Dhr96[1] mutation and maternal tudor[1] mutation increase life span and reduce the beneficial effects of mifepristone in mated female Drosophila.

Mating and receipt of male Sex Peptide hormone cause increased egg laying, increased midgut size and decreased life span in female Drosophila. Feeding mated females with the synthetic steroid mifepristone decreases egg production, reduces midgut size, and increases life span. Here, several gene mutations were assayed to investigate possible mechanisms for mifepristone action. Drosophila Dhr96 is a hormone receptor, and a key positive regulator of midgut lipid uptake and metabolism. Dhr96[1] null mutation increased female life span, and reduced the effects of mifepristone on life span, suggesting that Dhr96[1] mutation and mifepristone may act in part through the same mechanism. Consistent with this idea, lipidomics analysis revealed that mating increases whole-body levels of triglycerides and fatty-acids in triglycerides, and these changes are reversed by mifepristone. Maternal tudor[1] mutation results in females that lack the germ-line and produce no eggs. Maternal tudor[1] mutation increased mated female life span, and reduced but did not eliminate the effects of mating and mifepristone on life span. This indicates that decreased egg production may be related to the life span benefits of mifepristone, but is not essential. Mifepristone increases life span in w[1118] mutant mated females, but did not increase life span in w[1118] mutant virgin females. Mifepristone decreased egg production in w[1118] mutant virgin females, indicating that decreased egg production is not sufficient for mifepristone to increase life span. Mifepristone increases life span in virgin females of some, but not all, white[+] and mini-white[+] strains. Backcrossing of mini-white[+] transgenes into the w[1118] background was not sufficient to confer a life span response to mifepristone in virgin females. Taken together, the data support the hypothesis that mechanisms for mifepristone life span increase involve reduced lipid uptake and/or metabolism, and suggest that mifepristone may increase life span in mated females and virgin females through partly different mechanisms.

Markers and mechanisms of death in Drosophila.

Parameters correlated with age and mortality in Drosophila melanogaster include decreased negative geotaxis and centrophobism behaviors, decreased climbing and walking speed, and darkened pigments in oenocytes and eye. Cessation of egg laying predicts death within approximately 5 days. Endogenous green fluorescence in eye and body increases hours prior to death. Many flies exhibit erratic movement hours before death, often leading to falls. Loss of intestinal barrier integrity (IBI) is assayed by feeding blue dye ("Smurf" phenotype), and Smurf flies typically die within 0-48 h. Some studies report most flies exhibit Smurf, whereas multiple groups report most flies die without exhibiting Smurf. Transgenic reporters containing heat shock gene promoters and innate immune response gene promoters progressively increase expression with age, and partly predict remaining life span. Innate immune reporters increase with age in every fly, prior to any Smurf phenotype, in presence or absence of antibiotics. Many flies die on their side or supine (on their back) position. The data suggest three mechanisms for death of Drosophila. One is loss of IBI, as revealed by Smurf assay. The second is nervous system malfunction, leading to erratic behavior, locomotor malfunction, and falls. The aged fly is often unable to right itself after a fall to a side-ways or supine position, leading to inability to access the food and subsequent dehydration/starvation. Finally, some flies die upright without Smurf phenotype, suggesting a possible third mechanism. The frequency of these mechanisms varies between strains and culture conditions, which may affect efficacy of life span interventions.

A screen of small molecule and genetic modulators of life span in female Drosophila identifies etomoxir, RH5849 and unanticipated temperature effects.

