PERfect Day: reversible and dose-dependent control of circadian time-keeping in the mouse suprachiasmatic nucleus by translational switching of PERIOD2 protein expression

Abstract

The biological clock of the suprachiasmatic nucleus (SCN) orchestrates circadian (approximately daily) rhythms of behaviour and physiology that underpin health. SCN cell-autonomous time-keeping revolves around a transcriptional/translational feedback loop (TTFL) within which PERIOD (PER1,2) and CRYPTOCHROME (CRY1,2) proteins heterodimerise and suppress trans-activation of their encoding genes (Per1,2; Cry1,2). To explore its contribution to SCN time-keeping, we used adeno-associated virus–mediated translational switching to express PER2 (tsPER2) in organotypic SCN slices carrying bioluminescent TTFL circadian reporters. Translational switching requires provision of the non-canonical amino acid, alkyne lysine (AlkK), for protein expression. Correspondingly, AlkK, but not vehicle, induced constitutive expression of tsPER2 in SCN neurons and reversibly and dose-dependently suppressed pPer1-driven transcription in PER-deficient (Per1,2-null) SCN, illustrating the potency of PER2 in negative regulation within the TTFL. Constitutive expression of tsPER2, however, failed to initiate circadian oscillations in arrhythmic PER-deficient SCN. In rhythmic, PER-competent SCN, AlkK dose-dependently reduced the amplitude of PER2-reported oscillations as inhibition by tsPER2 progressively damped the TTFL. tsPER2 also dose-dependently lengthened the period of the SCN TTFL and neuronal calcium rhythms. Following wash-out of AlkK to remove tsPER2, the SCN regained TTFL amplitude and period. Furthermore, SCN retained their pre-washout phase: the removal of tsPER2 did not phase-shift the TTFL. Given that constitutive tsCRY1 can regulate TTFL amplitude and period, but also reset TTFL phase and initiate rhythms in CRY-deficient SCN, these results reveal overlapping and distinct properties of PER2 and CRY1 within the SCN, and emphasise the utility of translational switching to explore the functions of circadian proteins.