Central Circadian Clock Research Articles

Neuropeptide-dependent spike time precision and plasticity in circadian output neurons.

Circadian rhythms influence various physiological and behavioral processes such as sleep-wake cycles, hormone secretion, and metabolism. Circadian output neurons are a group of neurons that receive input from the central circadian clock located in the suprachiasmatic nucleus of the mammalian brain and transmit timing information to different regions of the brain and body, coordinating the circadian rhythms of various physiological processes. In Drosophila , an important set of circadian output neurons are called pars intercerebralis (PI) neurons, which receive input from specific clock neurons called DN1. These neurons can further be subdivided into functionally and anatomically distinctive anterior (DN1a) and posterior (DN1p) clusters. The neuropeptide diuretic hormones 31 (Dh31) and 44 (Dh44) are the insect neuropeptides known to activate PI neurons to control activity rhythms. However, the neurophysiological basis of how Dh31 and Dh44 affect circadian clock neural coding mechanisms underlying sleep in Drosophila is not well understood. Here, we identify Dh31/Dh44-dependent spike time precision and plasticity in PI neurons. We find that the application of synthesized Dh31 and Dh44 affects membrane potential dynamics of PI neurons in the precise timing of the neuronal firing through their synergistic interaction, possibly mediated by calcium-activated potassium channel conductance. Further, we characterize that Dh31/Dh44 enhances postsynaptic potentials in PI neurons. Together, these results suggest multiplexed neuropeptide-dependent spike time precision and plasticity as circadian clock neural coding mechanisms underlying sleep in Drosophila .

Transcriptomic Plasticity of the Circadian Clock in Response to Photoperiod: A Study in Male Melatonin-Competent Mice.

Seasonal daylength, or circadian photoperiod, is a pervasive environmental signal that profoundly influences physiology and behavior. In mammals, the central circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus where it receives retinal input and synchronizes, or entrains, organismal physiology and behavior to the prevailing light cycle. The process of entrainment induces sustained plasticity in the SCN, but the molecular mechanisms underlying SCN plasticity are incompletely understood. Entrainment to different photoperiods persistently alters the timing, waveform, period, and light resetting properties of the SCN clock and its driven rhythms. To elucidate novel candidate genes for molecular mechanisms of photoperiod plasticity, we performed RNA sequencing on whole SCN dissected from mice raised in long (light:dark [LD] 16:8) and short (LD 8:16) photoperiods. Fewer rhythmic genes were detected in mice subjected to long photoperiod, and in general, the timing of gene expression rhythms was advanced 4-6 h. However, a few genes showed significant delays, including Gem. There were significant changes in the expression of the clock-associated gene Timeless and in SCN genes related to light responses, neuropeptides, gamma aminobutyric acid (GABA), ion channels, and serotonin. Particularly striking were differences in the expression of the neuropeptide signaling genes Prokr2 and Cck, as well as convergent regulation of the expression of 3 SCN light response genes, Dusp4, Rasd1, and Gem. Transcriptional modulation of Dusp4 and Rasd1 and phase regulation of Gem are compelling candidate molecular mechanisms for plasticity in the SCN light response through their modulation of the critical NMDAR-MAPK/ERK-CREB/CRE light signaling pathway in SCN neurons. Modulation of Prokr2 and Cck may critically support SCN neural network reconfiguration during photoperiodic entrainment. Our findings identify the SCN light response and neuropeptide signaling gene sets as rich substrates for elucidating novel mechanisms of photoperiod plasticity. Data are also available at http://circadianphotoperiodseq.com/, where users can view the expression and rhythmic properties of genes across these photoperiod conditions.

