Experimental Gerontology 55 (2014) 70–79

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Differential modulation of clock gene expression in the suprachiasmatic nucleus, liver and heart of aged mice Marta Bonaconsa a,⁎, Giorgio Malpeli b, Angela Montaruli c, Franca Carandente c, Gigliola Grassi-Zucconi a, Marina Bentivoglio a a b c

Department of Neurological and Movement Sciences, University of Verona, Verona, Italy Department of Pathology and Diagnostics, University of Verona, Verona, Italy Department of Biomedical Sciences for Health, University of Milan, Milan, Italy

a r t i c l e

i n f o

Article history: Received 14 August 2013 Received in revised form 24 February 2014 Accepted 17 March 2014 Available online 24 March 2014 Section Editor: Christian Humpel Keywords: Aging Peripheral oscillators Circadian pacemaker Bmal1 Period genes Cryptochrome genes Rev-erbα Dbp Dec1

a b s t r a c t Studies on the molecular clockwork during aging have been hitherto addressed to core clock genes. These previous investigations indicate that circadian profiles of core clock gene expression at an advanced age are relatively preserved in the master circadian pacemaker and the hypothalamic suprachiasmatic nucleus (SCN), and relatively impaired in peripheral tissues. It remains to be clarified whether the effects of aging are confined to the primary loop of core clock genes, or also involve secondary clock loop components, including Rev-erbα and the clock-controlled genes Dbp and Dec1. Using quantitative real-time RT-PCR, we here report a comparative analysis of the circadian expression of canonical core clock genes (Per1, Per2, Cry1, Cry2, Clock and Bmal1) and non-core clock genes (Rev-erbα, Dbp and Dec1) in the SCN, liver, and heart of 3 month-old vs 22 month-old mice. The results indicate that circadian clock gene expression is significantly modified in the SCN and peripheral oscillators of aged mice. These changes are not only highly tissue-specific, but also involve different clock gene loops. In particular, we here report changes of secondary clock loop components in the SCN, changes of the primary clock loop in the liver, and minor changes of clock gene expression in the heart of aged mice. The present findings outline a track to further understanding of the role of primary and secondary clock loop components and their crosstalk in the impairment of circadian output which characterizes aging. © 2014 Published by Elsevier Inc.

1. Introduction The mammalian circadian system consists of multiple oscillators distributed throughout the organism and a central pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus which coordinates the entire system making it coherent. The molecular mechanisms underlying circadian function are similar in central and peripheral oscillators and consist of highly conserved genes, the so-called clock genes. Together with their protein products, clock genes are organized in transcription–translation feedback loops able to complete a cycle in ~24 h (Ko and Takahashi, 2006). The primary feedback loop, or core loop, consists of different components. The positive transcriptional activators CLOCK and BMAL1 (or its paralog NPAS2) heterodimerize and promote transcription of the Period genes (Per1 and Per2) and Cryptochrome genes (Cry1 ⁎ Corresponding author at: Department of Neurological and Movement Sciences, Medical School, Strada Le Grazie 8, 35134 Verona, Italy. Tel.: +39 045 8027155; fax: +39 045 8027163. E-mail address: [email protected] (M. Bonaconsa).

http://dx.doi.org/10.1016/j.exger.2014.03.011 0531-5565/© 2014 Published by Elsevier Inc.

and Cry2). The negative components include PER and CRY which heterodimerize, translocate back to the nucleus and repress their own transcription by acting on the CLOCK–BMAL1 complex (Sato et al., 2006). The CLOCK–BMAL1 complex is involved in an additional loop, interlocked with the core loop, that induces the expression of the nuclear hormone receptors Rev-erbα and Rorα. Their products, REV-ERBα and RORα, bind competitively to Bmal1 and determine the cycling of Bmal1 expression by suppressing and activating, respectively, its transcription (Guillaumond et al., 2005; Preitner et al., 2002). A key function of the molecular clock machinery is represented by the circadian output, whose efficacy depends on the coordinated circadian behavior and physiology of the entire organism (see for reviews Takahashi et al., 2008; Albrecht, 2012). The transcriptional regulation of this function is achieved not only directly via core loop transcription factors, but also indirectly via auxiliary loops interlocking with the core loop, which include the transcription factors Dbp and Dec1. These interlocking loops and their constituents, the so-called first order “clock-controlled genes” (CCGS) (Reppert and Weaver, 2002) play a crucial intermediate role in conveying circadian signals to downstream local clock-controlled genes, specific for each tissue (Liu et al., 2008).

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The cyclic transcription of Dbp is under the control of CLOCK– BMAL1, and is repressed by PER/CRY heterodimers. In turn, DBP can regulate the core clock machinery activating Per1 transcription. Dec1 rhythmic transcription is positively regulated by CLOCK–BMAL1, while DEC1, interacting with BMAL1 for E-box occupation, negatively acts on CLOCK/BMAL1 activity (Honma et al., 2002; Kawamoto et al., 2004; see also for review Bonaconsa et al., 2013). Evidence in experimental animals and in humans indicates that the circadian timing system is progressively modified with advancing age, as shown by the reduced amplitude, phase and period length of circadian rhythms, increased tendency towards internal desynchronization and deficient responsiveness to phase-shifting stimuli with loss of synchronization to the environment (Gibson et al., 2009; Hofman and Swaab, 2006). Circadian rhythms of locomotor activity, core body temperature, blood pressure, and the sleep–wake cycle are especially affected (Reppert and Weaver, 2002). The possibility that these agingrelated changes are due to impairment of the molecular clockwork in the SCN and/or in peripheral tissues has been repeatedly investigated. In the SCN, Per1 and Cry1 circadian rhythms have been reported to be preserved at an advanced age in mice (Weinert et al., 2001), rats (Asai et al., 2001), and hamsters (Kolker et al., 2003). A decrease of Per2 circadian oscillation has been reported in aged mice (Weinert et al., 2001), but not in rats (Asai et al., 2001) and hamsters (Kolker et al., 2003). An aging-related decline in Clock and Bmal1 circadian rhythm has been reported in hamsters (Kolker et al., 2003). Studies exploring the impact of aging on clock gene expression in peripheral tissues have indicated that Per1 expression is preserved in aged rats (Asai et al., 2001; Yamazaki et al., 2002). Moderate agingrelated changes of Per2 and Bmal1 circadian rhythms have been reported in the macaque pituitary gland (Sitzmann et al., 2010). Analyses conducted in rats at only two time points for 24 h have indicated a significant age-related decrease of Per1–3 mRNAs and unchanged Cry1–2, Clock and Bmal1 mRNAs in the liver, associated with Per1–3 decrease and Bmal1 increase in the heart (Claustrat et al., 2005). It remains to be clarified whether a definite dysfunction of the molecular clockwork occurs in the circadian impairment that characterizes aging. The matter is still open not only because the available data are limited and in some instances controversial, but also because they mainly focus on clock genes of the core loop, while information on the clock output function mediated by CCGS is limited. On this basis, using quantitative real-time RT-PCR, we here assessed in young and aged mice the circadian expression of canonical core clock genes (Per1–2, Cry1–2, Clock and Bmal1), of the transcription factor Rev-erbα which, as mentioned above, forms with Bmal1 a central regulatory loop, and of the first-order CCGS Dec1 and Dbp. In order to compare the clock properties of central and peripheral oscillators, we here investigated the SCN, liver and heart.

