Chronic jet lag impairs startle-induced locomotion in Drosophila

    Chronic jet lag impairs startle-induced locomotion in Drosophila Alexandra Vaccaro, Serge Birman, Andr´e Klarsfeld PII: DOI: Reference:

S0531-5565(16)30343-6 doi: 10.1016/j.exger.2016.09.012 EXG 9911

To appear in:

Experimental Gerontology

Received date: Revised date: Accepted date:

9 May 2016 11 September 2016 13 September 2016

Please cite this article as: Vaccaro, Alexandra, Birman, Serge, Klarsfeld, Andr´e, Chronic jet lag impairs startle-induced locomotion in Drosophila, Experimental Gerontology (2016), doi: 10.1016/j.exger.2016.09.012

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Chronic jet lag impairs startle-induced locomotion in Drosophila

Alexandra Vaccaro, Serge Birman and André Klarsfeld

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Genes Circuits Rhythms and Neuropathologies, Brain Plasticity Unit, CNRS, PSL Research University, ESPCI ParisTech, 10 rue Vauquelin, 75005 Paris, France Contact information: [email protected], +331 4079 5180, fax : +331 4079 4757

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(other email addresses: [email protected], [email protected])

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Running title: Locomotor aging in period mutants

ACCEPTED MANUSCRIPT Abstract1 Endogenous circadian clocks with ~24-h periodicity are found in most organisms from

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cyanobacteria to humans. Daylight synchronizes these clocks to solar time. In humans, shiftwork and jet lag perturb clock synchronization, and such perturbations, when repeated or

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chronic, are strongly suspected to be detrimental to healthspan. Here we investigated locomotor aging and longevity in Drosophila melanogaster with genetically or

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environmentally disrupted clocks. We compared two mutations in period (per, a gene essential for circadian rhythmicity in Drosophila), after introducing them in a common

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reference genetic background: the arrhythmic per01, and perT which displays robust short 16h rhythms. Compared to the wild type, both per mutants showed reduced longevity and

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decreased startle-induced locomotion in aging flies, while spontaneous locomotor activity was not impaired. The per01 phenotypes were generally less severe than those of perT,

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suggesting that chronic jet lag is more detrimental to aging than arrhythmicity in Drosophila.

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Interestingly, the adjustment of environmental light-dark cycles to the endogenous rhythms of the perT mutant fully suppressed the acceleration in the age-related decline of startle-

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induced locomotion, while it accelerated this decline in wild-type flies. Overall, our results show that chronic jet lag accelerates a specific form of locomotor aging in Drosophila, and that this effect can be alleviated by environmental changes that ameliorate circadian rhythm synchronization.

Keywords: Circadian clock, environmental synchronization, period gene, arrhythmia, lightdark cycle, healthspan, lifespan

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Abbreviations: ARLI (age-related locomotor impairment), LD (light-dark), PI (performance index), SING (startle-induced negative geotaxis) 1

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1. Introduction

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Circadian clocks, when synchronized to solar time, allow organisms to anticipate the

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many nycthemeral changes in their environment. At the molecular level, in flies as in humans, circadian clocks are based on negative feedback loops [1]. The best characterized one relies on transcription factors Clock (Clk) and Cycle (Cyc), which form a heterodimer and

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promote the transcription of two negative elements, period (per) and (in D. melanogaster)

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timeless (tim). Per and Tim also form a heterodimer that inhibits the activity of Clk/Cyc. This will eventually result in Per/Tim degradation and the release of Clk/Cyc from repression to

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start a new cycle. This loop enables a rhythmic expression of circadian clock mRNAs and proteins, with a period close to 24 h that is maintained under constant darkness.

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Under natural conditions, circadian clocks are precisely synchronized to the solar day,

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mainly by the accompanying light and, in ectotherms, temperature variations. Light is the strongest clock synchronizer. It acts through the visual system and, in most non-mammalian

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species including D. melanogaster, through the activation of the blue-light photoreceptor cryptochrome (Cry) [1]. Activated Cry binds to Tim and promotes its degradation, thereby resetting the clock at dawn. Organisms are generally fitter in LD cycles that are closer to their natural circadian period [2-6], a form of resonance that presumably minimizes the small but chronic jet lag between internal and external rhythms. The complete suppression of rhythmicity, either under sufficiently intense constant light [2] or because of a mutation in a clock gene [7-9], has detrimental effects on healthspan and longevity. However, there has been no direct

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ACCEPTED MANUSCRIPT comparison of age-related functional impairments between arrhythmic and jet-lagged conditions.

