Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans.

The response to insufficient oxygen (hypoxia) is orchestrated by the conserved hypoxia-inducible factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel with HIF to mediate the physiological hypoxia response. Here, we describe a hypoxia response pathway controlled by Caenorhabditis elegans nuclear hormone receptor NHR-49, an orthologue of mammalian peroxisome proliferator-activated receptor alpha (PPARα). We show that nhr-49 is required for animal survival in hypoxia and is synthetic lethal with hif-1 in this context, demonstrating that these factors act in parallel. RNA-seq analysis shows that in hypoxia nhr-49 regulates a set of genes that are hif-1-independent, including autophagy genes that promote hypoxia survival. We further show that nuclear hormone receptor nhr-67 is a negative regulator and homeodomain-interacting protein kinase hpk-1 is a positive regulator of the NHR-49 pathway. Together, our experiments define a new, essential hypoxia response pathway that acts in parallel with the well-known HIF-mediated hypoxia response.

A Novel Mechanism To Prevent H2S Toxicity in Caenorhabditis elegans.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule that can be cytoprotective, especially in conditions of ischemia/reperfusion injury. However, H2S is also toxic, and unregulated accumulation or exposure to environmental H2S can be lethal. In Caenorhabditis elegans, the hypoxia inducible factor (hif-1) coordinates the initial transcriptional response to H2S, and is essential to survive exposure to low concentrations of H2S. We performed a forward genetic screen to identify mutations that suppress the lethality of hif-1 mutant animals in H2S. The mutations we recovered are specific for H2S, as they do not suppress embryonic lethality or reproductive arrest of hif-1 mutant animals in hypoxia, nor can they prevent the death of hif-1 mutant animals exposed to hydrogen cyanide. The majority of hif-1 suppressor mutations we recovered activate the skn-1/Nrf2 transcription factor. Activation of SKN-1 by hif-1 suppressor mutations increased the expression of a subset of H2S-responsive genes, consistent with previous findings that skn-1 plays a role in the transcriptional response to H2S. Using transgenic rescue, we show that overexpression of a single gene, rhy-1, is sufficient to protect hif-1 mutant animals in H2S. The rhy-1 gene encodes a predicated O-acyltransferase enzyme that has previously been shown to negatively regulate HIF-1 activity. Our data indicate that RHY-1 has novel, hif-1 independent, function that promotes survival in H2S.

Fasting prevents hypoxia-induced defects of proteostasis in C. elegans.

Low oxygen conditions (hypoxia) can impair essential physiological processes and cause cellular damage and death. We have shown that specific hypoxic conditions disrupt protein homeostasis in C. elegans, leading to protein aggregation and proteotoxicity. Here, we show that nutritional cues regulate this effect of hypoxia on proteostasis. Animals fasted prior to hypoxic exposure develop dramatically fewer polyglutamine protein aggregates compared to their fed counterparts, indicating that the effect of hypoxia is abrogated. Fasting also reduced the hypoxia-induced exaggeration of proteostasis defects in animals that express Aß1-42 and in animals with a temperature-sensitive mutation in dyn-1, suggesting that this effect was not specific to polyglutamine proteins. Our data also demonstrate that the nutritional environment experienced at the onset of hypoxia dictates at least some aspects of the physiological response to hypoxia. We further demonstrate that the insulin/IGF-like signaling pathway plays a role in mediating the protective effects of fasting in hypoxia. Animals with mutations in daf-2, the C. elegans insulin-like receptor, display wild-type levels of hypoxia-induced protein aggregation upon exposure to hypoxia when fed, but are not protected by fasting. DAF-2 acts independently of the FOXO transcription factor, DAF-16, to mediate the protective effects of fasting. These results suggest a non-canonical role for the insulin/IGF-like signaling pathway in coordinating the effects of hypoxia and nutritional state on proteostasis.

Caenorhabditis elegans HIF-1 Is Broadly Required for Survival in Hydrogen Sulfide.

