[Autophagy 3:2, 93-95; March/April 2007]; ©2007 Landes Bioscience
Brief Report
Autophagy Regulates Ageing in C. elegans Eszter S. Hars1 Haiyan Qi1 Alexey G. Ryazanov1 Shengkan Jin1 Li Cai2 Chengcheng Hu3 Leroy F. Liu1,*
Abstract
1Department
Autophagy, a process for protein and organelle (e.g., mitochondria) degradation, has been suggested to be involved in the regulation of ageing.1‑6 The only genetic evidence linking autophagy to ageing comes from Melendez et al.5 who showed that in the nematode C. elegans, knocking down the expression of bec‑1 (T19E7.3; ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens the lifespan of daf‑2(e1370) (insulin/IGF‑1 receptor) mutant worms.5 This suggests that autophagy may play a role in the regulation of ageing through the conserved insulin/ IGF‑1 signaling pathway. However, Beclin1/ATG6 is also involved in other cellular functions in addition to autophagy.7‑12 In yeast, the bec‑1 ortholog ATG6 (VPS30) is required for endosome‑ to‑Golgi retrograde transport.9 In mammals, beclin 1 homozygous knockout mice have different phenotypes from other autophagy gene knockout mice. Whereas homozygous deletion of beclin 1 leads to embryonic lethality,10 atg5 homozygous knockout mice are born alive.11 In C. elegans, bec‑1 is essential during embryonic development and bec‑1 knock‑down causes morphological changes in the first generation offspring (F1 generation).12 Thus, the involvement of bec‑1 in other cellular functions could complicate the interpretation of the effect of autophagy on lifespan regulation in C. elegans. To provide stronger support for the role of autophagy in lifespan control, here we examined the effect of knocking down two additional C. elegans autophagy genes, atg‑7 and atg‑12 (orthologs of yeast ATG7 and ATG12, respectively) on the lifespan of both N2 (wild‑type) and daf‑2(e1370) mutant nematodes, both in the F1 and the parental (P) generation. ATG7 is an ubiquitin E1‑like protein required to conjugate ATG12 (an ubiquitin‑like protein) to ATG5. This process is essential for autophagy. When they reached the young adult stage, worms were fed with E. coli expressing double‑stranded RNA (dsRNA). Day 1 of adult lifespan was defined as the first day of the young adult stage. The effect of RNAi treatment on the lifespan of the P generation was minimal. Inhibition of autophagy by RNAi had no effect on lifespan in the N2 (wild‑type) strain and shortened the lifespan of the daf‑2(e1370) mutant strain only slightly (data not shown). We also measured lifespan of the offspring (F1) since a longer exposure to dsRNA is generally expected to cause a stronger phenotype. Accordingly, when they reached the young adult stage, worms were fed with E. coli expressing dsRNA as above. Eggs were collected and transferred to fresh plates (seeded with E. coli expressing dsRNA). The hatched worms (F1) were grown on E. coli expressing dsRNA and their lifespan monitored. The results obtained with the F1 generation are shown in Figure 1 (and Tables 1 and 2 for statistical analysis). The daf‑2(e1370) mutant strain exhibited an increase in lifespan compared to N2 (wild type) worm (p = 1.2 x 10‑6, log rank test) (Table 1). However, the extent of lifespan extension of the daf‑2(e1370) strain was smaller than reported,5 possibly due to our experimental conditions.
