Hypothesis

During sleep the brain does not merely clear waste; it performs an active synaptic triage in which recently active synapses are marked for either preservation or removal based on a phosphorylation‑dependent tag that integrates metabolic state (NAD+/SIRT1, AMPK/mTOR) and neuronal activity (CaMKII). This tag determines whether a synapse enters the glymphatic‑autophagic clearance pathway or is spared, shaping next‑day network architecture. Chronic sleep disruption prevents the completion of this triage, leading to accumulation of ‘zombie’ synapses—partially tagged, incompletely cleared connections that impair plasticity and promote neurodegeneration.

Mechanistic Rationale

1. Activity‑dependent tagging – Wakeful activity raises intracellular Ca2+, activating CaMKII, which phosphorylates synaptic scaffolding proteins (e.g., PSD‑95, Synapsin) on specific serine residues. This creates a ‘degron’ recognized by the LC3‑interacting motif of autophagosomes.
2. Metabolic gating – Sleep‑induced melatonin and AMPK activation suppress mTORC1, lowering the threshold for autophagic engulfment of tagged synapses. Concurrently, SIRT1 activation (via rising NAD+) deacetylates autophagy machinery, enhancing flux.
3. Glymphatic coupling – The interstitial fluid surge driven by AQP4 facilitates extracellular diffusion of phosphorylated synaptic fragments, allowing peripheral macrophage‑like cells (as shown in Drosophila) to phagocytose lipids and protein debris released from eliminated synapses.
4. Outcome – Synapses with low activity (low CaMKII phosphorylation) escape tagging and are preserved; highly active synapses that have completed their functional cycle are efficiently cleared, preventing excitotoxic accumulation.

Testable Predictions

• Prediction 1: In mice, optogenetic induction of hippocampal CA1 pyramidal cell firing during wakefulness will increase CaMKII‑dependent phosphorylation of PSD‑95. Sleep deprivation will reduce the subsequent sleep‑dependent decrease in this phospho‑signal, whereas pharmacological inhibition of CaMKII (e.g., with autocamtide‑2‑related inhibitory peptide) during sleep will restore normal phospho‑decline.
• Prediction 2: Using dynamic SILAC labeling of synaptic proteins, the half‑life of tagged PSD‑95 will be significantly shorter in rested mice (>2‑fold increase in degradation rate) compared to sleep‑deprived mice. Administration of an NAD+ precursor (NR) will rescue the degradation rate in sleep‑deprived animals by enhancing SIRT1 activity.
• Prediction 3: Mice subjected to chronic sleep restriction will show accumulation of PSD‑95‑positive, ubiquitinated synaptic fragments in the perivascular space (detected by immuno‑EM) and concomitant deficits in long‑term potentiation and spatial memory. Genetic knockdown of lysosomal LC3 in astrocytes will exacerbate this accumulation, confirming the autophagic arm of triage.
• Prediction 4: Administering rapamycin at the onset of the sleep phase (when mTOR is naturally low) will synergize with sleep‑dependent autophagy to accelerate clearance of tagged synapses, whereas daytime dosing will have no effect.

Falsifiability

If sleep deprivation does not alter the sleep‑dependent phosphorylation/dephosphorylation dynamics of activity‑tagged synaptic proteins, or if manipulating CaMKII phosphorylation fails to rescue synaptic clearance and cognitive deficits despite normal glymphatic flux, the core mechanistic link between activity tagging and sleep‑dependent synaptic triage would be refuted.

References

• Glymphatic clearance enhanced during sleep and driven by AQP4‑dependent water flux: https://doi.org/10.1084/jem.20211275
• Selective slow‑wave sleep disruption acutely increases cerebrospinal fluid amyloid‑β: https://doi.org/10.1093/brain/awx148
• Melatonin inhibits mTOR like rapamycin, activates p‑AMPK/ULK1 for autophagy: https://www.scientificarchives.com/article/impact-of-sleep-on-autophagy-and-neurodegenerative-disease-sleeping-your-mind-clear
• Sleep deprivation impairs autophagic clearance creating a positive feedback loop: https://pubmed.ncbi.nlm.nih.gov/41013508/
• Peripheral macrophage‑like cells migrate to the brain during sleep to clear lipids: https://pubmed.ncbi.nlm.nih.gov/41673150/
• NAD+ precursors boost NAD+ stores, inhibit JNK to reduce Aβ oligomers: https://www.scientificarchives.com/article/impact-of-sleep-on-autophagy-and-neurodegenerative-disease-sleeping-your-mind-clear