Neurofibromin Regulates Sleep Consolidation via Cholinergic Neurons in a Drosophila Model of Neurofibromatosis 1

Abstract

Neurofibromatosis type 1 (NF1) patients frequently present with fragmented, poor-quality sleep, but the neural circuits and molecular mechanisms underlying this deficit remain undefined. Using Drosophila, we show that loss of the NF1 ortholog dNf1 recapitulates the human phenotype: null mutants exhibit markedly reduced total sleep with blunted nighttime consolidation, reflected in fewer and shorter sleep bouts and elevated inter-bout activity, a recessive phenotype reproduced by independent null alleles and by pan-neuronal RNAi knockdown. Despite the broad expression of dNf1 across the adult CNS, including the mushroom bodies, ellipsoid body, dorsal fan-shaped body, and GABAergic neurons, cell-type-restricted knockdown showed the protein is dispensable for sleep regulation in each of these populations. Instead, dNf1 loss specifically in cholinergic neurons, the most abundant neuronal class in the fly brain, was sufficient to reproduce the deficient, fragmented sleep of null mutants, and restoring wild-type dNf1 to cholinergic neurons of null animals was both necessary and sufficient to rescue sleep. Mechanistically, cholinergic expression of the GAP-related domain (GRD) alone, which normalizes elevated Ras/MAPK signaling, fully restored sleep in nulls, whereas a catalytically dead GRD variant did not, establishing Ras/MAPK dysregulation in cholinergic neurons as the proximate cause. Two disease-associated missense mutations, dNf1C1045Y and dNf1R1861C, produced distinct, dose-graded sleep phenotypes. dNf1C1045Y showed severe deficits resembling the null and was resistant to pan-neuronal, but responded to cholinergic neuron-restricted rescue, consistent with a dominant-negative effect on dNf1 dimerization. In contrast, dNf1R1861C presented a milder phenotype that was largely reversible by wild-type dNf1 but only partially by the GRD domain, implicating an additional, GRD-independent mechanism. Collectively, these findings define cholinergic neurons as the key circuit through which Neurofibromin regulates sleep consolidation via Ras/MAPK signaling and reveal mutation-specific mechanisms that mirror the phenotypic heterogeneity seen in NF1 patients, with implications for genotype-informed understanding of sleep disturbance in this disorder.