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.

Clock knockdown attenuated reactive oxygen species-mediated senescence of chondrocytes through restoring autophagic flux.

AIMS: Articular cartilage degeneration has been recognized as the primary pathological change in osteoarthritis (OA). Mechanisms that govern the shift from cartilage homeostasis to OA remain unknown. Previous studies have reported that intrinsic circadian clock in chondrocytes could function to optimize cartilage repair/remodeling to optimum times of day, but little is known about its molecular mechanisms. This study attempted to investigate the potential role and mechanism of circadian gene Clock in OA pathology. MATERIALS AND METHODS: The expression of Clock in OA chondrocytes and cartilage was detected by qRT-PCR, western blot and immunohistochemistry. Temporal gene expression changes were analyzed using qRT-PCR in chondrocytes transfected with siClock following dexamethasone synchronization. In addition, the effect of Clock knockdown on senescent phenotypes and autophagic flux was evaluated in chondrocytes treated with siClock or siCntrl. KEY FINDINGS: The expression of Clock was up-regulated in OA cartilage from humans and mouse models. Clock knockdown had no influence on rhythmic expression of the downstream genes in primary chondrocytes. We also found that Clock knockdown elevated antioxidant enzyme activities, diminished reactive oxygen species (ROS) production and attenuated senescence of chondrocytes via restoring autophagic flux. SIGNIFICANCE: Clock knockdown can attenuate ROS-mediated senescence of chondrocytes through restoring autophagic flux in non-circadian manner, providing a potential therapeutic target for OA.

Identification of synthetic inhibitors for the DNA binding of intrinsically disordered circadian clock transcription factors.

Essential components of the human circadian clock, BMAL1 and CLOCK, which are intrinsically disordered transcription factors, were expressed and subjected to a fluorescent in vitro binding assay using an E-box DNA fragment. Screening of a chemical library identified 5,8-quinoxalinedione (1), which was found to inhibit binding of the heterodimer BMAL1/CLOCK to E-box at low micromolar concentrations.

The clock gene Gigantea 1 from Petunia hybrida coordinates vegetative growth and inflorescence architecture.

The gene GIGANTEA (GI) appeared early in land plants. It is a single copy gene in most plants and is found in two to three copies in Solanaceae. We analyzed the silencing of one GI copy, Petunia hybrida GI1 (PhGI1), by hairpin RNAs in Petunia in order to gain knowledge about its range of functions. Decreased transcript levels of PhGI1 were accompanied also by a reduction of PhGI2. They were further associated with increased time period between two consecutive peaks for PhGI1 and CHANEL (PhCHL), the orthologue of the blue light receptor gene ZEITLUPE (ZTL), confirming its role in maintaining circadian rhythmicity. Silenced plants were bigger with modified internode length and increased leaf size while flowering time was not altered. We uncovered a new function for PhGI1 as silenced plants showed reduction of flower bud number and the appearance of two flower buds in the bifurcation point, were normally one flower bud and the inflorescence meristem separate. Furthermore, one of the flower buds consistently showed premature flower abortion. Flowers that developed fully were significantly smaller as a result of decreased cell size. Even so the circadian pattern of volatile emission was unchanged in the silenced lines, flowers emitted 20% less volatiles on fresh weight basis over 24 hours and showed changes in the scent profile. Our results indicate a novel role of PhGI1 in the development of reproductive organs in Petunia. PhGI1 therefore represses growth in vegetative plant parts, maintains the typical cymose inflorescence structure, and inhibits premature flower abortion.

Pharmacological Interventions to Circadian Clocks and Their Molecular Bases.

Daily physiological rhythms are regulated by the body's internal timekeeper known as the circadian clock. Expression, post-translational modification, and degradation of clock proteins constituting the circadian clock are precisely controlled in a rhythmic manner. Perturbation of these processes by nature and nurture results in physiological dysfunction and diseases. Small-molecule modulators of clock or clock-related proteins can adjust clock functions, and thus represent a promising method of therapeutic treatment for a range of clock-related diseases. In this review, we will introduce the identification and development of small-molecule compounds that target clock proteins, as well as X-ray crystal structures of protein-compound complexes that facilitate the understanding of clock protein regulation and drug derivatization. Furthermore, we describe the effects of these compounds in a diseased setting and discuss the therapeutic potential of clock modulators.

