Circadian Gene cry Controls Glioblastoma Tumorigenesis through Modulation of myc Expression

Glioblastoma (GB) is the most frequent malignant brain tumor among adults and currently there is no effective treatment. It is a very aggressive tumor that grows fast and spreads through the brain causing the death of patients in 15 months. GB cells mutate frequently and generate a heterogeneous population of tumoral cells genetically distinct. Thus, the contribution of genes and signaling pathways relevant for GB progression is of great relevance. We use a Drosophila model of GB that reproduces the features of human GB, and describe the upregulation of the circadian gene cry in GB patients and in a Drosophila GB model. We study the contribution of cry to the expansion of GB cells, to the neurodegeneration caused by GB, and to premature death and determine that cry is required for GB progression. Moreover, we analyze the mechanisms that regulate cry expression by the PI3K pathway. Finally, we conclude that cry is necessary and sufficient to regulate myc expression in GB. These results contribute to the understanding of the signals that impulse GB malignancy and lethality and open novel opportunities for the treatment of GB patients.

A ROS-Ca2+ signalling pathway identified from a chemical screen for modifiers of sugar-activated circadian gene expression.

Sugars are essential metabolites for energy and anabolism that can also act as signals to regulate plant physiology and development. Experimental tools to disrupt major sugar signalling pathways are limited. We have performed a chemical screen for modifiers of activation of circadian gene expression by sugars to discover pharmacological tools to investigate and manipulate plant sugar signalling. Using a library of commercially available bioactive compounds, we identified 75 confident hits that modified the response of a circadian luciferase reporter to sucrose in dark-adapted seedlings. We validated the transcriptional effect on a subset of the hits and measured their effects on a range of sugar-dependent phenotypes for 13 of these chemicals. Chemicals were identified that appear to influence known and unknown sugar signalling pathways. Pentamidine isethionate (PI) was identified as a modifier of a sugar-activated Ca2+ signal that acts downstream of superoxide in a metabolic signalling pathway affecting circadian rhythms, primary metabolism and plant growth. Our data provide a resource of new experimental tools to manipulate plant sugar signalling and identify novel components of these pathways.

Polymethoxyflavones in Citrus Regulate Lipopolysaccharide-Induced Oscillating Decay of Circadian Rhythm Genes by Inhibiting Nlrp3 Expression

The aim of this study is to compare the regulatory abilities of citrus flavonoids on the oscillating expression of circadian genes. Seven varieties of citrus fruits and twenty-five citrus flavonoids were selected and evaluated. Per2 luciferase bioluminescence report system and serum shock were used to induce circadian gene expression in mouse microglia BV-2 cells. In vivo experiments were carried out using C57BL6/J mice to evaluate the regulation of flavonoids on the oscillatory expression of liver biorhythm genes. Lipopolysaccharide was used to interfere the gene oscillating expression. QRT-PCR was performed to detect the expression of circadian rhythm-related genes, including Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbβ, Rorα, Dbp, and Npas2. The results show that the polymethoxyflavones (PMFs) exerted stronger circadian gene regulatory capability, while the flavonoids containing glycosides showed no biological activity. Also, all tested flavonoids decreased LPS-induced nitric oxide release, but only polymethoxyflavones inhibited circadian rhythm disorder. PMFs inhibited Nlrp3 inflammasome-related genes and proteins, including Nlrp3, IL-1β, ASC, and Caspase1, while other flavonoids only affected IL-1β and Caspase1 expression. This mechanism was preliminarily verified using the Nlrp3 inhibitor INF39.

Bmal1 Downregulation Worsens Critical Limb Ischemia by Promoting Inflammation and Impairing Angiogenesis

Critical limb ischemia (CLI) is the most advanced clinical stage of peripheral vascular disease with high mobility and mortality. CLI patients suffer from lower extremity rest pain, ulceration, and gangrene caused by insufficient blood and oxygen supply. Seeking for effective biomarkers and therapeutic targets is of great significance for improving the life quality of CLI patients. The circadian clock has been reported to be involved in the progression of kinds of cardiovascular diseases. Whether and how circadian genes play a role in CLI remains unknown. In this study, by collecting femoral artery and muscle specimens of CLI patients who underwent amputation, we confirmed that the circadian gene Bmal1 is downregulated in the CLI femoral artery and ischemic distal lower limb muscle. Furthermore, we verified that Bmal1 affects CLI by regulating lipid metabolism, inflammation, and angiogenesis. A hindlimb ischemia model performed in wild-type and Bmal1−/− mice confirmed that Bmal1 disruption would lead to impaired angiogenesis. In vitro experiments indicated that the decreased expression of Bmal1 would increase ox-LDL uptake and impair endothelial cell functions, including proliferation, migration, and tube formation. As for mechanisms, Bmal1 represses inflammation by inhibiting lipid uptake and by activating IL-10 transcription and promotes angiogenesis by transcriptionally regulating VEGF expression. In conclusion, we provide evidence that the circadian gene Bmal1 plays an important role in CLI by inhibiting inflammation and promoting angiogenesis. Thus, Bmal1 may be an effective biomarker and a potential therapeutic target in CLI.

Dopamine adjusts the circadian gene expression of Per2 and Per3 in human dermal fibroblasts from ADHD patients

A link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different dopamine concentrations in human dermal fibroblast (HDF) cultures, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analyzed via qRT-PCR. We found no statistical significant effect in the actigraphy of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.

Norepinephrine influences the circadian clock in human dermal fibroblasts from study participants with a diagnosis of attention-deficit hyperactivity disorder

Attention-deficit hyperactivity disorder (ADHD) is characterized by changes to the circadian process. Many medications used to treat the condition, influence norepinephrine levels. Several studies have, in addition, reported that norepinephrine itself has an effect on circadian function. The aim of this study was to investigate the circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after norepinephrine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with an ADHD diagnosis. Circadian preference was evaluated with German Morningness–Eveningness Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different norepinephrine concentrations in HDF cultures, the rhythmicity of circadian gene expression was analyzed via qRT-PCR. The exposure of 1 µM norepinephrine to confluent cultures of human dermal fibroblasts from participants with a diagnosis of ADHD, was shown to dampen Per1 rhythmicity. The expression of Bmal1, Per1 and Per3 in control subjects was also influenced by incubation with 1 µM norepinephrine. Cultures from the ADHD group revealed no statistically significant overall differences in circadian gene expression, between cultures with and without norepinephrine incubation. Per3 expression showed a significant ZT × group interaction via mixed ANOVA. Per3 expression at ZT4 was significant higher in the group of control samples incubated with 1 µM norepinephrine, compared to the control group without norepinephrine. This effect was also shown in the control samples incubated with 1 µM norepinephrine and cultures from subjects with ADHD without norepinephrine incubation. Per3 expression differed between the healthy control group and the ADHD group without norepinephrine incubation at ZT28. The results of the present study illustrate that norepinephrine impacts on circadian function. In both groups, control group and cultures taken from subjects with ADHD, the expression of the periodic genes (Per1–3) was significantly influenced by incubation with norepinephrine.