Master Circadian Clock Research Articles

Targeting circadian transcriptional programs through a cis-regulatory mechanism in triple negative breast cancer.

Circadian clock genes are emerging targets in many types of cancer, but their mechanistic contributions to tumor progression are still largely unknown. This makes it challenging to stratify patient populations and develop corresponding treatments. In this work, we show that in breast cancer, the disrupted expression of circadian genes has the potential to serve as biomarkers. We also show that the master circadian transcription factors (TFs) BMAL1 and CLOCK are required for the proliferation of metastatic mesenchymal stem-like (mMSL) triple-negative breast cancer (TNBC) cells. Using currently available small molecule modulators, we found that a stabilizer of cryptochrome 2 (CRY2), the direct repressor of BMAL1 and CLOCK transcriptional activity, synergizes with inhibitors of proteasome, which is required for BMAL1 and CLOCK function, to repress a transcriptional program comprising circadian cycling genes in mMSL TNBC cells. Omics analyses on drug-treated cells implied that this repression of transcription is mediated by the transcription factor binding sites (TFBSs) features in the cis-regulatory elements (CRE) of clock-controlled genes. Through a massive parallel reporter assay, we defined a set of CRE features that are potentially repressed by the specific drug combination. The identification of cis -element enrichment might serve as a new concept of defining and targeting tumor types through the modulation of cis -regulatory programs, and ultimately provide a new paradigm of therapy design for cancer types with unclear drivers like TNBC.

Time-restricted feeding reveals a role for neural respiratory clocks in optimizing daily ventilatory-metabolic coupling in mice.

The master circadian clock, located in the suprachiasmatic nuclei (SCN), organizes the daily rhythm in minute ventilation (V̇e). However, the extent that the daily rhythm in V̇e is secondary to SCN-imposed O2 and CO2 cycles (i.e., metabolic rate) or driven by other clock mechanisms remains unknown. Here, we experimentally shifted metabolic rate using time-restricted feeding (without affecting light-induced synchronization of the SCN) to determine the influence of metabolic rate in orchestrating the daily V̇e rhythm. Mice eating predominantly at night exhibited robust daily rhythms in O2 consumption (V̇o2), CO2 production (V̇co2), and V̇e with similar peak times (approximately ZT18) that were consistent with SCN organization. However, feeding mice exclusively during the day separated the relative timing of metabolic and ventilatory rhythms, resulting in an approximately 8.5-h advance in V̇co2 and a disruption of the V̇e rhythm, suggesting opposing circadian and metabolic influences on V̇e. To determine if the molecular clock of cells involved in the neural control of breathing contributes to the daily V̇e rhythm, we examined V̇e in mice lacking BMAL1 in Phox2b-expressing respiratory cells (i.e., BKOP mice). The ventilatory and metabolic rhythms of predominantly night-fed BKOP mice did not differ from wild-type mice. However, in contrast to wild-type mice, exclusive day feeding of BKOP mice led to an unfettered daily V̇e rhythm with a peak time aligning closely with the daily V̇co2 rhythm. Taken together, these results indicate that both daily V̇co2 changes and intrinsic circadian time-keeping within Phox2b respiratory cells are predominant orchestrators of the daily rhythm in ventilation.NEW & NOTEWORTHY The master circadian clock organizes the daily rhythm in ventilation; however, the extent that this rhythm is driven by SCN regulation of metabolic rate versus other clock mechanisms remains unknown. We report that metabolic rate alone is insufficient to explain the daily oscillation in ventilation and that neural respiratory clocks within Phox2b-expressing cells additionally optimize breathing. Collectively, these findings advance our mechanistic understanding of the circadian rhythm in ventilatory control.