The circadian clock protein REVERBα inhibits pulmonary fibrosis development.

Pulmonary inflammatory responses lie under circadian control; however, the importance of circadian mechanisms in the underlying fibrotic phenotype is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERBα in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERBα in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERBα revealed regulation of the little-understood transcription factor TBPL1. Both REVERBα and TBPL1 altered integrinß1 focal-adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of 2 human cohorts. In the UK Biobank, circadian strain markers (sleep length, chronotype, and shift work) are associated with pulmonary fibrosis, making them risk factors. In a separate cohort, REVERBα expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERBα inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, thus suggesting that targeting of REVERBα could be a viable therapeutic approach.

An impaired intrinsic microglial clock system induces neuroinflammatory alterations in the early stage of amyloid precursor protein knock-in mouse brain.

BACKGROUND: Disturbances in clock genes affect almost all patients with Alzheimer's disease (AD), as evidenced by their altered sleep/wake cycle, thermoregulation, and exacerbation of cognitive impairment. As microglia-mediated neuroinflammation proved to be a driver of AD rather than a result of the disease, in this study, we evaluated the relationship between clock gene disturbance and neuroinflammation in microglia and their contribution to the onset of AD. METHODS: In this study, the expression of clock genes and inflammatory-related genes was examined in MACS microglia isolated from 2-month-old amyloid precursor protein knock-in (APP-KI) and wild-type (WT) mice using cap analysis gene expression (CAGE) deep sequencing and RT-PCR. The effects of clock gene disturbance on neuroinflammation and relevant memory changes were examined in 2-month-old APP-KI and WT mice after injection with SR9009 (a synthetic agonist for REV-ERB). The microglia morphology was studied by staining, neuroinflammation was examined by Western blotting, and cognitive changes were examined by Y-maze and novel object recognition tests. RESULTS: CLOCK/BMAL1-driven transcriptional negative feedback loops were impaired in the microglia from 2-month-old APP-KI mice. Pro-inflammatory genes in microglia isolated from APP-KI mice were significantly higher than those isolated from WT mice at Zeitgeber time 14. The expression of pro-inflammatory genes was positively associated with NF-κB activation and negatively associated with the BMAL1 expression. SR9009 induced the activation of microglia, the increased expression of pro-inflammatory genes, and cognitive decline in 2-month-old APP-KI mice. CONCLUSION: Clock gene disturbance in microglia is involved in the early onset of AD through the induction of chronic neuroinflammation, which may be a new target for preventing or slowing AD.

A PERK-miR-211 axis suppresses circadian regulators and protein synthesis to promote cancer cell survival.

The unfolded protein response (UPR) is a stress-activated signalling pathway that regulates cell proliferation, metabolism and survival. The circadian clock coordinates metabolism and signal transduction with light/dark cycles. We explore how UPR signalling interfaces with the circadian clock. UPR activation induces a 10 h phase shift in circadian oscillations through induction of miR-211, a PERK-inducible microRNA that transiently suppresses both Bmal1 and Clock, core circadian regulators. Molecular investigation reveals that miR-211 directly regulates Bmal1 and Clock via distinct mechanisms. Suppression of Bmal1 and Clock has the anticipated impact on expression of select circadian genes, but we also find that repression of Bmal1 is essential for UPR-dependent inhibition of protein synthesis and cell adaptation to stresses that disrupt endoplasmic reticulum homeostasis. Our data demonstrate that c-Myc-dependent activation of the UPR inhibits Bmal1 in Burkitt's lymphoma, thereby suppressing both circadian oscillation and ongoing protein synthesis to facilitate tumour progression.

CLOCK-BMAL1 regulate the cardiac L-type calcium channel subunit CACNA1C through PI3K-Akt signaling pathway.

