Life-span extension by dietary restriction is mediated by NLP-7 signaling and coelomocyte endocytosis in C. elegans

Abstract

Recent studies have shown that the rate of aging can be modulated by diverse interventions. Dietary restriction is the most widely used intervention to promote longevity; however, the mechanisms underlying the effect of dietary restriction remain elusive. In a previous study, we identified two novel genes, nlp-7 and cup-4, required for normal longevity in Caenorhabditis elegans. nlp-7 is one of a set of neuropeptide-like protein genes; cup-4 encodes an ion-channel involved in endocytosis by coelomocytes. Here, we assess whether nlp-7 and cup-4 mediate longevity increases by dietary restriction. RNAi of nlp-7 or cup-4 significantly reduces the life span of the eat-2 mutant, a genetic model of dietary restriction, but has no effect on the life span of long-lived mutants resulting from reduced insulin/IGF-1 signaling or dysfunction of the mitochondrial electron transport chain. The life-span extension observed in wild-type N2 worms by dietary restriction using bacterial dilution is prevented significantly in nlp-7 and cup-4 mutants. RNAi knockdown of genes encoding candidate receptors of NLP-7 and genes involved in endocytosis by coelomocytes also specifically shorten the life span of the eat-2 mutant. We conclude that two novel pathways, NLP-7 signaling and endocytosis by coelomocytes, are required for life extension under dietary restriction in C. elegans.

Figure 1.

Effect of RNAi of skn-1, nlp-7, or cup-4 on life span of long-lived mutants. A) Longevity of eat-2(ad465) mutant was significantly (P<0.01) suppressed by skn-1, nlp-7, or cup-4 knockdown. Mean life span of N2 and eat-2(ad465) was 19.3 and 24.1 d, respectively. Both mean and maximum life span of the eat-2(ad465) mutant were significantly shortened by RNAi of skn-1 (mean life span 14.8 d), nlp-7 (mean life span 20.9 d), or cup-4 (mean life span 20.2 d) (Supplemental Table S2). B, C) Life-span analysis of age-1(hx546) (B) and clk-1(e2519) (C) mutants fed with dsRNA of skn-1, nlp-7, or cup-4. Data are means of duplicates.

Figure 2.

Effect of DR on life span using bacterial dilution. A, B) Serial dilution of OP50 showed significant increases in both mean (A) and maximum (B) life span of wild-type N2 worms. However, maximum life span of nlp-7 and cup-4 mutants was not affected by DR, and mean life span of these mutants was only moderately increased in worms with 5 × 10⁸ bacteria/ml. Data are means ± se of 3 independent experiments. C) Life-span curves of N2, nlp-7(tm2984), nlp-7(tm2990), and cup-4(ok837) mutants were compared between AL (1×10¹⁰ bacteria/ml) and DR (5×10⁸ bacteria/ml).

Figure 3.

Knockout of nlp-7 or cup-4 reduces life span and resistance to oxidative stress. A, B) Life span of nlp-7(tm2984) (A) and nlp-7(tm2990) (B) (P<0.01) decreased significantly compared with wild-type N2 worms. RNAi of cup-4 on nlp-7 mutants showed no effect on life span of either nlp-7 mutant. However, a further decrease in life span of nlp-7(tm2990) (B) by RNAi of skn-1 (Supplemental Table S5) was evident. C) Mean life span of cup-4(ok837) (17.5 d) was shorter than that of N2 (22.1 d). skn-1 RNAi (mean life span 16.3 d) further decreased the life span of cup-4(ok837), while nlp-7 RNAi had no effect on the life span of cup-4(ok837) (Supplemental Table S5). D) Paraquat-resistance assay of nlp-7(tm2984), nlp-7(tm2990), and cup-4(ok837). Resistance to oxidative stress caused by paraquat was reduced in nlp-7 or cup-4 mutants. Data are means of duplicates.

Figure 4.

Fertility of nlp-7 and cup-4 transgenic worms. A) Total number of progeny produced by each strain. Fertility of zEx4101 carrying nlp-7 under hsp-16.2 promoter was similar to that of wild-type N2. However, zEx4104 carrying nlp-7 with its native promoter and zEx4105 carrying cup-4 with hsp-16.2 promoter produced less progeny compared with wild-type N2. B) Time-course distribution of number of progeny produced. Compared to wild-type N2, the curve was shifted to left in all transgenic strains tested, which suggests delayed production of progeny in nlp-7 and cup-4 transgenic worms. Data are means ± se.

Figure 5.

Quantitative RT-PCR of pha-4, nlp-7, and cup-4. Relative expression of each gene under DR was calculated compared to the expression in AL (percentage of AL). ama-1 was used a control for data normalization. Values are means ± se; n = 5–6. *P < 0.05.

Figure 6.

Possible mechanisms of DR-induced longevity in C. elegans. DR activates SKN-1 in two ASI neurons, which signals to coelomocytes to increase endocytosis required for proteins/organelle recycling under DR. Then, the neuronal signaling involving NLP-7, a worm ortholog of mammalian satiety factor, and the expression of PHA-4, a transcription factor mediating DR-induced longevity in C. elegans, are induced to extend life span in diet-restricted worms.