T. Keith Blackwell

A Conserved MST-FOXO Signaling Pathway Mediates Oxidative-Stress Responses and Extends Life Span

Oxidative stress influences cell survival and homeostasis, but the mechanisms underlying the biological effects of oxidative stress remain to be elucidated. Here, we demonstrate that the protein kinase MST1 mediates oxidative-stress-induced cell death in primary mammalian neurons by directly activating the FOXO transcription factors. MST1 phosphorylates FOXO proteins at a conserved site within the forkhead domain that disrupts their interaction with 14-3-3 proteins, promotes FOXO nuclear translocation, and thereby induces cell death in neurons. We also extend the MST-FOXO signaling link to nematodes. Knockdown of the C. elegans MST1 ortholog CST-1 shortens life span and accelerates tissue aging, while overexpression of cst-1 promotes life span and delays aging. The cst-1-induced life-span extension occurs in a daf-16-dependent manner. The identification of the FOXO transcription factors as major and evolutionarily conserved targets of MST1 suggests that MST kinases play important roles in diverse biological processes including cellular responses to oxidative stress and longevity.

Direct Inhibition of the Longevity-Promoting Factor SKN-1 by Insulin-like Signaling in C. elegans

Insulin/IGF-1-like signaling (IIS) is central to growth and metabolism and has a conserved role in aging. In C. elegans, reductions in IIS increase stress resistance and longevity, effects that require the IIS-inhibited FOXO protein DAF-16. The C. elegans transcription factor SKN-1 also defends against oxidative stress by mobilizing the conserved phase 2 detoxification response. Here we show that IIS not only opposes DAF-16 but also directly inhibits SKN-1 in parallel. The IIS kinases AKT-1, -2, and SGK-1 phosphorylate SKN-1, and reduced IIS leads to constitutive SKN-1 nuclear accumulation in the intestine and SKN-1 target gene activation. SKN-1 contributes to the increased stress tolerance and longevity resulting from reduced IIS and delays aging when expressed transgenically. Furthermore, SKN-1 that is constitutively active increases life span independently of DAF-16. Our findings indicate that the transcription network regulated by SKN-1 promotes longevity and is an important direct target of IIS.

MK2‐induced tristetraprolin:14‐3‐3 complexes prevent stress granule association and ARE‐mRNA decay

Stress granules (SGs) are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. SG‐associated proteins such as TIA‐1, TIAR and HuR bind to AU‐rich element (ARE)‐containing mRNAs and control their translation and stability. Here we show that tristetraprolin (TTP), an ARE‐binding protein that destabilizes ARE‐mRNAs, is recruited to SGs that are assembled in response to FCCP‐induced energy deprivation, but not arsenite‐induced oxidative stress. Exclusion of TTP from arsenite‐induced SGs is a consequence of MAPKAP kinase‐2 (MK2)‐induced phosphorylation at serines 52 and 178, which promotes the assembly of TTP:14‐3‐3 complexes. 14‐3‐3 binding excludes TTP from SGs and inhibits TTP‐dependent degradation of ARE‐containing transcripts. In activated RAW 264.7 macrophages, endogenous TTP:14‐3‐3 complexes bind to ARE‐RNA. Our data reveal the mechanism by which the p38‐MAPK/MK2 kinase cascade inhibits TTP‐mediated degradation of ARE‐containing transcripts and thereby contributes to lipopolysaccharide‐induced TNFα expression.

Condition‐adapted stress and longevity gene regulation by Caenorhabditis elegans SKN‐1/Nrf

Studies in model organisms have identified regulatory processes that profoundly influence aging, many of which modulate resistance against environmental or metabolic stresses. In Caenorhabditis elegans, the transcription regulator SKN‐1 is important for oxidative stress resistance and acts in multiple longevity pathways. SKN‐1 is the ortholog of mammalian Nrf proteins, which induce Phase 2 detoxification genes in response to stress. Phase 2 enzymes defend against oxygen radicals and conjugate electrophiles that are produced by Phase 1 detoxification enzymes, which metabolize lipophilic compounds. Here, we have used expression profiling to identify genes and processes that are regulated by SKN‐1 under normal and stress–response conditions. Under nonstressed conditions SKN‐1 upregulates numerous genes involved in detoxification, cellular repair, and other functions, and downregulates a set of genes that reduce stress resistance and lifespan. Many of these genes appear to be direct SKN‐1 targets, based upon presence of predicted SKN‐binding sites in their promoters. The metalloid sodium arsenite induces skn‐1‐dependent activation of certain detoxification gene groups, including some that were not SKN‐1‐upregulated under normal conditions. An organic peroxide also triggers induction of a discrete Phase 2 gene set, but additionally stimulates a broad SKN‐1‐independent response. We conclude that under normal conditions SKN‐1 has a wide range of functions in detoxification and other processes, including modulating mechanisms that reduce lifespan. In response to stress, SKN‐1 and other regulators tailor transcription programs to meet the challenge at hand. Our findings reveal striking complexity in SKN‐1 functions and the regulation of systemic detoxification defenses.

