Changes in lipid metabolism driven by steroid signalling modulate proteostasis in C. elegans.

Alzheimer's, Parkinson's and Huntington's diseases can be caused by mutations that enhance protein aggregation, but we still do not know enough about the molecular players of these pathways to develop treatments for these devastating diseases. Here, we screen for mutations that might enhance aggregation in Caenorhabditis elegans, to investigate the mechanisms that protect against dysregulated homeostasis. We report that the stomatin homologue UNC-1 activates neurohormonal signalling from the sulfotransferase SSU-1 in ASJ sensory/endocrine neurons. A putative hormone, produced in ASJ, targets the nuclear receptor NHR-1, which acts cell autonomously in the muscles to modulate polyglutamine repeat (polyQ) aggregation. A second nuclear receptor, DAF-12, functions oppositely to NHR-1 to maintain protein homeostasis. Transcriptomics analyses of unc-1 mutants revealed changes in the expression of genes involved in fat metabolism, suggesting that fat metabolism changes, controlled by neurohormonal signalling, contribute to protein homeostasis. Furthermore, the enzymes involved in the identified signalling pathway are potential targets for treating neurodegenerative diseases caused by disrupted protein homeostasis.

Physiological insight into the conserved properties of Caenorhabditis elegans acid-sensing degenerin/epithelial sodium channels.

Acid-sensing ion channels (ASICs) are members of the diverse family of degenerin/epithelial sodium channels (DEG/ENaCs). They perform a wide range of physiological roles in healthy organisms, including in gut function and synaptic transmission, but also play important roles in disease, as acidosis is a hallmark of painful inflammatory and ischaemic conditions. We performed a screen for acid sensitivity on all 30 subunits of the Caenorhabditis elegans DEG/ENaC family using two-electrode voltage clamp in Xenopus oocytes. We found two groups of acid-sensitive DEG/ENaCs characterised by being either inhibited or activated by increasing proton concentrations. Three of these acid-sensitive C. elegans DEG/ENaCs were activated by acidic pH, making them functionally similar to the vertebrate ASICs. We also identified three new members of the acid-inhibited DEG/ENaC group, giving a total of seven additional acid-sensitive channels. We observed sensitivity to the anti-hypertensive drug amiloride as well as modulation by the trace element zinc. Acid-sensitive DEG/ENaCs were found to be expressed in both neurons and non-neuronal tissue, highlighting the likely functional diversity of these channels. Our findings provide a framework to exploit the C. elegans channels as models to study the function of these acid-sensing channels in vivo, as well as to study them as potential targets for anti-helminthic drugs. KEY POINTS: Acidosis plays many roles in healthy physiology, including synaptic transmission and gut function, but is also a key feature of inflammatory pain, ischaemia and many other conditions. Cells monitor acidosis of their surroundings via pH-sensing channels, including the acid-sensing ion channels (ASICs). These are members of the degenerin/epithelial sodium channel (DEG/ENaC) family, along with, as the name suggests, vertebrate ENaCs and degenerins of the roundworm Caenorhabditis elegans. By screening all 30 C. elegans DEG/ENaCs for pH dependence, we describe, for the first time, three acid-activated members, as well as three additional acid-inhibited channels. We surveyed both groups for sensitivity to amiloride and zinc; like their mammalian counterparts, their currents can be blocked, enhanced or unaffected by these modulators. Likewise, they exhibit diverse ion selectivity. Our findings underline the diversity of acid-sensitive DEG/ENaCs across species and provide a comparative resource for better understanding the molecular basis of their function.

The diverse functions of the DEG/ENaC family: linking genetic and physiological insights.

The DEG/ENaC family of ion channels was defined based on the sequence similarity between degenerins (DEG) from the nematode Caenorhabditis elegans and subunits of the mammalian epithelial sodium channel (ENaC), and also includes a diverse array of non-voltage-gated cation channels from across animal phyla, including the mammalian acid-sensing ion channels (ASICs) and Drosophila pickpockets. ENaCs and ASICs have wide ranging medical importance; for example, ENaCs play an important role in respiratory and renal function, and ASICs in ischaemia and inflammatory pain, as well as being implicated in memory and learning. Electrophysiological approaches, both in vitro and in vivo, have played an essential role in establishing the physiological properties of this diverse family, identifying an array of modulators and implicating them in an extensive range of cellular functions, including mechanosensation, acid sensation and synaptic modulation. Likewise, genetic studies in both invertebrates and vertebrates have played an important role in linking our understanding of channel properties to function at the cellular and whole animal/behavioural level. Drawing together genetic and physiological evidence is essential to furthering our understanding of the precise cellular roles of DEG/ENaC channels, with the diversity among family members allowing comparative physiological studies to dissect the molecular basis of these diverse functions.

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock.

