Oxidized mtDNA sustains JNK/AP-1 signaling to lock senescent cells into a chronic SASP

Hypothesis

Oxidized mitochondrial DNA (ox-mtDNA) released during minority MOMP sustains JNK activation by inhibiting MAPK phosphatases, thereby prolonging AP-1–driven SASP and locking senescent cells into a chronic proinflammatory state.

Mechanistic Model

1. Source – Senescent cells undergo BAX/BAK‑dependent minority MOMP, releasing mtDNA fragments into the cytosol (4).
2. Oxidation – Elevated mitochondrial ROS in senescence generates 8‑oxoguanine and abasic lesions, making the released mtDNA highly oxidized (3).
3. Sensor – Ox-mtDNA binds directly to the cysteine‑rich catalytic domain of MAPK phosphatases DUSP1 and DUSP16, oxidizing their active‑site cysteines and rendering them inactive. This mechanism is distinct from cGAS‑STING and does not require IFN signaling.
4. Signal propagation – With DUSP activity suppressed, phosphorylated JNK (p‑JNK) accumulates, driving sustained AP‑1 (c‑Jun/c‑Fos) transcriptional activity.
5. Outcome – Persistent AP‑1 signaling maintains expression of SASP components (IL‑6, IL‑8, MMPs) even after the initial ROS burst has subsided, converting an acute damage signal into a chronic inflammatory program.

Experimental Plan

• Generate ox‑mtDNA – Treat isolated mitochondria with H₂O₂ or use mtDNA mutator (PolG^mut) mice to produce oxidized mtDNA; verify oxidation by 8‑OG ELISA.
• Delivery – Transfect cytosolic extracts of senescent fibroblasts with purified ox‑mtDNA or native mtDNA; include controls with DNase I treatment.
• Readouts – Measure p‑JNK (Western blot), AP‑1 luciferase reporter, and SASP cytokines (ELISA) over 24‑72 h.
• Phosphatase assay – Assess DUSP1/DUSP16 activity using a phosphatase‑substrate assay in lysates +/- ox‑mtDNA; rescue with DUSP1 overexpression.
• Genetic tests – Use DUSP1/16 double‑knockout cells to see if ox‑mtDNA adds no further JNK activation; use cGAS‑/‑ or STING‑/‑ cells to test pathway independence.
• In vivo – Introduce ox‑mtDNA into mouse liver via AAV‑mediated cytosolic expression; monitor p‑JNK levels, SASP markers, and tissue fibrosis.

Expected Outcomes and Falsifiability

• If ox‑mtDNA specifically prolongs p‑JNK and AP‑1 activity compared with native mtDNA, and this effect is abolished by DUSP1/16 overexpression or absent in DUSP1/16 KO cells, the hypothesis is supported.
• If ox‑mtDNA fails to enhance JNK signaling, or if the effect is completely dependent on cGAS‑STING signaling, the hypothesis is falsified.
• If antioxidant treatment (MitoQ) blocks ox‑mtDNA‑induced JNK activation without affecting mtDNA release, it would confirm that the oxidative modification, not merely the presence of mtDNA, is required.

This framework directly links the oxidative state of mtDNA to the duration of JNK/AP‑1 signaling, offering a testable mechanism by which the mitochondrial genome could drive the chronic phase of inflammaging—shifting the focus from nuclear editing to mitochondrial genome maintenance.