What are the mechanisms that control sleep?
Each night, we grow tired and enter sleep, a behavioral state in which we are largely disengaged from the external world. Humans and other animals have been sleeping for millions of years, yet both the purpose of sleep and the mechanisms that control this fundamental behavior remain poorly understood. Understanding these mechanisms is important, given that sleep is integral to human health and, that disruptions of sleep are associated with physiological and neurological disorders.
Sleep is an evolutionarily ancient behavior, and most animals that have been examined have a sleep state. Among these animals is the fruit fly, Drosophila melanogaster, an evolutionary cousin of humans and a model organism long favored by biologists due to its powerful genetic tools. By using the fly to identify mechanisms that control sleep, our studies have the potential to reveal fundamental biological mechanisms shared by other animals and to impact the diagnosis and treatment of human sleep disorders.
insomniac links the development and function of a sleep regulatory circuit.
(2021). Li Q, Jang H, Lim KY, Lessing A, Stavropoulos N. eLife 10:e65437.
Although many genes are known to influence sleep, when and how they impact sleep-regulatory circuits remain ill-defined. Here, we show that insomniac (inc), a conserved adaptor for the autism-associated Cul3 ubiquitin ligase, acts in a restricted period of neuronal development to impact sleep in adult Drosophila. The loss of inc causes structural and functional alterations within the mushroom body (MB), a center for sensory integration, associative learning, and sleep regulation. In inc mutants, MB neurons are produced in excess, develop anatomical defects that impede circuit assembly, and are unable to promote sleep when activated in adulthood. Our findings link neurogenesis and postmitotic development of sleep-regulatory neurons to their adult function and suggest that developmental perturbations of circuits that couple sensory inputs and sleep may underlie sleep dysfunction in neurodevelopmental disorders.
Cul3 and insomniac are required for rapid ubiquitination of postsynaptic targets and retrograde homeostatic signaling.
(2019). Kikuma K, Li X, Perry S, Li Q, Goel P, Chen C, Kim D, Stavropoulos N, Dickman D. Nature Communications 10(1):2998.
At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. Here, we design a forward genetic screen to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, we find that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, we show that Peflin, a Ca2+-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca2+ signaling and control of Cul3/Inc activity in the postsynaptic compartment. Our study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses.
Structural and behavioral analysis reveals that Insomniac impacts sleep by functioning as a Cul3 adaptor.
(2019). Li Q, Lim KYY, Stavropoulos N. bioRxiv.
The insomniac (inc) gene is required for normal sleep in Drosophila and encodes a conserved BTB protein that is a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase. Here we test whether Inc serves as a Cul3 adaptor by generating mutant forms of Inc and assessing their biochemical properties and physiological activity in vivo. We show that the N-terminal BTB domain of Inc is necessary and sufficient for Inc self-association and interactions with Cul3. Inc point mutations that weaken interactions with Cul3 impair the ability of Inc to rescue the sleep deficits of inc mutants, indicating that Cul3-Inc binding is critical for Inc function in vivo. Deletions of the conserved Inc C-terminus preserve Inc-Inc and Inc-Cul3 interactions but abolish Inc activity in vivo, implicating the Inc C-terminus as an effector domain that recruits Inc substrates. Mutation of a conserved C-terminal arginine similarly abolishes Inc function, suggesting that this residue is vital for the recruitment or ubiquitination of Inc targets. Mutation of the same residue in the human Inc ortholog KCTD17 is associated with myoclonic dystonia, indicating its functional importance in Inc family members. Finally, we show that Inc assembles into multimeric Cul3-Inc complexes in vivo and that depleting Cul3 causes accumulation of Inc, suggesting that Inc is negatively regulated by Cul3-dependent autocatalytic ubiquitination, a hallmark of Cullin adaptors. Our findings implicate Inc as a Cul3 adaptor and provide tools to identify the targets of Inc family proteins that impact sleep and neurological disorders.
Elementary sensory-motor transformations underlying olfactory navigation in walking fruit-flies.
(2018). Álvarez-Salvado E, Licata AM, Connor EG, McHugh MK, King BM, Stavropoulos N, Victor JD, Crimaldi JP, Nagel KI. eLife 7:e37815.
A bidirectional relationship between sleep and oxidative stress in Drosophila.
(2018). Hill VM, O'Connor RM, Sissoko GB, Irobunda IS, Leong S, Canman JC, Stavropoulos N, Shirasu-Hiza M. PLoS Biology 16(7): e2005206.
Conserved properties of Drosophila Insomniac link sleep regulation and synaptic function.
(2017). Li Q, Kellner DA, Hatch HAM, Yumita T, Sanchez S, Machold RP, Frank CA, Stavropoulos N. PLoS Genetics 13(5):e1006815.
Evaluation of Ligand-Inducible Expression Systems for Conditional Neuronal Manipulations of Sleep in Drosophila
(2016). Li Q, Stavropoulos N. G3: Genes | Genomes | Genetics 6(10):3351-3359.