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Deletion of microRNA-80 activates dietary restriction to extend C. elegans healthspan and lifespan.

Vora M, Shah M, Ostafi S, Onken B, Xue J, Ni JZ, Gu S, Driscoll M - PLoS Genet. (2013)

Bottom Line: 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.Under food limitation, lowered miR-80 levels directly or indirectly increase CBP-1 protein levels to engage metabolic loops that promote DR.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Nelson Biological Laboratories, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT
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 Caenorhabditis elegans microRNA-80 (mir-80) is a major regulator of the DR state. mir-80 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. mir-80 expression is generally high under ad lib feeding and low under food limitation, with most striking food-sensitive expression changes in posterior intestine. The acetyltransferase transcription co-factor cbp-1 and interacting transcription factors daf-16/FOXO and heat shock factor-1 hsf-1 are essential for mir-80(Δ) benefits. Candidate miR-80 target sequences within the cbp-1 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.

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Related in: MedlinePlus

mir-80 expression is generally high in the presence of food, but low when food is lacking.3A. Examples of expression of extrachromosomal bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli. Note that this transgenic line, typical of 4 lines that have the long mir-80 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 (Fig. S4B) so age pigments do not confound this analysis. 3B. Examples of expression of the bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli 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 mir-80 promoter fusion reporter line in food vs. food limitation. Fluorescence of overall bzEx207[Pmir-80LmCherry] line expression after 48 hrs on no-food plates. Food limitation in these studies was by dietary deprivation [28], but food dilution on solid NGM media [4] and food dilution in liquid media [4] induced similar changes in these lines (Fig. S4). 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 pmir-80LmCherry expression along the nematode body implicates posterior intestinal regions as a major site of regulation. We compared pmir-80LmCherry 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.
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pgen-1003737-g003: mir-80 expression is generally high in the presence of food, but low when food is lacking.3A. Examples of expression of extrachromosomal bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli. Note that this transgenic line, typical of 4 lines that have the long mir-80 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 (Fig. S4B) so age pigments do not confound this analysis. 3B. Examples of expression of the bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli 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 mir-80 promoter fusion reporter line in food vs. food limitation. Fluorescence of overall bzEx207[Pmir-80LmCherry] line expression after 48 hrs on no-food plates. Food limitation in these studies was by dietary deprivation [28], but food dilution on solid NGM media [4] and food dilution in liquid media [4] induced similar changes in these lines (Fig. S4). 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 pmir-80LmCherry expression along the nematode body implicates posterior intestinal regions as a major site of regulation. We compared pmir-80LmCherry 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.

Mentions: Fig. 1A. mir-80(Δ) has low intestinal age pigment levels compared to wild type during late adult life (day 11). We grew age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey) under standard conditions (20°C, on E. coli OP50-1) and scored animals for age pigment levels using a fluorimeter (n = 100 per strain/trial; day 11, as counted from the hatch; mir-80(Δ) is nDf53; mir-80(+) rescue transgene is nEx1457[18]). Age pigment fluorescence, which increases with age, is normalized to endogenous tryptophan fluorescence, which remains relatively constant with age [19], (AGE/TRP ratio ∼58% decreased in mir-80(Δ) 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[Pmir-80(+)] rescue p<0.12. In the rescued strain, age pigment levels might not reach WT levels due to mosaicism of the extrachromosomal transgene, the mir-80 transgene dose, or “sponge” effects of overexpression. Fig. 1B. mir-80(Δ) maintains youthful pharyngeal pumping in late adulthood. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey, nEx1457 ([18])) 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 eat-2(ad1116) 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. mir-80(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). Fig. 1C. mir-80(Δ) maintains youthful swimming vigor in late adulthood. We assayed age-synchronized animals, WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (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 mir-80(Δ) and WT swim similarly in young adult life, mir-80(Δ) mutants better maintain swimming prowess late in life, ∼69% increased body bend rate. Fig. 1D. mir-80(Δ) mutants have increased mean and maximum lifespans. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (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 Fig. S1. Statistics were calculated using the Log-rank Test. mir-80(Δ) mutants exhibit a significant extension in lifespan as compared to WT (p<0.0001) and transgenic expression of mir-80(+) reversed the longevity increase (p<.0001).


