans and Delays the Progression of Aging-Related Diseases in PD/AD Models via DAF-16.

Naringin is a dihydroflavonoid, which is rich in several plant species used for herbal medicine. It has a wide range of biological activities, including antineoplastic, anti-inflammatory, antiphotoaging, and antioxidative activities. So it would be interesting to know if naringin has an effect on aging and aging-related diseases. We examined the effect of naringin on the aging of Caenorhabditis elegans (C. elegans). Our results showed that naringin could extend the lifespan of C. elegans. Moreover, naringin could also increase the thermal and oxidative stress tolerance, reduce the accumulation of lipofuscin, and delay the progress of aging-related diseases in C. elegans models of AD and PD. Naringin could not significantly extend the lifespan of long-lived mutants from genes in insulin/IGF-1 signaling (IIS) and nutrient-sensing pathways, such as daf-2, akt-2, akt-1, eat-2, sir-2.1, and rsks-1. Naringin treatment prolonged the lifespan of long-lived glp-1 mutants, which have decreased reproductive stem cells. Naringin could not extend the lifespan of a null mutant of the fox-head transcription factor DAF-16. Moreover, naringin could increase the mRNA expression of genes regulated by daf-16 and itself. In conclusion, we show that a natural product naringin could extend the lifespan of C. elegans and delay the progression of aging-related diseases in C. elegans models via DAF-16.

GABAergic system's Injuries Induced by Sodium Sulfite in Caenorhabditis elegans Were Prevented by the Anti-Oxidative Properties of Dehydroepiandrosterone Sulfate.

Several pathophysiological processes involve Hypoxia conditions, where the nervous system is affected as well. We postulate that the GABAergic system is especially sensitive. Furthermore, drugs improving the resistance to hypoxia have been investigated, such as the neurosteroid dehydroepiandrosterone sulfate (DHEAS) which has shown beneficial effects in hypoxic processes in mammals; however, at the cellular level, its exact mechanism of action has yet to be fully elucidated. Here, we used a chemical hypoxia model through sodium sulfite (SS) exposure in Caenorhabditis elegans (C. elegans), a nematode whose response to hypoxia involves pathways and cellular processes conserved in mammals, and that allows study the direct effect of DHEAS without its conversion to sex hormones. This work aimed to determine the effect of DHEAS on damage to the GABAergic system associated with SS exposure in C. elegans. Worms were subjected to nose touch response (Not Assay) and observed in epifluorescence microscopy. DHEAS decreased the shrinkage response of Not Assay and the level of damage in GABAergic neurons on SS-exposed worms. Also, the enhanced nuclear localization of DAF-16 and consequently the overexpression of chaperone HSP-16.2 by hypoxia were significantly reduced in SS + DHEAS exposed worms. As well, DHEAS increased the survival rate of worms exposed to hydrogen peroxide. These results suggest that hypoxia-caused damage over the GABAergic system was prevented at least partially by DHEAS, probably through non-genomic mechanisms that involve its antioxidant properties related to its chemical structure.

SKN-1 is involved in combination of apple peels and blueberry extracts synergistically protecting against oxidative stress in Caenorhabditis elegans.

Increased consumption of fruits and vegetables is associated with reduced risk of age-related functional declines and chronic diseases, primarily attributed to their bioactive phytochemicals. Apples and blueberries are rich in phytochemicals with a wide range of biological activities and health benefits. Our previous research has shown the combination of apple peel extracts (APE) and blueberry extracts (BE) can synergistically promote the lifespan of Caenorhabditis elegans (C. elegans). The objectives of this study were to determine whether the extension of lifespan was involved in regulation of oxidative stress, and to explore the underlying mechanisms of action. The results showed that the combination of APE and BE could synergistically ameliorate oxidative stress by improving antioxidant enzyme activities and enhancing resistance to paraquat. Meanwhile, treatment with APE plus BE could down-regulate the overexpression of reactive oxygen species (ROS) and affect the expression of antioxidant related genes, including sod-3, cat-1, ctl-1, skn-1, mev-1 and isp-1. However, administration with APE plus BE abolished the extension of the lifespan of skn-1(zu135) mutants, and inhibited the expression of skn-1 downstream genes, including gcs-1, gst-4 and gst-7. In addition, supplementation with APE plus BE could promote the migration of SKN-1 into the nucleus, which eliminated improvement to ROS and paraquat. In conclusion, the combination of APE and BE could synergistically protect against oxidative stress in C. elegans via the SKN-1/Nrf2 pathway. This study provided the theoretical basis to explore the combination of phytochemicals in the prevention of aging regulated by oxidative stress.

