Rilmenidine extends lifespan and healthspan in Caenorhabditis elegans via a nischarin I1-imidazoline receptor

Abstract

Repurposing drugs capable of extending lifespan and health span has a huge untapped potential in translational geroscience. Here, we searched for known compounds that elicit a similar gene expression signature to caloric restriction and identified rilmenidine, an I1-imidazoline receptor agonist and prescription medication for the treatment of hypertension. We then show that treating Caenorhabditis elegans with rilmenidine at young and older ages increases lifespan. We also demonstrate that the stress-resilience, health span, and lifespan benefits of rilmenidine treatment in C. elegans are mediated by the I1-imidazoline receptor nish-1, implicating this receptor as a potential longevity target. Consistent with the shared caloric-restriction-mimicking gene signature, supplementing rilmenidine to calorically restricted C. elegans, genetic reduction of TORC1 function, or rapamycin treatment did not further increase lifespan. The rilmenidine-induced longevity required the transcription factors FOXO/DAF-16 and NRF1,2,3/SKN-1. Furthermore, we find that autophagy, but not AMPK signaling, was needed for rilmenidine-induced longevity. Moreover, transcriptional changes similar to caloric restriction were observed in liver and kidney tissues in mice treated with rilmenidine. Together, these results reveal a geroprotective and potential caloric restriction mimetic effect by rilmenidine that warrant fresh lines of inquiry into this compound.

Keywords: aging; autophagy; drug repurposing; longevity; mTOR; nischarin receptor.

FIGURE 1

Improved survival of C. elegans treated with rilmenidine. (a) Pooled survival curve showing significant lifespan extension in WT animals treated with rilmenidine at all concentrations, except 400 μM, compared to 1% DMSO vehicle control. (b) Bar graph showing quantified lifespan data in terms of mean lifespan (days) for each rilmenidine concentration (100–400 μM) treated WT, compared to DMSO vehicle control; 200 μM concentration of rilmenidine provides maximum lifespan increase (19%) in WT. (c) Pooled survival curve showing late‐life treatment with rilmenidine (200 μM) at day 12 of adulthood increased lifespan in WT compared to DMSO vehicle control. (d) Quantified data for lifespan assay showing percentage increase in mean lifespan of adult WT‐fed 200 μM rilmenidine at different times (day 1 or day 12 adulthood) compared to DMSO vehicle control. Error bars represent SEM; adjusted p‐value was derived from log‐rank test and Bonferroni correction. Kaplan–Meir survival analysis was performed on pooled data from at least three independent trials. Quantitative data and statistical analyses for the representative experiments are included in Table S1.

FIGURE 2

Effects of rilmenidine treatment on survival of CR‐associated mutants. Survival curves showing the inability of rilmenidine to extend life span in (a) DR1116 eat‐2(ad1116) mutants, (b) DR412 daf‐15(m81/+) mutants, (c) rapamycin‐treated WT and (d) GR1307 daf‐16(mgDf50), and LD1057 skn‐1(tm3411), but not in TG38 aak‐2(gt33) mutants. Raw data, quantitative data, additional trials, and statistical analyses for the representative experiments are included in Table S1.

FIGURE 3

Induced autophagy by rilmenidine perturbed polyQ aggregation. (a) Representative images of day 2 adult transgenic animals, expressing the intestinal specific autophagy reporter gene Pnhx‐2::mCherry::lgg‐1 showing increased autophagy, when exposed to varying concentrations of rilmenidine for 24 h compared to 1% DMSO vehicle. Arrows indicate autophagosome puncta formation. Scale bar = 20 μm. (b) The graph shows the interquartile distribution of the mean number of mCherry::LGG‐1 puncta in the posterior intestine of the animals in each condition. Error bars, upper: Q3 + 1.5*IQR; minimum: Q1–1.5*IQR. **p < 0.01, *p < 0.05; one‐way ANOVA followed by a Tukey's post hoc test. (c, d) Inhibition of autophagy abrogates the prolongevity effect of rilmenidine as shown by survival curves of WT animals fed either RNAi bacteria expressing an empty vector (L4440), or lgg‐1 (c) or bec‐1 (d) dsRNA from day 1 adulthood in the presence or absence of 200 μM rilmenidine. Kaplan–Meir survival analysis was performed on pooled data from at least three independent trials. Groups tested by log‐rank with Bonferroni correction; p < 0.05. Quantitative data and statistical analyses for the representative experiments are included in Table S1. (e) The graph depicts quantified data of Q40::YFP aggregates in body wall muscles per entire animal after treatment with differing rilmenidine concentrations for the indicated times from L1. Data represented as the pooled mean number of aggregates per animal. Error bars are ± SEM. Significance derived from two‐way repeated‐measures ANOVA p < 0.05.

