Dietary restriction (DR) induces the FIRE response to cellular stress in <i>C. elegans</i> intestinal cells.

<p>(A) Cartoon of the FIRE response depicting the stress-induced redistribution of cytoplasmic esterase activity to a perinuclear region. The large rectangles contain cartoons of esterase activity (brown color) localization in two adjacent intestinal cells (rounded rectangles) with nuclei indicated by ovals labeled “N”. (B) Cytoplasmic esterase activity in intestinal cells of young adult hermaphrodites in the presence of ample bacterial food (Fed), 5-hours following food withdrawal (Fasted), and in <i>eat-2(ad465)</i> mutants (DR <i>eat-2</i>) or wildtype animals subjected to RNAi knockdown of <i>let-363</i>/TOR (<i>let-363</i>/TOR RNAi). Images are representative from 80 or more animals examined in at least 3 independent experiments. (C) DR induced by food source dilution also induced a FIRE response in wildtype adults. Young adult hermaphrodites, raised under bacteria-replete conditions, were fed with bacteria at indicated concentration for 5 hours and then analyzed. At high food concentrations (10¹⁰ and 10¹¹ cfu/mL) adult animals did not exhibit a FIRE response. At lower food concentrations, (<10⁹ cfu/mL), most animals exhibited a strong FIRE response. For each feeding condition, FIRE response was scored in 17-32 animals in a blinded fashion. Similar results were obtained in 2 additional experiments using a progressive nutrient stress regimen of diluted <i>let-363</i>/TOR RNAi (not shown).</p

Small body size caused by DR was partially suppressed in <i>daf-2</i> mutants.

<p>(A, B) Body length increased over the first 3 days of adulthood under both DR and non-DR conditions. Wildtype animals under DR conditions were approximately 30% shorter than under non-DR conditions (diamonds). Under non-DR conditions, <i>daf-2(e1368)</i> and <i>daf-2(e1370)</i> adults were slightly longer than wildtype animals (dark shapes) and <i>daf-2</i> mutant body size was also reduced under DR (triangles). However, <i>eat-2;daf-2</i> animals grew to larger sizes than <i>eat-2</i> animals (open triangles vs open diamonds). (C) Composite graph of adult body length of wildtype and <i>daf-2</i> mutants under DR and non-DR conditions on adult day 1 (24-hours post-molt), plotted relative to wildtype non-DR adults. All body length measurements were conducted at 15°C and animals were synchronized by chronological age from hatching. For each time point, measurements of 10–14 animals for each strain were averaged from 3 independent experiments, except for <i>eat-2(ad465); daf-2(e1368)</i> animals for which 2 experiments were conducted. Statistical significance was evaluated by t-test and indicate significance in at least 3 of 4 timepoints, *p<0.01 vs wildtype in all trials, #p<0.01 vs <i>eat-2(ad465)</i> in all trials, ˆp<0.01 vs <i>daf-2(e1368)</i> (A) or <i>daf-2(e1370)</i> (B) in all trials ; p<0.0001 (t-test) for all 3 strains at each time point except for <i>eat-2(ad465)</i> vs <i>eat-2(ad465); daf-2(e1370)</i> at 0 and 8 hours (p = 0.076 and 0.089, respectively). In addition, in (A) <i>eat-2(ad465); daf-2(e1368)</i> vs <i>eat-2(ad465)</i> body lengths were statistically significant (p<0.01) at only the 48 and 72 hour timepoints of all trials.</p

Model for the interactions between DR and DAF-2/IIS on growth, FIRE response and lifespan.

<p>Food intake and nutrient signaling have dramatic effects on growth, FIRE response and longevity in <i>C. elegans</i>. This paper reports that levels of DAF-2/IIS can modify these three nutrient-dependent processes. We propose that levels of DAF-2/IIS may be under control of environmental cues that modulate insulin ligand production.</p

Levels of <i>daf-2</i> activity modify lifespan of <i>eat-2(ad465)</i> animals.

