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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

CBP-1 is critical for mir-80(Δ) healthspan benefits, and is a candidate direct binding target of miR-80.Fig. 6A. cbp-1(RNAi) in the mir-80(Δ) background reverses the DR Exmax 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 Exmax 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 mir-80(Δ) empty vector). cbp-1(RNAi) Exmax is comparable to that of ad lib wild type (p = 0.729). Note that cbp-1(RNAi) treatment of WT does not change Exmax (data not shown), so this effect is specific to the DR signature of mir-80(Δ). Fig. 6B. cbp-1(RNAi) in the mir-80(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. [19]. 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 mir-80(Δ)+empty vector RNAi). Note that cbp-1(RNAi) treatment of WT induces modest reduction of age pigment levels (p = 0.01, data not shown). Fig. 6C. mir-80(Δ) longevity is dependent on cbp-1. 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 cbp-1(RNAi) 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. cbp-1(RNAi) decreases the lifespan of mir-80(Δ) (p<0.0001 compared to vector control. Because RNAi knockdown is inefficient the nervous system (see [59]), the profound effects of cbp-1(RNAi) suggest that critical cbp-1/mir-80 regulation occurs outside of the C. elegans nervous system. Fig. 6D. The cbp-1 transcript includes two predicted binding sites for miR-80. Exon structure of cbp-1 is indicated by thick blue bars, introns in thin black lines (see WormBase for details). The rna22 algorithm [10], which searches for target sites outside the 3′UTR, predicts that miR-80 binds cbp-1 within the 5′ UTR and within exon 8. The potential alignments of miR-80 (red) to C. elegans cbp-1 (blue) sequences are indicated. Note that the seed match to the exon 8 region is a perfect 10 bp match for C. elegans, and that the target sequence is conserved in mouse and human CBP1 (see Fig. S7). Fig. 6E. Endogenous CBP-1 protein levels are increased in 7 day old mir-80(Δ) 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 mir-80(Δ), suggesting that additional regulatory controls are exerted on CBP-1 expression levels in development or early adulthood.
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pgen-1003737-g006: CBP-1 is critical for mir-80(Δ) healthspan benefits, and is a candidate direct binding target of miR-80.Fig. 6A. cbp-1(RNAi) in the mir-80(Δ) background reverses the DR Exmax 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 Exmax 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 mir-80(Δ) empty vector). cbp-1(RNAi) Exmax is comparable to that of ad lib wild type (p = 0.729). Note that cbp-1(RNAi) treatment of WT does not change Exmax (data not shown), so this effect is specific to the DR signature of mir-80(Δ). Fig. 6B. cbp-1(RNAi) in the mir-80(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. [19]. 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 mir-80(Δ)+empty vector RNAi). Note that cbp-1(RNAi) treatment of WT induces modest reduction of age pigment levels (p = 0.01, data not shown). Fig. 6C. mir-80(Δ) longevity is dependent on cbp-1. 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 cbp-1(RNAi) 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. cbp-1(RNAi) decreases the lifespan of mir-80(Δ) (p<0.0001 compared to vector control. Because RNAi knockdown is inefficient the nervous system (see [59]), the profound effects of cbp-1(RNAi) suggest that critical cbp-1/mir-80 regulation occurs outside of the C. elegans nervous system. Fig. 6D. The cbp-1 transcript includes two predicted binding sites for miR-80. Exon structure of cbp-1 is indicated by thick blue bars, introns in thin black lines (see WormBase for details). The rna22 algorithm [10], which searches for target sites outside the 3′UTR, predicts that miR-80 binds cbp-1 within the 5′ UTR and within exon 8. The potential alignments of miR-80 (red) to C. elegans cbp-1 (blue) sequences are indicated. Note that the seed match to the exon 8 region is a perfect 10 bp match for C. elegans, and that the target sequence is conserved in mouse and human CBP1 (see Fig. S7). Fig. 6E. Endogenous CBP-1 protein levels are increased in 7 day old mir-80(Δ) 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 mir-80(Δ), suggesting that additional regulatory controls are exerted on CBP-1 expression levels in development or early adulthood.

Mentions: To identify genes required for mir-80(Δ)-regulated DR, we used RNAi to knockdown genes previously implicated in DR lifespan benefits, hypothesizing that genes required for mir-80(Δ) DR should be needed for the Exmax shift and low age pigment levels typical of multiple DR states. Of the 18 genes we screened, we found that RNAi knockdown of transcription factors daf-16/FOXO, heat shock transcription factor hsf-1, and CREB binding protein homolog cbp-1 modulated both the Exmax shift and low age pigment levels of mir-80(Δ) (Tables S1, S2, Figs. 4A,B; 5A,B; 6A,B).


