Expression Of Clock Genes Per1 Research Articles

Glycyrrhizic acid treatment ameliorates anxiety-like behaviour via GLT1 and Per1/2-dependent pathways

Ethnopharmacological relevanceAs a traditional Chinese medicinal herb, Glycyrrhiza. UralensisFisch. (licorice root, chinese name: Gancao) has a variety of medicinal values and is widely used clinically. Its main active ingredient, glycyrrhizic acid (GA), is believed to have a neuroprotective effect. However, the underlying biological mechanisms of GA on stress-induced anxiety disorders are still unclear. Aim of the studyTo investigate the anti-anxiety effect of GA and its underlying mechanism. MethodsWe selected the anxiety model induced by repeated chronic restraint stress (CRS) for 2 h on each of 7 consecutive days. GA (4, 20, 100 mg/kg) was injected intraperitoneally once daily for 1 week. The potential GA receptors were identified using whole-cell patches and computer-assisted docking of molecules. High-throughput RNA sequencing, adeno-associated virus-mediated gene regulation, Western blotting, and RT-qPCR were used to assess the underlying molecular pathways. ResultsGA alleviate depression-like and anxiety-like behaviors in CRS mice. GA decreased synaptic transmission by facilitating glutamate reuptaking in mPFC. Meanwhile, long-term GA treatment increased the expression of clock genes Per1 and Per2. Suppressing both Per1 and Per2 abolished the anxiolytic effects of GA treatment. ConclusionOur study suggests that GA may be developed for the treatment of stress-induced anxiety disorders, and its mechanism is related to GLT1 and Per1/2-dependent pathways. This presents a novel approach to discovering potent therapeutic drugs.

Nonphotic entrainment of central and peripheral circadian clocks in mice by scheduled voluntary exercise under constant darkness.

In mammals, the central circadian pacemaker in the suprachiasmatic nucleus (SCN) entrains to an environmental light-dark (LD) cycle and organizes the temporal order of circadian rhythms in physiology and behavior. Previously, some studies have demonstrated that scheduled exercise could entrain the free-running behavior rhythm in nocturnal rodents. However, it remains unknown whether entrainment by scheduled exercise alters the internal temporal order of the behavioral circadian rhythms or clock gene expression in the SCN, extra-SCN brain regions, and peripheral organs when mice are entrained to the scheduled exercise under constant darkness (DD). In the present study, we examined circadian rhythms in locomotor activity and clock gene Per1 expression by bioluminescence reporter (Per1-luc) in the SCN, arcuate nucleus (ARC), liver, and skeletal muscle of mice entrained to an LD cycle, mice free-running under DD, and mice entrained to daily exposure to a new cage with a running wheel (NCRW) under DD. All mice showed a steady-state entrainment of behavioral circadian rhythms to NCRW exposure under DD in parallel with shortening of the α when compared with that under DD. The temporal order of behavioral circadian rhythms and the Per1-luc rhythms in the SCN and peripheral tissues but not in the ARC were maintained in the mice entrained to the NCRW and LD cycles; in contrast, the temporal order was altered in the mice under DD. The present findings reveal that the SCN entrains to daily exercise, and daily exercise reorganizes the internal temporal order of behavioral circadian rhythms and clock gene expression in the SCN and peripheral tissues.

Iron accumulation with age alters metabolic pattern and circadian clock gene expression through the reduction of AMP-modulated histone methylation

Iron accumulates with age in mammals, and its possible implications in altering metabolic responses are not fully understood. Here, we report that both high-iron diet and advanced age in mice consistently altered gene expression of many pathways, including those governing the oxidative stress response and the circadian clock. We used a metabolomic approach to reveal similarities between metabolic profiles and the daily oscillation of clock genes in old and iron-overloaded mouse livers. In addition, we show that phlebotomy decreased iron accumulation in old mice, partially restoring the metabolic patterns and amplitudes of the oscillatory expression of clock genes Per1 and Per2. We further identified that the transcriptional regulation of iron occurred through a reduction in AMP-modulated methylation of histone H3K9 in the Per1 and H3K4 in the Per2 promoters, respectively. Taken together, our results indicate that iron accumulation with age can affect metabolic patterns and the circadian clock.

Inhibitor of DNA binding 2 (Id2) Regulates Photic Entrainment Responses in Mice: Differential Responses of the Id2-/- Mouse Circadian System Are Dependent on Circadian Phase and on Duration and Intensity of Light.

