Tomatidine enhances lifespan and healthspan in C. elegans through mitophagy induction via the SKN-1/Nrf2 pathway

Lifespan of the nematode C. elegans quantifies organismal longevity that depends upon various aging processes31. We exposed N2 (wild type) C. elegans to tomatidine at concentrations of 0, 25, and 50 μM beginning at the L4 stage, and C. elegans survival was recorded. Tomatidine consistently extended lifespan at a concentration of 25 μM with (7.0 ± 0.8)% increase (Figs 1A and S1B), while there was no significant change of lifespan at 50 μM (Figure S1A). When assessing the survival curve further, it was noted that many C. elegans exposed to 50 μM tomatidine died at relatively early ages (7 to 13 days) compared to C. elegans not exposed to tomatidine, but those that survived this period of increased death had a longer maximal lifespan (Figure S1A). This biphasic dose – response effect of tomatidine is consistent with a hormesis-based mechanism of action17. We also noticed that 50 μM tomatidine exhibited some toxicity to C. elegans healthspan as well (data not shown), while no detectable toxicity was noticed with tomatidine concentrations of 25 μM or less. Based on these dose-dependent responses in lifespan, 25 μM tomatidine was selected as an optimal concentration for most subsequent experiments.

There is a dramatic physical decline in swimming ability, pharyngeal pumping, and maximum movement velocity as C. elegans age32,33. These behaviors reflect the overall muscle health of C. elegans, and any attenuation of these age-related declines due to an intervention would suggest anti-aging effect on muscle cells. Our data consistently showed age-dependent decreases of pharyngeal pumping and swimming movement in N2 C. elegans (Fig. 1B,C). C. elegans treated with 25 μM tomatidine exhibited enhanced pharyngeal pumping at multiple time points during their life (Fig. 1B). Although 25 μM tomatidine did not affect swimming ability in young (adult day 4) C. elegans, as they aged (adult day 12), tomatidine significantly delayed the age-related decrease of swimming movement scores (48% improvement compared with vehicle) (Fig. 1C). Maximum movement velocity in C. elegans has been suggested to decline with age and to correlate with longevity33. We verified the decrease of maximum movement velocity with age in N2 C. elegans, but found no significant differences in maximum movement velocity in tomatidine-treated C. elegans from adult days 4 to 10 compared with vehicle treatment (Figure S1C, and data not shown). Collectively, tomatidine is able to alleviate some features of aging-related physical decline in C. elegans, which indicates an improvement in healthspan.

We further asked whether the improvement of swimming and pharyngeal pumping implied inhibition of sarcopenia, so we performed phalloidin staining on worm muscle cells to assess actin filament structure34. We carefully analyzed the muscle morphology in day 4 and day 8 adult worms treated with or without tomatidine, and presented the data as percentage of muscle cells that possessed “no damage”, “minor damage”, or “major damage” (Figure S1E). Tomatidine had no significant effect on actin filament damage in adult day 4 worms. However, in day 8 worms, tomatidine decreased the percentage of muscle cells that exhibited severe damage: first repeat, 54% in N2 (veh) compared with 24% N2 (tomatidine); second repeat 15% in N2 (veh) vs. 7% N2 (tomatidine) (Figure S1F). Collectively, these data suggest that tomatidine inhibits sarcopenia not only in mice30 but also in C. elegans.

We further investigated whether tomatidine-induced improvement of muscle-dependent behaviors could be related to the preservation of muscle cells. We previously showed that pharynx muscle morphology reflects the aging state of the whole organism, and based on this mechanism in combination with machine-learning software, we analyzed the effects of tomatidine on age stage scoring of pharynx in accordance to the previous publications35,36. We defined C. elegans pharynx scoring from stage 0 (young) to stage 3 (old): stage 0 C. elegans are predominantly found in the first 2 days of adulthood and are developing fecundity. Both Stage-1 and Stage-2 C. elegans are actively laying eggs. Stage-3 C. elegans are predominantly found after egg laying has ceased, beginning as early as day 7 of adulthood. Administration of 10 μM or 25 μM tomatidine consistently lowered the age-states of individual C. elegans pharynx (Figs 1D,E and S1D). Notably, for 25 μM tomatidine-treated worms, there are more stage 2 pharynx compared to the Day 7 vehicle condition, but there are no stage 3 pharynx in this group while stage 3 pharynx is 5% in Day 7 vehicle condition. All these data suggest that tomatidine may attenuate age-related pharynx deterioration. These results suggest that tomatidine likely enhances pharyngeal pumping by preservation of youthful pharynx morphology.

