Hypothesis

Administering exogenous D‑β‑hydroxybutyrate (D‑BHB) at the transition to slow‑wave sleep will potentiate the brain’s nightly maintenance program by simultaneously enhancing HDAC3‑dependent autophagy and astrocytic glymphatic flow, resulting in measurable increases in toxic protein clearance and improved next‑day cognitive performance.

Mechanistic Rationale

• HDAC3 inhibition → autophagy: D‑BHB is a endogenous class I HDAC inhibitor, preferentially targeting HDAC3 in cortical neurons [1]. HDAC3 suppression drives transcription of Npas4, LC3B‑II and Beclin‑1 while lowering p62, thereby activating the autophagy‑lysosome pathway [2]
• Astrocyte‑mediated glymphatic boost: D‑BHB also activates the hydroxycarboxylic acid receptor 2 (HCAR2) on astrocytes [3], triggering cAMP‑PKC signaling that phosphorylates aquaporin‑4 (AQP4) and promotes its perivascular localization—a key determinant of glymphatic influx [5]
• Temporal synergy: Slow‑wave sleep is when endogenous glymphatic clearance peaks and autophagic flux is naturally upregulated [4]. Aligning exogenous D‑BHB exposure with this window should therefore amplify both pathways beyond their basal sleep‑associated activity.

Novel Insight

While prior work treats BHB as a passive energy substrate or a general HDAC inhibitor, we propose that its dual role—as a metabolic signal that directly modulates astrocytic water channels and as an epigenetic regulator of neuronal autophagy—creates a feed‑forward loop: cleared interstitial waste reduces inflammatory signaling, which further sustains HDAC3 inhibition via lowered NAD⁺ consumption, prolonging the autophagic state throughout the night.

Testable Predictions

1. Biomarker surge: In a within‑subject crossover design, participants receiving D‑BHB (0.3 g kg⁻¹) 15 min before scheduled sleep will show a ≥20 % increase in CSF‑derived autophagic markers (LC3B‑II/Beclin‑1 ratio) and a ≥15 % rise in glymphatic influx rate (measured by intrathecal Gadobutrol‑MRI) compared with placebo.
2. Protein clearance: PET imaging of amyloid‑β will reveal a ≥10 % greater overnight reduction in cortical SUVr in the D‑BHB condition.
3. Cognitive outcome: Next‑day psychomotor vigilance test (PVT) lapses will decrease by ≥15 % relative to baseline, correlating with the magnitude of autophagic/glymphatic changes.
4. Specificity: Co‑administration of a selective HCAR2 antagonist will blunt the glymphatic enhancement without affecting autophagic markers, whereas an HDAC3‑selective siRNA will attenuate the autophagy rise but leave glymphatic flow intact.

Falsifiability

If D‑BHB given at sleep onset fails to produce statistically significant changes in any of the primary biomarkers (autophagy, glymphatic influx, amyloid‑β clearance) or cognitive performance, the hypothesis that timed ketone exposure amplifies sleep‑dependent brain maintenance is refuted.

Implementation Sketch

• Population: 30 healthy adults, ages 20‑35, screened for normal sleep.
• Design: Double‑blind, randomized, placebo‑controlled crossover with two 2‑night sessions separated by ≥1 week.
• Intervention: Oral D‑BHB ketone ester vs. isocaloric maltodextrin placebo administered 15 min before lights‑out.
• Measurements:
  • CSF sampling via lumbar puncture at awakening for LC3B‑II, Beclin‑1, p62 (Western blot).
  • Glymphatic MRI: intrathecal Gadobutrol, dynamic contrast‑enhanced imaging to quantify influx rate (Ktrans).
  • Amyloid‑β PET (PiB) before sleep and after awakening.
  • Cognitive battery: PVT, n‑back, mood scales.
  • Plasma BHB levels to confirm pharmacokinetic timing.
• Analysis: Mixed‑effects models with session order as covariate; significance set at p<0.05 (two‑tailed).

By directly linking the timing of ketone availability to the brain’s endogenous repair cycle, this work could redefine nutritional strategies for neurodegeneration prevention and clarify whether the “autopsy” metaphor reflects a biochemically tractable, modifiable process.