Circadian clocks: It's time for chronobiology.

Circadian clocks are everywhere, yet we still have not translated the vast knowledge gained in the past 20 years on the properties of circadian clocks into practical applications. We are missing opportunities for improving quality of life, health, and sustainability.

Circadian Clock and Hypoxia.

The timing of life on Earth is remarkable: between individuals of the same species, a highly similar temporal pattern is observed, with shared periods of activity and inactivity each day. At the individual level, this means that over the course of a single day, a person alternates between two states. They are either upright, active, and communicative or they lie down in a state of (un)consciousness called sleep where even the characteristic of neuronal signals in the brain shows distinctive properties. The circadian clock governs both of these time stamps-activity and (apparent) inactivity-making them come and go consistently at the same approximate time each day. This behavior thus represents the meeting of two pervasive systems: the circadian clock and metabolism. In this article, we will describe what is known about how the circadian clock anticipates daily changes in oxygen usage, how circadian clock regulation may relate to normal physiology, and to hypoxia and ischemia that can result from pathologies such as myocardial infarction and stroke.

Rhythms in barriers and fluids: Circadian clock regulation in the aging neurovascular unit.

The neurovascular unit is where two very distinct physiological systems meet: The central nervous system (CNS) and the blood. The permeability of the barriers separating these systems is regulated by time, including both the 24 h circadian clock and the longer processes of aging. An endogenous circadian rhythm regulates the transport of molecules across the blood-brain barrier and the circulation of the cerebrospinal fluid and the glymphatic system. These fluid dynamics change with time of day, and with age, and especially in the context of neurodegeneration. Factors may differ depending on brain region, as can be highlighted by consideration of circadian regulation of the neurovascular niche in white matter. As an example of a potential target for clinical applications, we highlight chaperone-mediated autophagy as one mechanism at the intersection of circadian dysregulation, aging and neurodegenerative disease. In this review we emphasize key areas for future research.

A functional context for heterogeneity of the circadian clock in cells.

Characterization of circadian systems at the organism level-a top-down approach-has led to definition of unifying properties, a hallmark of the science of chronobiology. The next challenge is to use a bottom-up approach to show how the molecular workings of the cellular circadian clock work as building blocks of those properties. We review new studies, including a recently published PLOS Biology paper by Nikhil and colleagues, that show how programmed but also stochastic generation of variation in cellular circadian period explain important adaptive features of entrained circadian phase.

Deviations from temporal scaling support a stage-specific regulation for C. elegans postembryonic development.

BACKGROUND: After embryonic development, Caenorhabditis elegans progress through for larval stages, each of them finishing with molting. The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homologue. Nevertheless, each larval stage has a defined duration and entails specific events. Since the overall duration of development is controlled by numerous factors, we have asked whether different rate-limiting interventions impact all stages equally. RESULTS: We have measured the duration of each stage of development for over 2500 larvae, under varied environmental conditions known to alter overall developmental rate. We applied changes in temperature and in the quantity and quality of nutrition and analysed the effect of genetically reduced insulin signalling. Our results show that the distinct developmental stages respond differently to these perturbations. The changes in the duration of specific larval stages seem to depend on stage-specific events. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. CONCLUSIONS: Altogether, our results show that multiple factors fine tune developmental timing, impacting larval stages independently. Further understanding of the regulation of this process will allow modelling the mechanisms that control developmental timing.

Weekly and seasonal variation in the circadian melatonin rhythm in humans: A response.

We read with interest the commentary by Skeldon and Dijk about our article "Weekly, seasonal and chronotype-dependent variation of dim light melatonin onset." The discussion points raised by Skeldon and Dijk are currently among the most hotly debated in human circadian science. What external factors determine human phase of entrainment? How great is the contribution of natural versus artificial light and sun time versus social time? Our intra-individual data add to the still limited evidence from field studies in this matter. In their commentary, Skeldon and Dijk formulate two either-or hypotheses, postulating that humans entrain either solely to the natural light-dark cycle (sun time referenced by midday) (H1 ) or solely to the light selected by local clock time and social constraints (H2 ). Neither hypothesis accounts for the effect of season on human light exposure. We interpreted our findings along more complex lines, speculating that the 1-h earlier melatonin rise in summer found in our sample is likely the combined result of daylight saving time (DST)-induced behavioral advances and a stronger natural zeitgeber in summer (light exposure determined by social and seasonal factors, Horiginal ). Here, we show how the criticism by Skeldon and Dijk is based on two sentences quoted out of context (misrepresenting our hypothesis as H1 ) and that their hypothesis H2  leaves out important seasonal components in light exposure.

Weekly, seasonal, and chronotype-dependent variation of dim-light melatonin onset.