Sweetened caffeine drinking revealed behavioral rhythm independent of the central circadian clock in male mice

Caffeine consumption is associated with the evening chronotype, and caffeine administration in mice results in prolonged period of the circadian rhythm in locomotor activity. However, as caffeine is bitter, sweetened caffeine is preferred by humans and mice; yet, its impact on the circadian clock has not been explored. In this study, mice were provided with freely available sweetened caffeine to investigate its effects on behavioral rhythms and peripheral clocks. Mice that freely consumed sweetened caffeine shifted from nocturnal to diurnal activity rhythms. In addition to the light-dark entrained behavioral rhythm component, some animals exhibited free-running period longer than 24-h. Intraperitoneal administration of caffeine at the beginning of the light phase also acutely induced diurnal behavior. The behavioral rhythms with long period (26–30 h) due to sweetened caffeine were observed even in mice housed under constant light or with a lesioned central circadian clock located in the suprachiasmatic nucleus of the hypothalamus; however, the rhythmicity was unstable. PER2::LUCIFERASE rhythms in peripheral tissues, such as the kidney, as measured via in vivo whole-body imaging during caffeine consumption, showed reduced amplitude and desynchronized phases among individuals. These results indicate that consumption of sweetened caffeine induces diurnal and long-period behavioral rhythms irrespective of the central clock, causing desynchronization of the clock in the body.

AGAMOUS-LIKE 24 senses continuous inductive photoperiod in the inflorescence meristem to promote anthesis in chrysanthemum.

During the floral transition, many plant species including chrysanthemum (Chrysanthemum morifolium) require continuous photoperiodic stimulation for successful anthesis. Insufficient photoperiodic stimulation results in flower bud arrest or even failure. The molecular mechanisms underlying how continuous photoperiodic stimulation promotes anthesis are not well understood. Here, we reveal that in wild chrysanthemum (Chrysanthemum indicum), an obligate short-day (SD) plant, floral evocation is not limited to SD conditions. However, SD signals generated locally in the inflorescence meristem (IM) play a vital role in ensuring anthesis after floral commitment. Genetic analyses indicate that the florigen FLOWERING LOCUS T-LIKE3 (CiFTL3) plays an important role in floral evocation, but a lesser role in anthesis. Importantly, our data demonstrate that AGAMOUS-LIKE 24 (CiAGL24) is a critical component of SD signal perception in the IM to promote successful anthesis, and that floral evocation and anthesis are two separate developmental events in chrysanthemum. We further reveal that the central circadian clock component PSEUDO-RESPONSE REGULATOR 7 (CiPRR7) in the IM activates CiAGL24 expression in response to SD conditions. Moreover, our findings elucidate a negative feedback loop in which CiAGL24 and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (CiSOC1) modulate LEAFY (CiLFY) expression. Together, our results demonstrate that the CiPRR7-CiAGL24 module is vital for sustained SD signal perception in the IM to ensure successful anthesis in chrysanthemum.

Higher central circadian temperature amplitude is associated with greater metabolite rhythmicity in humans

Robust circadian rhythms are essential for optimal health. The central circadian clock controls temperature rhythms, which are known to organize the timing of peripheral circadian rhythms in rodents. In humans, however, it is unknown whether temperature rhythms relate to the organization of circadian rhythms throughout the body. We assessed core body temperature amplitude and the rhythmicity of 929 blood plasma metabolites across a 40-h constant routine protocol, controlling for behavioral and environmental factors that mask endogenous temperature rhythms, in 23 healthy individuals (mean [± SD] age = 25.4 ± 5.7 years, 5 women). Valid core body temperature data were available in 17/23 (mean [± SD] age = 25.6 ± 6.3 years, 1 woman). Individuals with higher core body temperature amplitude had a greater number of metabolites exhibiting circadian rhythms (R2 = 0.37, p = .009). Higher core body temperature amplitude was also associated with less variability in the free-fitted periods of metabolite rhythms within an individual (R2 = 0.47, p = .002). These findings indicate that a more robust central circadian clock is associated with greater organization of circadian metabolite rhythms in humans. Metabolite rhythms may therefore provide a window into the strength of the central circadian clock.