Efficient metabolism is important for health, and decrements in cell metabolism have been implicated in age-related diseases and longevity37,38. To test the effect of tomatidine on C. elegans metabolomes, adult D7 tomatidine or vehicle-treated whole body tissues of C. elegans were collected and subjected to ALEX-CIS GCTOF mass spectrometry with more than 150 metabolites identified (Fig. 1F and Table S1). Tomatidine affected the levels of several metabolites, and we chose to focus on metabolites relating to the TCA cycle and amino acid metabolism, due to their importance in energy production and maintenance of muscle mass, and therefore muscle performance. A reduction of many TCA intermediates, including citrate, isocitrate, and malate, coupled with decreased lactate production and steady glycolysis intermediates suggests that tomatidine enhances general levels of oxidative phosphorylation in C. elegans. Tomatidine-treated C. elegans exhibited increased levels of many free amino acids (leucine, threonine, isoleucine, tryptophan, proline, arginine, histidine, valine, methionine) suggesting an increased catabolism (Figure S2A). Collectively, these results demonstrate that tomatidine affects several metabolic pathways which may be integral to its anti-aging properties.

We next investigated the mechanism(s) by which tomatidine enhances lifespan and muscle-related healthspan. To this end, we performed whole-genome gene expression analysis using microarrays to elucidate potential pathways that might mediate the anti-aging action of tomatidine. Heatmap analysis suggested that tomatidine had significant effects on the profiles of expressed transcripts (Fig. 2A). Gene ontology (GO) analysis identified 36 GO signalling pathways upregulated and 25 GO signalling pathways downregulated (Fig. 2B and Table S2). Intriguingly, tomatidine had significant effects on mitochondria function, as evidenced by changes in several mitochondria-related signaling pathways, including those affecting the electron transport chain, the mitochondrial inner membrane space and matrix, and the metabolism of glutathione (Fig. 2B). Additionally, the GO analysis also suggests the improvement of other health-related signaling pathways, including pathways associated with cell fate regulation, metabolism, ROS regulation, and increased resistance to exogenous stress (response to gamma radiation). These data suggest that tomatidine affects mitochondrial function and ROS metabolism.

Given the significant changes of tomatidine on mitochondria-related signaling pathways, we examined how tomatidine modulates mitochondrial homeostasis in C. elegans, primary rat cortical neurons, and human cells. Since mitochondrial homeostasis is tightly regulated by mitochondrial biogenesis and quality control through mitophagy5,39, we first determined whether tomatidine changes mitochondrial content and morphology in C. elegans. The pharynx is an important organ composed of pharyngeal muscles that show age-dependent loss of function (pumping, Fig. 1B) and structure (Fig. 1E), which are attenuated by tomatidine. We therefore examined mitochondrial distribution in the pharynx using MitoTracker Red. There were more mitochondria in pharyngeal cells in tomatidine-treated C. elegans compared with vehicle control (Fig. 3A). We further verified tomatidine-induced mitochondrial biogenesis using a muscle specific pmyo-3::gfp strain (ccIs4251) (Fig. 3B). Since there is an age-dependent disorganization of mitochondrial network5, we took advantage of this myo-3 strain and used a semi-quantitative rating scale to evaluate the mitochondrial networks in a double-blind manner; the lowest value of one indicates highly impaired mitochondrial network, while a value of five signifies optimally organized mitochondrial network running in parallel with the myofilament lattice (see Methods). Tomatidine moderately increased the mitochondrial network morphology score (control, 3.4; tomatidine, 3.9) (Fig. 3B,C). Tomatidine-induced mitochondrial biogenesis was conserved from C. elegans to mammalian cells since treatment of primary rat cortical neurons with 4 μM tomatidine increased their mitochondrial content, mitochondrial membrane potential, and cellular ROS (Figure S2B–D). Tomatidine increased, in a concentration-dependent manner, the expression of several major mitochondrial proteins in a human neural cell line including the inner mitochondrial membrane protein COX-VI, HSP-60 (a mitochondrial protein chaperone), superoxide dismutase 2 (SOD2), and mitochondrial Complex IV-COX II (Fig. 3D). These data show that tomatidine affects mitochondrial content and quality.

