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.

: A) Survival curve of WT treated with moxonidine at different concentrations.
Statistics and raw data are in Supplementary Table 1 Fluorescence Intensity per number of pixels was measured to quantify uptake of beads.
Quantified data shown as mean + SEM. P-value determined by two-tailed Welch's t-test.   elegans PCR genotyping of WT and nish-1 deletion mutant following x4 outcrossing. Using oligonucleotides flanking the deletion site (external primer pairs) generated either a 1968 bp or a 1936 bp amplicon depending on oligonucleotide pair in WT or a correspondingly 873 bp reduced amplicon size in the genomic DNA of homozygous nish-1 mutants. Likewise, an oligonucleotide pair targeted to sequences within the CRISPR deletion site produced a 1558 bp amplicon in WT but no amplicon in homozygous nish-1 mutants. Control amplifications were conducted on the lgc-37 gene coded on chromosome III. Primer sequences are in Supplementary Table 2. C) nish-1 mutants and rilmenidine-fed WT do not exhibit delayed vulval development from the L3 stage. In the chart, developmental stages are listed on the Yaxis, and the number of hours after a 24h exposure to food from L1 is plotted on the x-axis.
The areas of the circles in the chart reflect the percentage of the population at each stage of development; n≥30 from 3 independent pooled trials for each time point. Statistical difference established by way of 2-way ANOVA wherein a mean development score could be ascertained through pre-L3 corresponding to a score of 0, Early L3 is 1 and Mid L3 a 3 etc. No groups displayed any significant difference in development rate.

Generation of UV-Killed OP50 E. coli
32 mL of unconcentrated live OP50 E. coli cultures were aliquoted to each 50 mL falcon tube, mixed with absolute ethanol (8 mL) to yield a 20% ethanol solution as described in Calvert et al., 2016 and pipetted into T160 flasks to a volume of 150 ml. Flasks were transferred to a UV-Linker machine (CL-1000 Ultraviolet crosslinker UVP) and irradiated for 120 minutes at 999,900 microjoules/cm 2 . 50 ml aliquots of UV-killed E. coli-ethanol solution were pelleted at 3000 rpm for 10 minutes and resuspended to a 10X concentrate in 5 mL Lennox LB broth.

Drug Treatment
All drug treatments, unless otherwise stated, were administered to C. elegans via the addition of the respective compound to NGM (Zheng et al. 2013). Aliquots of stock solutions of the drugs (rilmenidine and rapamycin) were prepared by dissolving them into DMSO solvent, such that the final volume in NGM agar did not exceed 1%. NGM agar was cooled to below 65°C after same-day autoclaving, and respective drugs were concomitantly added. Plates were gently swirled to homogenise the solution and left to dry for 1 hour in a hood. 60 µl of 10X concentrated UV-killed OP50 E. coli was then spotted to the centre of the plate and again the plates were left to dry for 30 minutes in a hood. Unless otherwise stated, plates were prepared one day before use.

Manual Lifespan Assay
Well-fed gravid hermaphrodites were L1 synchronised overnight at 20°C, after hypochlorite treatment. The next day, L1s were transferred to NGM agar plates seeded with 10X OP50 E.
coli and allowed to develop for 52 hours at 20°C until late L4/adult. At L4/adult molt, animals were transferred to 6-well lifespan assay plates, containing 3 ml of NGM per well with test compounds and 100 µg/ml 5-Fluoro-2'deoxyuridine (FUdR) (Calvert et al. 2016). Plates were maintained at 20°C in the dark, wrapped in parafilm to moderate humidity. Animals were counted every 2 to 3 days, and dead ones (failure to respond to no more than 3 "prods" with a platinum pick) were removed at each inspection. 60 μL of UV-killed OP50 was added three times a week to all plates until day 15 of the assay by which point low-food consumption negated the need for additional E. coli (Calvert et al. 2016