Thallium Toxicity in Caenorhabditis elegans: Involvement of the SKN-1 Pathway and Protection by S-Allylcysteine.

Monovalent thallium (Tl+) is a cation that can exert complex neurotoxic patterns in the brain by mechanisms that have yet to be completely characterized. To learn more about Tl+ toxicity, it is necessary to investigate its major effects in vivo and its ability to trigger specific signaling pathways (such as the antioxidant SKN-1 pathway) in different biological models. Caenorhabditis elegans (C. elegans) is a nematode constituting a simple in vivo biological model with a well-characterized nervous system, and high genetic homology to mammalian systems. In this study, both wild-type (N2) and skn-1 knockout (KO) mutant C. elegans strains subjected to acute and chronic exposures to Tl+ [2.5-35 µM] were evaluated for physiological stress (survival, longevity, and worm size), motor alterations (body bends), and biochemical changes (glutathione S-transferase regulation in a gst-4 fluorescence strain). While survival was affected by Tl+ in N2 and skn-1 KO (worms lacking the orthologue of mammalian Nrf2) strains in a similar manner, the longevity was more prominently decreased in the skn-1 KO strain compared with the wild-type strain. Moreover, chronic exposure led to a greater compromise in the longevity in both strains compared with acute exposure. Tl+ also induced motor alterations in both skn-1 KO and wild-type strains, as well as changes in worm size in wild-type worms. In addition, preconditioning nematodes with the well-known antioxidant S-allylcysteine (SAC) reversed the Tl+-induced decrease in survival in the N2 strain. GST fluorescent expression was also decreased by the metal in the nematode, and recovered by SAC. Our results describe and validate, for the first time, features of the toxic pattern induced by Tl+ in an in vivo biological model established with C. elegans, supporting an altered redox component in Tl+ toxicity, as previously described in mammal models. We demonstrate that the presence of the orthologous SKN-1 pathway is required for worms in evoking an efficient antioxidant defense. Therefore, the nematode represents an optimal model to reproduce mammalian Tl+ toxicity, where toxic mechanisms and novel therapeutic approaches of clinical value may be successfully pursued.

Arsenite-induced transgenerational glycometabolism is associated with up-regulation of H3K4me2 via inhibiting spr-5 in caenorhabditis elegans.

Arsenic (As) is a toxic element that is highly abundant in the environment. However, there has not been sufficient research into the mechanisms of arsenic-induced transgenerational effects. In biomedical and environmental toxicology research field, C. elegans are often used as the ideal model. In this study, F0 generation animals were cultured with arsenite, while subsequent generations animals (F1 - F6) were cultured in the absence of arsenic. Experiments were performed to examine the transgenerational glycometabolism and the associated mechanisms in all seven generations (F0 - F6) of C. elegans. Results show that arsenite exposure increased total glucose content but reduced glucose metabolites in F0 generation C. elegans. The total glucose content was also elevated in subsequent generations probably due to transgenerational downregulation of fgt-1. In addition, arsenite exposure induced transgenerational downregulation of histone demethyltransferase spr-5 and elevated histone dimethylation in F0 generation. This study highlights that single generation exposure to arsenite causes transgenerational changes in glycometabolism in C. elegans, which may be caused by downregulation of spr-5 and elevation of H3K4me2.

Tunable light and drug induced depletion of target proteins.

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.

Hibiscus sabdariffa L. extract prolongs lifespan and protects against amyloid-ß toxicity in Caenorhabditis elegans: involvement of the FoxO and Nrf2 orthologues DAF-16 and SKN-1.

