The genetic paradigms of dietary restriction fail to extend life span in cep-1(gk138) mutant of C. elegans p53 due to possible background mutations

Abstract

Dietary restriction (DR) increases life span and improves health in most model systems tested, including non-human primates. In C. elegans, as in other models, DR leads to reprogramming of metabolism, improvements in mitochondrial health, large changes in expression of cytoprotective genes and better proteostasis. Understandably, multiple global transcriptional regulators like transcription factors FOXO/DAF-16, FOXA/PHA-4, HSF1/HSF-1 and NRF2/SKN-1 are important for DR longevity. Considering the wide-ranging effects of p53 on organismal biology, we asked whether the C. elegans ortholog, CEP-1 is required for DR-mediated longevity assurance. We employed the widely-used TJ1 strain of cep-1(gk138). We show that cep-1(gk138) suppresses the life span extension of two genetic paradigms of DR, but two non-genetic modes of DR remain unaffected in this strain. We find that two aspects of DR, increased autophagy and up-regulation of the expression of cytoprotective xenobiotic detoxification program (cXDP) genes, are dampened in cep-1(gk138). Importantly, we find that background mutation(s) in the strain may be the actual cause for the phenotypic differences that we observed and cep-1 may not be directly involved in genetic DR-mediated longevity assurance in worms. Identifying these mutation(s) may reveal a novel regulator of longevity required specifically by genetic modes of DR.

Fig 1. In cep-1(gk138), genetic paradigms of Dietary Restriction fail to extend life span.

(A) The life span extension upon drl-1 KD in WT is suppressed when cep-1(gk138) is used. (B) The extended life span of eat-2(ad1116) is supressed in eat-2(ad1116);cep-1(gk138). (C) The extended life span of eat-2(ad465) is supressed in eat-2(ad465);cep-1(gk138). (D) The life span of daf-2(e1370) is partially suppressed when combined with cep-1(gk138), as in daf-2(e1370);cep-1(gk138). Life spans were performed at 20 °C. Details of life span are provided in S1 Table. Mantel-Cox log rank test using OASIS software available at http://sbi.postech.ac.kr/oasis [22] was used for statistical analysis. Life spans in 1B and 2B have identical controls as they were part of the same experiment (also see S1 Fig).

Fig 2. The life span extension on non-genetic modes of DR remain unaffected in cep-1(gk138).

(A) A bell-shaped curve is observed when WT or cep-1(gk138) worms are grown on different dilutions of bacterial feed and the average life span plotted against the OD₆₀₀. Data are presented as mean values ± SEM. N = 3 independent experiments. Two-way Annova was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001. (B) Supplementation of 2-DOG extends life span in cep-1(gk138) mutant similar to WT. Life span were performed at 20°C and details are provided in S1 Table. Mantel-Cox log rank test using OASIS software available at http://sbi.postech.ac.kr/oasis [22] was used for statistical analysis.

Fig 3. Cep-1(gk138) regulates autophagy during DR.

(A) Increased autophagosome formation in lgg-1::gfp grown on drl-1 RNAi is suppressed in cep-1(gk138); lgg-1::gfp. Quantification of GFP puncta for one of three independent experiments is shown. Data are presented as mean values ± SEM. No. of animals analysed, n≥16. Two-way Annova with Sidak’s multiple comparison tests was used for statistical analysis. ****P≤0.0001, ns = non-significant. (B) The increased autophagosome formation in eat-2(ad1116) is suppressed in eat-2(ad1116);cep-1(gk138). Quantification of GFP puncta for one of three independent experiments is shown. Data are presented as mean values ± SEM. No. of animals analysed, n≥17. Unpaired two-tailed t-test with Welch’s correction was used for statistical analysis. ****P≤0.0001. Source data is provided in S1 File. (C) Transcript levels of autophagy genes, lgg-1, bec- 1, unc-51, vps-34 in WT and cep-1(gk138) on control and drl-1 RNAi. Data are presented as mean values ± SEM. N≥3 independent experiments. Two-way Annova with Sidak’s multiple comparisons test was used for statistical analysis. **P≤0.01, ****P≤0.0001. (D) Western blot using anti-GFP antibody to detect the unmodified LGG-1 or PE-LGG-1. The lgg-1::gfp or cep-1(gk138);lgg-1::gfp worms were grown on control or drl-1 RNAi. Densitometric quantification of protein bands were performed using ImageJ and ratio of PE-LGG-1/LGG-1 is shown. β-ACTIN was used as a loading control. One representative blot out of the two independent experiments is shown. Experiments were performed at 20 °C. Uncropped blots are provided in S1 File (also see S2 Fig).

