Auriculasin from Flemingia philippinensis roots shows good therapeutic indexes on hyperactive behavior in zebrafish.

Previously, period1b-/- zebrafish mutants were used to establish an attention deficit hyperactivity disorder (ADHD) model, in which hyperactive behavior was found to be a typical characteristic of ADHD due to down-regulated dopamine levels. Here, we used five prenylated isoflavones from Flemingia philippinensis roots to study their therapeutic effects on hyperactivity behavior in period1b-/- zebrafish. Results of locomotor activity assay showed that auriculasin, one of the prenylated isoflavones, significantly reduced the hyperactivity behavior in period1b-/- zebrafish. Hormone measurement results showed that auriculasin increased melatonin and dopamine content. Results of quantitative real-time polymerase chain reaction showed that auriculasin down-regulated the expression of mao but up-regulated the expression of th and per1b. Thus, auriculasin demonstrated a potential biological effect on dopamine activity to inhibit hyperactivity behavior in the ADHD zebrafish model by regulating circadian clock gene per1b.

Circadian genes period1b and period2 differentially regulate inflammatory responses in zebrafish.

The circadian clock has been shown to regulate various immune processes in different animals. Our previous report demonstrated that the innate immune responses in zebrafish show significant rhythmicity that could be regulated by melatonin. Here, we used diurnal zebrafish to determine the role of circadian genes in the inflammatory responses. Our results indicate that circadian genes exhibit rhythmic oscillations in zebrafish leukocytes, and mutations of the clock genes period1b (per1b) and period2 (per2) considerably affect these oscillations. Using a wounded zebrafish inflammation model, we found that under constant dark conditions (DD), the expression of pro-inflammatory cytokines is significantly downregulated in per1b gene mutant zebrafish and significantly upregulated in the per2 gene mutant zebrafish. Furthermore, using real-time imaging technology, we found that the per1b gene markedly disturbs the rhythmic recruitment of neutrophils toward the injury, whereas the per2 gene does not show a significant effect. Taken together, our results reveal differential functions of the circadian genes per1b and per2 in the inflammatory responses, serving as evidence that circadian rhythms play a vital role in immune processes.

Circadian gene period1b regulates proinflammatory cytokine expression through NF-κB signalling in zebrafish.

The circadian clock plays a critical role in regulating the immune system. Our previous publication revealed that a mutation in the circadian gene period1b (per1b) in zebrafish significantly decreased proinflammatory gene expression, particularly under constant darkness (DD) conditions; however, the underlying mechanisms remain unclear. In this study, using per1b-null mutant zebrafish and a larval tail fin injury model, we observed that the loss of per1b resulted in the downregulation expression of proinflammatory cytokines, such as IL-6 and TNF-α, at protein level. Furthermore, the loss of per1b downregulated ERK phosphorylation and inhibited p65 phosphorylation, leading to reduced NF-κB activation, which could downregulate the expression of proinflammatory cytokines, such as IL-6 and TNF-α, in zebrafish. These results provided insight into the communication between the circadian clock and immune functions.

Circadian modulation of dopamine levels and dopaminergic neuron development contributes to attention deficiency and hyperactive behavior.

Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children and adults. While ADHD patients often display circadian abnormalities, the underlying mechanisms are unclear. Here we found that the zebrafish mutant for the circadian gene period1b (per1b) displays hyperactive, impulsive-like, and attention deficit-like behaviors and low levels of dopamine, reminiscent of human ADHD patients. We found that the circadian clock directly regulates dopamine-related genes monoamine oxidase and dopamine ß hydroxylase, and acts via genes important for the development or maintenance of dopaminergic neurons to regulate their number and organization in the ventral diencephalic posterior tuberculum. We then found that Per1 knock-out mice also display ADHD-like symptoms and reduced levels of dopamine, thereby showing highly conserved roles of the circadian clock in ADHD. Our studies demonstrate that disruption of a circadian clock gene elicits ADHD-like syndrome. The circadian model for attention deficiency and hyperactive behavior sheds light on ADHD pathogenesis and opens avenues for exploring novel targets for diagnosis and therapy for this common psychiatric disorder.

The circadian clock regulates autophagy directly through the nuclear hormone receptor Nr1d1/Rev-erbα and indirectly via Cebpb/(C/ebpß) in zebrafish.

Autophagy is a highly conserved intracellular degradation system, and recently was shown to display circadian rhythms in mice. The mechanisms underlying circadian regulation of autophagy, however, are still unclear. Here, we observed that numbers of autophagosomes and autolysosomes exhibit daily rhythms in the zebrafish liver, and cebpb/(c/ebpß) and various autophagy genes are rhythmically expressed in zebrafish larvae but significantly upregulated in per1b and TALEN-generated nr1d1/rev-erbα mutant fish, indicating that both Per1b and Nr1d1 play critical roles in autophagy rhythms. Luciferase reporter and ChIP assays show that the circadian clock directly regulates autophagy genes through Nr1d1, and also regulates transcription of cebpb through Per1b. We also found that fasting leads to altered expression of both circadian clock genes and autophagy genes in zebrafish adult peripheral organs. Further, transcriptome analysis reveals multiple functions of Nr1d1 in zebrafish. Taken together, these findings provide evidence for how the circadian clock regulates autophagy, imply that nutritional signaling affects both circadian regulation and autophagy activities in peripheral organs, and shed light on how circadian gene mutations act through autophagy to contribute to common metabolic diseases such as obesity.