A moderate static magnetic field promotes C. elegans longevity through cytochrome P450s

Ageing is co-regulated by genetic and environmental factors. Life on earth lives and evolves in a mild geomagnetic field. Yet, the biological effects of a moderate magnetic field on ageing and the underlying genetic mechanisms remain barely unknown. Here, we report that a moderate static magnetic field (SMF) extends the lifespan of Caenorhabditis elegans, a well-established model organism in ageing research. Consistently, the SMF-treated worms show improved motility and mitochondrial function when aged. We identified from the transcriptomic changes upon SMF treatment that the upregulation of three cytochrome P450 genes are required for SMF-induced longevity. Our findings thus reveal that proper SMF treatment could promote longevity through the well-conserved cytochrome P450 enzymes.

considered respectively as young and aged in our assays. At D3, the SMF of 50 mT exhibited a marginal effect by increasing worms' motility by 3.7%, whereas the other two showed no influences (Fig. 1a, Supplementary  Fig. S1b). The worms' motility at D10 was not changed upon the exposure to the SMFs of 50 mT or 100 mT ( Supplementary Fig. S1b). In contrast, the SMF of 10 mT significantly enhanced worms' motility by 21.5% at D10 (Fig. 1a, Supplementary Fig. S1b), indicating that a proper treatment of moderate SMF improves the health of aged worms. Stress resistance in young adults is tightly related to ageing 16 . We then examined the heat resistance of 10 mT SMF-treated worms at D1. 22.6% SMF-treated worms recovered from a 5.5 h heat shock at 35 °C, whereas the thermorecovery rate of untreated worms was 12.8% (Fig. 1b). Consistent with its positive effect on healthspan and heat resistance, the exposure to 10 mT SMF also extended the median lifespan by 18% and the maximum lifespan by 11% ( Fig. 1c and Supplementary Table 1). Taken together, SMF of 10 mT promotes the longevity of worms.

Moderate SMF alters the morphology and function of mitochondria.
A recent report shows that moderate SMF promotes mitochondrial respiration 8 . Since mitochondria are also critical in ageing 1 , we next checked the impact of SMF on mitochondria morphology in the body wall muscle (BWM) of worms at different ages. As reported 17,18 , mitochondria were in a tubular and well-organised network in young worms at day 1 of adulthood (D1), whereas they appeared to be swollen and fragmented in aged worms at D10 ( Fig. 2a and  b). 10 mT SMF treatment did not affect mitochondria morphology in D1 worms but suppressed the age-related changes in the mitochondria network ( Fig. 2a and b).
The morphology of mitochondria is tightly linked to its function. We then used Seahorse Mito Stress Test to measure mitochondria function in D1 and D8 worms by four benchmarks: basal OCR, ATP-linked respiration, spare capacity, and proton leak ( Fig. 2c and d). The four mitochondrial benchmarks respectively show the mitochondrial function under baseline conditions, its contribution to the cellular energy needs, its flexibility to respond to energetic demand, and its damage 19 . In agreement with its effect on mitochondria morphology, SMF of 10 mT significantly reduced basal OCR, ATP-linked respiration, and proton leak in aged worms ( Fig. 2e-h). Moreover, although SMF did not affect mitochondria morphology in the BWM of D1 worms ( Fig. 2a and b), it mildly suppressed basal OCR and remarkably increased spare capacity at D1 ( Fig. 2e and g). Proton leak was also slightly decreased upon SMF treatment at D1, albeit without statistical significance (P = 0.065) (Fig. 2h). Therefore, moderate SMF controls mitochondria in both young and aged worms.
Mitophagy is critical to the morphology and function of mitochondria and in turn to ageing 20,21 . The treatment of 10 mT SMF did not change the induction of mitophagy at D10 ( Supplementary Fig. S2), suggesting that mitophagy may not be involved in the SMF-induced regulation on mitochondria.
