The circadian clock gene period extends healthspan in aging Drosophila melanogaster

Introduction

Circadian clocks generate daily endogenous rhythms in behavior, physiological functions, and cellular activities, which are coordinated with external day/night cycles [1,2]. Circadian rhythms become impaired with age as evidenced by the dampening of daily oscillations in melatonin and other hormones and the disruption of night-time sleep in aged rodents and humans [3,4,5]. Remarkably, age-associated sleep fragmentation was also reported in Drosophila melanogaster[6], suggesting that effects of aging on circadian systems may be evolutionarily conserved. While aging impairs the circadian systems, there is also evidence that loss of circadian rhythms may, in turn, contribute to aging. Genetic disruption of circadian rhythms by knockout of specific clock genes leads to various age related pathologies and visible signs of premature aging in mice [7,8]. In addition, chronic jet-lag which disrupts the circadian clock, increases mortality in aged mice [9]. As extension of healthspan is of critical importance in aging human population, there is a need to elucidate how strong circadian clocks may support healthy aging.

The mechanisms linking circadian rhythms to the rate of aging and healthspan are not well understood. To address these mechanisms, we investigated whether disruption of the circadian clock affects response to homeostatic challenge and aggravates selected aging biomarkers in the model organism Drosophila melanogaster. We used a null mutation in the circadian clock gene period (per⁰¹) [10]; this gene is one of the four core clock genes that act in a negative auto-regulatory feedback loop generating daily endogenous rhythms [11,12]. The loss of per function disrupts behavioral and molecular rhythms in flies [10,11,13].

To compare lifespan and healthspan in flies with normal or disrupted circadian clock, we measured their ability to maintain ROS homeostasis during aging. We probed the health status of aging flies by exposing them to mild oxidative stress of 24h hyperoxia at increasingchronological ages, followed by assessment of the resulting oxidative damage and mortality hazards. Hyperoxia was chosen as a homeostatic challenge, because it directly leads to ROS production irrespective of age-related changes in food consumption and other physiological parameters [14].

We report that per⁰¹ flies have shortened healthspan as evidenced by their increased mortality hazard in response to homeostatic challenge during aging. This conclusion is also supported by accelerated functional senescence, and increased signs of neurodegeneration in per mutants compared to age-matched controls with an intact circadian clock. In addition, we show that the expression of per gene declines with age leading to disruption of the circadian regulatory network in old wild type flies.

Results

Discussion

This study demonstrates healthspan extending role of the clock gene period and suggest that functional circadian clocks may prevent premature aging in flies. Research on Drosophila has demonstrated that different genetic manipulations and environmental interventions can extend fly lifespan [18]. Less attention has been paid to healthspan, despite that extension of healthspan is of critical importance in aging human population. Here, we show that healthspan can follow different trajectories in flies which have similar lifespan under stress-free laboratory conditions. Healthspan is an important but poorly defined concept, and there is an ongoing debate whether model organisms, such as Drosophila, can help to characterize parameters that could detect differences in healthspan [19]. We demonstrate that a relatively mild exogenous stress of 24 h hyperoxia, which revealed health impairment of per⁰¹ mutant, could be established as a convenient method to probe fly healthspan in a search for mechanisms supporting healthy aging.

Here, we show that healthspan, measured as the ability to respond to homeostatic challenge is reduced in per⁰¹ flies. Exposure to mild oxidative stress in middle age significantly shortened life expectancy in per⁰¹ flies but, importantly, not in control flies. The lower capacity of per⁰¹ mutants to buffer short-term oxidative challenge was linked to greatly increased accumulation of oxidative damage during hyperoxia exposure. Thus, it appears that increased mortality hazard in hyperoxia-exposed per⁰¹ mutants may be caused by their impaired ability to clear the oxidative damage which is suggested to be one of the major causes of aging [20].

