Norepinephrine resets the clock of human dermal fibroblasts

Introduction Norepinephrine (NE) regulates important behavioural activities. Numerous studies linked the NE pathway with the pathophysiology of ADHD. Additionally, several animal studies demonstrated that alterations of the circadian clock are associated with the NE pathways. In this preliminary study we investigated the effect of NE stimulation on the circadian rhythm gene expression in human dermal fibroblasts (HDF).

Methods HDF were obtained via skin biopsy from healthy controls (HC) (3 men, 1 woman; 42.00 ± 15.38 years, mean ± SD; BMI: 29.10 ± 6.54 kg/m², mean ± SD) and volunteers suffering from ADHD (1 man, 3 women; 38.75 ± 8.15 years, mean ± SD; BMI: 24.97 ± 5.32 kg/m², mean ± SD). Cells were cultivated at 37 °C and 5% CO2. All participants completed the Multiple-Choice Word Test (IQ score: HC: 115.25 ± 10.04, mean ± SD; ADHD participants:110.25 ± 17.01, mean ± SD, n.s), German Morningness-Eveningness-Questionnaire (D-MEQ Score: HC: 50.00 ± 3.74, mean ± SD; ADHD participants: 53.50 ± 9.54, mean ± SD, n.s) and Wender Utah Rating Scale, German short-version (WURSk Score: HC: 13.75 ± 9.98, mean ± SD; ADHD participants: 31.75 ± 6.94, mean ± SD, p = 0.025). Synchronization was induced either with 1 µM NE or 0.1 µM dexamethasone (D) for 2 hours. Sampling was performed every forth hour starting after synchronization for a period of 28 hours. CLOCK, BMAL1, CRY1, PER1/2/3 gene expression was measured by qRT-PCR. Rhythmicity analysis was performed with CircWaveR software. Statistics were calculated using SPSSR.

Results HC presented an overweight BMI range, whereas the ADHD group had normal weight. Both groups showed a standard range IQ score. The WURSk score in the ADHD group is higher compared to the HC, like expected. Age and D-MEQ score of both groups are similar. Both groups showed an intermediate chronotype. Compared to D synchronization, NE induced CLOCK gene rhythms (p < 0.05, CircWave) whereas CRY1 and PER3 rhythms were dampened (p > 0.05, CircWave) in HC group.

In HC, gene expression was significantly different using NE compared to D, particularly, for time point ZT0 for CLOCK and BMAL1 (p < 0.05) and ZT16 for CRY1 (p = 0.007) and PER3 (p = 0.001). Additional, for NE synchronization in the ADHD group a phase advance was observed for CLOCK, BMAL1 and CRY1 · Furthermore, the expression of PER2 in ADHD group was different at ZT0 (p = 0.01) compared to HC.

Conclusion Our results suggest that NE stimulation may alter the circadian rhythm in HDF. Furthermore, both NE exposure and higher BMI might influence CLOCK gene rhythmicity.