Mammalian Circadian Oscillator Research Articles

Vasopressin: more than just an output of the circadian pacemaker? Focus on “Vasopressin receptor V1a regulates circadian rhythms of locomotor activity and expression of clock-controlled genes in the suprachiasmatic nuclei”

the timing of physiological events is carefully controlled so that incompatible processes do not compete and complementary functions coincide. Given the pivotal role of the hypothalamus in homeostatic regulation, the discovery that a master circadian pacemaker resides in this region was not

Circadian gene expression is resilient to large fluctuations in overall transcription rates

Mammalian circadian oscillators are considered to rely on transcription/translation feedback loops in clock gene expression. The major and essential loop involves the autorepression of cryptochrome (Cry1, Cry2) and period (Per1, Per2) genes. The rhythm-generating circuitry is functional in most cell types, including cultured fibroblasts. Using this system, we show that significant reduction in RNA polymerase II-dependent transcription did not abolish circadian oscillations, but surprisingly accelerated them. A similar period shortening was observed at reduced incubation temperatures in wild-type mouse fibroblasts, but not in cells lacking Per1. Our data suggest that mammalian circadian oscillators are resilient to large fluctuations in general transcription rates and temperature, and that PER1 has an important function in transcription and temperature compensation.

Angiotensin II regulates the activity of mouse suprachiasmatic nuclei neurons

Neuropeptide signaling plays key roles in coordinating cellular activity within the suprachiasmatic nuclei (SCN), site of the master circadian oscillator in mammals. The neuropeptide angiotensin II (ANGII) and its cognate receptor AT1, are both expressed by SCN cells, but unlike other SCN neurochemicals, very little is known about the cellular actions of ANGII within this circadian clock. We used multi-electrode, multiunit, extracellular electrophysiology, coupled with whole-cell voltage and current clamp techniques to investigate the actions of ANGII in mouse SCN slices. ANGII (0.001-10 microM) dose dependently stimulated and inhibited extracellularly recorded neuronal discharge in many SCN neurons ( approximately 60%). Both actions were blocked by pre-treatment with the AT1 receptor antagonist ZD7155 (0.03 microM), while suppressions but not activations were prevented by pre-treatment with the GABA A receptor antagonist bicuculline (20 microM). AT1 receptor blockade itself suppressed discharge in a subset ( approximately 30%) of SCN neurons, and this action was not blocked by bicuculline. In voltage-clamped SCN neurons (-70 mV), AT1 receptor activation dose-dependently enhanced the frequency of action potential-driven, GABA A receptor-mediated currents, but did not alter their responses to exogenously applied GABA. In current-clamped SCN neurons perfused with tetrodotoxin, ANGII induced a membrane depolarization with a concomitant decrease in input resistance. In conclusion we show that AT1 receptor activation by ANGII depolarizes SCN neurons and stimulates action potential firing, leading to increased GABA release in the mouse SCN. Additionally we provide the first evidence that endogenous AT1 receptor signaling tonically regulates the activities of some SCN neurons.

Mammalian Peripheral Circadian Oscillators Are Temperature Compensated

Mammalian Peripheral Circadian Oscillators Are Temperature Compensated

Systems Biology of Mammalian Circadian Clocks

Systems Biology is a natural extension of molecular biology and can be defined as biology after identification of key gene(s). Systems-biological research is hence seen as a multistage process, beginning with the comprehensive identification and quantitative analysis of individual system components and their networked interactions and leading to the ability to control existing systems toward the desired state and design new ones based on an understanding of structure and underlying dynamical principles. In this chapter, we take mammalian circadian clocks as a model system and describe systems-biological approaches, including the identification of clock-controlled genes, clock-controlled cis elements, and clock transcriptional circuits driven by functional genomics; the parameter change of clock components followed by quantitative measurement; and the dynamic and quantitative perturbation of the clock and its application to one of the fundamental but yet-unsolved questions: singularity behavior of clocks. As perspective for systems-biological investigations, we also introduce the system-level dynamical questions related to the core of clocks, including delay, nonlinearity, temperature-compensation and synchronization of mammalian circadian oscillator(s), and the system-level information problems related to clocks in the environment, including the internal representation of light change through perfect adaptation and internal representation of day length through photoperiodism in mammals.

β-TrCP1-Mediated Degradation of PERIOD2 Is Essential for Circadian Dynamics

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (beta-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous beta-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of beta-TrCP interaction-deficient PER2 variants--but not wild-type PER2--results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated beta-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally.

Optimal Internal Noise for Mammalian Circadian Oscillator

Based on a deterministic mammalian circadian oscillator proposed recently, we have constructed the corresponding mesoscopic stochastic model, and studied the effect of internal noise on the genetic oscillations of such a system. It is found that the stochastic genetic oscillations can show best performance at an optimal internal noise level via a mechanism of internal noise stochastic resonance. Furthermore, it is found that there exists a moderate system size that makes the stochastic model show effective oscillation at more extended region than the deterministic description, which indicates enhanced robustness as the result of internal noise. The potential biological application of such an effect is also discussed.