ID2 is a rhythmically expressed helix-loop-helix transcriptional repressor, and its deletion results in abnormal properties of photoentrainment. By examining parametric and nonparametric models of entrainment, we have started to explore the mechanism underlying this circadian phenotype. Id2-/- mice were exposed to differing photoperiods, and the phase angle of entrainment under short days was delayed 2 h as compared with controls. When exposed to long durations of continuous light, enhanced entrainment responses were observed after a delay of the clock but not with phase advances. However, the magnitude of phase shifts was not different in Id2-/- mice tested in constant darkness using a discrete pulse of saturating light. No differences were observed in the speed of clock resetting when challenged by a series of discrete pulses interspaced by varying time intervals. A photic phase-response curve was constructed, although no genotypic differences were observed. Although phase shifts produced by discrete saturating light pulses at CT16 were similar, treatment with a subsaturating pulse revealed a ~2-fold increase in the magnitude of the Id2-/- shift. A corresponding elevation of light-induced per1 expression was observed in the Id2-/- suprachiasmatic nucleus (SCN). To test whether the phenotype is based on a sensitivity change at the level of the retina, pupil constriction responses were measured. No differences were observed in responses or in retinal histology, suggesting that the phenotype occurs downstream of the retina and retinal hypothalamic tract. To test whether the phenotype is due to a reduced amplitude of state variables of the clock, the expression of clock genes per1 and per2 was assessed in vivo and in SCN tissue explants. Amplitude, phase, and period length were normal in Id2-/- mice. These findings suggest that ID2 contributes to a photoregulatory mechanism at the level of the SCN central pacemaker through control of the photic induction of negative elements of the clock.

Expression of Clock Genes Per1 , Per2 , and Per3 in Patients With Head and Neck Squamous Cell Carcinoma and Its Correlation With Clinical Factors

Expression of Clock Genes Per1 , Per2 , and Per3 in Patients With Head and Neck Squamous Cell Carcinoma and Its Correlation With Clinical Factors

Na⁺/Ca²⁺ Exchanger 2 (NCX2) in the Circadian Clock of the Rat Suprachiasmatic Nucleus: Colocalization with Neuropeptides and Daily Profiles of Gene Expression and Protein Levels.

The plasmalemmal Na⁺/Ca²⁺ changer (NCX) regulates intracellular Ca²⁺ by exchanging 3 Na⁺ for 1 Ca²⁺ in either the Ca²⁺ exit or Ca²⁺ entry mode. All three NCX isoforms NCX1, NCX2, and NCX3 are expressed in the rat brain, with isoform-specific differential distribution. In the central clock of suprachiasmatic nucleus (SCN), intracellular Ca²⁺ controls the circadian release of major neuropeptides, which are the arginine vasopressin (AVP), vasoactive intestinal peptide (VIP) and gastrin releasing peptide (GRP), and the NCX, most likely NCX1, rapidly clears depolarization-induced somatic Ca²⁺ influx. However, the role of NCX2 in the SCN remains unknown. This study aimed to investigate the colocalization of NCX2 with neuropeptides and daily expression profiles of NCX2 in mRNA and protein levels. Consistent with the restricted distribution of NCX2 in the retinorecipient ventral SCN, the immunostaining results showed colocalization of NCX2 with VIP, GRP and VIP/GRP in the ventral SCN, but not with AVP in the dorsal SCN, or markers for astrocyte and major input pathways. Importantly, the presynaptic marker Bassoon was found to colocalize with NCX2/GRP and NCX2/ VIP, indicating localization of both VIP/NCX2 and GRP/NCX2 at the presynaptic sites. Furthermore, real-time PCR and western blotting revealed no day-night difference in NCX2 mRNA and protein levels, in contrast to a robust circadian rhythm in the expression of clock genes Per1 and Per2. Together the results suggest a role of NCX2 in the regulation of the release of VIP and GRP.

Effects of feeding time on daily rhythms of neuropeptide and clock gene expression in the rat hypothalamus

Shiftworkers are exposed to several adverse health conditions, one being eating at night. Food consumption at an unnatural time-of-day is thought to be one of the main factors responsible for the increased risk of developing metabolic diseases, such as obesity and diabetes mellitus. The underlying mechanism is considered to include disruption of the circadian organization of physiology, leading to disruption of metabolism. When food is consumed at night, the hypothalamus, a brain region central to homeostasis, receives contradicting input from the central clock and the systemic circulation. This study investigated how timing of feeding affects hypothalamic function by studying, in different hypothalamic nuclei, expression of clock genes and key neuropeptide genes involved in energy metabolism, including orexin, melanin-concentrating hormone (MCH) and neuropeptide Y. Animals with food available ad libitum showed diurnal variation in the expression of clock genes Per1 and Per2 in the perifornical area and arcuate nucleus. Clock gene rhythms were lost in both nuclei when food was restricted to the light (i.e., sleep) period. Neuropeptide genes did not display significant daily variation in either feeding groups, except for orexin-receptor 1 in ad libitum animals. Analysis of genes involved in glutamatergic and GABAergic signaling did not reveal diurnal variation in expression, nor effects of feeding time. In conclusion, feeding at the ‘wrong’ time-of-day not only induces desynchronization between brain and body clocks but also within the hypothalamus, which may contribute further to the underlying pathology of metabolic dysregulation.

