Identification of evolutionarily conserved genetic regulators of cellular aging

Abstract

To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.

Fig. 1

Workflow for candidate gene identification and functional classification. First, gene expression data from 47 microarrays were processed to identify differentially expressed (DE) genes in experimental group 1 (EG1) and experimental group 2 (EG2). The selection of genes for functional classification and pathway analysis, starting from a total of 1566 genes, is shown (left arm). In the right arm of the diagram, the strategy to identify yeast genes for testing in lifespan analysis is depicted.

Fig. 2

Western blot analysis. Cell lysates were prepared from various model systems employed in this study: young and senescent human umbilical vein endothelial cells and renal proximal tubular epithelial cells, respectively; PFF treated with FCCP, AMP, and oligomycin for three days (A); mesenchymal stem cells cultured at atmospheric conditions of 3% or 20% oxygen; prostate stromal cells treated with either basic fibroblast growth factor (bFGF) or transforming growth factor beta 1 (TGFβ1) (B); CD8 T cells either isolated from a young or an elderly donor and sorted with respect to CD28 (C); Proteins were analyzed by immunoblotting using antibodies to EZH2, ALDH2, cystathionine beta-synthase, and IGFBP3, as indicated. For loading control, antibodies to β-actin and GAPDH were used as indicated.

Fig. 3

Functional analysis of selected candidate genes in yeast. Lifespan data for the nine yeast mutants with shortest lifespan (A) and for the seven mutants with lifespan extension (B) are shown. (*P < 0.05; **P < 0.01; ***P < 0.001).