Identification of circadian clock modulators from existing drugs
2018; Springer Nature; Volume: 10; Issue: 5 Linguagem: Inglês
10.15252/emmm.201708724
ISSN1757-4684
AutoresT. Katherine Tamai, Yusuke Nakane, Wataru Ota, Akane Kobayashi, M. Ishiguro, Naoya Kadofusa, Keisuke Ikegami, Kazuhiro Yagita, Yasufumi Shigeyoshi, Masaki Sudo, Taeko Nishiwaki‐Ohkawa, Ayato Sato, Takashi Yoshimura,
Tópico(s)Photoreceptor and optogenetics research
ResumoResearch Article17 April 2018Open Access Transparent process Identification of circadian clock modulators from existing drugs T Katherine Tamai T Katherine Tamai Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Yusuke Nakane Yusuke Nakane Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Wataru Ota Wataru Ota Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Akane Kobayashi Akane Kobayashi Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Masateru Ishiguro Masateru Ishiguro Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Naoya Kadofusa Naoya Kadofusa Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Keisuke Ikegami Keisuke Ikegami Department of Anatomy and Neurobiology, Kindai University Faculty of Medicine, Osaka, Japan Search for more papers by this author Kazuhiro Yagita Kazuhiro Yagita Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan Search for more papers by this author Yasufumi Shigeyoshi Yasufumi Shigeyoshi Department of Anatomy and Neurobiology, Kindai University Faculty of Medicine, Osaka, Japan Search for more papers by this author Masaki Sudo Masaki Sudo Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Taeko Nishiwaki-Ohkawa Taeko Nishiwaki-Ohkawa Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Ayato Sato Ayato Sato Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Takashi Yoshimura Corresponding Author Takashi Yoshimura [email protected] orcid.org/0000-0001-7018-9652 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Division of Seasonal Biology, National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author T Katherine Tamai T Katherine Tamai Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Yusuke Nakane Yusuke Nakane Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Wataru Ota Wataru Ota Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Akane Kobayashi Akane Kobayashi Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Masateru Ishiguro Masateru Ishiguro Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Naoya Kadofusa Naoya Kadofusa Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Keisuke Ikegami Keisuke Ikegami Department of Anatomy and Neurobiology, Kindai University Faculty of Medicine, Osaka, Japan Search for more papers by this author Kazuhiro Yagita Kazuhiro Yagita Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan Search for more papers by this author Yasufumi Shigeyoshi Yasufumi Shigeyoshi Department of Anatomy and Neurobiology, Kindai University Faculty of Medicine, Osaka, Japan Search for more papers by this author Masaki Sudo Masaki Sudo Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Taeko Nishiwaki-Ohkawa Taeko Nishiwaki-Ohkawa Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Ayato Sato Ayato Sato Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Search for more papers by this author Takashi Yoshimura Corresponding Author Takashi Yoshimura [email protected] orcid.org/0000-0001-7018-9652 Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Division of Seasonal Biology, National Institute for Basic Biology, Okazaki, Japan Search for more papers by this author Author Information T Katherine Tamai1, Yusuke Nakane1,2, Wataru Ota1,2, Akane Kobayashi1,2, Masateru Ishiguro1,2, Naoya Kadofusa1, Keisuke Ikegami3, Kazuhiro Yagita4, Yasufumi Shigeyoshi3, Masaki Sudo1, Taeko Nishiwaki-Ohkawa1,2, Ayato Sato1 and Takashi Yoshimura *,1,2,5,6 1Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan 2Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan 3Department of Anatomy and Neurobiology, Kindai University Faculty of Medicine, Osaka, Japan 4Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan 5Avian Bioscience Research Center, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan 6Division of Seasonal Biology, National Institute for Basic Biology, Okazaki, Japan *Corresponding author. Tel: +81 52 789 4056; E-mail: [email protected] EMBO Mol Med (2018)10:e8724https://doi.org/10.15252/emmm.201708724 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Chronic