Hypothesis

Supplemental butyrate-producing prebiotics enhance nocturnal glymphatic clearance by increasing aquaporin-4 (AQP4) polarization at astrocytic endfeet, thereby promoting selective removal of hyperphosphorylated tau and improving mood symptoms.

Mechanistic Rationale

Butyrate, a histone deacetylase inhibitor generated by fermenting bacteria, upregulates AQP4 transcription and facilitates its relocation to perivascular membranes [https://doi.org/10.1038/s41467-020-15119-w]. Polarized AQP4 expands interstitial space during NREM sleep, accelerating cerebrospinal fluid influx and efflux. Concurrently, butyrate reduces neuroinflammatory cytokines that otherwise disrupt perivascular fluid exchange [https://www.eurekalert.org/news-releases/1104277]. By tightening the glymphatic conduit, butyrate shifts the nightly "autopsy" from passive waste removal to an active triage that preferentially degrades pathogenic tau aggregates while sparing synaptic proteins essential for mood regulation.

Testable Predictions

1. Participants receiving a butyrate‑boosting prebiotic will show greater AQP4 polarization (measured via PET with an AQP4‑specific tracer) after a night of sleep compared with placebo.
2. Enhanced AQP4 polarization will correlate with increased intrathecal clearance of CSF‑tagged amyloid‑β/tau analogues (using MRI‑gadolinium clearance kinetics) during NREM sleep.
3. Improvements in glymphatic flux will mediate reductions in depression and anxiety scores (MADRS, HAM‑A) independent of changes in total sleep time.
4. Antibiotics that suppress butyrate producers will blunt the prebiotic‑induced AQP4 shift and abolish mood benefits.

Experimental Design

• Population: 60 adults aged 45‑65 with mild depressive symptoms (MADRS 10‑19) and self‑reported sleep disturbance.
• Design: Double‑blind, placebo‑controlled, crossover; 4‑week prebiotic (galactooligosaccharides + resistant starch) vs. maltodextrin placebo, 2‑week washout.
• Outcomes:
  • Primary: AQP4 PET signal change pre‑ vs. post‑intervention after a supervised night of polysomnography‑verified sleep.
  • Secondary: Glymphatic clearance rate (intrathecal gadolinium MRI), CSF phosphorylated‑tau and amyloid‑β levels, MADRS/HAM‑A scores, stool butyrate concentration (GC‑MS).
• Analysis: Mixed‑effects models testing interaction between treatment, AQP4 change, and glymphatic flux on mood scores; mediation analysis to assess whether glymphatic improvement accounts for mood effects.

If butyrate does not increase AQP4 polarization or glymphatic clearance, or if mood improvements occur without these neurobiological changes, the hypothesis is falsified. Conversely, a consistent chain from prebiotic → butyrate ↑ → AQP4 polarization ↑ → glymphatic flux ↑ → pathogenic protein ↓ → mood improvement would substantiate the proposed microbiome‑sleep‑clearance axis.