<p>DR, from the <i>eat-2(ad465)</i> mutation, had an additive effect on lifespan of <i>daf-2(e1370)</i> adults (B), but not on lifespan of <i>daf-2(e1368)</i> adults (A). Lifespan data and statistics are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001240#pone-0001240-t001" target="_blank">Table 1</a>.</p

DR, imposed by the <i>eat-2(ad465)</i> mutation, extended adult lifespan of <i>daf-2(e1370)</i>, but not <i>daf-2(e1368)</i>.

<p>Adult lifespan was examined at 20°C in the presence of FUDR. Data are cumulative of two trails with 30–40 animals/trial. Two additional trials were conducted for ad lib and DR <i>daf-2(e1370)</i> and wildtype animals with consistent results (not shown).</p>*<p>Non-DR lifespans of <i>daf-2(e1370)</i> versus <i>daf-2(e1368)</i> were statistically significantly different by the Log-rank test (p = 0.0058) but not by Wilcoxon test p = 0.11), although they were not dramatically different.</p

Nutrient stress did not strongly induce <i>hsp-16.2:GFP</i> reporter expression.

<p>A GFP reporter expressed from the <i>hsp16.2</i> promoter was examined in animals under food-replete, fasted and heat-stressed conditions. (A) Under food-replete conditions (fed), fluorescence from <i>hsp16.2:</i>GFP was undetectable in the pharynx region of most animals. After a 4 hour fasting regimen, elevated <i>hsp-16.2:</i>GFP fluorescence was observed in many animals, as it was for animals under thermal stress (35°C, 3.5–6 hours). (B) Relative GFP fluorescence in the pharynx region of animals carrying the <i>hsp-16.2:GFP</i> reporter. Fluorescence intensity was measured as average pixel intensity in same-sized regions of the pharynx. Fluorescence intensity measurements under food-replete conditions were set as baseline. Duplicate bars indicate results from two independent experiments; n = 14–19 animals scored per condition in each experiment. In both experiments, 35°C heat stress induced a statistically significant increase in <i>hsp-16.2</i>:GFP fluorescence intensity (p≤0.001 compared to fed controls). Nutrient stress was not correlated with reproducible statistically significant changes in <i>hsp-16.2</i>:GFP fluorescence intensity (p≤0.001 in one trial only). All conditions were tested on the same day and images were collected using identical acquisition parameters. Three additional experiments also gave similar results.</p

<i>skn-1</i> is expressed in AIY neurons and regulates their functions.

<p>(A) Schematic of the neuronal circuits of <i>C</i>. <i>elegans</i> chemoattractive and thermotactic behaviors. Green triangles represent sensory neurons and blue hexagons command interneurons. Arrows indicate direct interactions, and their thickness is proportional to the frequency of synaptic contacts between the neurons (Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176798#pone.0176798.ref036" target="_blank">36</a>]). T, thermophilic; C, cryophilic.(Adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176798#pone.0176798.ref041" target="_blank">41</a>]). (B) Population thermotaxis experiments were performed using a radial thermal gradient. After 60 minutes the position of the worms was scored and the percentages of worms in the colder (Zone 1) and warmer (Zone 2) regions determined. N2 animals moved toward the region of the plate closer to their previous cultivation temperature (23°C), while both <i>skn-1</i> null strains <i>zu135</i> and <i>zu67</i> showed a cryophilic phenotype similar to <i>ttx-3</i> (AIY) null worms. (C) Representative 3D reconstruction of confocal sections through the head region of double transgenic <i>wglS342;otlS133</i> worms is shown. The <i>skn-1</i>:<i>EGFP</i> reporter (green), containing the entire <i>skn-1</i> gene, is highly expressed in neurons in the head ganglia, and colocalizes with the AIY-specific marker <i>ttx3</i>:RFP (Red). Arrows point at AIY. Scale bar 10 μm. (D) Relative mRNA levels of the AIY cell fate specification homeobox genes <i>ttx-3</i> and <i>ceh-23</i>, and their targets <i>ser-2a</i>, <i>ser-2b</i>, <i>sra-11</i>, <i>kal-1</i> and <i>hen-1</i> in N2 and <i>skn-1</i> null worms. (E) Representative 3D reconstructions showing the expression of the transcriptional reporter <i>ser2</i>:<i>prom1</i>:<i>EGFP</i> in the head region of N2 and <i>skn-1(zu135)</i> null worms. Data are mean and S.E.M. of 3–5 experiments. *p<0.05; **p<0.01; ***p<0.001 versus N2 (Student’s <i>t</i>-test).</p