circadian disruption due to shift work or frequent travel across time zones leads to jet-lag and an increased risk of diabetes, cardiovascular disease, and cancer. The development of new pharmaceuticals to treat circadian disorders, however, is costly and hugely time-consuming. We therefore performed a high-throughput chemical screen of existing drugs for circadian clock modulators in human U2OS cells, with the aim of repurposing known bioactive compounds. Approximately 5% of the drugs screened altered circadian period, including the period-shortening compound dehydroepiandrosterone (DHEA; also known as prasterone). DHEA is one of the most abundant circulating steroid hormones in humans and is available as a dietary supplement in the USA. Dietary administration of DHEA to mice shortened free-running circadian period and accelerated re-entrainment to advanced light–dark (LD) cycles, thereby reducing jet-lag. Our drug screen also revealed the involvement of tyrosine kinases, ABL1 and ABL2, and the BCR serine/threonine kinase in regulating circadian period. Thus, drug repurposing is a useful approach to identify new circadian clock modulators and potential therapies for circadian disorders. Synopsis Chronic circadian misalignment has long term consequences on our health and leads to increased risk of developing diabetes, cardiovascular disease and cancer. Using a drug repurposing approach, dehydroepiandrosterone (DHEA) was identified as an important circadian clock modulator. Approximately 5% of the screened drugs altered circadian period. DHEA shortened circadian period in cells and tissues. When fed to mice, DHEA shortened circadian period and significantly reduced jet-lag. ABL1/2 tyrosine kinases and BCR serine/threonine kinase are involved in regulating circadian period. Drug repurposing is a useful approach to identify new circadian clock modulators. Introduction Circadian clocks are highly conserved, endogenous timers present in virtually all living organisms. These clocks regulate near 24-h rhythms in numerous behavioral and physiological processes, including our sleep–wake cycles and metabolism. In mammals, these daily rhythms are controlled by a central circadian pacemaker, the suprachiasmatic nucleus (SCN), located in the anterior hypothalamus (Ralph et al, 1990; Klein et al, 1991). Most peripheral tissues and cells also contain self-sustained circadian oscillators (Balsalobre et al, 1998; Yamazaki et al, 2000; Yoo et al, 2004), which are driven by transcriptional–translational feedback loops composed of circadian clock genes and proteins (Takahashi, 2015). The basic helix-loop-helix proteins CLOCK and BMAL1 heterodimerize to form a transcriptional activator complex and activate the PER and CRY repressor genes, whose protein products, in turn, repress their own transcription. Disruption of the circadian clock due to shift work or travel across time zones leads to circadian desynchrony, or jet-lag, and reflects a mismatch between the internal biological clock and external time cues (Arendt, 2009). Chronic circadian misalignment has long-term consequences on our health and often leads to an increased risk of diabetes, cardiovascular disease and cancer (Davidson et al, 2006; Scheer et al, 2009; Buxton et al, 2012). Recent circadian studies have identified several genes that by mutation, chemical inhibition or knockdown, significantly reduce jet-lag, including the vasopressin V1a and V1b receptors, casein kinase 1 epsilon (CK1ε), and salt inducible kinase 1 (Sik1; Jagannath et al, 2013; Yamaguchi et al, 2013; Pilorz et al, 2014). Injection of the neuropeptide vasoactive intestinal polypeptide (VIP) into the mouse SCN also accelerates re-entrainment to new light–dark (LD) cycles (An et al, 2013). However, there are currently no orally available drugs or supplements, except melatonin perhaps, to combat the complex behavioral and metabolic consequences of jet-lag. The development of new pharmaceuticals, from drug discovery to market approval, is costly and hugely time-consuming with a high rate of failure. Drug repurposing, or identifying new functions for existing compounds, is therefore a popular approach to fast track potential drugs through to clinical trials (Rennekamp & Peterson, 2015). We used a high-throughput chemical screening strategy to isolate new circadian clock modulators (Hirota et al, 2008; Isojima et al, 2009; Chen et al, 2011), with the