10 research outputs found
Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in <i>Caenorhabditis elegans</i>
<div><p>Mitochondrial dysfunction can increase oxidative stress and extend lifespan in <i>Caenorhabditis elegans</i>. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPR<sup>mt</sup>) and extends lifespan. Here we report that transaldolase (<i>tald-1</i>) deficiency impairs mitochondrial function <i>in vivo</i>, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H<sub>2</sub>O<sub>2</sub> levels. Lifespan extension from knockdown of <i>tald-1</i> is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (<i>fmo-2</i>). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.</p></div
Redox stress is downstream of transaldolase deficiency.
<p><b>(A)</b> H<sub>2</sub>O<sub>2</sub> levels increase from RNAi knockdown of <i>tald-1</i> or <i>cco-1</i> (N = 7 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(B)</b> NADPH levels decrease from RNAi knockdown of <i>tald-1</i> (N = 5+ biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(C)</b> RNAi knockdown of <i>tald-1</i> causes sensitivity to paraquat (PQ). Percent survival of N2 worms grown on RNAi bacteria and 10 mM PQ was measured over seven days. Survival analyses were performed at 25°C (N = 6 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p
The flavin-containing monooxygenase FMO-2 is upregulated in a HLH-30 and PMK-1 dependent fashion and regulates the lifespan extension from <i>tald-1(RNAi)</i>.
<p><b>(A)</b><i>fmo-2p</i>::<i>mCherry</i> reporter expression is increased by <i>tald-1(RNAi)</i> or BD in a HLH-30 and PMK-1 dependent fashion. BD animals were starved for 24 hours on FUDR plates prior to imaging. Scale bar, 200 μm. <b>(B)</b> Mean relative fluorescence of <i>fmo-2p</i>::<i>mCherry</i> reporter animals in the context of the <i>hlh-30(tm1978)</i> mutation. Fluorescence is calculated relative to N2 <i>EV(RNAi)</i> controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(C)</b> Mean relative fluorescence of <i>fmo-2p</i>::<i>mCherry</i> reporter animals in the context of the <i>pmk-1(km25)</i> mutation. Fluorescence is calculated relative to N2 <i>EV(RNAi)</i> controls (N = 5 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(D)</b> Gene expression of <i>fmo-2</i> is upregulated by <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i> (N = 11 biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(E)</b> Gene expression of <i>fmo-2</i> is upregulated by <i>tald-1(RNAi)</i> in a HLH-30 and PMK-1 dependent fashion (N = 3–6 biological replicates, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(F)</b> Percent of animals displaying HLH-30 nuclear localization. BD animals were starved for 8 hours on FUDR plates prior to imaging (N = 5 independent experiments, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). <b>(G)</b> FMO-2 is required for the lifespan extension from <i>tald-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 15.3±0.1 days, n = 341), N2 fed <i>tald-1(RNAi)</i> (mean 17.8±0.1 days, n = 353), <i>fmo-2(ok2147)</i> fed <i>EV(RNAi)</i> (mean 18±0.2 days, n = 314), <i>fmo-2(ok2147)</i> fed <i>tald-1(RNAi)</i> (mean 17.4±0.2 days, n = 382). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(H)</b> FMO-2 is partially required for the lifespan extension from <i>cco-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 15.7±0.1 days, n = 562), N2 fed <i>cco-1(RNAi)</i> (mean 23.3±0.2 days, n = 616), <i>fmo-2(ok2147)</i> fed <i>EV(RNAi)</i> (mean 18.3±0.1 days, n = 474), <i>fmo-2(ok2147)</i> fed <i>cco-1(RNAi)</i> (mean 20.5±0.2 days, n = 473). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. <b>(I)</b> Lifespan extension from <i>fmo-2</i> overexpression is not additive with <i>tald-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16.5±0.1 days, n = 453), N2 fed <i>tald-1(RNAi)</i> (mean 20.6±0.1 days, n = 421), <i>eft-3p</i>::<i>fmo-2</i> fed <i>EV(RNAi)</i> (mean 18.2±0.1 days, n = 439), <i>eft-3p</i>::<i>fmo-2</i> fed <i>tald-1(RNAi)</i> (mean 19.1±0.1 days, n = 435). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(J)</b> Lifespan extension from <i>fmo-2</i> overexpression is additive with <i>cco-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16.5±0.1 days, n = 453), N2 fed <i>cco-1(RNAi)</i> (mean 23.3±0.2 days, n = 259), <i>eft-3p</i>::<i>fmo-2</i> fed <i>EV(RNAi)</i> (mean 18.2±0.1 days, n = 439), <i>eft-3p</i>::<i>fmo-2</i> fed <i>cco-1(RNAi)</i> (mean 25.5±0.2 days, n = 352). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p
Inhibition of the pentose phosphate pathway activates the UPR<sup>mt</sup> and extends lifespan.
