Antisense-Induced Downregulation of Clock Genes in the Shell Region of the Nucleus Accumbens Reduces Binge Drinking in Mice.

INTRODUCTIONS: Binge drinking is a deadly pattern of alcohol consumption. Evidence suggests that genetic variation in clock genes is strongly associated with alcohol misuse; however, the neuroanatomical basis for such a relationship is unknown. The shell region of the nucleus accumbens (NAcSh) is well known to play a role in binge drinking. Hence, we examined whether clock genes in the NAcSh regulate binge drinking. METHODS: To address this question, 2 experiments were performed on male C57BL/6J mice. In the first experiment, mice exposed to alcohol or sucrose under the 4-day drinking-in-the-dark (DID) paradigm were euthanized at 2 different time points on day 4 [7 hours after light (pre-binge drinking) or dark (post-binge drinking) onset]. The brains were processed for RT-PCR to examine the expression of circadian clock genes (Clock, Per1, and Per2) in the NAcSh and suprachiasmatic nucleus (SCN). In the second experiment, mice were exposed to alcohol, sucrose, or water as described above. On day 4, 1 hour prior to the onset of alcohol exposure, mice were bilaterally infused with either a mixture of circadian clock gene antisense oligodeoxynucleotides (AS-ODNs; antisense group) or nonsense/random ODNs (R-ODNs; control group) through surgically implanted cannulas above the NAcSh. Alcohol/sucrose/water consumption was measured for 4 hours. Blood alcohol concentration was measured to confirm binge drinking. Microinfusion sites were histologically verified using cresyl violet staining. RESULTS: As compared to sucrose, mice euthanized post-binge drinking (not pre-binge drinking) on day 4 displayed a greater expression of circadian genes in the NAcSh but not in the SCN. Knockdown of clock genes in the NAcSh caused a significantly lower volume of alcohol to be consumed on day 4 than in the control treatment. No differences were found in sucrose or water consumption. CONCLUSIONS: Our results suggest that clock genes in the NAcSh play a crucial role in binge drinking.

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.

Role of Per3, a circadian clock gene, in embryonic development of mouse cerebral cortex.

Per3 is one of the primary components of circadian clock system. While circadian dysregulation is known to be involved in the pathogenesis of several neuropsychiatric diseases. It remains largely unknown whether they participate in embryonic brain development. Here, we examined the role of clock gene Per3 in the development of mouse cerebral cortex. In situ hybridization analysis revealed that Per3 is expressed in the developing mouse cortex. Acute knockdown of Per3 with in utero electroporation caused abnormal positioning of cortical neurons, which was rescued by RNAi-resistant Per3. Per3-deficient cells showed abnormal migration phenotypes, impaired axon extension and dendritic arbor formation. Taken together, Per3 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation.

Direct and indirect inhibition of the circadian clock protein Per1: effects on ENaC and blood pressure.

Circadian rhythms govern physiological functions and are important for overall health. The molecular circadian clock comprises several transcription factors that mediate circadian control of physiological function, in part, by regulating gene expression in a tissue-specific manner. These connections are well established, but the underlying mechanisms are incompletely understood. The overall goal of this study was to examine the connection among the circadian clock protein Period 1 (Per1), epithelial Na+ channel (ENaC), and blood pressure (BP) using a multipronged approach. Using global Per1 knockout mice on a 129/sv background in combination with a high-salt diet plus mineralocorticoid treatment, we demonstrated that loss of Per1 in this setting is associated with protection from hypertension. Next, we used the ENaC inhibitor benzamil to demonstrate a role for ENaC in BP regulation and urinary Na+ excretion in 129/sv mice. We targeted Per1 indirectly using pharmacological inhibition of Per1 nuclear entry in vivo to demonstrate altered expression of known Per1 target genes as well as a BP-lowering effect in 129/sv mice. Finally, we directly inhibited Per1 via genetic knockdown in amphibian distal nephron cells to demonstrate, for the first time, that reduced Per1 expression is associated with decreased ENaC activity at the single channel level.

Role of the clock gene Period3 in the human cell-autonomous circadian clock.