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Here, we used the D. melanogaster arrhythmic per01 [10] and short-period perT [11] alleles to study the effects of either circadian arrhythmia or conflicts between endogenous

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and environmental rhythmicity on locomotor aging and lifespan.

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2. Materials and Methods 2.1 Drosophila strains and culture conditions

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per01 is an arrhythmic null allele [10], while perT flies are strongly rhythmic, with a ~16-h period [11]. Both per mutations were induced by chemical mutagenesis. To limit the

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variability in genetic backgrounds, we first backcrossed the two per mutant strains, which carry a w+ allele [12], to the same w1118 strain. As the w and per loci are closely linked on the

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X chromosome, eye color allowed to reliably follow the mutant per alleles across six

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successive backcrosses. After each successive backcross to the w1118 strain, female heterozygous offspring, nominally w1118/per mutant, were used for the next backcross

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(always to w1118 males) to allow meiotic recombination between all parental chromosome pairs. After the final backcross, males and females offspring with per mutant phenotypes were selected by standard actimetric tests to establish backcrossed per mutant strains. Actimetric tests also allowed us to select wild-type recombinants (red-eyed with ~24h period) derived from female offspring of an additional cross between the w1118 strain and the final per01 backcrossed strain. These recombinants became our controls, with which the backcrossed per01 and perT mutant strains theoretically share at least 98% of their other loci, including the wild-type w allele. All strains were maintained on standard medium at 25°C under LD 12:12 (12h Light/12h Dark).

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ACCEPTED MANUSCRIPT 2.2 Lifespan assay Male flies were maintained on standard medium (at 25°C) under either LD 12:12 or

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LD 8:8 conditions from adult day 1 until death. 50 animals/bottle by triplicates were tested and the medium was changed every 3 days without using CO2. Each experiment was

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repeated twice. Light intensity at the level of the flies was in the range of 500-3000 lux (Osram 20W white fluorescent tubes). Temperature fluctuations in the two incubators were less than ± 0.6°C around the set value. Their mean temperatures differed by less than 0.3°C.

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Incubators were light-tight, and placed in a dark climatic room.

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2.3 Startle-induced locomotion assay

Male flies were aged under the same conditions as for the lifespan assays. SING assay

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and PI calculation were performed as described [13]. For each experimental point, flies were anesthetized with CO2 the day before the assay and split into five replicate groups of 10

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individuals each, which were transferred into vertical columns 20-25 min before the assay.

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The PI for each column was calculated as follows: ½[1 + (n top-nbot)/ntot], where ntot is the total number of flies in the column, ntop is the number of flies that have reached at least

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once the top of the column (above 22 cm) during a 1 min interval, and nbot is the number of flies that never left the bottom (below 4 cm). Plotted results are the means and standard errors of the means (SEM) obtained from 2 to 3 independent experiments performed at different times of the year. In order to evaluate ARLI, flies were tested weekly for 6 weeks, starting on day 10 post-eclosion. Dead flies were replaced by substitutes of the same age reared in the same incubator. 2.4 Locomotor activity rhythm monitoring Locomotor activity experiments were performed using commercial activity monitors (TriKinetics) placed in the same type of incubators used to assay lifespan. Young (10-day-

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ACCEPTED MANUSCRIPT old)or old (31-day-old) males fed on 5% sucrose-agar medium were maintained 5-6 days under LD 12:12 or LD 8:8 at 25°C, and then switched to at least 5-6 days of constant

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darkness, all at 25°C. Data analysis was performed with the FaasX software, as described previously [14]. All reported activity values are averages for 24-32 flies over 4-5 cycles. All

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behavioral experiments were performed at least twice with similar results. Note that male flies were used throughout for consistency, both within the present study and with previously published ones (e.g. [9, 12, 13]). It also simplifies aging experiments by avoiding

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the alterations in food medium resulting from egg-laying.

