During the International C. elegans Experiment First (ICE 1st) project7, we investigated the effect of spaceflight on the formation of aggregates of a 35-glutamine repeat (Q35) in C. elegans transgenically expressing the (CAG) 35 -yellow fluorescent protein (YFP) gene in muscle, which normally increases with advancing age8 (Fig. 1). Q35 aggregate formation expressed as the number of aggregates per worm was found to be lower in worms flown in space from the L1 larva stage and L4/young adult stage than in matched ground control worms (Fig. 2A, B). This difference may be because of the possible changes in growth rate induced by spaceflight. However, growth of worms has been reported to be unaffected by spaceflight7,9. Moreover, the number of aggregates per body length was lower in space-flown worms than in ground control worms (Fig. 2D, E). This indicated that the spaceflight-induced suppression of Q35 aggregates was not ascribed to the spaceflight-induced changes in growth rate. Numbers of Q35 aggregates per total YFP fluorescence intensity in each worm, an indicator of Q35 expression, was also lowered by spaceflight (Fig. 2F, G). This showed that the spaceflight-induced suppression of Q35 aggregates was not due to spaceflight-induced changes in Q35 expression. These results suggest that biomarkers of aging are expressed more slowly in space-flown C. elegans in than ground control worms.

Figure 1 Age-dependent increases in Q35 aggregate formation on ground: simulation of procedures of spaceflight experiment. (A) Eggs obtained by treatment of gravid-adult Q35–YFP transgenic worms with hypochlorite were allowed to hatch and maintained at L1 larva stage in CeMM for 7 days at 12°C. Then, temperature was shifted to 20°C. Worms grew to adult stage and started to lay eggs at 7 days after the temperature shift, on the other hand, the progenies produced by these worms did not grow to adult stage by 11 days after the temperature shift. Therefore, at 9 days after the temperature shift, adults seen in culture could be considered as the worms originally inoculated. (B) L4/young adult worms were incubated in CeMM with 40 μM FUdR for 7 days at 12°C and temperature was shifted to 20°C. Average numbers of Q35 aggregates/worm of more than 40 worms were plotted against time after the temperature shift to 20°C with ± S.E. Absent error bars indicate that errors fell within symbols. These figures show one of two trials, each of which gave similar results. Full size image

Figure 2 Spaceflight reduced Q35 aggregate formation. Q35-YFP transgenic worms were inoculated into CeMM with 40 μM FUdR at L4/young adult stage (A, D, F) or without FUdR at egg stage (B, E, G) and incubated on ground for 5 days at 12°C. Then they were space-flown or on ground for 2 days at 12°C and for next 9 days at 20°C. In B, E and G, only adult worms in the samples containing L1, L2 and adult stage worms were measured as adult worms were considered as the worms originally inoculated (See Legend for Fig. 1). Numbers of Q35 aggregates were counted from the photographs of worms obtained by a fluorescence microscope (as in (C)) using ImageJ software (Analyze Particles). (A, B) Numbers of Q35 aggregates per worm are shown. * vs ** and # vs ##: p<0.001 (Student's t test). (C) Photographs of YFP fluorescence of the worms flown in space from L1 stage and matched ground control are shown. Bars show 100 μm. (D, E) Numbers of Q35 aggregate formation per body length are shown. The lengths of individual worms were measured using the ImageJ software. * vs ** and # vs ##: p<0.001 (Student's t test). (F, G) Ratio of Q35 aggregate formation of space-flown worms to those of ground control worms, per total body fluorescence intensity are shown. Whole-worm YFP fluorescence was quantified using the measurement and analysis software VH-HIA5 (Keyence). * vs **: p<0.05 and # vs ##: p<0.001 (Student's t test). Numbers of worms examined were as follows, A, D and F: space-flown (n = 15) and ground control worms (n = 47); B, E and G: space-flown (n = 138) and ground control worms (n = 367). Data are expressed as the mean ± S.E. Full size image