Previous studies have shown that mouse Period3 (mPer3) is dispensable for the generation of autonomous oscillations in the circadian clock. However, human studies have suggested that human Period3 (hPer3) may have more important roles in the core clock machinery than mPer3. To investigate the role of hPer3 protein in the cell-autonomous circadian oscillator, we conducted gene knockout of the hPer3 gene in human bone osteosarcoma epithelial cells using genome-editing technology. We examined the circadian transcription of endogenous clock genes in hPer3-deficient cell clones and found that hPer3-deficient cells showed a phase advance in circadian transcription compared to wild-type cells. We subsequently transfected wild-type and mutant cells with an adenovirus carrying a luciferase gene whose expression was driven by a clock gene promotor, and monitored bioluminescence in real time. Cosinor analysis showed that the circadian period length in all hPer3-deficient cells was significantly shorter than that in wild-type cells, demonstrating that the phase advance in endogenous clock gene expression in hPer3-deficient cell clones was attributable to a shortened circadian period length rather than a phase shift. Together these findings are consistent with previous studies in mice lacking functional mPer3, indicating that the Per3 protein functions similarly in both mice and humans.

The auxin-inducible degradation system enables conditional PERIOD protein depletion in the nervous system of Drosophila melanogaster.

Tools that allow inducible and reversible depletion of target proteins are critical for biological studies. The plant-derived auxin-inducible degradation system (AID) enables the degradation of target proteins tagged with the AID motif. This system has been recently employed in mammalian cells as well as in Caenorhabditis elegans and Drosophila. To test the utility of the AID approach in the nervous system, we used circadian locomotor rhythms as a model and applied the AID method to temporally and spatially degrade PERIOD (PER), a critical pacemaker protein in Drosophila. We found that the period locus can be efficiently tagged with the AID motif by CRISPR/Cas9-based genome editing without disrupting PER function. Moreover, we demonstrated that the AID system could be used to induce rapid and efficient protein degradation in the nervous system as shown by effects on circadian and sleep behaviors. Furthermore, the protein degradation by AID was rapidly reversible after auxin removal. Together, our results show that the AID system provides a powerful tool for behavior studies in Drosophila.

The Circadian PER2 Enhancer Nobiletin Reverses the Deleterious Effects of Midazolam in Myocardial Ischemia and Reperfusion Injury.

BACKGROUND: Recently, we identified the circadian rhythm protein Period 2 (PER2) in robust cardioprotection from myocardial ischemia (MI). Based on findings that perioperative MI is the most common major cardiovascular complication and that anesthetics can alter the expression of PER2, we hypothesized that an anesthesia mediated downregulation of PER2 could be detrimental if myocardial ischemia and reperfusion (IR) would occur. METHODS AND RESULTS: We exposed mice to pentobarbital, fentanyl, ketamine, propofol, midazolam or isoflurane and determined cardiac Per2 mRNA levels. Unexpectedly, only midazolam treatment resulted in an immediate and significant downregulation of Per2 transcript levels. Subsequent studies in mice pretreated with midazolam using an in-situ mouse model for myocardial (IR)-injury revealed a significant and dramatic increase in infarct sizes or Troponin-I serum levels in the midazolam treated group when compared to controls. Using the recently identified flavonoid, nobiletin, as a PER2 enhancer completely abolished the deleterious effects of midazolam during myocardial IR-injury. Moreover, nobiletin treatment alone significantly reduced infarct sizes or Troponin I levels in wildtype but not in Per2-/- mice. Pharmacological studies on nobiletin like flavonoids revealed that only nobiletin and tangeritin, both found to enhance PER2, were cardioprotective in our murine model for myocardial IR-injury. CONCLUSION: We identified midazolam mediated downregulation of cardiac PER2 as an underlying mechanism for a deleterious effect of midazolam pretreatment in myocardial IR-injury. These findings highlight PER2 as a cardioprotective mechanism and suggest the PER2 enhancers nobiletin or tangeritin as a preventative therapy for myocardial IR-injury in the perioperative setting where midazolam pretreatment occurs frequently.

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.

The clock gene PER1 suppresses expression of tumor-related genes in human oral squamous cell carcinoma.

Abnormal expression of the clock gene PER1 is highly correlated with carcinogenesis and the development of malignant tumors. Here, we designed short hairpin RNAs (shRNAs) to effectively knock down PER1 in SCC15 human oral squamous cell carcinoma cells. shRNA-mediated PER1 knockdown promoted SCC15 cell growth, proliferation, apoptosis resistance, migration and invasion in vitro. PER1 knockdown also increased the cells' expression of KI-67, MDM2, BCL-2, MMP2 and MMP9 mRNA, and decreased expression of C-MYC, p53, BAX and TIMP-2. In BALB/c nu/nu nude mice subcutaneously injected with SCC15 cells, PER1 knockdown in the cells enhanced tumor development, leading to increased tumor weights and volumes. These results suggest that PER1 is an important tumor suppressor gene and may be a useful molecular target for the treatment of cancer.

