De novo synthesis of PERIOD initiates circadian oscillation in cultured mouse suprachiasmatic nucleus after prolonged inhibition of protein synthesis by cycloheximide.

The circadian oscillation is known to stop with prolonged inhibition of protein synthesis and to restart from a particular phase after the removal of inhibition. In order to know the underlying molecular mechanisms, the mouse suprachiasmatic nucleus was cultured and treated with a protein synthesis inhibitor, cycloheximide (CHX), for various durations. Circadian rhythms in Bmal1 expression and PER2 protein were monitored by means of bioluminescence reporters. Bmal1-LUC and PER2::LUC bioluminescence decreased to basal levels after CHX application. CHX washout restarted the circadian rhythms from a fixed phase when CHX treatment exceeded 18 h. mRNA of Per1, Per2 and Rev-erbα increased and reached high plateau levels in 18 h after CHX application, which continued for 48 h; whereas Bmal1 mRNA increased for the first 18 h but then decreased to the basal level. Immunoblot analysis showed a decreased PER2::LUC level at 24 h after CHX application, indicating that the transcription of Pers and Rev-erbα was disinhibited by CHX. CHX washout increased PER2::LUC bioluminescence and protein level in a few hours. High Per mRNA levels induced the rapid increases of their proteins, which might trigger the restarting of circadian oscillation. These findings indicate that the circadian oscillation is stopped by disinhibition of Per1 and Per2 transcriptions, and restarted upon the recovery of the PER mediated auto-feedback loop. De novo synthesis of PER protein is a key factor to initiate the circadian oscillation after prolonged inhibition of protein synthesis.