The essential but enigmatic functions of sleep1,2 must be reflected in molecular changes sensed by the brain’s sleep-control systems. In the fruitfly Drosophila, about two dozen sleep-inducing neurons3 with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need4, via the antagonistic regulation of two potassium conductances: the leak channel Sandman imposes silence during waking, whereas increased A-type currents through Shaker support tonic firing during sleep5. Here we show that oxidative byproducts of mitochondrial electron transport6,7 regulate the activity of dFB neurons through a nicotinamide adenine dinucleotide phosphate (NADPH) cofactor bound to the oxidoreductase domain8,9 of Shaker’s KVβ subunit, Hyperkinetic10,11. Sleep loss elevates mitochondrial reactive oxygen species in dFB neurons, which register this rise by converting Hyperkinetic to the NADP+-bound form. The oxidation of the cofactor slows the inactivation of the A-type current and boosts the frequency of action potentials, thereby promoting sleep. Energy metabolism, oxidative stress, and sleep—three processes implicated independently in lifespan, ageing, and degenerative disease6,12,13,14—are thus mechanistically connected. KVβ substrates8,15,16 or inhibitors that alter the ratio of bound NADPH to NADP+ (and hence the record of sleep debt or waking time) represent prototypes of potential sleep-regulatory drugs.
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Custom instrument control and analysis code used in this study is available from the corresponding author upon reasonable request.
The datasets generated during this study are available from the corresponding author upon reasonable request.
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We thank D. Pimentel for electrophysiology advice and C. Chintaluri and M. Murphy for discussions. N. Bonini, B. Dickson, B. Ganetzky, J. Hall, T. Holmes, K. Ito, H. Jacobs, L. Luo, J. Ng, J. Phillips, F. Rouyer, G. Rubin, R. Stocker, P. Taghert, Z. Yan, the Bloomington Stock Center, and the Vienna Drosophila Resource Center provided flies. This work was supported by grants (to G.M.) from the Wellcome Trust and the Gatsby Charitable Foundation. A.K. held postdoctoral fellowships from the Swiss National Science Foundation and EMBO; S.M.S. was a Commonwealth Scholar.
Nature thanks James Hodge, Yasuo Mori, Michael Palladino and the other anonymous reviewer(s) for their contribution to the peer review of this work.
A patent application has been filed by G.M., A.K., S.M.S. and Oxford University Innovation Ltd. on the basis of work described in this study.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Chronic or acute dFB-restricted perturbations of redox chemistry have no effect on waking locomotor activity or arousability.
a, Locomotor counts per waking minute of flies expressing R23E10-GAL4-driven SOD1 or pro-oxidant SOD1(A4V) do not differ from their respective parental controls (genotype effect: P > 0.2612, Kruskal–Wallis ANOVA with Dunn’s post-hoc test). b, The arousability of flies expressing R23E10-GAL4-driven SOD1 (left) or pro-oxidant SOD1(A4V) (right) does not differ from their respective parental controls (grey colours as in a) (genotype effects: P > 0.2487, vibrational force effects: P < 0.0001, vibrational force × genotype interactions: P > 0.9857, two-way ANOVA). Data are means ± s.e.m. of six trials per genotype (n = 16–32 flies each). c, Locomotor counts per waking minute of flies expressing R23E10-GAL4-driven miniSOG, with or without RNAi transgenes targeting KV channel subunits, and parental controls, in a custom video-tracking system31. Activity was monitored for 10 min before the photo-oxidation of miniSOG and then for a 30-min interval that included an initial 9-min exposure to blue light (genotype effect: P = 0.0827, illumination effect: P = 0.8059, illumination × genotype interaction: P = 0.3086, two-way repeated-measures ANOVA). Data are means ± s.e.m. n, number of flies (a, c) or trials (b). For statistical details see Supplementary Table 2.
Extended Data Fig. 2 Chronic perturbations of redox chemistry in cryptochrome- or pigment dispersing factor (PDF)-expressing clock neurons, Kenyon cells, or olfactory projection neurons have no impact on sleep.
a, Sleep in flies expressing cry-GAL4-driven SOD1 or SOD1(A4V) in clock neurons and parental controls. Kruskal–Wallis ANOVA with Dunn’s post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P > 0.1426). b, Sleep in flies expressing pdf-GAL4-driven SOD1 or SOD1(A4V) in clock neurons and parental controls. Kruskal–Wallis ANOVA with Dunn’s post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P > 0.1732). c, Sleep in flies expressing OK107-GAL4-driven SOD1 or SOD1(A4V) in KCs and parental controls. One-way ANOVA with Holm-Šídák’s post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P > 0.0603). d, Sleep in flies expressing GH146-GAL4-driven SOD1 or SOD1(A4V) in olfactory projection neurons and parental controls. Kruskal–Wallis ANOVA with Dunn’s post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P > 0.6901). Data are means ± s.e.m. n, number of flies. For statistical details see Supplementary Table 2.
Sleep in flies expressing two different R23E10-GAL4-driven cry-targeting RNAi transgenes and parental controls. One-way ANOVA with Holm-Šídák’s post-hoc test failed to detect significant differences of experimental flies from both of their respective parental controls (P > 0.1718). Data are means ± s.e.m. n, number of flies. For statistical details see Supplementary Table 2.
Extended Data Fig. 4 Blue illumination of miniSOG-negative dFB neurons has no effect on their electrical activity.
a–e, dFB neurons expressing R23E10-GAL4-driven CD8::GFP, before and after a 9-min exposure to blue light. Example voltage responses to current steps (a, sample sizes in b): illumination increases the input resistance (b, Rm; P = 0.0098, paired t-test) but not the membrane time constant (b, τm; P = 0.0723, paired t-test) and leaves unchanged the current–spike frequency function (c, left; current × genotype interaction; P = 0.9982, two-way repeated-measures ANOVA) and interspike interval distribution (c, right; P = 0.0947, Kolmogorov–Smirnov test). Example IA (normalized to peak) evoked by voltage steps to +40 mV (d, sample sizes in e): illumination leaves unchanged the IA amplitude (e; P = 0.8040, Wilcoxon test) and both inactivation time constants (e, τfast: P = 0.6387, τslow: P = 0.2958, Wilcoxon tests). *P < 0.05. Data are means ± s.e.m. n, number of cells. For statistical details see Supplementary Table 2.
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Kempf, A., Song, S.M., Talbot, C.B. et al. A potassium channel β-subunit couples mitochondrial electron transport to sleep. Nature 568, 230–234 (2019). https://doi.org/10.1038/s41586-019-1034-5
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