FIGURE 4

Characterization of NISH‐1. (a) Schematic diagram illustrating exons of nish‐1 gene, alongside translated protein motifs and the deleted regions in the homozygous nish‐1 mutant. (b–e) nish‐1 mutants exhibit reductions in body size compared to WT at day 1 of adulthood as shown by body area (defined as width x length). A two‐tailed t‐test was used for analysis; *p < 0.05. Representative pictures of WT and nish‐1 mutant captured in brightfield at 10X objective on a Zeiss Axio Observer following paralysis in 20 mM tetramisole. Animal heads are left. Scale bar: 200 μm. (f) Western blots showing MPK‐1 phosphorylation in WT, nish‐1 mutants, or nish‐1 rescue transgenic animals, in response to 24 h pharmacologic intervention. α‐tubulin was used as the loading control. Rilmenidine significantly increased MPK‐1 phosphorylation *(%CV vs FC = >1.5), however, neither efaroxan alone nor rilmenidine and efaroxan in combination significantly increased MPK‐1 phosphorylation, n/s (%CV vs FC = <1.5). Rilmenidine failed to reproducibly increase phosphorylation of MPK‐1 in nish‐1 mutants. n/s (%CV vs FC = <1.5). Transgenic rescue strains significantly increased MPK‐1 phosphorylation upon rilmenidine treatment. **(%CV vs FC = >2 in PHX945) and (%CV vs FC = >1.5 in PHX946). Data are quantified as log fold densitometric ratio change relative to α‐tubulin. Data are then expressed as log fold change compared to DMSO vehicle control. Coefficient of variation (%CV) defined as the percent standard deviation: mean ratio; the significant difference in groups if percent fold change is ×1.5* greater or ×2** than % CV.

FIGURE 5

nish‐1 is required for an extended lifespan and better health span. (a) Survival curve showing lifespan extension by rilmenidine in WT, but not in nish‐1 mutant (Statistics and raw data are in Table S1). (b) Rescue of nish‐1 enabled extension in lifespan by 200 μM rilmenidine in nish‐1 mutant. DB06 (=PHX893) is a nish‐1 knockout mutant and DB03 (=PHX945) and DB04 (=PHX946) are two rescue lines expressing wild‐type nish‐1 gene copies. The difference in control‐treated lifespan between DB03 and DB04 might be the random site of integration of the rescue transgene. Quantitative data and statistical analyses for the representative experiments are included in Table S1. (c) Thermotolerance: Rilmenidine increases the percentage survival of WT exposed to 37°C for 3 h and approximately 20 h recovery period, dependent on nish‐1. Bar graph represents mean % survival ± SEM from three independent trials of at least 150 animals per strain and/or condition. *p < 0.05 (one‐way ANOVA with Tukey post hoc comparisons). (d–f) Quantified data of body bends representing motility deterioration with age in WT, nish‐1 mutants, and two nish‐1 rescue mutants DB03 and DB04 in the presence or absence of rilmenidine. Rilmenidine at 200 μM significantly reduced age‐related motility deterioration in WT and nish‐1 rescue strains, but not in nish‐1 mutants. Data are represented as a pooled mean of 10 animals per time point and condition/genotype repeated over three independent trials overlaid on a box plot representing quartiles. Error bars, upper: Q3 + 1.5*IQR; minimum: Q1–1.5*IQR (adj. p < 0.05; two‐way ANOVA and Tukey post hoc).

FIGURE 6

Rilmenidine treatment in mice induces gene expression changes associated with a lifespan‐extending effect. (a) Association of the liver (top) and kidney (bottom) responses to rilmenidine with signatures of aging (left), lifespan extension (middle), and longevity‐related intracellular processes (right). The significance score was calculated as log₁₀(adjusted p‐value) corrected by a sign of regulation. Dotted lines represent the FDR threshold of 0.1. (b–e) GSEA enrichment plots with running normalized enrichment scores (top) and distributions (bottom) of selected up‐ (red) and downregulated (blue) gene signatures of lifespan‐extending interventions among genes perturbed by rilmenidine in the liver (b–d) and kidney (e). Perturbed genes were sorted based on the log₁₀(p‐value) of their differential expression between control and rilmenidine‐treated samples corrected by a sign of regulation. The resulting index was divided by the number of genes in the dataset. Dotted lines represent NES calculated for up‐ (red) and downregulated (blue) gene signatures. (f) Working model representing possible prolongevity signalling by rilmenidine to extend life span in C. elegans. NES, Normalized enrichment score; CR, Caloric restriction; GH, Growth hormone; mTOR, mammalian target of rapamycin.