Circadian Timekeeping System Research Articles

The Drosophila circadian clock gene cycle controls the development of clock neurons.

Daily behavioral and physiological rhythms are controlled by the brain's circadian timekeeping system, a synchronized network of neurons that maintains endogenous molecular oscillations. These oscillations are based on transcriptional feedback loops of clock genes, which in Drosophila include the transcriptional activators Clock (Clk) and cycle (cyc). While the mechanisms underlying this molecular clock are very well characterized, the roles that the core clock genes play in neuronal physiology and development are much less understood. The Drosophila timekeeping center is composed of ~150 clock neurons, among which the four small ventral lateral neurons (sLNvs) are the most dominant pacemakers under constant conditions. Here, we show that downregulating the clock gene cyc specifically in the Pdf-expressing neurons leads to decreased fasciculation both in larval and adult brains. This effect is due to a developmental role of cyc, as both knocking down cyc or expressing a dominant negative form of cyc exclusively during development lead to defasciculation phenotypes in adult clock neurons. Clk downregulation also leads to developmental effects on sLNv morphology. Our results reveal a non-circadian role for cyc, shedding light on the additional functions of circadian clock genes in the development of the nervous system.

Circadian rhythm disruption and endocrine-related tumors.

This review delved into the intricate relationship between circadian clocks and physiological processes, emphasizing their critical role in maintaining homeostasis. Orchestrated by interlocked clock genes, the circadian timekeeping system regulates fundamental processes like the sleep-wake cycle, energy metabolism, immune function, and cell proliferation. The central oscillator in the hypothalamic suprachiasmatic nucleus synchronizes with light-dark cycles, while peripheral tissue clocks are influenced by cues such as feeding times. Circadian disruption, linked to modern lifestyle factors like night shift work, correlates with adverse health outcomes, including metabolic syndrome, cardiovascular diseases, infections, and cancer. We explored the molecular mechanisms of circadian clock genes and their impact on metabolic disorders and cancer pathogenesis. Specific associations between circadian disruption and endocrine tumors, spanning breast, ovarian, testicular, prostate, thyroid, pituitary, and adrenal gland cancers, are highlighted. Shift work is associated with increased breast cancer risk, with PER genes influencing tumor progression and drug resistance. CLOCK gene expression correlates with cisplatin resistance in ovarian cancer, while factors like aging and intermittent fasting affect prostate cancer. Our review underscored the intricate interplay between circadian rhythms and cancer, involving the regulation of the cell cycle, DNA repair, metabolism, immune function, and the tumor microenvironment. We advocated for integrating biological timing into clinical considerations for personalized healthcare, proposing that understanding these connections could lead to novel therapeutic approaches. Evidence supports circadian rhythm-focused therapies, particularly chronotherapy, for treating endocrine tumors. Our review called for further research to uncover detailed connections between circadian clocks and cancer, providing essential insights for targeted treatments. We emphasized the importance of public health interventions to mitigate lifestyle-related circadian disruptions and underscored the critical role of circadian rhythms in disease mechanisms and therapeutic interventions.

From the Pineal Gland to the Central Clock in the Brain: Beginning of Studies of the Mammalian Biological Rhythms in the Institute of Physiology of the Czech Academy of Sciences.

The Institute of Physiology of the Czech Academy of Sciences (CAS) has been involved in the field of chronobiology, i.e., in research on temporal regulation of physiological processes, since 1970. The review describes the first 35 years of the research mostly on the effect of light and daylength, i.e., photoperiod, on entrainment or resetting of the pineal rhythm in melatonin production and of intrinsic rhythms in the central biological clock. This clock controls pineal and other circadian rhythms and is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. During the early chronobiological research, many original findings have been reported, e.g. on mechanisms of resetting of the pineal rhythm in melatonin production by short light pulses or by long exposures of animals to light at night, on modulation of the nocturnal melatonin production by the photoperiod or on the presence of high affinity melatonin binding sites in the SCN. The first evidence was given that the photoperiod modulates functional properties of the SCN and hence the SCN not only controls the daily programme of the organism but it may serve also as a calendar measuring the time of a year. During all the years, the chronobiological community has started to talk about "the Czech school of chronobiology". At present, the today´s Laboratory of Biological Rhythms of the Institute of Physiology CAS continues in the chronobiological research and the studies have been extended to the entire circadian timekeeping system in mammals with focus on its ontogenesis, entrainment mechanisms and circadian regulation of physiological functions. Key words: Pineal, Melatonin, AA-NAT rhythm, Light entrainment, Photoperiod, SCN clock.

