Sumino Yanase

Adaptive responses to oxidative damage in three mutants of Caenorhabditis elegans (age-1, mev-1 and daf-16) that affect life span

Oxidative damage shortens the life span of the nematode Caenorhabditis elegans (C. elegans), even in an age-1 mutant that is characterized by a long life and oxygen resistance. We found that daily short-term exposure (3 h) to hyperoxia further extended the life span of age-1, a phenomenon known as an adaptive response. age-1 also showed resistance to paraquat and heat. Acute hyperoxic treatment did not extend the life spans of wild type, daf-16 or mev-1. daf-16 mutant had a slightly shorter life span compared to wild type and was sensitive to heat and paraquat. The daf-16 phenotype resembles that of mev-1 showing a short life and oxygen sensitivity. We measured mRNA levels of superoxide dismutase genes (sod-1 through 4), catalase genes (clt-1 and ctl-2), known to encode anti-oxidant enzymes, and found they were elevated in age-1 young adults. On the other hand, in daf-16 and mev-1, the expression of sod-1, sod-2 and sod-3 genes was lower rather than in wild type. Conversely, ctl-1 and ctl-2 genes expression was significantly elevated in daf-16 and mev-1. This suggests that DAF-16, a forkhead/winged-helix transcription factor, whose expression is suppressed by AGE-1, phosphoinositide 3-kinase (PI3-kinase), regulates anti-oxidant genes as well as energy metabolism under atmospheric conditions. However, the level of gene expression of SOD and catalase was not elevated by short-term exposure to 90% oxygen in wild type, mev-1, daf-16 and even age-1. This suggests that SOD and catalase do not play a role in the adaptive response against oxidative stress under hyperoxia, at least under these experimental conditions.

The p38 signal transduction pathway participates in the oxidative stress-mediated translocation of DAF-16 to Caenorhabditis elegans nuclei

Much attention has focused on the insulin-like signaling pathway in Caenorhabditis elegans because of its pivotal role in life-span determination and oxidative stress resistance. The daf-16 gene encodes a fork-head transcription factor that is negatively regulated by this insulin-signaling pathway. The DAF-16 protein is translocated to the nucleus when animals were subjected to oxidative stress in the form of paraquat. This oxidative stress-mediated translocation was blocked by mutation of the p38-related sek-1 (MAPKK) mutant and DAF-16 instead remained cytoplasmic. The fact that DAF-16 translocation by oxidative stress is epistatic to sek-1 suggests that oxidative stress mediates regulation of DAF-16 through activating the p38 signal transduction pathway upstream of daf-16 so as to mobilize DAF-16 to the nucleus and activate transcription.

Effect of oxidative stress on translocation of DAF-16 in oxygen-sensitive mutants, mev-1 and gas-1 of Caenorhabditis elegans

Mutations in the mev-1 and gas-1 genes of the nematode Caenorhabditis elegans render animals hypersensitive to oxygen and paraquat, and lead to premature aging. We show that both mutants overproduce superoxide anion in isolated sub-mitochondrial particles, which probably explains their hypersensitivity to oxidative stress. The daf-16 gene encodes a fork-head transcription factor that is negatively regulated by an insulin-signaling pathway. In wild-type animals, the DAF-16 protein normally resides in the cytoplasm and only becomes translocated to nuclei upon activating stimuli such as oxidative stress. Conversely, DAF-16 resides constitutively in the nuclei of mev-1 and gas-1 mutants even under normal growth conditions. Supplementation of the antioxidant coenzyme Q(10) reversed this nuclear translocation of DAF-16. Since both gas-1 and mev-1 encode subunits of electron transport chain complexes, these data illustrate how mitochondrial perturbations can impact signal transduction pathways.

SOD-1 Deletions in Caenorhabditis elegans Alter the Localization of Intracellular Reactive Oxygen Species and Show Molecular Compensation

Superoxide dismutase (SOD) is an enzyme that catalytically removes the superoxide radical (*O2-) and protects organisms from oxidative damage during normal aging. We demonstrate that not only the cytosolic *O2- level but also the mitochondrial *O2- level increases in the deletion mutants of sod-1 gene encoding Cu/Zn SOD in Caenorhabditis elegans (C. elegans). Interestingly, this suggests that the activity of SOD-1, which so far has been thought to act mainly in cytoplasm, helps to control the detoxification of *O2- also in the mitochondria. We also found functional compensation by other SODs, especially the sod-5 gene, which was induced several fold in the mutants. Therefore, the possibility exists that the compensative expression of sod-5 gene in the sod-1 deficit is associated with the insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling pathway, which regulates longevity and stress resistance of C. elegans because the sod-5 gene may be a target of the pathway.

Oxidative stress pretreatment increases the X-radiation resistance of the nematode Caenorhabditis elegans

Pre-exposure of wild-type Caenorhabditis elegans to oxygen conferred a protective effect against the lethality imposed by subsequent X-irradiation. In contrast, two mutants (rad-1 and rad-2) that are UV and ionizing radiation hypersensitive but not oxygen sensitive, did not exhibit this adaptive response. To explore the molecular basis of protection, the expression of several key genes was examined using Northern blot analyses to measure mRNA levels. In the wild-type, expression of the heat shock protein genes, hsp16-1 and hsp16-48, increased dramatically after incubation under high oxygen. Expression of two superoxide dismutase genes (sod-1 and sod-3) was relatively unaffected. Unlike the wild-type, the basal levels of these four genes were significantly lower in the rad-1 and rad-2 mutants under atmospheric conditions. These genes were partially induced in response to oxidative stress. These data suggest that at least a portion of the hypersensitive phenotype of rad-1 and rad-2 may be attributed to inappropriate gene expression.

