Trametinib ameliorates aging-associated gut pathology in Drosophila females by reducing Pol III activity in intestinal stem cells.

Pharmacological therapies are promising interventions to slow down aging and reduce multimorbidity in the elderly. Studies in animal models are the first step toward translation of candidate molecules into human therapies, as they aim to elucidate the molecular pathways, cellular mechanisms, and tissue pathologies involved in the anti-aging effects. Trametinib, an allosteric inhibitor of MEK within the Ras/MAPK (Ras/Mitogen-Activated Protein Kinase) pathway and currently used as an anti-cancer treatment, emerged as a geroprotector candidate because it extended lifespan in the fruit fly Drosophila melanogaster. Here, we confirm that trametinib consistently and robustly extends female lifespan, and reduces intestinal stem cell (ISC) proliferation, tumor formation, tissue dysplasia, and barrier disruption in guts in aged flies. In contrast, pro-longevity effects of trametinib are weak and inconsistent in males, and it does not influence gut homeostasis. Inhibition of the Ras/MAPK pathway specifically in ISCs is sufficient to partially recapitulate the effects of trametinib. Moreover, in ISCs, trametinib decreases the activity of the RNA polymerase III (Pol III), a conserved enzyme synthesizing transfer RNAs and other short, non-coding RNAs, and whose inhibition also extends lifespan and reduces gut pathology. Finally, we show that the pro-longevity effect of trametinib in ISCs is partially mediated by Maf1, a repressor of Pol III, suggesting a life-limiting Ras/MAPK-Maf1-Pol III axis in these cells. The mechanism of action described in this work paves the way for further studies on the anti-aging effects of trametinib in mammals and shows its potential for clinical application in humans.

Long-lasting geroprotection from brief rapamycin treatment in early adulthood by persistently increased intestinal autophagy.

The licensed drug rapamycin has potential to be repurposed for geroprotection. A key challenge is to avoid adverse side effects from continuous dosing. Here we show that geroprotective effects of chronic rapamycin treatment can be obtained with a brief pulse of the drug in early adulthood in female Drosophila and mice. In Drosophila, a brief, early rapamycin treatment of adults extended lifespan and attenuated age-related decline in the intestine to the same degree as lifelong dosing. Lasting memory of earlier treatment was mediated by elevated autophagy in intestinal enterocytes, accompanied by increased levels of intestinal LManV and lysozyme. Brief elevation of autophagy in early adulthood itself induced a long-term increase in autophagy. In mice, a 3-month, early treatment also induced a memory effect, with maintenance similar to chronic treatment, of lysozyme distribution, Man2B1 level in intestinal crypts, Paneth cell architecture and gut barrier function, even 6 months after rapamycin was withdrawn.

Sexual identity of enterocytes regulates autophagy to determine intestinal health, lifespan and responses to rapamycin.

Pharmacological attenuation of mTOR presents a promising route for delay of age-related disease. Here we show that treatment of Drosophila with the mTOR inhibitor rapamycin extends lifespan in females, but not in males. Female-specific, age-related gut pathology is markedly slowed by rapamycin treatment, mediated by increased autophagy. Treatment increases enterocyte autophagy in females, via the H3/H4 histone-Bchs axis, whereas males show high basal levels of enterocyte autophagy that are not increased by rapamycin feeding. Enterocyte sexual identity, determined by transformerFemale expression, dictates sexually dimorphic cell size, H3/H4-Bchs expression, basal rates of autophagy, fecundity, intestinal homeostasis and lifespan extension in response to rapamycin. Dimorphism in autophagy is conserved in mice, where intestine, brown adipose tissue and muscle exhibit sex differences in autophagy and response to rapamycin. This study highlights tissue sex as a determining factor in the regulation of metabolic processes by mTOR and the efficacy of mTOR-targeted, anti-aging drug treatments.

A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing.

Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. Here we show that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organisation, induces intestinal autophagy through transcriptional regulation, and prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription, and alters chromatin organisation in the small intestine, suggesting that the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.

Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila.

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention.