nhl-2 modulates microRNA activity in Caenorhabditis elegans

TRIM-NHL proteins represent a large class of metazoan proteins implicated in development and disease. We demonstrate that a C. elegans TRIM-NHL protein, NHL-2, functions as a cofactor for the microRNA-induced silencing complex (miRISC) and thereby enhances the posttranscriptional repression of several genetically verified microRNA targets, including hbl-1 and let-60/Ras (by the let-7 family of microRNAs) and cog-1 (by the lsy-6 microRNA). NHL-2 is localized to cytoplasmic P-bodies and physically associates with the P-body protein CGH-1 and the core miRISC components ALG-1/2 and AIN-1. nhl-2 and cgh-1 mutations compromise the repression of microRNA targets in vivo but do not affect microRNA biogenesis, indicating a role for an NHL-2:CGH-1 complex in the effector phase of miRISC activity. We propose that the NHL-2:CGH-1 complex functions in association with mature miRISC to modulate the efficacy of microRNA:target interactions in response to physiological and developmental signals, thereby ensuring the robustness of genetic regulatory pathways regulated by microRNAs.

A redox-sensitive peroxiredoxin that is important for longevity has tissue- and stress-specific roles in stress resistance

Oxidative damage caused by reactive oxygen species (ROS) is implicated in many diseases and in aging. Removal of ROS by antioxidant enzymes plays an important part in limiting this damage. For instance, peroxiredoxins (Prx) are conserved, abundant, thioredoxin peroxidase enzymes that function as tumor suppressors. In addition to detoxifying peroxides, studies in single-cell systems have revealed that Prx act as chaperones and redox sensors. However, it is unknown in what manner the different activities of Prx influence stress resistance or longevity in the context of whole animals. Here, we reveal three distinct roles for the 2-Cys Prx, PRDX-2, in the stress resistance of the nematode worm Caenorhabditis elegans. (i) The thioredoxin peroxidase activity of PRDX-2 protects against hydrogen peroxide. (ii) Consistent with a chaperone activity for hyperoxidized PRDX-2, peroxide-induced oxidation of PRDX-2 increases resistance to heat stress. (iii) Unexpectedly, loss of PRDX-2 increases the resistance of C. elegans to some oxidative stress-causing agents, such as arsenite, apparently through a signaling mechanism that increases the levels of other antioxidants and phase II detoxification enzymes. Despite their increased resistance to some forms of oxidative stress, prdx-2 mutants are short-lived. Moreover, intestinal expression of PRDX-2 accounts for its role in detoxification of exogenous peroxide, but not its influence on either arsenite resistance or longevity, suggesting that PRDX-2 may promote longevity and protect against environmental stress through different mechanisms. Together the data reveal that in metazoans Prx act through multiple biochemical activities, and have tissue-specific functions in stress resistance and longevity.

Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis

During oogenesis, numerous messenger RNAs (mRNAs) are maintained in a translationally silenced state. In eukaryotic cells, various translation inhibition and mRNA degradation mechanisms congregate in cytoplasmic processing bodies (P bodies). The P body protein Dhh1 inhibits translation and promotes decapping-mediated mRNA decay together with Pat1 in yeast, and has been implicated in mRNA storage in metazoan oocytes. Here, we have investigated in Caenorhabditis elegans whether Dhh1 and Pat1 generally function together, and how they influence mRNA sequestration during oogenesis. We show that in somatic tissues, the Dhh1 orthologue (CGH-1) forms Pat1 (patr-1)-dependent P bodies that are involved in mRNA decapping. In contrast, during oogenesis, CGH-1 forms patr-1–independent mRNA storage bodies. CGH-1 then associates with translational regulators and a specific set of maternal mRNAs, and prevents those mRNAs from being degraded. Our results identify somatic and germ cell CGH-1 functions that are distinguished by the involvement of PATR-1, and reveal that during oogenesis, numerous translationally regulated mRNAs are specifically protected by a CGH-1–dependent mechanism.

