<i>hsf-1</i> is needed for the fluorimetric DR signature and longevity phenotypes of <i>mir-80</i>(Δ).

<p><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g005" target="_blank">Fig. 5A</a>. <i>hsf-1(RNAi)</i> in the <i>mir-80</i>(Δ) background reverses the DR Ex_max shift. We grew age-synchronized animals under standard RNAi feeding conditions (20°C, HT115) and measured age pigments at Day 4 (50 animals per RNAi clone). We recorded Ex_max as the highest peak detected by the Datamax software package suite (Horiba Scientific). Black bar, WT+ empty vector RNAi; red bar, <i>mir-80</i>(Δ)+empty vector RNAI; grey bar, <i>mir-80</i>(Δ)<i>+hsf-1(RNAi)</i>. Graphs represent cumulative data from 3 independent trials. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (** p<0.001). Note that <i>hsf-1(RNAi)</i> treatment of WT does not change Ex_max (data not shown). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g005" target="_blank">Fig. 5B</a>. <i>hsf-1(RNAi)</i> in the <i>mir-80</i>(Δ) background partially counters the low age pigment level phenotype of <i>mir-80</i>(Δ). We grew age-synchronized animals under standard conditions (20°C, HT115) and measured total age pigment fluorescence, normalized to total tryptophan fluorescence as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Gerstbrein1" target="_blank">[19]</a> (Day 4 post-hatching, 50 animals per RNAi clone). Black bar, WT+ empty vector RNAi; red bar, <i>mir-80</i>(Δ)+empty vector RNAi; grey bar, <i>mir-80</i>(Δ)<i>+hsf-1(RNAi)</i>. Graphs represent cumulative data from 3 independent trials. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (*** p<0.0001, * p<0.05 compared to <i>mir-80</i>(Δ) empty vector). Note that <i>hsf-1(RNAi)</i> treatment of WT does not change age pigment scores at day 4 (data not shown). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g005" target="_blank">Fig. 5C</a>. <i>hsf-1</i> is required for <i>mir-80</i>(Δ)-induced longevity. We grew age-synchronized animals under standard conditions with low levels of FUDR to prevent progeny production (20°C, OP50-1, 50 uM FuDR). At day 9, we placed 10 healthy animals per plate, ≥40 per strain per trial, and we scored viability as movement away from pick touch at the indicated days. The graphs represent data combined from 3 independent trials. Statistics are calculated using the Log-rank Test. Error bars indicate ± S.E.M. The <i>mir-80</i>(Δ); <i>hsf-1(sy441)</i> double mutant is shorter lived than <i>mir-80</i>(Δ) (p<0.0001). Because RNAi knockdown is inefficient the nervous system (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Calixto1" target="_blank">[59]</a>), the profound effects of <i>hsf-1(RNAi)</i> suggest that critical <i>hsf-1</i> and <i>mir-80</i> regulation occurs outside of the <i>C. elegans</i> nervous system.</p

<i>daf-16</i>/FOXO is needed for the fluorimetric DR signature and longevity phenotypes of <i>mir-80</i>(Δ).