The higher accrual of oxidative damage observed in per⁰¹ flies in normoxia and especially after hyperoxia could be influenced by a number of factors, with the primary suspect being higher production of endogenous ROS, which has been reported to increase in clock-disrupted flies [21] and mice [7]. Whether higher ROS is associated with decreased activity of ROS scavenging en- zymes remains to be determined. While microarray studies suggested that expression of superoxide dismutase and catalase may be controlled by the circadian clock in flies [22], qRT-PCR did not confirm such rhythm for catalase, but demonstrated that catalase activity is significantly lower in young clock-deficient flies [21]. It is currently unknown whether enzymes involved in protein repair are controlled by the circadian clock in animals, although such control was reported in plants [23]. Finally, excessive agglomeration of oxidatively damaged proteins in per⁰¹ flies could be related to impaired degradation as proteasome activity has been shown to decline with age in flies, and may be inhibited by PC and HNE [24,25]

As in humans, age-related functional declines such as disrupted sleep and decreased mobility are observed in Drosophila[6,26]. The negative geotaxis assay revealed significant impairment in climbing ability in aging per⁰¹ flies relative to age-matched controls suggesting that lack of per impairs physical performance during aging. Importantly, exacerbated mobility decline in per⁰¹ flies was associated with increased neuronal degeneration in the brain. Neurodegenerative effects in the form of vacuoles in the neuropil region were observed with higher frequency in 50-day old per⁰¹ mutants than in CS^p or per⁰¹{per⁺} flies with restored per function. The formation of vacuoles was previously linked to oxidative damage and accelerated aging in Drosophila with impaired carbonyl reductase gene [27], and in flies with Alzheimer-like phenotypes [28].

Our study suggests that functional circadian rhythms support healthy aging in flies. PER protein is the essential element of circadian clock and its absence disrupts molecular and cellular rhythms. We reported previously that young wild type flies have daily rhythms in ROS and PC levels, while in per⁰¹ flies levels of these deleterious compounds are significantly higher and arrhythmic [21]. We hypothesize that the circadian clock slows down the accumulation of oxidative damage in aging organisms by synchronizing the activities of enzymes involved in protein homeostasis. For example, microarray studies reported synchronous upregulation of several GST enzymes in flies [29], and it is known that glutathione participates in the conjugation of oxidized proteins [30]. In the absence of circadian clock, enzymes working in a specific pathway may become dysregulated leading to impaired removal of oxidative damage. However, we cannot exclude the possibility that per could affect efficiency of anti-oxidative defense systems independent of its role as a clock component, by acting in a pleiotropic non-circadian manner.

While loss of the circadian rhythms by disruption of the gene period accelerates aging, organisms with normal clocks also age. Our data demonstrate that at middle age per⁰¹ mutant shows aging phenotypes normally observed in chronologically older wild type flies, suggesting that clock gene activities may decline with age. Indeed, we demonstrate the amplitude of per mRNA oscillation is severely dampened in 50 day old flies and levels of per mRNA are significantly reduced at late night, when PER acts as essential element of clock negative feedback loop [11]. This suggests that circadian clocks and, consequently circadian rhythms are severely impaired in individuals of advanced age, which is consistent with declining strength of behavioral rhythms reported in aging flies [6]. While factors contributing to the decline of circadian rhythms in flies remain to be elucidated, oxidative stress is likely to be involved. We show here that oxidative damage accumulates to high levels even in wild type aging flies, and a previous report demonstrated that paraquat-induced oxidative stress, or decrease in FOXO expression, led to dampened per expression in Drosophila[31]. Decline in clock genes with age has been reported in zebrafish [32], rats [33] and most recently in rhesus monkey [34]. The intriguing similarities in the behavior of clock genes during aging between mammals, zebrafish, and flies warrants investigations of the mechanisms causing disruption of the circadian networks. Understanding these mechanisms will help to determine in future whether strong circadian clocks add water to the fountain of youth.

Materials and Methods

Fly rearing and l ife span analysis. Drosophila melanogaster were reared on yeast-cornmeal-molasses-agar diet (35g yeast/l) at 25°C in a 12-hour light/12-hour dark cycles; all experiments were performed 4-8 h after lights-on. The per⁰¹ mutant flies [10] were backcrossed 6 times to the Canton-S (CS) flies designated as CS^P. To rescue per-function, we used transgenic flies carrying a wild-type copy of per (designated as per^G) in a per⁰¹ background [35]. Males with two copies of per^G (y wper⁰¹;{per⁺:32.1};+ were crossed with per⁰¹;+;+ females, and F1 males containing one copy of rescue construct designated per⁰¹{per⁺} were used. We confirmed their rhythmic locomotor activity indicating rescue of circadian clock function.

