Mitochondria: Masters of Epigenetics.

Accumulating evidence argues that aging exerts a profound influence on epigenetics, and vice versa. A pair of studies by Merkwirth et al. and Tian et al. now provide insights on how mitochondrial stress experienced by C. elegans larvae propagates a specific and persistent epigenetic response that protects adult cells and extends lifespan.

Stalking the link between reproduction and aging: After decades of research, it still remains a mystery whether and how reproduction drives the process of aging: After decades of research, it still remains a mystery whether and how reproduction drives the process of aging.

The hypothesis that aging and number of offspring are linked with each other has attracted much attention and research, but evidence for it remains elusive.

Dehydration triggers ecdysone-mediated recognition-protein priming and elevated anti-bacterial immune responses in Drosophila Malpighian tubule renal cells.

BACKGROUND: Drosophila is a powerful model for the study of factors modulating innate immunity. This study examines the effect of water-loss dehydration on innate immune responsiveness in the Drosophila renal system (Malpighian tubules; MTs), and how this leads to elevated host defense and contributes to immunosenescence. RESULTS: A short period of desiccation-elevated peptidoglycan recognition protein-LC (PGRP-LC) expression in MTs, increased antimicrobial peptide (AMP) gene induction, and protected animals from bacterial infection. We show that desiccation increased ecdysone synthesis in MTs, while inhibition of ecdysone synthesis or ecdysone receptor expression, specifically within MTs, prevented induction of PGRP-LC and reduced protection from bacterial infection. Additionally, aged flies are constitutively water-stressed and have elevated levels of ecdysone and PGRP-LC. Conversely, adults aged at high relative humidity show less water loss and have reduced expression of PGRP-LC and AMPs. CONCLUSIONS: The Drosophila renal system is an important contributor to host defense and can modulate immune responses in an organ autonomous manner, responding to environmental changes such as desiccation. Desiccation primes immune responsiveness by elevating PGRP-LC expression specifically in MTs. In response to desiccation, ecdysone is produced in MTs and acts in a paracrine fashion to increase PGRP-LC expression, immune responsiveness, and improve host defense. This activity of the renal system may contribute to the immunosenescence observed in Drosophila.

Ecdysone Elicits Chronic Renal Impairment via Mineralocorticoid-Like Pathogenic Activities.

BACKGROUND/AIMS: Ecdysteroids are steroidal insect molting hormones that also exist in herbs. Ecdysteroid-containing adaptogens have been popularly used to improve well-being and by bodybuilders for muscle growth. However, the use of ecdysone in mammals is also associated with kidney growth and enlargement, indications of disturbed kidney homeostasis. The underlying pathogenic mechanism remains to be clarified. METHODS: Virtual screening tools were employed to identify compounds that are homologous to ecdysone and to predict putative ecdysone-interacting proteins. The kidney effect of ecdysone was examined in vitro and in vivo and compared with that of aldosterone. Cellular apoptosis was estimated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Cell motility was assessed by scratch-wound cell migration assay. Blood urea nitrogen was measured to evaluate renal function. Western immunblot analysis was employed to determine the expression profile of interested proteins. RESULTS: Computational molecular structure analysis revealed that ecdysone is highly homologous to aldosterone. Moreover, virtual screening based on compound-protein interaction profiles identified the Mineralocorticoid Receptor (MR) to potentially interact with ecdysone. Accordingly, to assess potential biological functions of ecdysone in mammals, ecdysone was applied to mineralocorticoid-sensitive inner medullar collecting duct cells. Ecdysone induced mesenchymal accumulation of extracellular matrix and epithelial dedifferentiation characterized by de novo expression of α-smooth muscle actin. In addition, ecdysone elicited cellular apoptosis and retarded cell motility, akin to the effect of aldosterone. In vivo, daily treatment of mice with ecdysone increased cell apoptosis in the kidney, impaired renal function and elicited early signs of renal fibrogenesis, marked by deposition of collagen and fibronectin in tubulointerstitium, reminiscent of the action of aldosterone. The MR signaling pathway is likely responsible for the cellular and pathobiological effects of ecdysone, as evidenced by strong ecdysone-induced MR nuclear translocation in renal tubular cells both in vitro and in vivo, while blockade of MR by concomitant spironolactone treatment largely abolished the detrimental effects of ecdysone. CONCLUSION: Our findings suggest that ecdysone induces mineralocorticoid-dependent activities that impair renal function and elicit renal injury.

Unraveling the Molecular Mechanism of Immunosenescence in Drosophila.

