The flight response impairs cytoprotective mechanisms by activating the insulin pathway.

An animal's stress response requires different adaptive strategies depending on the nature and duration of the stressor. Whereas acute stressors, such as predation, induce a rapid and energy-demanding fight-or-flight response, long-term environmental stressors induce the gradual and long-lasting activation of highly conserved cytoprotective processes1-3. In animals across the evolutionary spectrum, continued activation of the fight-or-flight response weakens the animal's resistance to environmental challenges4,5. However, the molecular and cellular mechanisms that regulate the trade-off between the flight response and long-term stressors are poorly understood. Here we show that repeated induction of the flight response in Caenorhabditis elegans shortens lifespan and inhibits conserved cytoprotective mechanisms. The flight response activates neurons that release tyramine, an invertebrate analogue of adrenaline and noradrenaline. Tyramine stimulates the insulin-IGF-1 signalling (IIS) pathway and precludes the induction of stress response genes by activating an adrenergic-like receptor in the intestine. By contrast, long-term environmental stressors, such as heat or oxidative stress, reduce tyramine release and thereby allow the induction of cytoprotective genes. These findings demonstrate that a neural stress hormone supplies a state-dependent neural switch between acute flight and long-term environmental stress responses and provides mechanistic insights into how the flight response impairs cellular defence systems and accelerates ageing.

A Change in the Ion Selectivity of Ligand-Gated Ion Channels Provides a Mechanism to Switch Behavior.

Behavioral output of neural networks depends on a delicate balance between excitatory and inhibitory synaptic connections. However, it is not known whether network formation and stability is constrained by the sign of synaptic connections between neurons within the network. Here we show that switching the sign of a synapse within a neural circuit can reverse the behavioral output. The inhibitory tyramine-gated chloride channel, LGC-55, induces head relaxation and inhibits forward locomotion during the Caenorhabditis elegans escape response. We switched the ion selectivity of an inhibitory LGC-55 anion channel to an excitatory LGC-55 cation channel. The engineered cation channel is properly trafficked in the native neural circuit and results in behavioral responses that are opposite to those produced by activation of the LGC-55 anion channel. Our findings indicate that switches in ion selectivity of ligand-gated ion channels (LGICs) do not affect network connectivity or stability and may provide an evolutionary and a synthetic mechanism to change behavior.

The ketone body ß-hydroxybutyrate ameliorates neurodevelopmental deficits in the GABAergic system of daf-18/PTEN Caenorhabditis elegans mutants.

A finely tuned balance between excitation and inhibition (E/I) is essential for proper brain function. Disruptions in the GABAergic system, which alter this equilibrium, are a common feature in various types of neurological disorders, including autism spectrum disorders (ASDs). Mutations in Phosphatase and Tensin Homolog (PTEN), the main negative regulator of the phosphatidylinositol 3-phosphate kinase/Akt pathway, are strongly associated with ASD. However, it is unclear whether PTEN deficiencies can differentially affect inhibitory and excitatory signaling. Using the Caenorhabditis elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) inputs regulate muscle activity, we found that daf-18/PTEN mutations impact GABAergic (but not cholinergic) neurodevelopment and function. This selective impact results in a deficiency in inhibitory signaling. The defects observed in the GABAergic system in daf-18/PTEN mutants are due to reduced activity of DAF-16/FOXO during development. Ketogenic diets (KGDs) have proven effective for disorders associated with E/I imbalances. However, the mechanisms underlying their action remain largely elusive. We found that a diet enriched with the ketone body ß-hydroxybutyrate during early development induces DAF-16/FOXO activity, therefore improving GABAergic neurodevelopment and function in daf-18/PTEN mutants. Our study provides valuable insights into the link between PTEN mutations and neurodevelopmental defects and delves into the mechanisms underlying the potential therapeutic effects of KGDs.