Supplemental Cellular Protection by a Carotenoid Extends Lifespan via Ins/IGF-1 Signaling in Caenorhabditis elegans

Astaxanthin (AX), which is produced by some marine animals, is a type of carotenoid that has antioxidative properties. In this study, we initially examined the effects of AX on the aging of a model organism C. elegans that has the conserved intracellular pathways related to mammalian longevity. The continuous treatments with AX (0.1 to 1 mM) from both the prereproductive and young adult stages extended the mean lifespans by about 16–30% in the wild-type and long-lived mutant age-1 of C. elegans. In contrast, the AX-dependent lifespan extension was not observed even in a daf-16 null mutant. Especially, the expression of genes encoding superoxide dismutases and catalases increased in two weeks after hatching, and the DAF-16 protein was translocated to the nucleus in the AX-exposed wild type. These results suggest that AX protects the cell organelle mitochondria and nucleus of the nematode, resulting in a lifespan extension via an Ins/IGF-1 signaling pathway during normal aging, at least in part.

Modulatory effect of ionizing radiation on food-NaCl associative learning: the role of γ subunit of G protein in Caenorhabditis elegans

Ionizing radiation (IR) is known to impair learning by suppressing adult neurogenesis in the hippocampus. However, in a mature nervous system, IR‐induced functional alterations that are independent of neurogenesis remain largely unknown. In the present study, we analyzed the effects of IR on a food‐NaCl associative learning paradigm of adult Caenorhabditis elegans that does not undergo neurogenesis. We observed that a decrease in chemotaxis toward NaCl occurs only after combined starvation and exposure to NaCl. Exposure to IR induced an additional decrease in chemotaxis immediately after an acute dose in the transition stage of the food‐NaCl associative learning. Strikingly, chronic irradiation induced negative chemo‐taxis in the exposed animals, i.e., the primary avoidance response. IR‐induced additional decreases in chemo‐taxis after acute and chronic irradiation were significantly suppressed in the gpc‐1 mutant, which was defective in GPC‐1 (one of the two y subunits of the heterotrimeric G‐protein). Chemotaxis to cAMP, but not to lysine and benzaldehyde, was influenced by IR during the food‐NaCl associative learning. Our novel findings suggest that IR behaves as a modulator in the food‐NaCl associative learning via C. elegans GPC‐1 and a specific neuronal network and may shed light on the modulatory effect of IR on learning.—Sakashita, T., Hamada, N., Ikeda, D. D., Yanase, S., Suzuki, M., Ishii, N., Kobayashi, Y. Modulatory effect of ionizing radiation on food‐NaCl associative learning: the role of γ subunit of G protein in Caenorhabditis elegans. FASEB J. 22, 713–720 (2008)

Molecular cloning of oxygen-inducible genes in Caenorhabditis elegans by RT-PCR differential display

Abstract To investigate the genetic response to oxidative stress in eukaryotic cells, we have applied the method RT-PCR differential display to the small, free-living nematode Caenorhabditis elegans (C. elegans) which was cultured under atmospheric or high oxygen concentrations. As a result, a new gene was cloned whose expression increased at high oxygen concentration.

Timing mechanism and effective activation energy concerned with aging and lifespan in the long-lived and thermosensory mutants of Caenorhabditis elegans.

The lifespans of many poikilothermic animals, including the nematode Caenorhabditis elegans, depend significantly on environmental temperature. Using long-living, thermosensory mutants of C. elegans, we tested whether the temperature dependency of the mean lifespan is compatible with the Arrhenius equation, which typically represents one of the chemical reaction rate theories. The temperature dependency of C. elegans was the Arrhenius type or normal, but daf-2(e1370) mutants were quite different from the others. However, taking into account the effect of the thermal denaturation of DAF-2 with the temperature, we showed that our analyzed results are compatible with previous ones. We investigated the timing mechanism of one parameter (the onset of biodemographic aging (t(0))) in the lifespan equation by applying the RNAi feeding method to daf-2 mutants in order to suppress daf-16 activity at different times during the life cycle. In summary, we further deepened the biological role of two elements, t(0) and z (the inverse of the aging rate), in the lifespan equation and mean lifespan formulated by our diffusion model z(2) = 4Dt(0), where z is composed of t(0) and D (the diffusion constant).

Impaired p53/CEP‐1 is associated with lifespan extension through an age‐related imbalance in the energy metabolism of C. elegans

In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP‐1 mediates the stress response, activates germ line apoptosis and regulates meiotic chromosome segregation. A reduction in its expression, which frequently occurs in mammalian cancer cells, extends lifespan and induces an adaptive response in C. elegans. However, these effects do not involve an increase in oxidative stress resistance. Here, we showed that intermittent exposure to hyperoxia, which induces oxidative stress resistance and lowers the production of ROS derived from mitochondrial respiration in C. elegans, slightly improved the lifespan extension of cep‐1 mutant. Interestingly, ATP levels were increased without an increase in oxygen consumption in cep‐1 mutant during aging. In the wild‐type, lactate levels and consequentially the lactate/pyruvate ratio decreased during aging in adults. Furthermore, the expression levels of mitochondrial respiration‐related sco‐1, which is a target of p53/CEP‐1, as well as those of gluconeogenesis regulation and mammalian sirtuin ortholog genes, were also increased in the aged and adaptive conditioned wild‐type animals. In contrast, the lactate/pyruvate ratio increased in cells of the cep‐1 mutant and was amplified by intermittent hyperoxia. These results suggest that impaired p53/CEP‐1 leads to an imbalance in the age‐related energy metabolic alteration between mitochondrial oxidative phosphorylation and aerobic glycolysis and plays an important role in the extension of both intact and adaptive lifespans.