<p><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g004" target="_blank">Fig. 4A</a>. Transcription factor <i>daf-16/FOXO</i> is required for the Ex_max shift phenotype in <i>mir-80(Δ)</i>. We reared age-synchronized animals under standard growth conditions (20°C, OP50-1) and measured age pigment spectral properties at Day 4 (50 animals per strain) for WT (black bar), <i>mir-80</i>(Δ) (red), <i>daf-16</i>(Δ) allele <i>mgDf50</i> (blue), and <i>mir-80</i>(Δ);<i>daf-16</i>(Δ) double mutant (grey). The same color coding is used for panels 4A–4D. We recorded Ex_max as the highest peak detected by the Datamax software package suite (Horiba Scientific). Graphs represent mean data from at least 3 independent trials. Data were compared using 2-tailed Student's T-test. <i>mir-80</i>(Δ) compared to WT * - p<0.05; <i>mir-80</i>(Δ);<i>daf-16</i>(Δ) double mutant compared to WT, ns. Deletion of <i>daf-16</i> reverses the Ex_max shift phenotype of <i>mir-80</i>(Δ). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g004" target="_blank">Fig. 4B</a>. <i>daf-16</i>/FOXO is required for low age pigment levels in <i>mir-80</i>(Δ). We grew age-synchronized animals under standard conditions (20°C, OP50-1) and measured total age pigment fluorescence, normalized to total tryptophan fluorescence as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Gerstbrein1" target="_blank">[19]</a> (Day 4, 50 animals per trial). Graphs represent mean data from at least 3 independent trials. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test. *** - p<0.0005, ** - p<0.005. The low age pigment accumulation phenotype of <i>mir-80</i>(Δ) is reversed in the <i>mir-80</i>(Δ);<i>daf-16</i>(Δ) double mutant on day 4 (shown here) as well as on day 9 (data not shown). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g004" target="_blank">Fig. 4C</a>. <i>daf-16</i> is required for the lifespan extension of <i>mir-80</i>(Δ). We grew age-synchronized animals under standard conditions (20°C, OP50-1). At day 9, we placed 10 healthy animals per plate, ≥40 per strain per trial, and we scored viability as movement away from pick touch on the indicated days. The graphs represent data combined from 3 independent trials. Statistics are calculated using the Log-rank Test. The <i>mir-80</i>(Δ);<i>daf-16</i>(Δ) double mutant is suppressed for the longevity phenotype of <i>mir-80</i>(Δ) (p<0.0001). We did not, however, observe dramatic overall changes in nuclear localization of DAF-16::GFP +/− <i>mir-80</i> (data not shown). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g004" target="_blank">Fig. 4D</a>. <i>mir-80</i>(Δ) lifespan can be further extended by <i>daf-2(RNAi)</i>. We placed age-synchronized <i>mir-80</i>(Δ) L1 larvae (Day 1) on empty vector control (pL4440) or <i>daf-2</i> RNAi plates under standard conditions (20°C). At day 9, we placed 10 healthy animals per plate, ≥40 per strain per trial, and we scored viability as movement away from pick touch at the indicated days. The graphs represent data combined from 3 independent trials. Statistics are calculated using the Log-rank Test. <i>daf-2(RNAi)</i> increases the lifespan of <i>mir-80</i>(Δ) vector control (p<0.005), but additive effects for <i>mir-80</i>(Δ)<i>+daf-2(RNAi)</i> above the <i>daf-2(RNAi)</i> level are not observed (p = 0.98). Note that data from these experiments also provide a general sense of how <i>mir-80</i>(Δ) compares to <i>daf-2</i> for lifespan extension; roughly we find <i>mir-80</i>(Δ) effects are slightly less than half those of <i>daf-2(rf)</i>, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737.s011" target="_blank">Table S4</a> for exact data from individual trials.</p

A model for miR-80 regulation of DR metabolism.

<p>In adults, when food is abundant, <i>mir-80</i> is expressed at a high level, and miR-80 binds to metabolic and signaling targets to down-regulate their expression. The <i>cbp-1</i> transcript, which includes two potential binding sites for miR-80, one in the 5′ UTR and one in exon 8 (exons thick dark blue lines, promoter lighter blue), and is essential for <i>mir-80</i>(Δ) benefits, is one candidate target (light blue represents relatively low CBP-1 concentration in food). When food is limiting, miR-80 levels drop, and translational repression of <i>cbp-1</i> could be relieved (dark blue circle represents higher concentration CBP-1). The CBP-1 protein associates with DAF-16 and HSF-1 to promote expression of genes required for DR metabolism and longevity. Note that although <i>cbp-1</i> is essential for <i>mir-80</i>(Δ) DR benefits, direct targeting remains to be proved and it is likely that additional targets help modulate the DR state. Since we cannot rule out that <i>daf-16</i>, <i>hsf-1</i>, and <i>cbp-1</i> disruptions make animals too generally sick to gain <i>mir-80</i>(Δ) benefits, alternative models are possible.</p

Deletion of microRNA-80 Activates Dietary Restriction to Extend <i>C. elegans</i> Healthspan and Lifespan

