Rhythmic Expression Of Clock Genes Research Articles

Nr1d1 affects autophagy in the skeletal muscles of juvenile Nile tilapia by regulating the rhythmic expression of autophagy-related genes

Autophagy is an important evolutionary conserved process in eukaryotic organisms for the turnover of intracellular substances. Recent studies revealed that autophagy displays circadian rhythms in mice and zebrafish. To date, there is no report focused on the rhythmic changes of autophagy in fish skeletal muscles upon nutritional deprivation. In this study, we examined the circadian rhythms of 158 functional genes in tilapia muscle in response to starvation. We found that 12 genes were involved in autophagy changed their rhythm after starvation. Among these genes, Atg4c, Bnip3la, Lc3a, Lc3b, Lc3c, and Ulk1a exhibited a daily rhythmicity in tilapia muscle, and Atg4b, becn1, bnip3la, bnip3lb, Lc3a, and ulk1b were significantly upregulated in response to starvation. The number of autophagosomes was dramatically increased in fasted fish, indicating that nutritional signals affect not only the muscular clock system but also its autophagy behavior. Administration of GSK4112, an activator of Nr1d1, altered rhythmic expression of both circadian clock genes and autophagy genes in tilapia muscle. Taken together, these findings provide evidence that nutritional deficiency affects both circadian regulation and autophagy activities in skeletal muscle.

Circadian rhythm of expression of core clock genes in the photosensitive pineal organ of catfish, Clarias gariepinus under different photoperiodic regimes

ABSTRACT Effects of different regimes of photoperiod (12L–12D, 16L–8D, 8L–16D, LL and DD) were studied on the circadian rhythm of expression of core clock genes (clock, bmal1, per1, per2, per3, cry1 and cry2) in the photosensitive pineal organ of air-breathing catfish, Clarias gariepinus during summer and winter seasons under both in vivo and in vitro conditions using qPCR. Our findings indicate that all major core clock genes are expressed in the photosensitive pineal organ of C. gariepinus, and exhibit significant circadian rhythm of their expression under both in vivo and in vitro conditions irrespective of photoperiods and seasons. However, photoperiods had season-dependent differential effects on mesor (24 h average), amplitude, and acrophase (peak) of the circadian rhythm of expression of the clock genes under both in vivo and in vitro conditions. Based on these findings, we suggest that the photosensitive pineal organ of the fish possesses internal autonomous clockwork composed of clock genes, and different photoperiods deferentially affect rhythm parameters of the rhythmic expression of the pineal clock genes in a season-dependent manner.

Keeping time in the lamina terminalis: Novel oscillator properties of forebrain sensory circumventricular organs.

Drinking behavior and osmotic regulatory mechanisms exhibit clear daily variation which is necessary for achieving the homeostatic osmolality. In mammals, the master clock in the brain's suprachiasmatic nuclei has long been held as the main driver of circadian (24 h) rhythms in physiology and behavior. However, rhythmic clock gene expression in other brain sites raises the possibility of local circadian control of neural activity and function. The subfornical organ (SFO) and the organum vasculosum laminae terminalis (OVLT) are two sensory circumventricular organs (sCVOs) that play key roles in the central control of thirst and water homeostasis, but the extent to which they are subject to intrinsic circadian control remains undefined. Using a combination of ex vivo bioluminescence and in vivo gene expression, we report for the first time that the SFO contains an unexpectedly robust autonomous clock with unusual spatiotemporal characteristics in core and noncore clock gene expression. Furthermore, putative single‐cell oscillators in the SFO and OVLT are strongly rhythmic and require action potential‐dependent communication to maintain synchrony. Our results reveal that these thirst‐controlling sCVOs possess intrinsic circadian timekeeping properties and raise the possibility that these contribute to daily regulation of drinking behavior.

Circadian disruption by shifting the light-dark cycle negatively affects bone health in mice.

