Mitochondrial Origins of Sleep Pressure

 

Mitochondrial Origins of Sleep Pressure

Source: Raffaele Sarnataro, Cecilia D. Velasco, Nicholas Monaco, Anissa Kempf & Gero Miesenböck (2025). "Mitochondrial origins of the pressure to sleep." Nature, DOI: 10.1038/s41586-025-09261-y.

Date of Publication: Received: 22 February 2024; Accepted: 9 June 2025; Published online: xx xx xxxx (Open access)

I.  Summary

This study proposes a novel, unifying theory for the fundamental cause of sleep, suggesting it is an "inescapable consequence of aerobic metabolism." Through single-cell transcriptomics and functional experiments in Drosophila flies, the researchers pinpoint specific sleep-control neurons (dorsal fan-shaped body neurons, dFBNs) where sleep pressure arises from a mismatch between mitochondrial electron supply and ATP demand. Prolonged waking leads to an oversupply of electrons to the mitochondrial respiratory chain in dFBNs, resulting in increased reactive oxygen species (ROS) and mitochondrial fragmentation. Sleep serves as a compensatory mechanism to restore mitochondrial function and prevent widespread damage. The study provides strong evidence that mitochondrial dynamics (fission and fusion) directly regulate sleep duration, depth, and neuronal excitability in these key sleep-promoting cells, establishing a physical interpretation for "sleep pressure."

II. Main Themes and Key Findings

1. Mitochondrial Respiration as the Proximate Cause of Sleep Pressure:

• The core hypothesis is that "Sleep, like ageing, may be an inescapable consequence of aerobic metabolism."
• Single-cell RNA sequencing of sleep-deprived Drosophila brains revealed that gene transcripts upregulated in dFBNs (sleep-control neurons) are "almost exclusively proteins with roles in mitochondrial respiration and ATP synthesis." This distinct transcriptomic signature of sleep loss was unique to dFBNs among the tested neuron types.
• Specifically, sleep loss led to upregulation of components of electron transport complexes (I-V) and enzymes of the tricarboxylic acid cycle, while genes involved in synaptic transmission were downregulated.
• This suggests that the growing need for sleep is not a global brain phenomenon, but rather originates in specialized neurons.

1. Electron Surplus and ATP Dynamics in dFBNs During Waking:

• During waking, dFBNs experience an "oversupply (relative to ATP demand) of electrons to CoQ," the mobile carrier in the respiratory chain.
• This electron surplus is exacerbated by high caloric intake during waking and reduced electrical activity/ATP consumption in dFBNs, which are inhibited during arousal.
• Measurements with genetically encoded ATP sensors showed "approximately 1.2-fold higher ATP concentrations in dFBNs… after a night of sleep deprivation than at rest."
• This electron oversupply increases the "probability of… non-enzymatic single-electron reductions of O2," leading to increased production of reactive oxygen species (ROS). dFBNs are described as an "effective early warning system against widespread damage" due to their predisposition to this electron leak.

1. Mitochondrial Fragmentation and Mitophagy as Consequences of Sleep Deprivation:

• Sleep deprivation causes significant morphological changes in dFBN mitochondria: reduced size, elongation, and branching.
• This fragmentation is accompanied by the relocation of Drp1 (dynamin-related protein 1), a key fission dynamin, from the cytosol to the mitochondrial surface.
• Sleep deprivation also stimulates increased "mitochondria–endoplasmic reticulum contacts," which are sites for fission/fusion machinery and lipid transfer, and "enhanced mitophagy" (clearance of dysfunctional mitochondria).
• Importantly, interventions that mitigate the electron surplus (e.g., expressing alternative oxidase, AOX) protected dFBN mitochondria from sleep loss-induced fragmentation, supporting the role of ROS as the "initial spark that triggers fission."

1. Direct Causal Link Between Mitochondrial Dynamics and Sleep:

• Experimentally manipulating mitochondrial fission and fusion in dFBNs directly altered sleep behavior:
• Fragmenting dFBN mitochondria (overexpressing Drp1 or depleting Opa1/Marf) "decreased sleep," "abolished the homeostatic response to sleep deprivation," and "reduced ATP concentrations in dFBNs."
• Promoting mitochondrial fusion (knocking down Drp1 or overexpressing Opa1/Marf) "increased baseline as well as rebound sleep" and "elevated the arousal threshold."
• These interventions also had "large and opposite behavioural consequences," correlating with established biophysical signatures of sleep pressure (e.g., altered current–spike frequency functions and somnogenic bursts in dFBNs).
• The study further identifies phosphatidic acid (PA) and proteins like mitoPLD (zucchini) and Mitoguardin (Miga) as crucial for mitochondrial fusion and sleep regulation, indicating a lipid-mediated component to mitochondrial dynamics.

