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The LATE ELONGATED HYPOCOTYL (LHY) transcription factor functions as part of the oscillatory mechanism of the Arabidopsis circadian clock. This paper reports the genome-wide analysis of its binding targets and reveals a role in the control of abscisic acid (ABA) biosynthesis and downstream responses. LHY directly repressed expression of 9-cis-epoxycarotenoid dioxygenase enzymes, which catalyse the rate-limiting step of ABA biosynthesis. This suggested a mechanism for the circadian control of ABA accumulation in wild-type plants. Consistent with this hypothesis, ABA accumulated rhythmically in wild-type plants, peaking in the evening. LHY-overexpressing plants had reduced levels of ABA under drought stress, whereas loss-of-function mutants exhibited an altered rhythm of ABA accumulation. LHY also bound the promoter of multiple components of ABA signalling pathways, suggesting that it may also act to regulate responses downstream of the hormone. LHY promoted expression of ABA-responsive genes responsible for increased tolerance to drought and osmotic stress but alleviated the inhibitory effect of ABA on seed germination and plant growth. This study reveals a complex interaction between the circadian clock and ABA pathways, which is likely to make an important contribution to plant performance under drought and osmotic stress conditions.

The accumulation of adenosine is strongly correlated with the need for sleep and the detection of sleep pressure is antagonised by caffeine. Caffeine also affects the circadian timing system directly and independently of sleep physiology, but how caffeine mediates these effects upon the circadian clock is unclear. Here we identify an adenosine-based regulatory mechanism that allows sleep and circadian processes to interact for the optimisation of sleep/wake timing in mice. Adenosine encodes sleep history and this signal modulates circadian entrainment by light. Pharmacological and genetic approaches demonstrate that adenosine acts upon the circadian clockwork via adenosine A 1 /A 2A receptor signalling through the activation of the Ca 2+  -ERK-AP-1 and CREB/CRTC1-CRE pathways to regulate the clock genes Per1 and Per2 . We show that these signalling pathways converge upon and inhibit the same pathways activated by light. Thus, circadian entrainment by light is systematically modulated on a daily basis by sleep history. These findings contribute to our understanding of how adenosine integrates signalling from both light and sleep to regulate circadian timing in mice. Sleep pressure and circadian rhythms influence one another. However, the regulatory mechanisms are unclear. Here, the authors show that adenosine A 1 /A 2A receptor antagonists, such as caffeine, shift circadian rhythms and enhance the effects of light, providing a molecular link between sleep pressure and circadian rhythm.

Mitochondrial dysfunction is present in the ovaries of patients with polycystic ovary syndrome (PCOS). Melatonin (MT) has shown promise in treating PCOS by improving mitochondrial dysfunction, though the underlying mechanisms remain unclear. In this study, we first assessed the levels of proteins associated with mitochondrial autophagy and dynamics in ovary granulosa cells (GCs) of PCOS patients and in the ovaries of DHEA-induced PCOS mice. We found abnormal expression of these proteins, indicating the presence of mitochondrial dysfunction in PCOS ovaries. Notably, the expression of the circadian gene Clock and melatonin synthetic enzymes were also decreased in the ovaries of PCOS patients. Studies have suggested a potential role of circadian rhythm genes in the pathogenesis and progression of PCOS. We subsequently observed that pretreatment with MT could ameliorate the abnormal levels of mitochondrial-related proteins, reverse the low expression of CLOCK, and reduce pyroptosis in PCOS ovaries. Given the potential interaction between MT and Clock, we focused on whether exogenous MT improves mitochondrial dysfunction in PCOS ovaries by regulating the expression of the circadian gene Clock. Through in vitro culture of the human ovarian granulosa cell line KGN, we further found that when CLOCK levels were inhibited, the beneficial effects of MT on abnormal mitochondrial autophagy, disturbed mitochondrial dynamics, and mitochondrial dysfunction in PCOS ovaries were not significant, and there was no notable improvement in ovary GCs pyroptosis. Our study suggests that MT may improve ovary mitochondrial dysfunction by regulating circadian gene Clock while also reducing GCs pyroptosis in PCOS.

