Using Translational Switching to Investigate the SCN Clockwork

Abstract

The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal clock driving circadian rhythms of physiology and behaviour that adapt mammals to environmental cycles. Timekeeping in the SCN is underpinned by a self-sustaining cell-autonomous transcriptional/translational delayed feedback loop (TTFL), whereby negative regulators inhibit their own transcription. The positive regulators CLOCK and BMAL drive transcription of the negative regulators Period1/2 and Cryptochrome1/2 via E-BOX regulatory sequences. PER/CRY mediated repression of BMAL/CLOCK closes the feedback loop allowing the cycle to begin again every 24 hours. In the SCN, synchronisation of the individual cell-autonomous clocks is achieved by circuit level signalling mediated via interneuronal neuropeptidergic interactions, as well as communication between astrocytes and neurons. The result is a robust SCN network that allows timekeeping to be maintained even when SCN are cultured as organotypic slices ex vivo. Thus, the SCN provides a highly tractable model for studying circadian timekeeping. In a formal sense, the TTFL motif is readily compatible with limit-cycle models, where the oscillation of key components (called state variables) entirely underpins the phase of the oscillation. In Neurospora and Drosophila the negative regulators Frequency (FRQ) and Period (Per) have been identified as state variables of their respective TTFLs. However, the identity of state variables in the SCN, or indeed if the simple limit cycle model is even compatible with SCN circadian oscillations, is less clear. Key to testing a component for a role as a state variable is reversible and conditional expression of a transgenic copy. By using genetic code expansion (GCE), it is possible to create translational switches whereby the translation of a transgene is conditional on the provision of a non-canonical amino acid. Incorporation of an ectopic amber stop codon (TAG) into the coding sequence of a gene of interest causes premature termination of translation and prevents generation of a functional protein. However, an orthogonal amino-acyl-tRNA synthetase/tRNACUA pair that specifically incorporates a non-canonical amino acid (ncAA) at the amber stop codon allows translational read-through when the amino acid is present. Delivery of this pair into the SCN, along with a translationally switchable gene of interest, can be achieved by use of adeno-associated virus (AAV) vectors, and ncAA can be added to culture medium. In this work I developed novel translationally switchable Bmal1 (tsBMAL1) and Period2 (tsPER2), as well as using a translationally switchable copy of CRY1 (tsCRY1) developed previously in the lab, to investigate the SCN clockwork. First, I show how generation of tsBMAL1 provided conditional and reversible control of transgenic expression in the SCN. Bmal1 knockout mice show behavioural arrhythmicity, however SCN slices from p10 mice showed low amplitude and highly variable oscillations. Reversible tsBMAL1 expression following AlkK provision reorganised the noisy oscillations and induced stable circadian rhythms. Then, by using tsCRY1 and tsPER2 constructs, I assessed the extent to which either CRY1 or PER2 could be considered state variables and the limits of the limit cycle model to describe circadian rhythmicity in organotypic SCN slices. I conclude that both CRY1 and PER2 display some of the hallmarks of state variables found in simpler limit cycle systems like the Neurospora clock but are also acting within a more complex cycle that underpins SCN timekeeping.