Hydrogen sulfide is common in the environment, and is also endogenously produced by animal cells. Although hydrogen sulfide is often toxic, exposure to low levels of hydrogen sulfide improves outcomes in a variety of mammalian models of ischemia-reperfusion injury. In Caenorhabditis elegans, the initial transcriptional response to hydrogen sulfide depends on the hif-1 transcription factor, and hif-1 mutant animals die when exposed to hydrogen sulfide. In this study, we use rescue experiments to identify tissues in which hif-1 is required to survive exposure to hydrogen sulfide. We find that expression of hif-1 from the unc-14 promoter is sufficient to survive hydrogen sulfide. Although unc-14 is generally considered to be a pan-neuronal promoter, we show that it is active in many nonneuronal cells as well. Using other promoters, we show that pan-neuronal expression of hif-1 is not sufficient to survive exposure to hydrogen sulfide. Our data suggest that hif-1 is required in many different tissues to direct the essential response to hydrogen sulfide.

Computational Analysis of Lifespan Experiment Reproducibility.

Independent reproducibility is essential to the generation of scientific knowledge. Optimizing experimental protocols to ensure reproducibility is an important aspect of scientific work. Genetic or pharmacological lifespan extensions are generally small compared to the inherent variability in mean lifespan even in isogenic populations housed under identical conditions. This variability makes reproducible detection of small but real effects experimentally challenging. In this study, we aimed to determine the reproducibility of C. elegans lifespan measurements under ideal conditions, in the absence of methodological errors or environmental or genetic background influences. To accomplish this, we generated a parametric model of C. elegans lifespan based on data collected from 5,026 wild-type N2 animals. We use this model to predict how different experimental practices, effect sizes, number of animals, and how different "shapes" of survival curves affect the ability to reproduce real longevity effects. We find that the chances of reproducing real but small effects are exceedingly low and would require substantially more animals than are commonly used. Our results indicate that many lifespan studies are underpowered to detect reported changes and that, as a consequence, stochastic variation alone can account for many failures to reproduce longevity results. As a remedy, we provide power of detection tables that can be used as guidelines to plan experiments with statistical power to reliably detect real changes in lifespan and limit spurious false positive results. These considerations will improve best-practices in designing lifespan experiment to increase reproducibility.

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development.

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.

Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide.

Hydrogen sulfide (H2S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H2S improves survival of ischemia/reperfusion. We have previously shown that exposure to H2S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H2S metabolism. SQRD-1 is generally considered important to detoxify H2S. Here, we show that SQRD-1 is also required to maintain protein translation in H2S. In sqrd-1 mutant animals, exposure to H2S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H2S or in hif-1(ia04) mutants that die when exposed to low H2S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H2S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H2S, but not in wild-type animals. We speculate that SQRD-1 activity in H2S may coordinate proteostasis responses in multiple cellular compartments.

Cell nonautonomous activation of flavin-containing monooxygenase promotes longevity and health span.

Stabilization of the hypoxia-inducible factor 1 (HIF-1) increases life span and health span in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in Caenorhabditis elegans through a cell nonautonomous signal to the intestine, which results in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This prolongevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and is necessary for DR-mediated life-span extension, which suggests that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple life span-extending interventions in mice, which suggests that these enzymes may play a critical role in promoting health and longevity across phyla.

Tissue-specific autophagy responses to aging and stress in C. elegans.

Cellular function relies on a balance between protein synthesis and breakdown. Macromolecular breakdown through autophagy is broadly required for cellular and tissue development, function, and recovery from stress. While Caenorhabditis elegans is frequently used to explore cellular responses to development and stress, the most common assays for autophagy in this system lack tissue-level resolution. Different tissues within an organism have unique functional characteristics and likely vary in their reliance on autophagy under different conditions. To generate a tissue-specific map of autophagy in C. elegans we used a dual fluorescent protein (dFP) tag that releases monomeric fluorescent protein (mFP) upon arrival at the lysosome. Tissue-specific expression of dFP::LGG-1 revealed autophagic flux in all tissues, but mFP accumulation was most dramatic in the intestine. We also observed variable responses to stress: starvation increased autophagic mFP release in all tissues, whereas anoxia primarily increased intestinal autophagic flux. We observed autophagic flux with tagged LGG-1, LGG-2, and two autophagic cargo reporters: a soluble cytoplasmic protein, and mitochondrial TOMM-7. Finally, an increase in mFP in older worms was consistent with an age-dependent shift in proteostasis. These novel measures of autophagic flux in C. elegans reveal heterogeneity in autophagic response across tissues during stress and aging.