Multiple Photopigments Entrain the Mammalian Circadian Oscillator

Circadian rhythms are entrained to the natural day:night cycle. Melanopsin expressed in retinal ganglion cells partially accounts for circadian photoentrainment. Dkhissi-Benyahya et al. demonstrate that medium wavelength opsin (MW-opsin) also plays an important role in the process. Furthermore, they develop a model explaining wavelength-dependent photoentrainment by melanopsin and MW-opsin.

Systems Biology of Mammalian Circadian Clocks

Systems Biology is a natural extension of molecular biology and can be defined as biology after identification of key gene(s). Systems-biological research is hence seen as a multistage process, beginning with the comprehensive identification and quantitative analysis of individual system components and their networked interactions and leading to the ability to control existing systems toward the desired state and design new ones based on an understanding of structure and underlying dynamical principles. In this chapter, we take mammalian circadian clocks as a model system and describe systems-biological approaches, including the identification of clock-controlled genes, clock-controlled cis elements, and clock transcriptional circuits driven by functional genomics; the parameter change of clock components followed by quantitative measurement; and the dynamic and quantitative perturbation of the clock and its application to one of the fundamental but yet-unsolved questions: singularity behavior of clocks. As perspective for systems-biological investigations, we also introduce the system-level dynamical questions related to the core of clocks, including delay, nonlinearity, temperature-compensation and synchronization of mammalian circadian oscillator(s), and the system-level information problems related to clocks in the environment, including the internal representation of light change through perfect adaptation and internal representation of day length through photoperiodism in mammals.

Differential effects of PER2 phosphorylation: molecular basis for the human familial advanced sleep phase syndrome (FASPS)

PERIOD (PER) proteins are central components within the mammalian circadian oscillator, and are believed to form a negative feedback complex that inhibits their own transcription at a particular circadian phase. Phosphorylation of PER proteins regulates their stability as well as their subcellular localization. In a systematic screen, we have identified 21 phosphorylated residues of mPER2 including Ser 659, which is mutated in patients suffering from familial advanced sleep phase syndrome (FASPS). When expressing FASPS-mutated mPER2 in oscillating fibroblasts, we can phenocopy the short period and advanced phase of FASPS patients' behavior. We show that phosphorylation at Ser 659 results in nuclear retention and stabilization of mPER2, whereas phosphorylation at other sites leads to mPER2 degradation. To conceptualize our findings, we use mathematical modeling and predict that differential PER phosphorylation events can result in opposite period phenotypes. Indeed, interference with specific aspects of mPER2 phosphorylation leads to either short or long periods in oscillating fibroblasts. This concept explains not only the FASPS phenotype, but also the effect of the tau mutation in hamster as well as the doubletime mutants (dbtS and dbtL ) in Drosophila.

Keeping Time

Circadian rhythms are thought to be generated by interlocked feedback loops of gene expression. Period (PER) proteins are essential components of the transcriptional feedback cycle and for phase-shifting the clock to different times of day, but the mechanism by which they repress transcription is not clear. Brown et al. have identified two PER-associated proteins, NONO and WDR5, that differentially modulate PER activity. NONO, an RNA-binding protein whose activity is essential for normal clock function, antagonizes PER-mediated repression. In contrast, WDR5, a histone methyltransferase adaptor, enhances PER-mediated repression. These regulatory proteins may impart resilience to the circadian oscillator in the face of fluctuations of clock components due to stochastic gene expression and environmental changes. S. A. Brown, J. Ripperger, S. Kadener, F. Fleury-Olela, F. Vilbois, M. Rosbash, U. Schibler, PERIOD1-associated proteins modulate the negative limb of the mammalian circadian oscillator. Science 308 , 693-696 (2005). [Abstract] [Full Text]

PERIOD1-Associated Proteins Modulate the Negative Limb of the Mammalian Circadian Oscillator

The clock proteins PERIOD1 (PER1) and PERIOD2 (PER2) play essential roles in a negative transcriptional feedback loop that generates circadian rhythms in mammalian cells. We identified two PER1-associated factors, NONO and WDR5, that modulate PER activity. The reduction of NONO expression by RNA interference (RNAi) attenuated circadian rhythms in mammalian cells, and fruit flies carrying a hypomorphic allele were nearly arrhythmic. WDR5, a subunit of histone methyltransferase complexes, augmented PER-mediated transcriptional repression, and its reduction by RNAi diminished circadian histone methylations at the promoter of a clock gene.