Automated Lifespan Measurements
Automated survival analysis was performed as described in (Statzer et al. 2020) employing the lifespan machine setup developed by Stroustrup and colleagues (Stroustrup et al. 2013).
Briefly, C. elegans were age-synchronized using bleach lysis and kept in L1 culture (52h, 20°C), placed on live OP50 until L4 stage, and subsequently transferred to plates containing the drug, FUdR (50 µg/ml) and dead OP50 from L4 to day 4. Lastly, the animals were moved to tight-fitting Petri dishes (BD Falcon Petri Dishes, 50 x 9 mm) containing the drug, dead bacteria, and FUdR and imaged until the end of life.
Rilmenidine hemifumarate was dissolved in 1% DMSO and supplemented to the agar just before pouring to yield 200 µM, 300 µM, and 400 µM final concentrations. When using dead bacteria, the agar was additionally supplemented with Nystatin (44 U/ml) and Penicillin-Streptomycin (50 U/mL).
Air-cooled Epson V800 scanners were utilized for all experiments operating at a scanning frequency of one scan per 30 minutes. To limit condensation, the tight-fitting plates were dried without lids in a laminar flow hood for 40 minutes before starting the experiment.
Furthermore, temperature probes (Thermoworks, Utah, U.S.) were used to monitor the temperature on the scanner flatbed and maintain 20.0°C. Animals that left the imaging area during the experiment were censored. Automated lifespan results were validated using manual lifespan measurements as described in (Ewald et al. 2016) by picking L4 from normal culturing plates onto the corresponding assay plates. Lifespans were calculated from the L4 stage (= day 0).

Feeding rates and Food Uptake
Larval stage, L4 C. elegans hermaphrodites were transferred on the freshly prepared OP50 seeded NGM plates containing rilmenidine (200uM) and FuDR (50uM). At day3, pharyngeal pumping rate was measured by counting pharynx movement per minute; considering only the animals moving on bacteria and constantly pumping for the measurements (Ewald et. al. 2016). The animals were then transferred on the plates containing RFP fluorescent beads diluted in OP50 (1:100) and let them feed for 30minutes (Venz et al. 2021). After feeding, animals were picked in M9 Buffer and washed thrice, anaesthetized in 50mM tetramisole and mounted on 2% agarose pads for imaging. The uptake of food was measured by the presence of beads in the intestine. The red fluorescence from beads was captured using an upright bright-field fluorescence microscope (Tritech Research, model: BX-51-F) at 10X. Experiment was performed in three biological replicates. Approximately 30 animals per condition were used for the analysis. A two-tailed Welch's t-test was used for statistics analysis.

Protein Extraction
Whole worm protein lysates were prepared as in (Hu et al. 2017) . ~500 day 1 adult worms per condition and genotype were transferred to either empty NGM plates to induce starvation (+ve control) or to UV-killed OP50 E.coli NGM plates containing requisite drugs or vehicle (1% DMSO). After 24 hours, worms were suspended in M9 buffer and centrifuged at 1000rpm for 2 minutes to yield a wet pellet. Worms were then re-suspended on ice in 50µl of RIPA buffer with added phosphatase inhibitors (Cell Signalling Technologies #9806) and complete EDTAfree protease inhibitor cocktail (Roche # 04693159001) before sonication: 10 sec ON/1 minute OFF, 14% amplitude 3 times until the vast majority of animals were dissolved. Lysates were then spun down twice at 13,000g/20minutes 4 o C and immediately frozen at -20 o C for no more than one week before use.

