Mitochondrial DNA Mutation Signatures Drive NLRP3 Inflammasome Activation via Impaired Mitophagy and Altered Mitochondrial‑Derived Peptide Signaling

Hypothesis

Specific combinations of mitochondrial DNA (mtDNA) mutations create a signature that impairs mitophagy and alters the release of mitochondrial-derived peptides (MDPs), which together amplify NLRP3 inflammasome activation through a dual-signal model: (1) increased cytosolic mtDNA acting as a DAMP via TLR9/NF‑κB and cGAS‑STING, and (2) deficient MDPs that normally restrain inflammasome activity.

Mechanistic Rationale

• mtDNA mutation combos such as m.c3256t, m.del652g, m.g13513a destabilize nucleoid proteins, promoting miMOMP and mtDNA efflux [2][3]
• These same combos reduce PINK1‑PRKN mediated mitophagy, causing accumulation of damaged mitochondria [5]
• Concurrently, mutant mtDNA transcripts generate aberrant MDPs (e.g., altered humanin or MOTS‑c) that lose their ability to inhibit NLRP3, as shown for MDPs suppressing inflammasome signaling [1]
• The loss of MDP‑mediated inhibition removes a brake on NLRP3, while excess cytosolic mtDNA provides the activation signal, creating a self‑reinforcing loop that drives IL‑6 secretion and inflammaging [4][6][7]

Testable Predictions

1. Cells harboring the defined mtDNA mutation combo will show higher cytosolic mtDNA levels and lower extracellular MDP concentrations compared with wild‑type cybrids.
2. Adding synthetic humanin or MOTS‑c to these cells will suppress NLRP3 activation and IL‑6 release despite persistent mtDNA release.
3. In vivo, mice engineered to carry the mutation combo will develop accelerated inflammaging that is rescued by MDP supplementation or by enhancing mitophagy (e.g., Urolithin A treatment).

Experimental Design

• Generate cybrid lines with the mutation combo using platelet‑derived ρ⁰ cells and patient mtDNA isolates.
• Measure cytosolic mtDNA by qPCR after subcellular fractionation; quantify MDPs in supernatant by targeted mass spectrometry.
• Assess NLRP3 activation via ASC speck formation and caspase‑1 cleavage; IL‑6 by ELISA.
• Treat subsets with humanin/MOTS‑c (100 nM) or mitophagy activators; repeat assays.
• Validate in a knock‑in mouse model carrying the human mtDNA combo; monitor serum IL‑6, grip strength, and histopathology over 12 months.

Potential Falsification

If cytosolic mtDNA levels do not differ between mutant and control cybrids, or if MDP supplementation fails to modulate NLRP3 activity, the hypothesis that specific mtDNA mutation signatures drive inflammaging via combined DAMP release and loss of MDP inhibition would be refuted.