The tau Mutation in the Syrian Hamster Differentially Reprograms the Circadian Clock in the SCN and Peripheral Tissues

The hypothalamic suprachiasmatic nuclei (SCN), the principal circadian oscillator in mammals, are synchronized to the solar day by the light-dark cycle, and in turn, they coordinate circadian oscillations in peripheral tissues. The tau mutation in the Syrian hamster is caused by a point mutation leading to a deficiency in the ability of Casein Kinase 1epsilon to phosphorylate its targets, including circadian PER proteins. How this accelerates circadian period in neural tissues is not known, nor is its impact on peripheral circadian oscillators established. We show that this mutation has no effect on per mRNA expression nor the nuclear accumulation of PER proteins in the SCN. It does, however, accelerate the clearance of PER proteins from the nucleus to an extent sufficient to explain the shortened circadian period of behavioral rhythms. The mutation also has novel, unanticipated consequences for circadian timing in the periphery, including tissue-specific phase advances and/or reduced amplitude of circadian gene expression. The results suggest that the tau mutation accelerates a specific phase, during mid-late subjective night of the SCN circadian feedback loop, rather than cause a global compression of the entire cycle. This reprogrammed output from the clock is associated with peripheral desynchrony, which in turn could account for impaired growth and metabolic efficiency of the mutant.

CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor α (PPARα) in mice

PPARalpha (peroxisome-proliferator-activated receptor alpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. In the present study, we show that circadian expression of mouse PPARalpha mRNA requires the basic helix-loop-helix PAS (Per-Arnt-Sim) protein CLOCK, a core component of the negative-feedback loop that drives circadian oscillators in mammals. The circadian expression of PPARalpha mRNA was abolished in the liver of homozygous Clock mutant mice. Using wild-type and Clock-deficient fibroblasts derived from homozygous Clock mutant mice, we showed that the circadian expression of PPARalpha mRNA is regulated by the peripheral oscillators in a CLOCK-dependent manner. Transient transfection and EMSAs (electrophoretic mobility-shift assays) revealed that the CLOCK-BMAL1 (brain and muscle Arnt-like protein 1) heterodimer transactivates the PPARalpha gene via an E-box-rich region located in the second intron. This region contained two perfect E-boxes and four E-box-like motifs within 90 bases. ChIP (chromatin immunoprecipitation) also showed that CLOCK associates with this E-box-rich region in vivo. Circadian expression of PPARalpha mRNA was intact in the liver of insulin-dependent diabetic and of adrenalectomized mice, suggesting that endogenous insulin and glucocorticoids are not essential for the rhythmic expression of the PPARalpha gene. These results suggested that CLOCK plays an important role in lipid homoeostasis by regulating the transcription of a key protein, PPARalpha.

Entrainment in a Model of the Mammalian Circadian Oscillator

To adapt the timing of processes regulated by the circadian clock to seasonally varying photoperiods, the phase relation between the circadian clock and dusk or dawn ("phase of entrainment") must be tightly adjusted. The authors use a mathematical model of the molecular mammalian circadian oscillator to investigate the influence of the free-running period (tau) and the shape of the PRC on the phase of entrainment. They find that a phase-dependent sensitivity ("gating") of light-induced period gene transcription enables a constant phase relation to dusk or dawn under different photoperiods. Depending on the freerunning period tau and on the shaping of the PRC by gating, the model circadian oscillator tracks either light onset or light offset under different photoperiods. The study indicates that the phase of entrainment of oscillating cells can be systematically controlled by regulating both gating and the free-running period tau.

Identification of neuromedin S and its possible role in the mammalian circadian oscillator system

The discovery of neuropeptides has resulted in an increased understanding of novel regulatory mechanisms of certain physiological phenomena. Here we identify a novel neuropeptide of 36 amino-acid residues in rat brain as an endogenous ligand for the orphan G protein-coupled receptor FM-4/TGR-1, which was identified to date as the neuromedin U (NMU) receptor, and designate this peptide 'neuromedin S (NMS)' because it is specifically expressed in the suprachiasmatic nuclei (SCN) of the hypothalamus. NMS shares a C-terminal core structure with NMU. The NMS precursor contains another novel peptide. NMS mRNA is highly expressed in the central nervous system, spleen and testis. In rat brain, NMS expression is restricted to the core of the SCN and has a diurnal peak under light/dark cycling, but remains stable under constant darkness. Intracerebroventricular administration of NMS in rats activates SCN neurons and induces nonphotic type phase shifts in the circadian rhythm of locomotor activity. These findings suggest that NMS in the SCN is implicated in the regulation of circadian rhythms through autocrine and/or paracrine actions.