Abstract 829: Phthalate exposure results in circadian rhythm-induced BBB dysfunction increasing the risk for breast cancer brain metastasis

Abstract Phthalates, classified as plasticizers, represent a group of chemicals pervasive in the general U.S. population, with notably higher levels in non-Hispanic Blacks compared to non-Hispanic Whites. These persistent organic pollutants (POPs) have been identified for their impact on the circadian rhythms of the brain and vascular system. Several studies indicate that phthalates can elevate neuroinflammation and reduce levels of the junctional protein ZO-1 in the hypothalamus, home to the master circadian clock known as the suprachiasmatic nucleus (SCN). The SCN regulates cellular and tissue circadian rhythms, including tight junction proteins essential for the blood-brain barrier's (BBB) main barrier function. Interestingly, experimental studies reveal that prolonged chronic circadian rhythm disruption (CRD) in mice leads to increased breast cancer metastasis. However, the effects of phthalates on CRD concerning brain cancer and metastasis remain poorly understood. Given their significant prevalence and biological impact, we hypothesize that phthalate exposure induces CRD through hypothalamic dysregulation, resulting in BBB dysfunction and an elevated risk of brain metastasis. Results. We developed an in vitro BBB model using endothelial cells and exposed them to an environmentally relevant mixture of five phthalates simulating chronic low-grade exposure. Analysis showed dysregulation of critical clock genes (Bmal1 and Clock), along with a substantial decrease in BBB tight junction proteins (claudin-5 and occludin) at the protein and mRNA levels. The tumor trans-endothelial migration assay using breast adenocarcinoma cells along with endothelial cells demonstrated an increased metastatic potential following phthalate exposure. Conclusion. These findings offer crucial insights into the molecular mechanisms underlying phthalate exposure, suggesting potential targets for future brain metastasis treatments. Additionally, they hold implications for the future regulation and management of phthalate usage and policy. This work was supported by the University of Miami U-LINK Resilience Challenge grant. Citation Format: Destiny Tiburcio, Sarah Adolphe, Ijeoma B. Akpu, Timea Teglas, Oandy Naranjo, Sophia H. George, Michal J. Toborek. Phthalate exposure results in circadian rhythm-induced BBB dysfunction increasing the risk for breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 829.

Deficiency of circadian clock gene Bmal1 exacerbates noncanonical inflammasome-mediated pyroptosis and lethality via Rev-erbα-C/EBPβ-SAA1 axis

Circadian arrhythmia has been linked to increased susceptibility to multiple inflammatory diseases, such as sepsis. However, it remains unclear how disruption of the circadian clock modulates molecular aspects of innate immune responses, including inflammasome signaling. Here, we examined the potential role of the circadian clock in inflammasome-mediated responses through myeloid-specific deletion of BMAL1, a master circadian clock regulator. Intriguingly, Bmal1 deficiency significantly enhanced pyroptosis of macrophages and lethality of mice under noncanonical inflammasome-activating conditions but did not alter canonical inflammasome responses. Transcriptome analysis of enriched peritoneal myeloid cells revealed that Bmal1 deficiency led to a marked reduction in Rev-erbα expression at steady state and a significant increase in serum amyloid A1 (SAA1) expression upon poly(I:C) stimulation. Notably, we found that the circadian regulator Rev-erbα is critical for poly(I:C)- or interferon (IFN)-β-induced SAA1 production, resulting in the circadian oscillation pattern of SAA1 expression in myeloid cells. Furthermore, exogenously applied SAA1 markedly increased noncanonical inflammasome-mediated pyroptosis of macrophages and lethality of mice. Intriguingly, our results revealed that type 1 IFN receptor signaling is needed for poly(I:C)- or IFN-β-induced SAA1 production. Downstream of the type 1 IFN receptor, Rev-erbα inhibited the IFN-β-induced association of C/EBPβ with the promoter region of Saa1, leading to the reduced transcription of Saa1 in macrophages. Bmal1-deficient macrophages exhibited enhanced binding of C/EBPβ to Saa1. Consistently, the blockade of Rev-erbα by SR8278 significantly increased poly(I:C)-stimulated SAA1 transcription and noncanonical inflammasome-mediated lethality in mice. Collectively, our data demonstrate a potent suppressive effect of the circadian clock BMAL1 on the noncanonical inflammasome response via the Rev-erbα-C/EBPβ-SAA1 axis.