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2.5 Statistics

For lifespan assays, survival curves were generated and compared using the log-rank

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(Mantel-Cox) test, and ~300-450 animals were tested per genotype and LD condition, in 2 (LD 8:8) or 3 (LD 12:12) independent cohorts of ~150 flies. For median lifespan, 75th

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percentile and maximum lifespan, means and SEM between the cohorts are reported in

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Table S1. For SING assays, the mean and SEM were calculated for each trial and two-way Anova with the post-hoc Tukey-Kramer test was used for statistical analysis. Total

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spontaneous locomotor activity was analyzed using one-way Anova with the post hoc TukeyKramer test. GraphPad Prism (GraphPad Software) was used for all statistical analyses.

3. Results and Discussion 3.1 The arrhythmic per01 mutation accelerates locomotor aging and decreases lifespan Startle-induced negative geotaxis (SING), which tests the flies' ability to climb up in response to a mechanical shock, is a common paradigm to monitor age-related locomotor impairment (ARLI) in D. melanogaster. Under standard LD 12:12 cycles, per01 mutants displayed significantly accelerated ARLI (Fig. 1A) and decreased lifespan (e.g. median of 46 ±

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ACCEPTED MANUSCRIPT 0.9 days compared to 54 ± 0.7 days for controls) (Fig. 1B, Tables S1 and S2). Although an accelerated locomotor decline has been previously observed in per01 flies, their lifespan was

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reported not to be reduced relative to controls, unless flies were subjected to 24 h hyperoxia in middle age [9]. This would apparently conflict with our results as well as with recently

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published ones [15]. However, the latter report showed that the effect of circadian mutations on lifespan, for both per01 and tim01, is very sensitive to yeast content in fly food. Either such an effect or the genetic background could be involved in these discrepancies.

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Lifespan reductions have been observed to be induced by arrhythmic mutations in

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per (here and [15]), tim [15], cyc [7] and Clk (our unpublished results), as well as for wildtype flies in constant light which stops the clock ([2] and our unpublished results). The

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simplest interpretation is that eliminating circadian rhythms decreases lifespan in Drosophila, which may also hold in rodents [8, 16]. It was recently shown that 6-month exposure to

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constant light affects many health parameters in rodents [17]. However, the relevant

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mechanisms, including the presumed link to rhythm perturbations, remain to be elucidated.

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3.2 The stronger acceleration of locomotor aging observed in the short-period perT mutant is completely rescued in adapted, short environmental cycles Another per mutant, perT, displays a short 16-h circadian period. In LD 12:12, these flies anticipate light-off much too early (Fig. S1E,F), and experience chronic jet lag, with a daily 8-h “time reset” (delay) of their clock. Under these conditions, their ARLI was more affected than that of arrhythmic per01 flies (Fig. 1A). In LD 8:8, which closely fits their endogenous period, perT mutants displayed strong, properly timed anticipation of light-off, similar to control flies in LD 12:12 (compare e.g. Fig. S2E with Fig. S1A). Conversely, these very short LD cycles prevented wild-type flies from anticipating the light transitions, so that their activity profiles now resembled those of per01

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ACCEPTED MANUSCRIPT (compare Fig. S2A,B with Fig. S2C,D). Under these conditions, SING declined faster for controls and slower for perT mutants (Fig. 1C and S3), such that performance of the perT mutants was now significantly higher than that of both control and per01 flies after 3 weeks

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of age (Fig. 1A). In LD 8:8, perT flies indeed performed as well as the wild-type controls in LD

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12:12 (Fig. S3C; see also below). In contrast, the LD regime had no significant effect on ARLI in per01 flies (Fig. S3E). As per01 flies have no clock, their insensitivity to the LD regime reinforces the conclusion that the locomotion defects observed in rhythmic perT or control

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flies resulted from a conflict, or lack of circadian resonance [2, 6], between endogenous and

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environmentally-imposed periods.

3.3 The short-period perT mutation reduces lifespan even in adapted, short environmental

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cycles

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In LD 12:12, perT mutants displayed reduced lifespan (Fig. 1B). As for ARLI, perT (with e.g. a median lifespan of 38 ± 0.3 days, Table S1) was significantly more affected than per01

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(Table S2). In LD 8:8, despite the significant locomotor rescue observed for perT flies, their

