Hypothesis

During NREM slow‑wave sleep the brain actively re‑acidifies lysosomes and upregulates cystatin B, converting the glymphatic influx into a controlled proteolytic triage that trims excess synapses; sleep loss blocks this reset, allowing cathepsin leakage to drive maladaptive synaptic pruning.

Mechanistic Basis

• Aging lysosomes lose membrane integrity, raising pH and triggering TRPML1‑mediated Ca2+ release that activates calpains, further damaging the membrane【https://pmc.ncbi.nlm.nih.gov/articles/PMC7388076/】.
• Cytosolic Ca2+ also activates calpains that cleave CSTB, diminishing its inhibitory potency on cathepsins B and L【https://pmc.ncbi.nlm.nih.gov/articles/PMC11753708/】.
• CSTB normally restrains chromatin‑associated cathepsin L, preventing aberrant histone degradation【https://www.frontiersin.org/journals/molecular-neuroscience/articles/10.3389/fnmol.2022.1069122/full】.
• Slow‑wave sleep coincides with a surge in glymphatic CSF‑interstitial exchange that delivers protons and ATP‑driven V‑ATPase activity to lysosomes, re‑acidifying them and inhibiting TRPML1 opening【https://pmc.ncbi.nlm.nih.gov/articles/PMC8202514/】.
• Concurrently, sleep‑dependent signaling (e.g., via melatonin‑MT1 receptors) boosts CSTB transcription, reinforcing the cathepsin brake【https://pubmed.ncbi.nlm.nih.gov/41271184/】.
• When this window is missed, leaked cathepsins degrade PSD‑95, MAP2 and other synaptic scaffolds, producing the synaptic loss seen in chronic sleep disruption.

Testable Predictions

1. Cortical lysosome pH will be lowest during early NREM and rise progressively with wakefulness.
2. CSTB protein levels will peak after 2–3 h of consolidated sleep and trough after prolonged wakefulness.
3. Pharmacological blockade of TRPML1 or genetic overexpression of CSTB in mice will prevent sleep‑deprivation‑induced cathepsin B/L activity and preserve synaptic protein levels.
4. Conversely, chemogenetic inhibition of V‑ATPase during sleep will mimic the lysosomal pH rise seen in wakefulness and exacerbate cathepsin‑mediated synaptic degradation.

Experimental Approach

• Record in vivo lysosomal pH using LysoSensor‑yellow/blue dye in Thy1‑LysoSensor mice across sleep‑wake states measured by EEG/EMG.
• Quantify CSTB and cathepsin activity via western blot and activity‑based probes in microdissected cortex at ZT2 (early sleep) vs ZT14 (late wake).
• Subject mice to 6 h sleep deprivation; treat subsets with ML‑SI1 (TRPML1 inhibitor) or AAV‑CSTB; assess cathepsin leakage (immunostaining for cytosolic cathepsin L) and dendritic spine density (DiOlistic labeling).
• Rescue experiments: administer cathepsin B/L inhibitor (Ca‑074Me) during deprivation to test whether synaptic loss is protease‑dependent.
• Statistical plan: two‑way ANOVA (state × treatment) with post‑hoc Tukey; n ≥ 8 per group for power > 0.8.

If predictions hold, sleep’s autopsy function is recast as a lysosomal quality‑control step that gates which neural architectures survive to the next day. Failure of this gate—through impaired re‑acidification or CSTB loss—converts a restorative process into a source of neurodegeneration.