Temperature-driven coordination of circadian transcriptional regulation.

The circadian clock is an evolutionarily-conserved molecular oscillator that enables species to anticipate rhythmic changes in their environment. At a molecular level, the core clock genes induce circadian oscillations in thousands of genes in a tissue-specific manner, orchestrating myriad biological processes. While previous studies have investigated how the core clock circuit responds to environmental perturbations such as temperature, the downstream effects of such perturbations on circadian regulation remain poorly understood. By analyzing bulk-RNA sequencing of Drosophila fat bodies harvested from flies subjected to different environmental conditions, we demonstrate a highly condition-specific circadian transcriptome: genes are cycling in a temperature-specific manner, and the distributions of their phases also differ between the two conditions. Further employing a reference-based gene regulatory network (Reactome), we find evidence of increased gene-gene coordination at low temperatures and synchronization of rhythmic genes that are network neighbors. We report that the phase differences between cycling genes increase as a function of geodesic distance in the low temperature condition, suggesting increased coordination of cycling on the gene regulatory network. Our results suggest a potential mechanism whereby the circadian clock mediates the fly's response to seasonal changes in temperature.

Dietary restriction fails to extend lifespan of Drosophila model of Werner syndrome.

Werner syndrome (WS) is a rare genetic disease in humans, caused by mutations in the WRN gene that encodes a protein containing helicase and exonuclease domains. WS is characterized by symptoms of accelerated aging in multiple tissues and organs, involving increased risk of cancer, heart failure, and metabolic dysfunction. These conditions ultimately lead to the premature mortality of patients with WS. In this study, using the null mutant flies (WRNexoΔ) for the gene WRNexo (CG7670), homologous to the exonuclease domain of WRN in humans, we examined how diets affect the lifespan, stress resistance, and sleep/wake patterns of a Drosophila model of WS. We observed that dietary restriction (DR), one of the most robust nongenetic interventions to extend lifespan in animal models, failed to extend the lifespan of WRNexoΔ mutant flies and even had a detrimental effect in females. Interestingly, the mean lifespan of WRNexoΔ mutant flies was not reduced on a protein-rich diet compared to that of wild-type (WT) flies. Compared to WT control flies, the mutant flies also exhibited altered responses to DR in their resistance to starvation and oxidative stress, as well as changes in sleep/wake patterns. These findings show that the WRN protein is necessary for mediating the effects of DR and suggest that the exonuclease domain of WRN plays an important role in metabolism in addition to its primary role in DNA-repair and genome stability.

Con-FLIC: concurrent measurement of feeding behaviors and food consumption in Drosophila at single-fly resolution.

Accurate quantification of food intake and feeding behaviors are essential for understanding various biological processes, such as metabolism and aging. Currently, no methods allow for the concurrent measurement of both parameters for the same individual flies. Here, we couple Con-Ex ( Con sumption- Ex cretion) and FLIC ( F ly L iquid-Food I nteraction C ounter), previously developed to measure food consumption and various feeding behaviors, respectively, into a single platform that we named Con-FLIC. Using starvation as a known condition that changes food intake and feeding behaviors, we validate that Con-FLIC enables concurrent measurement of feeding behaviors and food intake in Drosophila at a single-fly resolution. We expect that Con-FLIC will be an easy non-invasive option to quantify food consumption and feeding behaviors concurrently in the same individual flies.

Temperature-driven coordination of circadian transcriptome regulation.

The circadian rhythm is an evolutionarily-conserved molecular oscillator that enables species to anticipate rhythmic changes in their environment. At a molecular level, the core clock genes induce a circadian oscillation in thousands of genes in a tissue-specific manner, orchestrating myriad biological processes. While studies have investigated how the core clock circuit responds to environmental perturbations such as temperature, the downstream effects of such perturbations on circadian regulation remain poorly understood. By analyzing bulk-RNA sequencing of Drosophila fat bodies harvested from flies subjected to different environmental conditions, we demonstrate a highly condition-specific circadian transcriptome. Further employing a reference-based gene regulatory network (Reactome), we find evidence of increased gene-gene coordination at low temperatures and synchronization of rhythmic genes that are network neighbors. Our results point to the mechanisms by which the circadian clock mediates the fly's response to seasonal changes in temperature.

Dietary Restriction Impacts Peripheral Circadian Clock Output Important for Longevity in Drosophila.

