The MYST family histone acetyltransferase complex regulates stress resistance and longevity through transcriptional control of DAF-16/FOXO transcription factors.

The well-known link between longevity and the Sir2 histone deacetylase family suggests that histone deacetylation, a modification associated with repressed chromatin, is beneficial to longevity. However, the molecular links between histone acetylation and longevity remain unclear. Here, we report an unexpected finding that the MYST family histone acetyltransferase complex (MYS-1/TRR-1 complex) promotes rather than inhibits stress resistance and longevity in Caenorhabditis elegans Our results show that these beneficial effects are largely mediated through transcriptional up-regulation of the FOXO transcription factor DAF-16. MYS-1 and TRR-1 are recruited to the promoter regions of the daf-16 gene, where they play a role in histone acetylation, including H4K16 acetylation. Remarkably, we also find that the human MYST family Tip60/TRRAP complex promotes oxidative stress resistance by up-regulating the expression of FOXO transcription factors in human cells. Tip60 is recruited to the promoter regions of the foxo1 gene, where it increases H4K16 acetylation levels. Our results thus identify the evolutionarily conserved role of the MYST family acetyltransferase as a key epigenetic regulator of DAF-16/FOXO transcription factors.

The microRNA machinery regulates fasting-induced changes in gene expression and longevity in Caenorhabditis elegans.

Intermittent fasting (IF) is a dietary restriction regimen that extends the lifespans of Caenorhabditis elegans and mammals by inducing changes in gene expression. However, how IF induces these changes and promotes longevity remains unclear. One proposed mechanism involves gene regulation by microRNAs (miRNAs), small non-coding RNAs (∼22 nucleotides) that repress gene expression and whose expression can be altered by fasting. To test this proposition, we examined the role of the miRNA machinery in fasting-induced transcriptional changes and longevity in C. elegans We revealed that fasting up-regulated the expression of the miRNA-induced silencing complex (miRISC) components, including Argonaute and GW182, and the miRNA-processing enzyme DRSH-1 (the ortholog of the Drosophila Drosha enzyme). Our lifespan measurements demonstrated that IF-induced longevity was suppressed by knock-out or knockdown of miRISC components and was completely inhibited by drsh-1 ablation. Remarkably, drsh-1 ablation inhibited the fasting-induced changes in the expression of the target genes of DAF-16, the insulin/IGF-1 signaling effector in C. elegans Fasting-induced transcriptome alterations were substantially and modestly suppressed in the drsh-1 null mutant and the null mutant of ain-1, a gene encoding GW182, respectively. Moreover, miRNA array analyses revealed that the expression levels of numerous miRNAs changed after 2 days of fasting. These results indicate that components of the miRNA machinery, especially the miRNA-processing enzyme DRSH-1, play an important role in mediating IF-induced longevity via the regulation of fasting-induced changes in gene expression.

Octopamine enhances oxidative stress resistance through the fasting-responsive transcription factor DAF-16/FOXO in C. elegans.

Dietary restriction regimens lead to enhanced stress resistance and extended life span in many species through the regulation of fasting and/or diet-responsive mechanisms. The fasting stimulus is perceived by sensory neurons and causes behavioral and metabolic adaptations. Octopamine (OA), one of the Caenorhabditis elegans neurotransmitters, is involved in behavioral adaptations, and its levels are increased under fasting conditions. However, it remains largely unknown how OA contributes to the fasting responses. In this study, we found that OA administration enhanced organismal resistance to oxidative stress. This enhanced resistance was suppressed by a mutation of the OA receptors, SER-3 and SER-6. Moreover, we found that OA administration promoted the nuclear translocation of DAF-16, the key transcription factor in fasting responses, and that the OA-induced enhancement of stress resistance required DAF-16. Altogether, our results suggest that OA signaling, which is triggered by the absence of food, shifts the organismal state to a more protective one to prepare for environmental stresses.

Src family kinases suppress differentiation of brown adipocytes and browning of white adipocytes.