Dissociation of Per1 and Bmal1 circadian rhythms in the suprachiasmatic nucleus in parallel with behavioral outputs

The temporal order of physiology and behavior in mammals is primarily regulated by the circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of bioluminescence reporters, we monitored the circadian rhythms of the expression of clock genes Per1 and Bmal1 in the SCN of freely moving mice and found that the rate of phase shifts induced by a single light pulse was different in the two rhythms. The Per1-luc rhythm was phase-delayed instantaneously by the light presented at the subjective evening in parallel with the activity onset of behavioral rhythm, whereas the Bmal1-ELuc rhythm was phase-delayed gradually, similar to the activity offset. The dissociation was confirmed in cultured SCN slices of mice carrying both Per1-luc and Bmal1-ELuc reporters. The two rhythms in a single SCN slice showed significantly different periods in a long-term (3 wk) culture and were internally desynchronized. Regional specificity in the SCN was not detected for the period of Per1-luc and Bmal1-ELuc rhythms. Furthermore, neither is synchronized with circadian intracellular Ca2+ rhythms monitored by a calcium indicator, GCaMP6s, or with firing rhythms monitored on a multielectrode array dish, although the coupling between the circadian firing and Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respectively associated with the expression of activity onset and offset.

The clock gene PER1 plays an important role in regulating the clock gene network in human oral squamous cell carcinoma cells.

The various clock genes in normal cells, through their interaction, establish a number of positive and negative feedback loops that compose a network structure. These genes play an important role in regulating normal physiological activities. The expression of clock gene PER1 is decreased in many types of cancer. PER1 is highly correlated with the initiation and progression of cancer by regulating numerous downstream genes. However, it is still unclear whether the lower expression of PER1 in cancer can influence the expression of other clock genes in the clock gene network. In this study, we used short hairpin RNA interference to knock down PER1 effectively in SCC15 human oral squamous cell carcinoma cells. These cancer cells later were subcutaneously injected into the back of nude mice. We discovered that after PER1 knockdown, apoptosis was decreased and cell proliferation and in vivo tumor formation were enhanced. Quantitative real-time PCR result indicated that in vitro and in vivo cancer cells after PER1 knockdown, PER2, DEC1, DEC2, CRY1, CRY2 and NPAS2 were significantly down-regulated at the mRNA level, while PER3, TIM, RORα and REV-ERBα were significantly up-regulated. It prompts that the role of PER1 in carcinogenesis is exerted not only by regulating downstream genes, but also through the synergistic dysregulation of many other clock genes in the clock gene network.

Downregulation of the spinal dorsal horn clock gene Per1 expression leads to mechanical hypersensitivity via c-jun N-terminal kinase and CCL2 production in mice.

Disturbances of circadian rhythm and dysregulation of clock gene expression are involved in the induction of various neurological disorder states, including chronic pain. However, the relationship between the CNS circadian-clock gene system and nociception remains poorly defined. Significant circadian oscillations of Period (Per1, Per2), Bmal1 and Cryptochrome 1 (Cry1) mRNA expression have been observed in the lumbar spinal dorsal horn of naïve mice. The current study examined the expression of clock genes in the lumbar spinal dorsal horn of mice with neuropathic pain due to a partial sciatic nerve ligation (PSNL). Seven days after PSNL, the mice displayed a robust unilateral hind paw mechanical hypersensitivity. The normal circadian oscillations of Per1, Per2 and Cry1, but not Bmal1, mRNA expression were significantly suppressed in the ipsilateral lumbar spinal dorsal horn of PSNL mice 7days following surgery. The circadian expression of PER1 protein, in particular, was also significantly suppressed in the ipsilateral spinal dorsal horn of PSNL mice. Double-labeling immunohistochemistry revealed downregulation of PER1 in neurons and astrocytes, but not microglia. Knockdown of Per1 expression by intrathecal treatment with Per1 siRNA also induced mechanical hypersensitivity, phosphorylation of c-jun N-terminal kinase (JNK) and the upregulation of chemokine (C-C motif) ligand 2 (CCL2) production in the lumbar spinal dorsal horn. Per1 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of either the JNK inhibitor SP600125 or the selective CCL2 receptor (CCR2) antagonist RS504393, indicating that these intracellular messengers are crucial in mediating the mechanical hypersensitivity following the downregulation of PER1 expression. These results suggest that the downregulation of the spinal dorsal horn clock genes such as Per1 expressed could be crucial in the induction of neuropathic pain following peripheral nerve injury. Modulating clock gene Per1 expression could be a novel therapeutic strategy in alleviating neuropathic pain.