aim of repurposing known bioactive compounds. We tested over 1,000 small molecules from two chemical libraries, including US Food and Drug Administration (FDA)-approved drugs and the International Drug Collection (IDC), and we identified nearly 60 chemicals that altered circadian period, including the period-shortening molecule DHEA. When tested in mice in vivo, DHEA significantly shortened circadian period of locomotor rhythms and accelerated re-entrainment to advanced LD cycles, suggesting that DHEA might serve as a convenient over-the-counter treatment for jet-lag. Our chemical screen also identified several tyrosine kinase inhibitors that modulated clock function, implicating this class of kinases in the control of circadian period. Thus, drug repurposing is a useful strategy to identify potential treatments for circadian misalignment and new clock regulators. Results Identification of new circadian clock modulators Using a human osteosarcoma U2OS cell line stably expressing the clock reporter Bmal1-dLuc (Oshima et al, 2015), we screened over 1,000 molecules from an FDA-approved drug library and the IDC to identify new circadian clock modulators. To reduce the number of false positives, all compounds were tested in triplicate at two different concentrations (10 and 1 μM) in a 384-well plate format. Primary screening identified several potential hit compounds based on period change. Drugs that lengthened circadian period by 1 or more hours or shortened period by 0.5 or more hours (Fig 1A) were selected for additional screening, although some chemicals, whose effects were clearly dose-dependent but below threshold values, were also included. Of these 72 potential hit compounds, 59 were validated in a secondary screen for dose-dependency (Appendix Fig S1), with 46 chemicals lengthening and 13 compounds shortening circadian period. Thus, ~5% of drugs currently on the market or in clinical trials altered circadian period. This indicates that, depending on the dose and duration of treatment for other ailments, one "side effect" of these drugs might be perturbation of the circadian clock. Therapeutic classification revealed eight different categories of hit compounds, including (i) anti-cancer and immunosuppressive drugs, (ii) disinfectants and antiseptics, (iii) hormones and contraceptives, (iv) drugs targeting the central nervous system, (v) dermatological agents, (vi) gastrointestinal drugs, (vii) vitamins and minerals, and (viii) cardiovascular agents (Fig 1B). Importantly, some of these compounds have been previously identified in circadian drug screens (i.e., DNA damage agent, mitoxantrone; Hirota et al, 2008), whereas others affect pathways that have been implicated in regulating circadian rhythms (i.e., EGF receptor inhibitor, erlotinib; Kramer et al, 2001), thus validating our screening protocol. One obvious advantage of screening existing drugs is that they frequently have known mechanisms of action (Rennekamp & Peterson, 2015). All known targets of hit compounds identified in our screen are listed in Table EV1. In addition, the top six categories of drug targets are shown and include DNA, the androgen receptor (AR), DNA topoisomerase 2α, the nuclear receptor subfamily 1 group I member 2 (NR1I2), the retinoic acid receptor α (RARα)/retinoid X receptor β (RXRβ), and the tubulin β chain (Fig 1C). Figure 1. Chemical screening for circadian clock modulators US Food and Drug Administration (FDA)-approved drugs (left graphs) and the International Drug Collection (IDC) (right graphs) were screened in U2OS clock reporter cells at 10 μM (top graphs) and 1 μM (bottom graphs). Primary screening identified 72 potential hit compounds that lengthened (by 1 or more hours; at or above red line) or shortened (by 0.5 or more hours; at or below blue line) circadian period. Therapeutic classification of 59 hit compounds validated in a secondary screen for dose-dependency (data for all 59 hit compounds are shown in Appendix Fig S1, and all statistical information is shown in Appendix Table S2). Top six targets of hit compounds reported in the DrugBank or KEGG DRUG databases. Download figure Download PowerPoint DHEA primarily shortens circadian period Relatively few chemicals identified in our screen shortened circadian period, so we initially focused on characterizing some of these compounds. Interestingly, the steroid hormone DHEA and its derivative DHEA acetate showed clear