Brown adipocytes and beige adipocytes can expend energy, generate heat, and increase whole-body energy expenditure. The detailed mechanisms of adipogenesis and thermogenesis of these cells are still obscure. Here, we show that Src family kinases (SFKs) regulate both brown adipogenesis and browning of white adipocytes. To identify factors involved in brown adipogenesis, we first examined the effect of several chemical inhibitors on the differentiation of brown preadipocytes isolated from mouse brown adipose tissue (BAT) and found that treatment with PP2, the specific inhibitor of SFKs, promoted the differentiation. Another inhibitor of SFKs, PP1, also promoted the brown adipogenesis, whereas an inactive analogue of PP2, PP3, did not. Moreover, over-expression of C-terminal Src kinase (CSK), the negative regulator of SFKs, also promoted brown adipogenesis. Next, we examined the effect of inhibition of SFKs on the differentiation of white preadipocytes isolated from white adipose tissue (WAT). Our results showed that either PP2 treatment or CSK-over-expression generated Ucp1-positive beige adipocytes, thus inducing browning of white adipocytes. Finally, our analysis showed that the expression levels and activity of SFKs in WAT were much higher than in BAT. These results taken together suggest that SFKs regulate differentiation and browning of fat cells in vivo.

Mechanisms of lifespan extension by dietary restriction.

Aging is inevitable for almost all the organisms. Aging processes vary from one organism to another. It has been shown that many organisms have lifespan-regulation mechanisms in common. Dietary restriction is the most robust non-genetic interventions to ameliorate aging and aging-associated diseases in many organisms. Reducing overall calorie intake was thought to be a crucial factor for dietary restriction -induced longevity. However, recent studies indicate that both the meal timing and lowered intake of specific nutrients rather than overall calorie intake are important for lifespan regulation. These findings shed light on new aspects of mechanisms underlying dietary restriction -induced longevity. The genetic factors are also shown to be involved in dietary restriction-induced longevity. This review summarizes the dietary interventions and the genetic factors that mediate lifespan extension by dietary interventions.

Signalling through RHEB-1 mediates intermittent fasting-induced longevity in C. elegans.

Dietary restriction is the most effective and reproducible intervention to extend lifespan in divergent species. In mammals, two regimens of dietary restriction, intermittent fasting (IF) and chronic caloric restriction, have proven to extend lifespan and reduce the incidence of age-related disorders. An important characteristic of IF is that it can increase lifespan even when there is little or no overall decrease in calorie intake. The molecular mechanisms underlying IF-induced longevity, however, remain largely unknown. Here we establish an IF regimen that effectively extends the lifespan of Caenorhabditis elegans, and show that the low molecular weight GTPase RHEB-1 has a dual role in lifespan regulation; RHEB-1 is required for the IF-induced longevity, whereas inhibition of RHEB-1 mimics the caloric-restriction effects. RHEB-1 exerts its effects in part by the insulin/insulin growth factor (IGF)-like signalling effector DAF-16 in IF. Our analyses demonstrate that most fasting-induced upregulated genes require RHEB-1 function for their induction, and that RHEB-1 and TOR signalling are required for the fasting-induced downregulation of an insulin-like peptide, INS-7. These findings identify the essential role of signalling by RHEB-1 in IF-induced longevity and gene expression changes, and suggest a molecular link between the IF-induced longevity and the insulin/IGF-like signalling pathway.

Lactobacillus paracasei subsp. paracasei 2004 improves health and lifespan in Caenorhabditis elegans.

Recent research has highlighted the importance of the gut microbiome in regulating aging, and probiotics are interventions that can promote gut health. In this study, we surveyed several novel lactic acid bacteria to examine their beneficial effect on organismal health and lifespan in C. elegans. We found that animals fed some lactic acid bacteria, including L. acidophilus 1244 and L. paracasei subsp. paracasei 2004, grew healthy. Supplementation with the lactic acid bacterial strains L. acidophilus 1244 or L. paracasei subsp. paracasei 2004 significantly improved health, including food consumption, motility, and resistance to oxidative stressor, hydrogen peroxide. Our RNA-seq analysis showed that supplementation with L. paracasei subsp. paracasei 2004 significantly increased the expression of daf-16, a C. elegans FoxO homolog, as well as genes related to the stress response. Furthermore, daf-16 deletion inhibited the longevity effect of L. paracasei subsp. paracasei 2004 supplementation. Our results suggest that L. paracasei subsp. paracasei 2004 improves health and lifespan in a DAF-16-dependent manner.

Single housing of juveniles accelerates early-stage growth but extends adult lifespan in African turquoise killifish.