A common feature of the aging process is a decline in immune system performance. Extensive research has sought to elucidate how changes in adaptive immunity contribute to aging and to provide evidence showing that changes in innate immunity have an important role in the overall decline of net immune function. Drosophila is an emerging model used to address questions related to immunosenescence via research that integrates its capacity for genetic dissection of aging with groundbreaking molecular biology related to innate immunity. Herein, we review information on the immunosenescence of Drosophila and suggest its possible mechanisms that involve changes in insulin/IGF(insulin-like growth factor)-1 signaling, hormones such as juvenile hormone and 20-hydroxyecdysone, and feedback system degeneration. Lastly, the emerging role of microbiota on the regulation of immunity and aging in Drosophila is discussed.

Ecdysone triggered PGRP-LC expression controls Drosophila innate immunity.

Throughout the animal kingdom, steroid hormones have been implicated in the defense against microbial infection, but how these systemic signals control immunity is unclear. Here, we show that the steroid hormone ecdysone controls the expression of the pattern recognition receptor PGRP-LC in Drosophila, thereby tightly regulating innate immune recognition and defense against bacterial infection. We identify a group of steroid-regulated transcription factors as well as two GATA transcription factors that act as repressors and activators of the immune response and are required for the proper hormonal control of PGRP-LC expression. Together, our results demonstrate that Drosophila use complex mechanisms to modulate innate immune responses, and identify a transcriptional hierarchy that integrates steroid signalling and immunity in animals.

Total Solid-Phase Synthesis of Biologically Active Drosophila Insulin-Like Peptide 2 (DILP2).

In the fruit fly Drosophila melanogaster, there are eight insulin-like peptides (DILPs) with DILPs 1-7 interacting with a sole insulin-like receptor tyrosine kinase (DInR) while DILP8 interacts with a single G protein-coupled receptor (GPCR), Lgr3. Loss-of-function dilp mutation studies show that the neuropeptide DILP2 has a key role in carbohydrate and lipid metabolism as well as longevity and reproduction. A better understanding of the processes whereby DILP2 mediates its specific actions is required. Consequently we undertook to prepare DILP2 as part of a larger, detailed structure-function relationship study. Use of our well-established insulin-like peptide synthesis protocol that entails separate solid phase assembly of each of the A- and B-chains with selective cysteine S-protection followed by sequential S-deprotection and simultaneous disulfide bond formation produced DILP2 in good overall yield and high purity. The synthetic DILP2 was shown to induce significant DInR phosphorylation and downstream signalling, with it being more potent than human insulin. This peptide will be a valuable tool to provide further insights into its binding to the insulin receptor, the subsequent cell signalling and role in insect metabolism.

Activin signaling targeted by insulin/dFOXO regulates aging and muscle proteostasis in Drosophila.

Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling.

Fine-scale mapping of natural variation in fly fecundity identifies neuronal domain of expression and function of an aquaporin.

To gain insight into the molecular genetic basis of standing variation in fitness related traits, we identify a novel factor that regulates the molecular and physiological basis of natural variation in female Drosophila melanogaster fecundity. Genetic variation in female fecundity in flies derived from a wild orchard population is heritable and largely independent of other measured life history traits. We map a portion of this variation to a single QTL and then use deficiency mapping to further refine this QTL to 5 candidate genes. Ubiquitous expression of RNAi against only one of these genes, an aquaporin encoded by Drip, reduces fecundity. Within our mapping population Drip mRNA level in the head, but not other tissues, is positively correlated with fecundity. We localize Drip expression to a small population of corazonin producing neurons located in the dorsolateral posterior compartments of the protocerebrum. Expression of Drip-RNAi using both the pan-neuronal ELAV-Gal4 and the Crz-Gal4 drivers reduces fecundity. Low-fecundity RILs have decreased Crz expression and increased expression of pale, the enzyme encoding the rate-limiting step in the production of dopamine, a modulator of insect life histories. Taken together these data suggest that natural variation in Drip expression in the corazonin producing neurons contributes to standing variation in fitness by altering the concentration of two neurohormones.

Minibrain/Dyrk1a regulates food intake through the Sir2-FOXO-sNPF/NPY pathway in Drosophila and mammals.

Feeding behavior is one of the most essential activities in animals, which is tightly regulated by neuroendocrine factors. Drosophila melanogaster short neuropeptide F (sNPF) and the mammalian functional homolog neuropeptide Y (NPY) regulate food intake. Understanding the molecular mechanism of sNPF and NPY signaling is critical to elucidate feeding regulation. Here, we found that minibrain (mnb) and the mammalian ortholog Dyrk1a, target genes of sNPF and NPY signaling, [corrected] regulate food intake in Drosophila melanogaster and mice. In Drosophila melanogaster neuronal cells and mouse hypothalamic cells, sNPF and NPY modulated the mnb and Dyrk1a expression through the PKA-CREB pathway. Increased Dyrk1a activated Sirt1 to regulate the deacetylation of FOXO, which potentiated FOXO-induced sNPF/NPY expression and in turn promoted food intake. Conversely, AKT-mediated insulin signaling suppressed FOXO-mediated sNPF/NPY expression, which resulted in decreasing food intake. Furthermore, human Dyrk1a transgenic mice exhibited decreased FOXO acetylation and increased NPY expression in the hypothalamus, and [corrected] increased food intake. Our findings demonstrate that Mnb/Dyrk1a regulates food intake through the evolutionary conserved Sir2-FOXO-sNPF/NPY pathway in Drosophila melanogaster and mammals.

