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Redox Potential as an Interface between Sleep homeostasis and Circadian Rhythms

Final Report Summary - REDOXSLEEPCIRCADIAN (Redox potential as an interface between sleep homeostasis and circadian rhythms)

The timing and quality of both sleep and wakefulness are thought to result from the fine-tuned interaction of two processes, namely a homeostatic process that is activated by and counters the effects of sleep loss and a circadian process that determines the time of day at which sleep preferably occurs. The interaction between both processes allows us to stay awake and alert throughout the day and remain asleep through the night. Given that even minor misalignments between these two processes can significantly and negatively impact our daytime functioning, such as the alterations experienced during jet-lag and shift-work, the study of their interaction is of direct relevance for understanding the mechanisms determining cognitive performance and contributing to quality of life.

Even though both processes are widely thought to operate independently, our previous studies suggested that the genes that were essential to generate circadian rhythms, i.e. the clock genes, were also necessary for an intact homeostatic regulation of sleep. The transcriptional activity of the two core clock genes, CLOCK and NPAS2, directly depended on and affected intracellular metabolic state. The increases in the expression of the clock genes Per1 and Per2, whose transcription was controlled by CLOCK and NPAS2, and which we observed during sleep deprivation (SD), might signal deficits in intra-cellular energy charge incurred by prolonged wakefulness. We proposed that, from a sleep homeostatic perspective, the same genes that signalled circadian time of day information could also signal time spent awake. From a sleep functional perspective we proposed that cellular energy charge was the key regulated variable.

We also investigated the relationship between sleep need, circadian rhythms and energy charge to establish whether circadian rhythmicity was accompanied by changes in redox potential consistent with those reported to alter CLOCK and NPAS2 dependent transcriptional activation in vitro, as well as to establish the sleep-wake dependent and circadian changes in Per2 gene expression in the brain and peripheral tissues. Using redox genetic probes targeted to the mitochondria and the nucleus, along with a time-lapse imaging system, we were able to observe redox changes over 72 hours in transfected living fibroblasts. Ongoing work aimed at recording redox changes in different subcellular domains in parallel with circadian changes (Rev-erbalpha or Per2) within the same fibroblasts and in primary cortical neurons.

In parallel, since Per2 expression increased in the brain after SD, we focussed on the regulation of Per2 gene in the brain. Using Per2::luciferase mice, we demonstrated in all living mice that SD increased Per2 protein with different dynamics in the brain, particularly in the cerebral cortex, in the liver and in the kidney. We also remarked that extended periods of wakefulness affected Per2 ribonucleic acid (RNA) expression in the forebrain through both circadian and non-circadian mechanisms and via several SDs at different times of the day, while they always resulted in an increase of Per2 protein.

Thus, Per2 seemed to combine activity-induced and circadian aspects known to be central to the sleep-wake regulation. Our results were anticipated to gain insight into the dynamic coupling of the two aspects of the sleep in the brain and to increase our knowledge regarding the involvement of circadian genes in the sleep function. These data also illustrated that the dogma of the separation of circadian and homeostatic processes was no longer tenable, and suggested that the molecularly feedback machinery underlying circadian rhythm generation was equally suited to keep track and anticipate homeostatic sleep need.

By the time of the project completion we were now further investigating the role of the suprachiasmatic nucleus (SCN), i.e. the main circadian pacemaker, in the relationship between ‘clock’ genes and sleep and wakefulness.