2. Methods 2.1. Animals and tissue sampling Two groups of male Balb/c mice of different ages, 3 and 22 month-old, respectively (n = 24 per age group), were housed in the animal facility at a standard room temperature (21 ± 2 °C), under a 12 h/12 h light/dark (LD) cycle, and fed ad libitum. Life span of Balb/c mice is about 23 months (Jucker and Ingram, 1997). All efforts were made to avoid animal suffering and minimize the number of animals used. The experiments received approval by the Animal Care and Use Committee of the University of Verona and authorization by the Italian Ministry of Health, and were conducted under veterinarian assistance and according to the European Communities Council (86/609/EEC) directives. On the day of the experiment, the SCN, liver and heart were harvested from each mouse every 6 h for 24 h (n = 6 mice per age group and per time point), beginning at lights-on time (7 am), frozen in liquid nitrogen

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and stored at −80 °C until the assay. Infrared goggles were used for tissue sampling during the dark phase. The procedure to dissect the SCN was performed under the stereomicroscope. Coronal hypothalamic slices were cut with a scalpel between the anterior and posterior tips of the optic chiasm (about 800 μm–1 mm in thickness). The SCN was then carefully dissected out using the chiasm and third ventricle as landmarks, placed in lysis buffer (Qiagen, Hilden, Germany) and homogenized by pipetting. In preliminary studies aimed at standardizing this procedure, we verified that the expression of Per1 in the sampled SCN slice of young adult mice corresponded to that previously reported in the SCN (e.g. Yan et al., 1999; Zheng et al., 2001). Liver and heart tissue samples were homogenized using a drill head fitting the 0.2 PCR tubes, rapidly placed in liquid nitrogen and stored at −80 °C until use. 2.2. Quantitative real-time RT-PCR Total RNA was extracted from liver and heart tissue samples using TRIzol® (Life Technologies, Carlsbad, CA, USA). RNA was isolated from the SCN using the RNeasy Mini Kit (Qiagen), according to manufacturer's instructions. Genomic DNA contamination from RNA of all tissues was then removed by RNase-free DNase treatment using amplification grade DNase I (Invitrogen, Frederick, MD, USA) for 15 min at room temperature, inactivated by adding 2.5 mM EDTA, and finally incubated for 10 min at 65 °C. Reverse transcription of the DNase-treated RNA was performed in 20 μl reaction volumes using 250 ng of random primers, 1× RT buffer, 10 mM DTT, 0.5 mM of dNTP and 200 U of MoMLV reverse-transcriptase (SuperScript II). cDNA synthesis was performed at 42 °C for 1 h before inactivation at 70 °C for 15 min. Quantitative real-time RT-PCR was carried out using the StepOne Plus Real-Time System (Life Technologies, Foster City, CA, USA). Primer pairs (Table 1) were designed with the Primer Express software (Life Technologies). Specificity of each gene amplification was confirmed by analyzing the dissociation curves with the Sequence Detection System software. For 25 μl reaction, 4 ng of cDNA template was mixed with 400 nM of each primer and 1 × Platinum® qPCR SuperMix-UDG with ROX (Life Technologies). The reaction was allowed to proceed for 2 min at 50 °C for heat activation, for 2 min at 95 °C for denaturation, followed by 45 cycles at 95 °C for 15 s and 63 °C for 30 s for annealing/extension. 2.3. Data analysis For real-time RT-PCR data analysis, the comparative CT method (ΔΔCT) (Livak and Schmittigen, 2001) was used, as in our previous investigations on the SCN (Deng et al., 2010; Sadki et al., 2007). For each gene, the relative quantification was calculated using as endogenous reference GAPDH (CT REFERENCE ) and as calibrator the average of C T values obtained in young mice. Briefly, the ΔC T value (ΔC T = C T TEST GENE − C T REFERENCE ) was calculated for each sample and related to the calibrator sample (ΔΔCT = ΔCT TEST SAMPLE − ΔCT CALIBRATOR SAMPLE) to obtain a 2−ΔΔCT threshold corresponding to the

Table 1 Sequences of oligonucleotides used for real-time RT-PCR. Gene

Forward primer 5′ → 3′

Reverse primer 5′ → 3′

GAPDH Per1 Per2 Dbp Bmal1 Rev-erbα Clock Cry1 Cry2 Dec1

ACGGGAAGCTCACTGGCATGGCCTT CAGGCTAACCAGGAATATTACCAGC GGCTTCACCATGCCTGTTGT AATGACCTTTGAACCTGATCCCGCT GCAGTGCCACTGACTACCAAGA CGTTCGCATCAATCGCAACC CCTATCCTACCTTGGCCACACA CCCAGGCTTTTCAAGGAATGGAACA GGGACTCTGTCTATTGGCATCTG CTGAAGGATCTCCTACCC