<p><b>(A)</b> Diagram of both the oxidative and non-oxidative branches of the PPP. The oxidative branch produces NADPH, while the non-oxidative branch produces ribose-5-P and interconverts sugar carbon backbones. The white boxes contain enzyme names with the human gene listed above the <i>C</i>. <i>elegans</i> homolog. <b>(B)</b> PPP gene knockdown increases <i>hsp-6p</i>::<i>gfp</i> reporter expression. <b>(C)</b> Mean relative fluorescence of <i>hsp-6p</i>::<i>gfp</i> animals grown on PPP RNAi. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 4 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(D)</b> RNAi knockdown of PPP genes extends <i>C</i>. <i>elegans</i> lifespan. N2 fed <i>EV(RNAi)</i> (mean 17.4±0.1 days, n = 455), N2 fed <i>tald-1(RNAi)</i> (mean 19.9±0.2 days, n = 391), N2 fed <i>tkt-1(RNAi)</i> (mean 18.4±0.1 days, n = 461), N2 fed T25B9.9<i>(RNAi)</i> (mean 18.8±0.2 days, n = 311). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(E)</b> RNAi knockdown of <i>tald-1</i> extends lifespan independently of the UPR<sup>mt</sup>. N2 fed <i>EV(RNAi)</i> (mean 19.3±0.2 days, n = 192), N2 fed <i>tald-1(RNAi)</i> (mean 22.1±0.2 days, n = 251), <i>atfs-1(tm4525)</i> fed <i>EV(RNAi)</i> (mean 19.6±0.2 days, n = 230), <i>atfs-1(tm4525)</i> fed <i>tald-1(RNAi)</i> (mean 24.5±0.3 days, n = 228), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>EV(RNAi)</i> (mean 18.9±0.2 days, n = 205), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>tald-1(RNAi)</i> (mean 23.1±0.3 days, n = 220). Lifespans were performed at 20°C, with pooled data from two independent experiments shown. <b>(F)</b> RNAi knockdown of <i>cco-1</i> extends lifespan independently of the UPR<sup>mt</sup>. N2 fed <i>EV(RNAi)</i> (mean 19.3±0.2 days, n = 192), N2 fed <i>cco-1(RNAi)</i> (mean 32.3±0.5 days, n = 187), <i>atfs-1(tm4525)</i> fed <i>EV(RNAi)</i> (mean 19.6±0.2 days, n = 230), <i>atfs-1(tm4525)</i> fed <i>cco-1(RNAi)</i> (mean 29±0.6 days, n = 194), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>EV(RNAi)</i> (mean 18.9±0.2 days, n = 205), <i>atfs-1(tm4525);gcn-2(ok871)</i> fed <i>cco-1(RNAi)</i> (mean 32.6±0.5 days, n = 228). Lifespans were performed at 20°C, with pooled data from two independent experiments shown. <b>(G)</b> RNAi knockdown of <i>tald-1</i> extends lifespan only when knockdown occurs during development. N2 fed <i>EV(RNAi)</i> (mean 14.2±0.1 days, n = 361), N2 fed <i>tald-1(RNAi)</i> from hatching (mean 16.4±0.2 days, n = 468), N2 fed <i>tald-1(RNAi)</i> from L4 (mean 14.4±0.1 days, n = 330). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p
Transaldolase deficiency causes a starvation-like response that decreases animal fat content and rewires lipid metabolism gene expression.
