Hypothesis

Persistent JNK‑AP‑1 activity in aging results from miMOMP‑generated mitochondrial ROS that oxidatively inactivate cytosolic JNK phosphatases (MKPs), converting a transient stress signal into a self‑sustaining, bistable switch that maintains AP‑1‑dependent SASP.

Mechanistic Basis

• miMOMP, a hallmark of senescent cells, releases localized ROS without triggering apoptosis【1†https://doi.org/10.1101/gad.331272.119】.
• These ROS oxidize the catalytic cysteine of MAPK phosphatases (e.g., DUSP1/MKP1, DUSP16), reducing their activity toward phospho‑JNK【2†https://pmc.ncbi.nlm.nih.gov/articles/PMC12302471/】.
• Loss of phosphatase activity creates a positive feedback loop: sustained JNK phosphorylates c‑Jun, driving transcription of SASP cytokines (IL‑6, IL‑1β) and further mitochondrial ROS via NADPH oxidase activation【3†https://www.frontiersin.org/journals/aging-neuroscience/articles/10.3389/fnagi.2024.1453710/full】.
• The switch is bistable because phosphatase reactivation requires reduction of the oxidized cysteine, a process limited by declining thiol‑buffer capacity (e.g., glutathione) in aged tissues.
• Tissue‑specific outcomes arise from differential expression of antioxidant enzymes (Prdx3, Txn2) that scavenge miMOMP‑ROS before they reach cytosolic MKPs, explaining why JNK delays lifespan globally in Drosophila yet accelerates aging in gut epithelium.

Testable Predictions

1. Phosphatase oxidation correlates with chronic JNK – In senescent human fibroblasts, immunoprecipitation of DUSP1 will show increased cysteine‑sulfinic/sulfonic acid modifications proportional to miMOMP markers (cytochrome c‑positive macropores) and phospho‑JNK levels【4†https://pmc.ncbi.nlm.nih.gov/articles/PMC12033962/】.
2. Phosphatase rescue reverses SASP – Expressing oxidation‑resistant DUSP1 (Cys→Ser) in senescent cells will reduce phospho‑JNK, lower IL‑6/IL‑1β secretion, and attenuate MMP‑1 activity without affecting acute stress‑induced JNK peaks.
3. Antioxidant targeting of miMOMP ROS prevents the switch – Mitochondria‑targeted catalase (mCAT) or Prdx3 overexpression will block MKP oxidation, maintain JNK transientality, and extend healthspan in pro‑geroid mouse models.
4. Lipid peroxidation products modulate the switch – Exogenous 4‑HNE will mimic miMOMP‑ROS by inhibiting MKP activity; scavenging 4‑HNE with carnosine will prevent chronic JNK activation even when miMOMP is present.

Experimental Approach

• Use live‑cell FRET biosensors for JNK activity and roGFP2‑Orp1 for mitochondrial H₂O₂ to map temporal dynamics in primary human fibroblasts undergoing doxorubicin‑induced senescence.
• Apply proximity labeling (BioID) to identify oxidized MKPs in miMOMP‑positive versus ‑negative senescent cells.
• Employ CRISPR‑knockin of oxidation‑resistant DUSP1 alleles in mice and assess SASP burden, frailty index, and lifespan.

If miMOMP‑mediated phosphatase oxidation is required for the transient‑to‑chronic JNK transition, inhibiting this redox node should decouple mitochondrial damage from SASP production, offering a precise point of intervention to mitigate inflammaging without compromising acute stress responses.