Hypothesis

In aged basal forebrain cholinergic neurons, oxidative/nitrative stress induces S‑nitrosylation of the selective autophagy receptor p62/SQSTM1, shifting its oligomerization state toward a conformation that preferentially binds soluble cholinergic proteins (ChAT, TrkA) over insoluble protein aggregates. This reroutes the autophagy hierarchy, causing premature degradation of essential cholinergic machinery while aggregates persist, thereby driving the retrograde transport failure observed in aging.

Mechanistic Basis

• S‑nitrosylation of p62 at Cys138 promotes formation of higher‑order oligomers that expose its ubiquitin‑associated (UBA) domain, increasing affinity for mono‑ubiquitinated soluble substrates.
• Concurrently, oxidative damage to lysosomal membranes reduces cathepsin activity, creating a bottleneck that favors rapid turnover of readily accessible proteins before aggregates can be sequestered.
• The resulting imbalance depletes ChAT and TrkA faster than aggregates are cleared, weakening NGF‑TrkA signaling and retrograde endosome transport.

Testable Predictions

1. Aged BFCNs will show elevated p62‑SNO levels and increased p62 oligomer size compared with young neurons.
2. Preventing p62 S‑nitrosylation (Cys138Ser mutant) will preserve ChAT and TrkA protein levels without altering overall autophagic flux.
3. Enhancing lysosomal cathepsin activity (e.g., via TFEB overexpression) will rescue cholinergic function even when p62‑SNO is high, by alleviating the degradation bottleneck.

Experimental Approach

• isolate BFCNs from young and old mice; perform biotin‑switch assay to quantify p62‑SNO and native PAGE to assess oligomerization.
• express wild‑type or Cys138Ser p62 via AAV in aged BFCNs; measure ChAT, TrkA, and p62‑positive aggregates by immunoblot and immunofluorescence.
• assess NGF‑TrkA retrograde transport using live‑cell imaging of fluorescently tagged NGF in axons.
• manipulate lysosomal capacity with TFEB AAV or lysosomal acidification agents and evaluate whether cholinergic phenotypes are rescued.

If p62‑SNO drives the selective loss of cholinergic proteins, blocking this modification should uncouple autophagy activation from cholinergic decline, falsifying the hypothesis that generic autophagy enhancement is universally beneficial in aged BFCNs.