CATGAGGTCCACCACCCTGTTGCTG CACAGCCACAGAGAAGGTGTCCTGG GGAGTTATTTCGGAGGCAAGTGT GCTCCAGTACTTCTCATCCTTCTGT TCCTGGACATTGCATTGCAT GATGTGGAGTAGGTGAGGTC TCCCGTGGAGCAACCTAGAT TCTCATCATGGTCATCAGACAGAGG GTCACTCTAGCCCGCTTGGT GCTCCCCATTCTGTAAAGC

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level of expression of each gene normalized to an endogenous reference and relative to a calibrator. Every time series was analyzed for rhythm characterization by the single Cosinor procedure involving the fit of a cosine to the data by least-squares linear regression (Nelson et al., 1979). Rhythm characterization includes the average level of three parameters, calculated with 95% confidence limits: the MESOR (middle value of the fitted cosine representing a rhythm-adjusted mean), the amplitude (half the difference between the minimum and maximum of the fitted cosine function) and the acrophase (the peak time of the fitted cosine function, expressed as the lag in hours from the time of light onset). This procedure allows to test the null hypothesis that the amplitude of the cosine function is equal to zero. Rhythm detection was considered statistically significant at p ≤ 0.05. In addition, to evaluate whether the levels of transcripts differed between the age groups, two-way analysis of variance (ANOVA) was performed to identify the effect of time, age, and their interaction. Significant differences between young and aged mice at each time point were evaluated using the post-hoc Bonferroni test for pairwise comparisons following ANOVA. 3. Results To analyze the mechanism underlying age-related impairment of circadian clock function, we examined the 24 h patterns of mRNA expression of nine clock genes, including the six canonical genes of the core clock feedback loop (Per1, Per2, Cry1, Cry2, Clock, Bmal1) and three clock genes representative of the CCGS (Rev-erbα, Dbp and Dec1). As mentioned previously, the latter genes, components of clock gene secondary loops interlocking with the core loop, act as intermediaries in circadian signaling. The study was conducted in the SCN and peripherally in the liver and heart of young adult (3 month-old) and aged (22 month-old) mice, sampling the tissues every 6 h for 24 h. The results, expressed as mean ± standard error of the mean (SEM), are shown in Fig. 1, and the results of Cosinor analysis are shown in Fig. 2. Data on statistics performed with Cosinor and ANOVA analyses are listed in Tables 2 and 3, respectively. Data derived from Bonferroni post-hoc test following ANOVA, performed to reveal main effects of age at each time point, are shown in Table 4. 3.1. Transcripts in the SCN Cosinor analyses (Table 2) of clock gene expression in the master oscillator of young mice demonstrated significant 24 h rhythms in Per1 (p b 0.05), Per2 (p b 0.0001), Bmal1 (p b 0.0001), Rev-erbα (p b 0.001), Dbp (p b 0.05) and Dec1 (p b 0.0001) mRNAs, but not in Cry1, Cry 2 and Clock mRNAs. The acrophases of Per1, Rev-erbα, Dbp and Dec1 mRNAs were found in the light period, the acrophase of Per2 at the beginning of the dark phase (Figs. 1, 2; Table 2), while the acrophase of Bmal1 was seen in the dark period. The diurnal rhythm of Per1 occurred in the antiphase (i.e., with a difference of about 12 h) from that of Bmal1, and the Bmal1 rhythm in the antiphase from that of Rev-erbα. We also observed that the acrophase of Per1 expression occurred in the SCN approximately 5–6 h earlier than in peripheral tissues. In aged mice, diurnal rhythmicity was preserved in Per1 (p b 0.01), and in Per2 (p b 0.0001) expression with minor amplitude variation, but with a tendency to a phase advance in the timing of the peak with respect to young mice, in agreement with previous findings (Weinert et al., 2001). In addition, as in young mice, no significant diurnal rhythm of Cry2 and Clock expression was found in the aged mice. At variance with young mice, a significant oscillation of Cry1 expression (p b 0.05) was found instead in the aged mice. In contrast to young mice, in aged mice no diurnal rhythm was found in Bmal1, Dbp, and Dec1 mRNAs, while daily oscillation of Rev-erbα was still significant despite amplitude decrease. The two-way ANOVA (Table 3) evaluation of the expression of these clock genes in the SCN revealed a main effect of age for Per2 (F1,47 = 5.79, p b 0.05), Bmal1 (F1,47 = 14.73, p b 0.001), Rev-erbα

