Hypothesis

Age‑related decline in the autophagy receptor p62/SQSTM1 reorders the selective autophagy hierarchy, causing NBR1‑mediated degradation to dominate. This shift preferentially spares K63‑ubiquitinated extracellular matrix (ECM) remodeling enzymes while over‑degrading hyaluronan synthase (HAS) and procollagen chaperones, leading to the dense collagen‑rich, hyaluronan‑poor vocal fold lamina propria observed in presbyphonia.

Mechanistic Rationale

1. Receptor hierarchy determines substrate fate – p62/SQSTM1 binds ubiquitinated cargo via its UBA domain and recruits LC3 through LIR motifs, directing aggrephagy, mitophagy, and clearance of protein aggregates【1】. NBR1 can substitute but lacks certain domains, altering substrate preference【2】.
2. Aging attenuates p62 – p62/SQSTM1 levels fall with age, impairing aggrephagy and mitophagy and accelerating senescence phenotypes【1】. When p62 drops, NBR1 compensates, but its reduced affinity for K63‑linked ubiquitin chains results in selective accumulation of K63‑ubiquitinated TGF‑β1 and MMP regulators【4】.
3. ECM consequences – TGF‑β1 drives p62 degradation during fibroblast‑mediated ECM remodeling【4】, creating a feed‑forward loop: low p62 → excess TGF‑β1 signaling → further p62 loss. Concurrently, NBR1‑biased autophagy over‑degrades HAS2 and procollagen‑binding chaperones (e.g., HSP47), suppressing hyaluronan synthesis and proper collagen fibrillogenesis【5】.
4. Vocal fold phenotype – The net effect is a lamina propria with dense, disorganized collagen fibrils and sparse hyaluronan, exactly the histological signature of aged rat vocal folds【5】 and the biomechanical substrate of presbyphonia【6】.

Testable Predictions

• Prediction 1: In aged mouse vocal folds, p62/SQSTM1 protein levels will be inversely correlated with NBR1‑dependent autophagic flux (measured by LC3‑II turnover after lysosomal blockade) and directly correlated with HAS2 and procollagen chaperone abundance.
• Prediction 2: Genetic or pharmacological restoration of p62 (e.g., AAV‑mediated p62 overexpression in thyroarytenoid fibroblasts) will rescue HAS2 expression, increase hyaluronan deposition, and reduce collagen cross‑linking, improving vocal fold vibration metrics in vivo.
• Prediction 3: Selective knockdown of NBR1 in aged vocal folds will normalize the autophagy substrate profile, decreasing K63‑ubiquitinated TGF‑β1 accumulation without globally inhibiting autophagy.

Experimental Approach

1. Quantify receptor levels – Western blot and immunofluorescence for p62 and NBR1 in young (3 mo) vs aged (24 mo) mouse vocal folds.
2. Measure autophagic flux – Treat ex vivo vocal fold explants with bafilomycin A1 and assess LC3‑II accumulation via immunoblot; compare flux in p62‑overexpressing vs control samples.
3. Assess ECM enzymes – qPCR and ELISA for HAS2, procollagen‑I, MMP‑2/9, and TGF‑β1; ubiquitin‑remnant profiling to detect K63‑linked substrates.
4. Functional read‑outs – Atomic force microscopy for lamina propria stiffness; high‑speed videoendoscopy to evaluate vocal fold mucosal wave and fundamental frequency stability.
5. Intervention – AAV9‑p62 or shRNA‑NBR1 delivery to thyroarytenoid fibroblasts; assess histological (Masson’s trichrome, Alcian blue) and biochemical changes after 4 weeks.

Implications

If validated, this hypothesis reframes presbyphonia not as passive ECM decay but as an active autophagy‑receptor maladministration. It pinpoints the p62/NBR1 ratio as a therapeutic lever: restoring the autophagy “pecking order” could re‑balance ECM turnover, offering a novel strategy to combat age‑related voice loss.