The Mammalian Circadian Time-Keeping System.

Our physiology and behavior follow precise daily programs that adapt us to the alternating opportunities and challenges of day and night. Under experimental isolation, these rhythms persist with a period of approximately one day (circadian), demonstrating their control by an internal autonomous clock. Circadian time is created at the cellular level by a transcriptional/translational feedback loop (TTFL) in which the protein products of the Period and Cryptochrome genes inhibit their own transcription. Because the accumulation of protein is slow and delayed, the system oscillates spontaneously with a period of ∼24 hours. This cell-autonomous TTFL controls cycles of gene expression in all major tissues and these cycles underpin our daily metabolic programs. In turn, our innumerable cellular clocks are coordinated by a central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. When isolated in slice culture, the SCN TTFL and its dependent cycles of neural activity persist indefinitely, operating as "a clock in a dish". In vivo, SCN time is synchronized to solar time by direct innervation from specialized retinal photoreceptors. In turn, the precise circadian cycle of action potential firing signals SCN-generated time to hypothalamic and brain stem targets, which co-ordinate downstream autonomic, endocrine, and behavioral (feeding) cues to synchronize and sustain the distributed cellular clock network. Circadian time therefore pervades every level of biological organization, from molecules to society. Understanding its mechanisms offers important opportunities to mitigate the consequences of circadian disruption, so prevalent in modern societies, that arise from shiftwork, aging, and neurodegenerative diseases, not least Huntington's disease.

Circadian rhythms in auditory hallucinations and psychosis.

Circadian rhythms are imprinted in all organisms and influence virtually all aspects of physiology and behavior in adaptation to the 24-h day-night cycle. This recognition of a circadian timekeeping system permeating essentially all healthy functioning of body and mind quickly leads to the realization that, in turn, human ailments should be probed for the degree to which they are rooted in or marked by disruptions and dysregulations of circadian clock functions in the human body. In this review, we will focus on psychosis as a key mental illness and foremost one of its cardinal symptoms: auditory hallucinations. We will discuss recent empirical evidence and conceptual advances probing the potential role of circadian disruption in auditory hallucinations. Moreover, a dysbalance in excitation and inhibition within cortical networks, which in turn drive a disinhibition of dopaminergic signaling, will be highlighted as central physiological mechanism. Finally, we will propose two avenues for experimentally intervening on the circadian influences to potentially alleviate hallucinations in psychotic disorders.

Rhythmic transcription of Bmal1 stabilizes the circadian timekeeping system in mammals

In mammals, the circadian clock consists of transcriptional and translational feedback loops through DNA cis-elements such as E-box and RRE. The E-box-mediated core feedback loop is interlocked with the RRE-mediated feedback loop, but biological significance of the RRE-mediated loop has been elusive. In this study, we established mutant cells and mice deficient for rhythmic transcription of Bmal1 gene by deleting its upstream RRE elements and hence disrupted the RRE-mediated feedback loop. We observed apparently normal circadian rhythms in the mutant cells and mice, but a combination of mathematical modeling and experiments revealed that the circadian period and amplitude of the mutants were more susceptible to disturbance of CRY1 protein rhythm. Our findings demonstrate that the RRE-mediated feedback regulation of Bmal1 underpins the E-box-mediated rhythm in cooperation with CRY1-dependent posttranslational regulation of BMAL1 protein, thereby conferring the perturbation-resistant oscillation and chronologically-organized output of the circadian clock.

Treatment of Circadian Rhythm Sleep-Wake Disorders.