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lifespan remained lower than that of the control flies (Fig 1D, Tables S1 and S2). In fact, the lifespan of both per mutants was not altered by the LD regime, while the lifespan of the control was significantly decreased in LD 8:8, approximately to the level of the per01 mutant, but still above that of the perT mutant (Fig. 1D, Tables S1 and S2). The reduced lifespan of the perT mutant relative to control in both LD regimes may reflect a general frailty, possibly because its very short period is outside some physiologically acceptable range, as previously suggested [12]. Total and daytime spontaneous locomotor activity were on average very similar in old control and perT flies, maintained either in LD 12:12 or LD 8:8 (Fig. 2, Table S3). Decreased SING in per01 flies, as they age, was associated with an approximately 30% increase in spontaneous activity (Fig. 2A, B). Our results would

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ACCEPTED MANUSCRIPT thus tend to rule out a major alteration of muscular function in the per mutant flies, and indicate that chronic jet lag can aggravate specific aspects of aging such as ARLI. Focusing on

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a simple assay such as SING should help to identify specific mechanisms underlying the accelerated ARLI of clock-disrupted flies.

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3.4 Chronic jet lag and healthspan

Many experimental and epidemiological studies [2-4, 18-20] suggest that repeated or

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chronic jet lag is detrimental to healthspan. Recently, fertility was found to decrease in middle-aged female mice when they were phase-shifted back and forth once a week for

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several weeks, while the cry1 or cry2 mutants, with a circadian period close to 22.5 and 24.5 h, respectively, lost their fertility in middle age under standard LD conditions [5].

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Interestingly, that loss was reverted by rearing them for a few weeks in LD regimes adjusted

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to their endogenous periods [5]. Similarly, the age-dependent pathologies developed by short-period hamster mutants in standard LD cycles were absent when they were reared

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under an adjusted LD regime [4].

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Here, we also made the reciprocal comparison. Although LD 8:8 fully rescued the locomotor performance of the perT mutant up to that of the controls in LD 12:12 (Fig. S3C), the opposite did not hold: in LD 8:8, the controls did not perform as poorly as the perT mutant in LD 12:12 (Fig. S3D). Because control flies may be generally healthier than the perT mutant (as indicated by the lifespan data), they could be more resistant to chronic jet lag. Alternatively, daily 8-h clock delays could be more detrimental than daily 8-h advances. In contrast, isolated or spaced delays seem more challenging than advances [21]. Daily clock resetting, which is inevitable because circadian periods generally differ from 24 h, may thus impact organisms differently than isolated phase-shifts. This distinction is important, as daily clock resets are exacerbated in our 24/7 societies, leading to generalized "social jet lag" [22].

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Acknowledgements

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We thank François Rouyer for fly strains and the FaasX program, Honorine Lucchi and Audrey Lombi for technical help, Baya Chérif-Zahar for critical reading of the manuscript, and

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other GCRN team members for helpful discussions. This work was supported by funding from the Fondation de France (2012-00024435) and the Labex MemoLife (ANR-10-LABX-54

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MEMO LIFE) to SB. AV was supported by PhD fellowships from the ED3C/Université Pierre et

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Marie Curie and Fondation pour la Recherche Médicale (FDT20150532717).

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Figure Legends

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Figure 1. Locomotor aging and longevity of per mutants maintained in two different LD conditions. As compared to wild-type (ctrl) flies, ARLI of per01 and perT mutants was

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accelerated under standard LD 12:12 conditions (A), while their lifespan was reduced (B). Under LD 8:8 cycles, the perT mutant had a significantly slower locomotor decline than both

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wild-type and per01 flies (C), while its lifespan remained shorter (D). PI: Performance Index; *p<0.05, **p<0.01, ***p<0.001.

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Figure 2. Effects of genetic and environmental rhythmicity on spontaneous locomotor activity in young and old flies. Total locomotor activity in LD 12:12 (A) and in LD 8:8 (B), in

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young (initially 10-day-old) and older (initially 31-day-old) flies of the indicated genotypes.

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31 days was chosen because it is the age at which the spread in PI between the three strains was greatest in standard LD 12:12 (Fig. 1A), and at which the age-related decrease in survival

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was generally fastest (Fig. 1B, D). Note that the values displayed on the graphs represent

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averages over 4-5 cycles (see section 2.4). **p<0.01, ***p<0.001.

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References Allada, R. and B.Y. Chung, Circadian organization of behavior and physiology in Drosophila. Annu Rev Physiol, 2010. 72: p. 605-24.

Pittendrigh, C.S. and D.H. Minis, Circadian systems: longevity as a function of

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2.