(F1,47 = 32.29, p b 0.001), Dbp (F1,47 = 7.18, p b 0.05), and Dec1 (F1,47 = 9.07, p b 0.01) expression, and a significant time × age interaction for Per2 (F3,47 = 9.62, p b 0.001), Rev-erbα (F3,47 = 13.64, p b 0.001), and Dec1 (F3,47 = 12.55, p b 0.001) expression. 3.2. Transcripts in the liver Cosinor analyses (Table 2) of clock gene expression in the liver of young mice revealed significant 24 h rhythms in the expression of Per1 (p b 0.0001), Per2 (p b 0.0001), Cry1 (p b 0.01), Bmal1 (p b 0.0001), Rev-erbα (p b 0.001), Dbp (p b 0.0001), and Dec1 (p b 0.001), but not of Cry 2 and Clock mRNAs. The acrophases of Per1, Per2 and Cry1 expression occurred during the period of darkness, whereas the acrophases of Bmal1, Rev-erbα, Dbp and Dec1 expression were found during the light phase (Figs. 1, 2; Table 2). The same analysis in the aged mice revealed changes mostly in the expression of clock genes of the core loop, with relative preservation of the examined CCGS. In particular, a significant diurnal oscillation of Bmal1 (p b 0.05), Rev-erbα (p b 0.0001), Dbp (p b 0.0001) and Dec1 (p b 0.05) was preserved in the aged liver. On the other hand, a decrease of the amplitude of Per1, Per2, and Cry1 expression led to significant dampening of the respective rhythms. Among core clock genes, an exception was represented by the rhythm of Clock expression, which was non significant in both the liver and heart of young mice and was instead significant (p b 0.05) in these tissues of the aged mice. It is worth noting that, except for Per1, Per2 and Dec1 expression, all the other transcripts exhibited a general tendency towards an increased MESOR value. In the liver of the aged mice the MESOR value of Rev-erbα was about 1.5 times higher than in young mice. The two way ANOVA evaluation (Table 3) of the expression of clock gene transcripts in the liver revealed a main effect of age for Per1 (F1,47 = 7.42, p b 0.05), Cry1 (F1,47 = 7.98, p b 0.01), Clock (F1,47 = 5.94, p b 0.05), and Rev-erbα (F1,47 = 6.75, p b 0.05), and a significant time × age interaction for the expression of Per1 (F3,47 = 14.51, p b 0.001), Per2 (F3,47 = 3.28, p b 0.05), and Rev-erbα (F3,47 = 5.92, p b 0.01). 3.3. Transcripts in the heart Cosinor analyses (Fig. 2, Table 2) for the heart of young mice revealed significant 24 h rhythms in the expression of Per1 (p b 0.0001), Per2 (p b 0.0001), Cry1 (p b 0.05), Bmal1 (p b 0.0001), Rev-erbα (p b 0.01), Dbp (p b 0.001) and Dec1 (p b 0.01), but not of Cry 2 and Clock mRNAs. As in the liver, the acrophases of Per1, Per2 and Cry1 expression appeared during the period of darkness, whereas the acrophases of Bmal1, Rev-erbα, Dbp, and Dec1 expression were found during the light phase (Figs. 1, 2; Table 2). The daily rhythms of the analyzed clock genes, both those of the core loop and the CCGS, were mainly unchanged in the heart of the aged mice. An exception was represented by the rhythm of Clock expression that, as in the liver, was documented in the aged mice (p b 0.05) but not in young ones. As for the other analyzed genes, a significant rhythm of the expression of Per1 (p b 0.01), Per2 (p b 0.0001), Cry1 (p b 0.001), Bmal1 (p b 0.0001), Rev-erbα (p b 0.05), and Dbp (p b 0.0001) was preserved in the aged mice, with dampening of Cry2 and Dec1 expression. In addition, in the aged mice the values of MESOR of Per2 and Bmal1 transcripts exhibited a tendency towards a decrease, and those of Per1 and Dbp transcripts a tendency towards an increase. The two way-ANOVA evaluation (Table 3) of the expression of clock gene transcripts in the heart revealed a main effect of age for Per2 (F1,47 = 8.41, p b 0.01), Cry1 (F1,47 = 6.27, p b 0.05), Bmal1 (F1,47 = 4.48, p b 0.05), and Dbp (F1,47 = 13.78, p b 0.01), and a significant time × age interaction for the expression of Per1 (F3,47 = 1.23, p b 0.05), Per2 (F3,47 = 16.04, p b 0.001), Bmal1 (F3,47 = 10.83, p b 0.001), Rev-erbα (F3,47 = 3.31, p b 0.05), and Dec1 (F3,47 = 5.05, p b 0.01).

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SCN Per1

LIVER

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M. Bonaconsa et al. / Experimental Gerontology 55 (2014) 70–79

Transcript Per1

Acrophase (95%CL)

SCN

Per2 Cry1 Bmal1 Rev-erba Dbp Dec1 Per1

Liver

Per2 Cry1 Clock Bmal1 Rev-erba Dbp Dec1 Per1

Heart

Per2 Cry1 Clock Bmal1 Rev-erba Dbp Dec1 00

04

08

12

16

20

00

TIME (Hours After Light On) Fig. 2. Acrophase chart showing peaks of fitted 24 h cosine for the clock genes analyzed in the suprachiasmatic nucleus (SCN), liver and heart of young and aged mice sampled every 6 h (see also Table 2). The lateral bars indicate confidence limits (CL) of the acrophase when p was ≤0.05 from the non-zero amplitude. The gray column indicates the 12 h of darkness in the light/dark cycle. Open dots indicate aged mice and filled dots young mice.

4. Discussion The present study shows that aging is associated with changes of the clock gene network which show distinct features at central and peripheral levels. The findings obtained in the different analyzed tissues are discussed below. 4.1. Preservation of core clock loop rhythm and loss of secondary clock loop rhythm in the aged SCN A decline of humoral signaling, synaptic network (Kawakami et al., 1997; Palomba et al., 2008; Roozendaal et al., 1987), and electrical

intercellular phase synchronization (Farajnia et al., 2012; Nakamura et al., 2011) has been reported in the aging SCN, and in turn can lead to a weakened circadian output to downstream oscillators. Previous investigations (Aujard et al., 2001; Nakamura et al., 2011) have shown that the circadian output measured at the level of electrical activity rhythms in the SCN deteriorates during aging, and this precedes an alteration of the molecular clockwork measured by PER2 levels. However, the molecular clockwork can drive rhythms in the SCN neural activity (see for review Colwell, 2011). It is therefore crucial to identify the clock gene pathways involved in the aging-related weakening of SCN activity. The present results indicate that the 24 h profiles of the expression of the core feedback loop components Per1 and Per2 are preserved in the aged SCN, in agreement with previous data in different species (Asai et al., 2001; Kolker et al., 2003; Weinert et al., 2001; Yamazaki et al., 2002). Cry1 expression, previously reported as rhythmic and unaltered in the aged SCN (Asai et al., 2001), and as arrhythmic in both the young and aged SCN investigated at the two time points (Weinert et al., 2001), was found in our study to be rhythmic only in the aged SCN. This discrepancy with previous data could be ascribed to the sampled time points. On the other hand, Cry2 was here found to be arrhythmic in both young and aged mice. No age effect was found also in Clock expression, in accordance with a previous report in mice (Weinert et al., 2001), and at variance with an age-related decrease of Clock expression reported in hamsters (Kolker et al., 2003), which could be due to differences in the utilized experimental parameters and species. In contrast, the diurnal expression of Bmal1 transcript, robustly rhythmic in the young SCN, was here found to be arrhythmic in the SCN of aged mice. The dampening of Bmal1 oscillation, together with a decrease of its expression level especially during the period of darkness, is consistent with previous results in the hamster (Duncan et al., 2013; Kolker et al., 2003), and with data, based on immunohistochemistry, in middle-aged mice (Wyse and Coogan, 2010). Bmal1 represents a unique intersection in the complex network of core clock genes and CCGs. Essential in both input and output clock pathways, Bmal1 is the only gene whose deletion abolishes both behavioral and metabolic circadian rhythms (Bunger et al., 2000), as supported by several lines of evidence (see for review Bonaconsa et al., 2013). In the aged SCN Bmal1 expression could contribute to a normal generation of rhythms by the clock core loop. Noteworthy, in mice with targeted deletion of the gene encoding vasoactive intestinal peptide (VIP), the diurnal rhythms of Bmal1 expression in the SCN disappear and the amplitude decreases (Loh et al., 2011). Interestingly, the present results in the SCN of aged mice seem to parallel previous studies of reduced expression of VIP in the aged SCN (Roozendaal et al., 1987; review in Bonaconsa et al., 2013). Advanced age was here found to markedly affect the expression in the SCN also of Rev-erbα, with amplitude weakening and a phase advance (4 h) of the acrophase, though with a still significant rhythm. As with Bmal1, Rev-erbα plays a major role in the control of rhythmic transcription of the clock output (Preitner et al., 2002). The dampening of Rev-erbα expression we observed in the aged SCN could affect the rhythmic transcription of clock output genes. The expression of the CCGS Dbp and Dec1 became arrhythmic in the aged SCN. Besides the reciprocal control exerted by Dbp on the clock core loop (Ripperger and Schibler, 2006; Yamaguchi et al., 2000), this gene is a direct regulator of the clock output (Lopez-Molina et al., 1997). It is worth recalling that Dbp expression in the SCN has been involved in circadian behavior, concerning not only spontaneous

