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.

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.

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.