The past decade, it has become evident that circadian rhythms within metabolically active tissues are very important for physical health. However, although shift work has also been associated with an increased risk of fractures, circadian rhythmicity has not yet been extensively studied in bone. Here, we investigated which genes are rhythmically expressed in bone, and whether circadian disruption by shifts in light-dark cycle affects bone turnover and structure in mice. Our results demonstrate diurnal expression patterns of clock genes (Rev-erbα, Bmal1, Per1, Per2, Cry1, Clock), as well as genes involved in osteoclastogenesis, osteoclast proliferation and function (Rankl, Opg, Ctsk), and osteocyte function (c-Fos) in bone. Weekly alternating light-dark cycles disrupted rhythmic clock gene expression in bone and caused a reduction in plasma levels of procollagen type 1 amino-terminal propeptide (P1NP) and tartrate-resistant acidic phosphatase (TRAP), suggestive of a reduced bone turnover. These effects coincided with an altered trabecular bone structure and increased cortical mineralization after 15 weeks of light-dark cycles, which may negatively affect bone strength in the long term. Collectively, these results show that a physiological circadian rhythm is important to maintain bone health, which stresses the importance of further investigating the association between shift work and skeletal disorders.

Expression of miR-34a-5p is up-regulated in human colorectal cancer and correlates with survival and clock gene PER2 expression.

Colorectal cancer represents a leading cause of cancer death. MicroRNAs (miRNAs) are small non-coding RNA molecules that have been extensively studied in tumours, since changes in their levels can reveal patient prognosis. Cancer progression is also influenced by the circadian system whose functioning is based on the rhythmic expression of clock genes. Therefore, we performed macroarray screening of tumour and adjacent tissues in patients undergoing surgery for colorectal carcinoma. We identified 17 miRNAs showing expression that was more than 100 times higher in tumour tissue compared to adjacent tissue. From in silico analysis, miR-34a-5p was selected as showing a computer-predicted interaction with PER2. Real-time PCR revealed a negative correlation between expression of PER2 mRNA and miR-34a in patients with more advanced cancer stage. Expression of miR-34a was up-regulated in cancer tissue compared to adjacent tissue. High miR-34a expression was associated with better survival of patients. miR-34a showed lower expression levels in male patients with lymph node involvement, and a trend towards decreased expression in male patients with distant metastases. Male patients, but not female patients, with high expression of miR-34a and who were free of distant metastases and/or lymph node involvement showed better survival. Therefore, we proposed that expression of miR-34a was regulated in a sex-dependent manner and could be considered a marker of prognosis in earlier cancer stages in male patients.

Rhythmic Release of Corticosterone Induces Circadian Clock Gene Expression in the Cerebellum

Neurons of the cerebellar cortex contain a circadian oscillator, with circadian expression of clock genes being controlled by the master clock of the suprachiasmatic nucleus (SCN). However, the signaling pathway connecting the SCN to the cerebellum is unknown. Glucocorticoids exhibit a prominent SCN-dependent circadian rhythm, and high levels of the glucocorticoid receptor have been reported in the cerebellar cortex; we therefore hypothesized that glucocorticoids may control the rhythmic expression of clock genes in the cerebellar cortex. We here applied a novel methodology by combining the electrolytic lesion of the SCN with implantation of a micropump programmed to release corticosterone in a circadian manner mimicking the endogenous hormone profile. By use of this approach, we were able to restore the corticosterone rhythm in SCN-lesioned male rats. Clock gene expression in the cerebellum was abolished in rats with a lesioned SCN, but exogenous corticosterone restored the daily rhythm in clock gene expression in the cerebellar cortex, as revealed by quantitative real-time PCR and radiochemical in situ hybridization for the detection of the core clock genes Per1, Per2, and Arntl. On the contrary, exogenous hormone did not restore circadian rhythms in body temperature and running activity. RNAscope in situ hybridization further revealed that the glucocorticoid receptor colocalizes with clock gene products in cells of the cerebellar cortex, suggesting that corticosterone exerts its actions by binding directly to receptors in neurons of the cerebellum. However, rhythmic clock gene expression in the cerebellum was also detectable in adrenalectomized rats, indicating that additional control mechanisms exist. These data show that the cerebellar circadian oscillator is influenced by SCN-dependent rhythmic release of corticosterone.