Circadian rhythm sleep-wake disorders (CRSWDs) are a distinct class of sleep disorders caused by alterations to the circadian time-keeping system, its entrainment mechanisms, or a mismatch between the endogenous circadian rhythm and the external environment. The main clinical manifestations are insomnia and excessive daytime sleepiness that often lead to clinically meaningful distress or cause mental, physical, social, occupational, educational, or other functional impairment. CRSWDs are easily mistaken for insomnia or early waking up, resulting in inappropriate treatment. CRSWDs can be roughly divided into two categories, namely, intrinsic CRSWDs, in which sleep disturbances are caused by alterations to the endogenous circadian rhythm system due to chronic changes in the regulation or capture mechanism of the biological clock, and extrinsic circadian rhythm sleep-wake disorders, in which sleep disorders, such as jet lag or shift-work disorder, result from environmental changes that cause a mismatch between sleep-wakefulness times and internal circadian rhythms. Sleep diaries, actigraphy, and determination of day and night phase markers (dim light melatonin onset and core body temperature minimum) have all become routine diagnostic methods for CRSWDs. Common treatments for CRSWD currently include sleep health education, time therapy, light therapy, melatonin, and hypnotic drug therapy. Here, we review the progress in the epidemiology, etiology, diagnostic evaluation, diagnostic criteria, and treatment of intrinsic CRSWD, with emphasis on the latter, in the hope of bolstering the clinical diagnosis and treatment of CRSWDs.

Rhythmic transcription of Bmal1 stabilizes the circadian timekeeping system in mammals

Rhythmic transcription of Bmal1 stabilizes the circadian timekeeping system in mammals

Evolution of the repression mechanisms in circadian clocks

BackgroundCircadian (daily) timekeeping is essential to the survival of many organisms. An integral part of all circadian timekeeping systems is negative feedback between an activator and repressor. However, the role of this feedback varies widely between lower and higher organisms.ResultsHere, we study repression mechanisms in the cyanobacterial and eukaryotic clocks through mathematical modeling and systems analysis. We find a common mathematical model that describes the mechanism by which organisms generate rhythms; however, transcription’s role in this has diverged. In cyanobacteria, protein sequestration and phosphorylation generate and regulate rhythms while transcription regulation keeps proteins in proper stoichiometric balance. Based on recent experimental work, we propose a repressor phospholock mechanism that models the negative feedback through transcription in clocks of higher organisms. Interestingly, this model, when coupled with activator phosphorylation, allows for oscillations over a wide range of protein stoichiometries, thereby reconciling the negative feedback mechanism in Neurospora with that in mammals and cyanobacteria.ConclusionsTaken together, these results paint a picture of how circadian timekeeping may have evolved.

Uncovering the dynamics of a circadian-dopamine model influenced by the light–dark cycle

The neurotransmitter dopamine (DA) is known to be influenced by the circadian timekeeping system in the mammalian brain. We have previously created a single-cell differential equations model to understand the mechanisms behind circadian rhythms of extracellular DA. In this paper, we investigate the dynamics in our model and study different behaviors such as entrainment to the 24-hour light–dark cycle and robust periodicity versus decoupling, quasiperiodicity, and chaos. Imbalances in DA are often accompanied by disrupted circadian rhythms, such as in Parkinson’s disease, hyperactivity, and mood disorders. Our model provides new insights into the links between the circadian clock and DA. We show that the daily rhythmicity of DA can be disrupted by decoupling between interlocked loops of the clock circuitry or by quasiperiodic clock behaviors caused by misalignment with the light–dark cycle. The model can be used to further study how the circadian clock affects the dopaminergic system, and to help develop therapeutic strategies for disrupted DA rhythms.

Transcriptome-wide deregulation of gene expression by artificial light at night in tadpoles of common toads