In view of the intricate interplay between mitochondrial biogenesis and mitophagy5,39, we asked if tomatidine affects mitophagy. A series of novel techniques were applied to critically investigate mitophagy. A mitophagy reporter strain in C. elegans has been established recently whereby colocalization of LGG-1 (the C. elegans homolog of a key mammalian autophagy marker LC3) and mitochondrial outer membrane localized mitophagy receptor DCT-1 (a putative orthologue to the mammalian NIX/BNIP3L) would indicate mitochondria undergoing mitophagy5. C. elegans were treated with 25 μM tomatidine from the L4 stage, followed by imaging at adult day 4. The data showed that 25 μM tomatidine induced an increase of mitophagy in C. elegans muscle cells (Fig. 3E and quantification in S2E). We further asked whether tomatidine can also induce mitophagy in primary rat cerebral cortical neurons and human neural cells. A commercial kit was applied for mitophagy detection in which a pH-dependent increase of fluorescence detects incidence of “mitolysosomes” (which occur when mitochondria in autophagosomes fuse with acidic lysosomes) which can be detected as the colocalization of the ‘Mtphagy’ dye and a lysosome dye (see Methods for details). In line with an induction of mitophagy in C. elegans, tomatidine increased mitophagy incidence in rat cortical neurons as indicted by increased colocalization of ‘Mtphagy’ dye with the lysosome dye (Fig. 3F,G). Similarly, tomatidine also induced mitophagy in human neural cells (Fig. 3H,I). Additionally, we used a standard mt-mKeima mitophagy reporter to verify changes of mitophagy by tomatidine; the mt-mKeima exhibits green fluorescence (excitation 488 nm) in healthy mitochondria (pH ~8) and a red signal (excitation 561 nm) when mitochondria are in mitolysosomes40,41. As expected, tomatidine increased the mt-mKeima signal (561 nm) indicating higher mitophagy (Fig. 3J–K). In conclusion, tomatidine can induce mitophagy across species.

We further explored how tomatidine-induced increases in healthy mitochondria might affect overall mitochondrial respiratory chain function in the entire C. elegans by examining basal oxygen consumption rate (OCR) with Seahorse XF extracellular flux instrument. C. elegans were exposed to increasing doses of tomatidine from the L4 stage, and basal OCR was measured at adult days 1 and 4. There was a trend towards an age-dependent decrease of OCR between adult day 1 and 4C. elegans, and tomatidine increased basal OCR in adult day 4 worms compared age-matched vehicle-treated control C. elegans (Fig. 3L), suggesting increased mitochondrial quality. Taken together, these results suggest that tomatidine improves mitochondrial homeostasis through induction of mitochondrial biogenesis and mitophagy, and that this process is conserved across species from C. elegans to human cells.

Mitophagy plays a primary role in mitochondrial quality control, and impaired mitophagy causes neurodegeneration, dysfunctional cell metabolism, and even premature aging and shorter lifespan in laboratory animal models4,5,8,9,42. In C. elegans, the proteins PINK-1 and DCT-1 are two critical conserved executors of mitophagy, and may act on a similar genetic pathway to induce mitophagy5. To investigate the roles of mitophagy in tomatidine-related health benefits in wild type C. elegans, we knocked down dct-1 using RNA interference (RNAi) and examined changes in pharyngeal pumping (Fig. 4A). In older C. elegans (day 9), dct-1 knockdown exacerbated the age-dependent decrease of pharyngeal pumping. Importantly, dct-1 knockdown abolished tomatidine-induced increase of pumping measured in 5 and 9 day-old adults, unveiling an essential role of mitophagy induction in tomatidine-induced increase of pumping (Fig. 4A).