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)

CBP-1 is critical for mir-80(Δ) healthspan benefits, and is a candidate direct binding target of miR-80.Fig. 6A. cbp-1(RNAi) in the mir-80(Δ) background reverses the DR Exmax 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 Exmax 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 mir-80(Δ) empty vector). cbp-1(RNAi) Exmax is comparable to that of ad lib wild type (p = 0.729). Note that cbp-1(RNAi) treatment of WT does not change Exmax (data not shown), so this effect is specific to the DR signature of mir-80(Δ). Fig. 6B. cbp-1(RNAi) in the mir-80(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. [19]. 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 mir-80(Δ)+empty vector RNAi). Note that cbp-1(RNAi) treatment of WT induces modest reduction of age pigment levels (p = 0.01, data not shown). Fig. 6C. mir-80(Δ) longevity is dependent on cbp-1. 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 cbp-1(RNAi) 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. cbp-1(RNAi) decreases the lifespan of mir-80(Δ) (p<0.0001 compared to vector control. Because RNAi knockdown is inefficient the nervous system (see [59]), the profound effects of cbp-1(RNAi) suggest that critical cbp-1/mir-80 regulation occurs outside of the C. elegans nervous system. Fig. 6D. The cbp-1 transcript includes two predicted binding sites for miR-80. Exon structure of cbp-1 is indicated by thick blue bars, introns in thin black lines (see WormBase for details). The rna22 algorithm [10], which searches for target sites outside the 3′UTR, predicts that miR-80 binds cbp-1 within the 5′ UTR and within exon 8. The potential alignments of miR-80 (red) to C. elegans cbp-1 (blue) sequences are indicated. Note that the seed match to the exon 8 region is a perfect 10 bp match for C. elegans, and that the target sequence is conserved in mouse and human CBP1 (see Fig. S7). Fig. 6E. Endogenous CBP-1 protein levels are increased in 7 day old mir-80(Δ) 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 mir-80(Δ), suggesting that additional regulatory controls are exerted on CBP-1 expression levels in development or early adulthood.
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pgen-1003737-g006: CBP-1 is critical for mir-80(Δ) healthspan benefits, and is a candidate direct binding target of miR-80.Fig. 6A. cbp-1(RNAi) in the mir-80(Δ) background reverses the DR Exmax 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 Exmax 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 mir-80(Δ) empty vector). cbp-1(RNAi) Exmax is comparable to that of ad lib wild type (p = 0.729). Note that cbp-1(RNAi) treatment of WT does not change Exmax (data not shown), so this effect is specific to the DR signature of mir-80(Δ). Fig. 6B. cbp-1(RNAi) in the mir-80(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. [19]. 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 mir-80(Δ)+empty vector RNAi). Note that cbp-1(RNAi) treatment of WT induces modest reduction of age pigment levels (p = 0.01, data not shown). Fig. 6C. mir-80(Δ) longevity is dependent on cbp-1. 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 cbp-1(RNAi) 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. cbp-1(RNAi) decreases the lifespan of mir-80(Δ) (p<0.0001 compared to vector control. Because RNAi knockdown is inefficient the nervous system (see [59]), the profound effects of cbp-1(RNAi) suggest that critical cbp-1/mir-80 regulation occurs outside of the C. elegans nervous system. Fig. 6D. The cbp-1 transcript includes two predicted binding sites for miR-80. Exon structure of cbp-1 is indicated by thick blue bars, introns in thin black lines (see WormBase for details). The rna22 algorithm [10], which searches for target sites outside the 3′UTR, predicts that miR-80 binds cbp-1 within the 5′ UTR and within exon 8. The potential alignments of miR-80 (red) to C. elegans cbp-1 (blue) sequences are indicated. Note that the seed match to the exon 8 region is a perfect 10 bp match for C. elegans, and that the target sequence is conserved in mouse and human CBP1 (see Fig. S7). Fig. 6E. Endogenous CBP-1 protein levels are increased in 7 day old mir-80(Δ) 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 mir-80(Δ), suggesting that additional regulatory controls are exerted on CBP-1 expression levels in development or early adulthood.
Mentions: To identify genes required for mir-80(Δ)-regulated DR, we used RNAi to knockdown genes previously implicated in DR lifespan benefits, hypothesizing that genes required for mir-80(Δ) DR should be needed for the Exmax shift and low age pigment levels typical of multiple DR states. Of the 18 genes we screened, we found that RNAi knockdown of transcription factors daf-16/FOXO, heat shock transcription factor hsf-1, and CREB binding protein homolog cbp-1 modulated both the Exmax shift and low age pigment levels of mir-80(Δ) (Tables S1, S2, Figs. 4A,B; 5A,B; 6A,B).

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