Hypothesis

Premature activation of the mitochondrial autophagy receptor TAX1BP1 relative to aggregate receptors p62/NBR1 inverts the canonical cargo hierarchy, causing mitochondrial degradation before clearance of ubiquitin‑protein aggregates. This hierarchy inversion releases iron and ROS that promote aberrant phase separation of splicing factors, leading to sustained intron retention and a transcriptomic drift that underlies age‑related functional decline.

Mechanistic Basis

• Under proteotoxic stress, K63‑linked ubiquitination and phosphorylation normally favor p62/NBR1 condensates that sequester aggregates (see Receptor specificity arises from unique ubiquitin-binding domains, PB1 polymerization domains, and PTMs).
• TAX1BP1 is recruited to damaged mitochondria via its ubiquitin‑binding domain and can drive mitophagy even when other receptors are absent (TAX1BP1 is sufficient to promote mitophagy even when other major receptors are absent).
• We propose that age‑associated shifts in cellular redox state or kinase activity increase TAX1BP1 phosphorylation (e.g., TBK1-mediated) or alter its ubiquitin affinity, causing it to outcompete p62/NBR1 for limited FIP200/ULK1 complexes. Consequently, mitochondria are stripped while aggregates persist.
• Persistent K63‑ubiquitinated aggregates sequester splicing regulators such as SRSF2 and hnRNPA1, promoting their condensation into stress‑resistant granules that impair spliceosome assembly (K63-linked ubiquitination and phosphorylation favor proteotoxic aggregates under stress).
• Mitochondrial loss releases Fe2+ and ROS, which oxidize cysteine residues on splicing factors, further stabilizing aberrant condensates and driving intron retention.
• The resulting splicing dysregulation produces isoforms that diminish proteasome activity and autophagy receptor expression, creating a feed‑forward loop that locks the hierarchy in the inverted state.

Testable Predictions

1. In aged cells, phospho‑specific antibodies will show increased TAX1BP1 activation concurrent with decreased p62/NBR1 phosphorylation at known autophagy‑promoting sites.
2. Genetic mimicry of TAX1BP1 hyper‑phosphorylation (e.g., TAX1BP1-S/E mutants) in young cells will recapitulate early mitochondrial loss, aggregate accumulation, and increased intron retention.
3. Conversely, knock‑down of TAX1BP1 or expression of a ubiquitin‑binding deficient mutant in aged cells will restore aggregate clearance, reduce splicing factor sequestration, and improve healthspan.
4. Pharmacological inhibition of TBK1 or ROS scavengers will suppress the hierarchy inversion and rescue splicing fidelity.

Experimental Approach

• Use human fibroblasts or iPSC‑derived neurons from young (<30 y) and old (>70 y) donors. Measure phospho‑TAX1BP1, phospho‑p62, mitochondrial mass (MitoTracker), aggregate load (p62 puncta), and intron retention via RNA‑seq.
• Introduce CRISPR‑edited TAX1BP1 phospho‑mutants or ubiquitination‑deficient constructs; assess rescue of mitochondrial function, aggregate clearance, and splicing patterns.
• In Drosophila, overexpress phosphomimetic TAX1BP1 in neurons; track lifespan, climbing ability, and retinal neurodegeneration as readouts of splicing dysfunction.
• Validate causality by treating aged mice with TBK1 inhibitor (e.g., MRT67307) and evaluating autophagy flux (LC3‑II turnover), mitochondrial respiration, and spliceosome activity in brain tissue.

If the hierarchy inversion is a driver rather than a byproduct, correcting the receptor priority should ameliorate splicing defects and extend functional lifespan, falsifying the hypothesis that autophagy decline is merely quantitative loss of activity.