Hypothesis

Aging neurons convert dangerous misfolded proteins into chaperone‑rich aggregates that serve as reversible storage depots for select RNA‑binding proteins (RBPs). These RBPs remain functionally silent but intact within the aggregate matrix and can be liberated by disaggregase activity to restart translation during recovery phases.

Mechanistic Basis

1. Phase‑separation seeding – Stress‑induced low‑complexity regions of RBPs drive transient liquid droplets that mature into amyloid‑like fibrils, a process observed in both physiological stress granules and pathological aggregates 1.
2. Chaperone coating – Hsp70/Hsp90 and small Hsps bind the fibril surface, creating a protective shell that prevents further seeding while keeping incorporated RBPs in a near‑native conformation 2.
3. Post‑translational gating – Phosphorylation of RBPs lowers their affinity for the fibril core, acting as a molecular switch that favors release when kinase activity drops 3.
4. Disaggregase extraction – The HSP110‑HSP70‑HSP40 complex can thread polypeptides out of the fibril without dissolving the core, delivering RBPs to the cytosol in a refold‑competent state 4.

Testable Predictions

• P1: Elevating disaggregase levels in aged brains will decrease the fraction of chaperone‑rich aggregates without raising soluble oligomer levels.
• P2: Cytosolic concentrations of sequestered RBPs (e.g., TIA1, G3BP1) will rise after disaggregase boost, correlating with increased polysome‑associated RNA and puromycin incorporation.
• P3: Behavioral assays of hippocampal‑dependent memory will show improvement proportional to the restoration of translation rates.
• P4: Pharmacological inhibition of disaggregases will exacerbate translational deficits even when aggregate burden is unchanged.

Experimental Approach

• Use AAV9‑HSP110 to overexpress the disaggregase subunit in 18‑month‑old APP/PS1 mice and age‑matched controls.
• Fractionate brain lysates into soluble, chaperone‑rich aggregate, and insoluble pellet fractions; immunoblot for RBPs (TIA1, G3BP1, FUS) and aggregate markers (phospho‑tau, amyloid‑β).
• Perform polysome profiling and SUnSET puromycin labeling to quantify global translation.
• Assess cognition with Morris water maze and novel object recognition.
• Include groups treated with HSP110‑specific siRNA or the small‑inhibitor MKT-077 to test the converse prediction.
• Statistical analysis: two‑way ANOVA with age and treatment as factors; post‑hoc Tukey.

Interpretation

If P1‑P3 are satisfied, the data support the model that aggregates act as a tunable reservoir rather than inert waste, and that enhancing disaggregase activity can rescue proteostasis without increasing toxicity. Failure to observe RBP release or translational rescue despite aggregate reduction would falsify the hypothesis, indicating that aggregates are not a releasable store under these conditions.