<p><b>(A)</b> Intestinal fat staining decreases from RNAi knockdown of <i>tald-1</i> or <i>cco-1</i>. Oil Red O (ORO) staining was performed on day 3 from hatching animals propagated at 20°C. Scale bar, 50 μm. <b>(B)</b> Quantification of ORO staining within anterior intestine (N = 2 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(C)</b> RNAi knockdown of <i>tald-1</i> causes an increase in adipose triglyceride lipase ATGL-1 protein levels. Scale bar, 200 μm. <b>(D)</b> Mean relative fluorescence of ATGL-1::GFP signal in animals grown on <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i>. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 4 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(E)</b> RNAi knockdown of <i>tald-1</i> or <i>cco-1</i> causes a decrease in stearoyl-CoA desaturase <i>fat-7p</i>::<i>gfp</i> reporter expression. Scale bar, 200 μm. <b>(F)</b> Mean relative fluorescence of <i>fat-7p</i>::<i>gfp</i> reporter animals grown on <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i>. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(G)</b> Gene expression of starvation-responsive lipid metabolism genes is altered in <i>tald-1(RNAi)</i> animals. Log2 fold change calculated to emphasize the increases and decreases in gene expression levels from RNAi treatments (N = 6–8 independent experiments, error bars indicate s.e.m., paired student’s t-tests with Bonferroni’s correction). <b>(H)</b> RNAi knockdown of <i>tald-1</i> does not robustly extend lifespan of BD animals. N2 fed <i>EV(RNAi)</i> (mean 18.2±0.2 days, n = 161), N2 fed <i>tald-1(RNAi)</i> (mean 20.4±0.2 days, n = 151), BD animals developed on <i>EV(RNAi)</i> (mean 20.2±0.2 days, n = 123), BD animals developed on <i>tald-1(RNAi)</i> (mean 21.5±0.3 days, n = 150). Lifespans were performed at 25°C, with one experiment shown. <b>(I)</b> RNAi knockdown of <i>cco-1</i> extends lifespan dissimilar from BD. N2 fed <i>EV(RNAi)</i> (mean 18.2±0.2 days, n = 161), N2 fed <i>cco-1(RNAi)</i> (mean 24±0.3 days, n = 156), BD animals developed on <i>EV(RNAi)</i> (mean 20.2±0.2 days, n = 123), BD animals developed on <i>cco-1(RNAi)</i> (mean 27.1±0.3 days, n = 148). Lifespans were performed at 25°C, with one experiment shown. <b>(J)</b> RNAi knockdown of <i>tald-1</i> does not require NHR-49 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 17.5±0.1 days, n = 366), N2 fed <i>tald-1(RNAi)</i> (mean 20.1±0.1 days, n = 397), <i>nhr-49(nr2041)</i> fed <i>EV(RNAi)</i> (mean 11.5±0.1 days, n = 310), <i>nhr-49(nr2041)</i> fed <i>tald-1(RNAi)</i> (mean 12.9±0.1 days, n = 333). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(K)</b> RNAi knockdown of <i>cco-1</i> does not require NHR-49 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 17±0.1 days, n = 532), N2 fed <i>cco-1(RNAi)</i> (mean 22.6±0.2 days, n = 344), <i>nhr-49(nr2041)</i> fed <i>EV(RNAi)</i> (mean 11.1±0.1 days, n = 495), <i>nhr-49(nr2041)</i> fed <i>cco-1(RNAi)</i> (mean 15±0.1 days, n = 489). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p
Model of transaldolase deficiency mediated longevity.
<p>Reduced activity of the pentose phosphate pathway enzyme transaldolase has several consequences, including inhibition of mitochondrial respiration, induction of a mitochondrial stress response, alterations in redox homeostasis, and activation of a starvation-like metabolic response. Lifespan extension in response to transaldolase deficiency appears to be mediated by both MAPK signaling and HLH-30 mediated induction of autophagy and activation of FMO-2.</p
HLH-30 mediates the lifespan extension and autophagy gene expression from <i>tald-1(RNAi)</i>.