Within the same species, individuals exhibiting faster growth tend to have shorter lifespans, even if their fast growth arises from early-life pharmacological interventions. However, in vertebrates, the impact of the early-life environment on the growth rate and lifespan has not been fully elucidated. In this study, by utilizing the short-lived African turquoise killifish, which is suitable for a comprehensive life-stage analysis in a brief timeframe, we explored the effects of housing density during the juvenile stage on holistic life traits. As a result, we found that lower housing densities resulted in faster growth, but led to longer adult lifespan, which was contrary to the common notion. Furthermore, the single-housed adult fish displayed a longer egg-laying period than did their group-housed counterparts. Our transcriptome analysis also demonstrated that, in terms of internal transcriptional programs, the life stage progression and aging process of single-housed fish were slower than those of group-housed fish. Collectively, our results suggest that sharing housing with others in early life might influence whole-life attributes, potentially leading to specific life history traits beyond the typical relationship between the growth rate and lifespan.

Discovery of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives as non-peptidic selective SUMO-sentrin specific protease (SENP)1 inhibitors.

We developed 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivative 4 (GN6958) as a non-peptidic selective SUMO-sentrin specific protease (SENP)1 protease inhibitor based on the hypoxia inducible factor (HIF)-1α inhibitor 1 (GN6767). The direct interaction of compound 1 with SENP1 protein in cells was observed by the pull-down experiments using the biotin-tagged compound 2 coated on the streptavidin affinity column. Among the various 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives tested, compounds 3 and 4 suppressed HIF-1α accumulation in a concentration-dependent manner without affecting the expression level of tubulin protein in HeLa cells. Both compounds inhibited SENP1 protease activity in a concentration-dependent manner, and compound 4 exhibited more potent inhibition than compound 3. Compound 4 exhibited selective inhibition against SENP1 protease activity without inhibiting other protease enzyme activities in vitro.

Intertissue small RNA communication mediates the acquisition and inheritance of hormesis in Caenorhabditis elegans.

Environmental conditions can cause phenotypic changes, part of which can be inherited by subsequent generations via soma-to-germline communication. However, the signaling molecules or pathways that mediate intertissue communication remain unclear. Here, we show that intertissue small RNA communication systems play a key role in the acquisition and inheritance of hormesis effects - stress-induced stress resistance - in Caenorhabditis elegans. The miRNA-processing enzyme DRSH-1 is involved in both the acquisition and the inheritance of hormesis, whereas worm-specific Argonaute (WAGO) proteins, which function with endo-siRNAs, are involved only in its inheritance. Further analyses demonstrate that the miRNA production system in the neuron and the small RNA transport machinery in the intestine are both essential for its acquisition and that both the transport of small RNAs in the germline and the germline Argonaute HRDE-1 complex are required for its inheritance. Our results thus demonstrate that overlapping and distinct roles of small RNA systems in the acquisition and inheritance of hormesis effects.

Neuronal DAF-16-to-intestinal DAF-16 communication underlies organismal lifespan extension in C. elegans.

Previous studies have revealed the importance of inter-tissue communications for lifespan regulation. However, the inter-tissue network responsible for lifespan regulation is not well understood, even in a simple organism Caenorhabditis elegans. To understand the mechanisms underlying systemic lifespan regulation, we focused on lifespan regulation by the insulin/insulin-like growth factor-1 signaling (IIS) pathway; IIS reduction activates the DAF-16/FOXO transcription factor, which results in lifespan extension. Our tissue-specific knockdown and knockout analyses demonstrated that IIS reduction in neurons and the intestine markedly extended lifespan. DAF-16 activation in neurons resulted in DAF-16 activation in the intestine and vice versa. Our dual gene manipulation method revealed that intestinal and neuronal DAF-16 mediate longevity induced by daf-2 knockout in neurons and the intestine, respectively. In addition, the systemic regulation of intestinal DAF-16 required the IIS pathway in intestinal and neurons. Collectively, these results highlight the importance of the neuronal DAF-16-to-intestinal DAF-16 communication for organismal lifespan regulation.

Intestine-to-Germline Transmission of Epigenetic Information Intergenerationally Ensures Systemic Stress Resistance in C. elegans.

Changes in epigenetic states affect organismal homeostasis, including stress resistance. However, the mechanisms coordinating epigenetic states and systemic stress resistance remain largely unknown. Here, we identify the intestine-to-germline communication of epigenetic states, which intergenerationally enhances stress resistance in C. elegans. The alterations in epigenetic states by deficiency of the histone H3K4me3 modifier ASH-2 in the intestine or germline increase organismal stress resistance, which is abrogated by knockdown of the H3K4 demethylase RBR-2. Remarkably, the increase in stress resistance induced by ASH-2 deficiency in the intestine is abrogated by RBR-2 knockdown in the germline, suggesting the intestine-to-germline transmission of epigenetic information. This communication from intestine to germline in the parental generation increases stress resistance in the next generation. Moreover, the intertissue communication is mediated partly by transcriptional regulation of F08F1.3. These results reveal that intertissue communication of epigenetic information provides mechanisms for intergenerational regulation of systemic stress resistance.