Postnatal Constant Light Compensates Cryptochrome1 and 2 Double Deficiency for Disruption of Circadian Behavioral Rhythms in Mice under Constant Dark

Clock genes Cryptochrome (Cry1) and Cry2 are essential for expression of circadian rhythms in mice under constant darkness (DD). However, circadian rhythms in clock gene Per1 expression or clock protein PER2 are detected in the cultured suprachiasmatic nucleus (SCN) of neonatal Cry1 and Cry2 double deficient (Cry1 -/-/Cry2 -/-) mice. A lack of circadian rhythms in adult Cry1 -/-/Cry2 -/- mice is most likely due to developmentally disorganized cellular coupling of oscillating neurons in the SCN. On the other hand, neonatal rats exposed to constant light (LL) developed a tenable circadian system under prolonged LL which was known to fragment circadian behavioral rhythms. In the present study, Cry1 -/-/Cry2 -/- mice were raised under LL from postnatal day 1 for 7 weeks and subsequently exposed to DD for 3 weeks. Spontaneous movement was monitored continuously after weaning and PER2::LUC was measured in the cultured SCN obtained from mice under prolonged DD. Surprisingly, Chi square periodogram analysis revealed significant circadian rhythms of spontaneous movement in the LL-raised Cry1 -/-/Cry2 -/- mice, but failed to detect the rhythms in Cry1 -/-/Cry2 -/- mice raised under light-dark cycles (LD). By contrast, prolonged LL in adulthood did not rescue the circadian behavioral rhythms in the LD raised Cry1 -/-/Cry2 -/- mice. Visual inspection disclosed two distinct activity components with different periods in behavioral rhythms of the LL-raised Cry1-/-/Cry2-/- mice under DD: one was shorter and the other was longer than 24 hours. The two components repeatedly merged and separated. The patterns resembled the split behavioral rhythms of wild type mice under prolonged LL. In addition, circadian rhythms in PER2::LUC were detected in some of the LL-raised Cry1-/-/Cry2-/- mice under DD. These results indicate that neonatal exposure to LL compensates the CRY double deficiency for the disruption of circadian behavioral rhythms under DD in adulthood.

Clock Gene Expression in the Human Pituitary Gland

Pituitary function relies on strictly timed, yet plastic mechanisms, particularly with respect to the daytime-dependent coordination of hormone synthesis and release. In other systems, clock genes and their protein products are well-described candidates to anticipate the daily demands in neuroendocrine coupling and to manage cellular adaptation on changing internal or external circumstances. To elucidate possible mechanisms of time management, a total of 52 human autoptic pituitary glands were allocated to the 4 time-of-day groups, night, dawn, day, and dusk, according to reported time of death. The observed daytime-dependent dynamics in ACTH content supports a postmortem conservation of the premortem condition, and thus, principally validates the investigation of autoptic pituitary glands. Pituitary extracts were investigated for expression of clock genes Per1, Cry1, Clock, and Bmal1 and corresponding protein products. Only the clock gene Per1 showed daytime-dependent differences in quantitative real-time PCR analyses, with decreased levels observed during dusk. Although the overall amount in clock gene protein products PER1, CRY1, and CLOCK did not fluctuate with time of day in human pituitary, an indication for a temporally parallel intracellular translocation of PER1 and CRY1 was detected by immunofluorescence. Presented data suggest that the observed clock gene expression in human pituitary cells does not provide evidence for a functional intrinsic clockwork. It is suggested that clock genes and their protein products may be directly involved in the daytime-dependent regulation and adaptation of hormone synthesis and release and within homeostatic adaptive plasticity.

Simultaneous recording revealed differentially phased circadian rhythms in spontaneous firing and clock gene Per1 expression in the mouse suprachiasmatic nucleus

Simultaneous recording revealed differentially phased circadian rhythms in spontaneous firing and clock gene Per1 expression in the mouse suprachiasmatic nucleus

P3.111 Expression of clock genes Per1 and Bmal1 in total leukocytes in health and Parkinson's disease

P3.111 Expression of clock genes Per1 and Bmal1 in total leukocytes in health and Parkinson's disease

Resveratrol Regulates Circadian Clock Genes in Rat-1 Fibroblast Cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.