Lifespan Extension Via Dietary Restriction: Time to Reconsider the Evolutionary Mechanisms?

Dietary restriction (DR) is the most consistent environmental manipulation to extend lifespan. Originally thought to be caused by a reduction in caloric intake, recent evidence suggests that macronutrient intake underpins the effect of DR. The prevailing evolutionary explanations for the DR response are conceptualized under the caloric restriction paradigm, necessitating reconsideration of how or whether these evolutionary explanations fit this macronutrient perspective. In the authors' opinion, none of the current evolutionary explanations of DR adequately explain the intricacies of observed results; instead a context-dependent combination of these theories is suggested which is likely to reflect reality. In reviewing the field, it is proposed that the ability to track the destination of different macronutrients within the body will be key to establishing the relative roles of the competing theories. Understanding the evolution of the DR response and its ecological relevance is critical to understanding variation in DR responses and their relevance outside laboratory environments.

Sexual Dimorphisms in Innate Immunity and Responses to Infection in Drosophila melanogaster.

The sexes show profound differences in responses to infection and the development of autoimmunity. Dimorphisms in immune responses are ubiquitous across taxa, from arthropods to vertebrates. Drosophila melanogaster shows strong sex dimorphisms in immune system responses at baseline, upon pathogenic challenge, and over aging. We have performed an exhaustive survey of peer-reviewed literature on Drosophila immunity, and present a database of publications indicating the sex(es) analyzed in each study. While we found a growing interest in the community in adult immunity and in reporting both sexes, the main body of work in this field uses only one sex, or does not stratify by sex. We synthesize evidence for sexually dimorphic responses to bacterial, viral, and fungal infections. Dimorphisms may be mediated by distinct immune compartments, and we review work on sex differences in behavioral, epithelial, cellular, and systemic (fat body-mediated) immunity. Emerging work on sexually dimorphic aging of immune tissues, immune senescence, and inflammation are examined. We consider evolutionary drivers for sex differences in immune investment, highlight the features of Drosophila biology that make it particularly amenable to studies of immune dimorphisms, and discuss areas for future exploration.

Intestinal Fork Head Regulates Nutrient Absorption and Promotes Longevity.

Reduced activity of nutrient-sensing signaling networks can extend organismal lifespan, yet the underlying biology remains unclear. We show that the anti-aging effects of rapamycin and reduced intestinal insulin/insulin growth factor (IGF) signaling (IIS) require the Drosophila FoxA transcription factor homolog Fork Head (FKH). Intestinal FKH induction extends lifespan, highlighting a role for the gut. FKH binds to and is phosphorylated by AKT and Target of Rapamycin. Gut-specific FKH upregulation improves gut barrier function in aged flies. Additionally, it increases the expression of nutrient transporters, as does lowered IIS. Evolutionary conservation of this effect of lowered IIS is suggested by the upregulation of related nutrient transporters in insulin receptor substrate 1 knockout mouse intestine. Our study highlights a critical role played by FKH in the gut in mediating anti-aging effects of reduced IIS. Malnutrition caused by poor intestinal absorption is a major problem in the elderly, and a better understanding of the mechanisms involved will have important therapeutic implications for human aging.

Sex difference in pathology of the ageing gut mediates the greater response of female lifespan to dietary restriction.

Women live on average longer than men but have greater levels of late-life morbidity. We have uncovered a substantial sex difference in the pathology of the aging gut in Drosophila. The intestinal epithelium of the aging female undergoes major deterioration, driven by intestinal stem cell (ISC) division, while lower ISC activity in males associates with delay or absence of pathology, and better barrier function, even at old ages. Males succumb to intestinal challenges to which females are resistant, associated with fewer proliferating ISCs, suggesting a trade-off between highly active repair mechanisms and late-life pathology in females. Dietary restriction reduces gut pathology in aging females, and extends female lifespan more than male. By genetic sex reversal of a specific gut region, we induced female-like aging pathologies in males, associated with decreased lifespan, but also with a greater increase in longevity in response to dietary restriction.

Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila.

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.

Gender and longevity: why do men die earlier than women? Comparative and experimental evidence.

