Cellular mechanisms of circadian pacemaking: beyond transcriptional loops.

Circadian clocks drive the daily rhythms in our physiology and behaviour that adapt us to the 24-h solar and social worlds. Because they impinge upon every facet of metabolism, their acute or chronic disruption compromises performance (both physical and mental) and systemic health, respectively. Equally, the presence of such rhythms has significant implications for pharmacological dynamics and efficacy, because the fate of a drug and the state of its therapeutic target will vary as a function of time of day. Improved understanding of the cellular and molecular biology of circadian clocks therefore offers novel approaches for therapeutic development, for both clock-related and other conditions. At the cellular level, circadian clocks are pivoted around a transcriptional/post-translational delayed feedback loop (TTFL) in which the activation of Period and Cryptochrome genes is negatively regulated by their cognate protein products. Synchrony between these, literally countless, cellular clocks across the organism is maintained by the principal circadian pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. Notwithstanding the success of the TTFL model, a diverse range of experimental studies has shown that it is insufficient to account for all properties of cellular pacemaking. Most strikingly, circadian cycles of metabolic status can continue in human red blood cells, devoid of nuclei and thus incompetent to sustain a TTFL. Recent interest has therefore focused on the role of oscillatory cytosolic mechanisms as partners to the TTFL. In particular, cAMP- and Ca²⁺-dependent signalling are important components of the clock, whilst timekeeping activity is also sensitive to a series of highly conserved kinases and phosphatases. This has led to the view that the 'proto-clock' may have been a cytosolic, metabolic oscillation onto which evolution has bolted TTFLs to provide robustness and amplify circadian outputs in the form of rhythmic gene expression. This evolutionary ascent of the clock has culminated in the SCN, a true pacemaker to the innumerable clock cells distributed across the body. On the basis of findings from our own and other laboratories, we propose a model of the SCN pacemaker that synthesises the themes of TTFLs, intracellular signalling, metabolic flux and interneuronal coupling that can account for its unique circadian properties and pre-eminence.