Adaptation to glucose starvation is associated with molecular reorganization of the circadian clock in Neurospora crassa.

The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress.

Time restricted feeding modifies leukocyte responsiveness and improves inflammation outcome.

Time restricted eating, the dietary approach limiting food intake to a maximal 10-hour period of daytime is considered beneficial in metabolic dysfunctions, such as obesity and diabetes. Rhythm of food intake and parallel changes in serum nutrient levels are also important entrainment signals for the circadian clock, particularly in tissues involved in metabolic regulation. As both the metabolic state and the circadian clock have large impact on immune functions, we investigated in mice whether time restricted feeding (TRF) affects systemic inflammatory potential. TRF slackened the symptoms in K/BxN serum-transfer arthritis, an experimental model of human autoimmune joint inflammation. Compared to ad libitum conditions TRF reduced the expression of inflammatory mediators in visceral adipose tissue, an integrator and coordinator of metabolic and inflammatory processes. Furthermore, TRF strengthened the oscillation of peripheral leukocyte counts and alongside decreased the pool of both marginated and tissue leukocytes. Our data suggest that the altered leukocyte distribution in TRF mice is related to the attenuated expression of adhesion molecules on the surface of neutrophils and monocytes. We propose that TRF modifies both rhythm and inflammatory potential of leukocytes which contribute to the milder reactivity of the immune system and therefore time-restricted eating could serve as an effective complementary tool in the therapy of autoinflammatory processes.

A systems biological analysis of the ATF4-GADD34-CHOP regulatory triangle upon endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress-dependent accumulation of incorrectly folded proteins leads to activation of the unfolded protein response. The role of the unfolded protein response (UPR) is to avoid cell damage and restore the homeostatic state by autophagy; however, excessive ER stress results in apoptosis. Here we investigated the ER stress-dependent feedback loops inside one of the UPR branches by focusing on PERK-induced ATF4 and its two targets, called CHOP and GADD34. Our goal was to qualitatively describe the dynamic behavior of the system by exploring the key regulatory motifs using both molecular and theoretical biological techniques. Using the HEK293T cell line as a model system, we confirmed that the life-or-death decision is strictly regulated. We investigated the dynamic characteristics of the crucial elements of the PERK pathway at both the RNA and protein level upon tolerable and excessive levels of ER stress. Of particular note, inhibition of GADD34 or CHOP resulted in various phenotypes upon high levels of ER stress. Our computer simulations suggest the existence of two new feedback loops inside the UPR. First, GADD34 seems to have a positive effect on ATF4 activity, while CHOP inhibits it. We claim that these newly described feedback loops ensure the fine-tuning of the ATF4-dependent stress response mechanism of the cell.

Expression of the translation termination factor eRF1 is autoregulated by translational readthrough and 3'UTR intron-mediated NMD in Neurospora crassa.

Eukaryotic release factor 1 (eRF1) is a translation termination factor that binds to the ribosome at stop codons. The expression of eRF1 is strictly controlled, since its concentration defines termination efficiency and frequency of translational readthrough. Here, we show that eRF1 expression in Neurospora crassa is controlled by an autoregulatory circuit that depends on the specific 3'UTR structure of erf1 mRNA. The stop codon context of erf1 promotes readthrough that protects the mRNA from its 3'UTR-induced nonsense-mediated mRNA decay (NMD). High eRF1 concentration leads to inefficient readthrough, thereby allowing NMD-mediated erf1 degradation. We propose that eRF1 expression is controlled by similar autoregulatory circuits in many fungi and seed plants and discuss the evolution of autoregulatory systems of different translation termination factors.

Association of Social Jetlag With Sleep Quality and Autonomic Cardiac Control During Sleep in Young Healthy Men.