In humans, the most important zeitgeber for entrainment is light. Laboratory studies have shown that meaningful changes in light exposure lead to phase shifts in markers of the circadian clock. In natural settings, light is a complex signal varying with external conditions and individual behaviors; nonetheless, phase of entrainment is assumed to be fairly stable. Here, we investigated the influence of season and weekly schedule (as indicators of variation in light landscapes) on phase of entrainment. Using a within-subjects design (N = 33), we assessed dim-light melatonin onset (DLMO) as a circadian phase marker in humans, on workdays and work-free days, in summer (under daylight saving time) and in winter, while also estimating sleep times from actimetry. Our mixed-model regressions show that both season and weekly structure are linked with changes in phase of entrainment and sleep. In summer, both DLMO and sleep times were about 1 hour earlier compared to winter, and sleep duration was shorter. On work-free days, DLMO and sleep times were later, and their phase relationship differed more relative to workdays. All these effects were stronger in later chronotypes (those who habitually sleep late). Our results confirm that phase of entrainment is earlier when stronger zeitgebers are present (summer) and show that it relates to midday or midnight rather than sunrise or sunset. Additionally, they suggest that late chronotypes are capable of rapid phase shifts each week as they move between workdays and work-free days, stimulating interesting questions about the stability of circadian phase under natural conditions.

Strategies to decrease social jetlag: Reducing evening blue light advances sleep and melatonin.

The timing of sleep is under the control of the circadian clock, which uses light to entrain to the external light-dark cycle. A combination of genetic, physiological and environmental factors produces individual differences in chronotype (entrained phase as manifest in sleep timing). A mismatch between circadian and societal (e.g., work) clocks leads to a condition called social jetlag, which is characterized by changing sleep times over work and free days and accumulation of sleep debt. Social jetlag, which is prevalent in late chronotypes, has been related to several health issues. One way to reduce social jetlag would be to advance the circadian clock via modifications of the light environment. We thus performed two intervention field studies to describe methods for decreasing social jetlag. One study decreased evening light exposure (via blue-light-blocking glasses) and the other used increased morning light (via the use of curtains). We measured behaviour as well as melatonin; the latter in order to validate that behaviour was consistent with this neuroendocrinological phase marker of the circadian clock. We found that a decrease in evening blue light exposure led to an advance in melatonin and sleep onset on workdays. Increased morning light exposure advanced neither melatonin secretion nor sleep timing. Neither protocol led to a significant change in social jetlag. Despite this, our findings show that controlling light exposure at home can be effective in advancing melatonin secretion and sleep, thereby helping late chronotypes to better cope with early social schedules.

Insulin-like growth factor-1 acts as a zeitgeber on hypothalamic circadian clock gene expression via glycogen synthase kinase-3ß signaling.

Brain and muscle ARNT-like protein-1 (BMAL-1) is an important component of the cellular circadian clock. Proteins such as epidermal (EGF) or nerve growth factor (NGF) affect the cellular clock via extracellular signal-regulated kinases-1/2 (ERK-1/2) in NIH3T3 or neuronal stem cells, but no such data are available for the insulin-like growth factor-1 (IGF-1). The hypothalamus expresses receptors for all three growth factors, acts as a central circadian pacemaker, and releases hormones in a circadian fashion. However, little is known about growth factor-induced modulation of clock gene activity in hypothalamic cells. Here, we investigated effects of IGF-1, EGF, or NGF on the Bmal-1 promoter in two hypothalamic cell lines. We found that only IGF-1 but not EGF or NGF enhanced activity of the Bmal-1 promoter. Inhibition of ERK-1/2 activity did not affect IGF-1-induced Bmal-1 promoter activation and all three growth factors similarly phosphorylated ERK-1/2, questioning a role for ERK-1/2 in controlling BMAL-1 promoter activity. Of note, only IGF-1 induced sustained phosphorylation of glycogen synthase kinase-3ß (GSK-3ß). Moreover, the GSK-3ß inhibitor lithium or siRNA-mediated GSK-3ß knockdown diminished the effects of IGF-1 on the Bmal-1 promoter. When IGF-1 was used in the context of temperature cycles entraining hypothalamic clock gene expression to a 24-h rhythm, it shifted the phase of Bmal-1 promoter activity, indicating that IGF-1 functions as a zeitgeber for cellular hypothalamic circadian clocks. Our results reveal that IGF-1 regulates clock gene expression and that GSK-3ß but not ERK-1/2 is required for the IGF-1-mediated regulation of the Bmal-1 promoter in hypothalamic cells.

Peroxiredoxins are conserved markers of circadian rhythms.