<p><b>(A)</b> RNAi knockdown of <i>tald-1</i> increases nuclear localization of HLH-30 similarly to starvation. BD animals were starved for 8 hours on FUDR plates prior to imaging. Scale bar, 200 μm. <b>(B)</b> Percent of animals displaying HLH-30 nuclear localization. (N = 8 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(C)</b> HLH-30 is required for the lifespan extension from <i>tald-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16±0.1 days, n = 476), N2 fed <i>tald-1(RNAi)</i> (mean 19.2±0.1 days, n = 455), <i>hlh-30(tm1978)</i> fed <i>EV(RNAi)</i> (mean 12.9±0.1 days, n = 510), <i>hlh-30(tm1978)</i> fed <i>tald-1(RNAi)</i> (mean 12.9±0.1 days, n = 514). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(D)</b> HLH-30 is not required for the lifespan extension from <i>cco-1(RNAi)</i>. N2 fed <i>EV(RNAi)</i> (mean 16±0.1 days, n = 476), N2 fed <i>cco-1(RNAi)</i> (mean 24.3±0.2 days, n = 362), <i>hlh-30(tm1978)</i> fed <i>EV(RNAi)</i> (mean 12.9±0.1 days, n = 510), <i>hlh-30(tm1978)</i> fed <i>cco-1(RNAi)</i> (mean 19.2±0.1 days, n = 533). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(E)</b> Gene expression of autophagy genes is upregulated in <i>tald-1(RNAi)</i> animals (N = 6 biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(F)</b> HLH-30 is required for the upregulation of autophagy genes by <i>tald-1(RNAi)</i>. qRT-PCR was performed on RNA isolated from <i>hlh-30(tm1978)</i> animals (N = 3 biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(G)</b> Autophagic flux increases from RNAi knockdown of <i>tald-1</i> or <i>cco-1</i>. Western blot analysis was performed on protein lysates from <i>eft-3p</i>::<i>dFP</i>::<i>lgg-1</i> animals using an anti-GFP antibody to detect full-length dFP-LGG-1 and monomeric FP. An anti-α-tubulin antibody was used as a loading control. Three biological replicates for each RNAi treatment are presented. <b>(H)</b> Quantification of the dFP::LGG-1 ratiometric reporter. The intensity of monomeric FP to full-length dFP::LGG-1 was measured to determine autophagic flux (N = 5 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p
Transaldolase deficiency alters mitochondrial morphology and decreases <i>in vivo</i> mitochondrial respiration.
<p><b>(A)</b> Diagram depicting the posterior intestinal cells that were visualized for mitochondrial morphology. <b>(B)</b> Intestinal mitochondrial morphology is altered by <i>tald-1(RNAi)</i> and <i>cco-1(RNAi)</i>. The top panel represents a single 0.34 μm slice imaged using confocal microscopy, with a magnified area displayed in a white dotted box to highlight morphology differences. The bottom panel consists of a max intensity projection of five z-slices to emphasize mitochondrial content in these cells. Scale bar, 10 μm. <b>(C)</b> Quantification of percent mitochondrial area per cell. (N = 2 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(D)</b> Mitochondrial morphology changes from <i>tald-1(RNAi)</i> are regulated by DRP-1. RNAi treatments include <i>EV(RNAi)</i>, <i>tald-1(RNAi)</i> [50:50 with <i>EV(RNAi)</i>], <i>drp-1(RNAi)</i> [50:50 with <i>EV(RNAi)</i>], and <i>tald-1(RNAi)</i> [50:50 with <i>drp-1(RNAi)</i>]. Scale bar, 10 μm. <b>(E)</b> Oxygen consumption rate decreases with <i>tald-1(RNAi)</i> and <i>cco-1(RNAi)</i>. OCR was measured using the Seahorse XF Analyzer and normalized to animal number (N = 6 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(F)</b> P/O ratio (the ATP produced per oxygen atom reduced), <b>(G)</b> respiratory control index (State 3:State 4 rates), <b>(H)</b> malate-driven respiration (Complex I-IV), succinate-driven respiration (Complex II-IV), and TMPD/ascorbate-driven respiration (Complex IV) were measured using the OXPHOS assay on isolated mitochondria from RNAi treated animals. Respiratory rates were measured as rate of disappearance of oxygen (nmol[O<sub>2</sub>]) per minute per mg protein (N = 4 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). Also, in this figure, color coating of bars and lines reflect the legend in (C).</p
Lifespan extension from <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i> requires stress-activated MAPKs.
