Figure 1.
Knock-down of PHA-4, DAF-16, SKN-1, ATG-12, or SPHK-1 affect the extended longevity of hyl-1;lagr-1.
Cumulative survival curves of N2 and hyl-1;lagr-1 worms grown at 20°C subjected to either empty vector control bacteria (L4440) or the indicated RNAi from the early adult stage. (A) When subjected to atg-12 RNAi, the extended lifespan of hyl-1;lagr-1 is normalized to the extent of atg-12(RNAi) control animals, P = 0.3053. (B) When subjected to pha-4 RNAi, the extended lifespan of hyl-1;lagr-1 is normalized to the extent of pha-4(RNAi) control animals, P = 0.2369. (C) When subjected to daf-16 RNAi, the extended lifespan of hyl-1;lagr-1 is decreased beyond the extent of daf-16(RNAi) control animals, P = 0.0002. (D) When subjected to skn-1 RNAi, the extended lifespan of hyl-1;lagr-1 is normalized to the extent of skn-1(RNAi) control animals, P = 0.5476. (E) When subjected to daf-2 RNAi, hyl-1;lagr-1 lifespan is further extended compared to both hyl-1;lagr-1 control animals, P<0.0001, and daf-2(RNAi) control animals, P<0.0001. (F) When subjected to eat-2 RNAi, hyl-1;lagr-1 lifespan is decreased compared to hyl-1;lagr-1 control animals, P = 0.0002, while the lifespan of eat-2(RNAi) animals is extended compared to wild-type control animals, P<0.0001. (G) When subjected to aak-2 RNAi, hyl-1;lagr-1 lifespan is decreased compared to hyl-1;lagr-1 animals, P = 0.0009, while no lifespan effect is seen when comparing aak-2(RNAi) animals to wild-type control animals, P = 0.0975. (H) When subjected to sphk-1 RNAi, the extended lifespan of hyl-1;lagr-1 is normalized to the extent of sphk-1(RNAi) control animals, P = 0.8002. For additional details about these experiments, see Table 1.
Table 1.
Adult lifespan of hyl-1;lagr-1 and N2 control worms subjected to empty vector control or the indicated RNAi at 20°C.
Figure 2.
Autophagy is increased in hyl-1;lagr-1 and the response mechanism differs from that of wild type.
LGG-1 is part of autophagosomal membranes and widely used as an indicator of autophagy in C. elegans. Bars represent the mean number of LGG-1::GFP-containing puncta per seem cell in non-starved wild type and hyl-1;lagr-1 worms grown at 20°C subjected to either empty vector control bacteria (L4440) or the indicated RNAi. The number in each bar indicates the total number of seam cells observed. (A) Knock-down of atg-12 lowers the level of autophagy in both wild type and hyl-1;lagr-1. (B) Knock-down of pha-4 does not change the increased level of autophagy in hyl-1;lagr-1 but increases autophagy in wild type. (C) Knock-down of daf-16 lowers the increased level of autophagy in hyl-1;lagr-1 but increases autophagy in wild type. (D) Knock-down of skn-1 lowers the increased level of autophagy in hyl-1;lagr-1 but increases autophagy in wild type. (E) Knock-down of daf-2 increases autophagy to the same extent in wild type and hyl-1;lagr-1. (F) Knock-down of sphk-1 increases the level of autophagy in hyl-1;lagr-1 beyond wild type level. Statistical analyses were performed by unpaired two-tailed t-test (with Welch’s correction if variances were significantly different) using GraphPad Prism version 6.0 (GraphPad Software). The Bonferroni method was used to correct for multiple comparisons and P values below 0.0125 were considered statistically significant equivalent to a significance level of 0.05. (*) P≤0.0125, (**) P≤0.001, and (***) P≤0.0001. N used for analysis is the total number of worms observed for each treatment (23–45 worms, two trials). Mean ± SEM is shown.
Figure 3.
Phenotypes associated with lifespan extension and stress resistance are observed in hyl-1;lagr-1.
(A) Pumping rates of N2, hyl-1;lagr-1, and an eat-2 mutant. The latter displays a pronounced reduction in pumping rate and is commonly used as a genetic model for dietary restricted animals. Bars represent the mean number of pumps per minute. Compared to N2 displaying a mean pumping rate of 201±2 pumps/min, hyl-1;lagr-1 shows a 17.4% decrease with a mean pumping rate of 166±2, P<0.0001, while eat-2 shows a 67.2% decrease with a mean pumping rate of 66±3, P<0.0001. The data represents an mean ± SEM of 15 measurements in 5 worms of each genotype. (B) Quantification of fluorescent beads in the pharynx and the anterior part of the intestine following a feeding period of 30 minutes. Compared to N2, hyl-1;lagr-1 displays 59% less fluorescence, P = 0.0026. Mean ± SEM is shown, n indicates the number of worms. (C) Mean total brood size of N2 and hyl-1;lagr-1. Compared to N2 which displays a mean brood size of 304±9, hyl-1;lagr-1 shows a 33% decrease with a mean brood size of 204±6, P<0.0001. Mean ± SEM is shown, n = number of worms examined. (D) Survival curves of N2 and hyl-1;lagr-1 subjected to heat-shock at 37°C. Compared to N2, hyl-1;lagr-1 shows increased resistance, P = 0.0016. A total of 60 worms of each strain were assayed. Mean ± SD of 3 experiments is shown. N2-worms (6) and hyl-1;lagr-1 worms (22) were censored but are incorporated in the analysis until the time they were censored. (E) Bars represent mean median heat shock survival from the 3 experiments shown in D. Compared to N2 which has a median survival of 7 hours, hyl-1;lagr-1 displays a 29% increase in heat shock resistance with a median survival of 9 hours. Error bars represent ± SD.
Figure 4.
Lipidomic analysis reveals a modified sphingolipid composition in hyl-1;lagr-1.
Relative abundance of detected sphingolipid species showing significant changes in hyl-1;lagr-1. Sphingolipids containing C24-26 fatty acids and sphingomyelin species containing C16-18 fatty acids are lowered in hyl-1;lagr-1, while sphingolipids containing C21-22 fatty acids acids are more abundant. C. elegans sphingolipids predominately contain C17 long-chain bases, thus making the fatty acid chain length readily deducible. The number of carbon atoms indicated is without head groups. (A) Sphingomyelins containing C16-18 fatty acids are significantly reduced in hyl-1;lagr-1. (B) All significantly changed sphingolipid species containing C21 fatty acids are more abundant in hyl-1;lagr-1. (C) All significantly changed sphingolipid species containing C22 fatty acids are more abundant in hyl-1;lagr-1. (D) The only significantly changed sphingolipid species containing C23 fatty acids is more abundant in hyl-1;lagr-1. (E) All significantly changed sphingolipid species containing C24 fatty acids are less abundant in hyl-1;lagr-1. (F) All significantly changed sphingolipid species containing C25 fatty acids are less abundant in hyl-1;lagr-1. (G) All significantly changed sphingolipid species containing C26 fatty acids are less abundant in hyl-1;lagr-1. Statistical analyses were performed by one way analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism version 6.0 (GraphPad Software). Results from two biological experiments are shown for each strain. Mean ± SD is shown. All species shown are at least significant different at the P<0.05 level. The entirety of detected sphingolipid species can be seen in Figure S7 and Figure S8.