Hypothesis

Sleep-dependent glymphatic clearance actively removes excess pericentriolar material and regulates CDK2 activity at centrosomes, promoting declustering of supernumerary centrosomes. When sleep is disrupted, this nocturnal triage fails, allowing clustered extra centrosomes to persist, driving low‑grade chromosomal instability and increasing tumorigenic potential in aged somatic stem cells.

Mechanistic Rationale

1. Glymphatic flux and centrosome turnover – During sleep, interstitial CSF influx increases, facilitating clearance of protein aggregates. Centrosomal scaffolds such as pericentrin and CDK2‑regulated substrates are susceptible to glymphatic‑mediated removal when they become loosely attached after mitotic exit. This process is analogous to the autophagic clearance of damaged organelles but operates extracellularly via perivascular channels 1.

2. CDK2 activity gating – CDK2 phosphorylates centrosomal proteins (e.g., NEDD1, CPAP) to stabilize centrosome cohesion. Sleep is associated with reduced neuronal metabolic rate and lowered CDK2 activity in proliferative niches (e.g., intestinal stem cells). Low CDK2 permits dephosphorylation, loosening centrosomal cohesion and rendering extra centrosomes susceptible to glymphatic extraction 2.

3. Triage outcome – Centrosomes that are successfully cleared reset the spindle pole count, preventing the need for clustering. Those that escape clearance remain clustered via KIFC1/HSET, Aurora A/B, and CDK2 activity, leading to merotelic attachments and chronic CIN 3 4.

Testable Predictions

• Prediction 1: In young mice, enforced wakefulness will increase the proportion of intestinal stem cells harboring ≥3 centrosomes and show elevated KIFC1‑dependent clustering markers compared with sleep‑allowed controls.
• Prediction 2: Pharmacological enhancement of glymphatic flow (e.g., via intrathecal injection of hypertonic saline to boost AQP4‑dependent CSF influx) during sleep deprivation will rescue centrosome number to baseline levels 5.
• Prediction 3: CDK2 inhibition in sleep‑deprived animals will phenocopy the protective effect of glymphatic enhancement, reducing centrosome clustering and subsequent lagging chromosomes in anaphase.
• Prediction 4: Longitudinal chronic sleep disruption will accelerate tumorigenesis in a APC^Min/+ background, an effect abrogated by either glymphatic boosting or CDK2 inhibition.

Experimental Approach

• Use Pdgfrα‑CreERT2; Rosa26‑LSL‑tdTomato lineage‑tracing to label intestinal stem cells.
• Quantify centrosome number via pericentrin immunofluorescence and clustering via KIFC1 localization in crypts after 2 weeks of sleep fragmentation vs. ad libitum sleep.
• Measure glymphatic influx with intrathecal fluorescein‑isothiocyanate‑dextran and confocal imaging of perivascular spaces.
• Assess CIN through lagging chromosomes (CREST staining) and micronuclei formation.
• Monitor tumor burden in APC^Min/+ mice under the same regimens, with and without intrathecal AQP4 agonist or CDK2 inhibitor (e.g., CVT‑313).

Potential Implications

If validated, this hypothesis reframes sleep not merely as a passive restorative state but as an active genomic quality‑control mechanism that selectively prunes supernumerary centrosomes. It offers a mechanistic link between circadian disruption, centrosome‑driven CIN, and age‑related cancer, suggesting that therapies targeting glymphatic function or CDK2 could mitigate the oncogenic consequences of poor sleep.