Biological Sex Influences Daily Locomotor Rhythms in Mice Held Under Different Housing Conditions.

Daily rhythms are programmed by a central circadian clock that is modulated by photoperiod. Here, we recorded locomotor activity rhythms in C57Bl/6 or mPer2Luc mice of both sexes held under different housing conditions. First, we confirm that the structure of locomotor activity rhythms differs between male and female mice in both genetic backgrounds. Male mice exhibit a nightly "siesta," whereas female mice fluctuate between nights with and without a nightly siesta, which corresponds with changes in locomotor activity levels, circadian period, and vaginal cytology. The nightly siesta is modulated by the presence of a running wheel in both sexes but is not required for the infradian patterning of locomotor rhythms in females. Finally, photoperiodic changes in locomotor rhythms differed by sex, and females displayed phase-jumping responses earlier than males under a parametric photoentrainment assay simulating increasing day length. Collectively, these results highlight that sex and sex hormones influence daily locomotor rhythms under a variety of different environmental conditions.

Disruption of central and peripheral circadian clocks and circadian controlled estrogen receptor rhythms in night shift nurses in working environments.

Chronic disruption of circadian rhythms by night shift work is associated with an increased breast cancer risk. However, little is known about the impact of night shift on peripheral circadian genes (CGs) and circadian-controlled genes (CCGs) associated with breast cancer. Hence, we assessed central clock markers (melatonin and cortisol) in plasma, and peripheral CGs (PER1, PER2, PER3, and BMAL1) and CCGs (ESR1 and ESR2) in peripheral blood mononuclear cells (PBMCs). In day shift nurses (n = 12), 24-h rhythms of cortisol and melatonin were aligned with day shift-oriented light/dark schedules. The mRNA expression of PER2, PER3, BMAL1, and ESR2 showed 24-h rhythms with peak values in the morning. In contrast, night shift nurses (n = 10) lost 24-h rhythmicity of cortisol with a suppressed morning surge but retained normal rhythmic patterns of melatonin, leading to misalignment between cortisol and melatonin. Moreover, night shift nurses showed disruption of rhythmic expressions of PER2, PER3, BMAL1, and ESR2 genes, resulting in an impaired inverse correlation between PER2 and BMAL1 compared to day shift nurses. The observed trends of disrupted circadian markers were recapitulated in additional day (n = 20) and night (n = 19) shift nurses by measurement at early night and midnight time points. Taken together, this study demonstrated the misalignment of cortisol and melatonin, associated disruption of PER2 and ESR2 circadian expressions, and internal misalignment in peripheral circadian network in night shift nurses. Morning plasma cortisol and PER2, BMAL1, and ESR2 expressions in PBMCs may therefore be useful biomarkers of circadian disruption in shift workers.

Loss of temporal coherence in the circadian metabolome across multiple tissues during ageing in mice.

Circadian clock function declines with ageing, which can aggravate ageing-related diseases such as type 2 diabetes and neurodegenerative disorders. Understanding age-related changes in the circadian system at a systemic level can contribute to the development of strategies to promote healthy ageing. The goal of this study was to investigate the impact of ageing on 24-h rhythms in amine metabolites across four tissues in young (2months of age) and old (22-25 months of age) mice using a targeted metabolomics approach. Liver, plasma, the suprachiasmatic nucleus (SCN; the location of the central circadian clock in the hypothalamus) and the paraventricular nucleus (PVN; a downstream target of the SCN) were collected from young and old mice every 4h during a 24-h period (n= 6-7 mice per group). Differential rhythmicity analysis revealed that ageing impacts 24-h rhythms in the amine metabolome in a tissue-specific manner. Most profound changes were observed in the liver, in which rhythmicity was lost in 60% of the metabolites in aged mice. Furthermore, we found strong correlations in metabolite levels between the liver and plasma and between the SCN and the PVN in young mice. These correlations were almost completely abolished in old mice. These results indicate that ageing is accompanied by a severe loss of the circadian coordination between tissues and by disturbed rhythmicity of metabolic processes. The tissue-specific impact of ageing may help to differentiate mechanisms of ageing-related disorders in the brain versus peripheral tissues and thereby contribute to the development of potential therapies for these disorders.