To work optimally, the brain needs to delicately balance excitation and inhibition ­ that is, it must precisely control exactly when and how excitatory neurons (which activate the system) or inhibitory ones (which counteract these activating signals) are switched on. Neurological disorders can arise when this equilibrium is disrupted, for example when defects are present in an inhibitory signalling system that relies on a molecule known as GABA. More recently, a gene known as PTEN has also emerged as playing an important role during the development of the nervous system, yet exactly why this is the case has remained unclear. To explore this question, Giunti et al. focused on the neuromuscular system of the roundworm Caenorhabditis elegans, in which excitatory ('cholinergic') and inhibitory ('GABAergic') neurons control how muscles contract and relax. A range of biological approaches were used to assess the impact of PTEN deficiencies on this system. This revealed that mutations in this gene do not impact cholinergic activity; they did, however, lead to diminished GABAergic activity. Overall, this resulted in an increased ratio of excitatory to inhibitory activity in the system. Further work showed that, in the mutated worms, the suppression of inhibitory neurons was due to a specific protein being inactive during early development. This transcription factor is the worm equivalent of the human FOXO protein, and it helps to turn genes on and off during development. Its inactivity is linked to noticeable changes in the shape and activity of GABAergic neurons. In humans, medical ketogenic diets (which force the body to use fats rather than sugars as a source of energy) are known to improve conditions linked to imbalances in the excitatory and inhibitory systems. Giunti et al. therefore investigated whether a similar approach could mitigate some of the defects seen in PTEN mutants. Exposing these worms early in development to a type of molecule produced in ketogenic diets partly improved the state of their GABAergic neurons. Taken together, this work suggests a potential molecular basis for the association between PTEN and the balance between excitatory and inhibitory activity. As PTEN mutations are often found in individuals diagnosed with autism spectrum disorders, further research is necessary to validate these findings in mammals and explore their clinical relevance.

The neurohormone tyramine stimulates the secretion of an Insulin-Like Peptide from the intestine to modulate the systemic stress response in C. elegans.

The DAF-2/insulin/insulin-like growth factor signaling (IIS) pathway plays an evolutionarily conserved role in regulating reproductive development, lifespan, and stress resistance. In C. elegans , DAF-2/IIS signaling is modulated by an extensive array of insulin-like peptides (ILPs) with diverse spatial and temporal expression patterns. However, the release dynamics and specific functions of these ILPs in adapting to different environmental conditions remain poorly understood. Here, we show that the ILP, INS-3, plays a crucial role in modulating the response to different types of stressors in C. elegans . ins-3 mutants display increased resistance to both heat and oxidative stress; however, under favorable conditions, this advantage is countered by slower reproductive development. ins-3 expression in both neurons and the intestine is downregulated in response to environmental stressors. Conversely, the neurohormone tyramine, which is released during the acute flight response, triggers an upregulation in ins-3 expression. Moreover, we found that tyramine negatively impacts environmental stress resistance by stimulating the release of INS-3 from the intestine. The subsequent release of INS-3 systemically activates the DAF-2 pathway, resulting in the inhibition of cytoprotective mechanisms mediated by DAF-16/FOXO and HSF-1. These studies offer mechanistic insights into the brain-gut communication pathway that weighs adaptive strategies to respond to acute and long-term stress scenarios.

An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse.

Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the 'levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER.

How specific molecules can lead to overeating.

A molecular pathway involving compounds found in processed foods and biogenic amines increases food intake and aging in the roundworm C. elegans.

Contribution of subunits to Caenorhabditis elegans levamisole-sensitive nicotinic receptor function.

Caenorhabditis elegans muscle contains seven different nicotinic receptor (AChR) subunits, five of which have been shown to be components of adult levamisole-sensitive AChRs (L-AChRs). To elucidate the reason for such subunit diversity, we explore their functional roles in larva 1 (L1) muscle cells. Single-channel and macroscopic current recordings reveal that the α-type LEV-8 subunit is a component of native L1 L-AChRs but behaves as a nonessential subunit. It plays a key role in maintaining a low rate and extent of desensitization of L-AChRs. In the absence of the α-type ACR-8 subunit, L-AChR channel properties are not modified, thus indicating that ACR-8 is not a component of L1 L-AChRs. Together with our previous findings, this study reveals that L1 muscle cells express a main L-AChR type composed of five different subunits: UNC-38, UNC-63, UNC-29, LEV-1, and LEV-8. Analysis of a double lev-8; acr-8-null mutant, which shows an uncoordinated and levamisole-resistant phenotype, reveals that ACR-8 can replace LEV-8 in its absence, thus attributing a functional role to this subunit. Docking into homology modeled L-AChRs proposes that ACh forms the typical cation-π interaction, suggests why levamisole is less efficacious than ACh, and shows that ACR-8 can form activatable binding-sites, thus opening doors for elucidating subunit arrangement and anthelmintic selectivity.