Fig. 1. Relative expression of the transcripts in the suprachiasmatic nucleus (SCN), liver and heart of young adult (3 months) and aged (22 months) mice and their diurnal patterns at 4 time points (hours after lights on at 2, 8, 14, 20) for 24 h. The horizontal bar at the bottom of each graph represents the lighting conditions (12 h/12 h light/dark cycle, with Zeitgeber time, ZT, 0: lights-on time). Data from young animals are shown as solid lines, and those from aged animals as dashed lines. Open dots indicate aged mice and filled dots young mice. Bars represent the mean ± SEM of triplicate samples. The significance of the rhythm in gene expression was determined by Cosinor analysis (Table 2). When single Cosinor analysis detected a significant rhythm (p b 0.05), symbols in the graphics represent oscillation in young adult mice (solid line in the curve), in aged mice (dashed line in the curve), or in both age groups. The effect of time, age and interactions was analyzed by two-way ANOVA (see Table 3). The asterisks indicate significant differences between young and aged mice at each time point, as evaluated by Bonferroni post-hoc test following ANOVA (see Table 4).

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Table 2 Cosinor analysis of clock gene expression in the suprachiasmatic nucleus (SCN), liver and heart of mice of two age groups (young: 3 months; aged: 22 months). Tissue

Transcript

p

PR

MESOR (95% CL)

SCN young

Perl Per2 Cry1 Cry2 Clock Bmall Rev-erba Dbp Dec1 Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erba Dbp Dec1 Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erba Dbp Dec1 Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erba Dbp Dec1 Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erba Dbp Dec1 Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erba Dbp Dec1

b.05 b.0001 ns ns ns b.0001 b.001 b.05 b.0001 b.01 b.0001 b.05 ns ns ns b.05 ns ns b.0001 b.0001 b.01 ns ns b.0001 b.001 b.0001 b.001 ns ns ns ns b.05 b.05 b.0001 b.0001 b.05 b.0001 b.0001 b.05 ns ns b.0001 b.01 b.001 b.01 b.001 b.0001 b.001 ns b.05 b.0001 b.05 b.0001 ns

35 92 10 3 6 60 62 32 68 48 79 35 5 18 26 33 26 20 57 69 37 3 19 62 57 64 50 17 13 15 21 29 43 75 66 29 74 79 40 23 24 79 44 63 45 52 70 57 14 31 85 39 68 23

2.53 (2.03–3.03) 0.49 (0.46–0.52) 2.49 (2.12–2.86) 1.95 (1.52–2.38) 2.47 (2.16–2.77) 4.03 (3.39–4.67) 0.39 (0.33–0.45) 3.90 (3.35–4.44) 0.14 (0.12–0.16) 3.01 (2.65–3.37) 0.52 (0.48–0.56) 2.92 (2.57–3.26) 1.79 (1.59–2.00) 2.68 (2.39–2.97) 3.78 (3.40–4.15) 0.18 (0.15–0.22) 2.56 (2.10–3.02) 0.12 (0.11–0.13) 0.30 (0.15–0.44) 5.99 (4.96–7.02) 0.18 (0.14–0.23) 0.31 (0.20–0.42) 2.97 (2.20–3.73) 83.98 (59.32–108.64) 0.10 (0.06–0.13) 105.9 (62.5–149.4) 0.31 (0.24–0.39) 0.13 (0.05–0.20) 5.50 (4.42–6.58) 0.27 (0.22–0.33) 0.42 (0.32–0.52) 5.32 (3.40–7.23) 104.9 (76.8–133) 0.16 (0.12–0.20) 174.1 (109.5–238.6) 0.23 (0.16–0.30) 11.65 (8.72–14.59) 63.32 (47.26–79.38) 5.06 (4.65–5.46) 2.69 (2.17–3.22) 44.32 (34.82–59.82) 14.26 (10.56–17.97) 0.51 (0.34–0.68) 8.19 (4.66–11.73) 0.44 (0.37–0.50) 14.67 (11.99–17.34) 39.64 (33.04–46.25) 3.88 (3.35–4.41) 2.43 (2.08–2.78) 44.47 (37.30–51.65) 10.59 (9.32–11.85) 0.52 (0.38–0.67) 13.28 (9.65–16.91) 0.39 (0.34–0.45)

SCN aged

Liver young

Liver aged

Heart young

Heart aged

locomotor activity (Lopez-Molina et al., 1997), but also the homeostatic regulation of slow wave sleep (Franken et al., 2000; Mongrain et al., 2011). On the other hand, Dec1 interacts with core clock components (Honma et al., 2002), can be induced by a variety of exogenous and endogenous inputs, and its output targets diverse functions (Kato et al., 2010). Therefore, the present finding of a decrease of Dec1 expression in the aged SCN could reflect a weakened drive by systemic cues and/or by the output of a downstream clock network. Taken together the present data indicate that the core clock machinery, responsible for rhythm generation and maintenance, appears to be preserved in the aged SCN, while the secondary loops, represented by the Rev-erbα–Bmal loop and by the CCGs Dbp and Dec1, are instead markedly affected.