Biological Rhythms, Chrono-Nutrition, and Gut Microbiota: Epigenomics Insights for Precision Nutrition and Metabolic Health.

Circadian rhythms integrate a finely tuned network of biological processes recurring every 24 h, intricately coordinating the machinery of all cells. This self-regulating system plays a pivotal role in synchronizing physiological and behavioral responses, ensuring an adaptive metabolism within the environmental milieu, including dietary and physical activity habits. The systemic integration of circadian homeostasis involves a balance of biological rhythms, each synchronically linked to the central circadian clock. Central to this orchestration is the temporal dimension of nutrient and food intake, an aspect closely interwoven with the neuroendocrine circuit, gut physiology, and resident microbiota. Indeed, the timing of meals exerts a profound influence on cell cycle regulation through genomic and epigenetic processes, particularly those involving gene expression, DNA methylation and repair, and non-coding RNA activity. These (epi)genomic interactions involve a dynamic interface between circadian rhythms, nutrition, and the gut microbiota, shaping the metabolic and immune landscape of the host. This research endeavors to illustrate the intricate (epi)genetic interplay that modulates the synchronization of circadian rhythms, nutritional signaling, and the gut microbiota, unravelling the repercussions on metabolic health while suggesting the potential benefits of feed circadian realignment as a non-invasive therapeutic strategy for systemic metabolic modulation via gut microbiota. This exploration delves into the interconnections that underscore the significance of temporal eating patterns, offering insights regarding circadian rhythms, gut microbiota, and chrono-nutrition interactions with (epi)genomic phenomena, thereby influencing diverse aspects of metabolic, well-being, and quality of life outcomes.

The Suprachiasmatic Nucleus at 50: Looking Back, Then Looking Forward.

It has been 50 years since the suprachiasmatic nucleus (SCN) was first identified as the central circadian clock and 25 years since the last overview of developments in the field was published in the Journal of Biological Rhythms. Here, we explore new mechanisms and concepts that have emerged in the subsequent 25 years. Since 1997, methodological developments, such as luminescent and fluorescent reporter techniques, have revealed intricate relationships between cellular and network-level mechanisms. In particular, specific neuropeptides such as arginine vasopressin, vasoactive intestinal peptide, and gastrin-releasing peptide have been identified as key players in the synchronization of cellular circadian rhythms within the SCN. The discovery of multiple oscillators governing behavioral and physiological rhythms has significantly advanced our understanding of the circadian clock. The interaction between neurons and glial cells has been found to play a crucial role in regulating these circadian rhythms within the SCN. Furthermore, the properties of the SCN network vary across ontogenetic stages. The application of cell type-specific genetic manipulations has revealed components of the functional input-output system of the SCN and their correlation with physiological functions. This review concludes with the high-risk effort of identifying open questions and challenges that lie ahead.

The Roles of Circadian Clock Genes in Plant Temperature Stress Responses.