School of Public Health; Department of Biostatistics; Boston, Massachusettes USA *Correspondence to: Leroy F. Liu; Department of Pharmacology; University of Medicine and Dentistry of New Jersey—Robert Wood Johnson Medical School; 675 Hoes Lane; Piscataway, New Jersey 08854 USA; Tel.: 732.235.4592; Fax: 732.235.4073; Email:
[email protected] Original manuscript submitted: 09/25/06 Manuscript accepted: 11/30/06
IEN
Previously published online as an Autophagy E-Publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=3636
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3Harvard
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of Biomedical Engineering; Rutgers University; Piscataway, New
Jersey USA
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2Department
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of Pharmacology; University of Medicine and Dentistry of New Jersey—Robert Wood Johnson Medical School; Piscataway, New Jersey USA
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RIB
UT E
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The role of autophagy in ageing regulation has been suggested based on studies in C. elegans, in which knockdown of the expression of bec‑1 (ortholog of the yeast and mammalian autophagy genes ATG6/VPS30 and beclin 1, respectively) shortens lifespan of the daf‑2(e1370) mutant C. elegans. However, Beclin1/ATG6 is also known to be involved in other cellular functions in addition to autophagy. In the current study, we knocked down two other autophagy genes, atg‑7 and atg‑12, in C. elegans using RNAi. We showed that RNAi shortened the lifespan of both wild type and daf‑2 mutant C. elegans, providing strong support for a role of autophagy in ageing regulation.
SC
Key words
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ageing, autophagy, lifespan, atg-7, atg-12, bec-1, daf-2
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Acknowledgements
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We thank Dr. John Lenard (University of Medicine and Dentistry of New Jersey, Piscataway, NJ) for providing E. coli containing the empty vector (pPD129.36, L4440) (used as ctr.) as well as E. coli expressing bec-1, atg-7 and atg-12 dsRNA, and Dr. Monica Driscoll (Rutgers University, Piscataway, NJ) for providing E. coli expressing daf-16 dsRNA. This work was supported by NIH grants CA102463 (L.F.L.), CA39662 (L.F.L.), and AG19890 (A.G.R.), the Department of Defense grant BC045756 (H.Q.), and the ACS grant RSG-05-2065-01 (S.J.).
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Autophagy Regulates Ageing in C. elegans
In both the daf‑2(e1370) mutant and N2 (wild‑type) strains (F1 generation), knocking down either atg‑7 or atg‑12 caused statistically significant lifespan shortening compared to the control (ctr) (Fig. 1A and B, and see Table 1 for p values). In the daf‑2(e1370) strain, atg‑7 and atg‑12 RNAi nematodes had a mean lifespan of about 13 and 11 days, respectively, versus 18 days for the ctr worms. In the N2 strain, atg‑7 and atg‑12 RNAi nematodes had a mean lifespan of about 10 and 9 days, respectively, versus 14 days for the ctr strain. The effect of atg‑7 RNAi on the lifespan of both wild type and mutant worms appeared similar (see Fig. 1 and Table 1). However, the effect of atg‑12 on the lifespan of the N2 (wild type) strain appeared smaller than that on the lifespan of the daf‑2(e1370) mutant strain (Fig. 1 and Table 1). Using the Cox proportional hazard model (Table 2), we showed that the effect of atg‑12 RNAi treatment on the lifespan of wild type worms was smaller than that of daf‑2 worms (p = 0.129). For the last 50% and 20% surviving worms, this difference persisted and became statistically significant at the 0.05 level (p = 0.017 and 0.006, respectively, see Table 2), suggesting that the effect of atg‑12 RNAi on lifespan shortening is at least in part mediated through the insulin/IGF‑1 signaling pathway. Under the light microscope, atg‑7 and atg‑12 RNAi nematodes appeared normal, whereas bec‑1 Figure 1. The autophagy genes atg‑7 and atg‑12 are involved in lifespan control in N2 RNAi nematodes were slightly smaller and appeared (wild‑type) and daf‑2(e1370) mutant C. elegans. (A and B) show the lifespan of the F1 