Cellular life emerged ∼3.7 billion years ago. With scant exception, terrestrial organisms have evolved under predictable daily cycles owing to the Earth's rotation. The advantage conferred on organisms that anticipate such environmental cycles has driven the evolution of endogenous circadian rhythms that tune internal physiology to external conditions. The molecular phylogeny of mechanisms driving these rhythms has been difficult to dissect because identified clock genes and proteins are not conserved across the domains of life: Bacteria, Archaea and Eukaryota. Here we show that oxidation-reduction cycles of peroxiredoxin proteins constitute a universal marker for circadian rhythms in all domains of life, by characterizing their oscillations in a variety of model organisms. Furthermore, we explore the interconnectivity between these metabolic cycles and transcription-translation feedback loops of the clockwork in each system. Our results suggest an intimate co-evolution of cellular timekeeping with redox homeostatic mechanisms after the Great Oxidation Event ∼2.5 billion years ago.

Gene expression during zombie ant biting behavior reflects the complexity underlying fungal parasitic behavioral manipulation.

BACKGROUND: Adaptive manipulation of animal behavior by parasites functions to increase parasite transmission through changes in host behavior. These changes can range from slight alterations in existing behaviors of the host to the establishment of wholly novel behaviors. The biting behavior observed in Carpenter ants infected by the specialized fungus Ophiocordyceps unilateralis s.l. is an example of the latter. Though parasitic manipulation of host behavior is generally assumed to be due to the parasite's gene expression, few studies have set out to test this. RESULTS: We experimentally infected Carpenter ants to collect tissue from both parasite and host during the time period when manipulated biting behavior is experienced. Upon observation of synchronized biting, samples were collected and subjected to mixed RNA-Seq analysis. We also sequenced and annotated the O. unilateralis s.l. genome as a reference for the fungal sequencing reads. CONCLUSIONS: Our mixed transcriptomics approach, together with a comparative genomics study, shows that the majority of the fungal genes that are up-regulated during manipulated biting behavior are unique to the O. unilateralis s.l. genome. This study furthermore reveals that the fungal parasite might be regulating immune- and neuronal stress responses in the host during manipulated biting, as well as impairing its chemosensory communication and causing apoptosis. Moreover, we found genes up-regulated during manipulation that putatively encode for proteins with reported effects on behavioral outputs, proteins involved in various neuropathologies and proteins involved in the biosynthesis of secondary metabolites such as alkaloids.

Circadian regulation of olfaction and an evolutionarily conserved, nontranscriptional marker in Caenorhabditis elegans.

Circadian clocks provide a temporal structure to processes from gene expression to behavior in organisms from all phyla. Most clocks are synchronized to the environment by alternations of light and dark. However, many organisms experience only muted daily environmental cycles due to their lightless spatial niches (e.g., caves or soil). This has led to speculation that they may dispense with the daily clock. However, recent reports contradict this notion, showing various behavioral and molecular rhythms in Caenorhabditis elegans and in blind cave fish. Based on the ecology of nematodes, we applied low-amplitude temperature cycles to synchronize populations of animals through development. This entrainment regime reveals rhythms on multiple levels: in olfactory cued behavior, in RNA and protein abundance, and in the oxidation state of a broadly conserved peroxiredoxin protein. Our work links the nematode clock with that of other clock model systems; it also emphasizes the importance of daily rhythms in sensory functions that are likely to impact on organism fitness and population structure.

A circadian clock in Saccharomyces cerevisiae.

Circadian timing is a fundamental biological process, underlying cellular physiology in animals, plants, fungi, and cyanobacteria. Circadian clocks organize gene expression, metabolism, and behavior such that they occur at specific times of day. The biological clocks that orchestrate these daily changes confer a survival advantage and dominate daily behavior, for example, waking us in the morning and helping us to sleep at night. The molecular mechanism of circadian clocks has been sketched out in genetic model systems from prokaryotes to humans, revealing a combination of transcriptional and posttranscriptional pathways, but the clock mechanism is far from solved. Although Saccharomyces cerevisiae is among the most powerful genetic experimental systems and, as such, could greatly contribute to our understanding of cellular timing, it still remains absent from the repertoire of circadian model organisms. Here, we use continuous cultures of yeast, establishing conditions that reveal characteristic clock properties similar to those described in other species. Our results show that metabolism in yeast shows systematic circadian entrainment, responding to cycle length and zeitgeber (stimulus) strength, and a (heavily damped) free running rhythm. Furthermore, the clock is obvious in a standard, haploid, auxotrophic strain, opening the door for rapid progress into cellular clock mechanisms.

The circadian clock of the bacterium B. subtilis evokes properties of complex, multicellular circadian systems.

