Hypothesis

Aged cells actively suppress autophagy through the formation of a cytosolic phase‑separated hub that concentrates Rubicon and STK4/MST1, thereby sequestering BECN1 and ULK1 complexes. This hub is stabilized by age‑associated increases in oxidized phospholipids and serves as a rheostat that couples nutrient stress sensing to lysosomal capacity. Disrupting the hub with AAV‑delivered CRISPRi against Rubicon or STK4/MST1 will restore autophagic flux without over‑activating mTORC1, leading to improved tissue homeostasis and extended healthspan.

Rationale

• Rubicon rises with age and binds BECN1 to inhibit VPS34, blocking autophagosome nucleation [1].
• mTORC1 remains persistently active in senescent cells, phosphorylating ULK1 to prevent initiation [2].
• Young plasma restores autophagy in aged liver, indicating that the core machinery is intact when suppression is lifted [3].
• Genetic loss of Rubicon extends lifespan in worms and flies and protects against neurodegeneration models [4].
• Pharmacologic mTORC1 inhibition reactivates autophagy in aged fibroblasts and improves stress resistance [5].
• In CNS and heart, STK4/MST1 phosphorylates BECN1 and LC3B, adding a tissue‑specific layer of inhibition [6]. Together, these data suggest that aging does not merely exhaust autophagy components but actively assembles inhibitory complexes. Recent work on biomolecular condensates shows that multivalent proteins like Rubicon and STK4/MST1 can drive phase separation under conditions of altered lipid composition and oxidative stress, creating reservoirs that sequester essential autophagy factors.

Novel Mechanistic Insight

We propose that age‑linked accumulation of oxidized phosphatidylserine and cardiolipin promotes the nucleation of a Rubicon–STK4/MST1 condensate. Within this condensate, the local concentration of inhibitors exceeds the threshold needed to block both initiation (via ULK1 inhibition by mTORC1 that is recruited to the hub) and autophagosome‑lysosome fusion (via Rubicon–BECN1 sequestration). The hub thus functions as a bistable switch: low stress keeps it dissolved, allowing basal autophagy; high stress or aging drives its assembly, locking autophagy in an off state. This explains why lifting a single suppressor (e.g., Rubicon knockdown) only partially rescues flux, whereas simultaneous disruption of both arms yields synergistic restoration.

Predictions

1. In tissues from old mice, Rubicon and STK4/MST1 will colocalize in distinct cytosolic puncta that colocalize with markers of oxidized lipids (e.g., MDA‑modified phospholipids) and autophagy inhibitors.
2. Acute treatment with antioxidants or lipid‑scavenging agents will reduce puncta formation and increase autophagic flux, even without genetic manipulation.
3. AAV‑CRISPRi targeting either Rubicon or STK4/MST1 will decrease puncta number, but combined targeting will produce a greater than additive increase in LC3‑II turnover and p62 degradation.
4. Restoring flux via hub disruption will improve tissue‑specific phenotypes (e.g., grip strength in muscle, sinusoidal perfusion in liver, hippocampal LTP in brain) and extend median lifespan by ~15% relative to controls.
5. Over‑disruption (e.g., >80% knockdown of both proteins) will trigger excessive autophagic flux leading to cytotoxicity, establishing an upper therapeutic window.

Experimental Design

• Animals: 24‑month‑old C57BL/6J mice, both sexes.
• Vectors: AAV9‑shRNA‑Rubicon, AAV9‑shRNA‑STK4, AAV9‑scrambled control; optionally a dual‑vector co‑expressing both shRNAs.
• Delivery: Tail‑vein injection (1e11 vg) to achieve broad transduction; separate cohorts for liver‑detargeted (AAV8) and muscle‑preferring (AAV6) variants to test tissue specificity.
• Readouts (4 weeks post‑treatment):
  • Immunofluorescence colocalization of Rubicon, STK4/MST1, and MDA‑lipid puncta (confocal, Pearson’s r).
  • Autophagic flux assay: lysosomal inhibition with bafilomycin A1 ex vivo, LC3‑II and p62 immunoblotting.
  • Functional assays: grip strength, rotarod, serum ALT/AST, Morris water maze.
  • Longevity monitoring: survival curves up to 30 months.
• Controls: Young (3‑month) mice receiving scrambled AAV; old mice treated with rapamycin as pharmacological benchmark.

Potential Outcomes and Implications

If the hypothesis is correct, we will observe discrete inhibitor hubs in aged tissues, their dissolution upon antioxidant or CRISPRi treatment, and a dose‑dependent recovery of autophagy that translates into measurable healthspan gains. This would shift the therapeutic paradigm from merely over‑expressing autophagy components to precision‑editing of age‑dependent inhibitory condensates. Moreover, identifying the lipid triggers could open nutraceutical avenues (e.g., phospholipid‑targeted antioxidants) to prevent hub formation. Conversely, a lack of puncta colocalization or failure of CRISPRi to affect flux would falsify the model, prompting a re‑evaluation of whether suppression acts via soluble complexes rather than phase‑separated assemblies.