uneven. Nomarski optics confirmed that atg‑7 and generation of daf‑2(e1370) and N2 (wild type) worms, respectively. ctr. refers to empty vector, atg‑12 RNAi nematodes appeared normal, whereas used as a negative control for RNAi. daf‑16 RNAi, known to shorten the lifespan of daf‑2 bec‑1 nematodes showed an accumulation of large knock‑down worms,20 was used as a positive control. (C) shows the morphology at day 6 of vacuoles in the intestine (Fig. 1C) and enlarged germ adulthood of the N2 worms (at 24˚C), as observed by Nomarski optics. The gene targeted cells (not shown). This is consistent with phenotypic by RNAi is specified under each micrograph. Black arrows point to large vacuoles in the changes previously described.12 Importantly, we only intestine. observed these gross morphological changes in bec‑1 RNAi nematodes, but not in atg‑7 or atg‑12 RNAi nematodes. in various organisms.15‑18 The stronger lifespan shortening effect of Our current results provide strong support for a role of autophagy atg‑12 RNAi in daf‑2 mutants compared to wild type presented here in the extension of lifespan of C. elegans. The conserved insulin/ suggests that autophagy may contribute to lifespan regulation at least IGF‑1 signaling pathway, which negatively regulates autophagy in part through the insulin/IGF‑1 pathway. through TOR,13,14 is known to play a key role in lifespan regulation
Table 1
Statistical analysis of the survival data shown in Figure 1
Comparison
p‑value
mt_ctr vs mt_atg‑7
5.4 x 10‑9
mt_ctr vs mt_atg‑12
wt_ctr vs wt_atg‑7
2.1 x
4.7 x 10‑9
wt_ctr vs wt_atg‑12
1.1 x 10‑7
mt_ctr vs wt_ctr
1.2 x 10‑6
mt_atg‑7 vs wt_atg‑7
6.6 x 10‑10
mt_atg‑12 vs wt_atg_12
0.0011
p‑Value
Full data set wt vs. daf‑2
‑0.3441
0.2264
1.5197
0.1286
‑0.7228
0.3026
‑2.3886
0.0169
‑1.3544
0.4937
‑2.7431
0.0061
Last 50% surviving worms wt vs. daf‑2 Last 20% surviving worms
The survival data shown in Figure 1 were analyzed by the log‑rank (Cox‑Mantel) test.21 The p‑values for different treatment comparisons were calculated and shown above. mt, daf‑2 mutant; wt, wild type; ctr, control RNAi; atg‑7, atg‑7 RNAi; atg‑12, atg‑12 RNAi. For each treatment, 96 worms were used in the beginning of the experiment.
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The effect of atg‑12 RNAi on lifespan shortening of (N2) wild type and daf‑2 mutant worms; analysis with the Cox proportional hazard model22
Effect Error z‑Value Difference
10‑13
Table 2
wt vs. daf‑2
The survival data shown in Figure 1 were analyzed. The negative value in the Effect Difference indicates that the effect of atg‑12 RNAi on the lifespan of wild type worms was smaller than that of daf‑2 worms.
Autophagy
2007; Vol. 3 Issue 2
Autophagy Regulates Ageing in C. elegans
Methods The dsRNA clones were obtained from the library developed by Kamath et al.19 Based on the sequencing results, the genomic sequences in the RNAi constructs are as follows: atg‑7 (M7.5) 1238‑1669 bp; atg‑12 (B0336.8) 180 ‑ 357 bp; bec‑1 (T19E7.3) 82‑500 bp; and daf‑16 (R13H8.1) 1281‑ 1567 bp. For the knock‑down of bec‑1, atg‑7, and atg‑12 by RNA interference (RNAi) through feeding, we followed the protocol published by Kamath et al.19 (see also www.gurdon.cam.ac.uk/~ahringerlab/ data/RNAi_Feeding_Protocol.doc). Nematodes were cultured on agar plates seeded with E. coli (OP50) as the food source until they reached young adult stage, then transferred to plates seeded with E. coli expressing dsRNA. Lifespan was measured (P generation) and eggs were transferred to fresh dsRNA plates for experiments with the F1 generation. Worms were grown and lifespan was again measured (F1). Day 1 of adult lifespan was defined as the first day of the young adult stage. During reproductive growth, worms were transferred to fresh plates every two days. All experiments were performed at 15˚C, as in the Melendez study.5
20. Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, Kenyon C. Genes that act downstream of DAF‑16 to influence the lifespan of Caenorhabditis elegans. Nature 2003; 424:277‑83. 21. Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 1959; 22:719‑48. 22. Cox DR. Regression models and life tables. J Royal Statistical Society 1972; (Series B)34:187‑220.
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