Characterization and Modeling of Intermittent Locomotor Dynamics in Clock Gene-Deficient Mice

The scale-invariant and intermittent dynamics of animal behavior are attracting scientific interest. Recent findings concerning the statistical laws of behavioral organization shared between healthy humans and wild-type mice (WT) and their alterations in human depression patients and circadian clock gene (Period 2; Per2) mutant mice indicate that clock genes play functional roles in intermittent, ultradian locomotor dynamics. They also claim the clinical and biological importance of the laws as objective biobehavioral measures or endophenotypes for psychiatric disorders. In this study, to elucidate the roles of breakdown of the broader circadian regulatory circuit in intermittent behavioral dynamics, we studied the statistical properties and rhythmicity of locomotor activity in Per2 mutants and mice deficient in other clock genes (Bmal1, Clock). We performed wavelet analysis to examine circadian and ultradian rhythms and estimated the cumulative distributions of resting period durations during which locomotor activity levels are continuously lower than a predefined threshold value. The wavelet analysis revealed significant amplification of ultradian rhythms in the BMAL1-deficient mice, and instability in the Per2 mutants. The resting period distributions followed a power-law form in all mice. While the distributions for the BMAL1-deficient and Clock mutant mice were almost identical to those for the WT mice, with no significant differences in their parameter (power-law scaling exponent), only the Per2 mutant mice showed consistently and significantly lower values of the scaling exponent, indicating the increased intermittency in ultradian locomotor dynamics. Furthermore, based on a stochastic priority queuing model, we explained the power-law nature of resting period distributions, as well as its alterations shared with human depressive patients and Per2 mutant mice. Our findings lead to the development of a novel mathematical model for abnormal behaviors in psychiatric disorders.     

A Molecular Clock Regulates Angiopoietin-Like Protein 2 Expression

Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.     

Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation

Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the ROS scavenger N-acetylcysteine. Expression of Nox1 and Nox2 was increased upon differentiation of ATDC5 cells and primary mouse chondrocytes, whereas that of Nox4, which was relatively high initially, was decreased gradually during chondrogenesis. In developing limb, Nox1 and Nox2 were highly expressed in prehypertrophic and hypertrophic chondrocytes. However, Nox4 was highly expressed in proliferating chondrocytes and prehypertrophic chondrocytes. Depletion of Nox2 or Nox4 expression by RNA interference blocked both ROS generation and differentiation of ATDC5 cells, whereas depletion of Nox1 had no such effect. We also found that ATDC5 cells depleted of Nox2 or Nox4 underwent apoptosis. Further, inhibition of Akt phosphorylation along with subsequent activation of ERK was observed in the cells. Finally, depletion of Nox2 or Nox4 inhibited the accumulation of proteoglycan in primary chondrocytes. Taken together, our data suggest that ROS generated by Nox2 or Nox4 are essential for survival and differentiation in the early stage of chondrogenesis.     

IL-1β induces changes in expression of core circadian clock components PER2 and BMAL1 in primary human chondrocytes through the NMDA receptor/CREB and NF-κB signalling pathways

The circadian clock is a specialised cell signalling circuit present in almost all cells. It controls the timing of key cell activities such as proliferation and differentiation. In osteoarthritis, expression of two components of the circadian clock, BMAL1 and PER2 is altered in chondrocytes and this change has been causally linked with the increase in proliferation and altered chondrocyte differentiation in disease. IL-1β, an inflammatory cytokine abundant in OA joints, has previously been shown to induce changes in BMAL1 and PER2 expression in chondrocytes. The purpose of this study is to identify the mechanism involved.We found IL-1β treatment of primary human chondrocytes led to activation of NMDA receptors as evidenced by an increase in phosphorylation of GluN1 and an increase in intracellular calcium which was blocked by the NMDAR antagonist MK801. Levels of phosphorylated CREB were also elevated in IL-1β treated cells and this effect was blocked by co-treatment of cells with IL-1β and the NMDAR antagonist MK-801. Knockdown of CREB or inhibition of CREB activity prevented the IL-1β induced increase in PER2 expression in chondrocytes but had no effect on BMAL1. Phosphorylated p65 levels were elevated in IL-1β treated chondrocytes indicating increased NF-κB activation. Inhibition of NF-κB activity prevented the IL-1β induced reduction in BMAL1 expression and partially mitigated the IL-1β induced increase in PER2 expression in chondrocytes. These data indicate that the NMDAR/CREB and NF-κB signalling pathways regulate the core circadian clock components PER2 and BMAL1 in chondrocytes. Given that changes in expression of these clock components have been observed in a wide range of diseases, these findings may be broadly relevant for understanding the mechanism leading to circadian clock changes in pathology.     

Palmitate Inhibits SIRT1-Dependent BMAL1/CLOCK Interaction and Disrupts Circadian Gene Oscillations in Hepatocytes

Elevated levels of serum saturated fatty acid palmitate have been shown to promote insulin resistance, increase cellular ROS production, and trigger cell apoptosis in hepatocytes during the development of obesity. However, it remains unclear whether palmitate directly impacts the circadian clock in hepatocytes, which coordinates nutritional inputs and hormonal signaling with downstream metabolic outputs. Here we presented evidence that the molecular clock is a novel target of palmitate in hepatocytes. Palmitate exposure at low dose inhibits the molecular clock activity and suppresses the cyclic expression of circadian targets including Dbp, Nr1d1 and Per2 in hepatocytes. Palmitate treatment does not seem to alter localization or reduce protein expression of BMAL1 and CLOCK, the two core components of the molecular clock in hepatocytes. Instead, palmitate destabilizes the protein-protein interaction between BMAL1-CLOCK in a dose and time-dependent manner. Furthermore, we showed that SIRT1 activators could reverse the inhibitory action of palmitate on BMAL1-CLOCK interaction and the clock gene expression, whereas inhibitors of NAD synthesis mimic the palmitate effects on the clock function. In summary, our findings demonstrated that palmitate inhibits the clock function by suppressing SIRT1 function in hepatocytes.     

Glucocorticoids Affect 24 h Clock Genes Expression in Human Adipose Tissue Explant Cultures

Aims

to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock genes expression.

Subjects and Methods

VAT and SAT biopsies were obtained from morbid obese women (body mass index≥40 kg/m²) (n = 6). In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX) and AT explants treated with DEX (2 hours) were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR.

Results

CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element) was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements) in the SAT (situation not present in VAT). A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues.

Conclusions

24 h patterns in CLOCK and BMAL1 (positive clock elements) and PER2 (negative element) mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.