The master immune regulator NPR1 of Arabidopsis is a sensor of the plant’s redox state and regulates transcription of core circadian clock genes even in the absence of pathogen challenge. Immune system clocking on In plants — and in many other eukaryotic organisms — the circadian clock ensures daily rhythmic fluctuations in biological processes not just by affecting gene transcription but also by modifying the organism's redox state. The molecular mechanism by which redox rhythms are linked to the circadian clock and the biological significance of redox–circadian interactions remain unclear. Xinnian Dong and colleagues identify an unexpected regulator of such redox rhythms in Arabidopsis . They find that the master immune regulator NPR1 senses a plant's redox state and regulates transcription of both the morning and evening core circadian-clock genes, independently of pathogenic infection. This network architecture helps plants to 'gate' their immune responses to the morning, thus minimizing the effect on growth, which occurs during the night. Recent studies have shown that in addition to the transcriptional circadian clock, many organisms, including Arabidopsis , have a circadian redox rhythm driven by the organism’s metabolic activities 1 , 2 , 3 . It has been hypothesized that the redox rhythm is linked to the circadian clock, but the mechanism and the biological significance of this link have only begun to be investigated 4 , 5 , 6 , 7 . Here we report that the master immune regulator NPR1 (non-expressor of pathogenesis-related gene 1) of Arabidopsis is a sensor of the plant’s redox state and regulates transcription of core circadian clock genes even in the absence of pathogen challenge. Surprisingly, acute perturbation in the redox status triggered by the immune signal salicylic acid does not compromise the circadian clock but rather leads to its reinforcement. Mathematical modelling and subsequent experiments show that NPR1 reinforces the circadian clock without changing the period by regulating both the morning and the evening clock genes. This balanced network architecture helps plants gate their immune responses towards the morning and minimize costs on growth at night. Our study demonstrates how a sensitive redox rhythm interacts with a robust circadian clock to ensure proper responsiveness to environmental stimuli without compromising fitness of the organism.

The mammalian suprachiasmatic nucleus (SCN) forms not only the master circadian clock but also a seasonal clock. This neural network of ∼10,000 circadian oscillators encodes season-dependent day-length changes through a largely unknown mechanism. We show that region-intrinsic changes in the SCN fine-tune the degree of network synchrony and reorganize the phase relationship among circadian oscillators to represent day length. We measure oscillations of the clock geneBmal1, at single-cell and regional levels in cultured SCN explanted from animals raised under short or long days. Coupling estimation using the Kuramoto framework reveals that the network has couplings that can be both phase-attractive (synchronizing) and -repulsive (desynchronizing). The phase gap between the dorsal and ventral regions increases and the overall period of the SCN shortens with longer day length. We find that one of the underlying physiological mechanisms is the modulation of the intracellular chloride concentration, which can adjust the strength and polarity of the ionotropic GABAA-mediated synaptic input.We show that increasing day-length changes the pattern of chloride transporter expression, yielding more excitatory GABA synaptic input, and that blocking GABAAsignaling or the chloride transporter disrupts the unique phase and period organization induced by the day length. We test the consequences of this tunable GABA coupling in the context of excitation–inhibition balance through detailed realistic modeling. These results indicate that the network encoding of seasonal time is controlled by modulation of intracellular chloride, which determines the phase relationship among and period difference between the dorsal and ventral SCN.

ObjectivesThe circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration.MethodsClock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1.ResultsHere we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1β but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs.ConclusionsWe have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Background Circadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock. Results We found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events. Conclusions Here we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.

