Regulation of the Age‐Dependent Activity of the FOXO Transcription Factor DAF‐16 in Adult Caenorhabditis elegans Roundworms

The insulin and insulin‐like growth factor signaling (IIS) pathway regulates stress resistance and contributes to longevity in evolutionarily diverse species. In the roundworm Caenorhabditis elegans the lifespan of daf‐2 mutants, which lack functional insulin receptors, is twice that of wildtype animals. This phenotype is entirely dependent upon the FOXO transcription factor DAF‐16. In the absence of negative regulation through the IIS pathway, DAF‐16 upregulates genes important for stress resistance, defense, and repair. While it can be activated in response to environmental insults, in young animals DAF‐16 is normally inhibited. However, we and others have demonstrated that the activity of DAF‐16 increases in an age‐dependent manner. The mechanism by which DAF‐16 is regulated during aging has not yet been defined. Here we used a reverse genetic approach in C. elegans to investigate the function of genes that act upstream in the insulin signaling pathway to ask about their potential roles in modulating DAF‐16 activity in post‐reproductive adult animals. We tested the hypothesis that activation of DAF‐2 and the ensuing activation of the PDK‐1 kinase must be blocked in order to permit DAF‐16 to regulate its transcriptional targets. By analyzing the expression of a GFP‐labeled transcriptional reporter and by measuring gene expression levels by qRT‐PCR, we found that in an age‐dependent manner DAF‐18, an ortholog of mammalian PTEN that prevents PDK‐1 from being activated, is necessary for the increased expression of DAF‐16 targets over time. Further, we found that RNAi targeting daf‐18 reduced the ability of adult but not larval stage animals to resist infection by the bacterial pathogen Pseudomonas aeruginosa . This indicates that DAF‐18 is required for DAF‐16‐mediated innate immunity later in life. Our observations raise the intriguing possibility that an agonist engages DAF‐2 to initiate signaling through the IIS pathway at the same time that DAF‐16 is activated during adulthood. Previous studies established a role for micro RNAs (miRNAs) in regulating DAF‐2 activity, and we asked whether this could be true in the context of aging by knocking down the expression of alg‐2 , an argonaute protein involved in miRNA biogenesis. We reasoned that expression of a DAF‐2 ligand might be controlled post‐transcriptionally by miRNA. Our functional analysis implies a possible genetic interaction between alg‐2 and daf‐18 that is most evident in adult C. elegans , suggesting that their gene products may act in the same pathway that ultimately governs DAF‐16 function. Together, our results reveal elements of a transcriptional regulatory mechanism that is specific to adult animals and that involves evolutionarily conserved components, suggesting its general relevance to the aging process in diverse organisms.