RNAi screening implicates a SKN-1-dependent transcriptional response in stress resistance and longevity deriving from translation inhibition.

Caenorhabditis elegans SKN-1 (ortholog of mammalian Nrf1/2/3) is critical for oxidative stress resistance and promotes longevity under reduced insulin/IGF-1-like signaling (IIS), dietary restriction (DR), and normal conditions. SKN-1 inducibly activates genes involved in detoxification, protein homeostasis, and other functions in response to stress. Here we used genome-scale RNA interference (RNAi) screening to identify mechanisms that prevent inappropriate SKN-1 target gene expression under non-stressed conditions. We identified 41 genes for which knockdown leads to activation of a SKN-1 target gene (gcs-1) through skn-1-dependent or other mechanisms. These genes correspond to multiple cellular processes, including mRNA translation. Inhibition of translation is known to increase longevity and stress resistance and may be important for DR-induced lifespan extension. One model postulates that these effects derive from reduced energy needs, but various observations suggest that specific longevity pathways are involved. Here we show that translation initiation factor RNAi robustly induces SKN-1 target gene transcription and confers skn-1-dependent oxidative stress resistance. The accompanying increases in longevity are mediated largely through the activities of SKN-1 and the transcription factor DAF-16 (FOXO), which is required for longevity that derives from reduced IIS. Our results indicate that the SKN-1 detoxification gene network monitors various metabolic and regulatory processes. Interference with one of these processes, translation initiation, leads to a transcriptional response whereby SKN-1 promotes stress resistance and functions together with DAF-16 to extend lifespan. This stress response may be beneficial for coping with situations that are associated with reduced protein synthesis.

ATF-4 and hydrogen sulfide signalling mediate longevity in response to inhibition of translation or mTORC1.

Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H2S) production. This H2S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H2S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H2S levels, or enhancing mechanisms that H2S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition.

Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence.

In Caenorhabditis elegans, ablation of germline stem cells (GSCs) extends lifespan, but also increases fat accumulation and alters lipid metabolism, raising the intriguing question of how these effects might be related. Here, we show that a lack of GSCs results in a broad transcriptional reprogramming in which the conserved detoxification regulator SKN-1/Nrf increases stress resistance, proteasome activity, and longevity. SKN-1 also activates diverse lipid metabolism genes and reduces fat storage, thereby alleviating the increased fat accumulation caused by GSC absence. Surprisingly, SKN-1 is activated by signals from this fat, which appears to derive from unconsumed yolk that was produced for reproduction. We conclude that SKN-1 plays a direct role in maintaining lipid homeostasis in which it is activated by lipids. This SKN-1 function may explain the importance of mammalian Nrf proteins in fatty liver disease and suggest that particular endogenous or dietary lipids might promote health through SKN-1/Nrf.

Willow bark extract increases antioxidant enzymes and reduces oxidative stress through activation of Nrf2 in vascular endothelial cells and Caenorhabditis elegans.

Willow bark extract (WBE) is listed in the European Pharmacopoeia and has been traditionally used for treating fever, pain, and inflammation. Recent studies have demonstrated its clinical usefulness. This study investigated the antioxidative effects of WBE in human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. WBE prevented oxidative-stress-induced cytotoxicity of HUVECs and death of C. elegans. WBE dose-dependently increased mRNA and protein expression levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) target genes heme oxygenase-1, γ-glutamylcysteine ligase modifier and catalytic subunits, and p62 and intracellular glutathione (GSH) in HUVECs. In the nematode C. elegans, WBE increased the expression of the gcs-1::green fluorescent protein reporter, a well-characterized target of the Nrf2 ortholog SKN-1, in a manner that was SKN-1-dependent. WBE increased intranuclear expression and DNA binding of Nrf2 and the activity of an antioxidant response element (ARE) reporter plasmid in HUVECs. WBE-induced expression of Nrf2-regulated genes and increased GSH levels in HUVECs were reduced by Nrf2 and p38 small interfering (si) RNAs and by the p38-specific inhibitor SB203580. Nrf2 siRNA reduced the cytoprotective effect of WBE against oxidative stress in HUVECs. Salicin, a major anti-inflammatory ingredient of WBE, failed to activate ARE-luciferase activity, whereas a salicin-free WBE fraction showed intensive activity. WBE induced antioxidant enzymes and prevented oxidative stress through activation of Nrf2 independent of salicin, providing a new potential explanation for the clinical usefulness of WBE.

Mitochondrial SKN-1/Nrf mediates a conserved starvation response.

SKN-1/Nrf plays multiple essential roles in development and cellular homeostasis. We demonstrate that SKN-1 executes a specific and appropriate transcriptional response to changes in available nutrients, leading to metabolic adaptation. We isolated gain-of-function (gf) alleles of skn-1, affecting a domain of SKN-1 that binds the transcription factor MXL-3 and the mitochondrial outer membrane protein PGAM-5. These skn-1(gf) mutants perceive a state of starvation even in the presence of plentiful food. The aberrant monitoring of cellular nutritional status leads to an altered survival response in which skn-1(gf) mutants transcriptionally activate genes associated with metabolism, adaptation to starvation, aging, and survival. The triggered starvation response is conserved in mice with constitutively activated Nrf and may contribute to the tumorgenicity associated with activating Nrf mutations in mammalian somatic cells. Our findings delineate an evolutionarily conserved metabolic axis of SKN-1/Nrf, further establishing the complexity of this pathway.

TOR signaling and rapamycin influence longevity by regulating SKN-1/Nrf and DAF-16/FoxO.

The TOR kinase, which is present in the functionally distinct complexes TORC1 and TORC2, is essential for growth but associated with disease and aging. Elucidation of how TOR influences life span will identify mechanisms of fundamental importance in aging and TOR functions. Here we show that when TORC1 is inhibited genetically in C. elegans, SKN-1/Nrf, and DAF-16/FoxO activate protective genes, and increase stress resistance and longevity. SKN-1 also upregulates TORC1 pathway gene expression in a feedback loop. Rapamycin triggers a similar protective response in C. elegans and mice, but increases worm life span dependent upon SKN-1 and not DAF-16, apparently by interfering with TORC2 along with TORC1. TORC1, TORC2, and insulin/IGF-1-like signaling regulate SKN-1 activity through different mechanisms. We conclude that modulation of SKN-1/Nrf and DAF-16/FoxO may be generally important in the effects of TOR signaling in vivo and that these transcription factors mediate an opposing relationship between growth signals and longevity.

Role of microRNA processing in adipose tissue in stress defense and longevity.

Excess adipose tissue is associated with metabolic disease and reduced life span, whereas caloric restriction decreases these risks. Here we show that as mice age, there is downregulation of Dicer and miRNA processing in adipose tissue resulting in decreases of multiple miRNAs. A similar decline of Dicer with age is observed in C. elegans. This is prevented in both species by caloric restriction. Decreased Dicer expression also occurs in preadipocytes from elderly humans and can be produced in cells by exposure to oxidative stress or UV radiation. Knockdown of Dicer in cells results in premature senescence, and fat-specific Dicer knockout renders mice hypersensitive to oxidative stress. Finally, Dicer loss-of-function mutations in worms reduce life span and stress tolerance, while intestinal overexpression of Dicer confers stress resistance. Thus, regulation of miRNA processing in adipose-related tissues plays an important role in longevity and the ability of an organism to respond to environmental stress and age-related disease.