PT

1.

circadian resonance in Drosophila melanogaster. Proc Natl Acad Sci U S A, 1972. 69(6): p. 1537-9. 3.

von Saint Paul, U. and J. Aschoff, Longevity among blowflies Phormia terraenovae R.D.

4.

NU

kept in non-24-hour light-dark cycles. J. Comp. Physiol. , 1978. 127: p. 191-195. Martino, T.A., et al., Circadian rhythm disorganization produces profound

2008. 294(5): p. R1675-83. 5.

MA

cardiovascular and renal disease in hamsters. Am J Physiol Regul Integr Comp Physiol,

Takasu, N.N., et al., Recovery from Age-Related Infertility under Environmental Light-

ED

Dark Cycles Adjusted to the Intrinsic Circadian Period. Cell Rep, 2015. 12(9): p. 140713.

Ouyang, Y., et al., Resonating circadian clocks enhance fitness in cyanobacteria. Proc

PT

6.

Natl Acad Sci U S A, 1998. 95(15): p. 8660-4. Hendricks, J.C., et al., Gender dimorphism in the role of cycle (BMAL1) in rest, rest

CE

7.

regulation, and longevity in Drosophila melanogaster. J Biol Rhythms, 2003. 18(1): p.

8.

AC

12-25.

Kondratov, R.V., et al., Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock. Genes Dev, 2006. 20(14): p. 186873.

9.

Krishnan, N., et al., The circadian clock gene period extends healthspan in aging Drosophila melanogaster. Aging (Albany NY), 2009. 1(11): p. 937-48.

10.

Konopka, R.J. and S. Benzer, Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci U S A, 1971. 68(9): p. 2112-6.

11.

Konopka, R.J., et al., An ultrashort clock mutation at the period locus of Drosophila melanogaster that reveals some new features of the fly's circadian system. J Biol Rhythms, 1994. 9(3-4): p. 189-216.

11

ACCEPTED MANUSCRIPT 12.

Klarsfeld, A. and F. Rouyer, Effects of circadian mutations and LD periodicity on the life span of Drosophila melanogaster. J Biol Rhythms, 1998. 13(6): p. 471-8.

13.

Riemensperger, T., et al., A single dopamine pathway underlies progressive locomotor

14.

Klarsfeld, A., J.C. Leloup, and F. Rouyer, Circadian rhythms of locomotor activity in

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Drosophila. Behav Processes, 2003. 64(2): p. 161-175. 15.

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deficits in a Drosophila model of Parkinson disease. Cell Rep, 2013. 5(4): p. 952-60.

Katewa, S.D., et al., Peripheral Circadian Clocks Mediate Dietary RestrictionDependent Changes in Lifespan and Fat Metabolism in Drosophila. Cell Metab, 2016. 23(1): p. 143-54.

Dubrovsky, Y.V., W.E. Samsa, and R.V. Kondratov, Deficiency of circadian protein

NU

16.

CLOCK reduces lifespan and increases age-related cataract development in mice.

17.

Lucassen, E.A., et al., Environmental 24-hr Cycles Are Essential for Health. Curr Biol, 2016. 26(14): p. 1843-53.

Castanon-Cervantes, O., et al., Dysregulation of inflammatory responses by chronic

ED

18.

MA

Aging (Albany NY), 2010. 2(12): p. 936-44.

circadian disruption. J Immunol, 2010. 185(10): p. 5796-805. Libert, S., et al., Deviation of innate circadian period from 24 h reduces longevity in

PT

19.

mice. Aging Cell, 2012. 11(5): p. 794-800. Wang, X.S., et al., Shift work and chronic disease: the epidemiological evidence.

CE

20.

Occup Med (Lond), 2011. 61(2): p. 78-89. Davidson, A.J., et al., Chronic jet-lag increases mortality in aged mice. Curr Biol, 2006.

AC

21.

16(21): p. R914-6. 22.

Wittmann, M., et al., Social jetlag: misalignment of biological and social time. Chronobiol Int, 2006. 23(1-2): p. 497-509.

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ACCEPTED MANUSCRIPT Highlights Conflicting endogenous and external rhythms accelerate Drosophila locomotor aging.

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Chronic jet lag can have a stronger effect on locomotor aging than arrhythmia.

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Adjusting external LD cycles to short endogenous rhythms rescued locomotor aging.

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