Hypothesis

Mitochondrial DNA (mtDNA) released from stressed retinal pigment epithelium (RPE) activates the cytosolic DNA sensor cGAS‑STING pathway in neighboring microglia and macrophages, driving transcription of complement component C3 and alternative pathway factors. This positions mtDNA as an upstream initiator of complement overactivation rather than a parallel stressor.

Mechanistic Rationale

1. mtDNA release – Age‑associated mtDNA damage increases membrane permeability, allowing mtDNA to escape into the cytosol (1 2).
2. cGAS‑STING activation – Cytosolic mtDNA binds cGAS, producing 2’3’‑cGAMP, which stimulates STING‑TBK1‑IRF3 signaling. IRF3 induces interferon‑stimulated genes and, notably, the complement genes C3, CFB and CFD (3 4).
3. Complement amplification – Newly synthesized C3 is secreted, deposited on drusen‑like deposits, and fuels the alternative pathway cascade, leading to MAC formation and RPE loss (5 6).

This model predicts that blocking cGAS or STING will uncouple mtDNA stress from complement synthesis, even when mitochondrial damage persists.

Testable Predictions

• Prediction 1: In primary microglia, transfection of isolated mtDNA will increase C3 mRNA and protein secretion; this increase will be abolished by cGAS inhibitor (RU.521) or STING knockout.
• Prediction 2: RPE‑specific mtDNA mutator mice (PolG^D257A) will show elevated cytosolic mtDNA in adjacent microglia, heightened p‑STING and IRF3 nuclear translocation, and heightened C3 deposition; genetic deletion of STING in microglia (Cx3cr1‑Cre; Sting^fl/fl) will normalize C3 levels despite ongoing mtDNA damage.
• Prediction 3: Pharmacological STING inhibition (e.g., C‑176) in aged wild‑type mice will reduce complement‑driven lesions in laser‑induced choroidal neovascularization models without altering mtDNA mutation load.

Falsifiability

If complement C3 upregulation persists in microglia despite complete ablation of cGAS‑STING signaling (both pharmacologically and genetically), or if mtDNA release fails to induce IRF3 activation in these cells, the hypothesis that mtDNA drives complement activation via this pathway is false. Likewise, if STING deficiency does not ameliorate C3 deposition in mtDNA mutator mice, alternative upstream mechanisms must be sought.

Experimental Approach

• Isolate mtDNA from aged RPE, transfect BV2 microglia, measure cGAMP, p‑TBK1, IRF3, and C3 by ELISA and western blot.
• Generate Cx3cr1‑Cre; Sting^fl/fl mice crossed with PolG^D257A; assess retinal histology, complement deposition ( immunostaining for C3c), and functional outcomes (ERG).
• Treat aged mice with STING antagonist; quantify lesion area and mtDNA burden (qPCR for mtDNA in cytosolic fraction).

By directly linking mitochondrial danger signaling to the innate immune complement cascade, this hypothesis reframes mitochondrial genetics as a potential upstream regulator of retinal aging, opening therapeutic avenues that target mtDNA sensing rather than complement alone.