Biological clocks are fundamental to an organism's health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls the timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signalling, the generation of an ultradian oscillator, and its metabolic consequences.

Biological clocks regulate a myriad of processes that occur periodically, from sleeping and waking to how cells use nutrients and energy. One such clock is the one that controls intestinal movements and defecation in the nematode worm Caenorhabditis elegans, which consists of three muscle contractions occurring every 50 seconds. This rhythm is controlled by calcium and proton signalling in the cells of the intestine. The cells of the nematode intestine form a tube, through which gut contents pass. The inside of the tube is acidic, but acidity also plays a role on the outer face of the intestinal tube. In this area, nutrients are distributed and signals are conveyed to other tissues, such as muscles. In fact, acid ­ in the form of protons ­ secreted from the intestinal cells stimulates the muscles that contract in the biological clock that controls the worms' defecation. However, it is poorly understood how the worms control the release of these protons. Kaulich et al. identified two ion channels on the membranes of intestinal cells that become inhibited when the levels of acid surrounding them are high. These channels play distinct roles in controlling the contractions that move the contents of the roundworms' intestines along. The first channel contains a protein called ACD-5, and it is in the membrane of the intestinal cells that faces the inside of the intestinal tube. The second channel is formed by three proteins: FLR-1, ACD-3 and DEL-5. This channel is found on the other side of the intestinal cells, the region where nutrients are distributed and signals are conveyed to the rest of the body. To determine the role of each channel, Kaulich et al. genetically engineered the worms so they would not make the proteins that make up the channels, and imaged the live nematodes to see the effects of removing each channel. The inside of the intestines of worms lacking the ACD-5 containing channel was less acidic than that of normal worms, and the timing of the contractions that control defecation was also slightly altered. Removing the second channel (the one formed by three different proteins), however, had more dramatic effects: the worms were thin, developed more slowly, had less fat tissue and defecated very irregularly. Kaulich et al. imaged live worms to show that the second channel plays a major role in regulating oscillations in acidity both inside and outside cells, as well as controlling calcium levels. This demonstrates that this channel is responsible for the rhythmicity in the contractions that control defecation in the nematodes. Their findings provide important insights towards better understanding proton signalling and the role of acid-sensing ion channels in cellular contexts and biological clocks.

The Caenorhabditis elegans tmc-1 is involved in egg-laying inhibition in response to harsh touch.

The conserved family of Transmembrane channel-like (TMC) proteins has attracted significant interest since two members appear to be key components of the mammalian hair cell mechanotransducer involved in hearing. C. elegans expresses two TMC proteins, TMC-1 and TMC-2. TMC-1 is widely expressed in in both muscles and the nervous system. This wide expression pattern suggests that TMC-1 might serve different functions in the various neurons. TMC-1 has previously been shown to function in neurons, playing a role in chemosensation in the ASH neurons and mechanosensation in OLQ neurons, further supporting this hypothesis. tmc-1 is expressed in the high-threshold mechanosensory neuron, ALA. We show that tmc-1 mutants show defects in the ALA-dependent inhibition of egg-laying in response to a harsh mechanical stimulus.

Distinct roles for innexin gap junctions and hemichannels in mechanosensation.

Mechanosensation is central to a wide range of functions, including tactile and pain perception, hearing, proprioception, and control of blood pressure, but identifying the molecules underlying mechanotransduction has proved challenging. In Caenorhabditis elegans, the avoidance response to gentle body touch is mediated by six touch receptor neurons (TRNs), and is dependent on MEC-4, a DEG/ENaC channel. We show that hemichannels containing the innexin protein UNC-7 are also essential for gentle touch in the TRNs, as well as harsh touch in both the TRNs and the PVD nociceptors. UNC-7 and MEC-4 do not colocalize, suggesting that their roles in mechanosensory transduction are independent. Heterologous expression of unc-7 in touch-insensitive chemosensory neurons confers ectopic touch sensitivity, indicating a specific role for UNC-7 hemichannels in mechanosensation. The unc-7 touch defect can be rescued by the homologous mouse gene Panx1 gene, thus, innexin/pannexin proteins may play broadly conserved roles in neuronal mechanotransduction.

EFHC1, implicated in juvenile myoclonic epilepsy, functions at the cilium and synapse to modulate dopamine signaling.

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy.

Caenorhabditis elegans and the network control framework-FAQs.