Amplitude (95% CL)

Acrophase (95% CL)

1.15 (0.45–1.84) 0.25 (0.21–0.29)

07.50 (04.18–11.19) 13.44 (12.55–14.33)

2.44 (1.53–3.35) 0.22 (0.13–0.31) 1.15 (0.39–1.90) 0.08 (0.05–0.11) 1.08 (0.57–1.59) 0.21 (0.16–0.27) 0.79 (0.29–1.28)

20.56 (19.04–22.49) 08.33 (06.32–10.34) 08.18 (04.23–12.16) 07.49 (06.03–09.35) 07.14 (04.47–09.38) 13.50 (12.31–15.10) 07.04 (03.34–10.33)

0.08 (0.03–0.13)

04.43 (01.00–08.26)

0.52 (0.32–0.73) 4.25 (2.77–5.72) 0.11 (0.04–0.17)

13.51 (11.53–15.50) 14.00 (12.15–15.45) 19.49 (16.25–23.14)

90.98 (55.65–126.3) 0.13 (0.08–0.18) 168.5 (107.1–229.9) 0.23 (0.12–0.33)

23.50 (21.53–01.44) 08.47 (06.49–10.49) 08.26 (06.36–10.16) 11.13 (08.51–13.42)

3.56 (0.86–6.26) 63.6 (20.6–106.6) 0.20 (0.15–0.26) 265.2 (173.9–356.5) 0.13 (0.03–0.23) 13.65 (9.5–17.8) 81.06 (58.15–103.96) 0.82 (0.27–1.38)

23.53 (18.57–04.48) 22.41 (18.47–01.51) 05.49 (04.27–07.11) 07.32 (05.49–09.15) 08.32 (03.46–13.17) 12.57 (11.26–14.25) 13.35 (12.10–15.01) 21.28 (17.20–01.49)

16.22 (11.18–21.27) 0.44 (0.21–0.68) 12.82 (7.82–17.82) 0.18 (0.08–0.27) 8.2 (4.57–11.83) 29.34 (20.28–38.40) 1.81 (1.06–2.56)

23.50 (22.14–01.34) 08.11 (05.18–11.03) 07.35 (05.37–09.34) 07.40 (04.41–10.41) 10.37 (08.20–13.07) 11.26 (09.52–13.05) 17.54 (15.48–20.01)

14.46 (4.98–23.94) 9.06 (7.38–10.74) 0.30 (0.08–0.53) 16.17 (11.07–21.27)

21.44 (17.39–02.38) 21.13 (20.19–22.10) 04.11 (23.54–07.34) 07. 25 (05.52–08.54)

4.2. Loss of core clock loop rhythm and persistence of secondary clock loop rhythm in the aged liver Our findings indicate that the expression of clock genes in the aged liver exhibits a pattern distinct from that observed in the aged SCN. In particular, we here observed marked changes of the expression of key components of the clock core loop Per1, Per2 and Cry1 in the liver of aged mice, with amplitude dampening and loss of rhythmicity. The other component of the core loop, Clock, was found to be arrhythmic in the young liver, and rhythmic in the aged liver. These findings confirm and extend a previous study in rats of 13 months vs 27 months of age analyzed at two time points, i.e., 0–2 and 10–12 h after lights-on (Claustrat et al., 2005). Concerning other components of the clock gene auxiliary loops (Bmal1 and Rev-erbα), as well as Dbp and Dec1, the

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Table 3 Data of the statistical evaluation (two-way ANOVA) of transcript expression in the suprachiasmatic nucleus (SCN) and peripheral tissues. Tissue

SCN

Liver

Heart

Transcript

Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erbα Dbp Dec-1 Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erbα Dbp Dec-1 Per1 Per2 Cry1 Cry2 Clock Bmal1 Rev-erbα Dbp Dec-1

Main effect of time point

Main effect of age p

F (1,47)

p

F (3,47)

p

4.643 126.226 2.968 0.570 2.542 0.159 18.854 3.536 22.310 28.170 7.443 4.349 0.625 1.651 14.386 30.926 105.679 10.126 18.674 42.864 2.734 1.703 4.722 41.806 11.432 69.574 3.485

b0.01 b0.001 n.s. n.s. n.s. n.s. b0.001 b0.05 b0.001 b0.001 b0.01 b0.05 n.s. n.s. b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 n.s. n.s. b0.01 b0.001 b0.001 b0.001 b0.05

2.081 5.770 0.100 1.235 0.025 14.735 32.292 7.180 9.073 7.425 0.249 7.979 2.261 5.937 1.728 6.754 3.240 3.218 1.788 8.414 6.268 0.490 0.002 4.476 0.065 13.776 1.336

n.s. b0.05 n.s. n.s. n.s. b0.001 b0.001 b0.05 b0.01 b0.05 n.s. b0.01 n.s. b0.05 n.s. b0.05 n.s. n.s. n.s. b0.01 b0.05 n.s. n.s. b0.05 n.s. b0.01 n.s.