Mifepristone increases life span in female Drosophila melanogaster, and its molecular target(s) remain unclear. Here small molecule and genetic interventions were tested for ability to mimic mifepristone, or to decrease life span in a way that can be rescued by mifepristone. Etomoxir inhibits lipid metabolism, and significantly increased life span in virgin and mated females, but not males, at 50 µM concentration. Pioglitazone is reported to activate both mammalian PPARγ and its Drosophila homolog Eip75B. Pioglitazone produced minor and inconsistent benefits for female Drosophila life span, and only at the lowest concentrations tested. Ecdysone is a Drosophila steroid hormone reported to regulate responses to mating, and RH5849 is a potent mimic of ecdysone. RH5849 reduced virgin female life span, and this was partly rescued by mifepristone. Mifepristone did not compete with RH5849 for activation of an ecdysone receptor (EcR)-responsive transgenic reporter, indicating that the relevant target for mifepristone is not EcR. The conditional GAL4/GAL80ts system was used in attempt to test the effect of an Eip75B RNAi construct on female life span. However, the 29°C temperature used for induction reduced or eliminated mating-induced midgut hypertrophy, the negative life span effects of mating, and the positive life span effects of mifepristone. Even when applied after mating was complete, a shift to 29°C temperature reduced mating-induced midgut hypertrophy by half, and the life span effects of mating by 4.8-fold. Taken together, these results identify promising small molecules for further analysis, and inform the design of experiments involving the GAL4/GAL80ts system.

Mifepristone Increases Life Span in Female Drosophila Without Detectable Antibacterial Activity.

Mifepristone dramatically increases the life span of mated female Drosophila while reducing the expression of innate immune response genes. Previous results indicated that mifepristone also reduced the load of aero-tolerant bacteria in mated females. Experiments were conducted to further investigate the possible role of bacteria in mifepristone life span effects. Life span was assayed in flies grown from sterilized eggs on autoclaved media and in normally cultured controls in two independent assays. Sterilization increased mated female life span (+8.3% and +57%, respectively), and the effect of mifepristone was additive (+53% and +93%, respectively). High-throughput sequencing of 16S sequences revealed that sterilization reduced the abundance of multiple species and the classes Bacteroidia, Bacilli, Actinobacteria, and Cytophagia. By contrast, mifepristone caused no decreases and instead increased the abundance of three species. Five aero-tolerant bacterial species were cultured from extracts of mated female flies, including both Gram-positive and Gram-negative species (Acetobacter sicerae, Enterococcus faecalis, Lactobacillus plantarum, Serratia rubidea, and Paenibacillus glucanolyticus). There was no detectable effect of mifepristone on the growth of these bacteria in vitro, indicating that mifepristone does not have a direct antibiotic effect. To test if antibiotics could mimic the effects of mifepristone in vivo, mated female flies were treated throughout adult life span with high concentrations of the individual antibiotics doxycycline, ampicillin, kanamycin, and streptomycin, in replicate experiments. No significant effect on life span was observed for ampicillin, kanamycin, or streptomycin, and an inconsistent benefit was observed for doxycycline. Finally, supplementation of media with Enterococcus faecalis did not alter adult female life span in the presence or absence of mifepristone. Taken together, the results indicate the life span benefits of mifepristone are not due to an antibiotic effect.

Mifepristone Increases Life Span of Virgin Female Drosophila on Regular and High-fat Diet Without Reducing Food Intake.

Background: The synthetic steroid mifepristone is reported to have anti-obesity and anti-diabetic effects in mammals on normal and high-fat diets (HFD). We previously reported that mifepristone blocks the negative effect on life span caused by mating in female Drosophila melanogaster. Methods: Here we asked if mifepristone could protect virgin females from the life span-shortening effect of HFD. Mifepristone was assayed for effects on life span in virgin females, in repeated assays, on regular media and on media supplemented with coconut oil (HFD). The excrement quantification (EX-Q) assay was used to measure food intake of the flies after 12 days mifepristone treatment. In addition, experiments were conducted to compare the effects of mifepristone in virgin and mated females, and to identify candidate mifepristone targets and mechanisms. Results: Mifepristone increased life span of virgin females on regular media, as well as on media supplemented with either 2.5 or 5% coconut oil. Food intake was not reduced in any assay, and was significantly increased by mifepristone in half of the assays. To ask if mifepristone might rescue virgin females from all life span-shortening stresses, the oxidative stressor paraquat was tested, and mifepristone produced little to no rescue. Analysis of extant metabolomics and transcriptomics data suggested similarities between effects of mifepristone in virgin and mated females, including reduced tryptophan breakdown and similarities to dietary restriction. Bioinformatics analysis identified candidate mifepristone targets, including transcription factors Paired and Extra-extra. In addition to shortening life span, mating also causes midgut hypertrophy and activation of the lipid metabolism regulatory factor SREBP. Mifepristone blocked the increase in midgut size caused by mating, but did not detectably affect midgut size in virgins. Finally, mating increased activity of a SREBP reporter in abdominal tissues, as expected, but reporter activity was not detectably reduced by mifepristone in either mated or virgin females. Conclusion: Mifepristone increases life span of virgin females on regular and HFD without reducing food intake. Metabolomics and transcriptomics analyses suggest some similar effects of mifepristone between virgin and mated females, however reduced midgut size was observed only in mated females. The results are discussed regarding possible mifepristone mechanisms and targets.

