A cellular model for human daily behaviour

Abstract

All the biochemical, physiological or behavioural processes whose period is about 24 hours possess a circadian rhythm. In mammals circadian rhythms control almost all aspects of human daily behaviour and physiology. Dysregulation of circadian rhythms leads to several pathologies, such as depression, cancer and metabolic syndromes. Mammalian circadian system is organized in a hierarchic fashion: suprachiasmatic nucleus (SCN) is master clock and governs the circadian rhythms of all peripheral oscillators, virtually all the other cells of the body. The study of human circadian rhythms in subjects in vivo is expensive, time consuming and invading. However, since SCN and peripheral oscillators share the same circadian molecular machinery, it is possible to use peripheral oscillator as model to study molecular mechanisms of circadian rhythms. To visualize in real-time cellular circadian rhythms, fibroblasts were infected with a lentivirus coding for the circadian reporter firefly luciferase under a clock gene promoter (Bmal1). After the synchronization of circadian rhythms, the measurement of the light emitted by the cells gave a representation of fibroblast circadian oscillations. The aim of the thesis was to establish the use of human primary skin fibroblasts as a valuable model to study different aspects of human circadian rhythms. To address these questions three projects were designed. A first set of experiments aimed at validating human skin fibroblast model, ascertaining that this in vitro model parallels in vivo human circadian parameters. We found a very good correlation between the in vivo and the in vitro period length in the three groups of subjects (two sighted and one blind) recruited for this study. Interestingly, although the in vivo period obtained from the blind group was longer than the in vivo period obtained from the sighted groups, the in vitro period length from the three groups of subjects was similar, revealing that human skin fibroblasts are insensitive to the after-effects caused by light. In summary, human circadian period can be approximated by measurement in fibroblasts. In a second project human age-related circadian impairments were studied in the cellular skin fibroblasts model. Indeed sleep-wake cycle alterations and phase advancing of gene expression and behaviour can be found in elder individuals. To better understand the rebound of ageing on the circadian rhythms we characterized the period length of skin fibroblasts from young and elder persons. No differences in amplitude, phase and period length were found between cells from the two groups. However, in the presence of sera from older donors human fibroblasts showed a reduced period length and a shorter phase of entrainment compared to the same cells measured in the presence of sera from young donors. These differences are likely due to one or more thermolabile substances, since heat-inactivation of sera from older donors almost undid the reduction of the circadian period length. Thus, these results suggest that during ageing the molecular machinery of peripheral circadian clocks does not change per se, but some age-related circadian changes observed in vivo might be caused by circulating molecules. Human fibroblasts were also used to investigate the role of melatonin as zeitgeber on peripheral oscillators. Melatonin is secreted in a circadian fashion and was demonstrated to regulate the SCN firing rate and to entrain the sleep-wake cycle of most mammals and humans. The circadian presence of melatonin is well conserved in all biological fluids, suggesting that melatonin may be one of the molecules that the master clock uses to synchronize peripheral oscillators. This hypothesis was tested in damped fibroblasts, using a wide range of concentrations of melatonin to restore the amplitude of the rhythms. However, no increase of amplitude or phase shift of the rhythms was observed after treating cells with melatonin. Moreover, the application of the hormone to newly synchronized oscillators decreased their bioluminescence. In summary, the experiments demonstrated that melatonin does not play a direct role as peripheral oscillator zeitgeber. In conclusion, the studies of the present thesis succeeded in revealing three primary findings: first, fibroblast circadian rhythms parallel human circadian physiology, such as circadian period length. Second, apparently, during ageing the molecular components of peripheral circadian clocks in skin fibroblasts do not change per se, but some age-related circadian changes observed in vivo might be caused by one or more heat-sensitive substances present in the blood of older subjects. Finally, melatonin does not possess direct synchronizing properties on peripheral oscillators like fibroblasts. In total, the present thesis revealed that primary human skin fibroblasts are an easily accessible, cheap and reliable model to enlighten our understanding of human circadian mechanisms.