The Increase in Signaling by Kisspeptin Neurons in the Preoptic Area and Associated Changes in Clock Gene Expression That Trigger the LH Surge in Female Rats Are Dependent on the Facilitatory Action of a Noradrenaline Input.

In rodents, kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) of the preoptic area are considered to provide a major stimulatory input to the GnRH neuronal network that is responsible for triggering the preovulatory LH surge. Noradrenaline (NA) is one of the main modulators of GnRH release, and NA fibers are found in close apposition to kisspeptin neurons in the RP3V. Our objective was to interrogate the role of NA signaling in the kisspeptin control of GnRH secretion during the estradiol induced LH surge in ovariectomized rats, using prazosin, an α1-adrenergic receptor antagonist. In control rats, the estradiol-induced LH surge at 17 hours was associated with a significant increase in GnRH and kisspeptin content in the median eminence with the increase in kisspeptin preceding that of GnRH and LH. Prazosin, administered 5 and 3 hours prior to the predicted time of the LH surge truncated the LH surge and abolished the rise in GnRH and kisspeptin in the median eminence. In the preoptic area, prazosin blocked the increases in Kiss1 gene expression and kisspeptin content in association with a disruption in the expression of the clock genes, Per1 and Bmal1. Together these findings demonstrate for the first time that NA modulates kisspeptin synthesis in the RP3V through the activation of α1-adrenergic receptors prior to the initiation of the LH surge and indicate a potential role of α1-adrenergic signaling in the circadian-controlled pathway timing of the preovulatory LH surge.

EGR1 regulates hepatic clock gene amplitude by activating Per1 transcription.

The mammalian clock system is composed of a master clock and peripheral clocks. At the molecular level, the rhythm-generating mechanism is controlled by a molecular clock composed of positive and negative feedback loops. However, the underlying mechanisms for molecular clock regulation that affect circadian clock function remain unclear. Here, we show that Egr1 (early growth response 1), an early growth response gene, is expressed in mouse liver in a circadian manner. Consistently, Egr1 is transactivated by the CLOCK/BMAL1 heterodimer through a conserved E-box response element. In hepatocytes, EGR1 regulates the transcription of several core clock genes, including Bmal1, Per1, Per2, Rev-erbα and Rev-erbß, and the rhythm amplitude of their expression is dependent on EGR1's transcriptional function. Further mechanistic studies indicated that EGR1 binds to the proximal region of the Per1 promoter to activate its transcription directly. When the peripheral clock is altered by light or feeding behavior transposition in Egr1-deficient mice, the expression phase of hepatic clock genes shifts normally, but the amplitude is also altered. Our data reveal a critical role for EGR1 in the regulation of hepatic clock circuitry, which may contribute to the rhythm stability of peripheral clock oscillators.

miR-92a Corrects CD34+ Cell Dysfunction in Diabetes by Modulating Core Circadian Genes Involved in Progenitor Differentiation.

Autologous CD34(+) cells are widely used for vascular repair; however, in individuals with diabetes and microvascular disease these cells are dysfunctional. In this study, we examine expression of the clock genes Clock, Bmal, Per1, Per2, Cry1, and Cry2 in CD34(+) cells of diabetic and nondiabetic origin and determine the small encoding RNA (miRNA) profile of these cells. The degree of diabetic retinopathy (DR) was assessed. As CD34(+) cells acquired mature endothelial markers, they exhibit robust oscillations of clock genes. siRNA treatment of CD34(+) cells revealed Per2 as the only clock gene necessary to maintain the undifferentiated state of CD34(+) cells. Twenty-five miRNAs targeting clock genes were identified. Three of the miRNAs (miR-18b, miR-16, and miR-34c) were found only in diabetic progenitors. The expression of the Per2-regulatory miRNA, miR-92a, was markedly reduced in CD34(+) cells from individuals with DR compared with control subjects and patients with diabetes with no DR. Restoration of miR-92a levels in CD34(+) cells from patients with diabetes with DR reduced the inflammatory phenotype of these cells and the diabetes-induced propensity toward myeloid differentiation. Our studies suggest that restoring levels of miR-92a could enhance the usefulness of CD34(+) cells in autologous cell therapy.

The clock protein period 2 synchronizes mitotic expansion and decidual transformation of human endometrial stromal cells.

