Hypothesis

Combining senolytic clearance of dysfunctional astrocytes with time‑restricted eating (TRF) will amplify microglial lysosomal exocytosis during sleep, thereby increasing the extracellular removal of tau and amyloid‑β (Aβ) beyond what either intervention achieves alone.

Mechanistic Rationale

During slow‑wave sleep, the glymphatic system expands interstitial space, allowing cerebrospinal fluid to flush soluble aggregates [1]. Concurrently, microglia release lysosomal enzymes such as cathepsin B via lysosomal exocytosis, which decorates extracellular plaques and fibrils, marking them for perivascular drainage [2]. Senescent astrocytes impede this process by secreting TGF‑β1, which suppresses microglial TFEB activity and reduces lysosomal exocytosis [3,4]. Senolytics (e.g., dasatinib + quercetin) eliminate these astrocytes, relieving TGF‑β–mediated inhibition and restoring microglial cathepsin release.

TRF aligns fasting intervals with circadian peaks in AMPK activation, which phosphorylates and activates TFEB in microglia, potentiating lysosomal biogenesis and exocytosis [5,6]. Moreover, AMPK inhibition of mTORC1 in microglia counters the persistent mTORC1 signaling that renders senescent cells resistant to autophagy [4]. Thus, senolytics remove the cellular block, while TRF provides the metabolic signal to maximise microglial lysosomal output during the sleep window.

We propose that the synergistic increase in microglial cathepsin B release leads to greater opsonization of extracellular tau/Aβ, enhancing glymphatic clearance and reducing intracellular aggregate load via improved autophagic flux in neurons.

Experimental Design

Model: Aged (18‑month) APP/PS1 mice expressing a microglial pHluorin‑LAMP1 reporter to visualize lysosomal exocytosis in vivo. Groups (n=10 per group):

1. Vehicle control (ad libitum feeding)
2. Senolytics (dasatinib + querticin, 5 mg/kg + 50 mg/kg, twice weekly)
3. TRF (10‑hour feeding window aligned with dark phase)
4. Senolytics + TRF Intervention duration: 8 weeks. Readouts (collected at ZT2, peak slow‑wave sleep):

• Microglial lysosomal exocytosis rate (pHluorin‑LAMP1 fluorescence intensity per microglia, confocal imaging of cortex)
• Glymphatic influx (intrathecal Alexa‑647‑BSA tracer, quantified by MRI‑based clearance half‑time)
• Extracellular tau and Aβ levels (ELISA of cortical interstitial fluid collected via microdialysis)
• Neuronal autophagy flux (LC3‑II/I ratio and p62 Western blot of synaptoneurosomes)
• Cognitive performance (Y‑maze spontaneous alternation)

Predictions and Falsifiability

If the hypothesis is correct, the Senolytics + TRF group will show:

• A ≥30 % increase in microglial lysosomal exocytosis versus each monotherapy (p<0.01)
• A ≥25 % faster glymphatic tracer clearance compared with controls
• Significant reductions in extracellular tau and Aβ (≥20 % decrease) and concomitant improvements in neuronal autophagy flux and spatial working memory.

Failure to observe a synergistic enhancement of microglial exocytosis or glymphatic clearance in the combination group—i.e., no significant difference from the best single treatment—would falsify the hypothesis. Likewise, if senolytics do not reduce astrocytic TGF‑β1 levels or if TRF fails to increase microglial AMPK‑TFEB signaling, the proposed mechanistic link would be unsupported.