Juvenile hormone regulation of Drosophila aging.

BACKGROUND: Juvenile hormone (JH) has been demonstrated to control adult lifespan in a number of non-model insects where surgical removal of the corpora allata eliminates the hormone's source. In contrast, little is known about how juvenile hormone affects adult Drosophila melanogaster. Previous work suggests that insulin signaling may modulate Drosophila aging in part through its impact on juvenile hormone titer, but no data yet address whether reduction of juvenile hormone is sufficient to control Drosophila life span. Here we adapt a genetic approach to knock out the corpora allata in adult Drosophila melanogaster and characterize adult life history phenotypes produced by reduction of juvenile hormone. With this system we test potential explanations for how juvenile hormone modulates aging. RESULTS: A tissue specific driver inducing an inhibitor of a protein phosphatase was used to ablate the corpora allata while permitting normal development of adult flies. Corpora allata knockout adults had greatly reduced fecundity, inhibited oogenesis, impaired adult fat body development and extended lifespan. Treating these adults with the juvenile hormone analog methoprene restored all traits toward wildtype. Knockout females remained relatively long-lived even when crossed into a genotype that blocked all egg production. Dietary restriction further extended the lifespan of knockout females. In an analysis of expression profiles of knockout females in fertile and sterile backgrounds, about 100 genes changed in response to loss of juvenile hormone independent of reproductive state. CONCLUSIONS: Reduced juvenile hormone alone is sufficient to extend the lifespan of Drosophila melanogaster. Reduced juvenile hormone limits reproduction by inhibiting the production of yolked eggs, and this may arise because juvenile hormone is required for the post-eclosion development of the vitellogenin-producing adult fat body. Our data do not support a mechanism for juvenile hormone control of longevity simply based on reducing the physiological costs of egg production. Nor does the longevity benefit appear to function through mechanisms by which dietary restriction extends longevity. We identify transcripts that change in response to juvenile hormone independent of reproductive state and suggest these represent somatically expressed genes that could modulate how juvenile hormone controls persistence and longevity.

Insulin regulation of heart function in aging fruit flies.

Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. Here, we describe progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase dPTEN or the forkhead transcription factor dFOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology.

Combinatorial interventions in aging.

Insight on the underlying mechanisms of aging will advance our ability to extend healthspan, treat age-related pathology and improve quality of life. Multiple genetic and pharmacological manipulations extend longevity in different species, yet monotherapy may be relatively inefficient, and we have limited data on the effect of combined interventions. Here we summarize interactions between age-related pathways and discuss strategies to simultaneously retard these in different organisms. In some cases, combined manipulations additively increase their impact on common hallmarks of aging and lifespan, suggesting they quantitatively participate within the same pathway. In other cases, interactions affect different hallmarks, suggesting their joint manipulation may independently maximize their effects on lifespan and healthy aging. While most interaction studies have been conducted with invertebrates and show varying levels of translatability, the conservation of pro-longevity pathways offers an opportunity to identify 'druggable' targets relevant to multiple human age-associated pathologies.

Structural and biological properties of the Drosophila insulin-like peptide 5 show evolutionary conservation.

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel ß-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel ß-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.

A smell to die for.

How dietary restriction extends life span may involve anticipatory neurons. The odor of dietary yeast is sufficient to speed Drosophila aging while mutants of an odorant-binding protein extend life span.

The effects of a long-term psychosocial stress on reproductive indicators in the baboon.

Psychosocial stress is thought to negatively impact fecundity, but human studies are confounded by variation in nutrition and lifestyle. Baboons offer a useful model to test the effect of prolonged mild stress on reproductive indicators in a controlled setting. Following relocation from social groups to solitary housing, a previously documented stressful event for nonhuman primates, daily urine samples, tumescence, and menstrual bleeding were monitored in twenty baboons (Papio sp.) for 120-150 days. Specimens were assayed for estrone conjugates (E1C), pregnanediol-3-glucuronide (PDG), follicle-stimulating hormone (FSH), and cortisol. Linear mixed effects models examined (1) the effects of stress on frequency of anovulation, hormone levels, tumescence and cycle length, and (2) the relationship of cortisol with reproductive indicators. Despite cortisol levels indicative of stress, anovulation was negligible (1% in 102 cycles). PDG, FSH, cycle length, and tumescence declined during the first four cycles, but began recovery by the fifth. Cortisol was negatively associated with FSH but not associated with PDG, E1C or tumescence. Ovulation, E1C, and luteal phase length were not affected. Tumescence tracked changes in FSH and PDG, and thus may be a useful indicator of stress on the reproductive axis. Elevated cortisol was associated with reduced FSH, supporting a model of cortisol action at the hypothalamus rather than the gonad. After four to five menstrual cycles the reproductive indicators began recovery, suggesting adjustment to new housing conditions. In conclusion, individual housing is stressful for captive baboons, as reflected by cortisol and reproductive indicators, although ovulation, a relatively direct proxy for fecundity, is unaffected.