Hypoxia disrupts proteostasis in Caenorhabditis elegans.

Oxygen is fundamentally important for cell metabolism, and as a consequence, O2 deprivation (hypoxia) can impair many essential physiological processes. Here, we show that an active response to hypoxia disrupts cellular proteostasis - the coordination of protein synthesis, quality control, and degradation that maintains the functionality of the proteome. We have discovered that specific hypoxic conditions enhance the aggregation and toxicity of aggregation-prone proteins that are associated with neurodegenerative diseases. Our data indicate this is an active response to hypoxia, rather than a passive consequence of energy limitation. This response to hypoxia is partially antagonized by the conserved hypoxia-inducible transcription factor, hif-1. We further demonstrate that exposure to hydrogen sulfide (H2S) protects animals from hypoxia-induced disruption of proteostasis. H2S has been shown to protect against hypoxic damage in mammals and extends lifespan in nematodes. Remarkably, our data also show that H2S can reverse detrimental effects of hypoxia on proteostasis. Our data indicate that the protective effects of H2S in hypoxia are mechanistically distinct from the effect of H2S to increase lifespan and thermotolerance, suggesting that control of proteostasis and aging can be dissociated. Together, our studies reveal a novel effect of the hypoxia response in animals and provide a foundation to understand how the integrated proteostasis network is integrated with this stress response pathway.

Interactions between oxygen homeostasis, food availability, and hydrogen sulfide signaling.

The ability to sense and respond to stressful conditions is essential to maintain organismal homeostasis. It has long been recognized that stress response factors that improve survival in changing conditions can also influence longevity. In this review, we discuss different strategies used by animals in response to decreased O(2) (hypoxia) to maintain O(2) homeostasis, and consider interactions between hypoxia responses, nutritional status, and H(2)S signaling. O(2) is an essential environmental nutrient for almost all metazoans as it plays a fundamental role in development and cellular metabolism. However, the physiological response(s) to hypoxia depend greatly on the amount of O(2) available. Animals must sense declining O(2) availability to coordinate fundamental metabolic and signaling pathways. It is not surprising that factors involved in the response to hypoxia are also involved in responding to other key environmental signals, particularly food availability. Recent studies in mammals have also shown that the small gaseous signaling molecule hydrogen sulfide (H(2)S) protects against cellular damage and death in hypoxia. These results suggest that H(2)S signaling also integrates with hypoxia response(s). Many of the signaling pathways that mediate the effects of hypoxia, food deprivation, and H(2)S signaling have also been implicated in the control of lifespan. Understanding how these pathways are coordinated therefore has the potential to reveal new cellular and organismal homeostatic mechanisms that contribute to longevity assurance in animals.

Creating defined gaseous environments to study the effects of hypoxia on C. elegans.

Oxygen is essential for all metazoans to survive, with one known exception. Decreased O(2) availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms. The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O(2) concentrations without confounding effects since C. elegans obtain O(2) (and other gasses) by diffusion, as opposed to a facilitated respiratory system. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O(2) that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O(2). Hypoxia is a general term for "less than normal O(2)". Normoxia (normal O(2)) can also be difficult to define. We generally consider room air, which is 210,000 ppm O(2) to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O(2) concentrations from 5-12% (50,000-120,000 ppm O(2)). In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O(2). However, hif-1 does not play a role in the response to lower concentrations of O(2) (anoxia, operational definition <10 ppm O(2)). In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O(2). Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O(2). The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

HIF-1 and SKN-1 coordinate the transcriptional response to hydrogen sulfide in Caenorhabditis elegans.

Hydrogen sulfide (H2S) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in H2S are long-lived and thermotolerant. To identify mechanisms by which adaptation to H2S effects physiological functions, we have measured transcriptional responses to H2S exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H2S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H2S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H2S. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in H2S. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to H2S-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to H2S. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H2S that culminates in a global reorganization of protein homeostasis networks.