dose-dependent shortening of circadian period in U2OS cells (Fig 2A and Appendix Fig S1). Recent reports, however, have shown that high doses of DHEA (e.g., 100 μM) lengthen circadian period and decrease amplitude (Rey et al, 2016; Putker et al, 2017), which we have confirmed (Fig EV1). In humans, DHEA is a steroid hormone produced in the adrenal gland, gonads, and brain and is one of the most abundant circulating hormones. It functions as an androgen precursor and can be converted to testosterone or estrogen (Appendix Fig S2). To determine whether DHEA itself or one of its precursors or metabolites modulates circadian period, we treated U2OS cells with several endogenous steroid hormones and monitored bioluminescence. These results show that deoxycorticosterone (21-hydroxyprogesterone), 11-deoxycortisol, and testosterone lengthened circadian period (Appendix Fig S2). Some of these hormones were included in our primary drug screen, but were not identified as hit compounds, because their effects on circadian period were below threshold values. In contrast, progesterone and 17α-hydroxyprogesterone lengthened period by more than 1 h and were indeed classified as hit compounds (Appendix Figs S1 and S2). Interestingly, DHEA was the only endogenous steroid hormone tested that shortened period in U2OS cells. Notably, its metabolite DHEA sulfate had no effect on circadian period (Appendix Fig S2). Figure 2. DHEA shortens circadian period Chemical structure of DHEA (left) and its dose-dependent effects on circadian period in U2OS cells (histogram, right; luminescent traces, below). Data are the mean ± SEM from three independent experiments and were analyzed by one-way ANOVA, followed by a Dunnett's test (*P < 0.05, **P < 0.01). All statistical information is shown in Appendix Table S1. Effect of 20 μM DHEA on mouse embryonic fibroblasts (MEFs) and 50 μM DHEA on SCN and lung from mPer2Luc mice. Data are presented as the mean ± SEM of four or five independent experiments and were analyzed by a Welch's t-test (*P < 0.05, **P < 0.01). All statistical information is shown in Appendix Table S1. Representative double-plotted actograms of control (n = 8; left) and DHEA-treated (n = 14; middle) animals entrained in LD and transferred into DD. DHEA (0.5% w/w; vertical line) was administered in powdered food ˜1 week after transfer into DD for 6 days and then increased to 1.0% (w/w) for another 6 days. Animals were then returned to normal powdered food without drug for 1 week. Free-running period was calculated based on activity onset (left graph) or activity offset (right graph) and plotted as the mean ± SEM (far right). Data were analyzed by two-way ANOVA, followed by a Sidak's multiple comparisons test (*P < 0.05, **P < 0.01). All statistical information is shown in Appendix Table S1. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. High doses of DHEA lengthen circadian periodU2OS cells were treated with 0, 20, 50, and 100 μM DHEA in 0.2% DMSO. Histograms show effects on circadian period, and results are presented as the mean ± SEM (n = 6). Data were analyzed by one-way ANOVA, followed by a Dunnett's test (**P < 0.01). All statistical information is shown in Appendix Table S2. Download figure Download PowerPoint To determine whether the effects of DHEA on the circadian clock were cell type- or tissue-specific, we treated mPer2Luc mouse embryonic fibroblasts (MEFs) with DHEA. Similar to our observations in U2OS cells, DHEA shortened circadian period in MEFs (Fig 2B). We then prepared explant cultures of SCN and lung from mPer2Luc mice and treated them with DHEA. Although slightly higher concentrations were required, we observed significant shortening of circadian period in both of these tissues (Fig 2B). These results indicated that DHEA indeed shortens circadian period in cells and tissues, including the SCN. Numerous studies in mice have shown that DHEA can be administered orally (Milewich et al, 1995). Therefore, to test its effects in vivo, we added DHEA directly to powdered food and assessed circadian clock function by monitoring wheel-running activity. We also surgically implanted temperature devices into mice to measure body temperature rhythms. Mice were initially fed powdered food without drug and exposed to a light–dark cycle (12L:12D) for 1 or 2 weeks to entrain their circadian clocks (Fig 2C). Animals were then transferred into constant darkness (DD) for about 1 week to