To determine lifespan, 3-4 cohorts of 100 flies of each genotype were housed in 16 oz transparent plastic bottles inverted over 60 mm Petri-dishes containing 15 ml of diet. Diet was replaced on alternate days without anesthesia, and mortality was recorded daily. For hyperoxia exposure, males were transferred from cages to narrow vials with diet in groups of 25, and placed in a Plexiglas chamber filled with oxygen (100% medical grade) flowing at a constant rate (300ml/min) for 24 h. Control flies were transferred to narrow vials as above and kept under normoxia. Hyperoxia-treated and control flies were then either frozen for oxidative damage analysis or returned to cages and monitored for mortality.

Oxidative damage assays. The amount of protein carbonyls was assayed separately in 25 heads and bodies. Carbonyls were quantified after reaction with 2,4-dinitrophenylhydrazine (DNPH) as described previously [21] at 370 nm in a BioTek Synergy 2 plate reader. Results were expressed as nmol.mg^-1 protein using an extinction coefficient of 22,000 M^-1cm^-1. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) was assayed in heads and bodies by competitive enzyme-linked immunosorbent assay (ELISA) as described [36, 37]. Briefly, free HNE (Alpha Diagnostic, San Antonio, TX, USA) was conjugated to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein [38]. Wells in a 96-well plate were coated with 500 ng of HNE-GAPDH protein for 24h at 4°C, washed in PBS-Tween, and blocked with 1% BSA. A standard dose-response curve was developed from serial dilutions of HNE-GAPDH with polyclonal anti-HNE antibody (1:1000; Alpha Diagnostic). For samples, 10 μ;g of protein lysate was mixed with 1:1000 polyclonal rabbit anti-HNE antibody and added to wells in triplicate. Plates were incubated for 1 h, washed with buffer, incubated with 1:5000 secondary anti-rabbit antibody conjugated with horseradish peroxidase, washed, mixed with detection buffer TMB (Alpha Diagnostic), and read at OD 450nm in a BioTek plate reader.

Rapid iterative negative geotaxis (RING) assay. Vertical mobility was assayed using the RING method [15]. Briefly, 3 groups of 25 CS^p or per⁰¹ flies were transferred into empty narrow vials, which were loaded into the RING apparatus. After 3 minutes rest, the apparatus was rapped sharply on the table three times in rapid succession to initiate a negative geotaxis response. The flies' movements in tubes were videotaped and digital images captured 4 s after initiating the behavior. Five consecutive trials were interspersed with a 30s rest. The climbing performance was calculated and expressed as average height climbed in the 4 s interval. The performance of flies in a single vial was calculated as the average of 5 consecutive trials to generate n = 1.

Neuronal degeneration. Paraffin-embedded sections of heads were used to examine neurodegenerative defects. Fly heads of all genotypes were processed and sectioned in parallel, and microscopic pictures taken at the same level of the brain and the number and volume of vacuoles counted in double-blind experiments using described methods [39, 40].

Quantitative Real-Time PCR. 25 male heads were collected for each time point in triplicate, homogenized in TriReagent (Sigma), and RNA was isolated following manufacturer protocol. Samples were purified using the RNeasy mini kit (Qiagen) with on-column DNAse digestion (Qiagen). Synthesis of cDNA was achieved with Sprint RT Complete kit (Clontech) or iScript cDNA synthesis kit (Biorad). Real-time PCR was performed on Step-One Plus real-time machine (Applied Biosystems) in triplicate under default thermal cycling conditions with a dissociation curve step. Each reaction contained iTaq SYBR Green Supermix with ROX (Biorad), 0.6-1ng cDNA, 80nM primers (IDT Technologies). Primers sequences are available upon request. Data were analyzed using the standard 2^-∆∆CT method normalized to the gene rp49 and expressed relative to control samples at ZT0.

Statistical analyses. Life span and survival curves were plotted following Kaplan Meier survival analysis and statistical significance of curves assessed using the Log-Rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon test (GraphPad Prism v 5.0).Age-specific mortality was calculated using the Gompertz's model of population aging. Ln values of instantaneous mortality (μ_x) were plotted against chronological time. Mortality calculations and Gompertz-Makeham maximum likelihood estimates were done using WinModest V1.0.2 [41] and plotted on GraphPad Prism. For statistical analysis of biochemical results three-way ANOVA with post-hoc tests were performed using OpenStat (William G. Miller © 2009). Statistical analysis of locomotor assays was done with one and two-way ANOVA for comparison between ages and genotypes.

Supplementary Materials

Supplementary Table 1

Supplementary Table 2

Supplementary Table 3

Supplementary Figure 1