Capsaicin Ameliorates the Redox Imbalance and Glucose Metabolism Disorder in an Insulin-Resistance Model via Circadian Clock-Related Mechanisms.

Circadian rhythms are closely associated with metabolic homeostasis. Metabolic disorders can be alleviated by many bioactive components through controlling of clock gene expressions. Capsaicin has been demonstrated with many beneficial effects including anti-obesity and anti-insulin resistance activities, yet whether the rhythmic expression of circadian clock genes are involved in the regulation of redox imbalance and glucose metabolism disorder by capsaicin remains unclear. In this work, the insulin resistance was induced in HepG2 cells by treatment of glucosamine. Glucose uptake levels, reactive oxygen species, H2O2 production, and mitochondrial membrane potential (MMP) were measured with/without capsaicin cotreatment. The mRNA and protein expressions of core circadian clock genes were evaluated by RT-qPCR and western blot analysis. Our study revealed that circadian misalignment could be ameliorated by capsaicin. The glucosamine-induced cellular redox imbalance and glucose metabolism disorder were ameliorated by capsaicin in a Bmal1-dependent manner.

Rev-Erb\u03b1 and Photoreceptor Outer Segments modulate the Circadian Clock in Retinal Pigment Epithelial Cells

Retinal photoreceptor outer segments (POS) are renewed daily through phagocytosis by the adjacent retinal pigment epithelial (RPE) monolayer. Phagocytosis is mainly driven by the RPE circadian clock but the underlying molecular mechanisms remain elusive. Using ARPE-19 (human RPE cell-line) dispersed and monolayer cell cultures, we investigated the influence of cellular organization on the RPE clock and phagocytosis genes. PCR analysis revealed rhythmic expression of clock and phagocytosis genes in all ARPE-19 cultures. Monolayers had a tendency for higher amplitudes of clock gene oscillations. In all conditions ARNTL, CRY1, PER1-2, REV-ERBα, ITGB5, LAMP1 and PROS1 were rhythmically expressed with REV-ERBα being among the clock genes whose expression showed most robust rhythms in ARPE-19 cells. Using RPE-choroid explant preparations of the mPer2Luc knock-in mice we found that Rev-Erbα deficiency induced significantly longer periods and earlier phases of PER2-bioluminescence oscillations. Furthermore, early phagocytosis factors β5-Integrin and FAK and the lysosomal marker LAMP1 protein levels are rhythmic. Finally, POS incubation affects clock and clock-controlled phagocytosis gene expression in RPE monolayers in a time-dependent manner suggesting that POS can reset the RPE clock. These results shed some light on the complex interplay between POS, the RPE clock and clock-controlled phagocytosis machinery which is modulated by Rev-Erbα.

In vivo evaluation of the effect of lithium on peripheral circadian clocks by real-time monitoring of clock gene expression in near-freely moving mice

Lithium has been used as a mood stabilizer to treat human bipolar disorders for over half a century. Several studies have suggested the possibility that the efficacy of lithium treatment results in part from the amelioration of circadian dysfunction. However, the effect of lithium on clock gene expression has not yet been investigated in vivo because continuous measurement of gene expression in organs with high time resolution over a period of several days is difficult. To resolve this issue, we attached a small photo multiplier tube (PMT) tightly to the body surface of transgenic mice carrying a reporter gene such that the photon input window faced target organs such as the liver and kidney and succeeded in long-term continuous measurement of circadian gene expression in semi-freely moving mice over periods of several weeks. Using this simple method, we clearly showed that lithium causes circadian period elongation in peripheral clock gene expression rhythms in vivo. Further development of our detection system to maturity will aid a wide range of research fields in medicine and biology.