Artificial light at night (ALAN) affects numerous physiological and behavioural mechanisms in various species by potentially disturbing circadian timekeeping systems and modifying melatonin levels. However, given the multiple direct and indirect effects of ALAN on organisms, large-scale transcriptomic approaches are essential to assess the global effect of ALAN on biological processes. Moreover, although studies have focused mainly on variations in gene expression during the night in the presence of ALAN, it is necessary to investigate the effect of ALAN on gene expression during the day. In this study, we combined de novo transcriptome sequencing and assembly, and a controlled laboratory experiment to evaluate the transcriptome-wide gene expression response using high-throughput (RNA-seq) in Bufo bufo tadpoles exposed to ecologically relevant light levels. Here, we demonstrated for the first time that ALAN affected gene expression at night (3.5% and 11% of differentially expressed genes when exposed to 0.1 and 5 lx compared to controls, respectively), but also during the day (11.2% of differentially expressed genes when exposed to 5 lx compared to controls) with a dose-dependent effect. ALAN globally induced a downregulation of genes (during the night, 58% and 62% of the genes were downregulated when exposed to 0.1 and 5 lx compared to controls, respectively, and during the day, 61.2% of the genes were downregulated when exposed to 5 lx compared to controls). ALAN effects were detected at very low levels of illuminance (0.1 lx) and affected mainly genes related to the innate immune system and, to a lesser extend to lipid metabolism. These results provide new insights into understanding the effects of ALAN on organism. ALAN impacted the expression of genes linked to a broad range of physiological pathways at very low levels of ALAN during night-time and during daytime, potentially resulting in reduced immune capacity under environmental immune challenges.

Workshop report. Circadian rhythm sleep-wake disorders: gaps and opportunities.

This White Paper presents the results from a workshop cosponsored by the Sleep Research Society (SRS) and the Society for Research on Biological Rhythms (SRBR) whose goals were to bring together sleep clinicians and sleep and circadian rhythm researchers to identify existing gaps in diagnosis and treatment and areas of high-priority research in circadian rhythm sleep–wake disorders (CRSWD). CRSWD are a distinct class of sleep disorders caused by alterations of the circadian time-keeping system, its entrainment mechanisms, or a misalignment of the endogenous circadian rhythm and the external environment. In these disorders, the timing of the primary sleep episode is either earlier or later than desired, irregular from day-to-day, and/or sleep occurs at the wrong circadian time. While there are incomplete and insufficient prevalence data, CRSWD likely affect at least 800,000 and perhaps as many as 3 million individuals in the United States, and if Shift Work Disorder and Jet Lag are included, then many millions more are impacted. The SRS Advocacy Taskforce has identified CRSWD as a class of sleep disorders for which additional high-quality research could have a significant impact to improve patient care. Participants were selected for their expertise and were assigned to one of three working groups: Phase Disorders, Entrainment Disorders, and Other. Each working group presented a summary of the current state of the science for their specific CRSWD area, followed by discussion from all participants. The outcome of those presentations and discussions are presented here.

Shiftwork and Light at Night Negatively Impact Molecular and Endocrine Timekeeping in the Female Reproductive Axis in Humans and Rodents.

Shiftwork, including work that takes place at night (nightshift) and/or rotates between day and nightshifts, plays an important role in our society, but is associated with decreased health, including reproductive dysfunction. One key factor in shiftwork, exposure to light at night, has been identified as a likely contributor to the underlying health risks associated with shiftwork. Light at night disrupts the behavioral and molecular circadian timekeeping system, which is important for coordinated timing of physiological processes, causing mistimed hormone release and impaired physiological functions. This review focuses on the impact of shiftwork on reproductive function and pregnancy in women and laboratory rodents and potential underlying molecular mechanisms. We summarize the negative impact of shiftwork on female fertility and compare these findings to studies in rodent models of light shifts. Light-shift rodent models recapitulate several aspects of reproductive dysfunction found in shift workers, and their comparison with human studies can enable a deeper understanding of physiological and hormonal responses to light shifts and the underlying molecular mechanisms that may lead to reproductive disruption in human shift workers. The contributions of human and rodent studies are essential to identify the origins of impaired fertility in women employed in shiftwork.

Circadian Rhythms of Early Afterdepolarizations and Ventricular Arrhythmias in a Cardiomyocyte Model