Fig 4. Fat storage is differentially regulated by cep-1(gk138).

(A) Oil Red O staining of WT or cep-1(gk138) grown on control or drl-1 RNAi. Lowering of fat storage takes place in both the strains grown on drl-1 RNAi. Representative images (upper panel) and quantification (lower panel) for one of three independent experiments is shown. Images were captured at 400X magnification. Scale bar is 20 μm. Data are presented as mean values ± SEM. No. of animals analysed, n≥16. Two-way Annova with Sidak’s multiple comparisons test was used for statistical analysis. ****P≤0.0001. (B) Oil Red O staining of eat-2(ad1116) or eat-2(ad1116);cep-1(gk138). Lowering of fat storage in eat-2(ad1116) is attenuated in eat-2(ad1116);cep-1(gk138). Representative images (upper panel) and quantification (lower panel) for one of three independent experiments is shown. Images were captured at 400X magnification. Scale bar is 20 μm. Data are presented as mean values ± SEM. No. of animals analysed, n≥20. Unpaired two-tailed t-test with Welch’s correction was used for statistical analysis. ****P≤0.0001, *P≤0.05. Experiments were performed at 20 °C. Representative images presented in Fig 4B and S3 Fig are from the same experiment and thus, have the same control panels. Source data is provided in S1 File (also see S3 Fig).

Fig 5. The increased expression of cXDP genes in cep-1(gk138), undergoing genetic DR, is partially attenuated.

(A) The mRNA levels of cXDP genes are increased in WT grown on drl-1 RNAi, but partially suppressed in cep-1(gk138). Data are presented as mean values ± SEM. N≥3 independent experiments. Unpaired two-tailed t-test was used for statistical analysis. *P≤0.05, **P≤0.01, ns = not significant. (B) The increased levels of cXDP genes in eat-2(ad465) is reduced in eat-2(ad465);cep-1(gk138). Data are presented as mean values ± SEM. N≥3 independent experiments. Unpaired two-tailed t-test was used for statistical anaysis. *P≤0.05, ns = not significant. (C) Representative images of one of two independent experiments, showing increase in pcyp-35B1:gfp expression on drl-1 KD in WT, which abrogated in cep-1(gk138). n≥20. Multiple overlapping images were captured at 100X to cover the length of whole worm and then stitched together to generate a contiguous image. Arrow heads point towards the head of the worms. Experiments were performed at 20 °C.

Fig 6. Background mutation(s) in cep-1(gk138) may be responsible for suppressing life span of genetic paradigms of DR.

(A) Life span analysis of TJ1 cep-1(gk138) 11X backcrossed strain, (B) 12X backcrossed strain, (C) TJ1 strain reordered from CGC, (D) VC172 cep-1(gk138) 0X backcrossed strain, (E) cep-1(lg12501) and (F) cep-1(ep347) along with WT, grown on control or drl-1 RNAi. Life span was performed at 20°C and details are provided in S1 Table. Mantel-Cox log rank test using OASIS software available at http://sbi.postech.ac.kr/oasis [22] was used for statistical analysis. Life spans in Fig 6C-6D and 6E-6F have identical controls as they were part of the same experiment (also see S4 Fig).