BTBD9-driven IMPDH2 ubiquitination governs sleep homeostasis by gating adenosine metabolism in the basal forebrain

BTB domain containing 9 (BTBD9) is among the most reproducible genetic risk factors for sleep-related disorders, yet its molecular function in sleep regulation remains unclear. Here, we aimed to define the downstream substrate, core brain region, and molecular mechanism through which BTBD9 regulates sleep-wake homeostasis.

Population-scale genomic and brain expression quantitative trait locus (eQTL) datasets were integrated to infer the direction of BTBD9-associated genetic risk in sleep-related traits. Basal forebrain-specific neuronal manipulation of Btbd9 was combined with electroencephalography/electromyography (EEG/EMG)-based sleep analysis and fiberphotomerty to define the relevant brain locus and functional output. Ubiquitinomics, protein interaction, ubiquitination, and enzymatic assays were used to identify BTBD9-regulated substrates and determine how BTBD9-dependent modification affects metabolic enzyme activity. Basal forebrain-specific genetic disruption, receptor pharmacology, and adeno-associated virus (AAV)-delivered interfering peptides were further applied to test pathway necessity and reversibility in vivo.

human genetic analyses showed that BTBD9 variants associated with higher expression predicted a pro-arousal tendency. Consistently, neuron-specific deletion of Btbd9 in the mouse basal forebrain increased non-rapid eye movement (NREM) sleep and extracellular adenosine levels. Mechanistically, IMPDH2, the rate-limiting enzyme in de novo purine biosynthesis, was identified as a substrate of the Cullin-RING E3 ubiquitin ligase 3 (CRL3)^BTBD9 ubiquitin ligase. BTBD9-mediated ubiquitination at IMPDH2 lysine 195 (K195) relieved guanosine triphosphate (GTP)-dependent allosteric inhibition, thereby limiting adenosine accumulation and sleep pressure. Basal forebrain neuronal knockout of Impdh2 recapitulated the sleep-promoting effect of Btbd9 deletion, whereas adenosine A1 receptor (A1R) blockade attenuated this phenotype. AAV-delivered interfering peptides targeting the BTBD9-IMPDH2 interaction reduced IMPDH2 ubiquitination, elevated adenosine levels, increased NREM sleep, and rescued BTBD9 overexpression-induced abnormal wakefulness in vivo.

These findings identify a BTBD9-IMPDH2-adenosine pathway that links human genetic risk to basal forebrain control of sleep homeostasis. By defining BTBD9 as a molecular regulator of purine metabolism, this study provides a mechanistic framework for understanding and potentially targeting BTBD9-related sleep dysregulation.