Social jetlag (SJL), the difference in sleep timing between work and free days is a consequence of the discrepancy between the individual's circadian rhythm and the social clock. SJL is considered a chronic stress factor and has been linked to various health problems. In this field study, we examined for the first time the association between SJL and cardiac regulation during sleep. 33 healthy young men aged 20-26 years participated in the study. The median SJL was used as a cut-off value to assign the participants into two groups with either lower or higher SJL. As a marker of autonomic control we analyzed heart rate variability (HRV) and addressed intra-individual differences between workdays and free days. In subjects with higher SJL, pNN50, an indicator of vagal activity was lower in the first 3 h of sleep on workday as compared to free day (day × sleep block × group, p = 0.015), indicating a more adaptable regulation on free days, when subjects slept according to their own preference. However, in subjects with lower SJL, no HRV differences were found between the two nights. SJL showed correlation with the free day-workday differences of both pNN50 and another vagal index, RMSSD in the first 2 h of sleep (p = 0.023 and 0.047, respectively). In subjects with higher SJL, a different HF power on workdays and free days (p = 0.031) also indicated that a shift in sleep timing is accompanied by an altered parasympathetic activity in the first few hours of sleep. Furthermore, subjective sleep quality on workdays was negatively associated with SJL (p = 0.02), and subjects with higher SJL reported worse sleep quality on workday than on free day (p = 0.027). Taken together, our data call attention on the potential effect of SJL on sleep quality and vagal activity during sleep.

Circadian regulation of neutrophils: Control by a cell-autonomous clock or systemic factors?

BACKGROUND: The circadian time-measuring system enables the organism to anticipate and effectively respond to regular daily changes in the environment and is therefore a crucial factor of adaptation. A large body of epidemiological data underlines the circadian characteristics of human immune functions. Circadian control of neutrophil responsiveness contributes to daily changes in the pathology of both acute and chronic inflammation and may therefore time-dependently influence the outcome of therapeutic approaches. AIM: This review summarizes recent data on the role of the circadian clock in the control of immune responses, particularly of those linked to neutrophil activity, and possible mechanisms of the regulation. DISCUSSION: In the first section of this review we present the recent model of the mammalian molecular clock by introducing the main transcription-translation feedback loops and discussing the pace-setting role of post-translational modifications. The next sections summarize clinical, epidemiological and experimental data regarding the daily control of immune responses and studies analysing expression of clock components in various leukocytes and particularly, in human peripheral neutrophils. As the latter data indicate that expression of components of the cell-autonomous clock is relatively low in neutrophils, in the last section we review recent findings suggesting a role for systemic and local factors in the regulation of rhythmic neutrophil responses.

The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa.

Accumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2 RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock.

Circadian regulation of human peripheral neutrophils.

Neutrophils are the most abundant leukocytes in human blood. Beside being essential responders in bacterial and fungal infections, they also contribute to tissue reactions in many autoimmune and inflammatory diseases. Although several immune responses linked to neutrophil functions have been described to be rhythmic, the mechanism of the circadian regulation of these cells is still not understood. Characterization of the time-of-day-specific control of neutrophil responsiveness could help to better understand the pathomechanism of these inflammatory responses and design effective chronotherapy. Here we report that the time-dependent expression of core clock components in human neutrophils characteristically differs from that in mononuclear cells. Both the low expression and the reduced nuclear accumulation of the essential clock protein BMAL1 suggest that the molecular oscillator is down-regulated in neutrophils. By following the expression of the maturation marker Cxcr4 and morphological attributes (side-scattering properties and nuclear segmentation), we found that the distribution of young and aged cells within the peripheral neutrophil pool displays a daily rhythm. In addition, we detected synchronous fluctuations in the plasma level of the CXCR4 ligand CXCL12, an important regulator of cell trafficking within the bone marrow. We found that expression of another maturation marker, the core component of the superoxide generating NADPH oxidase, and parallelly, the superoxide producing capacity of neutrophils were also dependent on the time of the day. In line with this, number of opsonized bacteria engulfed by neutrophils also showed time-dependent differences, supporting that clearance of pathogens shows a daily rhythm. We suggest that maturation-dependent changes in neutrophil responsiveness rather than the cellular autonomous clock are involved in the daily regulation of human neutrophil functions.

Social jetlag negatively correlates with academic performance in undergraduates.