PURPOSE: Hibiscus sabdariffa L. is commonly used as an ingredient for herbal teas and food supplements. Several studies demonstrated the beneficial effects of Hibiscus sabdariffa L. extracts (HSE); however, the bioactive components and their mode of action still remain unclear. Caenorhabditis elegans (C. elegans) was used to study health-related effects and the underlying molecular mechanisms of HSE in this model organism as well as effects of hydroxycitric acid (HCA), a main compound of HSE, and its structural analogue isocitric acid (ICA). METHODS: Survival and locomotion were detected by touch-provoked movement. Thermotolerance was analysed using the nucleic acid stain SYTOX green, and intracellular ROS accumulation was measured via oxidation of H2DCF. Localisation of the transcription factors DAF-16 and SKN-1 was analysed in transgenic strains (DAF-16::GFP, SKN-1::GFP). The involvement of DAF-16 and SKN-1 was further investigated using loss-of-function strains as well as gene silencing by feeding RNAi-inducing bacteria. Protection against amyloid-ß toxicity was analysed using a transgenic strain with an inducible expression of human amyloid-ß peptides in body wall muscle cells (paralysis assay). RESULTS: HSE treatment resulted in a prominent extension of lifespan (up to 24%) and a reduction of the age-dependent decline in locomotion. HCA, a main compound of HSE increased lifespan too, but to a lesser extent (6%) while ICA was not effective. HSE and HCA did not modulate resistance against thermal stress conditions and did not exert antioxidative effects: HSE rather increased intracellular ROS levels, suggesting a pro-oxidative effect of the extract in vivo. HSE and HCA increased the nuclear localisation of the pivotal transcription factors DAF-16 and SKN-1 indicating an activation of these factors. Consistent with this result, lifespan prolongation by HSE was dependent on both transcription factors. In addition to the positive effect on lifespan, HSE treatment also elicited a (strong) protection against amyloid-ß induced toxicity in C. elegans in a DAF-16- and SKN-1-dependent manner. CONCLUSION: Our results demonstrate that HSE increases lifespan and protects against amyloid-ß toxicity in the model organism C. elegans. These effects were mediated, at least in parts via modulation of pathways leading to activation/nuclear localisation of DAF-16 and SKN-1. Since HCA, a main component of HSE causes only minor effects, additional bioactive compounds like flavonoids or anthocyanins as well as synergistic effects of these compounds should be investigated.

Recombinant Buckwheat Trypsin Inhibitor Improves the Protein and Mitochondria Homeostasis in Caenorhabditis elegans Model of Aging and Age-Related Disease.

BACKGROUND: With the acceleration of aging process in human society, improvements of the physical functionality and life quality in the elderly population are more meaningful than pure longevity. Buckwheat trypsin inhibitor is a low molecular weight polypeptide extracted from buckwheat, which is a beneficial food for improving the health in the elderly. OBJECTIVES: The aim of the current study was to evaluate the potential beneficial effects of recombinant buckwheat trypsin inhibitor (rBTI) on age-dependent function decline and the primary mechanism. METHOD: Day 10 N2 Caenorhabditis elegans and day 6 AM140 C. elegans cultured at 25°C were used as models of aging and age-related disease, respectively. Motor function was as an indicator of age-dependent function. ATP content and damage mitochondrial DNA mass were detected to assess mitochondrial damage and function by ATP Assay Kit and agarose gel electrophoresis, respectively. Soluble protein content was quantified by SDS polyacrylamide gel electrophoresis. Autophagy-related genes transcription levels, autophagy marker proteins lgg-1, and lysosomal content were analyzed to quantify autophagy levels by qRT-PCR, transgenic C. elegans, and lysosomal staining. Autophagy inhibitor chloroquine, daf-16 mutant, and RNA Interference were used to determine the roles of autophagy and DAF-16 in rBTI-mediated effects. RESULTS: In this study, we found that rBTI could decrease the proportions of insoluble protein and impaired mitochondria, finally reduce motility deficits in both models. Further study indicated that rBTI activated the autophagy, and the inhibition of autophagy reduced rBTI-mediated beneficial effects. Genetic analyses showed the transcriptional activity of DAF-16 was increased by rBTI and was required for rBTI-mediated beneficial effects. CONCLUSIONS: These data indicated that rBTI might promote the autophagy to alleviate the age-related functional decline via DAF-16 in C. elegans and suggested a potential role of rBTI as a nutraceutical for the improvement of age-related complications.