Western Blot Analysis
Total protein lysate concentrations were quantified using a bicinchoninic acid (BCA) assay (BioRAD) according to the manufacturer's protocol.
Protein lysates were heated for 5 minutes at 95°C, cooled on ice for 2 minutes and pelleted for 3 minutes at 13,000g. 25µg of protein was loaded per well into an 18 well 10%  (Gee et al. 2013) with gentle rocking. The following morning, blots were washed three times in TBST before being incubated for 1 hour with the secondary antibody IRDye® 800CW Goat anti-Rabbit IgG at a concentration of 1:10,00 in blocking buffer w/ 0.1% tween. This process was then repeated for the loading control, α-tubulin, using anti-alpha Tubulin (ab72910) and IRDye® 680RD Goat anti-Mouse IgG. Changes in ERK phosphorylation are often calculated using phospho-ERK:Total ERK ratios (Nykamp et al. 2008;Kao et al. 2004). However, given the frequent use of ERK as a loading control (Huang et al. 2004;Hwang et al. 2005), we followed previous examples in both worms (Villanueva-Chimal et al. 2017;Chen et al. 2008) and other models (Redshaw & Loughna 2012;Zhou et al. 2015)

Body Size Phenotyping
Synchronized day 1 C. elegans hermaphrodites cultured on live OP50 E. coli NGM plates were measured for body length and width in brightfield at 10X objective on a Zeiss Axio Observer following paralysis in 20 mM tetramisole. Between 10-20 animals per data point were used.
The animal's length was measured from the head (most visibly anterior buccal line) to a position where the tail tapered to a 10 ± 0.5 μm diameter (Petzold et al. 2011). Body width measurements were taken from the posterior vulval peak to the corresponding outer edge of the intestinal cuticle (Collins 2007). Measurements were made using the segmented lines function on Image J software normalized to the scale bar.

Developmental Measurements
Bleach synchronized animals that had been halted in L1 starvation for 24h were measured for development following exposure to food source (Schindler et al. 2014). ~ 500 L1s per genotype/condition were placed onto NGM plates containing rilmenidine and spotted with live OP50 E. coli and allowed to develop at 20°C. After 24 hours, measurement of development was conducted at different time points: 0 hours (24 hours exposure to the food source), 3 hours, 6 hours, and then 24 hours (48 hours exposure to the food source). Per trial and time point, 10 animals were immobilized and mounted onto glass slides in 20 mM solution of tetramisole hydrochloride and then imaged at a 10X objective for body length and 60X objective for vulval development in brightfield on a Zeiss Axio Observer. Vulval development was scored using visual identification of late larval stage vulval checkpoints detailed by Schindler (2014). Statistical significance of differences in vulval development was determined by Two-way ANOVA.

Motility Assay
NGM agar plates were firmly tapped onto the microscope to stimulate movement. In responding animals, not impeded by OP50, body bends were counted for 30 seconds in 10 animals for each condition at each time point (5, 10, and 15 days post-L4 molt) per trial across three independent trials to a total of 30 animals per genotype and condition. Mean deterioration in motility per genotype and/or condition was compared by Two-way of repeated ANOVA and Tukey post hoc correction whilst individual time-point comparisons were tested by student's ttest.

Thermotolerance and Recovery Assay
Well-fed gravid hermaphrodites were bleach synchronized and resultant embryos were allowed to grow for 52 hours at 20°C on NGM plates seeded with live OP50 E. coli. At the late-L4 stage, animals were transferred to seeded UV-killed OP50 E. coli NGM plates containing 200-400 µM rilmenidine, or vehicle for 24h. After 24h of the respective drug exposure, plates were upshifted to an incubator preset to 37°C to induce heat shock. After 3 hours, plates were moved to 20°C and scored for survival after a ~20h "recovery period (Kumsta et al. 2017). At least 100 animals per condition across three identically designed independent trials were scored as either dead or alive by their ability to respond to no more than 3 "prods" with a platinum pick. Percentage survival was calculated, and populations were compared by oneway ANOVA of variance with Tukey post hoc correction.