Discrepancies between sleep timing on workdays and weekends, also known as social jetlag (SJL), affect the majority of the population and have been found to be associated with increased health risk and health-impairing behaviors. In this study, we explored the relationship between SJL and academic performance in a sample of undergraduates of the Semmelweis University. We assessed SJL and other sleep-related parameters with the Munich ChronoType Questionnaire (MCTQ) (n = 753). Academic performance was measured by the average grade based on weekly test results as well as scores acquired on the final test (n = 247). The average mid-sleep point on free days in the Hungarian sample fits well the regression line plotted for longitudes within the Central European Time Zone and chronotypes, confirming that sunlight has a major impact on chronotype. Multivariate analysis showed negative effect of SJL on the weekly average grade (p = 0.028, n = 247) during the lecture term with its highly regular teaching schedules, while this association disappeared in the exam period (p = 0.871, n = 247) when students had no scheduled obligations (lower SJL). We also analyzed the relationship between the time of the weekly tests and academic performance and found that students with later sleep times on free days achieved worse in the morning (p = 0.017, n = 129), while the inverse tendency was observed for the afternoon test-takers (p = 0.10, n = 118). We did not find significant association between academic performance and sleep duration or sleep debt on work days. Our data suggest that circadian misalignment can have a significant negative effect on academic performance. One possible reason for this misalignment is socially enforced sleep times.

Interconnections of reactive oxygen species homeostasis and circadian rhythm in Neurospora crassa.

SIGNIFICANCE: Both circadian rhythm and the production of reactive oxygen species (ROS) are fundamental features of aerobic eukaryotic cells. The circadian clock enhances the fitness of organisms by enabling them to anticipate cycling changes in the surroundings. ROS generation in the cell is often altered in response to environmental changes, but oscillations in ROS levels may also reflect endogenous metabolic fluctuations governed by the circadian clock. On the other hand, an effective regulation and timing of antioxidant mechanisms may be crucial in the defense of cellular integrity. Thus, an interaction between the circadian timekeeping machinery and ROS homeostasis or signaling in both directions may be of advantage at all phylogenetic levels. RECENT ADVANCES: The Frequency-White Collar-1 and White Collar-2 oscillator (FWO) of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Several members of the ROS homeostasis were found to be controlled by the circadian clock, and ROS levels display circadian rhythm in Neurospora. On the other hand, multiple data indicate that ROS affect the molecular oscillator. CRITICAL ISSUES: Increasing evidence suggests the interplay between ROS homeostasis and oscillators that may be partially or fully independent of the FWO. In addition, ROS may be part of a complex cellular network synchronizing non-transcriptional oscillators with timekeeping machineries based on the classical transcription-translation feedback mechanism. FUTURE DIRECTIONS: Further investigations are needed to clarify how the different layers of the bidirectional interactions between ROS homeostasis and circadian regulation are interconnected.

Reactive oxygen species can modulate circadian phase and period in Neurospora crassa.

Reactive oxygen species (ROS) may serve as signals coupling metabolism to other cell functions. In addition to being by-products of normal metabolism, they are generated at elevated levels under environmental stress situations. We analyzed how reactive oxygen species affect the circadian clock in the model organism Neurospora crassa. In light/dark cycles, an increase in the levels of reactive oxygen species advanced the phase of both the conidiation rhythm and the expression of the clock gene frequency. Our results indicate a dominant role of the superoxide anion in the control of the phase. Elevation of superoxide production resulted in the activation of protein phosphatase 2A, a regulator of the positive element of the circadian clock. Our data indicate that even under nonstress conditions, reactive oxygen species affect circadian timekeeping. Reduction of their basal levels results in a delay of the phase in light/dark cycles and a longer period under constant conditions. We show that under entrained conditions the phase depends on the temperature and reactive oxygen species contribute to this effect. Our results suggest that the superoxide anion is an important factor controlling the circadian oscillator and is able to reset the clock most probably by activating protein phosphatase 2A, thereby modulating the activity of the White Collar complex.

Perivascular expression and potent vasoconstrictor effect of dynorphin A in cerebral arteries.

