Hypothesis

We hypothesize that in mammalian neurons the pathogenic impact of protein aggregates depends on the kinetic balance between toxic oligomer formation and their conversion into thermodynamically stable amyloid-like deposits, and that this balance is gated by lysosomal capacity. When lysosomes are functional, accelerating oligomer-to-fibril conversion sequesters harmful intermediates into inert inclusions, reducing cellular stress. When lysosomal degradation is impaired, the same acceleration produces long-lived, densely packed fibrils that obstruct axonal transport and sequester essential chaperones, thereby exacerbating neurodegeneration.

Mechanistic Rationale

Recent work in yeast and C. elegans shows that aggregates can act as a final containment strategy, walling off dangerous oligomers into highly ordered, thermodynamically stable structures[1][2]. In mammals, a wide range of disease‑linked proteins become detergent‑insoluble in early Alzheimer’s brains[3], yet it remains unclear whether these deposits represent successful sequestration or a downstream pathology.

We propose two sequential steps:

1. Oligomer sequestration – Small‑molecule nucleators or overexpressed chaperones promote rapid nucleation, shifting the equilibrium from soluble oligomers to insoluble fibrils. This lowers the concentration of metastable species that disrupt membranes and signaling.
2. Aggregate turnover – Lysosomal autophagy clears the resulting fibrils. If clearance proceeds, the net effect is protective. If lysosomal flux is insufficient (as occurs with age or in certain genetic backgrounds), fibrils accumulate, become physically obstructive, and begin to trap soluble factors such as Hsp70, PSD‑95, or kinases, altering their availability.

Thus, the same molecular maneuver can be protective or deleterious depending on the cellular degradation state.

Testable Predictions

• Prediction 1: In primary mouse neurons treated with an aggregation‑promoting compound (e.g., a polyphenol that nucleates tau fibrils), oligomer levels will drop and cell survival will increase only when lysosomal activity is pharmacologically boosted (e.g., with TFEB overexpression or rapamycin). Conversely, lysosomal inhibition (chloroquine) will abolish the benefit and increase markers of axonal damage.
• Prediction 2: Using live‑cell FRET reporters for tau oligomers versus fibrils, we will observe a rapid decline in oligomer signal followed by a gradual rise in fibril signal. Neurons with high lysosomal capacity will show a subsequent decline in fibril signal over 48 h, whereas lysosomal‑deficient neurons will retain a stable high fibril signal and display increased neurofilament accumulation in axons.
• Prediction 3: Genetic reduction of lysosomal genes (e.g., Lamp2a knockdown) in an Alzheimer’s mouse model will convert the protective effect of a tau‑nucleating virus into worsened cognitive performance, despite comparable reductions in soluble tau oligomers.

Experimental Approach

• Culture primary cortical neurons from wild‑type and lysosomal‑deficient mice.
• Treat with a well‑characterized tau nucleation peptide or small‑molecule inducer.
• Measure oligomer and fibril levels via conformation‑specific antibodies (TOC1 for oligomers, MC1 for fibrils) and filter‑trap assay.
• Assess lysosomal flux using LysoTracker and DQ‑BSA.
• Evaluate neuronal viability (LDH release, caspase‑3), axonal transport (mitochondrial motility), and sequestration of chaperones (co‑immunoprecipitation).
• In vivo, inject AAV‑mediated tau nucleator into 3×Tg‑AD mice with or without TFEB overexpression; monitor behavior (Morris water maze) and pathology.

Potential Outcomes and Interpretation

If the data show that enhancing fibril formation rescues neurons only when lysosomal clearance is competent, the hypothesis is supported. If fibril acceleration benefits neurons regardless of lysosomal state, the hypothesis is falsified, suggesting that aggregate stability alone suffices for protection. Conversely, if fibril promotion worsens outcomes even with enhanced lysosomal activity, the hypothesis would be refuted, indicating that the mere presence of fibrils is intrinsically harmful.

This framework reframes aggregation not as a uniform bad actor but as a tunable phase‑transition whose outcome hinges on the cell’s clearance capacity. Therapeutically, it argues for combination strategies that pair aggregation‑promoting agents with lysosomal activators, rather than attempting global inhibition of amyloid formation.