Sudden cardiac arrest is a malfunction of the heart’s electrical system, typically caused by ventricular arrhythmias, that can lead to sudden cardiac death (SCD) within minutes. Epidemiological studies have shown that SCD and ventricular arrhythmias are more likely to occur in the morning than in the evening, and laboratory studies indicate that these daily rhythms in adverse cardiovascular events are at least partially under the control of the endogenous circadian timekeeping system. However, the biophysical mechanisms linking molecular circadian clocks to cardiac arrhythmogenesis are not fully understood. Recent experiments have shown that L-type calcium channels exhibit circadian rhythms in both expression and function in guinea pig ventricular cardiomyocytes. We developed an electrophysiological model of these cells to simulate the effect of circadian variation in L-type calcium conductance. In our simulations, we found that there is a circadian pattern in the occurrence of early afterdepolarizations (EADs), which are abnormal depolarizations during the repolarization phase of a cardiac action potential that can trigger fatal ventricular arrhythmias. Specifically, the model produces EADs in the morning, but not at other times of day. We show that the model exhibits a codimension-2 Takens-Bogdanov bifurcation that serves as an organizing center for different types of EAD dynamics. We also simulated a two-dimensional spatial version of this model across a circadian cycle. We found that there is a circadian pattern in the breakup of spiral waves, which represents ventricular fibrillation in cardiac tissue. Specifically, the model produces spiral wave breakup in the morning, but not in the evening. Our computational study is the first, to our knowledge, to propose a link between circadian rhythms and EAD formation and suggests that the efficacy of drugs targeting EAD-mediated arrhythmias may depend on the time of day that they are administered.

Organization and molecular mechanism of insect circadian clocks

Life could not exist without organisms’ engineered ability to keep track of time on a 24-hour day-night cycle called a circadian rhythm. Insects display an impressive variety of daily rhythms, which are most evident in their behaviour. Circadian timekeeping systems that generate these daily rhythms of physiology and behaviour all involve three interacting elements: the timekeeper itself (i.e. the clock), inputs to the clock through which it entrains and otherwise responds to environmental cues such as light and temperature, and outputs from the clock through which it imposes daily rhythms on various physiological and behavioural parameters. The clocks control various behaviours, physiological functions, and developmental events, enabling adaptation to periodic environmental changes. Circadian clocks also function in time-compensation for celestial navigation and in the measurement of day or night length for photoperiodism. Molecular and physiological mechanisms are best understood for the optic-lobe and mid-brain circadian clocks, although there is no direct evidence that these clocks are involved in rhythmic phenomena other than circadian rhythms in daily events. Circadian clocks have also been localized in peripheral tissues. Circadian clocks are most well studied in Drosophila melanogaster. Drosophila flies spend their entire lives in small areas near the ground, and use their circadian brain clock to regulate daily rhythms of rest and activity, so as to organize their behaviour appropriately to the daily rhythms of their local environment. Migratory locusts and butterflies, on the other hand, spend substantial portions of their lives high up in the air migrating long distances and use their circadian brain clocks to provide time-compensation to their sun-compass navigational systems.

A Symphony of Signals: Intercellular and Intracellular Signaling Mechanisms Underlying Circadian Timekeeping in Mice and Flies.

The central pacemakers of circadian timekeeping systems are highly robust yet adaptable, providing the temporal coordination of rhythms in behavior and physiological processes in accordance with the demands imposed by environmental cycles. These features of the central pacemaker are achieved by a multi-oscillator network in which individual cellular oscillators are tightly coupled to the environmental day-night cycle, and to one another via intercellular coupling. In this review, we will summarize the roles of various neurotransmitters and neuropeptides in the regulation of circadian entrainment and synchrony within the mammalian and Drosophila central pacemakers. We will also describe the diverse functions of protein kinases in the relay of input signals to the core oscillator or the direct regulation of the molecular clock machinery.

Cognitive Impairments during the Transition to Working at Night and on Subsequent Night Shifts.

Demands of modern society force many work operations into the night when the internal circadian timekeeping system is promoting sleep. The combination of disturbed daytime sleep and circadian misalignment, which is common in overnight shift work, decreases cognitive performance, yet how performance may differ across multiple consecutive nights of shift work is not fully understood. Therefore, the primary aim of this study was to use a simulated night-shift protocol to examine the cognitive performance and ratings of sleepiness and clear-headedness across the hours of a typical daytime shift, a first night shift with an afternoon nap and extended wakefulness, and 2 subsequent overnight shifts. We tested the hypothesis that cognitive performance would be worse on the first night shift as compared with the baseline and subsequent nighttime shifts and that performance during nighttime shifts would be reduced as compared with the baseline daytime shift. Fifteen healthy adults (6 men) were studied in the 6-day in-laboratory protocol. Results showed that working during the night increased subjective sleepiness and decreased clear-headedness and performance on the Psychomotor Vigilance Task (i.e., slower median, fastest and slowest reaction times, and increased attentional lapses), Stroop color word task (decreased number of correct responses and slower median reaction time), and calculation addition performance task (decreased number attempted and correct). Furthermore, we observed limited evidence of sleepiness, clear-headedness, or performance adaptation across subsequent nights of simulated night work. Our findings demonstrate that night-shift work, regardless of whether it is the first night shift with a nap and extended wakefulness or subsequent night shifts, decreases performance and clear-headedness as compared with the day shift.