In-phasic cytosolic-nuclear Ca2+ rhythms in suprachiasmatic nucleus neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus is the master circadian clock in mammals. SCN neurons exhibit circadian Ca2+ rhythms in the cytosol, which is thought to act as a messenger linking the transcriptional/translational feedback loop (TTFL) and physiological activities. Transcriptional regulation occurs in the nucleus in the TTFL model, and Ca2+-dependent kinase regulates the clock gene transcription. However, the Ca2+ regulatory mechanisms between cytosol and nucleus as well as the ionic origin of Ca2+ rhythms remain unclear. In the present study, we monitored circadian-timescale Ca2+ dynamics in the nucleus and cytosol of SCN neurons at the single-cell and network levels. We observed robust nuclear Ca2+ rhythm in the same phase as the cytosolic rhythm in single SCN neurons and entire regions. Neuronal firing inhibition reduced the amplitude of both nuclear and cytosolic Ca2+ rhythms, whereas blocking of Ca2+ release from the endoplasmic reticulum (ER) via ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors had a minor effect on either Ca2+ rhythms. We conclude that the in-phasic circadian Ca2+ rhythms in the cytosol and nucleus are mainly driven by Ca2+ influx from the extracellular space, likely through the nuclear pore. It also raises the possibility that nuclear Ca2+ rhythms directly regulate transcription in situ.

Light Exposure as a Nonpharmacological Intervention for Alzheimer’s Disease

AbstractBackgroundIn recent years, significant advances have been made in our understanding of the non‐visible biological impact of light (Vetter et al, 2021). Many of these advances can be traced to the 2002 discovery of a previously unknown retinal photoreceptor known as the intrinsically photosensitive retinal ganglion cell, or ipRGC, which is now known to be connected directly to our master circadian clock in our superchiasmatic nucleus (SCN). Now a growing body of clinical and applied research has shown how light spectrum, intensity, and timing can be used to promote circadian entrainment, leading to significant improvements in human health outcomes, including for people with Alzheimer’s disease and related dementia (ADRD).MethodMany of these clinical studies have used lighting interventions in elder care facilities to evaluate the impact of lighting as a nonpharmacological intervention to improve mood, anxiety, sleep quality, falls and other health metrics for ADRD populations. These studies typically gather light exposure and health metric information during control periods or with control populations using traditional lighting approaches and compare results to periods or populations under “circadian‐enhanced” lighting conditions, which generally means brighter and bluer lighting during the day and dimmer and redder light in the evening and night hours.ResultThe results from these studies have consistently demonstrated significant improvements in circadian entrainment, sleep quality, mood, anxiety, falls and other health metrics for ADRD populations under circadian‐enhanced lighting conditions.ConclusionThis talk will provide an overview of our current understanding of how light exposure impacts human physiology. We will discuss several completed and ongoing clinical studies conducted with ADRD populations, including several studies on which the speaker has been the Principal Investigator. We will cover what questions remain in this area and what studies can be developed in the future to address them. Finally, we will close with a discussion of tools, products, building designs and daily routines that can be applied today by clinicians and caregivers that are most likely to improve the health and wellbeing of ADRD patients based on what is known today about the impact of light exposure.

Role of circadian clock system in the mitochondrial trans-sulfuration pathway and tissue remodeling.