BACKGROUND: Numerous literary data indicate that dynorphin A (DYN-A) has a significant impact on cerebral circulation, especially under pathophysiological conditions, but its potential direct influence on the tone of cerebral vessels is obscure. The aim of the present study was threefold: 1) to clarify if DYN-A is present in cerebral vessels, 2) to determine if it exerts any direct effect on cerebrovascular tone, and if so, 3) to analyze the role of κ-opiate receptors in mediating the effect. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analysis revealed the expression of DYN-A in perivascular nerves of rat pial arteries as well as in both rat and human intraparenchymal vessels of the cerebral cortex. In isolated rat basilar and middle cerebral arteries (BAs and MCAs) DYN-A (1-13) and DYN-A (1-17) but not DYN-A (1-8) or dynorphin B (DYN-B) induced strong vasoconstriction in micromolar concentrations. The maximal effects, compared to a reference contraction induced by 124 mM K(+), were 115±6% and 104±10% in BAs and 113±3% and 125±9% in MCAs for 10 µM of DYN-A (1-13) and DYN-A (1-17), respectively. The vasoconstrictor effects of DYN-A (1-13) could be inhibited but not abolished by both the κ-opiate receptor antagonist nor-Binaltorphimine dihydrochloride (NORBI) and blockade of G(i/o)-protein mediated signaling by pertussis toxin. Finally, des-Tyr(1) DYN-A (2-13), which reportedly fails to activate κ-opiate receptors, induced vasoconstriction of 45±11% in BAs and 50±5% in MCAs at 10 µM, which effects were resistant to NORBI. CONCLUSION/SIGNIFICANCE: DYN-A is present in rat and human cerebral perivascular nerves and induces sustained contraction of rat cerebral arteries. This vasoconstrictor effect is only partly mediated by κ-opiate receptors and heterotrimeric G(i/o)-proteins. To our knowledge our present findings are the first to indicate that DYN-A has a direct cerebral vasoconstrictor effect and that a dynorphin-induced vascular action may be, at least in part, independent of κ-opiate receptors.

Photoadaptation in Neurospora by competitive interaction of activating and inhibitory LOV domains.

Light responses and photoadaptation of Neurospora depend on the photosensory light-oxygen-voltage (LOV) domains of the circadian transcription factor White Collar Complex (WCC) and its negative regulator VIVID (VVD). We found that light triggers LOV-mediated dimerization of the WCC. The activated WCC induces expression of VVD, which then disrupts and inactivates the WCC homodimers by the competitive formation of WCC-VVD heterodimers, leading to photoadaptation. During the day, expression levels of VVD correlate with light intensity, allowing photoadaptation over several orders of magnitude. At night, previously synthesized VVD serves as a molecular memory of the brightness of the preceding day and suppresses responses to light cues of lower intensity. We show that VVD is essential to discriminate between day and night, even in naturally ambiguous photoperiods with moonlight.

Synchronization of the fungal and the plant circadian clock by light.

Circadian clocks are endogenous time keeping devices that provide temporal control of physiology in accordance with predicted daily changes in the environment. Photoentrainment is the process that synchronizes circadian clocks-and thereby clock-controlled gene expression and physiology-to the environmental day/night cycles. Light is primarily detected by specialized photoreceptors that are coupled--directly or through other signaling components--to the rhythm-generating oscillator. As a consequence, the expression, the activity or the stability of oscillator components are altered, resulting in a change of phase and/or pace of the oscillator. In this review our present knowledge about light absorption/transduction and light-induced modifications of oscillator components in Neurospora crassa and Arabidopsis thaliana is summarized. These systems provide a basis for understanding the molecular mechanisms of entrainment in the fungal and plant circadian systems.

Interlocked feedback loops of the circadian clock of Neurospora crassa.

Circadian clocks drive daily rhythms in physiology and behaviour, and thus allow organisms to better adapt to rhythmic changes in the environment. Circadian oscillators are cell-autonomous systems, which generate via transcriptional, post-transcriptional, translational and post-translational control mechanisms a daily activity-rhythm of a circadian transcription factor complex. According to recent models, this complex of transcription factors controls directly or indirectly expression of a large number of genes, and thus generates the potential to modulate physiological processes in a rhythmic fashion. The basic principles of the generation of circadian oscillation are similar in all eukaryotic systems. The circadian clock of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Focusing on the molecular properties, interactions and post-translational modifications of the core Neurospora clock proteins WHITE COLLAR-1, WHITE COLLAR-2, FREQUENCY and VIVID, this review summarizes our knowledge of the molecular basis of circadian time keeping in Neurospora. Moreover, we discuss the mechanisms by which environmental cues like light and temperature entrain and reset this circadian system.

Phosphorylation-dependent maturation of Neurospora circadian clock protein from a nuclear repressor toward a cytoplasmic activator.