measure free-running circadian period. Mice were then given either normal powdered food or powdered food mixed with 0.5% DHEA (w/w) for 6 days. This dose was then increased to 1% DHEA (w/w) for another 6 days. Importantly, these concentrations of DHEA have been previously tested in mice and shown to be safe (Milewich et al, 1995). Mice were then returned to normal powdered food until the end of the experiment. These results show that DHEA had a profound effect on behavior and appeared to shorten circadian period of both activity and body temperature rhythms in a dose-dependent manner (Figs 2C and EV2, respectively). Surprisingly, after return to normal powdered food, activity onset returned to its original phase (i.e., to the phase before drug treatment; Fig 2C). Although DHEA is known to readily cross the blood–brain barrier (Stárka et al, 2015), these data suggest that its effects on behavior were independent of the SCN clock and might be acting through extra-SCN circadian pacemaker(s) (Pezuk et al, 2010). Click here to expand this figure. Figure EV2. Effect of DHEA on circadian rhythms in body temperatureRepresentative double-plotted temperature records from control (n = 8; left) and DHEA-treated (n = 13; middle) animals. Free-running period (mean ± SEM) was determined (right) and analyzed by two-way ANOVA, followed by a Sidak's multiple comparisons test (**P < 0.01). All statistical information is shown in Appendix Table S2. Download figure Download PowerPoint Dietary administration of DHEA reduces jet-lag in mice By temporarily speeding up or slowing down the circadian clock, drugs that shorten or lengthen period, respectively, might accelerate re-entrainment to new light–dark (LD) cycles, thereby reducing jet-lag (Fig EV3). To test this hypothesis, we performed a jet-lag experiment and examined the effects of DHEA on re-entrainment to a 6-h phase-advanced LD cycle (Fig 3). We fed mice normal powdered food, as above, and exposed them to a LD cycle to synchronize their circadian clocks. After ~1 week, mice were given either normal powdered food or food mixed with DHEA, and the LD cycle was advanced by 6 h (Fig 3A). Chronic administration of DHEA led to a dramatic phase advance in activity onset compared to controls. In drug-treated mice, this advance occurred almost immediately to the new LD cycle, but surprisingly, subsequently appeared to free-run, even under LD conditions (Fig 3A). In some mice, this phase advance in activity onset appeared to stabilize ~4–6 h prior to lights off (Fig EV4). We wondered whether adjusting the treatment time of DHEA might be sufficient to reduce jet-lag to a 6-h phase-advanced LD cycle. Therefore, in the next experiment, treatment with DHEA was limited to the first 3 days of the new LD cycle (Fig 3B). Mice were then returned to normal food for the remainder of the experiment. These results show that mice given an acute dose of DHEA shifted much more rapidly to the new LD cycle and re-entrained within 1–3 days compared to the 6–7 days required for control mice (Fig 3B). In addition, body temperature rhythms in DHEA-treated animals appeared to shift and reset more rapidly to the new LD cycle compared to controls (the trough more so than the peak; Fig 3C). To determine whether acute DHEA treatment did, in fact, advance the circadian clock, we again treated mice with DHEA and exposed them to an advanced LD cycle for 3 days. Animals were then returned to normal food and transferred into DD (Fig 3D). These results show that the clock has indeed shifted more in DHEA-treated animals than in control mice. Thus, by adjusting the treatment time of DHEA to 3 days, it was possible to phase shift the circadian clock and accelerate re-entrainment to an advanced LD cycle, thereby reducing jet-lag with dietary DHEA. Click here to expand this figure. Figure EV3. Possible mechanisms of overcoming jet-lagTwo models have been proposed to explain the mechanisms by which circadian clocks are entrained to environmental LD cycles. In the "nonparametric model", entrainment is achieved by a phase shift caused by environmental stimuli. In contrast, the "parametric model" focuses on the angular velocity or rate of motion of the circadian clock, and entrainment is achieved by modulation of the free-running period (Daan & Aschof, 2001). Therefore, compounds that increase the magnitude of the phase shift are predicted to reduce jet-lag through a nonparametric