The suprachiasmatic nucleus (SCN) serves as the body’s master circadian clock that adaptively coordinates changes in physiology and behaviour in anticipation of changing requirements throughout the 24-h day–night cycle 1 – 4 . For example, the SCN opposes overnight adipsia by driving water intake before sleep 5 , 6 , and by driving the secretion of anti-diuretic hormone 7 , 8 and lowering body temperature 9 , 10 to reduce water loss during sleep 11 . These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase 12 – 16 . However, it is unknown whether osmo-sodium sensors require clock-output networks to drive homeostatic responses. Here we show that a systemic salt injection (hypertonic saline) given at Zeitgeber time 19—a time at which SCN VP (vasopressin) neurons are inactive—excited SCN VP neurons and decreased non-shivering thermogenesis (NST) and body temperature. The effects of hypertonic saline on NST and body temperature were prevented by chemogenetic inhibition of SCN VP neurons and mimicked by optogenetic stimulation of SCN VP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLT GAD ) relay this information to SCN VP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLT GAD neuron axon terminals excited SCN VP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLT GAD neurons blunted the effects of systemic hypertonic saline on NST and body temperature. Finally, we show that hypertonic saline significantly phase-advanced the circadian locomotor activity onset of mice. This effect was mimicked by optogenetic activation of the OVLT GAD → SCN VP pathway and was prevented by chemogenetic inhibition of OVLT GAD neurons. Collectively, our findings provide demonstration that clock time can be regulated by non-photic physiologically relevant cues, and that such cues can drive unscheduled homeostatic responses via clock-output networks. The authors demonstrate that clock time can be regulated by non-photic physiologically relevant cues and that such cues can drive unscheduled homeostatic responses via clock-output networks.

In mammals, the master circadian clock synchronizes daily rhythms of physiology and behavior with the day–night cycle. Failure of synchrony, which increases the risk for numerous chronic diseases, can be treated by phase adjustment of the circadian clock pharmacologically, for example, with melatonin, or a CK1δ/ε inhibitor. Here, using in silico experiments with a systems pharmacology model describing molecular interactions, and pharmacokinetic and behavioral experiments in cynomolgus monkeys, we find that the circadian phase delay caused by CK1δ/ε inhibition is more strongly attenuated by light in diurnal monkeys and humans than in nocturnal mice, which are common preclinical models. Furthermore, the effect of CK1δ/ε inhibition strongly depends on endogenous PER2 protein levels, which differs depending on both the molecular cause of the circadian disruption and the patient's lighting environment. To circumvent such large interindividual variations, we developed an adaptive chronotherapeutics to identify precise dosing regimens that could restore normal circadian phase under different conditions. Our results reveal the importance of photosensitivity in the clinical efficacy of clock‐modulating drugs, and enable precision medicine for circadian disruption. Synopsis The study identifies the sources of the inter‐ and intraspecies variability in the modulation of circadian phase by CK1δ/ε inhibition: photosensitivity and PER2 abundance, and proposes a personalized chronotherapy to treat circadian disruption. Light attenuates the effect of CK1δ/ε inhibition more strongly in diurnal monkeys than in nocturnal mice. The effect of CK1δ/ε inhibition becomes stronger as PER2 protein abundance increases, which leads to a large interindividual variability in circadian phase shift. To circumvent such large interindividual variability, an adaptive chronotherapeutic approach is developed, which identifies a personalized dosing time by tracking the patient's drug response. Graphical Abstract The study identifies the sources of the inter‐ and intraspecies variability in the modulation of circadian phase by CK1δ/ε inhibition: photosensitivity and PER2 abundance, and proposes a personalized chronotherapy to treat circadian disruption.

The suprachiasmatic nucleus (SCN) co-ordinates circadian behaviour and physiology in mammals. Its cell-autonomous circadian oscillations pivot around a well characterised transcriptional/translational feedback loop (TTFL), whilst the SCN circuit as a whole is synchronised to solar time by its retinorecipient cells that express and release vasoactive intestinal peptide (VIP). The cell-autonomous and circuit-level mechanisms whereby VIP synchronises the SCN are poorly understood. We show that SCN slices in organotypic culture demonstrate rapid and sustained circuit-level circadian responses to VIP that are mediated at a cell-autonomous level. This is accompanied by changes across a broad transcriptional network and by significant VIP-directed plasticity in the internal phasing of the cell-autonomous TTFL. Signalling via ERK1/2 and tuning by its negative regulator DUSP4 are critical elements of the VIP-directed circadian re-programming. In summary, we provide detailed mechanistic insight into VIP signal transduction in the SCN at the level of genes, cells and neural circuit. The suprachiasmatic nucleus (SCN) synchronises daily rhythms of behaviour and physiology to the light-dark cycle. Vasoactive intestinal peptide (VIP) is important for mediating SCN entrainment; however, the underlying mechanisms are incompletely understood. Here, the authors show that the effects of VIP on the SCN are mediated by ERK1/2 and DUSP4.