Stimulation of RAS-dependent ROS signaling extends longevity by modulating a developmental program of global gene expression.

We show that elevation of mitochondrial superoxide generation increases Caenorhabditis elegans life span by enhancing a RAS-dependent ROS (reactive oxygen species) signaling pathway (RDRS) that controls the expression of half of the genome as well as animal composition and physiology. RDRS stimulation mimics a program of change in gene expression that is normally observed at the end of postembryonic development. We further show that RDRS is regulated by negative feedback from the superoxide dismutase 1 (SOD-1)-dependent conversion of superoxide into cytoplasmic hydrogen peroxide, which, in turn, acts on a redox-sensitive cysteine (C118) of RAS. Preventing C118 oxidation by replacement with serine, or mimicking oxidation by replacement with aspartic acid, leads to opposite changes in the expression of the same large set of genes that is affected when RDRS is stimulated by mitochondrial superoxide. The identities of these genes suggest that stimulation of the pathway extends life span by boosting turnover and repair while moderating damage from metabolic activity.

ROS regulation of RAS and vulva development in Caenorhabditis elegans.

Reactive oxygen species (ROS) are signalling molecules whose study in intact organisms has been hampered by their potential toxicity. This has prevented a full understanding of their role in organismal processes such as development, aging and disease. In Caenorhabditis elegans, the development of the vulva is regulated by a signalling cascade that includes LET-60ras (homologue of mammalian Ras), MPK-1 (ERK1/2) and LIN-1 (an ETS transcription factor). We show that both mitochondrial and cytoplasmic ROS act on a gain-of-function (gf) mutant of the LET-60ras protein through a redox-sensitive cysteine (C118) previously identified in mammals. We show that the prooxidant paraquat as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS, inhibit the activity of LET-60rasgf on vulval development. In contrast, the antioxidant NAC and loss of sod-1, both of which decrease cytoplasmic H202, enhance the activity of LET-60rasgf. CRISPR replacement of C118 with a non-oxidizable serine (C118S) stimulates LET-60rasgf activity, whereas replacement of C118 with aspartate (C118D), which mimics a strongly oxidised cysteine, inhibits LET-60rasgf. These data strongly suggest that C118 is oxidized by cytoplasmic H202 generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibits LET-60ras. This contrasts with results in cultured mammalian cells where it is mostly nitric oxide, which is not found in worms, that oxidizes C118 and activates Ras. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-60ras acts on an as yet uncharacterized MPK-1-independent pathway. We also show that elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-60ras and downstream of LIN-1. Finally, we uncover a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Thus, there are at least three genetically separable pathways by which ROS regulates vulval development.

SK channel-mediated metabolic escape to glycolysis inhibits ferroptosis and supports stress resistance in C. elegans.

Metabolic flexibility is an essential characteristic of eukaryotic cells in order to adapt to physiological and environmental changes. Especially in mammalian cells, the metabolic switch from mitochondrial respiration to aerobic glycolysis provides flexibility to sustain cellular energy in pathophysiological conditions. For example, attenuation of mitochondrial respiration and/or metabolic shifts to glycolysis result in a metabolic rewiring that provide beneficial effects in neurodegenerative processes. Ferroptosis, a non-apoptotic form of cell death triggered by an impaired redox balance is gaining attention in the field of neurodegeneration. We showed recently that activation of small-conductance calcium-activated K+ (SK) channels modulated mitochondrial respiration and protected neuronal cells from oxidative death. Here, we investigated whether SK channel activation with CyPPA induces a glycolytic shift thereby increasing resilience of neuronal cells against ferroptosis, induced by erastin in vitro and in the nematode C. elegans exposed to mitochondrial poisons in vivo. High-resolution respirometry and extracellular flux analysis revealed that CyPPA, a positive modulator of SK channels, slightly reduced mitochondrial complex I activity, while increasing glycolysis and lactate production. Concomitantly, CyPPA rescued the neuronal cells from ferroptosis, while scavenging mitochondrial ROS and inhibiting glycolysis reduced its protection. Furthermore, SK channel activation increased survival of C. elegans challenged with mitochondrial toxins. Our findings shed light on metabolic mechanisms promoted through SK channel activation through mitohormesis, which enhances neuronal resilience against ferroptosis in vitro and promotes longevity in vivo.

Superoxide dismutases: Dual roles in controlling ROS damage and regulating ROS signaling.