Control is essential to the functioning of any neural system. Indeed, under healthy conditions the brain must be able to continuously maintain a tight functional control between the system's inputs and outputs. One may therefore hypothesize that the brain's wiring is predetermined by the need to maintain control across multiple scales, maintaining the stability of key internal variables, and producing behaviour in response to environmental cues. Recent advances in network control have offered a powerful mathematical framework to explore the structure-function relationship in complex biological, social and technological networks, and are beginning to yield important and precise insights on neuronal systems. The network control paradigm promises a predictive, quantitative framework to unite the distinct datasets necessary to fully describe a nervous system, and provide mechanistic explanations for the observed structure and function relationships. Here, we provide a thorough review of the network control framework as applied to Caenorhabditis elegans (Yan et al. 2017 Nature550, 519-523. (doi:10.1038/nature24056)), in the style of Frequently Asked Questions. We present the theoretical, computational and experimental aspects of network control, and discuss its current capabilities and limitations, together with the next likely advances and improvements. We further present the Python code to enable exploration of control principles in a manner specific to this prototypical organism.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'.

Recordings of Caenorhabditis elegans locomotor behaviour following targeted ablation of single motorneurons.

Lesioning studies have provided important insight into the functions of brain regions in humans and other animals. In the nematode Caenorhabditis elegans, with a small nervous system of 302 identified neurons, it is possible to generate lesions with single cell resolution and infer the roles of individual neurons in behaviour. Here we present a dataset of ~300 video recordings representing the locomotor behaviour of animals carrying single-cell ablations of 5 different motorneurons. Each file includes a raw video of approximately 27,000 frames; each frame has also been segmented to yield the position, contour, and body curvature of the tracked animal. These recordings can be further analysed using publicly-available software to extract features relevant to behavioural phenotypes. This dataset therefore represents a useful resource for probing the neural basis of behaviour in C. elegans, a resource we hope to augment in the future with ablation recordings for additional neurons.

Network control principles predict neuron function in the Caenorhabditis elegans connectome.

Recent studies on the controllability of complex systems offer a powerful mathematical framework to systematically explore the structure-function relationship in biological, social, and technological networks. Despite theoretical advances, we lack direct experimental proof of the validity of these widely used control principles. Here we fill this gap by applying a control framework to the connectome of the nematode Caenorhabditis elegans, allowing us to predict the involvement of each C. elegans neuron in locomotor behaviours. We predict that control of the muscles or motor neurons requires 12 neuronal classes, which include neuronal groups previously implicated in locomotion by laser ablation, as well as one previously uncharacterized neuron, PDB. We validate this prediction experimentally, finding that the ablation of PDB leads to a significant loss of dorsoventral polarity in large body bends. Importantly, control principles also allow us to investigate the involvement of individual neurons within each neuronal class. For example, we predict that, within the class of DD motor neurons, only three (DD04, DD05, or DD06) should affect locomotion when ablated individually. This prediction is also confirmed; single cell ablations of DD04 or DD05 specifically affect posterior body movements, whereas ablations of DD02 or DD03 do not. Our predictions are robust to deletions of weak connections, missing connections, and rewired connections in the current connectome, indicating the potential applicability of this analytical framework to larger and less well-characterized connectomes.

PACRG, a protein linked to ciliary motility, mediates cellular signaling.

Cilia are microtubule-based organelles that project from nearly all mammalian cell types. Motile cilia generate fluid flow, whereas nonmotile (primary) cilia are required for sensory physiology and modulate various signal transduction pathways. Here we investigate the nonmotile ciliary signaling roles of parkin coregulated gene (PACRG), a protein linked to ciliary motility. PACRG is associated with the protofilament ribbon, a structure believed to dictate the regular arrangement of motility-associated ciliary components. Roles for protofilament ribbon-associated proteins in nonmotile cilia and cellular signaling have not been investigated. We show that PACRG localizes to a small subset of nonmotile cilia in Caenorhabditis elegans, suggesting an evolutionary adaptation for mediating specific sensory/signaling functions. We find that it influences a learning behavior known as gustatory plasticity, in which it is functionally coupled to heterotrimeric G-protein signaling. We also demonstrate that PACRG promotes longevity in C. elegans by acting upstream of the lifespan-promoting FOXO transcription factor DAF-16 and likely upstream of insulin/IGF signaling. Our findings establish previously unrecognized sensory/signaling functions for PACRG and point to a role for this protein in promoting longevity. Furthermore, our work suggests additional ciliary motility-signaling connections, since EFHC1 (EF-hand containing 1), a potential PACRG interaction partner similarly associated with the protofilament ribbon and ciliary motility, also positively regulates lifespan.

Neuropeptidergic Signaling and Active Feeding State Inhibit Nociception in Caenorhabditis elegans.

Food availability and nutritional status are important cues affecting behavioral states. Here we report that, in Caenorhabditis elegans, a cascade of dopamine and neuropeptide signaling acts to inhibit nociception in food-poor environments. In the absence of food, animals show decreased sensitivity and increased adaptation to soluble repellents sensed by the polymodal ASH nociceptors. The effects of food on adaptation are affected by dopamine and neuropeptide signaling; dopamine acts via the DOP-1 receptor to decrease adaptation on food, whereas the neuropeptide receptors NPR-1 and NPR-2 act to increase adaptation off food. NPR-1 and NPR-2 function cell autonomously in the ASH neurons to increase adaptation off food, whereas the DOP-1 receptor controls neuropeptide release from interneurons that modulate ASH activity indirectly. These results indicate that feeding state modulates nociception through the interaction of monoamine and neuropeptide signaling pathways.

