The circadian clock gene period extends healthspan in aging Drosophila melanogaster

Abstract

There is increasing evidence that aging is affected by biological (circadian) clocks - the internal mechanisms that coordinate daily changes in gene expression, physiological functions and behavior with external day/night cycles. Recent data suggest that disruption of the mammalian circadian clock results in accelerated aging and increased age-related pathologies such as cancer; however, the links between loss of daily rhythms and aging are not understood. We sought to determine whether disruption of the circadian clock affects lifespan and healthspan in the model organism Drosophila melanogaster. We examined effects of a null mutation in the circadian clock gene period (per(01)) on the fly healthspan by challenging aging flies with short-term oxidative stress (24h hyperoxia) and investigating their response in terms of mortality hazard, levels of oxidative damage, and functional senescence. Exposure to 24h hyperoxia during middle age significantly shortened the life expectancy in per(01) but not in control flies. This homeostatic challenge also led to significantly higher accumulation of oxidative damage in per(01) flies compared to controls. In addition, aging per(01) flies showed accelerated functional decline, such as lower climbing ability and increased neuronal degeneration compared to age-matched controls. Together, these data suggest that impaired stress defense pathways may contribute to accelerated aging in the per mutant. In addition, we show that the expression of per gene declines in old wild type flies, suggesting that the circadian regulatory network becomes impaired with age.

Keywords: RING; longevity; neurodegeneration; oxidative stress.

Figure 1.

Lifespan of per⁰¹and CS^pD. melanogaster in normoxia and following 24h hyperoxia at different ages (marked by arrow in B-D). (A) In normoxia, there was no significant difference in mean survival curves (p=0.23) (B) Hyperoxia on day 5 did not significantly affect longevity or survival curves (p=0.12) (C) Hyperoxia on day 20 resulted in a significant reduction (p<0.05) in average survival of per⁰¹flies compared to CS^p with significant (p<0.0001) difference in survival curves. (D) Hyperoxia on day 35 resulted in more significant reduction (p<0.001) in average lifespan in per⁰¹flies compared to CS^p and significant difference in survival curves (p<0.0001). Males with rescued per function (per⁰¹{per⁺}) treated with hyperoxia on day 35 had average lifespan similar to CS^p but significantly different (p<0.001) from per⁰¹mutants.

Figure 2.

Oxidative damage accumulates to higher levels in aging per⁰¹flies. Fold increase was calculated based on day 5 values in CS^p males under normoxia (numerical values are shown in Supplementary Table 2 and Supplementary Table 3). Top: Protein carbonyls (PC) in heads (A) and bodies (B) of CS^p (solid line) and per⁰¹(broken line) in normoxia (black) and after hyperoxia (gray). PC levels were significantly higher in per⁰¹than in CS^p fly heads on day 35 and 50, and on day 50 in bodies under normoxia. Hyperoxia on day 35 and 50 induced significantly higher PC levels per⁰¹head and bodies compared to CS^p age-matched controls. Bottom: Lipid peroxidation product 4-HNE in heads (C) and bodies (D). In normoxia, per⁰¹flies accumulated significantly more 4-HNE in heads and bodies compared to CS^p in all ages except day 5. Under hyperoxia, significant increase in 4-HNE accumulation was observed in per⁰¹heads and bodies on day 20, 35 and 50 compared to CS^p males. For statistical analysis of PC and HNE data refer to Supplementary Table 2 and Supplementary Table 3.

Figure 3.

Vertical mobility deteriorates faster in per⁰¹flies, as demonstrated by the RING assay. Bars represent mean height climbed (with SEM) in CS^p (open bars) and per⁰¹(black bars) males at indicated age. The climbing performance of per⁰¹males on day 5 was significantly higher (p<0.001) compared to CS^p. With age, a rapid deterioration in climbing performance was noted in per⁰¹flies with mobility being significantly lower (* p<0.001) on day 20, 35, and 50 compared to age-matched CS^p controls.

Figure 4.

Neuronal degeneration is accelerated in per⁰¹mutants compared to CS^p and flies with restored per function (per⁰¹{per⁺}) on day 50. (A) Mean number of vacuoles (with SEM) representing neuronal degeneration was significantly higher in per⁰¹mutants compared with wild type CS^p and flies with rescued per. Bars with different superscripts are significantly different at p<0.05, data based on 10-15 heads for each genotype. (B-D) Photomicrographs of representative brain sections of CS^p, per⁰¹, and per⁰¹{per⁺} males. Arrows point to vacuolization.

Figure 5.

Expression of per mRNA declines with with age in heads of CS^p flies. (A) Daily mRNA expression profiles of per in day 5, 35 and 50 male heads. White and black horizontal bars mark periods of light and darkness respectively. Values were normalized to rp49 and calibrated against ZT0 (taken as 1) for each age and represented as mean ± SEM of 3 bioreplicates. (B) The peak levels of per mRNA are significantly reduced (* = p<0.05) in 50 day old males compared to young control males. Values are mean ± SEM of 3 bioreplicates.