Rapamycin restores ECM plasticity by activating TFEB‑driven chondroitin sulfate desulfation, not just bulk autophagy

Hypothesis

Rapamycin extends lifespan and healthspan by selectively enhancing the removal of aberrantly sulfated chondroitin sulfate proteoglycans (CSPGs) through a TFEB‑dependent transcriptional program that upregulates extracellular sulfatases, thereby resetting perineuronal net (PNN) ECM structure to a youthful state.

Mechanistic Rationale

1. mTORC1 inhibition activates TFEB – Rapamycin‑mediated mTORC1 suppression triggers nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy [4]. Beyond canonical autophagy, TFEB directly induces expression of arylsulfatases B, SULF1 and SULF2, enzymes that remove 4‑O‑ and 6‑O‑sulfate groups from chondroitin sulfate (CS) chains.
2. Targeted desulfation of pathogenic CSPGs – Aging astrocytes and microglia increase synthesis of CSPGs bearing elevated 4‑sulfation (C4S) and reduced 6‑sulfation (C6S) within PNNs [2][5]. These sulfation patterns increase CSPG affinity for amyloid‑β and inhibit neurite outgrowth. TFEB‑driven sulfatase upregulation preferentially hydrolyzes these excess sulfate groups, shifting the CS sulfation ratio toward a C6S‑rich, permissive ECM.
3. Functional readout – PNN plasticity – Desulfated CSPGs exhibit lower binding affinity for neurotoxic aggregates and reduced capacity to sterically hinder synaptic remodeling. Restoring a youthful CS sulfation profile should permit axon sprouting and dendritic spine turnover, directly linking ECM quality to cognitive resilience.
4. Distinction from generic autophagy – While bulk autophagy clears protein aggregates, it does not modify GAG fine structure. The hypothesis predicts that rapamycin’s longevity effect will be attenuated in cells lacking TFEB or specific sulfatases, even if lysosomal flux remains intact.

Testable Predictions

• Prediction 1: In rapamycin‑treated aged mice, nuclear TFEB levels in hippocampal astrocytes will correlate with increased mRNA and protein expression of ARSB, SULF1, and SULF2 (measure via immunofluorescence and qPCR).
• Prediction 2: CS extracted from rapamycin‑treated brains will show a significant decrease in the C4S/C6S ratio compared with vehicle controls, quantified by disaccharide analysis via LC‑MS/MS.
• Prediction 3: Conditional knockout of TFEB in forebrain astrocytes will abolish rapamycin‑induced CSPG desulfation and prevent the rescue of age‑related deficits in long‑term potentiation (LTP) and spatial memory, despite unchanged LC3‑II turnover.
• Prediction 4: Pharmacological inhibition of ARSB/SULF1/2 (using specific small‑molecule inhibitors) will block the beneficial effects of rapamycin on PNN permeability (assayed with fluorescent dextran diffusion) and neurogenesis, confirming the causal role of desulfation.

Falsification

If rapamycin extends lifespan and improves cognitive function without detectable changes in CSF sulfation patterns or without requiring TFEB/sulfatase activity, the hypothesis would be falsified. Conversely, demonstration that mTORC1 inhibition fails to alter CSPG sulfation yet still prolongs life would indicate that longevity stems from alternative mechanisms, refuting the ECM‑centric model.

Broader Implications

This model reframes rapamycin not merely as a mimetic of starvation but as a pharmacological reset‑switch for ECM "structural memory." By linking nutrient‑sensing pathways to GAG remodeling, it opens a dual‑target strategy: combine mTOR inhibition with sulfatase activators to synergistically restore matrix youthfulness in neurodegenerative diseases.