Further, these findings led us to propose a working hypothesis that the space environment changes the expression of genes involved in the control of aging. To identify the possible longevity-control genes, we first used the data from a DNA microarray experiment conducted to examine changes in gene expression in response to spaceflight10 (The data set was deposited in the Gene Expression Omnibus (GEO) database (accession number: GSE36358) and WormBase (www.wormbase.org.)), which showed that the expression of 48 genes increased by more than two-fold and that of 199 genes decreased to less than half in the spaceflight conditions relative to the ground control11. Among these genes, we noticed that eleven genes likely related to neuronal or endocrine signaling were down-regulated in space-flown worms. We confirmed these observations and quantitatively evaluated the extent of decreased mRNA expression using real-time RT-PCR (Fig. 3). Second, we examined the effects of the inactivation of these eleven genes by loss- or reduction-of-function mutations and/or feeding RNA interference (RNAi) on the lifespan under ground laboratory conditions. We found that the inactivation of each of seven genes among these eleven genes extended lifespan on NGM agar covered with killed or live bacteria as food (Fig. 4A, Supplementary Tables S1 and S2 online). These included gar-3, acetylcholine receptor; unc-17, acetylcholine transporter; cha-1, choline acetyltransferase; F57A8.4, rhodopsin-like G-protein coupled receptor (GPCR); glc-4, glutamate-gated chloride channel; shk-1, shaker family of potassium channel; and ins-35, insulin-like peptide. We also ascertained whether these lifespan extensions could be seen in the liquid culture medium (CeMM) that had been used to culture worms in the spaceflight experiment. The mutations in all of these genes, except unc-17(e245) whose lifespan could not be assessed because it could not grow in CeMM for presently unknown reasons, were also found to extend lifespan (Fig. 5).

Figure 3 The genes likely related to neuronal and endocrine signaling were down-regulated by spaceflight. Relative gene expression levels in the mixed population of space-flown or ground control N2 worms are shown. gar-3: acetylcholine receptor, unc-17: acetylcholine transporter, cha-1: choline acetyltransferase, F57A8.4: rhodopsin-like GPCR, glc-4: glutamate-gated chloride channel, shk-1: shaker family of potassium channel and ins-35: insulin-like peptide, lgc-54: ligand-gated ion channel, glr-1: glutamate receptor, sym-5: synthetic lethal with mec, sra-12: serpentine receptor. Real-time RT-PCR was performed in triplicate for one sample each from the spaceflight experiment and ground control. The mRNA levels of each gene were adjusted to that of gpd-2 (glyceraldehyde-3-phosphate dehydrogenase) mRNA, which was used as the internal standard. Bar graphs are ratios of the mRNA levels in spaceflight to those on ground (%). Data are expressed as the mean ± S.E. (n = 3). All of them were significantly different (p<0.05) compared with ground control (Student's t-test). Full size image

Figure 4 Lifespan of the mutants in the genes down-regulated by spaceflight. (A) The survival curves of N2 and the mutants in the genes that were down-regulated by spaceflight, on NGM with UV-killed OP50 are shown. The percentage of live worms is plotted against adult age. Lifespans of the mutants in the seven genes were longer than those of the wild type. Day 0 corresponds to the L4 molt. Data are one of two experiments, each of which gave similar results, detailed parameters of which are indicated in Supplementary Table S1. The survival curves of N2 and the mutants treated with daf-16 RNAi (B), skn-1 RNAi (C) and eat-2 RNAi (D) from the L1 stage until death. Data are one of two experiments detailed parameters of which are indicated in Supplementary Table S2 with the data of worms treated with mock-vector RNAi bacteria as control experiments. Full size image