Intermittent restraint stress induces circadian misalignment in the mouse bladder, leading to nocturia

Intermittent stress disrupts the circadian rhythm in clock genes such as Per2 only in peripheral organs without any effect on the central circadian clock in the suprachiasmatic nucleus. Here, the effect of restraint stress (RS) on circadian bladder function was investigated based on urination behavior and gene expression rhythms. Furthermore, PF670462 (PF), a Per2 phosphorylation enzyme inhibitor, was administered to investigate the effects on circadian bladder re-alignment after RS. Two-hour RS during the light (sleep) phase was applied to mice (RS mice) for 5 days. The following parameters were then examined: urination behaviors; clock gene expression rhythms and urinary sensory-related molecules such as piezo type mechanosensitive ion channel component 1 (Piezo1), transient receptor potential cation channel subfamily V member 4 (TRPV4), and Connexin26 (Cx26) in the bladder mucosa; Per2 expression in the excised bladder of Per2luciferase knock-in mice (Per2::luc); in vivo Per2 expression rhythms in the bladder of Per2::luc mice. Control mice did not show altered urination behavior in the light phase, whereas RS mice exhibited a higher voiding frequency and lower bladder capacity. In the bladder mucosa, RS mice also showed abrogated or misaligned Piezo1, TRPV4, Connexin26, and clock gene expression. The rhythmic expression of Per2 was also altered in RS mice both in excised- and in vivo bladder, compared with control mice. After PF administration, voiding frequency was reduced and bladder capacity was increased during the light phase in RS mice; the in vivo Per2 expression rhythm was also fully restored. Therefore, RS can alter circadian gene expression in the bladder during the light phase and might cause nocturia via changes in circadian bladder function due the dysregulation of clock genes. Amending the circadian rhythm therapeutically could be applied for nocturia.

Effect of melatonin on monochromatic light-induced changes in clock gene circadian expression in the chick liver

Light is the most prominent zeitgeber of the circadian system, which contains central and peripheral oscillators. Our previous studies found that light wavelength could influence the rhythms of melatonin synthesis and clock gene expression in the central oscillator of chicks. However, the effect of monochromatic light on the peripheral oscillator and the role of melatonin have yet to be clarified. In this study, 216 newly hatched chicks were divided into three groups (intact, sham operation and pinealectomy) and were raised under white (WL), red (RL), green (GL) or blue (BL) light for 14 days. Their plasma and livers were sampled at 6 time points with 4-h intervals. Plasma melatonin concentration and liver clock gene expression (cClock, cBmal1, cBmal2, cCry1, cCry2, cPer2, cPer3) were measured for circadian rhythm analysis. In intact and sham operation chicks under WL, all liver clock genes showed circadian expression along with oscillations in plasma melatonin. However, positive clock genes peaked at subjective night along with melatonin, while negative clock genes peaked at subjective day or the shifting time of day-night. Chick exposure to monochromatic light led to an unaltered circadian rhythmicity in plasma melatonin and liver clock genes; however, their rhythmic parameters were notably influenced. Compared to WL, GL enhanced the mesor and amplitude of melatonin and all kinds of clock genes, whereas RL had the opposite effect. Pinealectomy significantly decreased expression of liver clock genes, which was consistent with the reduction in plasma melatonin concentration, especially for the GL group, and resulted in the expression of liver clock genes showing low-mesor and low-amplitude oscillations as well as no statistically significant differences among the monochromatic light groups. Thus, we speculated that melatonin plays a key role in the effects of light wavelength on clock gene rhythm in the chick liver.

Mammary tumors compromise time-of-day differences in hypothalamic gene expression and circadian behavior and physiology in mice

Circadian rhythms influence various aspects of biology, including hormonal, immunological, and behavioral processes. These 24-hour oscillations are necessary to optimize cellular functions and to synchronize these processes with the environment. Breast cancer patients and survivors frequently report disruptions in circadian oscillations that adversely affect quality-of-life, including fragmented sleep-wake cycles and flattened cortisol rhythms, which are associated with negative behavioral comorbidities (e.g., fatigue). However, the potential causal role of tumor biology in circadian dysregulation has not been investigated. Here, we examined the extent to which sham surgery, non-metastatic mammary tumors, or mammary tumor removal in mice disrupts circadian rhythms in brain clock gene expression, locomotor behavior (free-running and entrained), and physiological rhythms that have been associated with cancer behavioral comorbidities. Tumors and tumor resection altered time-of-day differences in hypothalamic expression of eight circadian-regulated genes. The onset of activity in entrained running behavior was advanced in tumor-bearing mice, and the amplitude of free-running rhythms was increased in tumor-resected mice. Tumors flattened rhythms in circulating corticosterone and Ly6cHi monocytes which were largely restored by surgical tumor resection. This work implies that tumors alone may directly impact central and/or peripheral circadian rhythmicity in breast cancer patients, and that these effects may persist in cancer survivors, potentially contributing to behavioral comorbidities.

