Small nucleoli are a cellular hallmark of longevity

Abstract

Animal lifespan is regulated by conserved metabolic signalling pathways and specific transcription factors, but whether these pathways affect common downstream mechanisms remains largely elusive. Here we show that NCL-1/TRIM2/Brat tumour suppressor extends lifespan and limits nucleolar size in the major C. elegans longevity pathways, as part of a convergent mechanism focused on the nucleolus. Long-lived animals representing distinct longevity pathways exhibit small nucleoli, and decreased expression of rRNA, ribosomal proteins, and the nucleolar protein fibrillarin, dependent on NCL-1. Knockdown of fibrillarin also reduces nucleolar size and extends lifespan. Among wildtype C. elegans, individual nucleolar size varies, but is highly predictive for longevity. Long-lived dietary restricted fruit flies and insulin-like-peptide mutants exhibit small nucleoli and fibrillarin expression, as do long-lived dietary restricted and IRS1 knockout mice. Furthermore, human muscle biopsies from individuals who underwent modest dietary restriction coupled with exercise also display small nucleoli. We suggest that small nucleoli are a cellular hallmark of longevity and metabolic health conserved across taxa.

Figure 1. ncl-1 mediates DR and other forms of longevity.

(a) Longevity of eat-2(ad465) is abolished with the loss of ncl-1(e1942) (P<0.0001, three independent biological replicates). (b) ncl-1(e1942) is significantly shorter lived than N2 on bacterial dilution across 7 different concentrations (P<0.0001, three independent biological replicates). (c,d) ncl-1(e1942) is shorter lived than N2 on let-363/TOR and daf-2 RNAi (P<0.0001, three independent biological replicates). (e,f) glp-1(e2141) and isp-1(qm150) are significantly longer lived than glp-1;ncl-1 (P<0.0001, three independent biological replicates) and isp-1;ncl-1 (P=0.0016, three independent biological replicates) respectively. (g) ncl-1 RNAi significantly shortens the longevity of ife-2(ok306) (P<0.0001, two independent biological replicates) (h) Over-expression of ncl-1(+) in N2 for two independent extra-chromosomal transgenic arrays (dhEx1007, dhEx1008) increases lifespan (P<0.0001, three independent biological replicates). P-values calculated by log-rank test.

Figure 2. Nucleolar size inversely correlates with longevity.

(a) eat-2(ad465) animals have smaller nucleoli while ncl-1(e1942) and eat-2;ncl-1 animals possess larger nucleoli compared to N2. (20 worms imaged per replicate, 3 independent biological replicates) (b) Nucleolar size is reduced on bacterial food reduction with a corresponding increase in lifespan. (P<0.0001, log-rank test). (c,d) eat-2(ad465), TOR RNAi, isp-1(qm150), glp-1(e2141) and daf-2(e1370) animals possess smaller nucleoli while daf-2;daf-16 have nucleoli similar to N2 in the hypodermis and pharyngeal muscle (Error bars represent mean±s.d.) (e,f) Schematic illustrating the experiment, which shows that longer-lived worms exhibit small nucleoli and vice versa. (The graph depicts mean and s.d. Pearson correlation coefficient R²=0.93 is calculated using the entire data set, the line equation is y=−0.1483x+6.9148). Scale bar represents 5 μm. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, NS, non-significant, unpaired t-test.

Figure 3. Longevity mutants have reduced ribosome biogenesis.

(a) FIB-1::GFP is strongly down-regulated in eat-2(ad465) animals but up-regulated in ncl-1(e1942) and eat-2;ncl-1 double mutants (20 worms imaged per replicate, 3 independent biological replicates). (b–d) rRNA, RPS6, RPS15 and FIB-1 levels are increased in ncl-1(e1942) and reduced in eat-2(ad465), isp-1(qm150), glp-1(e2141), daf-2 RNAi and TOR RNAi and this effect is partially reversed by the loss of ncl-1. daf-2(e1370) also shows reduced rRNA levels compared to daf-2;daf-16(mu86) (Error bars represent mean±s.e.m.). (e) fib-1 RNAi extends lifespan of N2 (P=0.0004, log-rank test, three independent biological replicates) (f) fib-1 RNAi reduces the nucleolar size of N2 (Error bars represent mean±s.d.). Scale bar represents 20 μm. *P<0.05, **P<0.01, ****P<0.0001, unpaired t-test.

Figure 4. Smaller nucleoli associate with longevity in higher organisms.

(a,b) DR, dilp2-3,5 and Rapamycin treated D. melanogaster possess small nucleoli in intestine and fat body (Error bars represent mean±s.d.). (c,d) DR and IRS1 knockout mice show reduced nucleolar size in kidney tissue compared to ad libitum fed mice and wildtype (Error bars represent mean±s.d.). (e,f) Muscle biopsies from humans undergoing DR and exercise exhibit small nucleoli. Scale bars represent 10 μm (a,c) and 20 μm (e). *P<0.05 paired t-test, ****P<0.0001 unpaired t-test.