Previous studies from our laboratory revealed that the gaseous molecule hydrogen sulfide (H2S), a metabolic product of epigenetics, involves trans-sulfuration pathway for ensuring metabolism and clearance of homocysteine (Hcy) from body, thereby mitigating the skeletal muscle's pathological remodeling. Although the master circadian clock regulator that is known as brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (i.e., BMAL 1) is associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock remains unexplored. We hypothesize that alterations in the functioning of circadian clock during sleep and wake cycle affect skeletal muscle's biology. To test this hypothesis, we measured serum matrix metalloproteinase (MMP) activities using gelatin gels for analyzing the MMP-2 and MMP-9. Further, employing casein gels, we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type and cystathionine β synthase-deficient (CBS-/+) mice strains were treated with H2S and subjected to measurement of trans-sulfuration factors from skeletal muscle tissues. The results suggested highly robust activation of MMPs in the wake mice versus sleep mice, which appears somewhat akin to the "1-carbon metabolic dysregulation", which takes place during remodeling of extracellular matrix during muscular dystrophy. Interestingly, the levels of trans-sulfuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in CBS-/+ mice. However, treatment with H2S mitigated the attenuation of the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock in trans-sulfuration pathway that affects skeletal muscle remodeling and mitochondrial regeneration.

Effect of circadian rhythm on NAD and other metabolites in human brain.

Nicotinamide Adenine Dinucleotide (NAD) plays a central role in the master circadian clock of the brain (the suprachiasmatic nuclei, SCN) as demonstrated in many model organisms. NAD acts as an enzyme co-factor and substrate and its modulation was found to be tightly regulated to the periodicity of the cycles. However, in human brain, the effect of the circadian rhythm (CR) on the metabolism of the SCN and other brain regions is poorly understood. We conducted a magnetic resonance spectroscopy (MRS) study at a high magnetic field, measuring the occipital brain NAD levels and other metabolites in two different morning and afternoon diurnal states in 25 healthy participants. Salivary cortisol levels were determined to confirm that the experiment was done in two chronologically different physiological conditions, and a behavioral test of risk-taking propensity was administered. Overall, we found that the CR did not significantly affect NAD levels in the occipital brain region. The other brain metabolites measured, including lactate, were not significantly affected by the CR either, except for taurine. The CR did impact risk-taking behavior and salivary cortisol level, confirming that the participants were in two circadian different behavioral and physiological states in the morning and in the afternoon. Measurement of the CR effect on NAD and taurine levels in other brain regions might provide stronger effects.

Can the Clocks Tick Together Despite the Noise? Stochastic Simulations and Analysis

The suprachiasmatic nucleus (SCN), also known as the circadian master clock, consists of a large population of oscillator neurons. Together, these neurons produce a coherent signal that drives the body’s circadian rhythms. What properties of the cell-to-cell communication allow the synchronization of these neurons, despite a wide range of environmental challenges such as fluctuations in photoperiods? To answer that question, we present a mean-field description of globally coupled neurons modeled as Goodwin oscillators with standard Gaussian noise. Provided that the initial conditions of all neurons are independent and identically distributed, any finite number of neurons becomes independent and has the same probability distribution in the mean-field limit, a phenomenon called propagation of chaos. This probability distribution is a solution to a Vlasov–Fokker–Planck type equation, which can be obtained from the stochastic particle model. We study, using the macroscopic description, how the interaction between external noise and intercellular coupling affects the dynamics of the collective rhythm, and we provide a numerical description of the bifurcations resulting from the noise-induced transitions. Our numerical simulations show a noise-induced rhythm generation at low noise intensities, while the SCN clock is arrhythmic in the high noise setting. Notably, coupling induces resonance-like behavior at low noise intensities, and varying coupling strength can cause period locking and variance dissipation even in the presence of noise.

Long-term circadian disruption shortens life span and dampens blood pressure diurnal rhythms in stroke-prone spontaneously hypertensive rats.