In vivo assay and modelling of protein and mitochondrial turnover during aging.

To maintain homoeostasis, cells must degrade damaged or misfolded proteins and synthesize functional replacements. Maintaining a balance between these processes, known as protein turnover, is necessary for stress response and cellular adaptation to a changing environment. Damaged mitochondria must also be removed and replaced. Changes in protein and mitochondrial turnover are associated with aging and neurodegenerative disease, making it important to understand how these processes occur and are regulated in cells. To achieve this, reliable assays of turnover must be developed. Several methods exist, including pulse-labelling with radioactive or stable isotopes and strategies making use of fluorescent proteins, each with their own advantages and limitations. Both cell culture and live animals have been used for these studies, in systems ranging from yeast to mammals. In vivo assays are especially useful for connecting turnover to aging and disease. With its short life cycle, suitability for fluorescent imaging, and availability of genetic tools, Drosophila melanogaster is particularly well suited for this kind of analysis.

Metabolic Signatures of Life Span Regulated by Mating, Sex Peptide, and Mifepristone/RU486 in Female Drosophila melanogaster.

Mating and transfer of male sex peptide (SP), or transgenic expression of SP, causes inflammation and decreased life span in female Drosophila. Mifepristone rescues these effects, yielding dramatic increases in life span. Here targeted metabolomics data were integrated with further analysis of extant transcriptomic data. Each of 7 genes positively correlated with life span were expressed in the brain or eye and involved regulation of gene expression and signaling. Genes negatively correlated with life span were preferentially expressed in midgut and involved protein degradation, amino acid metabolism, and immune response. Across all conditions, life span was positively correlated with muscle breakdown product 1/3-methylhistidine and purine breakdown product urate, and negatively correlated with tryptophan breakdown product kynurenic acid, suggesting a SP-induced shift from somatic maintenance/turnover pathways to the costly production of energy and lipids from dietary amino acids. Some limited overlap was observed between genes regulated by mifepristone and genes known to be regulated by ecdysone; however, mifepristone was unable to compete with ecdysone for activation of an ecdysone-responsive transgenic reporter. In contrast, genes regulated by mifepristone were highly enriched for genes regulated by juvenile hormone (JH), and mifepristone rescued the negative effect of JH analog methoprene on life span in adult virgin females. The data indicate that mifepristone increases life span and decreases inflammation in mated females by antagonizing JH signaling downstream of male SP. Finally, mifepristone increased life span of mated, but not unmated, Caenorhabditis elegans, in 2 of 3 trials, suggesting possible evolutionary conservation of mifepristone mechanisms.

Incorporating antagonistic pleiotropy into models for molecular replicators.