Longitudinal Metabolic Impacts of Perinatal Exposure to Phthalates and Phthalate Mixtures in Mice.

Developmental exposures to phthalates are suspected to contribute to risk of metabolic syndrome. However, findings from human studies are inconsistent, and long-term metabolic impacts of early-life phthalate and phthalate mixture exposures are not fully understood. Furthermore, most animal studies investigating metabolic impacts of developmental phthalate exposures have focused on diethylhexyl phthalate (DEHP), whereas newer phthalates, such as diisononyl phthalate (DINP), are understudied. We used a longitudinal mouse model to evaluate long-term metabolic impacts of perinatal exposures to three individual phthalates, DEHP, DINP, and dibutyl phthalate (DBP), as well as two mixtures (DEHP+DINP and DEHP+DINP+DBP). Phthalates were administered to pregnant and lactating females through phytoestrogen-free chow at the following exposure levels: 25 mg of DEHP/kg of chow, 25 mg of DBP/kg of chow, and 75 mg of DINP/kg of chow. One male and female per litter (n = 9 to 13 per sex per group) were weaned onto control chow and followed until 10 months of age. They underwent metabolic phenotyping at 2 and 8 months, and adipokines were measured in plasma collected at 10 months. Longitudinally, females perinatally exposed to DEHP only had increased body fat percentage and decreased lean mass percentage, whereas females perinatally exposed to DINP only had impaired glucose tolerance. Perinatal phthalate exposures also modified the relationship between body fat percentage and plasma adipokine levels at 10 months in females. Phthalate-exposed males did not exhibit statistically significant differences in the measured longitudinal metabolic outcomes. Surprisingly, perinatal phthalate mixture exposures were statistically significantly associated with few metabolic effects and were not associated with larger effects than single exposures, revealing complexities in metabolic effects of developmental phthalate mixture exposures.

Early life exposure of a biocide, CMIT/MIT causes metabolic toxicity via the O-GlcNAc transferase pathway in the nematode C. elegans.

Unusual cases of fatal lung injury, later determined to be a result of exposure to chemicals used as humidifier disinfectants, were reported among Korean children from 2006 to 2011. This resulted in considerable study of the pulmonary toxicity of humidifier disinfectant chemicals to establish the causal relationship between exposure and lung disease. However, the systemic toxicity of the former and health effects other than lung disease are not fully understood. Here, we investigated the effect of 5-chloro-2-methyl-4-isothiazoline-3-one and 2-methyl-4-isothiazolin-3-one (CMIT/MIT), among the humidifier disinfectants used in the accidents, on the development of metabolic toxicity in the model organism, Caenorhabditis elegans using an exposure scenario comparison. We screened the potential of CMIT/MIT to induce metabolic toxicity using C. elegans oga-1(ok1207) and ogt-1(ok1474) mutants. We also performed a pathway analysis based on C. elegans transcription factor RNAi library screening to identify the underlying toxicity mechanisms. Finally, to understand the critical window of exposure for metabolic toxicity, responses to exposure during different periods in the life cycles of the worms were compared. We determined that CMIT/MIT could induce metabolic toxicity through O-linked N-acetylglucosamine transferase and early life seems to be the critical window for exposure for metabolic toxicity for this substance. The O-linked N-acetylglucosamine transferase pathway is conserved from worms to humans; our results thus insinuate that early-life exposure to CMIT/MIT could cause metabolic health problems during adult life in humans. We therefore suggest that a systemic toxicity approach should be considered to comprehensively understand the adverse health effects of humidifier disinfectant misuse.

Bark Extract of the Amazonian Tree Endopleura uchi (Humiriaceae) Extends Lifespan and Enhances Stress Resistance in Caenorhabditis elegans.

