PCH-2 regulates Caenorhabditis elegans lifespan

Abstract

Components or downstream targets of many signaling pathways such as Insulin/IGF-1 and TOR, as well as genes involved in cellular metabolism and bioenergetics can extend worm lifespan 20% or more. The C. elegans gene pch-2 and its homologs, including TRIP13 in humans, have been studied for their functions in cell mitosis and meiosis, but have never been implicated in lifespan regulation. Here we show that over-expression of TRIP13 in human fibroblasts confers resistance to environmental stressors such as UV radiation and oxidative stress. Furthermore, pch-2 overexpression in C. elegans extends worm lifespan, and enhances worm survival in response to various stressors. Conversely, reducing pch-2 expression with RNAi shortens worm lifespan. Additional genetic epistasis analysis indicates that the molecular mechanism of pch-2 in worm longevity is tied to functions of the sirtuin family, implying that pch-2 is another chromatin regulator for worm longevity. These findings suggest a novel function of the pch-2 gene involved in lifespan determination.

Figure 1. Pch-2 is an evolutionarily conserved gene and its expression declines with tissue aging across species

(A) Sequences alignment of pch-2 genes cross species ranging from yeast, worm, fly and rodent, to human. (B) Pch-2 mRNA expression changes with age in human, mouse and worm. * indicates p <0.05.

Figure 2. TRIP13 over-expression human fibroblast cells are resistant to stressors of oxidation, apoptosis and DNA damage

(A) After 600uM H₂O₂ treatment, the TRIP13 over-expression fibroblast cells showed enhanced cell numbers when compared to control cells, wild type (WT) and empty vector (p<0.0001). (B) TRIP13 over-expression fibroblasts were treated with 10uM etoposide for 3 days, and exhibited higher cell numbers following treatment (p<0.001). (C) TRIP13 cells demonstrated significantly higher resistance after UV radiation when compared to the control groups (p<0.01). (Relative cell survival was as compared to numbers of cells plated originally, prior to culturing and stressors.) * indicates significant differences between groups.

Figure 3. Pch-2 over-expression extends lifespan and enhances stress-resistance in C. elegans

(A) genotyping of pch-2 shows a band at 1670bp indicating the transgene in the pch-2 over-expressing line. (B) qRT-PCR validation of pch-2 over-expression worm lines by using transgene-specific primers shows increased pch-2 gene expression (p<0.01). (C) lifespan measurement was conducted for both WT (GFP-expressing, n=126) and pch-2 over-expressing (n=140) animals. Both median lifespan and maximum lifespan of pch-2 over-expression lines show a 25% extension when compared to WT lines (p<0.001, p values were derived from student t test and log-rank test). (D) After 4mM paraquat treatment, the median survival of pch-2 over-expressing lines (n=33) was increased by 40%, and the maximum survival of pch-2 over-expressing lines was increased 150% (n=30) (p<0.001). (E) The median survival of pch-2 over-expressing lines (n=49) was increased 45% after UV radiation when compared to WT (n=45) (p<0.001). (F) With heat shock, the median survival of pch-2 over-expressing lines (n=62) was increased by 85% when compared to WT (n=52) (p<0.001).

Figure 4. Inhibition of pch-2 and sir-2 expression by RNAi impacts lifespan

(A) A shortened lifespan, both median (62%, 64%) and maximum (70%, 78%), was caused by pch-2 and sir2 specific RNAi (n=81, 65) as compared to RNAi vector lines (n=63) (p <0.0001). (B) In WT worms, RNAi for pch-2 resulted in significantly reduced expression by ~ 70% as validated by qRT-PCR, but did not have a significant effect on sir-2 transcripts. (C) Similarly, in WT worms, RNAi for sir-2 resulted a significant reduction of sir-2 expression about 50% by qRT-PCR validation, and without notable effect on pch-2 expression (*p <0.001).

Figure 5. Inhibition of pch-2 by RNAi impacts lifespan of pch-2 over-expressing C. elegans

(A) A shortened lifespan, both median (30%) and maximum (56%), was induced by pch-2-specific RNAi in C. elegans that were engineered to over-express pch-2 (n=101), as compared to RNAi vector lines (n=60) (p <0.0001). (B) qRT-PCR validated the RNAi effect, indicating a significant reduction (~70%) of pch-2 transcripts in pch-2 over-expressing worms treated with RNAi. ***p <0.001.

Figure 6. RNAi inhibition of both pch-2 in sir-2.1 over-expressing worms, and sir-2.1 in pch-2 over-expressing worms, shortens lifespan

(A) Purple curves show lifespans of sir-2.1 over-expressing worms, while red curves show lifespans of pch-2 over-expressing worms. In sir-2 over-expressing worms, pch-2 RNAi significantly decreased lifespan (56% and 75% decrease in median and maximum lifespan, respectively). In pch-2 over-expressing worms, sir-2.1 RNAi induced a 57% and 70% decrease in median and maximum lifespans (p <0.0001 for both effects). (B) qRT-PCR validated the RNAi effect, indicating a significant reduction (~90%) of pch-2 transcipts in sir-2 over-expressing worms that were treated with pch-2 RNAi. (C) qRT-PCR validating a significant reduction (~70%) of sir-2 transcripts in pch-2 over-expressing worms that were treated with sir-2 RNAi. *p <0.001.

Figure 7. Proposed interactions of pch-2 and sir-2 in aging in C. elegans

Based on our data and the literature, we propose that pch-2 may, similar to sir-2, affect the aging process of C. elegans. Briefly, pch-2 may play roles in surveillance of DNA damage and chromatin regulation, in concert with sir-2. Reduction of pch-2 expression induces genome instability and transcriptional dysregulation, and, therefore, may promote aging in C. elegans.