To further investigate the roles of mitophagy in tomatidine-induced health benefits, we assessed mitophagy-depleted worms for stress resistance. N2 C. elegans were sensitive to mitochondrial complex I inhibitor rotenone-induced death with 21% survival. Tomatidine treatment doubled the survival of rotenone-treated worms to over 40% (Fig. 4B). The protective effect of tomatidine against rotenone-induced mitochondrial stress was abolished in both dct-1- and pink-1-deficient C. elegans. Tomatidine treatment also protected wild type C. elegans, but not dct-1- and pink-1-deficient ones, against heat stress (Fig. 4C). These findings indicate that PINK-1/DCT-1-related mitophagy contributes to the health-promoting and stress resistance effects of tomatidine.

To elucidate the signaling pathway(s) by which tomatidine stimulates mitophagy, we used fluorescent reporter techniques and human HEK-293 cells to determine whether tomatidine affected the promoter activity of several transcriptional activators that regulate mitochondrial homeostasis. These include the longevity and autophagy-regulating protein SIRT1, the master regulator of mitochondrial biogenesis PGC-1α, and the anti-oxidant nuclear factor E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway 8,26,43. While tomatidine did not affect SIRT1 or PGC-1α promotor activities (Figure S3A,B), tomatidine induced a significant increase of Nrf2/ARE reporter activity in a concentration-dependent manner (Fig. 5A).

Because tomatidine increased Nrf2/ARE reporter activity, we performed immunoblot analysis to measure levels of several proteins encoded by Nrf2-responsive genes in human neural cells exposed to tomatidine. Upon activation by ROS, Nrf2 translocates from the cytoplasm to the nucleus where it binds to the ARE region to transcriptionally activate genes which encode antioxidant proteins, including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO1)43,44,45. Human neural cells exposed to tomatidine exhibited increased amounts of nuclear Nrf2, and increased levels of NQO-1 and HO-1 (Fig. 5B), indicating that tomatidine activates the Nrf2-ARE pathway. In control cells with normal levels of Nrf2, tomatidine induced a 20% increase in levels of ROS (Fig. 5C). Basal levels of ROS were increased in Nrf2-deficient cells, and tomatidine caused a large increase in ROS levels in the Nrf2-deficient cells (Fig. 5C). Moreover, tomatidine-induced mitophagy was attenuated in human neural cells in which Nrf2 was knocked down, and in cells treated with the antioxidant N-acetylcysteine (Fig. 5D), indicating that tomatidine stimulates mitophagy by a mechanism involving ROS-dependent activation of Nrf2. Collectively, these data suggest that tomatidine can induce activation of the Nrf2/ARE pathway, contributing to mitophagy induction.

To investigate whether activation of the Nrf2/ARE pathway in mammalian cells is also conserved at the organismal level, we examined the effect of tomatidine on the null C. elegans skn-1 (zu135) mutant strain (the C. elegans skn-1 gene is orthologous to the mammalian Nrf2 gene). When pharyngeal pumping was assessed, 25 μM tomatidine consistently enhanced pumping rate in adult wild type C. elegans (Fig. 5E). However, skn-1 mutation compromised tomatidine-induced increase of pumping rate, indicating that tomatidine requires Skn-1 activity to enhance muscular function. It is possible that tomatidine may activate other stress response pathways key to mitochondrial stress, such as the mitochondrial unfolded protein response (UPR^mt). Knocking down atfs-1, a key gene in regulating UPR^mt expression46, compromised tomatidine-induced increase of pumping rate (Fig. 5F). This suggests that UPR^mt may play a positive role in tomatidine-induced health benefits. Taken together, these data indicate that tomatidine induces mitophagy and preserves cell function during aging, possibly through multiple stress response pathways, such as the Nrf2/Skn-1 pathway and the activation of UPR^mt.

C. elegans were grown on standard nematode growth medium (NGM) and maintained following standard protocols as described previously62. The Bristol strain N2 (wild type), CB5600 ccIs4251[pSAK2 (myo-3p::gfp-lacz(NLS)) + psak4 (myo-3p::mitochondrial gfp)I, and skn-1(zu135) were obtained from the Caenorhabditis Genetics Center (CGC). The mitophagy reporter strain N2;Ex(pmyo-3::dsred::lgg-1; p_myo-2GFP)Ex(pdct-1::dct-1::gfp; pRF4) was from Prof. N. Tavernarakis (University of Crete, Greece)5. All C elegans were cultured at 25 °C on solid NGM agar plates with OP50 food source unless otherwise noted62.