Superoxide dismutases (SODs) are universal enzymes of organisms that live in the presence of oxygen. They catalyze the conversion of superoxide into oxygen and hydrogen peroxide. Superoxide anions are the intended product of dedicated signaling enzymes as well as the byproduct of several metabolic processes including mitochondrial respiration. Through their activity, SOD enzymes control the levels of a variety of reactive oxygen species (ROS) and reactive nitrogen species, thus both limiting the potential toxicity of these molecules and controlling broad aspects of cellular life that are regulated by their signaling functions. All aerobic organisms have multiple SOD proteins targeted to different cellular and subcellular locations, reflecting the slow diffusion and multiple sources of their substrate superoxide. This compartmentalization also points to the need for fine local control of ROS signaling and to the possibility for ROS to signal between compartments. In this review, we discuss studies in model organisms and humans, which reveal the dual roles of SOD enzymes in controlling damage and regulating signaling.

Proteostasis or Aging: Let the CHIPs Fall Where They May.

The conserved E3 ubiquitin ligase CHIP/CHN-1 contributes to proteostasis by ubiquitylating HSP70 and HSP90-interacting proteins. In a recent issue of Cell,Tawo et al. (2017) show that CHIP/CHN-1 also directly ubiquitylates the insulin receptor INSR/DAF-2 to regulate its turnover. These findings suggest an unexpected interpretation of the effects of altered proteostasis on survival.

Making a splash with splicing.

In a recent Nature paper, Heintz et al. identify a splicing factor (SFA-1) that is crucial for the longevity conferred by dietary restriction and the TORC1 pathway modulation in C. elegans.

The Multilayer Connectome of Caenorhabditis elegans.

Connectomics has focused primarily on the mapping of synaptic links in the brain; yet it is well established that extrasynaptic volume transmission, especially via monoamines and neuropeptides, is also critical to brain function and occurs primarily outside the synaptic connectome. We have mapped the putative monoamine connections, as well as a subset of neuropeptide connections, in C. elegans based on new and published gene expression data. The monoamine and neuropeptide networks exhibit distinct topological properties, with the monoamine network displaying a highly disassortative star-like structure with a rich-club of interconnected broadcasting hubs, and the neuropeptide network showing a more recurrent, highly clustered topology. Despite the low degree of overlap between the extrasynaptic (or wireless) and synaptic (or wired) connectomes, we find highly significant multilink motifs of interaction, pinpointing locations in the network where aminergic and neuropeptide signalling modulate synaptic activity. Thus, the C. elegans connectome can be mapped as a multiplex network with synaptic, gap junction, and neuromodulator layers representing alternative modes of interaction between neurons. This provides a new topological plan for understanding how aminergic and peptidergic modulation of behaviour is achieved by specific motifs and loci of integration between hard-wired synaptic or junctional circuits and extrasynaptic signals wirelessly broadcast from a small number of modulatory neurons.

Changes in Postural Syntax Characterize Sensory Modulation and Natural Variation of C. elegans Locomotion.

Locomotion is driven by shape changes coordinated by the nervous system through time; thus, enumerating an animal's complete repertoire of shape transitions would provide a basis for a comprehensive understanding of locomotor behaviour. Here we introduce a discrete representation of behaviour in the nematode C. elegans. At each point in time, the worm's posture is approximated by its closest matching template from a set of 90 postures and locomotion is represented as sequences of postures. The frequency distribution of postural sequences is heavy-tailed with a core of frequent behaviours and a much larger set of rarely used behaviours. Responses to optogenetic and environmental stimuli can be quantified as changes in postural syntax: worms show different preferences for different sequences of postures drawn from the same set of templates. A discrete representation of behaviour will enable the use of methods developed for other kinds of discrete data in bioinformatics and language processing to be harnessed for the study of behaviour.

A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans.

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force-posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.

The voltage-gated anion channels encoded by clh-3 regulate egg laying in C. elegans by modulating motor neuron excitability.

CLC-2 is a hyperpolarization-activated, inwardly rectifying chloride channel. Although the properties of the CLC-2 channel have been well characterized, its function in vivo is not well understood. We have found that channels encoded by the Caenorhabditis elegans CLC-2 homolog clh-3 regulate the activity of the spontaneously active hermaphrodite-specific neurons (HSNs), which control the egg-laying behavior. We identified a gain-of-function mutation in clh-3 that increases channel activity. This mutation inhibits egg laying and inhibits HSN activity by decreasing its excitability. Conversely, loss-of-function mutations in clh-3 lead to misregulated egg laying and an increase in HSN excitability, indicating that these channels modulate egg laying by limiting HSN excitability. clh-3-encoded channels are not required for GABAA-receptor-mediated inhibition of the HSN. However, they require low intracellular chloride for HSN inhibition, indicating that they inhibit excitability directly by mediating chloride influx. This mechanism of CLH-3-dependent modulation may be conserved in other neurons in which the driving force favors chloride influx.

Mitochondrial oxidative stress alters a pathway in Caenorhabditis elegans strongly resembling that of bile acid biosynthesis and secretion in vertebrates.