Measurement of Autophagy
Gravid hermaphrodites were bleach synchronized and resultant embryos were allowed to grow for 52 hours at 20°C on NGM plates seeded with live OP50 E. coli. At the late-L4 stage, animals were transferred to either empty NGM plates to induce starvation (+ve control) or to UV-killed OP50 E. coli NGM plates containing the requisite drug or vehicle (1% DMSO). Animals were then incubated at 20°C for 24 hours to maximize drug absorption and efficacy (Zheng et al. 2013). After 24 hours, approximately 10 day-1 adults per condition were imaged at a 10X objective on a Zeiss Axio Observer using a 150 ms exposure time. In total, at least 25 images across three independent trials were collected and pooled for each condition (Morselli et al. 2010;Eisenberg et al. 2009). Levels of autophagy induction were quantified using Zen

PolyQ Protein Aggregation Assay
Gravid hermaphrodites were bleached, and synchronized L1s were transferred to NGM plates containing rilmenidine or DMSO. After incubation for the indicated periods of time, approximately 30 animals per condition across three trials were imaged under a Zeiss Axio Observer microscope at 10X objective. The number of polyQ40::YFP aggregates in body wall muscle was counted using ZenBlue software with set histogram parameters (black: 500 gamma:1.0 white: 5000). Approximately 50 animals across three independent trials were randomly selected for each treatment group and scored for the number of aggregates. Groups were compared by two-factor repeated measures ANOVA and Tukey posthoc correction.

RNA-sequencing Data Processing
RNAseq data of gene expression changes induced by rilmenidine in mouse liver after 1 month of oral administration was obtained from (Tyshkovskiy et al. 2019 Quality filtering and adapter removal were performed using Trimmomatic (version 0.32). Processed/cleaned reads were then mapped with STAR (version 2.5.2b) and counted via featureCounts (version 1.5). To filter out genes with a low number of reads, we left only genes with at least 6 reads in at least 66.6% of the samples separately for each tissue. Filtered data was then passed to RLE normalization (Anders & Huber 2010). Differentially expressed genes between control and treated mice were identified using edgeR. For each gene, we calculated the p-value of its logFC in response to rilmenidine compared to control independently for every tissue. We then converted them to log 10 (p-value) corrected by the sign of regulation, calculated as: , where pv and lfc are p-value and logFC of a certain gene, respectively, and sgn is signum function (is equal to 1, -1 and 0 if the value is positive, negative and zero, respectively). The resulting pre-ranked list of genes was used to identify significant associations with the biomarkers of aging, lifespan extension and specific intracellular functions.

In-silico Association Analysis
To identify functions enriched by genes perturbed by rilmenidine, we performed gene set enrichment analysis (GSEA) (Subramanian et al. 2005) on a pre-ranked list of genes, obtained as described earlier. REACTOME, KEGG, and GO BP from Molecular Signature Database (MSigDB) have been used as gene sets for GSEA. Only functions related to the processes identified in C. elegans (insulin, FOXO, ERK and mTOR signaling, autophagy and proteolysis) were chosen for the analysis.
To identify associations between rilmenidine's effect in mice and biomarkers of lifespan-extending interventions and aging, we employed a GSEA-based algorithm developed in our previous study (Tyshkovskiy et al. 2019). First, for every trait, we specified significant genes, using the FDR threshold of 0.05. Among the remaining genes, we selected 1000 genes with the highest absolute logFC and divided them into up-and downregulated genes. These lists were considered as gene sets. Signatures of lifespan-extending interventions were taken from (Tyshkovskiy et al. 2019), while signatures of aging in liver and kidney were identified from GSE132040 dataset derived by Tabula Muris Consortium (Schaum et al. 2020). Then we calculated normalized enrichment scores (NES) separately for the up-and downregulated lists of each gene set as described in (Lamb 2006) and defined the final NES as a mean of the two.
To calculate the statistical significance of the NES, we performed a permutation test where we randomly selected gene sets of the same size. p-value of association was calculated as the frequency of observed final NES being bigger by absolute value than random final NES obtained from 5,000 random permutations. The resulting p-values were further adjusted for multiple hypothesis testing using Benjamini-Hochberg (BH) procedure (Benjamini & Hochberg 1995). Association was considered significant if the adjusted p-value was smaller than 0.1. To visualize the results of the association analysis, we converted adjusted p-values into significance scores as: . , where adj.pv and NES are BH adjusted p-value and final NES, respectively.