Environmental cues such as light and timing of food intake influence molecular clocks that produce circadian rhythmicity of many biological functions. The master circadian clock is entrained by light input and synchronizes with peripheral clocks in every organ of the body. Careers that require rotating shift work schedules predispose workers to a constant desynchronization of these biological clocks and are associated with increased risk of cardiovascular disease. We used a stroke-prone spontaneously hypertensive rat model exposed to a known biological desynchronizer, chronic environmental circadian disruption (ECD), to test the hypothesis that it would accelerate the time to stroke onset. We then investigated whether time-restricted feeding could delay stroke onset and evaluated its usefulness as a countermeasure when combined with the constant disruption of the light cycle. We found that phase advancing of the light schedule accelerated stroke onset. Restricting food access time to 5 h/day regardless of lighting profoundly delayed stroke onset in both standard 12-h:12-h light/dark or ECD-lighting conditions compared with ad libitum feeding; however, acceleration by ECD versus control lighting conditions was still observed. Since hypertension is a precursor to stroke in this model, we assessed blood pressure in a small cohort longitudinally using telemetry. Mean daily systolic and diastolic blood pressure increased in a similar manner across rats in control and ECD conditions, thus hypertension was not grossly accelerated to cause earlier strokes. However, we observed intermittent dampening of rhythms after each shift of the light cycle reminiscent of a relapsing-remitting nondipping state. Our results suggest that constant disruption of environmental rhythms may be associated with an increased risk of cardiovascular complications in the presence of cardiovascular risk factors.NEW & NOTEWORTHY This stroke-prone spontaneously hypertensive rat model significantly delayed stroke onset with the timed food restriction intervention. Blood pressure recordings in this same model were continuous through the 3 mo and showed dampened systolic rhythms after each shift in the lighting schedule.

Astrocytic control of extracellular GABA drives circadian timekeeping in the suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) is the master mammalian circadian clock. Its cell-autonomous timing mechanism, a transcriptional/translational feedback loop (TTFL), drives daily peaks of neuronal electrical activity, which in turn control circadian behavior. Intercellular signals, mediated by neuropeptides, synchronize and amplify TTFL and electrical rhythms across the circuit. SCN neurons are GABAergic, but the role of GABA in circuit-level timekeeping is unclear. How can a GABAergic circuit sustain circadian cycles of electrical activity, when such increased neuronal firing should become inhibitory to the network? To explore this paradox, we show that SCN slices expressing the GABA sensor iGABASnFR demonstrate a circadian oscillation of extracellular GABA ([GABA]e) that, counterintuitively, runs in antiphase to neuronal activity, with a prolonged peak in circadian night and a pronounced trough in circadian day. Resolving this unexpected relationship, we found that [GABA]e is regulated by GABA transporters (GATs), with uptake peaking during circadian day, hence the daytime trough and nighttime peak. This uptake is mediated by the astrocytically expressed transporter GAT3 (Slc6a11), expression of which is circadian-regulated, being elevated in daytime. Clearance of [GABA]e in circadian day facilitates neuronal firing and is necessary for circadian release of the neuropeptide vasoactive intestinal peptide, a critical regulator of TTFL and circuit-level rhythmicity. Finally, we show that genetic complementation of the astrocytic TTFL alone, in otherwise clockless SCN, is sufficient to drive [GABA]e rhythms and control network timekeeping. Thus, astrocytic clocks maintain the SCN circadian clockwork by temporally controlling GABAergic inhibition of SCN neurons.

Sex-specific differences in the circadian pattern of action potential firing by rat suprachiasmatic nucleus vasopressin neurons.