Endopleura uchi (Huber) Cuatrec (Humiriaceae), known as uxi or uxi-amarelo in Brazil, is an endemic tree of the Amazon forest. In traditional medicine, its stem bark is used to treat a variety of health disorders, including cancer, diabetes, arthritis, uterine inflammation, and gynecological infections. According to HPLC analysis, the main constituent of the bark extract is the polyphenol bergenin. In the current study, we demonstrate by in vitro and in vivo experiments the antioxidant potential of a water extract from the stem bark of E. uchi. When tested in the model organism Caenorhabditis elegans, the extract enhanced stress resistance via the DAF-16/FOXO pathway. Additionally, the extract promoted an increase in the lifespan of the worms independent from caloric restriction. It also attenuated the age-related muscle function decline and formation of polyQ40 plaques, as a model for Huntington's disease. Thus, these data support anti-aging and anti-oxidant properties of E. uchi, which has not yet been described. More studies are needed to assess the real benefits of E. uchi bark for human health and its toxicological profile.

Cannabinoids Stimulate the TRP Channel-Dependent Release of Both Serotonin and Dopamine to Modulate Behavior in C. elegans.

Cannabis sativa alters sensory perception and exhibits potential medicinal benefits. In mammals, cannabinoids activate two canonical receptors, CB1/CB2, as well additional receptors/ion channels whose overall contributions to cannabinoid signaling have yet to be fully assessed. In Caenorhabditis elegans, the endogenous cannabinoid receptor agonist, 2-arachidonoylglycerol (2-AG) activates a CB1 ortholog, NPR-19, to modulate behavior (Oakes et al., 2017). In addition, 2-AG stimulates the NPR-19 independent release of both serotonin (5-HT) and dopamine (DA) from subsets of monoaminergic neurons to modulate locomotory behaviors through a complex monoaminergic signaling pathway involving multiple serotonin and dopamine receptors. 2-AG also inhibits locomotion in remodeled monoamine receptor mutant animals designed to measure the acute release of either 5-HT or DA, confirming the direct effects of 2-AG on monoamine release. 2-AG-dependent locomotory inhibition requires the expression of transient receptor potential vanilloid 1 (TRPV1) and TRPN-like channels in the serotonergic or dopaminergic neurons, respectively, and the acute pharmacological inhibition of the TRPV1-like channel abolishes both 2-AG-dependent 5-HT release and locomotory inhibition, suggesting the 2-AG may activate the channel directly. This study highlights the advantages of identifying and assessing both CB1/CB2-dependent and independent cannabinoid signaling pathways in a genetically tractable, mammalian predictive model, where cannabinoid signaling at the molecular/neuronal levels can be correlated directly with changes in behavior.SIGNIFICANCE STATEMENT This study is focused on assessing CB1/CB2-independent cannabinoid signaling in a genetically tractable, whole-animal model where cannabinoid signaling at the molecular/neuronal levels can be correlated with behavioral change. Caenorhabditis elegans contains a cannabinoid signaling system mediated by a canonical cannabinoid receptor, NPR-19, with orthology to human CB1/CB2 (Oakes et al., 2017). The present study has characterized an NPR-19-independent signaling pathway that involves the cannabinoid-dependent release of both serotonin and dopamine and the expression of distinct TRP-like channels on the monoaminergic neurons. Our work should be of interest to those studying the complexities of CB1/CB2-independent cannabinoid signaling, the role of TRP channels in the modulation of monoaminergic signaling, and the cannabinoid-dependent modulation of behavior.

The transcription factor NHR-8: A new target to increase ivermectin efficacy in nematodes.