Aging renders desynchronization between clock and immune genes in male Wistar rat kidney: chronobiotic role of curcumin.

Suprachiasmatic nucleus (SCN) contains the central clock that orchestrate circadian rhythms in physiology and behavior in mammals. Tightly interlocked transcriptional and translational feedback loops (TTFLs) comprising of various clock genes such as Clock, Bmal1, Periods, Cryptochromes etc. in the SCN, send the timing signals to peripheral clocks that governs local metabolism with similar TTFLs. Peripheral clocks in kidney regulates several circadian rhythms like blood pressure, immunity etc. However, aging leads to circadian and inflammatory disorders in kidney. Though there are increasing evidences on age associated perturbations, studies elucidating the rhythmic expression of clock and immune genes across aging in kidney are obscure. We therefore studied changes in daily rhythms of clock and immune genes in kidney. In this study we measured mRNA expression of clock genes rBmal1, rPer1, rPer2, rCry1, rCry2, rRev-erbα, rRorα, and inflammatory genes rNfκb1, rTnfα, rIl6, rTlr4 and rTlr9 in 3, 12 and 24months male Wistar rat kidney using qRT-PCR. From our study, we did not observe significant changes in clock genes expression except rRorα, but immune genes showed significant phase alterations as well as increase in mean 24h levels. Pearson correlation analysis of data showed desynchronization between immune and clock genes expression. We further studied the effect of administration of curcumin which has anti-aging, anti-inflammatory, anti-oxidant etc. properties, and evaluated its chronobiotic properties. We here report differential effects of curcumin administration on daily rhythms of clock and immune genes expression.

Circadian neurogenetics of mood disorders.

Mood state alterations are often accompanied by disruptions of daily rhythms of physiology. Circadian rhythms of physiology are controlled by a central clock harbored in the suprachiasmatic nucleus (SCN), which is functionally dependent on the rhythmic expression of several clock genes. The molecular clockwork has been identified in other extra-SCN brain regions, some of which are implicated in the regulation of motivational and emotional states, although their specific circadian role is not fully known. In mood disorders, alterations of the molecular clock have been reported. Thus, functional expression of circadian genes in the brain is compromised in mood diseases. In the present review, we describe the current evidence that implicates the clock gene alterations as an important factor in the development of mood-related disorders. Furthermore, we describe the possible role of other brain clocks, beyond the SCN, in the circadian control of mood. The comprehension of the circadian neural and genetic mechanisms underlying mood alterations might guide towards the identification of optimal drug and non-drug therapies for the cure of depression and other mood disorders.

Defining the Independence of the Liver Circadian Clock

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained invivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

MON-227 Roles of Clock Expression Modulated by BMP-7 in Ovarian Steroidogenesis by Human Granulosa Cells