Drosophila germ-line modulation of insulin signaling and lifespan.

Ablation of germ-line precursor cells in Caenorhabditis elegans extends lifespan by activating DAF-16, a forkhead transcription factor (FOXO) repressed by insulin/insulin-like growth factor (IGF) signaling (IIS). Signals from the gonad might thus regulate whole-organism aging by modulating IIS. To date, the details of this systemic regulation of aging by the reproductive system are not understood, and it is unknown whether such effects are evolutionarily conserved. Here we report that eliminating germ cells (GCs) in Drosophila melanogaster increases lifespan and modulates insulin signaling. Long-lived germ-line-less flies show increased production of Drosophila insulin-like peptides (dilps) and hypoglycemia but simultaneously exhibit several characteristics of IIS impedance, as indicated by up-regulation of the Drosophila FOXO (dFOXO) target genes 4E-BP and l (2)efl and the insulin/IGF-binding protein IMP-L2. These results suggest that signals from the gonad regulate lifespan and modulate insulin sensitivity in the fly and that the gonadal regulation of aging is evolutionarily conserved.

Aging Regulated Through a Stability Model of Insulin/Insulin Growth Factor Receptor Function.

Mutations of the insulin-like receptor in Drosophila extend lifespan. New research suggests this receptor operates in two modes. The first extends lifespan while slowing reproduction and reducing growth. The second strongly extends lifespan without impairing growth or reproduction; it confers longevity assurance. The mutation that confers longevity assurance resides in the kinase insert domain, which contains a potential SH2 binding site for substrate proteins. We apply a recent model for the function of receptor tyrosine kinases to propose how insulin receptor structure can modulate aging. This concept hypothesizes that strong insulin-like ligands promote phosphorylation of high threshold substrate binding sites to robustly induce reproduction, which impairs survival as a consequence of trade-offs. Lower levels of receptor stimulation provide less kinase dimer stability, which reduces reproduction and extends lifespan by avoiding reproductive costs. Environmental conditions that favor diapause alter the expression of insulin ligands to further repress the stability of the interacting kinase domains, block phosphorylation of low threshold substrates and thus induce a unique molecular program that confers longevity assurance. Mutations of the insulin receptor that block low-phosphorylation site interactions, such as within the kinase insert domain, can extend lifespan while maintaining overall dimer stability. These flies are long-lived while maintaining reproduction and growth. The kinase insert domain of Drosophila provides a novel avenue from which to seek signaling of the insulin/insulin-like growth factor system of humans that modulate aging without impacting reproduction and growth, or incurring insulin resistance pathology.

Aging modulated by the Drosophila insulin receptor through distinct structure-defined mechanisms.

Mutations of the Drosophila melanogaster insulin/IGF signaling system slow aging, while also affecting growth and reproduction. To understand this pleiotropy, we produced an allelic series of single codon substitutions in the Drosophila insulin receptor, InR. We generated InR substitutions using homologous recombination and related each to emerging models of receptor tyrosine kinase structure and function. Three mutations when combined as trans-heterozygotes extended lifespan while retarding growth and fecundity. These genotypes reduced insulin-stimulated Akt phosphorylation, suggesting they impede kinase catalytic domain function. Among these genotypes, longevity was negatively correlated with egg production, consistent with life-history trade-off theory. In contrast, one mutation (InR353) was located in the kinase insert domain, a poorly characterized element found in all receptor tyrosine kinases. Remarkably, wild-type heterozygotes with InR353 robustly extended lifespan without affecting growth or reproduction and retained capacity to fully phosphorylate Akt. The Drosophila insulin receptor kinase insert domain contains a previously unrecognized SH2 binding motif. We propose the kinase insert domain interacts with SH2-associated adapter proteins to affect aging through mechanisms that retain insulin sensitivity and are independent of reproduction.

SIR2 calls upon the ER.

Resveratrol induces longevity in C. elegans through the action of SIR2. Recently published work shows that resveratrol induces genes of the unfolded protein stress response of the endoplasmic reticulum. Paradoxically, these stress genes are repressed by SIR2, suggesting that resveratrol increases life span by inhibiting this SIR2 action.