Frequency (FRQ) is a central component of interconnected negative and positive limbs of feedback loops of the circadian clock of Neurospora. In the negative limb, FRQ inhibits its transcriptional activator White Collar Complex (WCC) and in the positive limb, FRQ supports accumulation of WCC. We show that these conflicting functions are confined to distinct subcellular compartments and coordinated in temporal fashion. Inactivation of the transcriptional activator WCC requires nuclear FRQ and occurs early after the onset of FRQ expression. Support of WCC accumulation requires cytosolic FRQ and occurs on a post-translational level, when high amounts of FRQ have accumulated. The transcriptional function of FRQ in the negative loop and its post-translational function in the positive loop are independent and associated with distinct regions of FRQ. Phosphorylation of FRQ at the PEST-2 region triggers its maturation from a nuclear repressor toward a cytoplasmic activator.

Sec14 homology domain targets p50RhoGAP to endosomes and provides a link between Rab and Rho GTPases.

Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.

Transcriptional regulation of the Neurospora circadian clock gene wc-1 affects the phase of circadian output.

WHITE COLLAR-1 (WC-1) is the limiting component of the White Collar Complex (WCC) controlling expression of the Neurospora circadian clock protein Frequency (FRQ). Accumulation of WC-1 is supported by FRQ on a post-transcriptional level. Here, we show that transcription of wc-1 is organized in a complex way. Three promoters drive transcription of wc-1. Pdist is dependent on WCC. Pprox is independent of WCC in darkness, but inducible by light in a WCC-dependent manner. A third promoter, Pint, is located in the wc-1 open reading frame and promotes expression of an amino-terminally truncated WC-1 isoform of unknown function. Expression of wc-1 by Pdist or Pprox alone, or by a heterologous promoter, affects the entrained phase of circadian conidiation and the response of Neurospora to light. Our results indicate that transcriptional regulation of wc-1 is required to modulate the circadian phase of clock output.

Transcriptional feedback of Neurospora circadian clock gene by phosphorylation-dependent inactivation of its transcription factor.

The circadian clock protein Frequency (FRQ) feedback-regulates its own expression by inhibiting its transcriptional activator, White Collar Complex (WCC). We present evidence that FRQ regulates the bulk of WCC through modulation of its phosphorylation status rather than via direct complex formation. In the absence of FRQ, WCC is hypophosphorylated and transcriptionally active, while WCC is hyperphosphorylated and transcriptionally inactive when FRQ is expressed. The phosphorylation status of WCC changes rhythmically over a circadian cycle. Dephosphorylation and activation of WCC depend on protein phosphatase 2A (PP2A), and WCC is a substrate of PP2A in vitro. Hypophosphorylated WCC binds to the clock box of the frq promoter even in the presence of FRQ, while binding of hyperphosphorylated WCC is compromised even when FRQ is depleted. We propose that negative feedback in the circadian clock of Neurospora is mediated by FRQ, which rhythmically promotes phosphorylation of WCC, functionally equivalent to a cyclin recruiting cyclin-dependent kinase to its targets.

Dual role of phagocytic NADPH oxidase in bacterial killing.

The classical model of bacterial killing by phagocytic cells has been recently challenged by questioning the toxic effect of oxygen products and attributing the fundamental role to K(+) ions in releasing antimicrobial proteins within the phagosome. In the present study we followed O(2)(*-) production, changes of membrane potential, K(+) efflux, and bacterial killing in the presence of increasing concentrations of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. Efficiency of bacterial killing was assessed on the basis of bacterial survival measured by a new semiautomated method. Very low rates of O(2)(*-) production were accompanied by significant membrane depolarization and K(+) release and parallel improvement of bacterial killing. When O(2)(*-) production exceeded 20% of its maximal capacity, no further change was detected in the membrane potential and only minimal further K(+) efflux occurred, yet bacterial survival decreased parallel to the increase of O(2)(*-) production. The presented results indicate that both electrophysiological changes (depolarization and consequent ion movements) and the chemical effect of reactive oxygen species play a significant role in the killing of certain pathogens. The observation that an increase of membrane depolarization can compensate for decreased O(2)(*-) production may be important for potential therapeutic applications.