IF systemic administration of an adeno-associated virus serotype 9 (AAV9) vector encoding a mitochondria-targeted, OMA1-cleavage-site-mutated OPA1 long-form variant (designated OPA1-S1mut) — in which the primary OMA1 recognition site at the S1 cleavage domain is ablated by conservative alanine substitution — driven by a senescence-selective CDKN2A/p16INK4a bidirectional promoter, is delivered intravenously (1×10¹¹ vector genomes/kg, single tail-vein injection) to aged (22–24 month) male C57BL/6J mice,

THEN the following measurable outcomes will be observed within 12 weeks post-injection:

• ≥40% reduction in the S-OPA1/L-OPA1 ratio in liver, skeletal muscle, and lung tissues (western blot densitometry),
• ≥50% reduction in cytoplasmic mtDNA abundance in p16⁺ sorted cells (cytoplasmic fractionation + mtDNA qPCR for mt-Co1 and mt-Nd1),
• ≥35% reduction in phospho-STING (S366) and phospho-IRF3 (S396) protein levels in aged tissues (ELISA/immunohistochemistry),
• ≥30% reduction in plasma SASP cytokines IL-6 and CXCL1 (multiplex Luminex),
• Measurable improvement in grip strength and rotarod performance relative to AAV-GFP vehicle controls,

BECAUSE the following causal chain operates:

1. In aged and senescent cells, loss of mitochondrial membrane potential activates the stress-sensing metalloprotease OMA1, which cleaves L-OPA1 at the S1 site, converting the membrane-tethered long form to the soluble short form (S-OPA1) and thereby collapsing the cristae tight-junction architecture that normally sequesters mtDNA nucleoids against the inner membrane. (Senescence-associated OPA1 loss and cristae remodeling described mechanistically in the literature synthesis provided)[https://doi.org/10.1101/gad.331272.119]

2. Cristae unfolding caused by L-OPA1 oligomer dissolution releases mtDNA nucleoids into the mitochondrial matrix lumen and ultimately facilitates their escape into the cytoplasm — a step mechanistically enabled by the accompanying loss of respiratory supercomplex integrity and inner membrane curvature. (Mitochondrial structural dysfunction promotes cytoplasmic chromatin and inflammation)[https://doi.org/10.1101/gad.331272.119]

3. Cytosolic mtDNA directly ligates and activates the innate immune sensor cGAS, producing cGAMP, which activates STING → TBK1 → IRF3 → type-I interferon and NF-κB-driven SASP transcription (IL-6, CXCL1, IL-8). Concurrently, cristae disruption destabilizes respiratory supercomplexes, elevating mtROS that phosphorylates IκB, amplifying NF-κB nuclear translocation independently of the cGAS axis. (Mitochondria-to-nucleus ROS-JNK-53BP1 axis drives cytoplasmic chromatin and senescent inflammation)[https://doi.org/10.1101/gad.331272.119]

4. A parallel, previously underappreciated damage pathway is activated: the mitochondria-regulated p53–CCF (cytoplasmic chromatin fragment) circuit, in which mitochondrial dysfunction promotes the export of nuclear chromatin fragments that also serve as cGAS ligands, meaning cristae c...

SENS category: LysoSENS

Key references: • doi.org/10.1101/gad.331272.119] • doi.org/10.1101/2023.11.20.567963] • doi.org/10.1101/2024.05.23.595605] • doi.org/10.1101/2024.05.29.595206] • doi.org/10.1101/2024.05.29.595206