Moderate SMF modulates the expression of cytochrome P450s. To explore how SMF prolongs worm longevity and alters mitochondria activity (Figs. 1 and 2), we first examined AMPK, a pivotal metabolic regulator controlling mitochondria 17,18,22 . AMPK activation can be monitored by phosphorylation at its conserved Thr172 23 . In contrast to our hypothesis, SMF of 10 mT did not change the level of p-AMPK in worms at D1 ( Supplementary Fig. S3), implying that AMPK may not be involved in SMF-induced longevity. For clues of the molecular mechanism underlying SMF treatment, we next profiled the 10 mT SMF-induced transcriptomic changes in freshly moulted adults (D0). 826 and 494 genes were respectively up-and down-regulated by SMF with a change bigger than twofold ( Fig. 3a and Supplementary Table 2). Analysis using WormCat indicates that the upregulated genes are enriched in stress responses ( Fig. 3b and Supplementary Table 3), which are critical to longevity 24,25 . Consistent with its effect on mitophagy ( Supplementary Fig. S2), 10 mT SMF did not change the expression of pink-1, pdr-1, and dct-1, three critical mitophagy genes, by either RNA-Seq or RT-qPCR results (Supplementary Fig. S4 and Supplementary Table 2) 20 . Instead, a group of cytochrome P450 (CYP) genes aroused our interest among the upregulated genes regulating stress resistance. The CYP genes are closely related   (Fig. 3c). oPOSSUM-3 predicted lin-14 as the transcription factor driving the CYP genes, based on the over-represented conserved transcription factor binding sites in their promoters 27 . However, mutating lin-14 did not block the SMF-induced upregulation of CYP genes ( Supplementary Fig. S5). Worms were reported to sense GMF through tax-4 in neurons 13 . We then examined whether SMF increased the expression of CYP genes through tax-4. Whereas mutating tax-4 did not affect the upregulation of cyp-14A3 upon SMF treatment, it could suppress the SMF-induced increase of cyp-14A2, cyp-34A9, and cyp-34A10 ( Supplementary Fig. S5), implying that neuronal network could be involved in the response to SMF.
The upregulation of cytochrome P450s are required for SMF-induced longevity. We then explored the role of CYPs in SMF-induced longevity. Since CYPs are involved in proper mitochondria function 26 , we first checked the SMF-induced changes of mitochondria morphology. Under the control treatment of luc2 RNAi, SMF of 10 mT suppressed the deterioration of the mitochondria network ( Fig. 4a and b), as we observed ( Fig. 2a and b). RNAi against cyp-14A2 or cyp-34A10 abrogated the effect of 10 mT SMF on mitochondria morphology, whereas inhibiting cyp-14A3 showed no impact, and cyp-34A9 RNAi further disrupted the mitochondria network ( Fig. 4a and b). We then focused on cyp-14A2, cyp-34A9, and cyp-34A10, which block SMFinduced changes of mitochondria network, and examined their functions in longevity. The lifespans of these  Table 1). Moreover, when these CYPs were suppressed by RNAi, SMF of 10 mT no longer increased worms thrashing rates at D10 (Fig. 4d). The improved heat resistance upon SMF treatment was consistently abrogated by RNAi against the CYPs (Fig. 4e). Therefore, the three CYPs are essential to SMF-induced longevity.

Discussion
In this study, we used the nematode C. elegans as a model to explore the biological effect of static magnetic field (SMF) on ageing. Our data indicate that a moderate static magnetic field of around 10 mT induce significant transcriptomic changes in worms, alleviates the deterioration of mitochondria in ageing, and extends both the health span and life span of worms. A group of cytochrome P450 (CYP) genes play a critical role in SMF-induced longevity (Fig. 4f). Environmental cue is critical to ageing. Whereas food, temperature, and light have been extensively studied in the biology of ageing 28,29 , the role of the magnetic field remains unknown. All organisms on earth de facto live and evolve in the geomagnetic field (GMF). Magnetic fields have also been reported to have various biological effects 3,5 . It is natural that ageing, one of the most important genetic traits, could also be regulated by the magnetic field. Intervention of the magnetic field thus could alter the signalling network evolved in adaptation to GMF and modulate ageing. Indeed, we found that a moderate SMF of 10 mT promotes a series of longevity phenotypes, including motility, heat resistance, and lifespan (Fig. 1). Similar with a previous report 14 , we did not observe obvious defects, such as body size and brood size, in 10 mT SMF-treated adult worms. Nevertheless, we can not exclude that the SMF of 10 mT could cause some subtle negative effects on worms.