The suprachiasmatic nucleus (SCN) of the hypothalamus serves as the master circadian clock in mammals. Most SCN neurons express the inhibitory neurotransmitter GABA (gamma amino butyric acid) along with a peptide cotransmitter. Notably, the neuropeptides vasopressin (VP) and vasoactive intestinal peptide (VIP) define two prominent clusters within the SCN: those located in the ventral core (VIP) and those forming the dorsomedial "shell" of the nucleus (VP). Axons emerging from VP neurons in the shell are thought to mediate much of the SCN's output to other brain regions as well as VP release into the cerebrospinal fluid (CSF). Previous work has shown that VP release by SCN neurons is activity dependent and SCN VP neurons fire action potentials at a higher rate during the light phase. Accordingly, CSF VP levels are higher during daytime. Interestingly, the amplitude of the CSF VP rhythm is greater in males than females, suggesting the existence of sex differences in the electrical activity of SCN VP neurons. Here we investigated this hypothesis by performing cell-attached recordings from 1070 SCN VP neurons across the entire circadian cycle in both sexes of transgenic rats that express green fluorescent protein (GFP) driven by the VP gene promoter. Using an immunocytochemical approach we confirmed that >60% of SCN VP neurons display visible GFP. Recordings in acute coronal slices revealed that VP neurons display a striking circadian pattern of action potential firing, but the characteristics of this activity cycle differ in males and females. Specifically, neurons in males reached a significantly higher peak firing frequency during subjective daytime compared to females and the acrophase occurred ~1 h earlier in females. Peak firing rates in females were not significantly different at various phases of the estrous cycle.

The circadian clock in the piriform cortex intrinsically tunes daily changes of odor-evoked neural activity

The daily activity in the brain is typically fine-tuned by the circadian clock in the local neurons as well as by the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. In the olfactory response, odor-evoked activity in the piriform cortex (PC) and olfactory behavior retain circadian rhythmicity in the absence of the SCN, yet how the circadian rhythm in the PC is achieved independently of the SCN remains elusive. Here, to define neurons regulating the circadian rhythm of the odor-evoked activity in the PC, we knocked out the clock gene Bmal1 in a host of specific neurons along the olfactory circuit. We discovered that Bmal1 knockout in the PC largely abolishes the circadian rhythm of the odor-evoked activity. We further showed that isolated PC exhibits sustained circadian rhythms of the clock gene Per2 expression. Quantitative PCR analysis revealed that expression patterns of multiple genes involved in neural activity and synaptic transmission exhibit circadian rhythm in the PC in a BMAL1-dependent manner. Our findings indicate that BMAL1 acts intrinsically in the PC to control the circadian rhythm of the odor-evoked activity in the PC, possibly through regulating expression patterns of multiple genes involved in neural activity and transmission.

Bright Morning Lighting Enhancing Parasympathetic Activity at Night: A Pilot Study on Elderly Female Patients with Dementia without a Pacemaker.

Exposure to bright morning light (BML) entrains the master circadian clock, modulates physiological circadian rhythms, and reduces sleep-wake disturbances. However, its impact on the autonomic nervous system at night remains unclear. Here, we investigated the effects of BML exposure on parasympathetic nervous system (PSNS) and sympathetic nervous system (SNS) activity at night in elderly women. This nonrandomized controlled pilot study included female participants aged ≥ 60 years who were diagnosed with a type of dementia or cognitive disorder, excluding individuals with pacemakers. The treatment group was exposed to 2500 lx of BML, whereas the control group was exposed to 200 lx of general lighting. We measured heart rate variability to quantify ANS activity. The treatment group displayed significant increases in high-frequency (HF) power (Roy's largest root = 1.62; p < 0.001) and nonsignificant decreases in normalized low-frequency (LF%) power. The corresponding nonsignificant decreases in the low-frequency/high-frequency (LF/HF) ratio and cognitive function were correlated with PSNS activity (Roy's largest root = 1.41; p < 0.001), which improved severe dementia. BML exposure reduced SNS activity and enhanced PSNS activity at night in female participants, which improved cognitive function. Thus, BML therapy may be a useful clinical tool for alleviating cognitive decline.

Top-down and bottom-up alterations of connectivity patterns of the suprachiasmatic nucleus in chronic insomnia disorder.