Deletion of microRNA-80 activates dietary restriction to extend C. elegans healthspan and lifespan.

Vora M, Shah M, Ostafi S, Onken B, Xue J, Ni JZ, Gu S, Driscoll M - PLoS Genet. (2013)

mir-80 expression is generally high in the presence of food, but low when food is lacking.3A. Examples of expression of extrachromosomal bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli. Note that this transgenic line, typical of 4 lines that have the long mir-80 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 (Fig. S4B) so age pigments do not confound this analysis. 3B. Examples of expression of the bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli 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 mir-80 promoter fusion reporter line in food vs. food limitation. Fluorescence of overall bzEx207[Pmir-80LmCherry] line expression after 48 hrs on no-food plates. Food limitation in these studies was by dietary deprivation [28], but food dilution on solid NGM media [4] and food dilution in liquid media [4] induced similar changes in these lines (Fig. S4). 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 pmir-80LmCherry expression along the nematode body implicates posterior intestinal regions as a major site of regulation. We compared pmir-80LmCherry 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.
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pgen-1003737-g003: mir-80 expression is generally high in the presence of food, but low when food is lacking.3A. Examples of expression of extrachromosomal bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli. Note that this transgenic line, typical of 4 lines that have the long mir-80 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 (Fig. S4B) so age pigments do not confound this analysis. 3B. Examples of expression of the bzEx207 [Pmir-80LmCherry] line grown in the presence of unlimited E. coli 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 mir-80 promoter fusion reporter line in food vs. food limitation. Fluorescence of overall bzEx207[Pmir-80LmCherry] line expression after 48 hrs on no-food plates. Food limitation in these studies was by dietary deprivation [28], but food dilution on solid NGM media [4] and food dilution in liquid media [4] induced similar changes in these lines (Fig. S4). 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 pmir-80LmCherry expression along the nematode body implicates posterior intestinal regions as a major site of regulation. We compared pmir-80LmCherry 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.
Mentions: Fig. 1A. mir-80(Δ) has low intestinal age pigment levels compared to wild type during late adult life (day 11). We grew age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey) under standard conditions (20°C, on E. coli OP50-1) and scored animals for age pigment levels using a fluorimeter (n = 100 per strain/trial; day 11, as counted from the hatch; mir-80(Δ) is nDf53; mir-80(+) rescue transgene is nEx1457[18]). Age pigment fluorescence, which increases with age, is normalized to endogenous tryptophan fluorescence, which remains relatively constant with age [19], (AGE/TRP ratio ∼58% decreased in mir-80(Δ) 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[Pmir-80(+)] rescue p<0.12. In the rescued strain, age pigment levels might not reach WT levels due to mosaicism of the extrachromosomal transgene, the mir-80 transgene dose, or “sponge” effects of overexpression. Fig. 1B. mir-80(Δ) maintains youthful pharyngeal pumping in late adulthood. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey, nEx1457 ([18])) 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 eat-2(ad1116) 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. mir-80(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). Fig. 1C. mir-80(Δ) maintains youthful swimming vigor in late adulthood. We assayed age-synchronized animals, WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (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 mir-80(Δ) and WT swim similarly in young adult life, mir-80(Δ) mutants better maintain swimming prowess late in life, ∼69% increased body bend rate. Fig. 1D. mir-80(Δ) mutants have increased mean and maximum lifespans. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (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 Fig. S1. Statistics were calculated using the Log-rank Test. mir-80(Δ) mutants exhibit a significant extension in lifespan as compared to WT (p<0.0001) and transgenic expression of mir-80(+) reversed the longevity increase (p<.0001).

Bottom Line: 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.Under food limitation, lowered miR-80 levels directly or indirectly increase CBP-1 protein levels to engage metabolic loops that promote DR.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Nelson Biological Laboratories, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT
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 Caenorhabditis elegans microRNA-80 (mir-80) is a major regulator of the DR state. mir-80 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. mir-80 expression is generally high under ad lib feeding and low under food limitation, with most striking food-sensitive expression changes in posterior intestine. The acetyltransferase transcription co-factor cbp-1 and interacting transcription factors daf-16/FOXO and heat shock factor-1 hsf-1 are essential for mir-80(Δ) benefits. Candidate miR-80 target sequences within the cbp-1 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.

Show MeSH
Related in: MedlinePlus