mechanism, whereas period-shortening and period-lengthening compounds (i.e., compounds that affect angular velocity) might reduce jet-lag either by accelerating or slowing down the clock, thus accelerating re-entrainment to travel eastward or westward, respectively. Download figure Download PowerPoint Figure 3. Dietary administration of DHEA reduces jet-lag Effect of chronic DHEA administration (1.0% w/w; vertical line) on wheel-running activity in mice during re-entrainment to a 6-h phase-advanced LD cycle. Representative double-plotted actograms of control (n = 12; left) and DHEA-treated (n = 11; middle) animals. Activity onset (mean ± SEM) during a 6-h phase advance was plotted (right panel) and analyzed by a Welch's t-test (**P < 0.01). All statistical information is shown in Appendix Table S1. Effect of acute DHEA (3 days of 1.0% w/w) on wheel-running activity in mice during a 6-h phase-advanced LD cycle. Representative double-plotted actograms of control (n = 10; left) and DHEA-treated (n = 11; middle) animals. Activity onset during a 6-h phase-advanced LD cycle (mean ± SEM) (right panel). Data were analyzed by a Welch's t-test (*P < 0.05, **P < 0.01). All statistical information is shown in Appendix Table S1. Effect of acute DHEA (3 days of 1.0% w/w; red bar above) on body temperature rhythms. After surgical implantation of temperature devices, body temperature was measured for the duration of the experiment. Data are shown for 2.5 days of entrainment to a LD cycle and then 7.5 days of exposure to a 6-h phase advance LD cycle. Data represent the mean of each treatment. Effect of acute DHEA (3 days of 1.0% w/w) and exposure to a 6-h phase-advanced LD cycle (3 days) and then return to normal food and transfer into DD. Representative double-plotted actograms of control (n = 10; left) and DHEA-treated (n = 11; middle) animals. Activity onset was plotted during a 6-h phase-advanced LD cycle and then transfer into DD (mean ± SEM) (right panel). Data were analyzed by a Welch's t-test (*P < 0.05, **P < 0.01). All statistical information is shown in Appendix Table S1. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Chronic DHEA leads to a stable phase advance in activity onset in some miceDouble-plotted actograms of two DHEA-treated mice from the experiment shown in Fig 3A. DHEA was administered in powdered food (1.0% w/w; vertical line on the right) during re-entrainment to a 6-h phase-advanced LD cycle. These results show that, in some mice, chronic DHEA treatment leads to a relatively stable phase advance in activity onset ~4–6 h prior to lights off. Download figure Download PowerPoint Involvement of ABL and BCR kinases in the circadian clockwork Two tyrosine kinase inhibitors were identified in our screen, dasatinib and nilotinib, that lengthened and shortened circadian period, respectively, and both were effective in the nanomolar range (Fig 4A). These drugs are second-generation inhibitors of the BCR-ABL tyrosine kinase and approved for treatment of chronic myelogenous leukemia (CML; Molica et al, 2017). Most cases of CML are caused by a chromosomal translocation, called the Philadelphia chromosome, in which the break point cluster (BCR) gene on chromosome 22 and the Abelson (ABL) non-receptor type tyrosine kinase gene on chromosome 9 are fused, resulting in the chimeric oncogene, BCR-ABL (Faderl et al, 1999). Although much is known about the consequences of the BCR-ABL fusion protein in CML, the function of BCR and ABL in intact animals is not well understood (Schwartzberg et al, 1989; Tybulewicz et al, 1991; Voncken et al, 1996). Figure 4. Inhibition of ABL and BCR kinases modulates circadian period Dose-dependent effects of BCR-ABL tyrosine kinase inhibitors dasatinib and nilotinib on Bmal1-dLuc rhythms in U2OS cells. Luminescent traces from one of three or four independent experiments are shown. Circadian period was determined by curve fitting. Data are the mean ± SEM of three or four independent experiments and were analyzed by one-way ANOVA, followed by a Dunnett's test (**P < 0.01). All statistical information is shown in Appendix Table S1 Effect of siRNA-mediated knockdown of ABL, BCR, and SRC on circadian rhythms: Bmal1-dLuc rhythms (left) and circadian period (right). Data are presented as the mean ± SEM of three or four independent experiments (*P < 0.05, **P < 0.01, Welch's t-test). All sta
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