A Cys-loop mutation in the Caenorhabditis elegans nicotinic receptor subunit UNC-63 impairs but does not abolish channel function.

The nematode Caenorhabditis elegans is an established model organism for studying neurobiology. UNC-63 is a C. elegans nicotinic acetylcholine receptor (nAChR) α-subunit. It is an essential component of the levamisole-sensitive muscle nAChR (L-nAChR) and therefore plays an important role in cholinergic transmission at the nematode neuromuscular junction. Here, we show that worms with the unc-63(x26) allele, with its αC151Y mutation disrupting the Cys-loop, have deficient muscle function reflected by impaired swimming (thrashing). Single-channel recordings from cultured muscle cells from the mutant strain showed a 100-fold reduced frequency of opening events and shorter channel openings of L-nAChRs compared with those of wild-type worms. Anti-UNC-63 antibody staining in both cultured adult muscle and embryonic cells showed that L-nAChRs were expressed at similar levels in the mutant and wild-type cells, suggesting that the functional changes in the receptor, rather than changes in expression, are the predominant effect of the mutation. The kinetic changes mimic those reported in patients with fast-channel congenital myasthenic syndromes. We show that pyridostigmine bromide and 3,4-diaminopyridine, which are drugs used to treat fast-channel congenital myasthenic syndromes, partially rescued the motility defect seen in unc-63(x26). The C. elegans unc-63(x26) mutant may therefore offer a useful model to assist in the development of therapies for syndromes produced by altered function of human nAChRs.

1-Mesityl-3-(3-Sulfonatopropyl) Imidazolium Protects Against Oxidative Stress and Delays Proteotoxicity in C. elegans.

Due to the increase in life expectancy worldwide, age-related disorders such as neurodegenerative diseases (NDs) have become more prevalent. Conventional treatments comprise drugs that only attenuate some of the symptoms, but fail to arrest or delay neuronal proteotoxicity that characterizes these diseases. Due to their diverse biological activities, imidazole rings are intensively explored as powerful scaffolds for the development of new bioactive molecules. By using C. elegans, our work aims to explore novel biological roles for these compounds. To this end, we have tested the in vivo anti-proteotoxic effects of imidazolium salts. Since NDs have been largely linked to impaired antioxidant defense mechanisms, we focused on 1-Mesityl-3-(3-sulfonatopropyl) imidazolium (MSI), one of the imidazolium salts that we identified as capable of improving iron-induced oxidative stress resistance in wild-type animals. By combining mutant and gene expression analysis we have determined that this protective effect depends on the activation of the Heat Shock Transcription Factor (HSF-1), whereas it is independent of other canonical cytoprotective molecules such as abnormal Dauer Formation-16 (DAF-16/FOXO) and Skinhead-1 (SKN-1/Nrf2). To delve deeper into the biological roles of MSI, we analyzed the impact of this compound on previously established C. elegans models of protein aggregation. We found that MSI ameliorates ß-amyloid-induced paralysis in worms expressing the pathological protein involved in Alzheimer's Disease. Moreover, this compound also delays age-related locomotion decline in other proteotoxic C. elegans models, suggesting a broad protective effect. Taken together, our results point to MSI as a promising anti-proteotoxic compound and provide proof of concept of the potential of imidazole derivatives in the development of novel therapies to retard age-related proteotoxic diseases.

Drug discovery: Insights from the invertebrate Caenorhabditis elegans.

Therapeutic drug development is a long, expensive, and complex process that usually takes 12-15 years. In the early phases of drug discovery, in particular, there is a growing need for animal models that ensure the reduction in both cost and time. Caenorhabditis elegans has been traditionally used to address fundamental aspects of key biological processes, such as apoptosis, aging, and gene expression regulation. During the last decade, with the advent of large-scale platforms for screenings, this invertebrate has also emerged as an essential tool in the pharmaceutical research industry to identify novel drugs and drug targets. In this review, we discuss the reasons why C. elegans has been positioned as an outstanding cost-effective option for drug discovery, highlighting both the advantages and drawbacks of this model. Particular attention is paid to the suitability of this nematode in large-scale genetic and pharmacological screenings. High-throughput screenings in C. elegans have indeed contributed to the breakthrough of a wide variety of candidate compounds involved in extensive fields including neurodegeneration, pathogen infections and metabolic disorders. The versatility of this nematode, which enables its instrumentation as a model of human diseases, is another attribute also herein underscored. As illustrative examples, we discuss the utility of C. elegans models of both human neurodegenerative diseases and parasitic nematodes in the drug discovery industry. Summing up, this review aims to demonstrate the impact of C. elegans models on the drug discovery pipeline.

Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.

Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

Coupling of agonist binding to channel gating in an ACh-binding protein linked to an ion channel.

Neurotransmitter receptors from the Cys-loop superfamily couple the binding of agonist to the opening of an intrinsic ion pore in the final step in rapid synaptic transmission. Although atomic resolution structural data have recently emerged for individual binding and pore domains, how they are linked into a functional unit remains unknown. Here we identify structural requirements for functionally coupling the two domains by combining acetylcholine (ACh)-binding protein, whose structure was determined at atomic resolution, with the pore domain from the serotonin type-3A (5-HT3A) receptor. Only when amino-acid sequences of three loops in ACh-binding protein are changed to their 5-HT3A counterparts does ACh bind with low affinity characteristic of activatable receptors, and trigger opening of the ion pore. Thus functional coupling requires structural compatibility at the interface of the binding and pore domains. Structural modelling reveals a network of interacting loops between binding and pore domains that mediates this allosteric coupling process.

Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans.

Mutations in pre-synaptic voltage-gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic and decreased GABAergic transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin-dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.

Co-option of neurotransmitter signaling for inter-organismal communication in C. elegans.

Biogenic amine neurotransmitters play a central role in metazoan biology, and both their chemical structures and cognate receptors are evolutionarily conserved. Their primary roles are in cell-to-cell signaling, as biogenic amines are not normally recruited for communication between separate individuals. Here, we show that in the nematode C. elegans, a neurotransmitter-sensing G protein-coupled receptor, TYRA-2, is required for avoidance responses to osas#9, an ascaroside pheromone that incorporates the neurotransmitter, octopamine. Neuronal ablation, cell-specific genetic rescue, and calcium imaging show that tyra-2 expression in the nociceptive neuron, ASH, is necessary and sufficient to induce osas#9 avoidance. Ectopic expression in the AWA neuron, which is generally associated with attractive responses, reverses the response to osas#9, resulting in attraction instead of avoidance behavior, confirming that TYRA-2 partakes in the sensing of osas#9. The TYRA-2/osas#9 signaling system represents an inter-organismal communication channel that evolved via co-option of a neurotransmitter and its cognate receptor.

Diisopropylphenyl-imidazole (DII): A new compound that exerts anthelmintic activity through novel molecular mechanisms.

Nematode parasites cause substantial morbidity to billions of people and considerable losses in livestock and food crops. The repertoire of effective anthelmintic compounds for treating these parasitoses is very limited, as drug development has been delayed for decades. Moreover, resistance has become a global concern in livestock parasites and is an emerging issue for human helminthiasis. Therefore, anthelmintics with novel mechanisms of action are urgently needed. Taking advantage of Caenorhabditis elegans as an established model system, we here screened the nematicidal potential of novel imidazolium and imidazole derivatives. One of these derivatives, diisopropylphenyl-imidazole (DII), is lethal to C. elegans at both mature and immature stages. This lethal effect appears to be specific because DII concentrations which prove to be toxic to C. elegans do not induce significant lethality on bacteria, Drosophila melanogaster, and HEK-293 cells. Our analysis of DII action on C. elegans mutant strains determined that, in the adult stage, null mutants of unc-29 are resistant to the drug. Muscle expression of this gene completely restores DII sensitivity. UNC-29 has been largely reported as an essential constituent of the levamisole-sensitive muscle nicotinic receptor (L-AChR). Nevertheless, null mutants in unc-63 and lev-8 (essential and non-essential subunits of L-AChRs, respectively) are as sensitive to DII as the wild-type strain. Therefore, our results suggest that DII effects on adult nematodes rely on a previously unidentified UNC-29-containing muscle AChR, different from the classical L-AChR. Interestingly, DII targets appear to be different between larvae and adults, as unc-29 null mutant larvae are sensitive to the drug. The existence of more than one target could delay resistance development. Its lethality on C. elegans, its harmlessness in non-nematode species and its novel and dual mechanism of action make DII a promising candidate compound for anthelmintic therapy.