In mammals, the circadian pacemaker is known to control and integrate physiological rhythms via the coordination of oscillating peripheral clocks that reside in various organs, through the biologic actions of various neurochemical and/or hormonal transmitters. The expression of clock genes has been demonstrated in tissues composing the axis of the hypothalamic-pituitary-gonadal system. The roles of hypothalamic oscillators to produce the biological timings and rhythms in the reproductive axis have been recognized. Results of recent studies have shown that clock genes are expressed in the ovary, and accumulated findings have demonstrated a significant interrelationship between clock gene expression and follicular development. It has been reported that the rhythms of clock gene expression are undetectable in primordial and preantral follicles but can be detected at the timing of early antral stages of follicles and then become clearer at the late antral stages to preovulatory follicles. In the ovarian follicles, clock genes are expressed in granulosa cells, theca cells, oocytes and stromal cells composing the developing follicles. Dysfunction of clock gene expression in the ovary and/or desynchronicity between ovarian clocks and extra-ovarian circadian oscillators may cause various reproductive abnormalities. However, the regulatory mechanism and the physiological roles of the intrinsic clock activity in the ovary have remained uncertain. In the preset study, functional link between clock gene expression and ovarian steroidogenesis was studied using human granulosa KGN cells. Similarities between the oscillating patterns of mRNA and protein expression of Bmal1 and Clock and that of Per2 and Cry1 were demonstrated during 24-h culture of KGN cells in the presence of forskolin. Among the interrelationships between the expression levels of clock and steroidogenic factors, Clock mRNA had a strongly positive correlation with P450arom and a negative correlation with 3βHSD. Knockdown of Clock gene by siRNA resulted in a significant reduction of estradiol production by inhibiting P450arom expression, while it induced a significant increase of progesterone production by upregulating 3βHSD in KGN cells treated with forskolin. Moreover, BMP-7 had an enhancing effect on the expression of Clock mRNA and protein in KGN cells. Collectively, the expression levels of Clock, being upregulated by forskolin and BMP-7, were functionally linked to estradiol production and progesterone suppression by human granulosa cells.

SUN-020 Analyses Of Clock Gene Expressions In Peripheral Leukocytes In Hypercortisolism

Background: Hypercortisolism causes metabolic disorders such as hyperglycemia and dyslipidemia. Recent studies have shown relationships between impairment of peripheral circadian clock function and the development of metabolic diseases. However, the association between phenotypes and peripheral circadian regulations in overt hypercortisolism and subclinical hypercortisolism clinically has not been elucidated. This study examined peripheral clock-related gene expressions of patients with hypercortisolism in regulation of involvement of glucocorticoid receptors. Methods: Blood samples were obtained from four patients with hypercortisolism including one patient of Cushing syndrome due to adrenal tumor, two patients of Cushing disease, and one patient due to ectopic ACTH producing tumor and five comparatively young volunteers as normal subjects at 08:00 and 20:00. Peripheral bone mononuclear cells (PBMCs) were extracted by Ficoll density gradient methods. Transcript levels of clock genes (Clock, Bmal1, Per1,Cry1, and ROR), glucocorticoid receptor (GR) and GILZ were determined by real-time quantitative PCR. Results: Serum cortisol levels were significantly higher in samples from 08:00 than 20:00 in normal subjects (p<0.05) and these levels from patients of hypercortisolism were significantly higher than normal subjects (p<0.01). Diurnal variation of cortisol levels in the patients was not observed. The expressions of Clock and Bmal1 mRNA in normal subjects showed higher at 20:00 than at 08:00 and Per1 mRNA was significantly lower at 20:00 than 08:00 (p<0.05), exhibiting rhythmicity in the PBMCs of 5 normal subjects. The expression levels of these clock-associated mRNAs and GR were significantly (p<0.01) lower in the patients than in normal subjects at 08:00 and 20:00. Moreover, the amplitudes of mRNA expression rhythms of clock-related genes tended to diminish in the patients. Multivariate analysis showed a positive correlation between Clock and Bmal1 in normal subjects and between Per1 and Cry1 significantly (p <0.01, p<0.01), and a same correlation was observed in hypercortisolem (P <0.01, p <0.001). Clock-associated genes and GR expressions showed a significant negative correlation with cortisol levels. Conclusions: These results suggest that rhythmic mRNA expression of clock genes is impaired in PBMCs of patients of hypercortisolism. Interestingly, molecules forming dimerization at the same phase in the circadian loops showed coordinated gene expressions in hypercortisolism. The expressions of the peripheral clock genes in the patients were markedly reduced, which was thought to be caused by a remarkable cortisol excess. Expressions of peripheral clock genes may be useful markers in predicting metabolic complications and therapeutic effects as well as leading to elucidate the pathology of metabolic disorders in subclinical hypercortisolism.