In modern cells, chromosomal genes composed of DNA encode multi-subunit protein/RNA complexes that catalyze the replication of the chromosome and cell. One prevailing theory for the origin of life posits an early stage involving self-replicating macromolecules called replicators, which can be considered genes capable of self-replication. One prevailing theory for the genetics of aging in humans and other organisms is antagonistic pleiotropy, which posits that a gene can be beneficial in one context, and detrimental in another context. We previously reported that the conceptual simplicity of molecular replicators facilitates the generation of two simple models involving antagonistic pleiotropy. Here a third model is proposed, and each of the three models is presented with improved definition of the time variable. Computer simulations were used to calculate the proliferation of a hypothetical two-subunit replicator (AB), when one of the two subunits (B) exhibits antagonistic pleiotropy, leading to an advantage for B to be unstable. In model 1, instability of B yields free A subunits, which in turn stimulate the activity of other AB replicators. In model 2, B is lost and sometimes replaced by a more active mutant form, B'. In model 3, B becomes damaged and loses activity, and its instability allows it to be replaced by a new B. For each model, conditions were identified where instability of B was detrimental, and where instability of B was beneficial. The results are consistent with the hypothesis that antagonistic pleiotropy can promote molecular instability and system complexity, and provide further support for a model linking aging and evolution.

The proteasome beta 5 subunit is essential for sexually divergent adaptive homeostatic responses to oxidative stress in D. melanogaster.

Our studies center on the physiological phenomenon of adaptive homeostasis in which very low, signaling levels of an oxidant can induce transient expansion of the baseline homeostatic range of protective mechanisms, resulting in transient stress protection. The 20S proteasome is a major element of such inducible defense enzymes against oxidative stress but the relative importance of each of its three proteolytic subunits, ß1, ß2, and ß5, is only poorly understood. We focused the present studies on determining the role of the ß5 subunit in adaptation, survival, and lifespan. Decreased expression of the 20S proteasome ß5 subunit (with RNAi) blocked the adaptive increase in the catalytic activities of the 20S proteasome response to signaling levels of H2O2 in female flies. Similarly, female-specific adaptive increases in survival following H2O2 pretreatment and subsequent toxic challenge was blocked. In contrast, direct overexpression of the 20S proteasome ß5 subunit enabled an increased 20S proteasome proteolytic response, but prevented further adaptive homeostatic increases through H2O2 signaling, indicating there is a maximum 'ceiling' to the adaptive response. Males showed no adaptive change in proteasomal levels or activity whatsoever with H2O2 pretreatment and exhibited no significant impact upon the other 2 proteolytic subunits of the proteasome. However, chronic loss of the ß5 subunit led to shortened lifespan in both sexes. Our exploration of the importance of the 20S proteasome ß5 subunit in adaptive homeostasis highlights the interconnection between signal transduction pathways and regulated gene expression in sexually divergent responses to oxidative stimulation.

Analysis of Drosophila melanogaster Lifespan.

The laboratory fruit fly Drosophila melanogaster is one of the leading models for the study of aging. Whereas several behavioral and physiological biomarkers of aging have been identified for Drosophila, lifespan remains the most robust measure of aging rate. Aging and lifespan can be modulated by genetic alterations, as well as by drugs and dietary components, to reveal basic and conserved mechanisms of aging. Here methods are presented for Drosophila lifespan assay, including media preparation, supplementation of media with various drugs, culturing of the flies, passaging flies and recording deaths, and the analysis of lifespan data.

Sex differences in the response to oxidative and proteolytic stress.

Sex differences in diseases involving oxidative and proteolytic stress are common, including greater ischemic heart disease, Parkinson disease and stroke in men, and greater Alzheimer disease in women. Sex differences are also observed in stress response of cells and tissues, where female cells are generally more resistant to heat and oxidative stress-induced cell death. Studies implicate beneficial effects of estrogen, as well as cell-autonomous effects including superior mitochondrial function and increased expression of stress response genes in female cells relative to male cells. The p53 and forkhead box (FOX)-family genes, heat shock proteins (HSPs), and the apoptosis and autophagy pathways appear particularly important in mediating sex differences in stress response.

Behavioral and molecular markers of death in Drosophila melanogaster.