Circadian clocks are pervasive throughout nature, yet only recently has this adaptive regulatory program been described in nonphotosynthetic bacteria. Here, we describe an inherent complexity in the Bacillus subtilis circadian clock. We find that B. subtilis entrains to blue and red light and that circadian entrainment is separable from masking through fluence titration and frequency demultiplication protocols. We identify circadian rhythmicity in constant light, consistent with the Aschoff's rule, and entrainment aftereffects, both of which are properties described for eukaryotic circadian clocks. We report that circadian rhythms occur in wild isolates of this prokaryote, thus establishing them as a general property of this species, and that its circadian system responds to the environment in a complex fashion that is consistent with multicellular eukaryotic circadian systems.

A Unified Model for Entrainment by Circadian Clocks: Dynamic Circadian Integrated Response Characteristic (dCiRC).

Circadian rhythms are ubiquitous and are observed in all biological kingdoms. In nature, their primary characteristic or phenotype is the phase of entrainment. There are two main hypotheses related to how circadian clocks entrain, parametric and non-parametric models. The parametric model focuses on the gradual changes of the clock parameters in response to the changing ambient condition, whereas the non-parametric model focuses on the instantaneous change of the phase of the clock in response to the zeitgeber. There are ample empirical data supporting both models. However, only recently has a unifying model been proposed, the circadian integrated response characteristic (CiRC). In the current study, we developed a system of ordinary differential equations, dynamic CiRC (dCiRC), that describes parameters of circadian rhythms and predicts the phase of entrainment in zeitgeber cycles. dCiRC mathematically extracts the underlying information of velocity changes of the internal clock that reflects the parametric model and the phase shift trajectory that reflects the non-parametric model from phase data under entraining conditions. As a proof of concept, we measured clock parameters of 26 Neurospora crassa ecotypes in both cycling and constant conditions using dCiRC. Our data showed that the morning light shortens the period of the clock while the afternoon light lengthens it. We also found that individual ecotypes have different strategies of integrating light effects to accomplish the optimal phase of entrainment, a model feature that is consistent with our knowledge of how circadian clocks are organized and encoded. The unified model dCiRC will provide new insights into how circadian clocks function under different zeitgeber conditions. We suggest that this type of model may be useful in the advent of chronotherapies.

Keeping an eye on circadian time in clinical research and medicine.

BACKGROUND: Daily rhythms are observed in humans and almost all other organisms. Most of these observed rhythms reflect both underlying endogenous circadian rhythms and evoked responses from behaviours such as sleep/wake, eating/fasting, rest/activity, posture changes and exercise. For many research and clinical purposes, it is important to understand the contribution of the endogenous circadian component to these observed rhythms. CONTENT: The goal of this manuscript is to provide guidance on best practices in measuring metrics of endogenous circadian rhythms in humans and promote the inclusion of circadian rhythms assessments in studies of health and disease. Circadian rhythms affect all aspects of physiology. By specifying minimal experimental conditions for studies, we aim to improve the quality, reliability and interpretability of research into circadian and daily (i.e., time-of-day) rhythms and facilitate the interpretation of clinical and translational findings within the context of human circadian rhythms. We describe protocols, variables and analyses commonly used for studying human daily rhythms, including how to assess the relative contributions of the endogenous circadian system and other daily patterns in behaviours or the environment. We conclude with recommendations for protocols, variables, analyses, definitions and examples of circadian terminology. CONCLUSION: Although circadian rhythms and daily effects on health outcomes can be challenging to distinguish in practice, this distinction may be important in many clinical settings. Identifying and targeting the appropriate underlying (patho)physiology is a medical goal. This review provides methods for identifying circadian effects to aid in the interpretation of published work and the inclusion of circadian factors in clinical research and practice.

Genetic and molecular characterization of a cryptochrome from the filamentous fungus Neurospora crassa.

In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the cryptochrome from the filamentous fungus Neurospora crassa and demonstrate that Neurospora cry encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). The cry transcript and CRY protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulated, with a peak abundance opposite in phase to frq. Neither deletion nor overexpression of cry appears to perturb the free-running circadian clock. However, cry disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays (EMSA), we show that CRY is capable of binding single- and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of cry under our laboratory conditions.

Circadian effects on stroke outcome - Did we not wake up in time for neuroprotection?

The occurrence of stroke in humans peaks in the morning. A recent study revealed that time of day mitigates the therapeutic impact of neuroprotective paradigms. These findings might not only explain the previous failure of translation of neuroprotective therapies but inspire new paradigms in stroke chronopathophysiology research. Taking chronotype into account may complement the many factors that influence efficacy of experimental therapies in stroke.