The SMF of 10 mT is several hundred times stronger than GMF. Therefore, its effect on ageing does not necessary mean that GMF could promote longevity but may rather be from an artificial modulation of the signalling pathways naturally responsive to GMF. Nevertheless, it will still be interesting to check animal ageing in the magnetic shield condition to figure out the role of GMF in longevity. Meanwhile, SMF of 10 mT is common in outer space, suggesting that the extreme conditions in space may interact with the molecular network evolved on earth and produce unexpected biological effects. Testing organismal ageing in space is definitely of great interest.
As in the case of other environmental factors, a proper dosage of SMF treatment is crucial to its anti-ageing effect. SMF of 200 mT was shown to shorten the lifespan of worms 14 . Among the three SMFs in our test, only the SMF of 10 mT affects ageing ( Fig. 1 and Supplementary Fig. S1). Similarly, a recent report showed that 0.3 T SMF is more potent than 0.6 T SMF in inducing cytokine secretion of CD8 + T cells 8 . The dose-dependent feature of  Fig. 1b  www.nature.com/scientificreports/ SMF-induced biological effects makes it intriguing to find optimal SMF treatment in the future. Due to technical limitation, we were unable to test SMFs below 10 mT in this study. It will be interesting to check animals ageing in lower SMFs (e.g., 1 and 5 mT). It also raises attention for future studies because it is technically challenging to make an SMF with even intensity in a three-dimensional (3D) space. Therefore, C. elegans could be an excellent model in studying the SMF-induced longevity effect. In addition to its advantages in ageing research, lab-cultured worms on plates can be viewed as living in a 2D manner, facilitating the adjustment and maintenance of its SMF treatment. When it comes to other organisms such as mice, the spatial distribution of SMF should be considered when interpreting the biological effect of SMF. As with other ageing interventions, SMF of 10 mT induces a remarkable transcriptomic change. In addition to the CYP genes highlighted in this study, the differentially expressed genes upon SMF treatment are enriched in multiple gene sets related to ageing. For example, E3 ligases and innate immunity genes, which are essential in protein homeostasis and pathogen defence 30,31 , were also upregulated by SMF (Supplementary Table 3). Therefore, SMF could induce longevity through a complex genetic signalling network.
In the SMF-regulated genes, our data show that three CYPs, cyp-14A2, cyp-34A9 and cyp-34A10, are upregulated by SMF and essential to SMF-induced longevity (Figs. 3 and 4). Blocking their function via either genetic mutation or RNAi completely abolishes the anti-ageing phenotypes upon SMF treatment, including mitochondrial morphological changes, improved heat resistance, extended healthspan and lifespan (Fig. 4). CYPs are well known in drug metabolism 32,33 . cyp-34A9 and cyp-34A10 were shown to catalyse tolbutamide metabolism 34 . More importantly, CYPs are important enzymes in the oxidation of endogenous compounds such as lipids 35 . cyp-14A2 was reported to metabolise eicosapentaenoic acid (EPA), the predominant polyunsaturated fatty acid of C. elegans 36 . Therefore, the three SMF-upregulated CYPs could promote longevity through detoxification and lipid metabolism.
CYPs have intensive interaction with mitochondria. Mitochondria is a major cellular organelle of CYPs localisation 26 . The mitochondrial electron transport system serves as an electron donor for mitochondrial CYPs catalytic activity, whereas the products of CYP-dependent metabolism affect mitochondrial functions 26,35 . CYP2U1, the mammalian ortholog of the three CYPs in SMF-induced longevity, is localised in mitochondria and controls mitochondrial morphology 37 . As the three CYPs also regulate the morphological changes of mitochondria during worm ageing (Fig. 4), it will be interesting to clarify their interaction with mitochondria further and to pursue the potential role of CYP2U1 in mammal ageing.