Fly movement was tracked through 3-dimensional (3D) space as the fly died, using either reflected visible light, reflected infrared (IR) light, or fly GFP fluorescence. Behaviors measured included centrophobism, negative geotaxis, velocity, and total activity. In addition, frequency of directional heading changes (FDHC) was calculated as a measure of erratic movement. Nine middle-aged flies were tracked as they died during normal aging, and fifteen young flies were tracked as they died from dehydration/starvation stress. Episodes of increased FDHC were observed 0-8 h prior to death for the majority of the flies. FDHC was also increased with age in flies with neuronal expression of a human Abeta42 protein fragment associated with Alzheimer's disease. Finally, green autofluorescence appeared in the eye and body immediately prior to and coincident with death, and fluorescence of GFP targeted to the retina increased immediately prior to and coincident with death. The results suggest the potential utility of FDHC, green autofluorescence, and retinal GFP as markers of neuronal malfunction and imminent death.

Effects of light on aging and longevity.

Increasing evidence suggests an important role for light in regulation of aging and longevity. UV radiation is a mutagen that can promote aging and decrease longevity. In contrast, NIR light has shown protective effects in animal disease models. In invertebrates, visible light can shorten or extend lifespan, depending on the intensity and wavelength composition. Visible light also impacts human health, including retina function, sleep, cancer and psychiatric disorders. Possible mechanisms of visible light include: controlling circadian rhythms, inducing oxidative stress, and acting through the retina to affect neuronal circuits and systems. Changes in artificial lighting (e.g., LEDs) may have implications for human health. It will be important to further explore the mechanisms of how light affects aging and longevity, and how light affects human health.

Models of Replicator Proliferation Involving Differential Replicator Subunit Stability.

Several models for the origin of life involve molecules that are capable of self-replication, such as self-replicating polymers composed of RNA or DNA or amino acids. Here we consider a hypothetical replicator (AB) composed of two subunits, A and B. Programs written in Python and C programming languages were used to model AB replicator abundance as a function of cycles of replication (iterations), under specified hypothetical conditions. Two non-exclusive models describe how a reduced stability for B relative to A can have an advantage for replicator activity and/or evolution by generating free A subunits. In model 1, free A subunits associate with AB replicators to create AAB replicators with greater activity. In simulations, reduced stability of B was beneficial when the replication activity of AAB was greater than two times the replication activity of AB. In model 2, the free A subunit is inactive for some number of iterations before it re-creates the B subunit. A re-creates the B subunit with an equal chance of creating B or B', where B' is a mutant that increases AB' replicator activity relative to AB. In simulations, at moderate number of iterations (< 15), a shorter survival time for B is beneficial when the stability of B is greater than the inactive time of A. The results are consistent with the hypothesis that reduced stability for a replicator subunit can be advantageous under appropriate conditions.

Sex-specific adaptive homeostasis in D. melanogaster depends on increased proteolysis by the 20S Proteasome: Data-in-Brief.

Adaptive homeostasis enables rapid cellular signaling, leading to transcriptional and translational modifications (Davies, 2016) [1]. The Proteasome is one of the main cellular proteolytic enzymes that plays an essential role in the rapid clearance of oxidatively damaged cellular proteins, and is highly responsive to oxidative stress. Upon exposure to even very low, signaling levels of oxidants, the predominant form of the Proteasome becomes the ATP-independent 20S proteasome that enables rapid clearance of damaged proteins. Subsequently there is also a concurrent upregulation of de novo 20S proteasome synthesis. These cellular adaptations not only ensure effective and efficient removal of damaged proteins, but prepare cells to better cope with future, more severe oxidative insults. Male and female Drosophila melanogaster fruit flies were pretreated with an adaptive amount of an oxidant (10 µM hydrogen peroxide or 0.5 µM paraquat) to assess the changes in proteolytic capacity and the role of the 20S proteasome. Additionally, the adaptive signaling by non-damaging amounts of hydrogen peroxide or paraquat) were used to assess changes in male and female fruit flies, following a subsequent more toxic amount of the two oxidants. Further analysis and detailed results about the adaptive role of the 20S proteasome in multiple D. melanogaster strains can be found in "Sexual Dimorphism in Oxidant-Induced Adaptive Homeostasis in Multiple Wild-Type D. melanogaster Strains" (Pomatto et al., 2018) [2].