Plants monitor day length and memorize changes in temperature signals throughout the day, creating circadian rhythms that support the timely control of physiological and metabolic processes. The DEHYDRATION-RESPONSE ELEMENT-BINDING PROTEIN 1/C-REPEAT BINDING FACTOR (DREB1/CBF) transcription factors are known as master regulators for the acquisition of cold stress tolerance, whereas PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is involved in plant adaptation to heat stress through thermomorphogenesis. Recent studies have shown that circadian clock genes control plant responses to temperature. Temperature-responsive transcriptomes show a diurnal cycle and peak expression levels at specific times of throughout the day. Circadian clock genes play essential roles in allowing plants to maintain homeostasis by accommodating temperature changes within the normal temperature range or by altering protein properties and morphogenesis at the cellular level for plant survival and growth under temperature stress conditions. Recent studies revealed that the central oscillator genes CIRCADIAN CLOCK ASSOCIATED 1/LATE ELONGATED HYPOCOTYL (CCA1/LHY) and PSEUDO-RESPONSE REGULATOR5/7/9 (PRR5/7/9), as well as the EVENING COMPLEX (EC) genes REVEILLE4/REVEILLE8 (REV4/REV8), were involved in the DREB1 pathway of the cold signaling transcription factor and regulated the thermomorphogenesis gene PIF4. Further studies showed that another central oscillator, TIMING OF CAB EXPRESSION 1 (TOC1), and the regulatory protein ZEITLUPE (ZTL) are also involved. These studies led to attempts to utilize circadian clock genes for the acquisition of temperature-stress resistance in crops. In this review, we highlight circadian rhythm regulation and the clock genes involved in plant responses to temperature changes, as well as strategies for plant survival in a rapidly changing global climate.

Behavior and physiology in female Cricetulus barabensis are associated with the expression of circadian genes.

The circadian clock regulates the behavior, physiology, and metabolism of mammals, and these characteristics, such as sleep-wake cycles, exercise capacity, and hormone levels, exhibit circadian rhythms. Light signaling is the main stimulator of the mammalian circadian system. The photoperiod regulates the reproductive cycle of seasonal breeding animals, and the circadian clock plays a pivotal role in this process. However, the role of the clock in coordinating animal behavior and physiology in response to photoperiodic changes needs further investigation. The present study investigated the changes and correlation of behavioral activities, physiological indicators, and gene expression in female striped hamsters (Cricetulus barabensis) within 24 h under a 12L:12D photoperiod. We found that the daily rhythms of sleep-wake and open field were significant in hamsters. The expression of clock genes, melatonin receptor genes, and genes involved in general metabolism oscillated significantly in central and peripheral tissues (brain, hypothalamus, liver, ovary, and thymus) and was significantly associated with behavior and physiology. Our results revealed that the neuroendocrine system regulated the rhythmicity of behavior and physiology, and central and peripheral clock genes (Bmal1, Clock, Per1, Per2, Cry1, and Cry2), melatonin receptor genes (MT1, MT2, and GPR50), and metabolizing genes (SIRT1, FGF21, and PPARα) played important roles. Our results suggest that central and peripheral circadian clocks, melatonin receptors, and genes involved in general metabolism may play key roles in maintaining circadian behavior and metabolic homeostasis in striped hamsters. Our results may have important implication for rodent pest control.

Circadian Control of Lipid Metabolism.

To ensure optimum health and performance, lipid metabolism needs to be temporally aligned to other body processes and to daily changes in the environment. Central and peripheral circadian clocks and environmental signals such as light provide internal and external time cues to the body. Importantly, each of the key organs involved in insect lipid metabolism contains a molecular clockwork which ticks with a varying degree of autonomy from the central clock in the brain. In this chapter, we review our current knowledge about peripheral clocks in the insect fat body, gut and oenocytes, and light- and circadian-driven diel patterns in lipid metabolites and lipid-related transcripts. In addition, we highlight selected neuroendocrine signaling pathways that are or may be involved in the temporal coordination and control of lipid metabolism.

Influences of Desflurane on the Central and Hepatic Circadian Clock Persist at 24 Hours Following Anaesthesia in Rats

Influences of Desflurane on the Central and Hepatic Circadian Clock Persist at 24 Hours Following Anaesthesia in Rats

Mammalian circadian clock proteins form dynamic interacting microbodies distinct from phase separation.

Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro using recombinant proteins or in cells that overexpress protein, the physiological relevance of LLPS for endogenous protein is often unclear. PERIOD, the intrinsically disordered domain-rich proteins, are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Circadian clock studies often rely on experiments that overexpress clock proteins. Here, we show that when Per2 transgene was stably expressed in cells, PER2 protein formed nuclear phosphorylation-dependent slow-moving LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing nuclear microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by protein overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins are a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian clock studies.