C. elegans are protected from lethal hypoxia by an embryonic diapause.

At least 100 mammalian species exhibit embryonic diapause, where fertilized embryos arrest development in utero until suitable seasonal or nutritional environments are encountered. Delaying maternal investments in producing offspring allows these animals to utilize limited resources to survive while searching for better conditions and ensures that progeny are not produced when they are unlikely to survive. In addition, embryos may be protected from external environmental vicissitudes while in utero. Here we demonstrate embryonic diapause in C. elegans, and show that this diapause protects embryos from otherwise lethal hypoxia. Diapausing embryos in utero require san-1 to survive, indicating that hypoxia-induced embryonic diapause may be mechanistically related to suspended animation. Furthermore, we show that neuronal HIF-1 activity in the adult dictates the O(2) tension at which embryonic diapause is engaged. We suggest that the maternal perception of hypoxia stimulates a response to protect embryos in utero by inducing diapause, a natural form of suspended animation. This response is likely to be an important strategy to improve offspring survival in harsh conditions and allow adults to find environments more suitable for reproductive success.

How adolescents experience smoking cessation.

In this study we develop a model of how youth experience smoking cessation attempts. We followed 15 adolescent smokers twice monthly over three months. Through six semistructured interviews, we explored participants' subjective experiences of making a "quit" attempt. We analyzed transcript data using grounded theory procedures, beginning with open coding, axial coding, construction of matrices, and development of a preliminary theory or model of this phenomenon. We found that only emotionally compelling and inescapable quit reasons were truly motivating. Few parents actively supported their child during quit attempts; smoking friends and other peers undermined them. All successful quitters established new, nonsmoking friends and completely redefined themselves. The quit experience was physically uncomfortable, emotionally distressful, and socially isolating. Greater motivation, mature problem-solving skills, and a willingness to supplant their smoking friends characterized successful quitters. Further research is needed to test this model's efficacy in the adolescent population.

Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans.

Hydrogen sulfide (H(2)S) is naturally produced in animal cells. Exogenous H(2)S has been shown to effect physiological changes that improve the capacity of mammals to survive in otherwise lethal conditions. However, the mechanisms required for such alterations are unknown. We investigated the physiological response of Caenorhabditis elegans to H(2)S to elucidate the molecular mechanisms of H(2)S action. Here we show that nematodes exposed to H(2)S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. Instead, animals exposed to H(2)S are thermotolerant and long-lived. These phenotypes require SIR-2.1 activity but are genetically independent of the insulin signaling pathway, mitochondrial dysfunction, and caloric restriction. These studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H(2)S increases thermotolerance and lifespan in nematodes are conserved and that studies using C. elegans may help explain the beneficial effects observed in mammals exposed to H(2)S.

Tree-ring isotope records of tropical cyclone activity.

The destruction wrought by North Atlantic hurricanes in 2004 and 2005 dramatically emphasizes the need for better understanding of tropical cyclone activity apart from the records provided by meteorological data and historical documentation. We present a 220-year record of oxygen isotope values of alpha-cellulose in longleaf pine tree rings that preserves anomalously low isotope values in the latewood portion of the ring in years corresponding with known 19th and 20th century landfalling/near-coastal tropical storms and hurricanes. Our results suggest the potential for a tree-ring oxygen isotope proxy record of tropical cyclone occurrence extending back many centuries based on remnant pine wood from protected areas in the southeastern U.S.

Subdomain organization and catalytic residues of the F factor TraI relaxase domain.

TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.

Evidence for a monomeric intermediate in the reversible unfolding of F factor TraM.

F factor TraM is essential for efficient bacterial conjugation, but its molecular function is not clear. Because the physical properties of TraM may provide clues to its role in conjugation, we have characterized the TraM oligomerization equilibrium. We show that the reversible unfolding transition is non-two-state, indicating the presence of at least one intermediate. Analytical ultracentrifugation experiments indicate that the first phase of unfolding involves dissociation of the tetramer into folded monomers, which are subsequently unfolded to the denatured state in the second phase. Furthermore, we show that a C-terminal domain isolated by limited proteolysis is tetrameric in solution, like the full-length protein, and that its loss of structure correlates with dissociation of the TraM tetramer. Unfolding of the individual domains indicates that the N- and C-terminal regions act cooperatively to stabilize the full-length protein. Together, these experiments suggest structural overlap of regions important for oligomerization and DNA binding. We propose that modulating the oligomerization equilibrium of TraM may regulate its essential activity in bacterial conjugation.