<p><b>(A)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the JNK MAPK JNK-1. N2 fed <i>EV(RNAi)</i> (mean 17.2±0.1 days, n = 506), N2 fed <i>tald-1(RNAi)</i> (mean 20.4±0.1 days, n = 500), <i>jnk-1(gk7)</i> fed <i>EV(RNAi)</i> (mean 17±0.1 days, n = 582), <i>jnk-1(gk7)</i> fed <i>tald-1(RNAi)</i> (mean 18.1±0.1 days, n = 488). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. <b>(B)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the JNK MAPK JNK-1. N2 fed <i>EV(RNAi)</i> (mean 16.9±0.1 days, n = 494), N2 fed <i>cco-1(RNAi)</i> (mean 22.7±0.2 days, n = 431), <i>jnk-1(gk7)</i> fed <i>EV(RNAi)</i> (mean 16.1±0.1 days, n = 594), <i>jnk-1(gk7)</i> fed <i>cco-1(RNAi)</i> (mean 19.9±0.2 days, n = 408). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(C)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the JNK MAPK KGB-1. N2 fed <i>EV(RNAi)</i> (mean 15±0.1 days, n = 630), N2 fed <i>tald-1(RNAi)</i> (mean 18.7±0.1 days, n = 657), <i>kgb-1(um3)</i> fed <i>EV(RNAi)</i> (mean 13.1±0.1 days, n = 580), <i>kgb-1</i> fed <i>tald-1(RNAi)</i> (mean 11.9±0.1 days, n = 600). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(D)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the JNK MAPK KGB-1. N2 fed <i>EV(RNAi)</i> (mean 15±0.1 days, n = 630), N2 fed <i>cco-1(RNAi)</i> (mean 23.2±0.2 days, n = 511), <i>kgb-1(um3)</i> fed <i>EV(RNAi)</i> (mean 13.1±0.1 days, n = 580), <i>kgb-1</i> fed <i>cco-1(RNAi)</i> (mean 15.8±0.2 days, n = 501). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(E)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the p38 MAPK PMK-1. N2 fed <i>EV(RNAi)</i> (mean 16.8±0.1 days, n = 494), N2 fed <i>tald-1(RNAi)</i> (mean 19.3±0.1 days, n = 460), <i>pmk-1(km25)</i> fed <i>EV(RNAi)</i> (mean 14.3±0.1 days, n = 514), <i>pmk-1(km25)</i> fed <i>tald-1(RNAi)</i> (mean 14±0.1 days, n = 525). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(F)</b> RNAi knockdown of <i>cco-1</i> does not require the p38 MAPK PMK-1 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 16±0.1 days, n = 575), N2 fed <i>cco-1(RNAi)</i> (mean 22.3±0.2 days, n = 448), <i>pmk-1(km25)</i> fed <i>EV(RNAi)</i> (mean 13.8±0.1 days, n = 609), <i>pmk-1(km25)</i> fed <i>cco-1(RNAi)</i> (mean 18.7±0.1 days, n = 535). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(G)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the MAP3K NSY-1. N2 fed <i>EV(RNAi)</i> (mean 14.6±0.1 days, n = 542), N2 fed <i>tald-1(RNAi)</i> (mean 17.2±0.1 days, n = 599), <i>nsy-1(ag3)</i> fed <i>EV(RNAi)</i> (mean 14.9±0.1 days, n = 473), <i>nsy-1(ag3)</i> fed <i>tald-1(RNAi)</i> (mean 14.4±0.1 days, n = 508). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(H)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the MAP3K NSY-1. N2 fed <i>EV(RNAi)</i> (mean 14.6±0.1 days, n = 542), N2 fed <i>cco-1(RNAi)</i> (mean 22.5±0.2 days, n = 454), <i>nsy-1(ag3)</i> fed <i>EV(RNAi)</i> (mean 14.9±0.1 days, n = 473), <i>nsy-1(ag3)</i> fed <i>cco-1(RNAi)</i> (mean 18.5±0.2 days, n = 458). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>.</p