0.653 9.655 0.236 0.373 0.521 0.679 13.642 1.975 12.556 14.508 3.282 1.660 2.008 0.876 2.319 5.923 1.744 1.803 1.230 16.036 2.834 2.261 1.414 10.828 3.306 1.132 5.049

n.s. b0.001 n.s n.s. n.s. n.s. b0.001 n.s. b0.001 b0.001 b0.05 n.s. n.s. n.s. n.s. b0.01 n.s. n.s. b0.05 b0.001 n.s. n.s. n.s. b0.001 b0.05 n.s. b0.01

present data indicate that the circadian rhythm of the expression of these genes is preserved in the aged liver. As many other peripheral organs, the liver represents an autonomous oscillator whose circadian entrainment depends on signals from the SCN, on feeding cues and other systemic cues (review in Schmutz et al., 2012). The independence of the liver as an autonomous oscillator is shown by the finding that about 90% of transcripts is abolished when mouse hepatocyte clocks are turned off, indicating that the remaining 10%, which include Per2 mRNA, are driven by systemic cues such as hormones, metabolites and body temperature (Kornmann et al., 2007). In the aged liver, Per2 arrhythmicity, revealed by the present study, could represent a direct consequence of the impairment of the circadian output at the level of the SCN, in contrast with the preservation of diurnal rhythms of Bmal1, Rev-erbα, Dbp and Dec1, which are likely to be independent from the central regulation. A previous study on the expression of these clock genes at two time points (ZT2 and ZT14) has reported a persistence of Bmal1 expression and a decrease of Dbp and Dec1 expression in the aged liver (Oishi et al., 2011). It is also of relevance that Clock expression, which plays a key role in the regulation

Table 4 Significance values (p) of relative expression of transcripts from young versus aged mice in the suprachiasmatic nucleus (SCN) and peripheral tissues, as obtained by Bonferronicorrected post-hoc t-tests following ANOVA, performed separately for each time point. Only significant p values (corresponding to asterisks in Fig. 1) are listed. Tissue

Transcript

Young vs aged Post-hoc t-test p

SCN

Rev-erbα

Liver

Per1 Per2 Rev-erbα Per1 Per2 Bmal1

Heart

Time × age interaction

F (3,47)

ZT8 ZT14 ZT20 ZT14 ZT14 ZT2 ZT2 ZT14 ZT2

0.002 0.011 0.002 0.006 0.001 0.005 0.009 0.001 0.001

of circadian output genes in the liver (Oishi et al., 2003), was here found to be rhythmic in the aged liver. This may indicate that under conditions of disrupted signaling of the aged SCN, the self-sustained oscillator in the aged liver could become entrained by different systemic cues. The present finding that the Rev-erbα circadian rhythm exhibits at an advanced age increased robustness and transcript level in the aged liver is of special interest considering that this gene regulates Bmal1 expression (Preitner et al., 2002; Sato et al., 2004). Concerning liver functions, it is worth recalling that Rev-erbα is also an important modulator of circadian output metabolic pathways, including lipid and bile acid metabolism, adipogenesis, gluconeogenesis, as well as of the inflammatory response (Duez and Staels, 2009). Also Dbp plays a key role in the regulation of enzymes involved in detoxification and drug metabolism (Gachon et al., 2006). Furthermore, Dbp can adjust its own expression phase according to the photoperiod, so that the transcription of metabolic and detoxification enzymes can be activated prior to food intake (Stratmann et al., 2010). We here showed that the diurnal rhythm of Dec1 expression, involved in the fine regulation and robustness of the molecular clock (Nakashima et al., 2008) and primarily in hepatic metabolism (Noshiro et al., 2009), is preserved in the aged liver. Microarray analyses comparing the liver of Dec1-null mice and their wild-type counterpart have identified 42 target genes of Dec1, including insulin-like growth factor-binding protein 1, and important components of the innate immune system (Grechez-Cassiau et al., 2004), pointing to a key role of Dec1 in liver functions. The data we obtained indicate that the expression of clock genes in the aged liver on one hand reflects an impairment of the SCN and downstream circadian signaling, and on the other hand points to the liver as autonomous oscillator whose role in the metabolic homeostasis of the entire organism is preserved at an advanced age. 4.3. Preservation of core clock loop and secondary clock loop rhythm in the aged heart The clockwork machinery of the aged heart was here found to exhibit specific features, different from those of the aged SCN and

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liver. In particular, the diurnal rhythm of the expression of the core loop clock genes Per1, Per2, Cry1 and Bmal1 was preserved in the aged heart, though with severe amplitude dampening of Per1, Per2 and Bmal1 expression. The present data on the diurnal rhythm of Clock expression, significant only in the aged heart, and Cry2 expression, arrhythmic in both the young and aged hearts, are similar to those obtained in the liver. Concerning clock genes of the auxiliary loops, the diurnal rhythm of Rev-erbα and Dbp expression persisted in the aged heart, though with a significant reduction of Rev-erbα oscillation amplitude, whereas the diurnal oscillation of Dec1 was lost. These findings are supported by previous analyses limited to two time points. In particular, a tendency towards a decrease of Per expression and an increased Bmal1 expression was found in the heart of aged rats (Claustrat et al., 2005). Persistence of Bmal1, Dbp and Dec1 expression was reported in the heart of aged mice (Oishi et al., 2011). No significant differences in the rhythm of expression of Per2 and Bmal1, as well as that of Dbp was found in the heart of aged vs young mice (Durgan et al., 2011). The function of the heart as peripheral oscillator is due to the circadian clock which operates within myocytes (Durgan et al., 2005) and influences myocardial contractile function, metabolism, and gene expression (Bray et al., 2008; Young, 2006). At least 13% of genes expressed in the mouse heart exhibit significant circadian rhythmicity (Martino et al., 2004). Among clock genes, Per2 has been especially involved in the pathophysiology of the cardiovascular system as mediator of vascular senescence, angiogenesis and endothelial function (Viswambharan et al., 2007; Wang et al., 2008). Bmal1 plays a key role in the regulation of blood pressure and heart rate, as shown by the disruption of the circadian oscillation of these functions in mice with targeted Bmal1 deletion (Curtis et al., 2007). It is worth noting that Per2 and Bmal1 amplitudes in the aged heart have been here found to be dampened, but with preservation of their diurnal rhythm. The above findings indicate that the molecular clockwork is mostly preserved in the aged heart, though with a tendency towards amplitude dampening presumably associated with an attenuation of systemic cues. 4.4. Timing of clock gene expression at an advanced age Despite the tissue-specific alterations in the expression of clock genes reported above, the present results indicate that some key parameters of circadian rhythm organization are preserved at an advanced age. First, the temporal distribution of circadian gene expression, typically phase-delayed in peripheral tissues by 3–6 h compared to the SCN (Balsalobre et al., 1998; Lopez-Molina et al., 1997; Zylka et al., 1998) was here found to be unchanged in the aged liver and heart. In addition, the expression of the analyzed genes showed in the aged mice a tendency towards a phase advance in the 4 h range. For example, Rev-erbα acrophase exhibited a 3 h advance in the aged liver (from 8.48 h to 5.49 h) and a 4 h advance in the aged heart (from 8.11 h to 4.11 h). During aging, phase advance is widely documented for several body rhythms, including the sleep–wake cycle, hormonal release, and body temperature (reviews in Monk, 2005; Weinert, 2000). Furthermore, the temporal sequence of the expression of interlocked genes, such as Per1 and Bmal1, and Bmal1 and Rev-erbα, typically cycling in antiphase (Nishide et al., 2006; Oishi et al., 1998; Yagita et al., 2001) was here found to be preserved at an advanced age. Finally, the level of expression of genes such as Per1, Bma1, Rev-erbα and Dbp, typically higher in peripheral tissues than in the SCN (Dibner et al., 2010; Gachon et al., 2004; Lopez-Molina et al., 1997), was mainly preserved in the analyzed aged tissues. 4.5. Concluding remarks Our findings indicate that in the aged SCN the decline of the rhythmic function of secondary loops is likely to impair the circadian