An independent comparison of the Novolytix salivary melatonin radioimmunoassay with the new Novolytix salivary melatonin enzyme-linked immunosorbent assay.

The dim light melatonin onset (DLMO) is the current gold standard biomarker of the timing of the central circadian clock in humans and is often assessed from saliva samples. To date, only one commercially available salivary melatonin assay is considered accurate at the low daytime levels required to accurately detect the DLMO (Novolytix RIA RK-DSM2). The aim of this study was to conduct the first independent evaluation of a newly improved enzyme-linked immunosorbent assay (ELISA; Novolytix MLTN-96) and compare it with the recommended radioimmunoassay (RIA)-both in terms of melatonin concentrations and derived DLMOs. Twenty participants (15 females, 18-59 years old) provided saliva samples every 30 min in dim light starting 6 h before their habitual bedtime, yielding a total of 260 saliva samples. Both the RIA and ELISA yielded daytime melatonin concentrations <2 pg/mL, indicating adequate accuracy to detect the DLMO. The melatonin concentrations from the two assays were highly correlated (r = .94, p < .001), although the RIA yielded lower levels of melatonin concentration than the ELISA, on average by 0.70 pg/mL (p = .006). Seventeen DLMOs were calculated from the melatonin profiles and the DLMOs from both assays were not statistically different (p = .36) and were highly correlated (r = .97, p < .001). Two DLMOs derived from the RIA occurred more than 30 min earlier than the DLMO derived from the ELISA. These results indicate that the new Novolytix ELISA is an appropriate assay to use if the Novolytix RIA is not feasible or available.

Small-molecule CEM3 strengthens single-cell oscillators in the suprachiasmatic nucleus.

A robust endogenous clock is required for proper function of many physiological processes. The suprachiasmatic nucleus (SCN) constitutes our central circadian clock and allows us to adapt to daily changes in the environment. Aging can cause a decline in the amplitude of circadian rhythms in SCN and peripheral clocks, which contributes to increased risk of several chronic diseases. Strengthening clock function would therefore be an effective strategy to improve health. A high-throughput chemical screening has identified clock-enhancing molecule 3 (CEM3) as small molecule that increases circadian rhythm amplitude in cell lines and SCN explants. It is, however, currently not known whether CEM3 acts by enhancing the amplitude of individual single-cell oscillators or by enhancing synchrony among neurons. In view of CEM3's potential, it is of evident importance to clarify the mode of action of CEM3. Here, we investigated the effects of CEM3 on single-cell PERIOD2::LUCIFERASE rhythms in mouse SCN explants. CEM3 increased the amplitude in approximately 80%-90% of the individual cells in the SCN without disrupting the phase and/or period of their rhythms. Noticeably, CEM3's effect on amplitude is independent of the cell's initial amplitude. These findings make CEM3 a potential therapeutic candidate to restore compromised amplitude in circadian rhythms and will boost the development of other molecular approaches to improve health.

Expression of the vesicular GABA transporter within neuromedin S+ neurons sustains behavioral circadian rhythms.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the site of a central circadian clock that orchestrates overt rhythms of physiology and behavior. Circadian timekeeping requires intercellular communication among SCN neurons, and multiple signaling pathways contribute to SCN network coupling. Gamma-aminobutyric acid (GABA) is produced by virtually all SCN neurons, and previous work demonstrates that this transmitter regulates coupling in the adult SCN but is not essential for the nucleus to sustain overt circadian rhythms. Here, we show that the deletion of the gene that codes for the GABA vesicular transporter Vgat from neuromedin-S (NMS)+ neurons-a subset of neurons critical for SCN function-causes arrhythmia of locomotor activity and sleep. Further, NMS-Vgat deletion impairs intrinsic clock gene rhythms in SCN explants cultured ex vivo. Although vasoactive intestinal polypeptide (VIP) is critical for SCN function, Vgat deletion from VIP-expressing neurons did not lead to circadian arrhythmia in locomotor activity rhythms. Likewise, adult SCN-specific deletion of Vgat led to mild impairment of behavioral rhythms. Our results suggest that while the removal of GABA release from the adult SCN does not affect the pacemaker's ability to sustain overt circadian rhythms, its removal from a critical subset of neurons within the SCN throughout development removes the nucleus ability to sustain circadian rhythms. Our findings support a model in which SCN GABA release is critical for the developmental establishment of intercellular network properties that define the SCN as a central pacemaker.

