Hypothesis

Sleep provides a privileged window for microglia to phagocytose neurons harboring somatic copy number variations (CNVs) or persistent DNA lesions. During wakefulness, neuronal activity generates metabolic stress and DNA double‑strand breaks; sleep‑associated downregulation of synaptic firing reduces this burden and simultaneously upregulates microglial complement‑mediated phagocytic pathways (e.g., C1q, C3, TREM2). When sleep is chronically restricted, this microglial surveillance is blunted, allowing CNV‑bearing neurons to survive and accumulate, thereby driving genomic instability that correlates with cognitive decline.

Predictions

1. CNV burden rises with sleep loss – Mice subjected to chronic sleep fragmentation (4 h/night for 4 weeks) will show a significant increase in cortical somatic CNV density measured by single‑cell whole‑genome sequencing compared with ad libitum controls.
2. Microglial phagocytosis is required – Genetic ablation of TREM2 or pharmacological blockade of complement C3 will exacerbate the CNV increase under sleep fragmentation, whereas agonistic activation of TREM2 (e.g., with an agonist antibody) will rescue the phenotype.
3. DNA damage markers fluctuate across the sleep–wake cycle – Immunostaining for γH2AX and 53BP1 foci will peak during the early wake period and reach their nadir during mid‑sleep, an oscillation that is dampened in sleep‑deprived animals.
4. Human relevance – In a crossover study, healthy adults undergoing one night of total sleep deprivation will exhibit elevated circulating neuron‑derived extracellular vesicles carrying DNA fragments with copy‑number gains, detectable by droplet digital PCR.

Experimental Approach

• Animal model – Use adult Cx3cr1‑CreER; Rosa26‑tdTomato mice to label microglia; apply chronic sleep fragmentation via gentle handling or automated treadmill. Control groups receive undisturbed sleep.
• Single‑cell genomics – Isolate cortical neurons and microglia, perform single‑cell CNV calling (e.g., using CopyKat) to quantify burden per cell type.
• Phagocytosis assay – Inject pH‑rodo‑labeled synaptosomes intracerebroventricularly; quantify microglial uptake via flow cytometry and confocal microscopy.
• DNA damage imaging – Perform immunofluorescence for γH2AX and 53BP1 on coronal sections at zeitgeber times ZT2, ZT8, ZT14, ZT20.
• Human pilot – Recruit 12 healthy volunteers; collect blood before and after 24 h wakefulness; isolate neuron‑derived L1CAM+ extracellular vesicles; assay for CNV‑specific PCR amplicons (e.g., APP duplication) using ddPCR.

Potential Outcomes and Interpretation

If predictions hold, the data would support a causal role for sleep‑dependent microglial phagocytosis in purging genomically compromised neurons, positioning sleep as an active genome‑surveillance mechanism rather than a passive restorative state. Failure to observe CNV accumulation or microglial dependence would falsify the hypothesis and suggest that sleep‑genomic instability links are mediated primarily by upstream pleiotropic factors.

References (inline citations)

• Population studies show a U‑shaped relationship between sleep duration and CNV burden 1.
• Only a small fraction of CNV effects on cognition is mediated by sleep, indicating pleiotropy 2.
• GWAS of sleep traits highlight neuronal and synaptic pathways, not genome‑maintenance genes 3.
• Persistent DNA lesions in tissue‑resident macrophages drive neurodegeneration, suggesting a clearance role for microglia 4.
• CNVs in late‑onset Alzheimer’s patients regulate gene expression and associate with age of death, confirming functional impact 5.