<div><p>Caloric/dietary restriction (CR/DR) can promote longevity and protect against age-associated disease across species. The molecular mechanisms coordinating food intake with health-promoting metabolism are thus of significant medical interest. We report that conserved <i>Caenorhabditis elegans</i> microRNA-80 (<i>mir-80</i>) is a major regulator of the DR state. <i>mir-80</i> deletion confers system-wide healthy aging, including maintained cardiac-like and skeletal muscle-like function at advanced age, reduced accumulation of lipofuscin, and extended lifespan, coincident with induction of physiological features of DR. <i>mir-80</i> expression is generally high under <i>ad lib</i> feeding and low under food limitation, with most striking food-sensitive expression changes in posterior intestine. The acetyltransferase transcription co-factor <i>cbp-1</i> and interacting transcription factors <i>daf-16/FOXO</i> and heat shock factor-1 <i>hsf-1</i> are essential for <i>mir-80</i>(Δ) benefits. Candidate miR-80 target sequences within the <i>cbp-1</i> transcript may confer food-dependent regulation. Under food limitation, lowered miR-80 levels directly or indirectly increase CBP-1 protein levels to engage metabolic loops that promote DR.</p></div

<i>mir-80</i>(Δ) exhibits multiple features of healthy aging.

<p><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g001" target="_blank">Fig. 1A</a>. <i>mir-80</i>(Δ) has low intestinal age pigment levels compared to wild type during late adult life (day 11). We grew age-synchronized WT (black), <i>mir-80</i>(Δ) (red), and <i>mir-80</i>(Δ); Ex[P<i>mir-80(+)</i>] (grey) under standard conditions (20°C, on <i>E. coli</i> OP50-1) and scored animals for age pigment levels using a fluorimeter (n = 100 per strain/trial; day 11, as counted from the hatch; <i>mir-80</i>(Δ) is <i>nDf53; mir-80(+)</i> rescue transgene is <i>nEx1457 </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-AlvarezSaavedra1" target="_blank">[18]</a>). Age pigment fluorescence, which increases with age, is normalized to endogenous tryptophan fluorescence, which remains relatively constant with age <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Gerstbrein1" target="_blank">[19]</a>, (AGE/TRP ratio ∼58% decreased in <i>mir-80(Δ)</i> vs. wild type). Graphs represent mean data from at least 3 independent trials. Data were compared using the One-way ANOVA followed by Newman-Keuls multiple comparison test, *** - p<0.0005, * - p<0.05; WT to Ex[P<i>mir-80(+)</i>] rescue p<0.12. In the rescued strain, age pigment levels might not reach WT levels due to mosaicism of the extrachromosomal transgene, the <i>mir-80</i> transgene dose, or “sponge” effects of overexpression. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g001" target="_blank">Fig. 1B</a>. <i>mir-80</i>(Δ) maintains youthful pharyngeal pumping in late adulthood. We assayed age-synchronized WT (black), <i>mir-80</i>(Δ) (red), and <i>mir-80</i>(Δ); Ex[P<i>mir-80(+)</i>] (grey, <i>nEx1457</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-AlvarezSaavedra1" target="_blank">[18]</a>)) for pharyngeal pumping rates on Day 5 (left) and Day 11 (right) (30 s interval, n = 10/trial, 3 trials). For day 5, we included the <i>eat-2(ad1116)</i> mutant (blue), impaired for pharyngeal pumping to ∼30% WT rate, as a negative control. In this assay we compared healthy appearing animals (most vigorous locomotion). Graph is of cumulative data from 3 independent trials. Data were compared using the One-way ANOVA followed by Newman-Keuls multiple comparison test. * - p<0.05; ** - p<0.005, *** - p<0.0005. <i>mir-80</i>(Δ) pumping rate is modestly higher than WT at day 5 (p = 0.023), but note that relative pumping differences at Day 5 are small compared to differences at Day 11 (∼44% increase). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g003" target="_blank">Fig. 1C</a>. <i>mir-80</i>(Δ) maintains youthful swimming vigor in late adulthood. We assayed age-synchronized animals, WT (black), <i>mir-80</i>(Δ) (red), and <i>mir-80(Δ)</i>; Ex[P<i>mir-80(+)</i>] (grey) for swimming mobility at Day 5 and Day 11 post-hatching (n≥30, 3 independent trials are combined in presented data). Data were compared using 2-tailed Student's T-test, *** - p<0.0001. Although <i>mir-80</i>(Δ) and WT swim similarly in young adult life, <i>mir-80</i>(Δ) mutants better maintain swimming prowess late in life, ∼69% increased body bend rate. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g001" target="_blank">Fig. 1D</a>. <i>mir-80</i>(Δ) mutants have increased mean and maximum lifespans. We assayed age-synchronized WT (black), <i>mir-80</i>(Δ) (red), and <i>mir-80</i>(Δ); Ex[<i>Pmir-80(+)</i>] (grey) animals grown under standard conditions (20°C, OP50-1) for viability (movement away from worm pick by gentle touch) at the indicated days. We initiated trials with relatively vigorous animals on day 9 from the hatch (10 animals per plate, ≥25 per strain per trial, 3 independent trials, which are combined here). Data from individual trials are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737.s001" target="_blank">Fig. S1</a>. Statistics were calculated using the Log-rank Test. <i>mir-80</i>(Δ) mutants exhibit a significant extension in lifespan as compared to WT (p<0.0001) and transgenic expression of <i>mir-80(+)</i> reversed the longevity increase (p<.0001).</p