The heterodimerized transcription factors CLOCK-BMAL1 regulate the cardiomyocyte circadian rhythms. The L-type calcium currents play important role in the cardiac electrogenesis and arrhythmogenesis. Whether and how the CLOCK-BMAL1 regulate the cardiac L-type calcium channels are yet to be determined. The functions of the L-type calcium channels were evaluated with patch clamping techniques. Recombinant adenoviruses of CLOCK and BMAL1 were used in the expression experiments. We reported that the expressions and functions of CACNA1C (the α-subunit of the L-type calcium channels) showed circadian rhythms, with the peak at zeitgeber time 3 (ZT3). The endocardial action potential durations 90 (APD90) were correspondingly longer at ZT3. The protein levels of the phosphorylated Akt at threonine 308 (pAkt T308) also showed circadian rhythms. Overexpressions of CLOCK-BMAL1 significantly reduced the levels of CACNA1C while increasing the levels of pAkt T308 and pik3r1. Furthermore, the inhibitory effects of CLOCK-BMAL1 on CACNA1C could be abolished by the Akt inhibitor MK2206 or the PDK1 inhibitor GSK2334470. Collectively, our findings suggested that the expressions of the cardiac CACNA1C were under the CLOCK-BMAL1 regulation, probably through the PI3K-Akt signal pathway.

A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells.

AIM: To determine the impact of a functional human islet clock on insulin secretion and gene transcription. METHODS: Efficient circadian clock disruption was achieved in human pancreatic islet cells by small interfering RNA-mediated knockdown of CLOCK. Human islet secretory function was assessed in the presence or absence of a functional circadian clock by stimulated insulin secretion assays, and by continuous around-the-clock monitoring of basal insulin secretion. Large-scale transcription analysis was accomplished by RNA sequencing, followed by quantitative RT-PCR analysis of selected targets. RESULTS: Circadian clock disruption resulted in a significant decrease in both acute and chronic glucose-stimulated insulin secretion. Moreover, basal insulin secretion by human islet cells synchronized in vitro exhibited a circadian pattern, which was perturbed upon clock disruption. RNA sequencing analysis suggested alterations in 352 transcript levels upon circadian clock disruption. Among them, key regulators of the insulin secretion pathway (GNAQ, ATP1A1, ATP5G2, KCNJ11) and transcripts required for granule maturation and release (VAMP3, STX6, SLC30A8) were affected. CONCLUSIONS: Using our newly developed experimental approach for efficient clock disruption in human pancreatic islet cells, we show for the first time that a functional ß-cell clock is required for proper basal and stimulated insulin secretion. Moreover, clock disruption has a profound impact on the human islet transcriptome, in particular, on the genes involved in insulin secretion.

hCLOCK Causes Rho-Kinase-Mediated Endothelial Dysfunction and NF-κB-Mediated Inflammatory Responses.

BACKGROUND: The human Circadian Locomotor Output Cycle protein Kaput (CLOCK) gene was originally discovered as a regulator of essential human daily rhythms. This seemingly innocuous gene was then found to be associated with a multitude of human malignancies, via several biochemical pathways. We aimed to further investigate the role of hCLOCK in the hypoxia-oxidative stress response system at the biochemical level. METHODS: Expression levels of Rho GTPases were measured in normoxic and hypoxic states. The effect of hCLOCK on the hypoxic response was evaluated with the use of a retroviral shRNA vector system, a Rho inhibitor, and a ROS scavenger by analyzing expression levels of hCLOCK, Rho GTPases, and NF-κB pathway effectors. Finally, in vitro ROS production and tube formation in HUVECs were assessed. RESULTS: Hypoxia induces ROS production via hCLOCK. hCLOCK activates the RhoA and NF-κB signaling pathways. Conversely, inhibition of hCLOCK deactivates these pathways. Furthermore, inhibition of RhoA or decreased levels of ROS attenuate these pathways, but inhibition of RhoA does not lead to decreased levels of ROS. Overall findings show that hypoxia increases the expression of hCLOCK, which leads to ROS production, which then activates the RhoA and NF-κB pathways. CONCLUSION: Our findings suggest that hypoxic states induce vascular oxidative damage and inflammation via hCLOCK-mediated production of ROS, with subsequent activation of the RhoA and NF-κB pathways.

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.

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.

Nuclear receptors rock around the clock.