Resistance to the anthelmintic macrocyclic lactone ivermectin (IVM) has a great impact on the control of parasitic nematodes. The mechanisms by which nematodes adapt to IVM remain to be deciphered. We have identified NHR-8, a nuclear hormone receptor involved in the xenobiotic response in Caenorhabditis elegans, as a new regulator of tolerance to IVM. Loss-of-function nhr-8(ok186) C. elegans mutants subjected to larval development assays and electropharyngeogram measurements, displayed hypersensitivity to IVM, and silencing of nhr-8 in IVM-resistant worms increased IVM efficacy. In addition, compared to wild-type worms, nhr-8 mutants under IVM selection pressure failed to acquire tolerance to the drug. In addition, IVM-hypersensitive nhr-8(ok186) worms displayed low transcript levels of several genes from the xenobiotic detoxification network and a concomitant low Pgp-mediated drug efflux activity. Interestingly, some pgp and cyp genes known to impact IVM tolerance in many nematode species, were down regulated in nhr-8 mutants and inversely upregulated in IVM-resistant worms. Moreover, pgp-6 overexpression in nhr-8(ok186) C. elegans increased tolerance to IVM. Importantly, NHR-8 function was rescued in nhr-8(ok186) C. elegans with the homolog of the parasitic nematode Haemonchus contortus, and silencing of Hco-nhr-8 by RNAi on L2 H. contortus larvae increased IVM susceptibility in both susceptible and resistant H. contortus isolates. Thus, our data show that NHR-8 controls the tolerance and development of resistance to IVM in C. elegans and the molecular basis for this relates to the NHR-8-mediated upregulation of IVM detoxification genes. Since our results show that Hco-nhr-8 functions similarly to Cel-nhr-8, this study helps to better understand mechanisms underlying failure in drug efficacy and open perspectives in finding new compounds with NHR-8 antagonist activity to potentiate IVM efficacy.

3-Methyl-3-buten-1-ol (isoprenol) confers longevity and stress tolerance in Caenorhabditis elegans.

The present investigation demonstrates the longevity-promoting effects of 3-methyl-3-buten-1-ol (isoprenol) in the animal model Caenorhabditis elegans that might be served as a lead nutraceutical in geriatric research. Our results showed that 0.5 mM isoprenol extended the mean lifespan of worms by 25% in comparison to control worms. Isoprenol also significantly enhanced survival of the worms under various stress conditions. It was found that the longevity-promoting effects of isoprenol were associated with improved age-associated physiological behaviour and reduced intracellular reactive oxygen species (ROS) accumulation. Finally, studies with gene-specific mutants revealed the involvement of pro-longevity transcription factors (TFs) DAF-16 and SKN-1 with simultaneous over-expression of GST-4 and SOD-3 in isoprenol treated worms. In silico analysis revealed the binding affinity of isoprenol with DAF-16 and SKN-1 TFs. Together, the findings suggest that isoprenol is able to enhance the lifespan of C. elegans and embarks its potential in the developments of formulations for age-related ailments.

Blockade and reversal of swimming-induced paralysis in C. elegans by the antipsychotic and D2-type dopamine receptor antagonist azaperone.

The catecholamine neurotransmitter dopamine (DA) exerts powerful modulatory control of physiology and behavior across phylogeny. Perturbations of DA signaling in humans are associated with multiple neurodegenerative and behavioral disorders, including Parkinson's disease, attention-deficit/hyperactivity disorder, addiction and schizophrenia. In the nematode C. elegans, DA signaling regulates mating behavior, learning, food seeking and locomotion. Previously, we demonstrated that loss of function mutations in the dat-1 gene that encodes the presynaptic DA transporter (DAT-1) results in a rapid cessation of movement when animals are placed in water, termed Swimming Induced Paralysis (Swip). Loss of function mutations in genes that support DA biosynthesis, DA vesicular packaging and DA action at the extrasynaptic D2-type DA receptor DOP-3 suppress Swip in dat-1 animals, consistent with paralysis as arising from excessive DA signaling. Although animals grown on the vesicular monoamine transporter antagonist reserpine diminish Swip, the drug must be applied chronically, can impact the signaling of multiple biogenic amines, and has been reported to have penetrant, off-target actions. Here, we demonstrate that the antipsychotic drug azaperone potently and rapidly suppresses Swip behavior in either dat-1 mutants, as well as in wildtype animals treated with the DAT-1 antagonist nisoxetine, with genetic experiments consistent with DOP-3 antagonism as the mechanism of Swip suppression. Reversal of Swip in previously paralyzed dat-1 animals by azaperone application demonstrates an otherwise functionally-intact swimming circuit in these mutants. Finally, whereas azaperone suppresses DA-dependent Swip, the drug fails to attenuate the DA-independent paralysis induced by ßPEA, aldicarb or genetic disruption of γ-aminobutyric acid (GABA) signaling. We discuss our findings with respect to the use of azaperone as a potent and selective tool in the identification and analysis of presynaptic mechanisms that regulate DA signaling.