Lifespan analysis was performed as mentioned previously8,37,38. About 8–10 adult D1 C. elegans were transferred to 4 plates and allowed to lay eggs for 5 hours to give rise to synchronous population. After hatching, L4 stage nematodes were transferred to respective condition plate with either 10, 25, or 50 μM of tomatidine (all with 50 μM FuDR). Animals were scored as dead or alive every other day. Missing worms were censored from the lifespan count. Significance was determined by log rank test, and mean was calculated by averaging the days of C. elegans death for each condition. Figures display a smoothed Kaplan Meier survival curve of the pooled populations.

Luciferase assays were performed as described elsewhere26,43. The ARE-bla construct was used for Nrf2/ARE reporter assay43; the PGC-1α-2kb (the Renilla luciferase-expressing plasmid/pRL-TK used as an internal control.) was for PGC-1α promoter activity detection26.

SIRT1 promoter activity was detected by a pTA-Luc SIRT1 promoter (-202), a gift from Dr. Toren Finkel63. In Brief, plasmids were transfected to HEK-293 cells for 24 h, cells were then treated with different concentrations of tomatidine for 24 h. Luciferase activity was measured using a Dual-Luciferease-Reporter System (Promega), and luciferase activity for each well as normalized to the internal renilla luciferase activity.

These methods are detailed elsewhere64,65. Briefly, for swimming movement, nematodes from a treatment group were randomly selected and transferred to a 6 cm petri dish containing 1 mL of M9 buffer. They were allowed to acclimate for ~10 s, then movements were scored for 30 seconds. Maximum velocity was determined as described previously33. N2 worms were exposed to designated concentrations of tomatidine (0, 10, 25, and 50 μM) beginning at L4 stage and maximum velocity of movement was determined on designated days. In brief, worms for imaging were moved to physical assay plate void of bacterial lawn. The recording system was a stereomicroscope (Unitron 2850), a charge-coupled device camera (Infinity 2, Lumenera), and imaging software. Four to eight nematodes were placed in one plate and recorded for 30 s, followed by data analysis using ImageJ and wrMTrck Batch (www.phage.dk/plugins↗).

For measurements of pharyngeal pumping (contractions) C. elegans were synchronized and raised to adults as mentioned in the longevity assay methodology. At designated ages pharynx contractions were manually counted for 30 s. Experiments were blinded and two or more researchers quantified pumping and swimming to decrease bias. Significance was calculated by student t-test.

We imaged anesthetized worms, immobilized between an agarose pad and a number 1 coverslip, with an NA 1.4, 60x objective on an inverted Nikon Eclipse E800 microscope. The CCD output was saved as a stack and opened with ImageJ, for animals of a given cohort (drug treatment, age, etc.). A custom script was used to center a region of interest around the terminal bulb of each individual animal; the 300 × 300 pixel ROI was saved as a TIFF files, named systematically and later used to score the worms apparent age, as described35.

Basal oxygen consumption was measured per previous methods66 using a Seahorse XF96 instrument (Seahorse Bioscience Inc.). In brief, around 200 L4 nematodes/group were transferred to designated drug plates, followed by OCR detection on adult days 1 or 4. On the day of the experiment, all the C. elegans were collected using M9 buffer washing, followed by washing with M9 buffer twice. Nematodes were then re-suspended and transferred in 96-well standard Seahorse plates (#100777–004) (10~15 worms per well), and basal OCR was measured six times. After the experiments, nematodes numbers were counted, followed by data normalization to the number of nematodes in each individual well. We had ten replicates for each group, and repeated the experiment twice.

We used a myo-3::gfp reporter strain for mitochondrial networking morphology analysis in the body wall muscles. Worms were treated with the designated drugs from L4 stage for 4 days followed by imaging with a Zeiss confocal microscopy. We imaged over 15 worms/group each time (repeated two to three times). Mitochondrial network was evaluated in a double-blinded manner. A scoring scale of 1 to 5 was used, where a score of 5 denotes a perfectly organized mitochondrial network with healthy mitochondria running parallel with the myofilament lattice. For highly fragmented and disorganized mitochondrial network morphology, a score of 1 was given.