Mammalian bile acids (BAs) are oxidized metabolites of cholesterol whose amphiphilic properties serve in lipid and cholesterol uptake. BAs also act as hormone-like substances that regulate metabolism. The Caenorhabditis elegans clk-1 mutants sustain elevated mitochondrial oxidative stress and display a slow defecation phenotype that is sensitive to the level of dietary cholesterol. We found that: 1) The defecation phenotype of clk-1 mutants is suppressed by mutations in tat-2 identified in a previous unbiased screen for suppressors of clk-1. TAT-2 is homologous to ATP8B1, a flippase required for normal BA secretion in mammals. 2) The phenotype is suppressed by cholestyramine, a resin that binds BAs. 3) The phenotype is suppressed by the knock-down of C. elegans homologues of BA-biosynthetic enzymes. 4) The phenotype is enhanced by treatment with BAs. 5) Lipid extracts from C. elegans contain an activity that mimics the effect of BAs on clk-1, and the activity is more abundant in clk-1 extracts. 6) clk-1 and clk-1;tat-2 double mutants show altered cholesterol content. 7) The clk-1 phenotype is enhanced by high dietary cholesterol and this requires TAT-2. 8) Suppression of clk-1 by tat-2 is rescued by BAs, and this requires dietary cholesterol. 9) The clk-1 phenotype, including the level of activity in lipid extracts, is suppressed by antioxidants and enhanced by depletion of mitochondrial superoxide dismutases. These observations suggest that C. elegans synthesizes and secretes molecules with properties and functions resembling those of BAs. These molecules act in cholesterol uptake, and their level of synthesis is up-regulated by mitochondrial oxidative stress. Future investigations should reveal whether these molecules are in fact BAs, which would suggest the unexplored possibility that the elevated oxidative stress that characterizes the metabolic syndrome might participate in disease processes by affecting the regulation of metabolism by BAs.

Lipid transport and signaling in Caenorhabditis elegans.

The strengths of the Caenorhabditis elegans model have been recently applied to the study of the pathways of lipid storage, transport, and signaling. As the lipid storage field has recently been reviewed, in this minireview we (1) discuss some recent studies revealing important physiological roles for lipases in mobilizing lipid reserves, (2) describe various pathways of lipid transport, with a particular focus on the roles of lipoproteins, (3) debate the utility of using C. elegans as a model for human dyslipidemias that impinge on atherosclerosis, and (4) describe several systems where lipids affect signaling, highlighting the particular properties of lipids as information-carrying molecules. We conclude that the study of lipid biology in C. elegans exemplifies the advantages afforded by a whole-animal model system where interactions between tissues and organs, and functions such as nutrient absorption, distribution, and storage, as well as reproduction can all be studied simultaneously.

Tyramine: a new receptor and a new role at the synapse.

Biogenic amines are typically regarded as neuromodulators rather than fast-acting neurotransmitters. In this issue of Neuron, Pirri et al. report the characterization of LGC-55 in C. elegans, the first identified tyramine-gated chloride channel. This study suggests that the roles of classical and trace biogenic amines in all organisms may need to be reconsidered.

The anti-neurodegeneration drug clioquinol inhibits the aging-associated protein CLK-1.

The development of neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington disease is strongly age-dependent. Discovering drugs that act on the high rate of aging in older individuals could be a means of combating these diseases. Reduction of the activity of the mitochondrial enzyme CLK-1 (also known as COQ7) slows down aging in Caenorhabditis elegans and in mice. Clioquinol is a metal chelator that has beneficial effects in several cellular and animal models of neurodegenerative diseases as well as on Alzheimer disease patients. Here we show that clioquinol inhibits the activity of mammalian CLK-1 in cultured cells, an inhibition that can be blocked by iron or cobalt cations, suggesting that chelation is involved in the mechanism of action of clioquinol on CLK-1. We also show that treatment of nematodes and mice with clioquinol mimics a variety of phenotypes produced by mutational reduction of CLK-1 activity in these organisms. These results suggest that the surprising action of clioquinol on several age-dependent neurodegenerative diseases with distinct etiologies might result from a slowing down of the aging process through action of the drug on CLK-1. Our findings support the hypothesis that pharmacologically targeting aging-associated proteins could help relieve age-dependent diseases.

Oxygen homeostasis: how the worm adapts to variable oxygen levels.

A recent study has found that activation of the hypoxia-inducible transcription factor HIF in the nematode Caenorhabditis elegans can alter oxygen preferences and dramatically re-wire the neuronal circuit required for avoiding high oxygen levels.

Evolutionary conservation of drug action on lipoprotein metabolism-related targets.