The importance of the suprachiasmatic nucleus (SCN, also called the master circadian clock) in regulating sleep and wakefulness has been confirmed by multiple animal research. However, human studies of SCN in vivo are still nascent. Recently, the development of resting-state functional magnetic resonance imaging (fMRI) has made it possible to study SCN-related connectivity changes in patients with chronic insomnia disorder (CID). Hence, this study aimed to explore whether sleep-wake circuitry (i.e., communication between the SCN and other brain regions) is disrupted in human insomnia. Forty-two patients with CID and 37 healthy controls (HCs) underwent fMRI scanning. Resting-state functional connectivity (rsFC) and Granger causality analysis (GCA) were performed to find abnormal functional and causal connectivity of the SCN in CID patients. In addition, correlation analyses were conducted to detect associations between features of disrupted connectivity and clinical symptoms. Compared to HCs, CID patients showed enhanced rsFC of the SCN-left dorsolateral prefrontal cortex (DLPFC), as well as reduced rsFC of the SCN-bilateral medial prefrontal cortex (MPFC); these altered cortical regions belong to the "top-down" circuit. Moreover, CID patients exhibited disrupted functional and causal connectivity between the SCN and the locus coeruleus (LC) and the raphe nucleus (RN); these altered subcortical regions constitute the "bottom-up" pathway. Importantly, the decreased causal connectivity from the LC-to-SCN was associated with the duration of disease in CID patients. These findings suggest that the disruption of the SCN-centered "top-down" cognitive process and "bottom-up" wake-promoting pathway may be intimately tied to the neuropathology of CID.

Activation of mGluR1 negatively modulates glutamate-induced phase shifts of the circadian pacemaker in the mouse suprachiasmatic nucleus

In mammals, photic information delivered to the suprachiasmatic nucleus (SCN) via the retinohypothalamic tract (RHT) plays a crucial role in synchronizing the master circadian clock located in the SCN to the solar cycle. It is well known that glutamate released from the RHT terminals initiates the synchronizing process by activating ionotropic glutamate receptors (iGluRs) on retinorecipient SCN neurons. The potential role of metabotropic glutamate receptors (mGluRs) in modulating this signaling pathway has received less attention. In this study, using extracellular single-unit recordings in mouse SCN slices, we investigated the possible roles of the Gq/11 protein-coupled mGluRs, mGluR1 and mGluR5, in photic resetting. We found that mGluR1 activation in the early night produced phase advances in neural activity rhythms in the SCN, while activation in the late night produced phase delays. In contrast, mGluR5 activation had no significant effect on the phase of these rhythms. Interestingly, mGluR1 activation antagonized phase shifts induced by glutamate through a mechanism that was dependent upon CaV1.3 L-type voltage-gated Ca2+ channels (VGCCs). While both mGluR1-evoked phase delays and advances were inhibited by knockout (KO) of CaV1.3 L-type VGCCs, different signaling pathways appeared to be involved in mediating these effects, with mGluR1 working via protein kinase G in the early night and via protein kinase A signaling in the late night. We conclude that, in the mouse SCN, mGluR1s function to negatively modulate glutamate-evoked phase shifts.

Circadian Rhythm, Hypothalamo-Pituitary Adrenal Axis, and Immunity: Physiological and Pathological Examples

All living organisms; from single-celled microorganisms to humans, they have to adapt to changing environmental conditions to maintain their survival processes. Circadian rhythm is one of the most important mechanism that associated with this adaptation processes. There are biological clocks in the body, which are related to the circadian rhythm and have a hierarchical organization. The master circadian clock is located in the suprachiasmatic nucleus (SCN) of hypothalamus. SCN maintain body rhythms in synchronous with the light-dark cycle in the external environment. There are also peripheral oscillators that work in coordination with SCN. Neurological, endocrinological, and immunological functions in the body are under the influence of circadian and seasonal rhythms. Melatonin and cortisol (corticosterone in animals) are among the most important hormones that show circadian rhythm in the body. The body adapts to daily and seasonal changes with biological rhythms regulated by biological clocks. It is well known that the immune system is affected by the external environment. Changes in endocrine system, hypothalaomo-pituitary adrenal (HPA) axis, and immune system are marked, especially depending on the seasonal changes. Therefore, the immune system has close relationship with the circadian rhythm. Understanding relationship between physiological regulation of the circadian rhythm, HPA axis and immune activity is important for to keep our body in healthy conditions and struggle with the diseases as well. In current review, the interaction and relationship of genes and proteins related to the circadian rhythm with HPA axis and immune system parameters are discussed with both physiological and pathological examples.