Imprinting: When Early Life Memories Make Food Smell Bad.

A recent study has found that pathogen exposure early in the life of the nematode Caenorhabditis elegans leads to a long-lasting aversion that requires distinct sets of neurons for the formation and retrieval of the imprinted memory.

Relationship between alpha 7 nAChR and apoptosis in human lymphocytes.

The presence of nicotinic receptors (nAChRs) in blood cells has been demonstrated. However, little is known about their functional roles. We have detected mRNA of alpha7 nAChR in peripheral human lymphocytes and determined that its expression is highly variable among individuals and within the same individual at different times. Upregulation of alpha7 is systematically observed after incubation of lymphocytes with nicotine or alpha-bungarotoxin. In addition, the incubation with these drugs decreases the percentage of apoptotic cells induced by the exposure to cortisol. Our results suggest that alpha7 nAChRs are involved in the modulation of cortisol-induced apoptosis.

Activation of single nicotinic receptor channels from Caenorhabditis elegans muscle.

Nicotinic acetylcholine receptors (nAChRs) are pentameric neurotransmitter-gated ion channels that mediate synaptic transmission throughout the nervous system in vertebrates and invertebrates. Caenorhabditis elegans is a nonmammalian model for the study of the nervous system and a model of parasitic nematodes. Nematode muscle nAChRs are of considerable interest because they are targets for anthelmintic drugs. We show single-channel activity of C. elegans muscle nAChRs for the first time. Our results reveal that in the L1 larval stage acetylcholine (ACh) activates mainly a levamisole-sensitive nAChR (L-AChR). A single population of 39 pS channels, which are 5-fold more sensitive to levamisole than ACh, is detected. In contrast to mammalian nAChRs, open durations are longer for levamisole than for ACh. Studies in mutant strains reveal that UNC-38, UNC-63, and UNC-29 subunits are assembled into a single L-AChR in the L1 stage and that these subunits are irreplaceable, suggesting that they are vital for receptor function throughout development. Recordings from a strain mutated in the LEV-1 subunit show a main population of channels with lower conductance (26 pS), prolonged open durations, and reduced sensitivity to levamisole. Thus, although LEV-1 is preferentially incorporated into native L-AChRs, receptors lacking this subunit can still function. No single-channel activity from levamisole-insensitive nAChRs is detected. Thus, during neuromuscular transmission in C. elegans, the majority of ACh-activated current flows through L-AChRs. This study contributes to the understanding of the molecular mechanisms underlying functional diversity of the nAChR family and offers an excellent strategy to test novel antiparasitic drugs.

Molecular determinants of pyrantel selectivity in nicotinic receptors.

Nicotinic receptors (acetylcholine receptors, AChRs) play key roles in synaptic transmission throughout the nervous system. AChRs mediate neuromuscular transmission in nematodes, and they are targets for antiparasitic drugs. The anthelmintic agents levamisole and pyrantel, which are potent agonists of nematode muscle AChRs, are partial agonists of mammalian muscle AChRs. To further explore the structural basis of the differential activation of AChR subtypes by anthelmintics, we studied the activation of alpha7 AChRs using the high-conductance form of the alpha7-5-hydroxytryptamine-3A receptor, which is a good model for pharmacological studies involving the extracellular region of alpha7. Macroscopic and single-channel current recordings show that levamisole is a weak agonist of alpha7. It is interesting that pyrantel is a more potent agonist of alpha7 than acetylcholine (ACh). To identify determinants of this differential activation, we replaced residues of the complementary face of the binding site by the homologous residues in the muscle epsilon subunit and evaluated changes in activation. The mutation Q57G does not affect the activation by either ACh or levamisole. However, it increases EC50 values and decreases the maximal response to pyrantel. Kinetic analysis shows that gating of the mutant channel activated by pyrantel is profoundly impaired. The decreased sensitivity of alpha7-Q57G to pyrantel agrees with its weak action at muscle AChRs, indicating that when glycine occupies position 57, as in the mammalian muscle AChR, pyrantel behaves as a partial agonist. Thus, position 57 located at the complementary face of the binding site plays a key role in the selective activation of AChRs by pyrantel.