Sex differences in lifespan exist world-wide, with women outliving men by more than a decade in some countries. The gender gap is not a uniquely human phenomenon; most sexually reproducing species examined show sex differences in patterns of ageing, yet a comprehensive explanation does not exist. Here, we discuss how ageing responds to natural selection on traits that arise as a consequence of sexuality. Sexual dimorphisms in vertebrates are mediated by sex-steroids, such as androgens and oestrogens, and we examine their regulation of biological processes that can affect ageing and lifespan. The sexes can respond differently to dietary restriction and altered activity of nutrient-sensing pathways, with females showing a greater plasticity for life extension. We suggest that the cross-regulation of steroid hormone and nutrient-sensing signalling pathways is a promising process for further study in understanding the biological basis for the gender gap.

The Drosophila larva as a tool to study gut-associated macrophages: PI3K regulates a discrete hemocyte population at the proventriculus.

Immune cells not only patrol the body in the circulation but also importantly, associate with specific tissues, such as the intestinal epithelium. The complex interactions between immune cells and their target tissues are difficult to study and simple, genetically tractable models are lacking. Here, we present the first thorough characterization of gut-associated macrophages in Drosophila larvae. We analyze their gene expression, morphology, development and lineage and importantly, demonstrate that they are functional (phagocytic) macrophages. We test their regulation by phosphoinositide 3-kinase (PI3K) and show evidence that this pathway regulates the population size of gut hemocytes and their phagocytic activity, reminiscent of recent findings in mammalian colitis models. Our data suggest that PI3K signaling modifies the adhesive properties of hemocytes, a possible mechanism for gut-hemocyte regulation. These results demonstrate the potential of the Drosophila larva as a simple tool to uncover mechanisms regulating recruitment and maintenance of innate immune cells at their target tissues.

Drosophila hemocyte migration: an in vivo assay for directional cell migration.

This protocol describes an in vivo assay for random and directed hemocyte migration in Drosophila. Drosophila is becoming an increasingly powerful model system for in vivo cell migration analysis, combining unique genetic tools with translucency of the embryo and pupa, which allows direct imaging and traceability of different cell types. In the assay we present here, we make use of the hemocyte response to epithelium wounding to experimentally induce a transition from random to directed migration. Time-lapse confocal microscopy of hemocyte migration in untreated conditions provides a random cell migration assay that allows identification of molecular mechanisms involved in this complex process. Upon laser-induced wounding of the thorax epithelium, a rapid chemotactic response changes hemocyte migratory behavior into a directed migration toward the wound site. This protocol provides a direct comparison of cells during both types of migration in vivo, and combined with recently developed resources such as transgenic RNAi, is ideal for forward genetic screens.

An Fgf8-dependent bistable cell migratory event establishes CNS asymmetry.

Neuroanatomical and functional asymmetries are universal features of the vertebrate CNS, but how asymmetry is generated is unknown. Here we show that zebrafish fgf8 mutants do not elaborate forebrain asymmetries, demonstrated by the failure of the parapineal nucleus to migrate from its initial midline position to the left side of the brain. Local provision of Fgf8 restores the asymmetric migration of parapineal cells, usually to the left, irrespective of the location of the Fgf8 source. This laterality bias is due to left-sided Nodal signaling and when the bias in Nodal signaling is removed, parapineal cells migrate toward the source of Fgf8 protein. This study presents a mechanism for breaking neuroanatomical symmetry through Fgf8-dependent regulation of bistable left- or right-sided migration of the parapineal. The combined action of Fgf and Nodal signals ensures the establishment of neuroanatomical asymmetries with consistent laterality.

Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.

The mechanisms that establish behavioral, cognitive, and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate across the midline to form this left-sided nucleus. The parapineal subsequently innervates the left habenula, which derives from ventral epithalamic cells adjacent to the parapineal precursors. Ablation of cells in the left ventral epithalamus can reverse laterality in wild-type embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1 can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes. From these data, we propose that laterality is determined by a competitive interaction between the left and right epithalamus and that Nodal signaling biases the outcome of this competition.