Implantation requires coordinated interactions between the conceptus and surrounding decidual cells, but the involvement of clock genes in this process is incompletely understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK (circadian locomotor output cycles kaput) and brain muscle arnt-like 1 and repressors encoded by PER (Period) and Cryptochrome genes. We show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Down-regulation occurred between 12 and 24 hours following differentiation and coincided with reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter. RNA sequencing revealed that premature inhibition of PER2 by small interfering RNA knockdown leads to a grossly disorganized decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators among the 1121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of 70 midluteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure (Spearman rank test, ρ = -0.3260; P = 0.0046). Thus, PER2 synchronizes endometrial proliferation with initiation of aperiodic decidual gene expression; uncoupling of these events may cause recurrent pregnancy loss.

Chronic consumption of dietary proanthocyanidins modulates peripheral clocks in healthy and obese rats.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

A positive role for PERIOD in mammalian circadian gene expression.

In the current model of the mammalian circadian clock, PERIOD (PER) represses the activity of the circadian transcription factors BMAL1 and CLOCK, either independently or together with CRYPTOCHROME (CRY). Here, we provide evidence that PER has an entirely different function from that reported previously, namely, that PER inhibits CRY-mediated transcriptional repression through interference with CRY recruitment into the BMAL1-CLOCK complex. This indirect positive function of PER is consistent with previous data from genetic analyses using Per-deficient or mutant mice. Overall, our results support the hypothesis that PER plays different roles in different circadian phases: an early phase in which it suppresses CRY activity, and a later phase in which it acts as a transcriptional repressor with CRY. This buffering effect of PER on CRY might help to prolong the period of rhythmic gene expression. Additional studies are required to carefully examine the promoter- and phase-specific roles of PER.

Chronic exposure to low doses of pharmaceuticals disturbs the hepatic expression of circadian genes in lean and obese mice.

Drinking water can be contaminated with pharmaceuticals. However, it is uncertain whether this contamination can be harmful for the liver, especially during obesity. Hence, the goal of our study was to determine whether chronic exposure to low doses of pharmaceuticals could have deleterious effects on livers of lean and obese mice. To this end, lean and ob/ob male mice were treated for 4 months with a mixture of 11 drugs provided in drinking water at concentrations ranging from 10 to 106 ng/l. At the end of the treatment, some liver and plasma abnormalities were observed in ob/ob mice treated with the cocktail containing 106 ng/l of each drug. For this dosage, a gene expression analysis by microarray showed altered expression of circadian genes (e.g. Bmal1, Dbp, Cry1) in lean and obese mice. RT-qPCR analyses carried out in all groups of animals confirmed that expression of 8 different circadian genes was modified in a dose-dependent manner. For some genes, a significant modification was observed for dosages as low as 10²-10³ ng/l. Drug mixture and obesity presented an additive effect on circadian gene expression. These data were validated in an independent study performed in female mice. Thus, our study showed that chronic exposure to trace pharmaceuticals disturbed hepatic expression of circadian genes, particularly in obese mice. Because some of the 11 drugs can be found in drinking water at such concentrations (e.g. acetaminophen, carbamazepine, ibuprofen) our data could be relevant in environmental toxicology, especially for obese individuals exposed to these contaminants.

Egr1 regulates lithium-induced transcription of the Period 2 (PER2) gene.

A growing body of evidence suggests that the circadian molecular system is involved in the pathogenic and therapeutic mechanisms underlying bipolar disorders. Lithium, a representative mood stabilizer, has been reported to induce the Period 2 (PER2) gene; however, the underlying molecular mechanisms require further study. We found that lithium upregulated PER2 expression at the transcriptional level in neuronally differentiated SH-SY5Y human neuroblastoma cells. Promoter reporter analyses using serial deletions of the PER2 promoter revealed that two early growth response 1 (Egr1)-binding sites (EBS) between positions -180 and -100 are required for maximal activation of the PER2 promoter by lithium. Ectopic expression of Egr1 enhanced lithium-induced PER2 promoter activity, while a point mutation in EBS abolished it. Electrophoretic mobility shift assays and chromatin immunoprecipitation indicated that Egr1 bound directly to the PER2 promoter. Stimulation of the extracellular-signal regulated kinase (ERK)1/2/Elk1 pathway by lithium was functionally linked to PER2 expression through Egr1 induction, and lithium-induced PER2 expression was strongly attenuated by depletion of Egr1 by siRNA. Lithium also upregulated the expression of Per2 and Egr1 in mouse frontal cortex. Induction of Per2 by lithium was attenuated in Egr1(-/-) mice. In conclusion, lithium stimulates PER2 transcription through the ERK/Elk1/Egr1 pathway in neuronal cells, indicating a connection between the ERK-Egr1 pathway and a circadian gene system in the mechanism of action of lithium.

Mutual antagonism between circadian protein period 2 and hepatitis C virus replication in hepatocytes.