In summary, this study reveals the role of SMF in worm ageing and identifies a set of CYPs as essential genes for SMF-induced longevity. However, it remains a puzzle how SMF triggers this biological effect. Studies in animal navigation show that some animals, such as birds and butterflies, can sense magnetic fields [5][6][7] . C. elegans was also proposed to have magneto sensitive neurons for geomagnetic orientation 13 , implying that SMF might regulate worm ageing through these neurons. Consistently, we found that mutating tax-4 could affect the SMFdependent upregulation of three CYP genes. Moreover, recent studies on cellular effects of SMFs, together with our findings, highlight the role of mitochondria 8,9 , suggesting mitochondria could be an SMF sensor in the cell. Exploring the transcription factors driving the transcriptomic changes upon SMF treatment is another exciting issue to pursue. It is intriguing and essential to study how worms sense SMF alteration and turn on longevity response in the future.

Methods
Worm culture and RNAi interference. Caenorhabditis elegans were grown with standard techniques on NGM agar plates seeded OP50 at 20 °C, unless otherwise noted 38 . The Worm strains used in this study are listed in Supplementary Table 4. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). For synchronization, eggs laid in the desired time window (4 h to O/N) were collected unless otherwise noted.
RNAi experiments were performed as described 39 .  Heat resistance assay. To assess worms heat resistance, thermorecovery was measured as reported 41 . In brief, worms were heat shocked at 35 °C for 5.5 h and then grown at 20 °C for 24 h to recover. Worms able to crawl away without abnormal, jerky movement or paralysis upon prodding were classified as 'recovered' . Fewer than 30 worms were on each plate during the assay.

Permanent magnetic plates. N35
Seahorse assay. As reported, oxygen consumption was measured using a Seahorse XFe24 Analyzer (Agilent Technologies) 42  RNA-Seq and data analysis. RNA libraries were prepared by BGI and sequenced on a BGISEQ-500 sequencer. Raw sequencing reads were cleaned by removing adaptor sequences, reads with poly-N sequences, and low-quality reads. Approximately 21.6 million clean reads were mapped to the C. elegans reference genome (WBcel235) using HISAT/Bowtie2 tools. Normalisation was performed after data were mapped, and then FPKM (fragments per kilobase per million mapped reads) was calculated using RESM software. Genes with an FPKM bigger than 0.15 were considered as detected. The genes up-or down-regulated more than two-fold upon SMF treatment were considered differentially expressed. Gene set enrichment was analysed using WormCat by default settings 24 . Significance scores were reported as Fisher's exact test P values. Terms were considered significant if the WormCat reported P-value score was smaller than 0.05. Western blotting. Synchronized worms were grown to day 1 of adulthood and harvested in 4 × SDS gelloading buffer (Takara, Cat# 9173). After three rounds of freeze and thaw, worms were further lysed by incubation at 95 °C for 5 min. Proteins were separated by reducing SDS-PAGE and transferred to PVDF membranes. Membranes were blotted with antibodies against p-AMPK (CST, Cat# 4188 s, dilution: 1:1000) and α-tubulin (Sigma-Aldrich, Cat# T5168, dilution: 1:2000). Anti-mouse secondary antibody conjugated with horseradish peroxidase (Life Technologies, Cat# G21040, 1:5000) was used to detect anti-α-tubulin, and anti-rabbit secondary antibody (Life Technologies, Cat# G21234, 1:5000) was used for detecting anti-p-AMPK primary antibodies. Signals of western blotting were captured by a Tanon™ 5200 Chemiluminescent Imaging System and measured using Adobe Photoshop. Background signals were subtracted as reported 43 . Statistical analysis. Results are presented as Mean ± SD unless otherwise noted. Statistical tests were performed as indicated using GraphPad Prism (GraphPad software). Detailed statistics information was shown in Supplementary Table 1.