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output, whereas at peripheral level a deterioration of core clock gene expression prevails, with dampening of the rhythm, as in the liver, or of amplitude, as in the heart. The aging process leads to a chronic low-grade inflammatory condition, with increase of pro-inflammatory mediators at the periphery and in the brain, including the SCN (review in Bonaconsa et al., 2013). Pro-inflammatory cytokines impair clock gene expression (Cavadini et al., 2007; Gast et al., 2012). Dysregulation of the aged molecular clockwork could, at least in part, be ascribed to this mechanism, whose impact on different tissues remains to be investigated and is of crucial importance for the understanding of body aging. Our investigation spurs further inquiries and needs to be extended in future studies. Increasing the sampled time points (from 4 to 6) would lead to a finer phase resolution and a more precise evaluation of possible differences of diurnal gene expression. Experiments under constant light conditions could also provide finer details on the rhythm endogenous nature, though they frequently do not lead to statistically significant differences (Sothern et al., 2009; Takekida et al., 2000). Further studies are needed also to investigate whether and how the observed age-related changes of the molecular clockwork could be sensitive to pharmacological treatments or manipulations based on environmental cues. Even considering these limitations, the present findings point to a general picture of frailty, rather than to severe impairment and diffuse disruption, of the circadian system molecular clockwork at an advanced age. Conflict of interest The authors declare no conflicts of interest. Acknowledgments The authors are grateful to E. Moccheggiani (INRCCA, Ancona, Italy) for his help in obtaining aged animals, and to anonymous Experimental Gerontology reviewers for their helpful criticisms. The support of intramural funds of the University of Verona is gratefully acknowledged. Thanks are due to Valeria Colavito for her help in statistical analyses. References Albrecht, U., 2012. Timing to perfection: the biology of central and peripheral circadian clocks. Neuron 74, 246–260. Asai, M., Yoshinobu, Y., Kaneko, S., Mori, A., Nikaido, T., Moriya, T., Akiyama, M., Shibata, S., 2001. Circadian profile of Per gene mRNA expression in the suprachiasmatic nucleus, paraventricular nucleus, and pineal body of aged rats. J. Neurosci. Res. 66, 1133–1139. Aujard, F., Herzog, E.D., Block, G.D., 2001. Circadian rhythms in firing rate of individual suprachiasmatic nucleus neurons from adult and middle-aged mice. Neuroscience 106, 255–261. Balsalobre, A., Damiola, F., Schibler, U., 1998. A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93, 929–937. Bonaconsa, M., Colavito, V., Pifferi, F., Aujard, F., Schenker, E., Dix, S., Grassi-Zucconi, G., Bentivoglio, M., Bertini, G., 2013. Cell clocks and neuronal networks: neuron ticking and synchronization in aging and aging-related neurodegenerative disease. Curr. Alzheimer. Res. 10, 597–608. Bray, M.S., Shaw, C.A., Moore, M.W., Garcia, R.A., Zanquetta, M.M., Durgan, D.J., Jeong, W.J., Tsai, J.Y., Bugger, H., Zhang, D., Rohrwasser, A., Rennison, J.H., Dyck, J.R., Litwin, S.E., Hardin, P.E., Chow, C.W., Chandler, M.P., Abel, E.D., Young, M.E., 2008. Disruption of the circadian clock within the cardiomyocyte influences myocardial contractile function, metabolism, and gene expression. Am. J. Physiol. Heart Circ. Physiol. 294, 1036–1047. Bunger, M.K., Wilsbacher, L.D., Moran, S.M., Clendenin, C., Radcliffe, L.A., Hogenesch, J.B., Simon, M.C., Takahashi, J.S., Bradfield, C.A., 2000. Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103, 1009–1017. Cavadini, G., Petrzilka, S., Kohler, P., Jud, C., Tobler, I., Birchler, T., Fontana, A., 2007. TNF-α suppresses the expression of clock genes by interfering with E-box-mediated transcription. Proc. Natl. Acad. Sci. U. S. A. 104, 12843–12848. Claustrat, F., Fournier, I., Geelen, G., Brun, J., Corman, B., Claustrat, B., 2005. Aging and circadian clock gene expression in peripheral tissues in rats. Pathol. Biol. (Paris) 53, 257–260. Colwell, C.S., 2011. Linking neural activity and molecular oscillations in the SCN. Nat. Rev. Neurosci. 12, 553–569.

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Differential modulation of clock gene expression in the suprachiasmatic nucleus, liver and heart of aged mice.

Studies on the molecular clockwork during aging have been hitherto addressed to core clock genes. These previous investigations indicate that circadia...
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