Clock mechanisms for seasonal adaptation: Morning and evening oscillators in the suprachiasmatic nucleus

The circadian behavioral rhythms show seasonal changes not only in animals in the field but also in humans in urban environments. In nocturnal rodents the activity time is compressed or decompressed in response to day lengths. A two-oscillator model has been proposed to explain this photoperiodic response; an evening (E) oscillator is entrained to a dusk signal and drives activity onset, while a morning (M) oscillator is entrained to a dawn signal and controls the end of the active period. However, the location and the oscillation mechanism for these oscillators remain to be elucidated. Recent progress in understanding molecular circadian clock mechanisms and bioluminescent reporters for clock gene expressions enabled us to monitor the E and M clocks’ tick separately. We measured clock gene Per1 expression levels continuously from individual cells as well as tissue explants of the hypothalamic suprachiasmatic nucleus (SCN) using transgenic mice carrying a luciferase reporter gene (Per1-luc mice) which were kept in different photoperiods. We found that there are two regionally specific oscillatory cell groups in the SCN that regulate activity onset and end separately in response to photoperiods, which we assume correspond to E and M oscillators. In addition, a third group of oscillatory cells was identified in the SCN only in a long photoperiod. These oscillatory cell networks change dynamically their clock gene expression pattern depending on environmental lights. Similar mechanisms may underlie seasonal changes in sleep time and melatonin rhythms of humans.

Insight Into the Circadian Clock Within Rat Colonic Epithelial Cells

The gastrointestinal tract exhibits diurnal rhythms in many physiologic functions. These rhythms are driven by food intake but are also preserved during food deprivation, suggesting the presence of endogenous circadian rhythmicity. The aim of the study was to provide insight into the circadian core clock mechanism within the rat colon. Moreover, the potency of a restricted feeding regime to shift the circadian clock in the colon was tested. The question of whether the colonic clock drives circadian expression in NHE3, an electroneutral Na(+)/H(+) exchanger, was also addressed. Daily profiles in expression of clock genes Per1, Per2, Cry1, Bmal1, Clock, and Rev-erbalpha, and the NHE3 transporter were examined by reverse transcriptase-polymerase chain reaction and their mRNA levels, as well as PER1 and BMAL1 protein levels, were localized in the colonic epithelium by in situ hybridization and immunocytochemistry, respectively. Expression of Per1, Per2, Cry1, Bmal1, Clock, Rev-erbalpha, and NHE3, as well as PER1 and BMAL1 protein levels, exhibited circadian rhythmicity in the colon. The rhythms were in phase with those in the liver but phase-delayed relative to the master clock in the suprachiasmatic nucleus. Restricted feeding entrained the clock in the colon, because rhythms in clock genes as well as in NHE3 expression were phase-advanced similarly to the clock in the liver. The rat colon harbors a circadian clock. The colonic clock is likely to drive rhythmic NHE3 expression. Restricted feeding resets the colonic clock similarly to the clock in the liver.

Separate oscillating cell groups in mouse suprachiasmatic nucleus couple photoperiodically to the onset and end of daily activity

The pattern of circadian behavioral rhythms is photoperiod-dependent, highlighted by the conservation of a phase relation between the behavioral rhythm and photoperiod. A model of two separate, but mutually coupled, circadian oscillators has been proposed to explain photoperiodic responses of behavioral rhythm in nocturnal rodents: an evening oscillator, which drives the activity onset and entrains to dusk, and a morning oscillator, which drives the end of activity and entrains to dawn. Continuous measurement of circadian rhythms in clock gene Per1 expression by a bioluminescence reporter enabled us to identify the separate oscillating cell groups in the mouse suprachiasmatic nucleus (SCN), which composed circadian oscillations of different phases and responded to photoperiods differentially. The circadian oscillation in the posterior SCN was phase-locked to the end of activity under three photoperiods examined. On the other hand, the oscillation in the anterior SCN was phase-locked to the onset of activity but showed a bimodal pattern under a long photoperiod [light-dark cycle (LD)18:6]. The bimodality in the anterior SCN reflected two circadian oscillatory cell groups of early and late phases. The anterior oscillation was unimodal under intermediate (LD12:12) and short (LD6:18) photoperiods, which was always phase-lagged behind the posterior oscillation when the late phase in LD18:6 was taken. The phase difference was largest in LD18:6 and smallest in LD6:18. These findings indicate that three oscillating cell groups in the SCN constitute regionally specific circadian oscillations, and at least two of them are involved in photoperiodic response of behavioral rhythm.

Postnatal ontogenesis of the circadian clock within the rat liver

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.