1. Sleep as a Restorative Process for Mitochondrial Health:

• The morphological changes in mitochondria (fragmentation, increased contacts, enhanced mitophagy) observed after sleep deprivation are reversible after recovery sleep, with mitochondrial volume, shape, and branch length rebounding "above baseline values."
• The upregulation of mitochondrial protein transcripts after sleep deprivation is interpreted as potentially prefiguring "the proliferation and fusion of mitochondria during subsequent recovery sleep," suggesting a compensatory response to organelle damage and preparation for restoration.

III. Supporting Evidence and Mechanisms

• Transcriptomics: Used single-cell RNA sequencing (scRNA-seq) to profile transcriptomes of dFBNs in rested vs. sleep-deprived flies, identifying specific upregulation of mitochondrial metabolism genes.
• ATP Measurement: Genetically encoded ATP sensors (iATPSnFR and ATeam) showed elevated ATP in dFBNs after sleep deprivation and acute increases upon arousal-mediated inhibition.
• Electron Flux Manipulation:Introduction of Alternative Oxidase (AOX), which provides an "exit route for surplus electrons" from the CoQ pool, "relieved the basal pressure to sleep" and counteracted fragmentation.
• Overexpression of Uncoupling Proteins (Ucp4A/C), which "short-circuit the proton electrochemical gradient," "decreased sleep" by increasing electron demand.
• Light-driven proton pump (mito-dR), which powers ATP synthesis with photons, made NADH-derived electrons "redundant" and "precipitated sleep."
• Mitochondrial Morphometry: Confocal laser-scanning microscopy (CLSM) and optical photon reassignment microscopy (OPRM) were used to image and quantify mitochondrial size, shape, branching, and number, demonstrating fragmentation upon sleep deprivation and recovery during sleep.
• Drp1 Relocation: Immunostaining showed increased localization of Drp1 to mitochondria after sleep deprivation, indicating active fission.
• Mitophagy and ER Contact Sites: SPLICS (Split-GFP-based Contact Site Sensors) and mito-QC (ratiometric mitophagy sensor) confirmed increased ER-mitochondria contacts and enhanced mitophagy after sleep deprivation.
• Genetic Manipulations: Targeted overexpression or RNAi knockdown of key mitochondrial fission (Drp1) and fusion (Opa1, Marf) genes in dFBNs showed direct dose-dependent effects on sleep.
• Electrophysiology: dFBN excitability (current–spike frequency functions, bursting) was altered by manipulating mitochondrial dynamics, consistent with changes in sleep pressure.

IV. Broader Implications and Connections

• Evolutionary Basis of Sleep: The study posits sleep serves an "ancient metabolic purpose," linking its emergence to the "Cambrian explosion of multicellular life" and the rise of power-hungry nervous systems following increases in atmospheric oxygen. The allometric power law relating sleep to O2 consumption in mammals further supports this.
• Mitochondrial Disease: The authors note that an "overwhelming sense of tiredness... is in fact a common symptom of human mitochondrial disease," reinforcing the relevance of their findings to human health.
• Parallel to Hunger Regulation: The discussion draws a striking parallel between sleep pressure and hunger, noting that "the mitochondria of orexigenic neurons expressing agouti-related protein (AgRP) and of anorexigenic neurons expressing pro-opiomelanocortin undergo antiphasic cycles of fission and fusion" coupled to energy balance. This suggests a conserved metabolic origin for fundamental homeostatic drives.
• Physical Interpretation of Sleep Pressure: The research provides a concrete biophysical basis for the abstract concept of "sleep pressure," grounding it in "mismatches between NADH supply and ATP demand."
• Distinction from Other Sleep Theories: While acknowledging other functions of sleep (e.g., synaptic homeostasis, memory consolidation), this study focuses on a more fundamental, metabolic origin that may underpin these later-evolved roles.

Conclusion:

This groundbreaking study significantly advances our understanding of sleep's fundamental purpose, moving beyond merely correlative observations to establish a causal link between mitochondrial function in specific sleep-control neurons and the physiological need for sleep. By demonstrating that an electron surplus in dFBN mitochondria during waking drives sleep pressure through increased ROS and mitochondrial fragmentation, and that sleep serves to reverse these processes, the research offers a powerful new framework for understanding sleep homeostasis and its evolutionary roots in aerobic metabolism.

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