Figure 5 The survival curves of N2 and the mutants in the genes that were down-regulated by spaceflight, in liquid CeMM. The percentage of live worms is plotted against adult age. Day 0 corresponds to the L4 molt. Mean adult lifespan ± S.E. (day), number of assayed worms and statistical significance with N2 wild type are: N2: 49.5 ± 0.7, n = 364; gar-3(gk337): 62.3 ± 1.1, n = 205, p<0.001; ins-35(ok3297): 53.5 ± 1.3, n = 159, p<0.01; glc-4(ok212): 59.0 ± 0.9, n = 286, p<0.001; shk-1(ok1581): 55.2 ± 0.8, n = 204, p<0.001; F57A8.4 (tm4341): 68.1 ± 1.2, n = 179, p<0.001 and cha-1(p1152): 70.5 ± 1.1, n = 155, p<0.001. Full size image

To further explore the mechanism of lifespan extension by the inactivation of each of these genes, we investigated whether lifespan extensions are mediated through the DAF-16/FOXO transcription factor, which is a key factor in lifespan extension by reduction of insulin/IGF-1 like signaling (IIS)12, or through SKN-1, an Nrf-like xenobiotic-response factor, which is the other key factor in lifespan extension both by reduction of IIS13 and by dietary-restriction signaling14. Lifespan extensions caused by mutations either in ins-35, glc-4, unc-17, or shk-1 were totally abolished by daf-16 RNAi inactivation, whereas mutations either in gar-3, F57A8.4, or cha-1 still lived longer than wild-type worms under daf-16 RNAi (Fig. 4B, Supplementary Table S2 online). These results suggest that INS-35, GLC-4, UNC-17 and SHK-1 control lifespan thorough IIS/DAF-16 signaling. Further, the skn-1 RNAi completely abolished the lifespan extension induced by mutations in ins-35, glc-4, and shk-1, whereas mutants in unc-17, gar-3, F57A8.4 and cha-1 still lived longer than wild-type worms under skn-1 RNAi (Fig. 4C, Supplementary Table S2 online). These results suggest that INS-35, GLC-4 and SHK-1 control lifespan through SKN-1. To determine whether these lifespan extensions are mediated through dietary-restriction signaling, we examined the lifespan under the inactivation of eat-2, which induces feeding impairment-based dietary restriction15. The eat-2 RNAi further enhanced the extension of lifespan by mutations in ins-35, glc-4, unc-17, or gar-3, whereas the eat-2 RNAi shortened the lifespan of mutants of shk-1, cha-1, or F57A8.4 (Fig. 4D and Supplementary Table S2 online). These results suggest that SHK-1, CHA-1 and the F57A8.4 protein share a common lifespan control mechanism with dietary-restriction signaling.

In order to explore the involvement of these seven genes in the suppression of Q35 aggregate formation during spaceflight, we examined the effect of RNAi inactivation of some of these genes on Q35 aggregation. RNAi inactivation of gar-3, cha-1, and shk-1 reduced Q35 aggregation (Fig. 6), suggesting that GAR-3, CHA-1 and SHK-1 control Q35 aggregate formation.

Figure 6 The effect of RNAi of the genes on Q35 aggregate formation. The feeding RNAi of the designated genes against Q35-YFP transgenic worms was conducted as described in Methods for RNAi. L3 larval worms were placed on RNAi-expressing or mock-vector control bacteria. Worms were incubated for 2 days at 20°C until they became gravid adults. The gravid adults were transferred to fresh RNAi-expressing bacteria lawns and allowed to lay eggs for 2 hours and removed and eggs were allowed to grow for subsequent assays. Aggregates of Q35-YFP were counted at 7 days after the treatment was started. Numbers of Q35 aggregates per worm are shown. Data are expressed as the mean ± S.E. * vs **: p<0.001 (Student's t test). Full size image

We investigated whether these seven genes relate to the formation of dauer larvae, a long-lived growth arrest state under harsh environmental conditions. Mutation and RNAi of ins-35 and mutation of shk-1 were found to enhance pheromone-induced dauer formation, whereas mutations in glc-4, unc-17, or F57A8.4 suppressed it (Fig. 7A, B). These results suggest that INS-35 and SHK-1 may be related to dauer-associated life maintenance. Alternatively, some of these five genes may be involved in sensory perception or signaling related to dauer induction.