CEP-1, the Caenorhabditis elegans p53 homolog, mediates opposing longevity outcomes in mitochondrial electron transport chain mutants

Abstract

Caenorhabditis elegans CEP-1 and its mammalian homolog p53 are critical for responding to diverse stress signals. In this study, we found that cep-1 inactivation suppressed the prolonged lifespan of electron transport chain (ETC) mutants, such as isp-1 and nuo-6, but rescued the shortened lifespan of other ETC mutants, such as mev-1 and gas-1. We compared the CEP-1-regulated transcriptional profiles of the long-lived isp-1 and the short-lived mev-1 mutants and, to our surprise, found that CEP-1 regulated largely similar sets of target genes in the two mutants despite exerting opposing effects on their longevity. Further analyses identified a small subset of CEP-1-regulated genes that displayed distinct expression changes between the isp-1 and mev-1 mutants. Interestingly, this small group of differentially regulated genes are enriched for the "aging" Gene Ontology term, consistent with the hypothesis that they might be particularly important for mediating the distinct longevity effects of CEP-1 in isp-1 and mev-1 mutants. We further focused on one of these differentially regulated genes, ftn-1, which encodes ferritin in C. elegans, and demonstrated that it specifically contributed to the extended lifespan of isp-1 mutant worms but did not affect the mev-1 mutant lifespan. We propose that CEP-1 responds to different mitochondrial ETC stress by mounting distinct compensatory responses accordingly to modulate animal physiology and longevity. Our findings provide insights into how mammalian p53 might respond to distinct mitochondrial stressors to influence cellular and organismal responses.

Figure 1. CEP-1 mediates the longevity and development of two mitochondrial mutants in C. elegans.

(A) cep-1 mutation partially suppresses isp-1 mutant longevity as the cep-1;isp-1 double mutant lifespan is shorter than that of the isp-1 single mutant. (B) cep-1 mutation restores the mev-1 mutant lifespan as the lifespans of two cep-1;mev-1 isolates (L1, L32) are similar to that of wt. (C) The percentage of worms at each developmental stage was quantified for wt, cep-1, isp-1, cep-1;isp-1, mev-1, and cep-1;mev-1 mutant worms after 60 hr of growth from the embryonic stage at 20°C. (D) The average number of progeny production for each line was calculated from 5 to 10 worms. The isp-1 and mev-1 mutants produce significantly less progeny than wt. The cep-1;isp-1 and cep-1;mev-1 double mutants display significantly lower brood sizes than their respective single mutant controls (*p<0.05, **p<0.0005). The error bars represent standard errors. Statistical analysis was performed using a two-tailed t-test.

Figure 2. CEP-1 mediates reduced physiological germline apoptosis in the isp-1 mutant.

Physiological levels of apoptosis were quantified by counting the number of apoptotic corpses per gonad arm in various C. elegans strains. The corpses were counted using DIC microscopy at 63× magnification 48 hr post L4. The data represent the average of at least 3 independent experimental replicates (n≥15 gonad arms for each) ± standard error. Statistical analysis was done using the Mann-Whitney U-test. ***p<0.001.

Figure 3. CEP-1-regulated transcriptomes in isp-1 and mev-1 mutants.

(A) CEP-1-regulated genes in isp-1 and mev-1 mutants are largely similar. Hierarchical single linkage gene clustering was performed, and the dendrogram shows the clustered relationship of individual arrays. The numbers on the dendrogram represent the correlation coefficients between arrays. Yellow: upregulated, Blue: downregulated, Black: no change. (B) The expression of CEP-1-regulated genes that were significantly changed in isp-1 and mev-1 mutants, identified by SAM analysis, are represented in the Venn diagram. (C) DAVID functional annotation of similarly and differentially expressed CEP-1-regulated genes in isp-1 and mev-1 mutants. The numbers represent the enrichment scores for each group (score>1.3 is considered as significant). Several examples of aging and metabolic genes are listed.

Figure 4. CEP-1-regulated ferritin induction partially mediates the extended lifespan of isp-1 mutants.

(A) Pftn-1::gfp expression in wt, cep-1, isp-1, cep-1;isp-1, mev-1, and cep-1;mev-1 mutant worms. Scale bar = 100 µm. (B, C) The lifespans of wt, cep-1, isp-1, cep-1;isp-1, mev-1 and cep-1;mev-1 mutant worms treated with ftn-1 and ftn-2 double RNAi. L4440 is a treatment control.

Figure 5. CEP-1 mediates the longevity and development of several mitochondrial mutants in C. elegans.

(A) cep-1 mutation fully suppresses the long lifespan of the nuo-6 mutant. (B) cep-1 mutation does not suppress clk-1 mutant longevity as the lifespans of two cep-1;clk-1 double mutant isolates (L4, L6) are similar to that of the clk-1 single mutant. (C) cep-1 mutation partially restores gas-1 mutant lifespan as two isolates of cep-1;gas-1 (L22, L34) live longer than the gas-1 single mutant. (D) The percentage of worms at each developmental stage was quantified as described in Fig. 1C. cep-1 deletion has little impact on nuo-6 and gas-1 mutant development.

Figure 6. ftn-1 is differentially expressed in various ETC mutants and mediates their lifespan outcomes.

(A, B) RNAi-mediated knockdown of ftn-1 attenuates the long life of the nuo-6 mutant but does not impact the lifespan of the short-lived gas-1 mutant. (C) qRT-PCR results of ftn-1 expression levels in various ETC mutants. The two-tailed student t-tests were performed to determine significant difference in ftn-1 expression levels with and without CEP-1 in each ETC mutant background.

Figure 7. Expression of differentially regulated CEP-1 targets in other ETC mutants.

(A–I) These genes are differentially regulated by CEP-1 between isp-1 and mev-1 mutants. The relative expression of each gene was normalized to act-1 and wt. The average log2 ratio between ETC mutants with and without cep-1 from three independent experiments are plotted. The error bars represent standard errors. Two-tailed t-tests were performed to determine significant differences of the expression of CEP-1 gene targets between the long-lived isp-1(qm150) and nuo-6(qm200) mutants and the short-lived mev-1(kn1) and gas-1(fc21) mutants. *p<0.05, ** p<0.01, *** p<0.001.

Figure 8. Expression of similarly regulated CEP-1 targets in other ETC mutants.

(A–K) These genes are similarly regulated by CEP-1 between isp-1 and mev-1 mutants. The relative expression of each gene was normalized to act-1 and wt. The average log2 ratio between ETC mutants with and without cep-1 from three independent experiments are plotted. The error bars represent standard errors. Two-tailed t-tests were performed to determine significant differences of the expression of CEP-1 target genes between the long-lived isp-1(qm150) and nuo-6(qm200) mutants and the short-lived mev-1(kn1) and gas-1(fc21) mutants. * p<0.05, ** p<0.01, *** p<0.001.