Specific SKN-1/Nrf Stress Responses to Perturbations in Translation Elongation and Proteasome Activity

SKN-1, the Caenorhabditis elegans Nrf1/2/3 ortholog, promotes both oxidative stress resistance and longevity. SKN-1 responds to oxidative stress by upregulating genes that detoxify and defend against free radicals and other reactive molecules, a SKN-1/Nrf function that is both well-known and conserved. Here we show that SKN-1 has a broader and more complex role in maintaining cellular stress defenses. SKN-1 sustains expression and activity of the ubiquitin-proteasome system (UPS) and coordinates specific protective responses to perturbations in protein synthesis or degradation through the UPS. If translation initiation or elongation is impaired, SKN-1 upregulates overlapping sets of cytoprotective genes and increases stress resistance. When proteasome gene expression and activity are blocked, SKN-1 activates multiple classes of proteasome subunit genes in a compensatory response. SKN-1 thereby maintains UPS activity in the intestine in vivo under normal conditions and promotes survival when the proteasome is inhibited. In contrast, when translation elongation is impaired, SKN-1 does not upregulate proteasome genes, and UPS activity is then reduced. This indicates that UPS activity depends upon presence of an intact translation elongation apparatus; and it supports a model, suggested by genetic and biochemical studies in yeast, that protein synthesis and degradation may be coupled processes. SKN-1 therefore has a critical tissue-specific function in increasing proteasome gene expression and UPS activity under normal conditions, as well as when the UPS system is stressed, but mounts distinct responses when protein synthesis is perturbed. The specificity of these SKN-1–mediated stress responses, along with the apparent coordination between UPS and translation elongation activity, may promote protein homeostasis under stress or disease conditions. The data suggest that SKN-1 may increase longevity, not only through its well-documented role in boosting stress resistance, but also through contributing to protein homeostasis.

Dauer-independent insulin/IGF-1-signalling implicates collagen remodelling in longevity

Interventions that delay ageing mobilize mechanisms that protect and repair cellular components, but it is unknown how these interventions might slow the functional decline of extracellular matrices, which are also damaged during ageing. Reduced insulin/IGF-1 signalling (rIIS) extends lifespan across the evolutionary spectrum, and in juvenile Caenorhabditis elegans also allows the transcription factor DAF-16/FOXO to induce development into dauer, a diapause that withstands harsh conditions. It has been suggested that rIIS delays C. elegans ageing through activation of dauer-related processes during adulthood, but some rIIS conditions confer robust lifespan extension unaccompanied by any dauer-like traits. Here we show that rIIS can promote C. elegans longevity through a program that is genetically distinct from the dauer pathway, and requires the Nrf (NF-E2-related factor) orthologue SKN-1 acting in parallel to DAF-16. SKN-1 is inhibited by IIS and has been broadly implicated in longevity, but is rendered dispensable for rIIS lifespan extension by even mild activity of dauer-related processes. When IIS is decreased under conditions that do not induce dauer traits, SKN-1 most prominently increases expression of collagens and other extracellular matrix genes. Diverse genetic, nutritional, and pharmacological pro-longevity interventions delay an age-related decline in collagen expression. These collagens mediate adulthood extracellular matrix remodelling, and are needed for ageing to be delayed by interventions that do not involve dauer traits. By genetically delineating a dauer-independent rIIS ageing pathway, our results show that IIS controls a broad set of protective mechanisms during C. elegans adulthood, and may facilitate elucidation of processes of general importance for longevity. The importance of collagen production in diverse anti-ageing interventions implies that extracellular matrix remodelling is a generally essential signature of longevity assurance, and that agents promoting extracellular matrix youthfulness may have systemic benefit.

SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans.

The mammalian Nrf/CNC proteins (Nrf1, Nrf2, Nrf3, p45 NF-E2) perform a wide range of cellular protective and maintenance functions. The most thoroughly described of these proteins, Nrf2, is best known as a regulator of antioxidant and xenobiotic defense, but more recently has been implicated in additional functions that include proteostasis and metabolic regulation. In the nematode Caenorhabditis elegans, which offers many advantages for genetic analyses, the Nrf/CNC proteins are represented by their ortholog SKN-1. Although SKN-1 has diverged in aspects of how it binds DNA, it exhibits remarkable functional conservation with Nrf/CNC proteins in other species and regulates many of the same target gene families. C. elegans may therefore have considerable predictive value as a discovery model for understanding how mammalian Nrf/CNC proteins function and are regulated in vivo. Work in C. elegans indicates that SKN-1 regulation is surprisingly complex and is influenced by numerous growth, nutrient, and metabolic signals. SKN-1 is also involved in a wide range of homeostatic functions that extend well beyond the canonical Nrf2 function in responses to acute stress. Importantly, SKN-1 plays a central role in diverse genetic and pharmacologic interventions that promote C. elegans longevity, suggesting that mechanisms regulated by SKN-1 may be of conserved importance in aging. These C. elegans studies predict that mammalian Nrf/CNC protein functions and regulation may be similarly complex and that the proteins and processes that they regulate are likely to have a major influence on mammalian life- and healthspan.