Mitochondrial Origin of Sleep Pressure

Authors: Raffaele Sarnataro, Cecilia D. Velasco, Nicholas Monaco, Anissa Kempf & Gero Miesenböck Journal: Nature | Vol 645 | 18 September 2025

Background and Significance

Sleep pressure is the process variable of sleep homeostasis, but it has long lacked a physical explanation. Although prolonged wakefulness correlates with many changes in the brain—neuronal firing patterns, synaptic strength, subcellular organization, metabolite concentrations, metabolic and gene-expression programs—it has been unclear whether these are causes or consequences of sleep need.

This study provides the first evidence for a mitochondrial origin of sleep pressure, directly linking mitochondrial function to sleep regulation, offering molecular-level evidence connecting sleep and energy metabolism, and suggesting new therapeutic targets for sleep and metabolic disorders.

Methods

The researchers characterized transcriptomes of single cells isolated from rested and sleep-deprived fruit flies, focusing on dFBN sleep-control neurons.

• Single-cell RNA-seq (10X Chromium): fly brains dissociated to single-cell suspensions; GFP-positive dFBNs isolated by FACS; differential expression and GO enrichment analysis
• Mitochondrial morphology: optical photon reassignment microscopy (OPRM) and confocal laser scanning microscopy (CLSM)
• Mitochondria–ER contacts: SPLICSshort sensor
• Mitophagy: mito-QC ratiometric sensor
• Electrophysiology: whole-cell patch clamp recordings from dFBNs in vivo
• ATP measurement: genetically encoded sensors (iATPSnFR, ATeam) with two-photon imaging
• Behavior: Trikinetics activity monitoring; sleep deprivation by mechanical perturbation or thermogenetics

Key Findings

• Transcriptional changes: After sleep deprivation, dFBNs selectively upregulate transcripts encoding mitochondrial respiratory chain complexes I–IV, ATP synthase (complex V), the ATP-ADP carrier sesB, and TCA cycle enzymes, while downregulating genes for synaptic assembly, vesicle release, and presynaptic homeostatic plasticity. This signature is unique to dFBNs (absent in antennal lobe projection neurons and Kenyon cells).
• Mitochondrial morphology: Sleep deprivation reduces mitochondrial size, elongation, and branching while increasing their number—indicating fission, accompanied by Drp1 redistribution to mitochondrial surfaces. These changes reverse after recovery sleep and are blunted by electron overflow channels (e.g., AOX) in the respiratory chain.
• ER contacts and mitophagy: Sleep deprivation increases mitochondria–ER contact sites and mitophagy, supporting mitochondrial biogenesis via phospholipid transfer.
• Mitochondrial dynamics modulate sleep: Inducing or blocking mitochondrial fission/fusion in dFBNs bidirectionally alters sleep and neuronal excitability—hyperfusion increases excitability and sleep; fragmentation decreases both.
• ATP and sleep pressure: ATP in dFBNs rises after forced wakefulness because wake-mediated inhibition reduces ATP consumption, increasing mitochondrial electron leak. Uncoupling electron flux from ATP synthesis relieves sleep pressure, whereas mismatching electron supply to ATP demand (via light-driven proton pumps) promotes sleep.

Mechanism

Mitochondrial electron leak

During wakefulness, dFBN mitochondria are prone to electron leak because:

• High caloric intake with reduced neuronal electrical activity keeps ATP reserves full
• Low ATP demand yields high proton-motive force and high ATP:ADP ratio
• High NADH:NAD+ ratio increases electron supply
• These conditions overfill the CoQ pool, risking superoxide and ROS formation

Hyperkinetic as a leak sensor

Hyperkinetic, the β subunit of the Shaker voltage-gated potassium channel, regulates dFBN electrical activity. It is an unusual aldoketoreductase with tightly bound NADPH whose redox state reflects the fate of electrons entering the respiratory chain. dFBNs indirectly gauge mitochondrial electron leak by counting reductions of lipid-peroxidation-derived carbonyls at Hyperkinetic's active site—an early warning system against damaging electron leak.

Fission–fusion feedback

• Fission (Drp1-mediated): fragments mitochondria, lowering excitability and sleep
• Fusion (Opa1/Marf-mediated): networks mitochondria, raising excitability and sleep
• Phosphatidic acid (produced by mitoPLD from cardiolipin) is key to fusion; Mitoguardin (Miga) stabilizes mitoPLD and transfers phospholipids to mitochondria

Metabolic origin of sleep

Sleep may be an inevitable consequence of aerobic metabolism—an innovation maximizing extractable free energy from electron transfer after two major rises in atmospheric oxygen (2.4 billion and 0.75–0.57 billion years ago), enabling the Cambrian explosion of multicellular life. Energy-hungry nervous systems brought the need for sleep. Sleep and hunger may share a similar mitochondrial origin: electrons flowing through respiratory chains of their feedback controllers determine when balance must be restored, like sand in an hourglass.

Conclusions and Outlook

Conclusions:

• Sleep pressure arises from a mismatch between mitochondrial electron supply and ATP demand, causing electron leak and ROS
• Mitochondrial dynamics directly regulate sleep and the electrical properties of sleep-control neurons
• Sleep may be an inevitable consequence of aerobic metabolism, sharing metabolic ground with aging

Significance:

• First physical explanation of sleep pressure, tying sleep to basic cellular metabolism
• New molecular pathways for understanding sleep regulation
• Potential therapeutic targets via mitochondrial dynamics or electron transport chain modulation

Future directions: validating mechanisms in mammals, linking mitochondrial dysfunction to sleep quality, developing drugs targeting mitochondrial dynamics, exploring sleep–metabolic disease mechanisms, and studying age-related mitochondrial effects on sleep.