EV‑mediated miRNA suppression of autophagy as an adaptive response to lysosomal fragility in aging

Hypothesis

In aged tissues autophagy is actively suppressed not only by mTORC1 hyperactivity but also by senescence‑derived extracellular vesicles (EVs) that deliver specific inhibitory microRNAs to recipient cells. These miRNAs target key autophagy initiation genes (e.g., ULK1, ATG13, WIPI2) and reinforce a lysosomal‑protective state that limits cathepsin leakage from destabilized organelles.

Mechanistic Rationale

Senescent cells accumulate with age and exhibit persistent mTORC1 signaling, which drives both autophagy inhibition and the secretion of pro‑inflammatory EVs [3][5]. Recent work shows that EV cargo is altered in senescence, suggesting a non‑local mechanism for autophagy suppression [5]. We propose that a subset of EV‑packaged miRNAs (e.g., miR‑34a, miR‑146a, miR‑21) directly bind the 3′UTRs of autophagy‑initiating transcripts, reducing their translation. Simultaneously, age‑related lysosomal membrane fragility increases the risk of cathepsin release upon autophagic engulfment of damaged mitochondria. By dampening autophagy initiation, the cell avoids a catastrophic proteolytic burst that could amplify oxidative stress and senescence. This creates a feedback loop: suppressed autophagy leads to further accumulation of damaged organelles, which sustains senescence and EV production.

Testable Predictions

1. Inhibiting EV release (e.g., with GW4869) in aged mice will restore autophagy markers (LC3‑II/I ratio, p62 degradation) in distant tissues without altering mTORC1 activity.
2. Transfecting young fibroblasts with EVs isolated from senescent donor cells will decrease ULK1 and WIPI2 protein levels and reduce autophagic flux, an effect rescued by antisense oligonucleotides against the candidate miRNAs.
3. Overexpressing a miRNA sponge for miR‑34a in aged hearts will increase autophagy flux and reduce senescence markers, even when mTORC1 remains active.
4. Inducing lysosomal membrane permeabilization (e.g., with LLOMe) in cultured aged cells will potentiate autophagy suppression only when EV‑derived miRNAs are present, indicating a synergistic protective response.

Experimental Design

• In vivo: Treat aged (24‑month) mice with GW4869 or neutral sphingomyelinase inhibitor for 2 weeks. Harvest liver, heart, and muscle. Assess autophagy (Western blot for LC3‑II, p62), mTORC1 signaling (p‑S6K), and senescence (SA‑β‑gal, p16). Compare to vehicle‑treated controls.
• In vitro: Collect EVs from irradiated human senescent fibroblasts (validated by p16 and SASP). Characterize miRNA content by small‑RNA seq. Add EVs to young fibroblast cultures with or without miRNA inhibitors. Measure autophagic flux using mCherry‑GFP‑LC3 reporter and qPCR for autophagy genes.
• Rescue: Transduce aged mouse hearts with AAV9 expressing a miR‑34a sponge. Evaluate autophagy flux, lysosomal integrity (LysoTracker), and cardiac function (echocardiography) after 4 weeks.

Potential Outcomes and Falsifiability

If EV‑mediated miRNA transfer is a key suppressor, then blocking EV release or neutralizing the specific miRNAs will significantly rescue autophagy flux and reduce senescence biomarkers, independent of mTORC1 status. Conversely, if autophagy restoration does not occur despite effective EV inhibition, the hypothesis would be falsified, indicating that other mechanisms (e.g., irreversible oxidative damage to ATG proteins) dominate. Demonstrating that lysosomal permeabilization exacerbates suppression only in the presence of inhibitory miRNAs would further support the protective‑adaptive framing, whereas a lack of interaction would refute the proposed synergy.