The phosphatidylinositol transfer protein PITP-1 facilitates fast recovery of eating behavior after hypoxia in the nematode Caenorhabditis elegans.

Among the fascinating adaptations to limiting oxygen conditions (hypoxia) is the suppression of food intake and weight loss. In humans, this phenomenon is called high-altitude anorexia and is observed in people suffering from acute mountain syndrome. The high-altitude anorexia appears to be conserved in evolution and has been seen in species across the animal kingdom. However, the mechanism underlying the recovery of eating behavior after hypoxia is still not known. Here, we show that the phosphatidylinositol transfer protein PITP-1 is essential for the fast recovery of eating behavior after hypoxia in the nematode Caenorhabditis elegans. Unlike the neuroglobin GLB-5 that accelerates the recovery of eating behavior through its function in the oxygen (O2 )-sensing neurons, PITP-1 appears to act downstream, in neurons that express the mod-1 serotonin receptor. Indeed, pitp-1 mutants display wild-type-like O2 -evoked-calcium responses in the URX O2 -sensing neuron. Intriguingly, loss-of-function of protein kinase C 1 (PKC-1) rescues pitp-1 mutants' recovery after hypoxia. Increased diacylglycerol (DAG), which activates PKC-1, attenuates the recovery of wild-type worms. Together, these data suggest that PITP-1 enables rapid recovery of eating behavior after hypoxia by limiting DAG's availability, thereby limiting PKC activity in mod-1-expressing neurons.

Regulation of Neuronal Oxygen Responses in C. elegans Is Mediated through Interactions between Globin 5 and the H-NOX Domains of Soluble Guanylate Cyclases.

Soluble guanylate cyclases (sGCs) are gas-binding proteins that control diverse physiological processes such as vasodilation, platelet aggregation, and synaptic plasticity. In the nematode Caenorhabditis elegans, a complex of sGCs, GCY-35 and GCY-36, functions in oxygen (O2) sensing. Previous studies suggested that the neuroglobin GLB-5 genetically interacts with GCY-35, and that the inhibitory effect of GLB-5 on GCY-35 function is necessary for fast recovery from prolonged hypoxia. In this study, we identified mutations in gcy-35 and gcy-36 that impact fast recovery and other phenotypes associated with GLB-5, without undermining sGC activity. These mutations, heb1 and heb3, change conserved amino acid residues in the regulatory H-NOX domains of GCY-35 and GCY-36, respectively, and appear to suppress GLB-5 activity by different mechanisms. Moreover, we observed that short exposure to 35% O2 desensitized the neurons responsible for ambient O2 sensing and that this phenomenon does not occur in heb1 animals. These observations may implicate sGCs in neuronal desensitization mechanisms far beyond the specific case of O2 sensing in nematodes. The conservation of functionally important regions of sGCs is supported by examining site-directed mutants of GCY-35, which suggested that similar regions in the H-NOX domains of O2 and NO-sensing sGCs are important for heme/gas interactions. Overall, our studies provide novel insights into sGC activity and regulation, and implicate similar structural determinants in the control of both O2 and NO sensors. Significance statement: Soluble guanylate cyclases (sGCs) control essential and diverse physiological processes, including memory processing. We used Caenorhabditis elegans to explore how a neuroglobin inhibits a complex of oxygen-sensing sGCs, identifying sGC mutants that resist inhibition. Resistance appears to arise by two different mechanisms: increased basal sGC activity or disruption of an interaction with neuroglobin. Our findings demonstrate that the inhibition of sGCs by neuroglobin is essential for rapid adaptation to either low or high oxygen levels, and that similar structural regions are key for regulating both oxygen and nitric oxide sensors. Based on our structural and functional analyses, we present the hypothesis that neuroglobin-sGC interactions may be generally important for adaptation processes, including those in organisms with more complex neurological functions.

GLOBIN-5-dependent O2 responses are regulated by PDL-1/PrBP that targets prenylated soluble guanylate cyclases to dendritic endings.

