Statins Induce a DAF-16/Foxo-dependent Longevity Phenotype via JNK-1 through Mevalonate Depletion in C. elegans

Abstract

Statins belong to the most pre-scribed cholesterol lowering drugs in western countries. Their competitive inhibition of the HMG-CoA reductase causes a reduction in the mevalonate pool, resulting in reduced cholesterol biosynthesis, impaired protein prenylation and glycosylation. Recently, a cohort study showed a decreased mortality rate in humans between age 78-90 going along with statin therapy, which is independent of blood cholesterol levels. As C. elegans harbors the mevalonate pathway, but is cholesterol-auxotroph, it is particularly suitable to study cholesterol-independent effects of statins on aging-associated phenotypes. Here, we show that low doses of lovastatin or a mild HMG-CoA reductase knockdown via hmgr-1(RNAi) in C. elegans substantially attenuate aging pigment accumulation, which is a well-established surrogate marker for biological age. Consistently, for two statins we found dosages, which prolonged the lifespan of C. elegans. Together with an observed reduced fertility, slower developmental timing and thermal stress resistance this complex of outcomes point to the involvement of DAF-16/hFOXO3a, the master regulator of stress resistance and longevity. Accordingly, prolonged low-dose statin exposure leads to an increased expression of jnk-1, a known activator of DAF-16. Moreover, the beneficial effects of statins on aging pigments and lifespan depend on DAF-16 and JNK-1, as shown in epistasis analyses. These effects can be reverted by mevalonate supplementation. In conclusion, we describe a lifespan extension in C. elegans, which is conferred via two well-conserved stress-related factors (JNK-1, DAF-16) and results from mevalonate depletion.

Keywords: C. elegans; insulin/IGF-1 like signaling; lifespan; mevalonate pathway; statins.

Figure 1.

Low dose lovastatin restrains progeny production and development but enhances thermal stress resistance in C. elegans. (A) Lovastatin treatment reduces total progeny production. Total progeny was determined over five days starting with L4-individuals randomly distributed to permanent treatment with three lovastatin concentrations (25, 50 and 100 µM) or DMSO. Shown is the percentage of progeny relative to the control population. All bars represent mean+SEM from three independent experiments, each with at least 14 individuals being analyzed (n = 3, N ≥ 42). (B) Continuous lovastatin treatment slightly delays development by trend. The percentage of developmental stages within lovastatin treated (25, 50 and 100 µM) and DMSO-treated control populations were determined 72 h after hatching. Bars represent mean value of three independent experiments with at least 20 individuals each (n = 3, N ≥ 60). There is a trend in the lovastatin group for a slower development with increasing statin dose. (C) Lovastatin does not confer resistance against H₂O₂-induced oxidative stress in C. elegans. The surviving fractions of lovastatin (100 µM, 24 h) or DMSO pre-incubated populations are indistinguishable after exposure to different H₂O₂ concentrations (2, 4 and 6 mM). Shown is the fraction of surviving animals in the population after four hours. Dots/triangles represent the mean from three independent experiments with at least 20 individuals each (n = 3, N ≥ 60). (D) Lovastatin robustly increases thermal stress resistance of C. elegans. After incubation of the nematodes for five or six hours at 37°C, the surviving fraction of lovastatin pre-incubated populations (100 µM, 24 h) is significantly higher as compared to DMSO-treated control population. Shown is the fraction of surviving animals in the respective population after the indicated incubation time at 37°C. Each dot/triangle represents the mean value from three independent experiments with at least 20 individuals each (n = 3, N ≥ 60). *p≤0.05 (1way ANOVA with uncorrected Fisher´s LSD post hoc test).

Figure 2.