Intermittent Restraint Stress Induces Circadian Misalignment in the Mouse Bladder, Leading to Nocturia

Background: Intermittent stress disrupts the circadian rhythm in clock genes such as Per2 only in peripheral organs without any effect on the central nervous system. Here, the effect of restraint stress (RS) on circadian bladder function was investigated based on urination behavior and gene expression rhythms. Furthermore, PF670462 (PF), a Per2 phosphorylation enzyme inhibitor, was administered to investigate the effects on circadian bladder re-alignment after RS. Method: Two-hour RS during the light phase was applied to mice (RS mice) for 5 days. The following parameters were then examined: urination behaviors; clock gene expression rhythms and urinary sensory-related molecules; Per2 expression in the excised bladder of Per2::luc mice; in vivo Per2 expression rhythms in the bladder of Per2::luc mice. Findings: Control mice did not show altered urination behavior in the light phase, whereas RS mice exhibited a higher voiding frequency and lower bladder capacity. In the bladder mucosa, RS mice also showed abrogated or misaligned urinary sensory-related molecules and clock gene expression. The rhythmic expression of Per2 was also altered in RS mice both in excised- and in vivo bladder, compared with control mice. After PF administration, voiding frequency was reduced and bladder capacity was increased during the light phase in RS mice; the in vivo Per2 expression rhythm was also fully restored. Interpretation: RS can alter circadian gene expression in the bladder during the light phase and might cause nocturia via changes in circadian bladder function due the dysregulation of clock genes. Amending the circadian rhythm therapeutically could be applied for nocturia. Funding Statement: This work was supported financially by Suzuki Urinary Medicine Promotion Foundation and Astellas Pharma Inc. Declaration of Interests: The authors declare that they have no conflicts of interest with the contents of this article. Ethics Approval Statement: All procedures were conducted in accordance with the Guiding Principles in the Care and Use of Animals in the Field of the Physiologic Society of Japan and the policies of the Institutional Animal Care and Use Committee.

Modulation of NMDA-Mediated Clock Resetting in the Suprachiasmatic Nuclei of mPer2 Luc Mouse by Endocannabinoids.

Light entrains the master circadian clock in the suprachiasmatic nucleus (SCN) predominantly through glutamatergic signaling via NMDA receptors. The magnitude and the direction of resulting phase shifts depend on timing of the photic stimulus. Previous reports based on behavioral and electrophysiological data suggested that endocannabinoids (EC) might reduce the ability of the SCN clock to respond to light. However, there is little direct evidence for the involvement of EC in entrainment of the rhythmic clock gene expression in the SCN. We have used luminescence recording of cultured SCN slices from mPer2Luc mice to construct a complete phase response curve (PRC) for NMDA receptor activation. The results demonstrated that NMDA administration phase-shifts the PER2 rhythm in a time-specific manner. A stable “singularity,” in the course of which the clock seemingly stops while the overall phase is caught between delays and advances, can occur in response to NMDA at a narrow interval during the PER2 level decrease. NMDA-induced phase delays were affected neither by the agonist (WIN 55,212-2 mesylate) nor by the antagonist (rimonabant hydrochloride) of EC receptors. However, the agonist significantly reduced the NMDA-induced phase advance of the clock, while the antagonist enhanced the phase advance, causing a shift in the sensitivity window of the SCN to NMDA. The modulation of EC signaling in the SCN had no effect by itself on the phase of the PER2 rhythm. The results provide evidence for a modulatory role of EC in photic entrainment of the circadian clock in the SCN.

Ultraviolet-C (UVC) ray acts as a synchronizing cue for circadian rhythm control in murine fibroblast

Ultraviolet-C (UVC) electromagnetic radiation is the most damaging type of the UV radiation and causes many cellular and physiological responses. UVC has been using for sterilization and disinfection, and the risk of exposure to the UVC is increasing. Here, we determined the effect of the UVC on the cellular circadian clock system. UVC irradiation synchronized the biological clock system and induced time-dependent expression of clock genes including Clock, Cry1, and Per1. The rhythmic expression of clock genes is also followed by time-dependent mRNA degradation or non-canonical translation initiation of clock genes. Furthermore, we show a translocation of PERIOD1 (PER1) protein after UVC irradiation, which mediates the rhythmic feedback loop of clock genes. Our results suggest that UVC can synchronize the circadian clock system, and induces rhythmic expression of clock genes via time-dependent transcription, post-transcription, and post-translational modification.