Aerobic animals constantly monitor and adapt to changes in O2 levels. The molecular mechanisms involved in sensing O2 are, however, incompletely understood. Previous studies showed that a hexacoordinated globin called GLB-5 tunes the dynamic range of O2-sensing neurons in natural C. elegans isolates, but is defective in the N2 lab reference strain (McGrath et al., 2009; Persson et al., 2009). GLB-5 enables a sharp behavioral switch when O2 changes between 21 and 17%. Here, we show that GLB-5 also confers rapid behavioral and cellular recovery from exposure to hypoxia. Hypoxia reconfigures O2-evoked Ca(2+) responses in the URX O2 sensors, and GLB-5 enables rapid recovery of these responses upon re-oxygenation. Forward genetic screens indicate that GLB-5's effects on O2 sensing require PDL-1, the C. elegans ortholog of mammalian PrBP/PDE6δ protein. In mammals, PDE6δ regulates the traffic and activity of prenylated proteins (Zhang et al., 2004; Norton et al., 2005). PDL-1 promotes localization of GCY-33 and GCY-35, atypical soluble guanylate cyclases that act as O2 sensors, to the dendritic endings of URX and BAG neurons, where they colocalize with GLB-5. Both GCY-33 and GCY-35 are predicted to be prenylated. Dendritic localization is not essential for GCY-35 to function as an O2 sensor, but disrupting pdl-1 alters the URX neuron's O2 response properties. Functional GLB-5 can restore dendritic localization of GCY-33 in pdl-1 mutants, suggesting GCY-33 and GLB-5 are in a complex. Our data suggest GLB-5 and the soluble guanylate cyclases operate in close proximity to sculpt O2 responses.

Ferritin is regulated by a neuro-intestinal axis in the nematode Caenorhabditis elegans.

Iron is vital for the life of most organisms. However, when dysregulated, iron can catalyze the formation of oxygen (O2) radicals that can destroy any biological molecule and thus lead to oxidative injury and death. Therefore, iron metabolism must be tightly regulated at all times, as well as coordinated with the metabolism of O2. However, how is this achieved at the whole animal level is not well understood. Here, we explore this question using the nematode Caenorhabditis elegans. Exposure of worms to O2 starvation conditions (i.e. hypoxia) induces a major upregulation in levels of the conserved iron-cage protein ferritin 1 (ftn-1) in the intestine, while exposure to 21% O2 decreases ftn-1 level. This O2-dependent inhibition is mediated by O2-sensing neurons that communicate with the intestine through neurotransmitter and neuropeptide signalling, and requires the activity of hydroxylated HIF-1. By contrast, the induction of ftn-1 in hypoxia appears to be HIF-1-independent. This upregulation provides protection against Pseudomonas aeruginosa bacteria and oxidative injury. Taken together, our studies uncover a neuro-intestine axis that coordinates O2 and iron responses at the whole animal level.

Characterization of gene expression associated with the adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity.

Oxygen (O2) is a double-edged sword to cells, for while it is vital for energy production in all aerobic animals and insufficient O2 (hypoxia) can lead to cell death, the reoxygenation of hypoxic tissues may trigger the generation of reactive oxygen species (ROS) that can destroy any biological molecule. Indeed, both hypoxia and hypoxia-reoxygenation (H/R) stress are harmful, and may play a critical role in the pathophysiology of many human diseases, such as myocardial ischemia and stroke. Therefore, understanding how animals adapt to hypoxia and H/R stress is critical for developing better treatments for these diseases. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from H/R stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to Pseudomonas aeruginosa pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Mechanisms of defense against products of cysteine catabolism in the nematode Caenorhabditis elegans.

Cysteine catabolism presents cells with a double-edged sword. On the one hand, cysteine degradation provides cells with essential molecules such as taurine and sulfide. The formation of sulfide in cells is thought to regulate important and diverse physiological processes including blood circulation, synaptic activity and inflammation. On the other hand, the catabolism of cysteine by gut microbiota can release high levels of sulfide that may underlie the development or relapse of ulcerative colitis, an inflammatory bowel disease affecting millions of people worldwide. Here, we have used the nematode C. elegans to explore how cells tolerate high levels of sulfide produced by cysteine degradation in bacteria. We have identified mutations in genes coding for thioredoxin family proteins, mitochondrial proteins, and collagens that confer tolerance to sulfide toxicity. Exposure to sulfide induces the unfolded protein response in the endoplasmic reticulum and mitochondria. Moreover, our results suggest that sulfide toxicity is mediated by reactive oxygen species (ROS). Indeed, pre-treatment of worms with antioxidants increases their tolerance to sulfide toxicity. Intriguingly, sub-toxic levels of the superoxide generator paraquat can also increase the tolerance of worms to sulfide. Therefore, it appears that activation of ROS detoxification pathway prior to the exposure to sulfide, can increase the tolerance to sulfide toxicity. Our results suggest that these detoxification pathways are mediated by the hypoxia inducible factor HIF-1. Finally, we show that sulfide resistance varies among wild C. elegans and other nematode species, suggesting that tolerance to sulfide was naturally selected in certain habitats.