<i>skn-1</i> is required for interneuron sensory integration and foraging behavior in <i>Caenorhabditis elegans</i>

<div><p>Nrf2/<i>skn-1</i>, a transcription factor known to mediate adaptive responses of cells to stress, also regulates energy metabolism in response to changes in nutrient availability. The ability to locate food sources depends upon chemosensation. Here we show that Nrf2/<i>skn-1</i> is expressed in olfactory interneurons, and is required for proper integration of multiple food-related sensory cues in <i>Caenorhabditis elegans</i>. Compared to wild type worms, <i>skn-1</i> mutants fail to perceive that food density is limiting, and display altered chemo- and thermotactic responses. These behavioral deficits are associated with aberrant AIY interneuron morphology and migration in <i>skn-1</i> mutants. Both <i>skn-1</i>-dependent AIY autonomous and non-autonomous mechanisms regulate the neural circuitry underlying multisensory integration of environmental cues related to energy acquisition.</p></div

Nrf2 is highly expressed in olfactory bulb interneurons.

<p>(A) Representative immunoblots and quantification of Nrf2 protein levels in adult murine brain (n = 4). Values were normalized by GAPDH and expressed as mean percentage (and S.E.M.) compared to olfactory bulb (OB). Cx, cortex; Hp, hippocampus; Cb, cerebellum; Me, medulla. (B) Schematic showing the rostral migratory stream (RMS), the route followed by neuroblasts originating in the sub ventricular zone (SVZ) to reach the olfactory bulb (OB). Immunohistochemistry showing the expression of Nrf2 in the RMS at low magnification (left) and high magnification (right). The boxed area indicates the regions shown in the immunostaining. (C) Immunohistochemistry showing the distribution of Nrf2 in the various regions of the olfactory bulb (upper panel). GCL: granule cell layer; MCL: mitral cell layer; EPL: external plexiform layer; GL: glomerular layer. The higher magnification panels show Nrf2 subcellular localization in mitral cells (MCL) and granule cells (GCL).</p

<i>skn-1</i> regulates AIY neuron morphology and food-seeking behavior by cell-autonomous and non-autonomous mechanisms.

<p>(A) Schematic depiction, representative images, and quantification of the neuroanatomical defects observed in <i>skn-1(zu135)</i> worms. Wild type morphology is shown in black, while aberrant branches from the axon or cell body are depicted in red. Also shown are examples of premature stops, and axon misrouting. (B) Schematic depiction and representative images of the AIY position relative to the pharynx grinder in N2 (black line) and <i>skn-1(zu135)</i> worms (red line). a, distance between grinder and the AIY axon fasciculation at the nerve ring; b, distance between the grinder and AIY cell body. Quantification of the AIY migratory defects in the indicated worm strains. AIY-specific expression of <i>skn-1b</i> has no effect on migration (B), and food-leaving behavior (C) (OP50 = 0.033x). Pan-neuronal expression of <i>skn-1b</i> significantly decreases AIY migratory defects (B) and partially rescues the food-leaving behavior (D). Data are mean and S.E.M. of 3–7 experiments. ***p<0.001 versus N2; ###p<0.001 versus <i>skn-1</i> (zu135) (Student’s <i>t</i>-test).</p