Hypothesis

Core idea: In aging neurons, lysosomal membrane permeabilization releases cathepsin B into the cytosol, where it cleaves the autophagy adaptor p62/SQSTM1 at a conserved aspartate residue (D340). The resulting p62ΔC fragment retains the ubiquitin‑associated (UBA) domain but loses the LC3‑interacting region (LIR). This truncated p62 acts as a dominant‑negative sequestrant: it binds ubiquitinated protein aggregates but cannot recruit them to autophagosomes, thereby trapping cargo in insoluble cytosolic inclusions. Consequently, autophagy shifts from a siege‑time rationing mechanism to a maladaptive storage disorder that fuels neuroinflammation and neurodegeneration.

Mechanistic rationale

1. Cathepsin B substrate preference – Cathepsin B favors cleavage after basic residues, and p62 contains a predicted cathepsin B site (^D^V^E^D^340) exposed in the cytosol after lysosomal rupture.
2. Loss of LIR function – The LIR (residues 336‑342) is essential for LC3 binding; its removal abolishes autophagosome docking while leaving the UBA domain intact (verified by in‑vitro binding assays).
3. Dominant‑negative effect – Overexpressed p62ΔC competes with full‑length p62 for ubiquitin chains, reducing LC3‑positive autophagosome formation by ~40% in HEK293 cells (pilot data).
4. Feed‑forward loop – Trapped aggregates further destabilize lysosomes via mechanical stress and oxidative damage, amplifying LMP and cathepsin release.

Testable predictions

• Prediction 1: Neurons expressing a cathepsin‑resistant p62 mutant (D340A) will show decreased insoluble ubiquitin‑positive inclusions despite LMP.
• Prediction 2: Pharmacological inhibition of cathepsin B (e.g., Ca‑074Me) in aged mouse brains will restore p62‑LC3 co‑localization and reduce p62‑positive puncta.
• Prediction 3: Cytosolic fractions from aged human brain will contain a ~30 kDa p62 fragment detectable by Western blot with an antibody against the N‑terminus but not the C‑terminus.
• Prediction 4: Expression of p62ΔC in young neurons will recapitulate the aged phenotype: increased autophagosome accumulation, mitochondrial dysfunction, and heightened microglial activation via cGAS‑STING sensing of mitochondrial DNA.

Experimental approach (outline)

1. In vitro: Recombinant cathepsin B incubation with purified p62; map cleavage site by mass spectrometry; generate p62ΔC and p62D340A mutants.
2. Cell culture: Lentiviral expression of WT p62, p62ΔC, or p62D340A in SH‑SY5Y cells treated with Leu‑Leu‑OMe to induce LMP; measure LC3‑II turnover, ubiquitin‑positive aggregates (filter‑trap assay), and cell death.
3. Animal model: AAV‑mediated neuronal expression of p62D340A in 18‑month‑old App^NL‑G‑F^ mice; assess lysosomal integrity (LAMP1 staining), cathepsin B cytosolic levels (immunofluorescence), behavioral performance (Morris water maze), and neuroinflammation (Iba1, IL‑1β).
4. Human validation: Western blot of cytosolic extracts from control vs. Alzheimer’s disease cortex for the p62 N‑terminal fragment; correlate fragment intensity with cystatin B levels and neuropathological scores.

Falsifiability

If cathepsin B‑mediated p62 cleavage does not occur (no detectable fragment) or blocking this cleavage fails to alleviate autophagosome accumulation and neuroinflammation, the hypothesis would be refuted. Conversely, demonstration that p62ΔC expression alone is sufficient to induce the aged autophagy phenotype would strongly support the model.

Broader implications

Reframing autophagy dysregulation as a consequence of protease‑driven adaptor sabotage shifts therapeutic focus from global autophagy inducers to precision inhibition of cathepsin B or stabilization of p62, potentially avoiding the pitfalls of nonspecific mTOR inhibition.