Inositol 1,4,5-trisphosphate signalling regulates the avoidance response to nose touch in Caenorhabditis elegans.

When Caenorhabditis elegans encounters an unfavourable stimulus at its anterior, it responds by initiating an avoidance response, namely reversal of locomotion. The amphid neurons, ASHL and ASHR, are polymodal in function, with roles in the avoidance responses to high osmolarity, nose touch, and both volatile and non-volatile repellents. The mechanisms that underlie the ability of the ASH neurons to respond to such a wide range of stimuli are still unclear. We demonstrate that the inositol 1,4,5-trisphosphate receptor (IP(3)R), encoded by itr-1, functions in the reversal responses to nose touch and benzaldehyde, but not in other known ASH-mediated responses. We show that phospholipase Cbeta (EGL-8) and phospholipase Cgamma (PLC-3), which catalyse the production of IP(3), both function upstream of ITR-1 in the response to nose touch. We use neuron-specific gene rescue and neuron-specific disruption of protein function to show that the site of ITR-1 function is the ASH neurons. By rescuing plc-3 and egl-8 in a neuron-specific manner, we show that both are acting in ASH. Imaging of nose touch-induced Ca(2+) transients in ASH confirms these conclusions. In contrast, the response to benzaldehyde is independent of PLC function. Thus, we have identified distinct roles for the IP(3)R in two specific responses mediated by ASH.

Caveolin-2 is required for apical lipid trafficking and suppresses basolateral recycling defects in the intestine of Caenorhabditis elegans.

Caveolins are plasma membrane-associated proteins that colocalize with, and stabilize caveolae. Their functions remain unclear although they are known to be involved in specific events in cell signaling and endocytosis. Caenorhabditis elegans encodes two caveolin genes, cav-1 and cav-2. We show that cav-2 is expressed in the intestine where it is localized to the apical membrane and in intracellular bodies. Using the styryl dye FM4-64 and BODIPY-labeled lactosylceramide, we show that the intestinal cells of cav-2 animals are defective in the apical uptake of lipid markers. These results suggest parallels with the function of caveolins in lipid homeostasis in mammals. We also show that CAV-2 depletion suppresses the abnormal accumulation of vacuoles that result from defective basolateral recycling in rme-1 and rab-10 mutants. Analysis of fluorescent markers of basolateral endocytosis and recycling suggest that endocytosis is normal in cav-2 mutants and thus, that the suppression of basolateral recycling defects in cav-2 mutants is due to changes in intracellular trafficking pathways. Finally, cav-2 mutants also have abnormal trafficking of yolk proteins. Taken together, these data indicate that caveolin-2 is an integral component of the trafficking network in the intestinal cells of C. elegans.

Inositol 1,4,5-trisphosphate signaling regulates mating behavior in Caenorhabditis elegans males.

Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP(3)) is required at several points during mating. Disruption of IP(3) receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-beta, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.

IRI-1, a LIN-15B homologue, interacts with inositol-1,4,5-triphosphate receptors and regulates gonadogenesis, defecation, and pharyngeal pumping in Caenorhabditis elegans.

Inositol-1,4,5-triphosphate receptors (IP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores. They are central to a wide range of cellular responses. IP(3)Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed. Signaling through IP(3) and IP(3)Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis. To further elucidate the molecular basis of the diversity of IP(3)R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1. We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B. Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1. In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle. Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate. Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted.

A direct interaction between IP(3) receptors and myosin II regulates IP(3) signaling in C. elegans.

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signalling machinery as an important mechanism for the regulation of calcium signalling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signalling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP(3)), mediated by the IP(3) receptor (IP(3)R). We show that C. elegans IP(3)Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. We defined the interaction sites on both the IP(3)R and MYO-1. To test the effect of disrupting the interaction in vivo we overexpressed interacting fragments of both proteins in C. elegans. This decreased the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP(3)-mediated process [15]. Other IP(3)-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP(3)Rs and myosin are required for maintaining the specificity of IP(3) signalling in C. elegans and probably more generally.

Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis.

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP(3) is to regulate release of Ca(2+) from intracellular stores through IP(3) receptors (IP(3)Rs). We describe a system for the transient disruption of IP(3) signaling in the model organism Caenorhabditis elegans. The IP(3) binding domain of the C. elegans IP(3)R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP(3) binding domain acting as an IP(3) "sponge." Disruption of IP(3) signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP(3)-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP(3) signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP(3)R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP(3) signaling through the IP(3)R and thus determine that IP(3)-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.