<i>mir-80</i> expression is generally high in the presence of food, but low when food is lacking.

<p>3A. Examples of expression of extrachromosomal <i>bzEx207</i> [P<i>_mir-80L</i>mCherry] line grown in the presence of unlimited <i>E. coli</i>. Note that this transgenic line, typical of 4 lines that have the long <i>mir-80</i> promoter region, exhibits substantial reporter expression in the first two cells of the intestine (indicated by white+sign) and in the posterior intestine (white bracket). Lower level expression is evident in several other tissues. Animals are adult day 6, but we find no bleed through of signals using red/green filter sets (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737.s004" target="_blank">Fig. S4B</a>) so age pigments do not confound this analysis. 3B. Examples of expression of the <i>bzEx207</i> [P<i>_mir-80L</i>mCherry] line grown in the presence of unlimited <i>E. coli</i> until young adulthood and then switched to no food for 48 hours. 6 day old adults are aligned with anterior to the left, posterior gut region indicated by white bracket. Most posterior gut fluorescence is markedly diminished, although expression in the anterior two intestinal cells, the central egg laying muscles, and the very posterior gut remains high. 3C. Quantitation of fluorescence signals for a <i>mir-80</i> promoter fusion reporter line in food vs. food limitation. Fluorescence of overall <i>bzEx207</i>[P<i>_mir-80L</i>mCherry] line expression after 48 hrs on no-food plates. Food limitation in these studies was by dietary deprivation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Kaeberlein2" target="_blank">[28]</a>, but food dilution on solid NGM media <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Greer1" target="_blank">[4]</a> and food dilution in liquid media <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Greer1" target="_blank">[4]</a> induced similar changes in these lines (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737.s004" target="_blank">Fig. S4</a>). Graph represents spectrofluorimeter measurements of fluorescence levels (whole body) for at least 50 animals per DR regimen. Pairwise comparisons were made using Two-tailed Students' T-test. *** - p<0.0005. Same exposure times were used for complementary panels. 3D. Analysis of food-regulated expression of p<i>_mir-80L</i>mCherry expression along the nematode body implicates posterior intestinal regions as a major site of regulation. We compared p<i>_mir-80L</i>mCherry signals in transgenic ZB3042 grown either in the presence of food (blue) or switched to no food for 24 hrs (red) (measured at day 4, n = 39). We used the ImageJ program to create a 25 pixel segmented line covering the animal and measured mean fluorescence intensity along the body, dividing the length into 12 equal bins and plotting the mean fluorescence intensity at each point. Representative animals are depicted above with the approximate body positions indicated (H = head, P = pharynx, V = vulva, T = tail). Note that although food regulation is apparent in most of the body, food-regulated expression changes in the regions of the mid- and posterior intestine are most dramatic. Error bars indicate standard error for each bin measurement.</p

CBP-1 is critical for <i>mir-80</i>(Δ) healthspan benefits, and is a candidate direct binding target of miR-80.