1-[4-(N-Benzylamino)phenyl]-3-phenylurea derivatives as a new class of hypoxia-inducible factor-1alpha inhibitors.

A series of 1-[4-(N-benzylamino)phenyl]-3-phenylurea derivatives 2a-r were synthesized as HIF-1alpha inhibitors. Among the compounds synthesized, compound 2k was found to be a potent inhibitor against HIF-1alpha accumulation under hypoxic condition and inhibited the hypoxia-induced HIF-1 transcriptional activity in HEK293 cells (IC(50)=7.2 microM). Furthermore, compound 2k suppressed the hypoxia-induced secretion of VEGF in HeLa cells (IC(50)=15 microM).

Molecular mechanisms regulating lifespan and environmental stress responses.

Throughout life, organisms are subjected to a variety of environmental perturbations, including temperature, nutrient conditions, and chemical agents. Exposure to external signals induces diverse changes in the physiological conditions of organisms. Genetically identical individuals exhibit highly phenotypic variations, which suggest that environmental variations among individuals can affect their phenotypes in a cumulative and inhomogeneous manner. The organismal phenotypes mediated by environmental conditions involve development, metabolic pathways, fertility, pathological processes, and even lifespan. It is clear that genetic factors influence the lifespan of organisms. Likewise, it is now increasingly recognized that environmental factors also have a large impact on the regulation of aging. Multiple studies have reported on the contribution of epigenetic signatures to the long-lasting phenotypic effects induced by environmental signals. Nevertheless, the mechanism of how environmental stimuli induce epigenetic changes at specific loci, which ultimately elicit phenotypic variations, is still largely unknown. Intriguingly, in some cases, the altered phenotypes associated with epigenetic changes could be stably passed on to the next generations. In this review, we discuss the environmental regulation of organismal viability, that is, longevity and stress resistance, and the relationship between this regulation and epigenetic factors, focusing on studies in the nematode C. elegans.

Cholesterol regulates DAF-16 nuclear localization and fasting-induced longevity in C. elegans.

Cholesterol has attracted significant attention as a possible lifespan regulator. It has been reported that serum cholesterol levels have an impact on mortality due to age-related disorders such as cardiovascular disease. Diet is also known to be an important lifespan regulator. Dietary restriction retards the onset of age-related diseases and extends lifespan in various organisms. Although cholesterol and dietary restriction are known to be lifespan regulators, it remains to be established whether cholesterol is involved in dietary restriction-induced longevity. Here, we show that cholesterol deprivation suppresses longevity induced by intermittent fasting, which is one of the dietary restriction regimens that effectively extend lifespan. We also found that cholesterol is required for the fasting-induced upregulation of transcriptional target genes such as the insulin/IGF-1 pathway effector DAF-16 and that cholesterol deprivation suppresses the long lifespan of the insulin/IGF-1 receptor daf-2 mutant. Remarkably, we found that cholesterol plays an important role in the fasting-induced nuclear accumulation of DAF-16. Moreover, knockdown of the cholesterol-binding protein NSBP-1, which has been shown to bind to DAF-16 in a cholesterol-dependent manner and to regulate DAF-16 activity, suppresses both fasting-induced longevity and DAF-16 nuclear accumulation. Furthermore, this suppression was not additive to the cholesterol deprivation-induced suppression, which suggests that NSBP-1 mediates, at least in part, the action of cholesterol to promote fasting-induced longevity and DAF-16 nuclear accumulation. These findings identify a novel role for cholesterol in the regulation of lifespan.

Environmental stresses induce transgenerationally inheritable survival advantages via germline-to-soma communication in Caenorhabditis elegans.