BACKGROUND: Hepatitis C virus (HCV) infects approximately 3% of the world population and is the leading cause of liver disease, impacting hepatocyte metabolism, depending on virus genotype. Hepatic metabolic functions show rhythmic fluctuations with 24-h periodicity (circadian), driven by molecular clockworks ticking through translational-transcriptional feedback loops, operated by a set of genes, called clock genes, encoding circadian proteins. Disruption of biologic clocks is implicated in a variety of disorders including fatty liver disease, obesity and diabetes. The relation between HCV replication and the circadian clock is unknown. METHODS: We investigated the relationship between HCV core infection and viral replication and the expression of clock genes (Rev-Erbα, Rorα, ARNTL, ARNTL2, CLOCK, PER1, PER2, PER3, CRY1 and CRY2) in two cellular models, the Huh-7 cells transiently expressing the HCV core protein genotypes 1b or 3a, and the OR6 cells stably harboring the full-length hepatitis C genotype 1b replicon, and in human liver biopsies, using qRT-PCR, immunoblotting, luciferase assays and immunohistochemistry. RESULTS: In Huh-7 cells expressing the HCV core protein genotype 1b, but not 3a, and in OR6 cells, transcript and protein levels of PER2 and CRY2 were downregulated. Overexpression of PER2 led to a consistent decrease in HCV RNA replicating levels and restoration of altered expression pattern of a subset of interferon stimulated genes (ISGs) in OR6 cells. Furthermore, in liver biopsies from HCV genotype 1b infected patients, PER2 was markedly localized to the nucleus, consistent with an auto-inhibitory transcriptional feedback loop. CONCLUSIONS: HCV can modulate hepatic clock gene machinery, and the circadian protein PER2 counteracts viral replication. Further understanding of circadian regulation of HCV replication and rhythmic patterns of host-hosted relationship may improve the effectiveness of HCV antiviral therapy. This would extend to hepatic viral infections the current spectrum of chronotherapies, implemented to treat metabolic, immune related and neoplastic disease.

Aging-like circadian disturbances in folate-deficient mice.

The elderly population shows various circadian disturbances, including dampened amplitude of rhythmicity and decreased responsiveness to light. The common poor folate status in the elderly might account for these aging-related circadian disturbances. To test this hypothesis, we investigated whether folate deficiency in mice affects circadian oscillations of the master clock in the suprachiasmatic nuclei, and the shifting responses to light. Mice fed a diet without folate for 6 weeks displayed markedly reduced (4.5-fold) erythrocyte folate concentration and increased (2.3-fold) homocysteinemia compared with control mice. Folate deficiency decreased the circadian amplitude of vasopressin and the clock protein PERIOD 2 (PER2) in the master clock, slowed the rate of re-entrainment of behavioral rhythms after delayed light-dark cycle and reduced light-induced phase-delays, without detectable morphologic changes in the retina, such as the number of melanopsinergic ganglion cells, that might have impaired photodetection. In conclusion, folate deficiency and consecutive hyperhomocysteinemia led to dampened PER2 and vasopressin oscillations in the master clock and reduced responsiveness to photic resetting, which constitute hallmarks of aging effects on circadian rhythmicity.

Time-dependent repression of mPer2 expression in the suprachiasmatic nucleus by inhalation anesthesia with sevoflurane.

Some anesthetics can affect gene expression. Previously, we reported that sevoflurane anesthesia drastically and reversibly repressed the expression of mouse Per2 (mPer2), a core clock gene in the suprachiasmatic nucleus (SCN). In the current study, we examined the time-dependent effect of sevoflurane on mPer2 expression and its interactions with the circadian rest/activity rhythm of mice. During certain hours of the day, mice were anesthetized with 2.5% sevoflurane in 40% oxygen for 4h. The expression level of mPer2 in the SCN was measured by in situ hybridization using a radiolabeled cRNA probe. Anesthesia during the morning hours showed the greatest repressive effect on mPer2 expression. Sevoflurane anesthesia repressed mPer2 expression during the conditions of light/dark and constant dark, and the light conditions modified the repression rate under anesthesia. Moreover, anesthesia in the morning also repressed mPer2 expression the following day. This dominant effect of anesthesia in the morning indicates that sevoflurane anesthesia affects the onset of mPer2 transcription. Behavior analysis revealed that the anesthetic treatment also induced a phase-delay in the rest/activity rhythm. However, no time-dependent effects of anesthesia on the circadian rest/activity rhythm were observed. Further investigation into the molecular events caused by anesthesia are required to explain atypical clinical signs observed in patients after surgical procedures, such as fatigue, sleep disorder, mood alteration and delirium.