Circadian clocks may mediate lifespan extension by caloric or dietary restriction (DR). We find that the core clock transcription factor Clock is crucial for a robust longevity and fecundity response to DR in Drosophila. To identify clock-controlled mediators, we performed RNA-sequencing from abdominal fat bodies across the 24 h day after just 5 days under control or DR diets. In contrast to more chronic DR regimens, we did not detect significant changes in the rhythmic expression of core clock genes. Yet we discovered that DR induced de novo rhythmicity or increased expression of rhythmic clock output genes. Network analysis revealed that DR increased network connectivity in one module comprised of genes encoding proteasome subunits. Adult, fat body specific RNAi knockdown demonstrated that proteasome subunits contribute to DR-mediated lifespan extension. Thus, clock control of output links DR-mediated changes in rhythmic transcription to lifespan extension.

Roles of peripheral clocks: lessons from the fly.

To adapt to and anticipate rhythmic changes in the environment such as daily light-dark and temperature cycles, internal timekeeping mechanisms called biological clocks evolved in a diverse set of organisms, from unicellular bacteria to humans. These biological clocks play critical roles in organisms' fitness and survival by temporally aligning physiological and behavioral processes to the external cues. The central clock is located in a small subset of neurons in the brain and drives daily activity rhythms, whereas most peripheral tissues harbor their own clock systems, which generate metabolic and physiological rhythms. Since the discovery of Drosophila melanogaster clock mutants in the early 1970s, the fruit fly has become an extensively studied model organism to investigate the mechanism and functions of circadian clocks. In this review, we primarily focus on D. melanogaster to survey key discoveries and progresses made over the past two decades in our understanding of peripheral clocks. We discuss physiological roles and molecular mechanisms of peripheral clocks in several different peripheral tissues of the fly.

Mechanisms of Lifespan Regulation by Calorie Restriction and Intermittent Fasting in Model Organisms.

Genetic and pharmacological interventions have successfully extended healthspan and lifespan in animals, but their genetic interventions are not appropriate options for human applications and pharmacological intervention needs more solid clinical evidence. Consequently, dietary manipulations are the only practical and probable strategies to promote health and longevity in humans. Caloric restriction (CR), reduction of calorie intake to a level that does not compromise overall health, has been considered as being one of the most promising dietary interventions to extend lifespan in humans. Although it is straightforward, continuous reduction of calorie or food intake is not easy to practice in real lives of humans. Recently, fasting-related interventions such as intermittent fasting (IF) and time-restricted feeding (TRF) have emerged as alternatives of CR. Here, we review the history of CR and fasting-related strategies in animal models, discuss the molecular mechanisms underlying these interventions, and propose future directions that can fill the missing gaps in the current understanding of these dietary interventions. CR and fasting appear to extend lifespan by both partially overlapping common mechanisms such as the target of rapamycin (TOR) pathway and circadian clock, and distinct independent mechanisms that remain to be discovered. We propose that a systems approach combining global transcriptomic, metabolomic, and proteomic analyses followed by genetic perturbation studies targeting multiple candidate pathways will allow us to better understand how CR and fasting interact with each other to promote longevity.

Control of apoptosis by Drosophila DCAF12.

Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. Here, we report the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. We find that DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD.

Genetics of Circadian Rhythms.

Nearly all organisms exhibit time-dependent behavior and physiology across a 24-hour day known as circadian rhythms. These outputs are manifestations of endogenous cyclic gene expression patterns driven by the activity of a core transcription/translation feedback loop. Cyclic gene expression determines highly tissue-specific functional activity regulating such processes as metabolic state, endocrine activity, and neural excitability. Entrainment of these cellular clocks is achieved through exogenous daily inputs, such as light and food. Dysregulation of the transcription/translation feedback loop has been shown to result in a wide range of disorders and diseases driving increased interest in circadian therapies.

The functional costs and benefits of dietary restriction in Drosophila.

Dietary restriction (DR) extends lifespan in an impressively wide array of species spanning three eukaryotic kingdoms. In sharp contrast, relatively little is known about the effects of DR on functional senescence, with most of the work having been done on mice and rats. Here we used Drosophila melanogaster to test the assumption that lifespan extension through DR slows down age-related functional deterioration. Adult virgin females were kept on one of three diets, with sucrose and yeast concentrations ranging from 7% to 11% to 16% (w/v). Besides age-specific survival and fecundity, we measured starvation resistance, oxidative stress resistance, immunity, and cold-stress resilience at ages 1, 3, 5, and 7 weeks. We confirmed that DR extends lifespan: median lifespans ranged from 38 days (16% diet) to 46 days (11% diet) to 54 days (7% diet). We also confirmed that DR reduces fecundity, although the shortest-lived flies only had the highest fecundity when males were infrequently available. The most striking result was that DR initially increased starvation resistance, but strongly decreased starvation resistance later in life. Generally, the effects of DR varied across traits and were age dependent. We conclude that DR does not universally slow down functional deterioration in Drosophila. The effects of DR on physiological function might not be as evolutionarily conserved as its effect on lifespan. Given the age-specific effects of DR on functional state, imposing DR late in life might not provide the same functional benefits as when applied at early ages.

Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body.

In Drosophila melanogaster, ageing is slowed when insulin-like signalling is reduced: life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (chico) are mutated, or when insulin-producing cells are ablated. But we have yet to resolve when insulin affects ageing, or whether insulin signals regulate ageing directly or indirectly through secondary hormones. Caenorhabditis elegans lifespan is also extended when insulin signalling is inhibited in certain tissues, or when repressed in adult worms, and this requires the forkhead transcription factor (FOXO) encoded by daf-16 (ref. 6). The D. melanogaster insulin-like receptor mediates phosphorylation of dFOXO, the equivalent of nematode daf-16 and mammalian FOXO3a. We demonstrate here that dFOXO regulates D. melanogaster ageing when activated in the adult pericerebral fat body. We further show that this limited activation of dFOXO reduces expression of the Drosophila insulin-like peptide dilp-2 synthesized in neurons, and represses endogenous insulin-dependent signalling in peripheral fat body. These findings suggest that autonomous and non-autonomous roles of insulin signalling combine to control ageing.

Different age-specific demographic profiles are generated in the same normal-lived Drosophila strain by different longevity stimuli.

We review the empirical data obtained with our normal-lived Ra control strain of Drosophila and show that this one genome is capable of invoking at least three different responses to external stimuli that induce the animal to express one of three different extended longevity phenotypes, each of which arises from one of three different antagonistic molecular mechanisms of stress resistance. The phenotypes are distinguished by different age-specific mortality patterns. Depending on the selected mechanism, the genome may respond by expressing a delayed onset of senescence (type 1), an increased early survival (type 2), or an increased late survival (type 3) phenotype, suggesting their different demographic effects. We suggest that the different demographic effects stem in part from the differential ability of each selection regime to reallocate the organism's energy from reproduction to somatic maintenance. These data document the complexity of the aging process and argue for a relationship between molecular mechanisms and longevity phenotypes.

Genomic plasticity, energy allocations, and the extended longevity phenotypes of Drosophila.

The antagonistic pleiotropy theory of the evolution of aging is shown to be too simple to fully apply to the situation in which Drosophila are selected directly for delayed female fecundity and indirectly for extended longevity. We re-evaluated our own previously reported selection experiments using previously unreported data, as well as new data from the literature. The facts that led to this re-evaluation were: (1) the recognition that there are at least three different extended longevity phenotypes; (2) the existence of metabolic and mitochondrial differences between normal- and long-lived organisms; and most importantly; (3) the observation that animals selected for extended longevity are both more fecund and longer-lived than their progenitor control animals. This latter observation appears to contradict the theory. A revised interpretation of the events underlying the selection process indicates that there is a two-step change in energy allocations leading to a complex phenotype. Initial selection first allows the up-regulation of the antioxidant defense system genes and a shift to the use of the pentose shunt. This is later followed by alterations in mitochondrial fatty acid composition and other changes necessary to reduce the leakage of H(2)O(2) from the mitochondria into the cytosol. The recaptured energy available from the latter step is diverted from somatic maintenance back into reproduction, resulting in animals that are both long-lived and fecund. Literature review suggests the involvement of mitochondrial and antioxidant changes are likely universal in the Type 1 extended longevity phenotype.

Metabolic alterations and shifts in energy allocations are corequisites for the expression of extended longevity genes in Drosophila.

Evolutionary theories suggest that the expression of extended longevity depends on the organism's ability to shift energy from reproduction to somatic maintenance. New data led us to reexamine our older data and integrate the two into a larger picture of the genetic and metabolic alterations required if the animal is to live long. Our Ra normal-lived control strain can express any one of three different extended longevity phenotypes, only one of which involves significant and proportional increases in both mean and maximum longevity and thus a delayed onset of senescence. This phenotype is dependent on the up-regulation of the antioxidant defense system (ADS) genes and enzymes. Animals that express this phenotype typically have a pattern of altered specific activities in metabolically important enzymes, suggesting they are necessary to support the NAD+/NADP+ reducing system required for the continued high ADS enzyme activities. Fecundity data suggests that the energy required for this higher level of somatic maintenance initially came from a reduced egg production. This was only transient, however, for the females significantly increased their fecundity in later generations while still maintaining their longevity. The energy required for this enhanced fecundity was probably obtained from an increased metabolic efficiency, for the mitochondria of the La long-lived strain are metabolically more efficient and have a lower leakage of reactive oxygen species (ROS) to the cytosol. Selection pressures that do not lead to these shifts in energy allocations result in extended longevity phenotypes characterized by increased early survival or increased late survival but not by a delayed onset of senescence.