Hypothesis The extent of mitochondrial outer membrane permeabilization (miMOMP) acts as a quantitative rheostat that determines the duration and outcome of JNK/AP‑1 signaling. Limited miMOMP releases modest amounts of mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS‑STING pathway just sufficiently to prolong JNK phosphorylation via TBK1‑mediated scaffold stabilization, producing a transient senescent state that can be reversed when mitochondrial stress is alleviated. When miMOMP exceeds a critical threshold, cytosolic mtDNA surpasses the activation threshold for cGAS‑STING, triggering robust TBK1‑IRF3 signaling and concomitant NF‑κB‑driven IL‑1β secretion. IL‑1β then engages its receptor on the same cell (autocrine) and neighboring cells, activating downstream MAPK cascades that sustain JNK/AP‑1 activity independently of the original mitochondrial trigger. This creates a bistable switch: below the threshold, JNK/AP‑1 activity decays upon mitoROS scavenging (e.g., MitoQ) and cells can revert; above the threshold, the IL‑1β‑cGAS‑STING‑JNK loop locks cells into a permanent senescent, SASP‑high state that drives inflammaging.

Key Predictions

1. Graded induction of miMOMP (e.g., using titrated BAX/BAK activators) will produce a linear increase in cytosolic mtDNA measured by qPCR, which correlates with the duration of phospho‑JNK (p‑JNK) signal after wash‑out of the inducer.
2. Pharmacological or genetic inhibition of cGAS or STING will shorten p‑JNK persistence and increase the proportion of cells that regain proliferation after MitoQ treatment, without affecting the initial JNK activation peak.
3. Neutralizing IL‑1β (with antibodies or IL‑1R antagonist) will break the autocrine loop, causing rapid decline of p‑JNK and SASP markers even in cells that experienced high miMOMP, restoring a reversible senescent phenotype.
4. In vivo, tissue‑specific differences in basal antioxidant capacity will shift the miMOMP threshold for irreversible senescence; tissues with lower glutathione (e.g., brain) will show lower miMOMP needed to trigger the persistent loop.

Experimental Approach

• Use a inducible, mitochondria‑targeted BAX construct to generate controllable miMOMP levels in cultured human fibroblasts.
• Quantify cytosolic mtDNA (qPCR for mt‑ND1), p‑JNK (Western blot/flow cytometry), and SASP components (ELISA for IL‑6, IL‑8, MMP‑3) over time after inducer removal.
• Apply MitoQ, cGAS inhibitor (RU.521), STING antagonist (H‑151), and IL‑1β neutralizing antibody in separate conditions.
• Assess reversibility via EdU incorporation and SA‑β‑gal staining.
• Validate findings in murine models of tissue‑specific injury (e.g., liver CCl4 vs. brain ischemia) by measuring miMOMP (cytochrome c‑GFP release), mtDNA, and inflammaging markers.

Falsifiability If inhibiting cGAS/STING or IL‑1β does not alter the persistence of JNK/AP‑1 signaling or the reversibility of senescence despite robust miMOMP, the proposed feedback loop is not required. Conversely, if transient JNK activation persists irrespective of mtDNA levels or downstream cGAS‑STING/IL‑1β signaling, the hypothesis would be refuted.