Sexual Dimorphism and Aging Differentially Regulate Adaptive Homeostasis.

External and internal stimuli cause modifications to gene and biochemical pathways. In turn, demonstrating that biological systems continuously make short-term adaptations both to set-points, and to the range of "normal" capacity, due to mild conditional changes, or to subtoxic, nondamaging levels of chemical agents. This is termed as "Adaptive Homeostasis," defined with the following: "The transient expansion or contraction of the homeostatic range in response to exposure to sub-toxic, nondamaging, signaling molecules or events, or the removal or cessation of such molecules or events." Research from several laboratories, including our own, found that adaptive homeostasis declines with age in organisms as diverse as worms, flies, and mammals, and decreases with senescence in mammalian cell cultures. We suggest that diminishing adaptive homeostasis may play a causal role as a factor responsible for the aging phenotype. Furthermore, although studies of humans, animals, and model organisms are often limited to a single sex, and cell culture studies may even be conducted with lines whose donor's sex was unknown, studies reveal distinct sexual dimorphism in adaptive homeostasis. Interestingly, although young males and females may exhibit dramatic differences in adaptive capacities and/or preferences, these distinctions are lost with age as adaptive homeostasis patterns converge.

Sexual dimorphism in oxidant-induced adaptive homeostasis in multiple wild-type D. melanogaster strains.

Sexual dimorphism includes the physical and reproductive differences between the sexes, including differences that are conserved across species, ranging from the common fruit fly, Drosophila melanogaster, to humans. Sex-dependent variations in adaptive homeostasis, and adaptive stress responses may offer insight into the underlying mechanisms for male and female survival differences and into differences in chronic disease incidence and severity in humans. Earlier work showed sex-specific differences in adaptive responses to oxidative stressors in hybrid laboratory strains of D. melanogaster. The present study explored whether this phenomenon is also observed in wild-type D. melanogaster strains Oregon-R (Or-R) and Canton-S (Ca-S), as well as the common mutant reference strain w[1118], in order to better understand whether such findings are descriptive of D. melanogaster in general. Flies of each strain were pretreated with non-damaging, adaptive concentrations of hydrogen peroxide (H2O2) or of different redox cycling agents (paraquat, DMNQ, or menadione). Adaptive homeostasis, and changes in the expression of the Proteasome and overall cellular proteasomal proteolytic capacity were assessed. Redox cycling agents exhibited a male-specific adaptive response, whereas H2O2 exposure provoked female-specific adaptation. These findings demonstrate that different oxidants can elicit sexually dimorphic adaptive homeostatic responses in multiple fly strains. These results (and those contained in a parallel study [1]) highlight the need to address sex as a biological variable in fundamental science, clinical research, and toxicology.

Sex-Specific Gene Expression and Life Span Regulation.

Aging-related diseases show a marked sex bias. For example, women live longer than men yet have more Alzheimer's disease and osteoporosis, whereas men have more cancer and Parkinson's disease. Understanding the role of sex will be important in designing interventions and in understanding basic aging mechanisms. Aging also shows sex differences in model organisms. Dietary restriction (DR), reduced insulin/IGF1-like signaling (IIS), and reduced TOR signaling each increase life span preferentially in females in both flies and mice. Maternal transmission of mitochondria to offspring may lead to greater control over mitochondrial functions in females, including greater life span and a larger response to diet. Consistent with this idea, males show greater loss of mitochondrial gene expression with age.

The age- and sex-specific decline of the 20s proteasome and the Nrf2/CncC signal transduction pathway in adaption and resistance to oxidative stress in Drosophila melanogaster.

Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C (Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H2O2) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female D. melanogaster (fruit-flies). Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H2O2, and blocking adaptive increases in proteasome expression also prevented full adaptation. We find, however, that this adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H2O2 adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keep1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.