Modeling Feedback Loops of the Mammalian Circadian Oscillator

The suprachiasmatic nucleus governs daily variations of physiology and behavior in mammals. Within single neurons, interlocked transcriptional/translational feedback loops generate circadian rhythms on the molecular level. We present a mathematical model that reflects the essential features of the mammalian circadian oscillator to characterize the differential roles of negative and positive feedback loops. The oscillations that are obtained have a 24-h period and are robust toward parameter variations even when the positive feedback is replaced by a constantly expressed activator. This demonstrates the crucial role of the negative feedback for rhythm generation. Moreover, it explains the rhythmic phenotype of Rev-erbα −/− mutant mice, where a positive feedback is missing. The interplay of negative and positive feedback reveals a complex dynamics. In particular, the model explains the unexpected rescue of circadian oscillations in Per2 Brdm1 / Cry2 −/− double-mutant mice ( Per2 Brdm1 single-mutant mice are arrhythmic). Here, a decrease of positive feedback strength associated with mutating the Per2 gene is compensated by the Cry2 −/− mutation that simultaneously decreases the negative feedback strength. Finally, this model leads us to a testable prediction of a molecular and behavioral phenotype: circadian oscillations should be rescued when arrhythmic Per2 Brdm1 mutant mice are crossed with Rev- erbα −/− mutant mice.

The gut–brain peptide neuromedin U is involved in the mammalian circadian oscillator system

Immunohistochemical analysis revealed the presence of a gut–brain peptide, neuromedin U (NMU), in the suprachiasmatic nucleus (SCN), which is the site of the master circadian oscillator. The expression of NMU mRNA exhibited a circadian rhythm, with the peak expression in the SCN occurring at CT4–8 h. The two NMU-binding receptors (NMU-R1 and NMU-R2) were also expressed in the SCN, but their phase angles were different. Intracerebroventricular injection (ICV) of NMU induced the expression of Fos protein in the SCN cells and caused a phase-dependent phase shift of the circadian locomotor activity rhythm. The magnitude of the phase shift was dose dependent. This NMU-induced phase shift was of the nonphotic type. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed increases in the expression in the SCN of immediate early genes, such as c-fos, NGFI-A, NGFI-B, and JunB. Furthermore, ICV injection of NMU increased the expression of Per1, but not Per2, in the SCN. These results indicate that NMU may play some important role in the circadian oscillator by exerting an autocrine or paracrine action in the SCN.

Role of VIP-neurons in the hypothalamic suprachiasmatic nucleus in the control of blood glucose

The hypothalamic suprachiasmatic nucleus (SCN), a master circadian oscillator in mammals, contains VIP-neurons. In our study on the mechanism of the central regulation of glucose metabolism in rats, we obtained following results: 1) intracranial injection of either 2-deoxy-D-glucose (2DG) or VIP elicited hyperglycemia by enhancing neural activities of the sympathetic nerves and by the suppression of the insulin secretion and enhances of secretions of adrenaline and glucagon; 2) bilateral lesions of the SCN eliminated the hyperglycemia and sympathetic excitation induced by intracranial injection of 2DG, and intracranial administration of VIP restored the 2DG-hyperglycemia; 3) infusion of VIP-antisense oligo in the SCN reduced the VIP content in the SCN and abolished the 2DG-hyperglycemia, and intracranial injection of VIP restored the 2DG-hyperglycemia in rats infused the VIP-antisense oligo; 4) intrapancreatic injection of pseudorabies virus (PRV, Bartha), which is retrogradedly transported, caused the transfer of PRV to VIP-neurons in the SCN, and denervations of both the sympathetic and parasympathetic nerves innervating the pancreas eliminated the retrograde transport of PRV to VIP-neurons in the SCN. These findings suggest that VIP-neurons in the SCN regulate the blood glucose level through the enhancement of the sympathetic activity.

A model of the mammalian circadian oscillator including the REV-ERBalpha module.

Many cellular and physiological processes have been shown to display a rhythm of about 24 hours. This so-called circadian rhythm is based on a system of interlocked negative and positive molecular feedback loops. Here we extend a previous model of the circadian oscillator by including REV-ERBalpha as an additional component. This new model will allow us to investigate the function of an additional negative feedback loop via REV-ERBalpha. We obtain circadian oscillations with the correct period and phase relations between clock components. Parameter variations that correspond to clock-gene mutations reproduce experimental results: With parameter variations mimicking the Bmal1(-/-) and the Per2(Brdm1) mutation the oscillations cease to exist. In contrast, the system shows sustained oscillations if we use a parameter set that reflects the Rev-erbalpha mutation. The model also accounts for the differential effect of the Cry1(-/-) and Cry2(-/-) mutations on the circadian period. The simulations of the extended model indicate that the original model is robust with respect to the incorporation of the additional component. Depending on the kinetics of the Per2/Cry transcriptional activation by BMAL1, an increasing BMAL1 expression leads to either an increase or decrease of the clock period. This indicates that overexpression experiments could help to characterize the impact of BMAL1 on Per2/Cry transcription.