Inhibition of HMG-CoA reductase decelerates aging pigment accumulation and extends lifespan in C. elegans. (A) Aging pigments constantly rise during aging of wild type C. elegans. After six, ten and twelve days of incubation with DMSO the autofluorescence was determined (DAPI filter: extinction 360-370 nm; emission 420-460 nm). Shown are the relative fluorescence units (RFU) of at least 12 individuals from three independent experiments as measured after background correction using ImageJ software for quantification (n = 3, N ≥ 36). Test for statistical difference was performed using 1way ANOVA with uncorrected Fisher´s LSD post hoc test *p<0.05. (B) Low dose treatment with lovastatin robustly reduces aging pigment accumulation rate. After six, ten and twelve days of permanent treatment with different lovastatin concentrations (25, 50, 100 µM) or DMSO (vehicle control) the autofluorescence was determined (DAPI filter: extinction 360-370 nm; emission 420-460 nm) of at least 30 individuals from three independent experiments (n = 3, N ≥ 90). Expressed is the relative fluorescence normalized to the respective control. (C) Genetic inhibition of HMG-CoA reductase phenocopies pharmacological effects on aging pigment accumulation. After twelve days on dilutions of hmgr-1(RNAi) (1:8, 1:16, 1:24, 1:32) the autofluorescence was determined (DAPI filter: extinction 360-370 nm; emission 420-460 nm). Expressed is the relative fluorescence normalized to the respective control. All bars represent mean + SEM from three independent experiments with at least 10 individuals (n = 3, N ≥ 30). Test for statistical difference was performed using 1way ANOVA with uncorrected Fisher´s LSD post hoc test *p<0.05. (D) Treatment with lovastatin extends the post-reproductive lifespan of C. elegans. Synchronized nematode populations were subdivided to DMSO (control), 25, 50 or 100 µM lovastatin on day eight of adulthood after reaching the post-reproductive stage. Shown are Kaplan-Meier survival plots for the time of treatment with lovastatin from three independent trials. The log rank post-hoc test showed statistical difference in comparison to the control for all lovastatin treatment groups (n = 3, N ≥ 80; *p≤0.05).

Figure 3.

DAF-16 is the key factor in conferring statin effects regarding aging. (A) HMG-CoA inhibition enhances jnk-1 mRNA expression. Expression levels of different candidate genes were investigated in wild-type C. elegans populations after 24 h treatment with: (I) vector control RNAi, (II) hmgr-1(RNAi) diluted 1:1, (III) hmgr-1(RNAi) diluted 1:8, (IV) hmgr-1(RNAi) diluted 1:64 or (V) 100 μM lovastatin. Shown are the expression changes for hmgr-1 and jnk-1 mRNA. Hmgr-1 expression is reduced in case of hmgr-1(RNAi) in dilutions 1:1 (about 50 %) and 1:8 (about 38 %). Of interest is the fact that jnk-1 mRNA is more abundant after hmgr-1(RNAi) diluted 1:8 and 100 µM lovastatin treatment, as these treatments also decrease the aging pigment accumulation. Shown is the mean ± SEM from three technical replicates (cDNA from about 2000 individuals). (B) Lovastatin enhances nuclear localization of DAF-16::GFP in dependence upon JNK-1. Subcellular DAF-16::GFP localization was investigated after 24 h incubation with lovastatin (25, 50, 100 µM) or 0.1 % DMSO in the wild-type (wt)- or jnk-1(gk7) populations. Bars represent results from three independent experiments with at least 15 individuals each (n = 3, N ≥ 45). Test for statistical difference was performed using ordinary 1way ANOVA with uncorrected Fisher´s LSD post hoc test *p<0.05. (C) Lovastatin does not affect aging pigment accumulation in daf-16, jnk-1 or daf-2 knockout populations. Wild-type-, daf-16(mu86)-, jnk-1(gk7) and daf-2(e1370) populations were continuously treated with lovastatin (100 µM) or 0.1 % DMSO. After twelve days the autofluorescence was determined (DAPI filter: extinction 360-370 nm; emission 420-460 nm). Shown is the relative fluorescence of mutants as normalized to the wild type control population (= 1.0). Bars represent the mean + SEM from three independent experiments and at least 30 individuals. Test for statistical difference was performed using ordinary 1way ANOVA with uncorrected Fisher´s LSD post hoc test *p<0.05. (D) The lovastatin effect on lifespan in C. elegans depends on jnk-1 and daf-16. Synchronized nematode populations were subdivided to 0.1 % DMSO (control) or 100 µM lovastatin on day one of adulthood. Shown are Kaplan-Meier survival plots for the time of treatment with lovastatin from three independent trials with at least 60 individuals each (n = 3, N ≥ 180. The log rank post-hoc test showed statistical difference in comparison to the control for daf-2(e1370) (*p≤0.05).