Circadian rhythms characterize almost every aspect of human physiology, endocrinology, xenobiotic detoxification, cell growth, and behavior. Modern lifestyles that disrupt our normal circadian rhythms are increasingly thought to contribute to various disease conditions ranging from depression and metabolic disorders to cancer. This self-sustained time-keeping system is generated and maintained by an endogenous molecular machine, the circadian clock, which is a transcriptional mechanism composed of the transcription factors CLOCK and BMAL and their co-repressors, PER and CRY. Nuclear receptors (NRs) represent a large family of hormone-sensitive transcriptional regulators involved in a myriad of biological processes such as development, energy metabolism, reproduction, inflammation, and tissue homeostasis. Recent studies point not only to NR regulation by the clock, but also to NR regulation of the clock itself. Here, we discuss recent studies that functionally and mechanistically implicate NRs as key components of both the universal and adaptive circadian clock mechanisms. As proven pharmacological targets, nuclear receptors are promising targets for therapeutic control of many pathological conditions associated with the disruption of circadian rhythm.

Downregulation of core clock gene Bmal1 attenuates expression of progesterone and prostaglandin biosynthesis-related genes in rat luteinizing granulosa cells.

Ovarian circadian oscillators have been implicated in the reproductive processes of mammals. However, there are few reports regarding the detection of ovarian clock-controlled genes (CCGs). The present study was designed to unravel the mechanisms through which CCG ovarian circadian oscillators regulate fertility, primarily using quantitative RT-PCR and RNA interference against Bmal1 in rat granulosa cells. Mature granulosa cells were prepared from mouse Per2-destabilized luciferase (dLuc) reporter gene transgenic rats. A real-time monitoring system of Per2 promoter activity was employed to detect Per2-dLuc oscillations. The cells exposed to luteinizing hormone (LH) displayed clear Per2-dLuc oscillations and a rhythmic expression of clock genes (Bmal1, Per1, Per2, Rev-erbα, and Dbp). Meanwhile, the examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Lhcgr, and p53) showed rhythmic transcript profiles except for Hsd3b2, indicating that these rhythmic expression genes may be CCGs. Notably, Bmal1 small interfering (si)RNA treatment significantly decreased both the amplitude of Per2-dLuc oscillations and Bmal1 mRNA levels compared with nonsilencing RNA treatment in luteinizing granulosa cells. Depletion of Bmal1 by siRNA decreased the transcript levels of clock genes (Per1, Per2, Rev-erbα, and Dbp) and examined ovarian genes (Star, Cyp19a1, Cyp11a1, Ptgs2, Hsd3b2, and Lhcgr). Accordingly, knockdown of Bmal1 also inhibited the synthesis of progesterone and prostaglandin E2, which are associated with crucial reproductive processes. Collectively, these data suggest that ovarian circadian oscillators regulate the synthesis of steroid hormones and prostaglandins through ovarian-specific CCGs in response to LH stimuli. The present study provides new insights into the physiologic significance of Bmal1 related to fertility in ovarian circadian oscillators.

Transforming growth factor-beta inhibits the expression of clock genes.

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-ß) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-ß2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-ß2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-ß2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-ß2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients.

Melatonin attenuates clock gene cryptochrome1, which may aggravate mouse anti-type II collagen antibody-induced arthritis.

Very recently, the circadian rhythm was proved to play an important role in the pathogenesis of arthritis. The role of melatonin in the development and progress of rheumatoid arthritis has been implicated for decades. This study was aimed to investigate the effect of melatonin on the expression of circadian clock genes in mouse anti-type II collagen antibody-induced arthritis (CIA). Mice were divided into 3 groups: control, CIA, and CIA + melatonin treatment (MLT). Both mRNA and protein levels of circadian clock gene Cryptochrome1 (Cry1) were markedly decreased in CIA + MEL group compared with those in control and CIA groups. MLT increased paw thickness. Histologic and X-ray assessment also revealed increased infiltration of inflammatory cells, synovial hyperplasia, and the destruction of articular cartilage and bone by MLT. The concentrations of anti-type II collagen antibody in CIA + MEL group mice were significantly higher than those in control and CIA groups (P < 0.05). Serum concentrations of TNF-α (P < 0.005) and IL-6 (P < 0.05) in CIA + MLT group were also increased. Taken together, these results implicate that clock gene Cry1 may be involved in the aggravation of MLT-mediated arthritis in mice anti-type II collagen antibody-induced arthritis.