Additional file 1: of Single swim sessions in C. elegans induce key features of mammalian exercise

Changes in specific oxidative stress response transcripts accompany swim exercise in C. elegans. (A–L) qPCR results in N2 animals before and at different time points post-exercise for oxidative stress reporter genes (n = 5 independent trials). Note the different y-axis scales between figure panels (particularly K and L). hsp-16.2 and hsp-16.41 are documented to be induced under both oxidative stress and heat shock. We calculated relative expression by normalization to reference genes followed by normalization to the time point before exercise. We used paired two-tailed Student’s t tests to compare relative expression of control versus exercise samples at each time point. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (PDF 260 kb

Additional file 7: of Single swim sessions in C. elegans induce key features of mammalian exercise

Primers used for qPCR. (DOCX 16 kb

CeleST reveals novel information on aging phenotypes.

<p><b>A–J</b>, Age-associated changes in swimming parameters in wild-type adults. <b>A</b>, Wave initiation rate; <b>B</b>, Body wave number; <b>C</b>, Asymmetry; <b>D</b>, Stretch; <b>E</b>, Attenuation; <b>F</b>, Reverse swimming; <b>G</b>, Curling; <b>H</b>, Travel speed; <b>I</b>, Brush stroke; and <b>J</b>, Activity index. from 9 independent trials, for each age day 4 and day 11. Data for ages ranging from day 4 to day 20 are presented in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s004" target="_blank">Figure S4</a>. <b>K–M</b>, CeleST reports great differences in graceful agers vs. poor agers for measures that change with age. We selected animals that appeared to have robust crawling capacity (Class A, graceful agers) and those that had decrepit crawling capacity (Class C, poor agers) at day 11 and compared swim behavior. <b>K</b>, Activity index; <b>L</b>, Asymmetry; <b>M</b>, Attenuation. from 3 independent trials, for each class. Data for all ten measures in this series are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s005" target="_blank">Figure S5</a>. <b>N</b>, Locomotory changes under life-extending and progeric insulin signaling pathway manipulation suggest complex influences of signaling over the lifetime. Activity index, WT: blue line (middle dashed line); long lived <i>age-1(hx546)</i>: green (top line); short-lived <i>daf-16(mgDf50)</i>: red (bottom line). Note that the <i>age-1</i> mutant has a higher activity index in young adult life as compared to WT, which suggests differences in swim performance are not limited to aging. Also, at day 15, WT and <i>age-1</i> scores appear increased, which we suggest reflects the preferential death of the poorest swimmers, rather than an actual increase in average swimming of individuals. in each data point from 4 independent trials. Data on all measures are presented in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s006" target="_blank">Figure S6</a>. Error bars show SEM, *** , **** .</p

Examples of ten CeleST measure outputs for an individual <i>C. elegans</i> swim trial.

<p>All measures reflect analysis of a 30/sec, with <b>C–G</b> calculated from analysis of radius of curvature over 12 body segments (curvature plot vs. time example is in <b>B</b>). For <b>C–G</b> and <b>M–O</b>, instantaneous values are plotted in black; the median value for each swim is drawn in red, and the 10–90 percentile range of values is shown in gray; median and range over the 30 s trial are listed on the right. Note that although this panel demonstrates analysis of measurements of a single animal swimming, the CeleST program score multiple animals simultaneously and can compile data from thousands of individual swim trials (examples in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi-1003702-g003" target="_blank">Figure 3</a>). <b>A</b>, Scored animal at three indicated time points in the video, with the curvature measure superimposed on the body; color key shown in <b>B</b>. <b>B</b>, Curvature heat map of an individual swim trial. Map is of curvature at a given body point (Y axis) as a function of time (X axis), with head curvature score at the top and tail curvature at the bottom on the Y axis, deep bend in one direction dark blue, and in the other direction dark red. Lines indicate posture of the animal depicted at that time point in panel <b>A</b>. Note that the posture of an individual at any point in time could be reconstructed from the measure of curvature over body position. Further details on each parameter measurement <b>C–O</b> are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s009" target="_blank">Text S1</a>. Informative videos (Videos S2 01–10) that feature extreme examples for each measure can be found on <a href="http://celest.mbb.rutgers.edu" target="_blank">http://celest.mbb.rutgers.edu</a>.</p

Summary of CeleST components and usage.

<p>Input files are videos of multiple swimming <i>C. elegans</i>. Files are stored in a database that records identifying features (strain, date, etc.) to permit easy selection of animals to be compared by analysis. After selection of animals to be compared, swimmers are automatically tracked from videos, and computation from curvature data or posture is used to score ten swim measures in 30 second swim trials (see description in text). Measures from the scored animals are compiled and can be exported in several alternative data analysis formats, including dot plots, line graphs, histograms and two dimensional comparison (ellipses indicate the principal directions and the standard deviations of the data). Statistical analysis is automated. A dynamic demonstration of CeleST tracking and computing of measures can be found in Video S1 1–4 on <a href="http://celest.mbb.rutgers.edu" target="_blank">http://celest.mbb.rutgers.edu</a>.</p

CeleST analysis reveals features of <i>C. elegans</i> swimming considerable individuality, gait preference, and reverse swimming.

<p><b>A–C</b>, <i>C. elegans</i> exhibit diverse swimming abilities, despite genetic and environmental homogeneity. CeleST can plot scores for two parameters against each other, for example: <b>A</b>, Travel speed vs. Asymmetry; <b>B</b>, Body wave number vs. Activity index; <b>C</b>, Brush stroke vs. Stretch. Data for WT 4-day old animals from 9 independent trials are plotted. <b>D</b>, <i>C. elegans</i> swim at specific wave initiation rates. We plotted in the form of line histograms the distribution of median Wave initiation rates (WIR) in wild-type animals as occurs over a 30 second interval. WIR values are binned to integers and the plot line delineates the contour of the bins in the histogram. X axis is median WIR, Y axis is the number of individuals exhibiting the indicated WIR. Data in this panel are combined to represent 3,372 animals ranging from 4 to 20 days old from 9 independent trials to emphasize the peaked distribution. Although animals in this large population do swim over the range of possible <u>median</u> WIRs (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s001" target="_blank">Figure S1F</a> for example), CeleST analysis reveals an unexpected bias for particular “gaits” in a subset of the population (about 14% total appear in favored WIRs). Older animals swim at lower median WIRs than young adult animals, but the preferred WIRs remain. WIR distributions for specific individual ages are depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s001" target="_blank">Figure S1</a>. Note that mean WIR rates do not exhibit a distribution bias (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s001" target="_blank">Figure S1F</a>), so this study emphasizes the value of also considering median scores in swim behavioral analysis. <b>E–G</b>, For brief periods, swimming animals reverse, with the tail initiating the body wave. Reverse swimming is illustrated in Videos S3 and S4 on <a href="http://celest.mbb.rutgers.edu" target="_blank">http://celest.mbb.rutgers.edu</a>. In 4-day old animals, the <i>glr-1(ky176)</i> mutant, lacking a neuronal glutamate receptor, reversal frequency is increased relative to WT (<b>E</b>), although the trend to increased time spent in reverse is not statistically significant () (<b>F</b>). Unexpectedly, <i>glr-1</i> mutants swim more symmetrically than WT at day 4 (<b>G</b>). from 3 independent trials for each strain. Data for all 10 measures, young and old age are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003702#pcbi.1003702.s002" target="_blank">Figure S2</a>. Error bars show SEM, **** .</p

<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

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> 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