Cul3 and the BTB Adaptor Insomniac Are Key Regulators of Sleep Homeostasis and a Dopamine Arousal Pathway in Drosophila

Abstract

Sleep is homeostatically regulated, such that sleep drive reflects the duration of prior wakefulness. However, despite the discovery of genes important for sleep, a coherent molecular model for sleep homeostasis has yet to emerge. To better understand the function and regulation of sleep, we employed a reverse-genetics approach in Drosophila. An insertion in the BTB domain protein CG32810/insomniac ( inc) exhibited one of the strongest baseline sleep phenotypes thus far observed, a ∼10 h sleep reduction. Importantly, this is coupled to a reduced homeostatic response to sleep deprivation, consistent with a disrupted sleep homeostat. Knockdown of the INC-interacting protein, the E3 ubiquitin ligase Cul3, results in reduced sleep duration, consolidation, and homeostasis, suggesting an important role for protein turnover in mediating INC effects. Interestingly, inc and Cul3 expression in post-mitotic neurons during development contributes to their adult sleep functions. Similar to flies with increased dopaminergic signaling, loss of inc and Cul3 result in hyper-arousability to a mechanical stimulus in adult flies. Furthermore, the inc sleep duration phenotype can be rescued by pharmacological inhibition of tyrosine hydroxylase, the rate-limiting enzyme for dopamine biosynthesis. Taken together, these results establish inc and Cul3 as important new players in setting the sleep homeostat and a dopaminergic arousal pathway in Drosophila.

Author Summary

Sleep is an essential behavior that encompasses roughly a third of our lives; however, the underlying function remains a mystery. The fruit fly has emerged as an important model system for understanding sleep behavior, exhibiting several behavioral and genetic similarities with mammalian sleep, including consolidated immobility, an elevation of arousal threshold to a range of stimuli, homeostatic drive, and manipulation by proven stimulants and sedatives. We tested disruptions of candidate sleep genes and identified a gene called insomniac that exhibits one of the strongest and most robust sleep phenotypes to date, including a suppressed homeostatic response to sleep deprivation. We find similar phenotypes for a gene previously shown to interact with inc and a known regulator of protein degradation, Cul3, linking sleep homeostasis to protein turnover. Importantly, we find that insomniac functions in a known arousal system in the brain, as defined by the neurotransmitter dopamine. This work provides an important insight into the genetic basis of sleep homeostasis with the discovery of a new molecular component of a dopaminergic arousal pathway. Given the conservation of fly and mammalian systems, these studies may lead to new insights into the molecules that mediate sleep homeostasis and arousal in humans.