Impact of adult attention deficit hyperactivity disorder and medication status on sleep/wake behavior and molecular circadian rhythms.

Attention deficit hyperactivity disorder (ADHD) is a common neuropsychiatric condition that has been strongly associated with changes in sleep and circadian rhythms. Circadian rhythms are near 24-h cycles that are primarily generated by an endogenous circadian timekeeping system, encoded at the molecular level by a panel of clock genes. Stimulant and non-stimulant medication used in the management of ADHD has been shown to potentially impact on circadian processes and their behavioral outputs. In the current study, we have analyzed circadian rhythms in daily activity and sleep, and the circadian gene expression in a cohort of healthy controls (N = 22), ADHD participants not using ADHD-medication (N = 17), and participants with ADHD and current use of ADHD medication (N = 17). Rhythms of sleep/wake behavior were assessed via wrist-worn actigraphy, whilst rhythms of circadian gene expression were assessed ex-vivo in primary human-derived dermal fibroblast cultures. Behavioral data indicate that patients with ADHD using ADHD-medication have lower relative amplitudes of diurnal activity rhythms, lower sleep efficiency, more nocturnal activity but not more nocturnal wakenings than both controls and ADHD participants without medication. At the molecular level, there were alterations in the expression of PER2 and CRY1 between ADHD individuals with no medication compared to medicated ADHD patients or controls, whilst CLOCK expression was altered in patients with ADHD and current medication. Analysis of fibroblasts transfected with a BMAL1:luc reporter showed changes in the timing of the peak expression across the three groups. Taken together, these data support the contention that both ADHD and medication status impact on circadian processes.

Circuit development in the master clock network of mammals.

Daily rhythms are generated by the circadian timekeeping system, which is orchestrated by the master circadian clock in the suprachiasmatic nucleus (SCN) of mammals. Circadian timekeeping is endogenous and does not require exposure to external cues during development. Nevertheless, the circadian system is not fully formed at birth in many mammalian species and it is important to understand how SCN development can affect the function of the circadian system in adulthood. The purpose of the current review is to discuss the ontogeny of cellular and circuit function in the SCN, with a focus on work performed in model rodent species (i.e., mouse, rat, and hamster). Particular emphasis is placed on the spatial and temporal patterns of SCN development that may contribute to the function of the master clock during adulthood. Additional work aimed at decoding the mechanisms that guide circadian development is expected to provide a solid foundation upon which to better understand the sources and factors contributing to aberrant maturation of clock function.

Modulation of circadian rhythms through estrogen receptor signaling.

Circadian rhythms are physiological and behavioral processes that exhibit a 24-hr cycle. These daily rhythms are essential for living organisms to align their behavior and physiology with the environment to increase the likelihood of survival. In mammals, circadian rhythms synchronize with the environment primarily by the suprachiasmatic nucleus, a hypothalamic brain region that integrates exogenous and endogenous timing cues. Sex steroid hormones, including estrogens, are thought to modulate sexually dimorphic behaviors through developmental programming of the brain (i.e., organization), as well as acute receptor signaling during adulthood (i.e., activation). Importantly, there are known sex differences in the expression of circadian locomotor activity and molecular organization of the suprachiasmatic nucleus, likely due, in part, to the actions of circulating estrogens. Circadian locomotor rhythms, which are coordinated by the suprachiasmatic nucleus, have been shown to be regulated by developmental and adult levels of circulating estrogens. Further, increasing evidence suggests that estrogens can modulate expression of circadian clock genes that are essential for orchestration of circadian rhythms by the suprachiasmatic nucleus. In this review, we will discuss the organizational and activational modulation of the circadian timekeeping system by estrogens through estrogen receptor signaling.