Genetic analysis has shown that the slower than normal rhythmic defecation behavior of the clk-1 mutants of Caenorhabditis elegans is the result of altered lipoprotein metabolism. We show here that this phenotype can be suppressed by drugs that affect lipoprotein metabolism, including drugs that affect HMG-CoA reductase activity, reverse cholesterol transport, or HDL levels. These pharmacological effects are highly specific, as these drugs affect defecation only in clk-1 mutants and not in the wild-type and do not affect other behaviors of the mutants. Furthermore, drugs that affect processes not directly related to lipid metabolism show no or minimal activity. Based on these findings, we carried out a compound screen that identified 190 novel molecules that are active on clk-1 mutants, 15 of which also specifically decrease the secretion of apolipoprotein B (apoB) from HepG2 hepatoma cells. The other 175 compounds are potentially active on lipid-related processes that cannot be targeted in cell culture. One compound, CHGN005, was tested and found to be active at reducing apoB secretion in intestinal Caco-2 cells as well as in HepG2 cells. This compound was also tested in a mouse model of dyslipidemia and found to decrease plasma cholesterol and triglyceride levels. Thus, target processes for pharmacological intervention on lipoprotein synthesis, transport, and metabolism are conserved between nematodes and vertebrates, which allows the use of C. elegans for drug discovery.

What keeps C. elegans regular: the genetics of defecation.

Caenorhabditis elegans exhibits a repertoire of behaviors that can be studied by genetic, anatomical and pharmacological approaches. Defecation is one of the simpler behaviors, involving a small number of muscles, a couple of neurons and only one neurotransmitter. This simplicity enables the precise characterization of the cells and genes required for executing the behavior and has made the defecation behavior a powerful model for investigating the genetic basis of nervous system function, muscle differentiation, rhythmic behaviors and oscillatory calcium signaling, and the metabolic and environmental regulation of behavior. Our review highlights how the function of a system even this simple results from the integration of many aspects of an organism's biology and involves the action of diverse genes.

Uncoupling the pleiotropic phenotypes of clk-1 with tRNA missense suppressors in Caenorhabditis elegans.

clk-1 encodes a demethoxyubiquinone (DMQ) hydroxylase that is necessary for ubiquinone biosynthesis. When Caenorhabditis elegans clk-1 mutants are grown on bacteria that synthesize ubiquinone (UQ), they are viable but have a pleiotropic phenotype that includes slowed development, behaviors, and aging. However, when grown on UQ-deficient bacteria, the mutants arrest development transiently before growing up to become sterile adults. We identified nine suppressors of the missense mutation clk-1(e2519), which harbors a Glu-to-Lys substitution. All suppress the mutant phenotypes on both UQ-replete and UQ-deficient bacteria. However, each mutant suppresses a different subset of phenotypes, indicating that most phenotypes can be uncoupled from each other. In addition, all suppressors restore the ability to synthesize exceedingly small amounts of UQ, although they still accumulate the precursor DMQ, suggesting that the presence of DMQ is not responsible for the Clk-1 phenotypes. We cloned six of the suppressors, and all encode tRNA(Glu) genes whose anticodons are altered to read the substituted Lys codon of clk-1(e2519). To our knowledge, these suppressors represent the first missense suppressors identified in any metazoan. The pattern of suppression we observe suggests that the individual members of the tRNA(Glu) family are expressed in different tissues and at different levels.

Specification of muscle neurotransmitter sensitivity by a Paired-like homeodomain protein in Caenorhabditis elegans.

The effects of neurotransmitters depend on the receptors expressed on the target cells. In Caenorhabditis elegans, there are two types of GABA receptors that elicit opposite effects: excitatory receptors that open cation-selective channels, and inhibitory receptors that open anion-selective channels. The four non-striated enteric muscle cells required for the expulsion step of the defecation behavior are all sensitive to GABA: the sphincter muscle expresses a classical GABA-sensitive chloride channel (UNC-49) and probably relaxes in response to GABA, while the other three cells express a cation-selective channel (EXP-1) and contract. Here we show that the expression of the exp-1 gene is under the control of dsc-1, which encodes a Paired-like homeodomain protein, a class of transcription factors previously associated with the terminal differentiation of neurons in C. elegans. dsc-1 mutants have anatomically normal enteric muscles but are expulsion defective. We show that this defect is due to the lack of expression of exp-1 in the three cells that contract in response to GABA. In addition, dsc-1, but not exp-1, affects the periodicity of the behavior, revealing an unanticipated role for the enteric muscles in regulating this ultradian rhythm.

Redox regulation of germline and vulval development in Caenorhabditis elegans.

In vitro studies have indicated that reactive oxygen species (ROS) and the oxidation of signaling molecules are important mediators of signal transduction. We have identified two pathways by which the altered redox chemistry of the clk-1 mutants of Caenorhabditis elegans acts in vivo on germline development. One pathway depends on the oxidation of an analog of vertebrate low density lipoprotein (LDL) and acts on the germline through the Ack-related tyrosine kinase (ARK-1) kinase and inositol trisphosphate (IP3) signaling. The other pathway is the oncogenic ras signaling pathway, whose action on germline as well as vulval development appears to be modulated by cytoplasmic ROS.