Hormesis is a biological phenomenon, whereby exposure to low levels of toxic agents or conditions increases organismal viability. It thus represents a beneficial aspect of adaptive responses to harmful environmental stimuli. Here we show that hormesis effects induced in the parental generation can be passed on to the descendants in Caenorhabditis elegans. Animals subjected to various stressors during developmental stages exhibit increased resistance to oxidative stress and proteotoxicity. The increased resistance is transmitted to the subsequent generations grown under unstressed conditions through epigenetic alterations. Our analysis reveal that the insulin/insulin-like growth factor (IGF) signalling effector DAF-16/FOXO and the heat-shock factor HSF-1 in the parental somatic cells mediate the formation of epigenetic memory, which is maintained through the histone H3 lysine 4 trimethylase complex in the germline across generations. The elicitation of memory requires the transcription factor SKN-1/Nrf in somatic tissues. We propose that germ-to-soma communication across generations is an essential framework for the transgenerational inheritance of acquired traits, which provides the offspring with survival advantages to deal with environmental perturbation.

Lifespan-regulating genes in C. elegans.

The molecular mechanisms underlying the aging process have garnered much attention in recent decades because aging is the most significant risk factor for many chronic diseases such as type 2 diabetes and cancer. Until recently, the aging process was not considered to be an actively regulated process; therefore, discovering that the insulin/insulin-like growth factor-1 signaling pathway is a lifespan-regulating genetic pathway in Caenorhabditis elegans was a major breakthrough that changed our understanding of the aging process. Currently, it is thought that animal lifespans are influenced by genetic and environmental factors. The genes involved in lifespan regulation are often associated with major signaling pathways that link the rate of aging to environmental factors. Although many of the major mechanisms governing the aging process have been identified from studies in short-lived model organisms such as yeasts, worms and flies, the same mechanisms are frequently observed in mammals, indicating that the genes and signaling pathways that regulate lifespan are highly conserved among different species. This review summarizes the lifespan-regulating genes, with a specific focus on studies in C. elegans.

A fasting-responsive signaling pathway that extends life span in C. elegans.

Intermittent fasting is one of the most effective dietary restriction regimens that extend life span in C. elegans and mammals. Fasting-stimulus responses are key to the longevity response; however, the mechanisms that sense and transduce the fasting stimulus remain largely unknown. Through a comprehensive transcriptome analysis in C. elegans, we find that along with the FOXO transcription factor DAF-16, AP-1 (JUN-1/FOS-1) plays a central role in fasting-induced transcriptional changes. KGB-1, one of the C. elegans JNKs, acts as an activator of AP-1 and is activated in response to fasting. KGB-1 and AP-1 are involved in intermittent fasting-induced longevity. Fasting-induced upregulation of the components of the SCF E3 ubiquitin ligase complex via AP-1 and DAF-16 enhances protein ubiquitination and reduces protein carbonylation. Our results thus identify a fasting-responsive KGB-1/AP-1 signaling pathway, which, together with DAF-16, causes transcriptional changes that mediate longevity, partly through regulating proteostasis.

Suppression of hypoxia-induced HIF-1alpha accumulation by VEGFR inhibitors: Different profiles of AAL993 versus SU5416 and KRN633.

The hypoxia-inducible factor (HIF) is a heterodimeric basic helix-loop-helix transcriptional factor and the activated HIF plays pivotal roles in various pathological conditions, including inflammation and cancer. HIF-1alpha overexpression has been observed in many common human cancers, including brain, breast, colon, lung, ovary, and prostate, and HIF-mediated genes, such as vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and insulin-like growth factor (IGF)-1, are associated with tumor angiogenesis, metastasis, and invasion. Therefore, the pro-oncogenic protein HIF is a novel target of cancer therapy. We examined the effects of VEGFR inhibitors, AAL993, SU5416, and KRN633, on suppression of HIF-1alpha accumulation under the hypoxic condition. We found that VEGFR tyrosine kinase inhibitors, AAL993, SU5416, and KRN633, possess dual functions: inhibition of VEGFR signaling and HIF-1alpha expression under the hypoxic condition. The detailed mechanistic study indicated that SU5416 and KRN633 suppressed HIF-1alpha expression through inhibition of both Akt and ERK phosphorylation signaling pathways, whereas AAL993 suppressed HIF-1alpha expression through ERK inhibition without affecting Akt phosphorylation.

de novo design and synthesis of N-benzylanilines as new candidates for VEGFR tyrosine kinase inhibitors.

N-Benzylanilines were designed and synthesized as vascular endothelial growth factor (VEGF)-2 inhibitors using de novo drug design systems based on the X-ray structure of VEGFR-2 kinase domain. Among compounds synthesized, compound showed the most potent inhibitory activity toward VEGFR-2 (KDR) tyrosine kinase and its IC(50) value was 0.57 microM.