Deletion of microRNA-80 activates dietary restriction to extend C. elegans healthspan and lifespan.
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.
Affiliation: Department of Molecular Biology and Biochemistry, Nelson Biological Laboratories, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America.
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.
- Caenorhabditis elegans/genetics*/physiology
- Caenorhabditis elegans Proteins/genetics*/metabolism
- Caloric Restriction*
- Forkhead Transcription Factors
- Gene Expression Regulation
- Histone Acetyltransferases/genetics/metabolism
- Sequence Deletion
- Signal Transduction
- Transcription Factors/genetics/metabolism
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pgen-1003737-g002: mir-80(Δ) exhibits multiple characteristics typical of DR animals.Fig. 2A. The mir-80(Δ) mutant exhibits the DR Exmax shift. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey) 4 day old animals grown under standard conditions (20°C, OP50-1). We used a spectrofluorimeter to scan transparent animals (n = 100 per strain/trial) to generate excitation/emission profiles as in ; wavelength of excitation at maximal fluorescence is indicated. Graphs represent mean data from at least 3 independent trials. Data were compared One-way ANOVA followed by Newman-Keuls multiple comparison test, *** - p<0.0005. mir-80(Δ) exhibits a significantly down-shifted Exmax (p<0.0005) as compared to wild type under conditions of abundant food, a feature unique to DR . Fig. 2B. The mir-80(Δ) mutant exhibits low age pigment levels early in life, as occurs in C. elegans DR. We assayed age-synchronized WT (black), mir-80(Δ) (red), and mir-80(Δ); Ex[Pmir-80(+)] (grey) 4 day old animals grown under standard conditions (20°C, OP50-1). We scanned animals (n = 100 per strain) for fluorescence over a range of wavelengths, and normalized age pigment fluorescence (AGE) to tryptophan (TRP) fluorescence as in  for comparison. Graphs represent mean data from at least 3 independent trials. Data were compared using One-way ANOVA followed by Newman-Keuls multiple comparison test, *** - p<0.0005, ** - p<0.005. mir-80(Δ) exhibits low age pigment levels as compared to wild type (p<0.0005) early in life, which is true of all DR conditions previously tested (although not unique to DR). In these assays, levels were on average 66% lower in mir-80(Δ). Fig. 2C. mir-80(Δ) mutants are physically smaller than WT, typical of animals in DR. We measured age-synchronized WT (black) and mir-80(Δ) (red) 4 day old animals (examples at the right) grown under standard conditions (20°C, OP50-1) by imaging animals (WT n = 77, mir-80(Δ) n = 88) under DIC under low magnification. We measured using the segmented line tool in the ImageJ software by drawing a line across the length of the animal, and converted length in pixels to uM using a stage micrometer to assess image scale. We compared data using 2-tailed Student's T-test, *** - p<0.0005. mir-80(Δ) mutants are ∼10% shorter and look thinner than WT reared under the same conditions, typical of the scrawny appearance of animals in DR, example comparison on the right. Although size varies somewhat and is not as quantitative a measure as age pigment scores, we have used the scrawny appearance to identify likely mir-80(Δ) homozygotes in crosses. Fig. 2D. mir-80(Δ) mutants exhibit reduced fertility and an extended reproductive lifespan. We assayed egg production in age-synchronized WT (black), mir-80(Δ) (red), and DR mutant eat-2(ad1116) (blue) grown under standard conditions (20°C, OP50-1; parent n = 10, 3 independent trials). eat-2 is one trial so bars are not provided. Data were compared using 2-tailed Student's T-test. Early in adult life, mir-80(Δ) produce a reduced number of live births per day (33% decrease, p<0.05 for Day 3; 180% increase, p<0.001 Day 4–6) and exhibit a prolonged reproductive lifespan (through Day 8 for mir-80(Δ) as compared to WT Day 6, p<0.001). The constitutive DR mutant, eat-2 experiences a shift in reproductive lifespan (compared to WT, p<0.001) that is similar to mir-80(Δ). Fig. 2E. SKN-1-GFP, a molecular reporter of DR, is upregulated in mir-80(Δ) in the presence of food. SKN-1::GFP expression in the two ASI neurons is a molecular signal of some DR . We constructed strains that included an integrated rescuing skn-1-gfp fusion gene expressed from the native skn-1 promoter, Is007[skn-1-gfp], and measured at Day 7, 20°C, growth in OP50-1 (white arrows). WT animals show low levels of ASI expression (36% with very weak expression in only one ASI), while DR-constitutive eat-2(ad1116) animals display constitutive expression of SKN-1-GFP in the ASI neurons (92% in 1 or 2 neurons, strong expression). 95% of mir-80(Δ) have 1–2 ASIs expressing at this timepoint. These data support that mir-80(Δ) mutants are in DR even when reared in the presence of ample food.
To ask whether mir-80(Δ) might act via a DR mechanism to extend healthspan and lifespan, we tested mir-80(Δ) mutants for phenotypic features of the DR state (Fig. 2). Our previous in vivo studies of fluorescent age pigments revealed that transparent C. elegans under DR have a unique fluorimetric “signature” that is distinct from spectral properties of both WT and long-lived mutants induced by other longevity pathways , , . A spectrofluorimeter excitation/emission direct scan of WT reports a characteristic excitation maximum (Exmax) for age pigments at ∼345 nm. However, under all DR-inducing conditions we previously tested (multiple feeding-impaired mutants , liquid feeding of WT , , limiting bacterial concentrations for WT , complete removal of food , treatment with DR-mimetic drug metformin ), we noted a downward shift in Exmax. Thus, the age pigment Exmax shift indicates a DR-like state. We found that mir-80(Δ) consistently exhibits the DR Exmax shift despite growth in the presence of abundant food (Fig. 2A) and has low age pigment levels, even at young age (day 4, p<0.0005, Fig. 2B, about 66% lower in these studies), the latter of which also characteristic of DR mutants. Thus, mir-80(Δ) exhibits the spectral signature of DR despite the presence of food, consistent with mir-80(Δ) being a DR constitutive mutant.