Circadian clock factors regulate the first condensation reaction of fatty acid synthesis in Arabidopsis

The circadian clock regulates temporal metabolic activities, but how it affects lipid metabolism is poorly understood. Here, we show that the central clock regulators LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) regulate the initial step of fatty acid (FA) biosynthesis in Arabidopsis. Triacylglycerol (TAG) accumulation in seeds was increased in LHY-overexpressing (LHY-OE) and decreased in lhycca1 plants. Metabolic tracking of lipids in developing seeds indicated that LHY enhanced FA synthesis. Transcript analysis revealed that the expression of genes involved in FA synthesis, including the one encoding β-ketoacyl-ACP synthase III (KASIII), was oppositely changed in developing seeds of LHY/CCA1-OEs and lhycca1. Chromatin immunoprecipitation, electrophoretic mobility shift, and transactivation assays indicated that LHY bound and activated the promoter of KASIII. Furthermore, phosphatidic acid, a metabolic precursor to TAG, inhibited LHY binding to KASIII promoter elements. Our data show a regulatory mechanism for plant lipid biosynthesis by the molecular clock.

Internal circadian misallignment in a mouse model of chemotherapy induced fatigue

BackgroundCancer survivors can experience long lasting fatigue resulting in a lower quality of life. How chemotherapy treatment contributes to this fatigue is poorly understood. Previously we have shown in a mouse model of cancer related fatigue that doxorubicin treatment induces fatigue-like symptoms related to disturbed circadian rhythms. However, the specific components of the circadian regulatory circuitry affected by doxorubicin treatment remained unclear. Therefore we investigated the role of the central circadian clock, the suprachiasmatic nucleus (SCN), in chemotherapy-induced fatigue. MethodsWe measured circadian controlled behavior and multiunit neuronal activity in the SCN in freely moving mice exhibiting fatigue-like behavior after doxorubicin treatment under both light–dark (LD) and constant dark (DD) conditions. Additionally, we assessed the expression of inflammation related genes in spleen and kidney as potential inducers of CRF. ResultsDoxorubicin treatment significantly reduced both the running wheel activity and time spent using the running wheel for over five weeks after treatment. In contrast to the pronounced effects on behavior and neuronal activity of doxorubicin on circadian rhythms, peripheral inflammation markers only showed minor differences, five weeks after the last treatment. Surprisingly, the circadian SCN neuronal activity under both LD and DD conditions was not affected. However, the circadian timing of neuronal activity in peri-SCN areas (the brain areas surrounding SCN) and circadian rest-activity behavior was strongly affected by doxorubicin, suggesting that the output of the SCN was altered. The reduced correlation between the SCN neuronal activity and behavioral activity after doxorubicin treatment, suggests that the information flow from the SCN to the periphery was disturbed. ConclusionOur preclinical study suggests that chemotherapy-induced fatigue disrupts the circadian rhythms in peripheral brain areas and behavior downstream from the SCN, potentially leading to fatigue like symptoms. Our data suggest that peripheral inflammation responses are less important for the maintenance of fatigue. Chronotherapy that realigns circadian rhythms could represent a non-invasive way to improve patient outcomes following chemotherapy.