RNA interference (RNAi) was performed as we did previously67. RNAi feeding constructs for L4440 (control), dct-1, pink-1, and atfs-1 were derived from two RNAi feeding libraries (Dr. Sige Zou’s laboratory (previously form NIA) and the other one from Open Biosystems [the E. coli host strain is HT115 (DE3) (Fire Lab)]). RNAi was performed with worms maintained on the RNAi from egg laying stage throughout the experiment67.

Whole worm tissue metabolomics were assessed with a method as described previously67. To evaluate changes of metabolome in N2 (veh) and N2 (tomatidine) worms, whole body tissues of adult Day 7 worms were collected and subjected to ALEX-CIS GCTOF mass spectrometry, UC Davis Genome Center Core Facilities. Four replicates were prepared for each group. Data were then normalized to internal control, followed by heat-map and TCR cycle analysis.

Stress assays were conducted as described previously5. Briefly, C. elegans were treated with different doses of tomatidine, followed by exogenous stress exposure including either 25 μM rotenone for two days or exposure to 37 °C for 7 h. Nematodes were then evaluated for survival. Significance was calculated by student t-test.

N2 C. elegans from L4 stage were exposed to either vehicle (1% DMSO) or 25 μM tomatidine (Sigma-Aldrich, USA). On adult day 7, nematodes where collected by washing off the plates with M9 buffer from four independent experiments. Samples were stored at −80 °C in lysis reagent (Qiagen Cat. no. 75142). Crude extracts were prepared by bead beating packed nematode pellets with the Mini-Beadbeater 8 (Biospec Products, Oklahoma), and total RNA isolated using RNeasy Mini Kit on the QIAcube per the manufacturer’s protocol (Qiagen, California). RNA concentration and quality was measured by Nanodrop (ThermoFisher, Waltham, MA USA) and the Agilent Bioanalyzer RNA 6000 Chip (Agilent, Santa Clara, CA). Two-hundred ng total RNA was labeled using the Agilent Low-Input QuickAmp Labeling Kit, and purified and quantified per the manufacturer’s instructions. A total of 825 ng Cy3-labeled cRNA was hybridized for 17 hours to Agilent C. elegans 4 × 44 K oligo microarrays. Following posthybridization rinses, arrays were scanned using an Agilent SureScan microarray Scanner at 3 micron resolution, and hybridization intensity data extracted from the scanned images using Agilent’s Feature Extraction Software. Raw microarray hybridization intensity data from four separate experiments were log-transformed. Three separate probe preparations were used individually or combined in equal amounts for the fourth hybridization. Raw microarray data were log transformed to yield z-scores. The z-ratio was calculated as the difference between the observed gene z-scores for the experimental and the control comparisons, and dividing by the standard deviation associated with the distribution of these differences. Z-ratio values of ±1.5 were used as cut-off values and calculated using a 5% false discovery rate (FDR) threshold. The complete set was tested for gene set enrichment using parametric analysis of gene set enrichment. For each pathway z-score, a p-value was computed using JMP 6.0 software to test for the statistical significance.

Mitophagy in nematodes was measured using a mitophagy reporter strain N2;Ex(pmyo-3::dsred::lgg-1;pdct-1::dct-1::gfp). Nematodes were exposed to tomatidine at L4 stage, followed by imaging of muscle cells at adult D4. A total of 15~20 nematodes/group were imaged in three independent replicates. Colocalization coefficient between DSRED::DCT1 and LGG1::GFP were analyzed using the Zeiss LSM Image Examiner.

For mitophagy evaluation in cultured cells, we used different cell systems, including primary rat cortical neurons, human SH-SY5Y neural cells, and the mt-mKeima HeLa cells. A commercial mitophagy kit was used for mitophagy detection in both neurons and SH-SY5Y cells (Dojindo Laboratories). Imaging of mt-mKeima cells (gift from Dr. Richard Youle) was performed as reported by Nuo and colleagues41, using different settings for GFP (alkaline condition, excitation 458 nm and emission 615 nm) and RFP (acidic condition, excitation 561 nm and emission 615 nm).

For the worm lifespan analysis, the log rank test was used for statistical analysis. For two group comparison, two tailed student t-test was applied. GraphPad Prism 5 (GraphPad Software, Inc.) was used for statistical analyses. The p values < 0.05 were considered statistically significant.