Molecular dynamics of circadian timing in a mouse model of human sleep disorder

We have known for some time that daily clocks regulate rhythmical behaviour of sleep and wake in man and other animals. These daily rhythms are endogenous as they free-run in constant conditions, and do not require synchronization to external factors such as light and dark, and are therefore termed circadian ('around a day'). The major rhythm generator of the body resides within the hypothalamus of the brain, and is termed the suprachiasmatic nucleus (or SCN). The SCN has the unique properties that it will continue to oscillate when cultured in laboratory conditions. Genes regulating the circadian clock have been cloned and we know that a key feature regulating timing is how the protein products of these genes cycle in real-time around the cell. This is regulated in part by a class of enzymes called kinases, which add phosphate bonds to the protein (phosphorylation) thereby affecting its activity. One of the best known mutations of the circadian clock is a kinase (CK1e) and was discovered in hamsters over 20 years ago, causing a shortening of circadian period. This was termed the tau mutation, since the term tau is used by circadian biologists to denote period. Mutations in the same or similar kinase systems are known to induce sleep disorders in man. We have re-made this mutation in mice and shown that it accelerates behavioural activity cycles to a similar extent as hamsters. Our proposed work now aims to study how this kinase mutation accelerates the circadian clock, both in the brain and in peripheral body clocks as well. Our earlier research using hamsters has shown that the circadian clock may be accelerated by an abrupt change in phase at a specific time of day, due to accelerated turnover in the nucleus of the cell of core clock proteins. This is equivalent in mechanical terms to a gear box missing a few key cogs, causing it to jump to a new position at each rotation. We aim to test this idea in the mouse by studying protein movements in real-time using new transgenic animals which we aim to create in which key clock proteins are tagged with a fluorescent marker. These types of studies can only be addressed in mice, as these are the only animals in which it is possible to make such genetic modifications. We will use these animals to define how the kinase acts on its target proteins by studying which areas (domains) of the protein are phosphorylated by this kinase. By crossing our clock protein-tagged mice to the tau mutant animals, we will be able to define how tau accelerates period and on which proteins it acts. This is important as a description of how this is achieved could in the longer term lead to the development of novel drugs impacting on sleep and wake cycles in man. Some of core genes involved in the generation of circadian rhythms have been deleted from the genome of mice by genetic modification techniques. These so-called knock-out mice are still rhythmic as other residual clock elements are sufficient to drive behaviour. We will cross our tau mutant mice into these knock out mice and define which of the knock-outs exhibits shortening of wheel-running activity cycles. This will tell us whether tau can act in the absence of a specific gene and also the extent to which it can shorten period. By this means we aim to define which clock gene proteins are likely targets for regulation of behavioural activity cycles by tau. Finally, we aim to capitalize from the fact that the SCN and other tissues continue to oscillate in culture. We will monitor activity of cultured tissues using specialized reporters of clock genes which generate light (luciferase reporters). By monitoring light levels with specialized photon recording equipment, we will be able to examine how the circadian clock regulates timing in tissues, and their responses to stimuli which can re-set the clock.

Circadian clocks are pervasive regulators of metabolism. Current models of the clock posit auto-regulatory feedback loops in which complexes of their own protein products suppress transcriptional activity of the Per and Cry genes. It is thought that circadian period is determined by complex formation, dissolution and proteosomal degradation, and that phosphorylation controls these rate-limiting processes. Nevertheless, we do not know how circadian proteins turnover in different cellular compartments in real time, how and when they interact, nor how phosphorylation regulates their mobility. This project will capitalize on our creation in mice of the circadian tau mutation, which resides in the catalytic domain of casein kinase (CK)1e, a regulator of clock proteins. The mice exhibit remarkable shortening of behavioural cycles in vivo and acceleration of the clock monitored in vitro by SCN electrical firing rhythms and luciferase imaging of SCN slices and peripheral tissues. We shall characterize how this mutation accelerates period by defining circadian patterns of clock gene mRNA and protein, and studying the re-setting behaviour of the clock, using in vivo and in vitro assays reporting clock gene action with luciferase gene reporters and SCN firing rates. We will define essential clock targets for CK1e by crossing tau mutant animals into mice lacking PER1 or 2 and CRY1 or 2, thereby establishing which components are required for period shortening by tau. We will define the specific phospho-residues within PER targeted by CK1e and raise antibodies for their characterization. We will engineer novel circadian reporter mice using YFP and CFP tagged to PER2 and CRY1. By using single- and dual-wavelength fluorescence and FRET real-time cellular imaging of SCN tissue in culture, we shall determine how these proteins interact over the course of the circadian cycle, and thence define the impact of the tau mutant on their behaviour, both individually and in complex. Joint with BB/E023223/1