Circadian regulator BMAL1::CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle

Hepatocellular carcinoma (HCC) remains a global health challenge whose incidence is growing worldwide. Previous evidence strongly supported the notion that the circadian clock controls physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Despite the progress, cellular and molecular mechanisms underpinning this HCC-clock crosstalk remain unknown. Addressing this knowledge gap, we show here that although the human HCC cells Hep3B, HepG2, and Huh7 displayed variations in circadian rhythm profiles, all cells relied on the master circadian clock transcription factors, BMAL1 and CLOCK, for sustained cell growth. Down-regulating Bmal1 or Clock in the HCC cells induced apoptosis and arrested cell cycle at the G2/M phase. Mechanistically, we found that inhibiting Bmal1/Clock induced dysregulation of the cell cycle regulators Wee1 and p21 which cooperatively contribute to tumor cell death. Bmal1/Clock knockdown caused downregulation of Wee1 that led to apoptosis activation and upregulation of p21 which arrested the cell cycle at the G2/M phase. Collectively, our results suggest that the circadian clock regulators BMAL1 and CLOCK promote HCC cell proliferation by controlling Wee1 and p21 levels, thereby preventing apoptosis and cell cycle arrest. Our findings shed light on cellular impact of the clock proteins for maintaining HCC oncogenesis and provide proof-of-principle for developing cancer therapy based on modulation of the circadian clock.

The impact of biological clock and sex hormones on the risk of disease.

Molecular clocks are responsible for defining 24-h cycles of behaviour and physiology that are called circadian rhythms. Several structures and tissues are responsible for generating these circadian rhythms and are named circadian clocks. The suprachiasmatic nucleus of the hypothalamus is believed to be the master circadian clock receiving light input via the optic nerve and aligning internal rhythms with environmental cues. Studies using both in vivo and in vitro methodologies have reported the relationship between the molecular clock and sex hormones. The circadian system is directly responsible for controlling the synthesis of sex hormones and this synthesis varies according to the time of day and phase of the estrous cycle. Sex hormones also directly interact with the circadian system to regulate circadian gene expression, adjust biological processes, and even adjust their own synthesis. Several diseases have been linked with alterations in either the sex hormone background or the molecular clock. So, in this chapter we aim to summarize the current understanding of the relationship between the circadian system and sex hormones and their combined role in the onset of several related diseases.

Circadian disruption and cancer- and treatment-related symptoms.

Cancer patients experience a number of co-occurring side- and late-effects due to cancer and its treatment including fatigue, sleep difficulties, depressive symptoms, and cognitive impairment. These symptoms can impair quality of life and may persist long after treatment completion. Furthermore, they may exacerbate each other's intensity and development over time. The co-occurrence and interdependent nature of these symptoms suggests a possible shared underlying mechanism. Thus far, hypothesized mechanisms that have been purported to underlie these symptoms include disruptions to the immune and endocrine systems. Recently circadian rhythm disruption has emerged as a related pathophysiological mechanism underlying cancer- and cancer-treatment related symptoms. Circadian rhythms are endogenous biobehavioral cycles lasting approximately 24 hours in humans and generated by the circadian master clock - the hypothalamic suprachiasmatic nucleus. The suprachiasmatic nucleus orchestrates rhythmicity in a wide range of bodily functions including hormone levels, body temperature, immune response, and rest-activity behaviors. In this review, we describe four common approaches to the measurement of circadian rhythms, highlight key research findings on the presence of circadian disruption in cancer patients, and provide a review of the literature on associations between circadian rhythm disruption and cancer- and treatment-related symptoms. Implications for future research and interventions will be discussed.