<p><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g006" target="_blank">Fig. 6A</a>. <i>cbp-1(RNAi)</i> in the <i>mir-80</i>(Δ) background reverses the DR Ex_max shift. We grew age-synchronized animals under standard RNAi feeding conditions (20°C, HT115) and measured age pigments at Day 4 (50 animals per RNAi clone). We recorded Ex_max as the highest peak detected by the Datamax software package suite (Horiba Scientific). Graphs represent cumulative data from 3 independent trials. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (** p<0.001, * p≤0.055 compared to <i>mir-80</i>(Δ) empty vector). <i>cbp-1(RNAi)</i> Ex_max is comparable to that of <i>ad lib</i> wild type (p = 0.729). Note that <i>cbp-1(RNAi)</i> treatment of WT does not change Ex_max (data not shown), so this effect is specific to the DR signature of <i>mir-80</i>(Δ). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g006" target="_blank">Fig. 6B</a>. <i>cbp-1(RNAi)</i> in the <i>mir-80</i>(Δ) background partially reverses low age pigment levels. We grew age-synchronized animals under standard conditions (20°C, HT115) and measured total age pigment fluorescence at day 4 (50 animals per RNAi clone), normalized to total tryptophan fluorescence as in ref. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Gerstbrein1" target="_blank">[19]</a>. Graphs represent cumulative data from 3 independent trials. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (** p<0.05, * p<0.1 compared to <i>mir-80(Δ)+</i>empty vector RNAi). Note that <i>cbp-1(RNAi)</i> treatment of WT induces modest reduction of age pigment levels (p = 0.01, data not shown). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g006" target="_blank">Fig. 6C</a>. <i>mir-80</i>(Δ) longevity is dependent on <i>cbp-1</i>. We placed age-synchronized L1 larvae on empty vector control (pL4440) plates under standard conditions (20°C) until Day 4 (day 1 of adult life) at which time animals were moved to either empty vector control (L4440) or <i>cbp-1(RNAi)</i> plates. At day 9, we placed 10 healthy animals per plate (≥40 per strain per trial), and we scored viability as movement away from pick touch on the indicated days. The graphs represent data combined from 3 independent trials. Statistics are calculated using the Log-rank Test. <i>cbp-1(RNAi)</i> decreases the lifespan of <i>mir-80</i>(Δ) (p<0.0001 compared to vector control. Because RNAi knockdown is inefficient the nervous system (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Calixto1" target="_blank">[59]</a>), the profound effects of <i>cbp-1(RNAi)</i> suggest that critical <i>cbp-1</i>/<i>mir-80</i> regulation occurs outside of the <i>C. elegans</i> nervous system. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g006" target="_blank">Fig. 6D</a>. The <i>cbp-1</i> transcript includes two predicted binding sites for miR-80. Exon structure of <i>cbp-1</i> is indicated by thick blue bars, introns in thin black lines (see WormBase for details). The rna22 algorithm <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737-Rottiers1" target="_blank">[10]</a>, which searches for target sites outside the 3′UTR, predicts that miR-80 binds <i>cbp-1</i> within the 5′ UTR and within exon 8. The potential alignments of miR-80 (red) to <i>C. elegans cbp-1</i> (blue) sequences are indicated. Note that the seed match to the exon 8 region is a perfect 10 bp match for <i>C. elegans</i>, and that the target sequence is conserved in mouse and human CBP1 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen.1003737.s007" target="_blank">Fig. S7</a>). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003737#pgen-1003737-g006" target="_blank">Fig. 6E</a>. Endogenous CBP-1 protein levels are increased in 7 day old <i>mir-80(Δ)</i> mutants. We grew age-synchronized animals under standard conditions (20°C, OP50-1) and extracted total protein at Day 7 (100 animals per strain) for Western blot analysis (top). Graphs represent CBP-1 levels for each strain normalized to own TUB-1 levels. Error bars represent ±S.E.M. Data were compared using 2-tailed Student's T-test (** p<0.005). The graphs represent data combined from 3 independent trials. We noted that during young adulthood, native levels of CBP-1 seemed comparable to WT in <i>mir-80</i>(Δ), suggesting that additional regulatory controls are exerted on CBP-1 expression levels in development or early adulthood.</p