Defining a novel role for cell cycle repression in promoting circadian rhythms

Circadian rhythms time organismal behaviours across the day-night cycle. A key function of the circadian clock is to regulate the onset and offset of sleep, a behaviour essential for neurological function. Perturbed sleep-wake patterns are frequently observed in patients with neurodevelopmental disorders, creating significant burdens for their carers. More broadly, sleep defects in healthy adults, in aged humans, and in patients with neurodegenerative disorders, represent an increasing socio-economic problem. Hence, there is an urgent need to understand the molecular mechanisms that promote circadian rhythmicity, and thus healthy sleep patterns. Circadian rhythms are highly conserved across metazoan species, and genetically tractable model organisms have yielded important insights into their molecular underpinnings. Here, we utilise the fruit fly, Drosophila, as such a model, and present robust preliminary data that defines a protein called Rbf as a novel and unexpected regulator of circadian rhythms and sleep timing. Rbf plays well-known roles in suppressing DNA synthesis and G1-S phase progression during the cell cycle, and a conserved orthologue is present in humans (RBL2). We reveal a previously unappreciated role for Rbf in post-mitotic circadian (clock) neurons that no longer undergo cell division. We show that loss of Rbf in clock neurons strongly disrupts circadian locomotor rhythms in adult flies. Additional cell-specific manipulations suggest a direct effect of Rbf on the molecular clock, and that Rbf is required in multiple clock neuron subtypes for rhythmic locomotor behaviour. To define cellular roles of Rbf in post-mitotic neurons, we utilised transcriptomic approaches. These revealed that loss of Rbf dramatically upregulates expression of DNA synthesis genes in mature neurons. These transcriptional changes correlate with non-uniform increases in DNA content and nuclear size across adult fly neurons, suggesting that specific neuronal subtypes in the fly brain are particularly vulnerable to genome replication (polyploidy) following loss of Rbf. Based on our preliminary data and prior studies, we therefore hypothesise that loss of Rbf similarly induces genome replication in a non-uniform manner across clock neurons. This, in turn, causes the molecular clock in distinct clock neuron subsets to run at different paces, leading to network desynchronisation, circadian arrhythmicity, and altered sleep timing. Through our Work Packages we will robustly test this hypothesis, thus defining the molecular mechanisms through which Rbf promotes circadian rhythmicity. Our recent study shows that human patients with mutations in the Rbf orthologue RBL2 exhibit disrupted sleep patterns, demonstrating an important and conserved role for this protein across evolution, with direct relevance to human health. More broadly, cell-cycle dysregulation is increasingly recognised as an important feature of the ageing brain and in neurodegenerative disorders. By examining how cell cycle disruption in mature clock neurons impacts circadian rhythmicity, our work therefore has relevance to understanding sleep disruption in these medically and socio-economically important conditions. Finally, by showing that Rbf continues to suppress cell cycle gene expression after cell cycle exit, our work reveals a new mechanism by which neurons maintain their post-mitotic identity across the lifespan – a process fundamental to neurological function.