Complement C3 drives a mitochondrial‑autophagy feedback loop that amplifies retinal degeneration in aging, and its loss unmasks alternative inflammatory pathways

Hypothesis

We hypothesize that sub‑RPE C3 deposition initiates a self‑reinforcing cycle: C3‑mediated opsonization of damaged mitochondria activates microglial phagocytosis, which generates reactive oxygen species (ROS) that further impair mitochondrial function and suppress autophagy flux. The resulting accumulation of defective mitochondria amplifies oxidative stress, promotes additional C3 synthesis by retinal microglia and monocytes, and drives terminal MAC formation, leading to RPE and photoreceptor loss. In the absence of C3, this loop is broken, but the loss of C3‑dependent opsonization shifts microglial clearance toward a pro‑inflammatory phenotype that relies on C5a‑mediated signaling, explaining why CFH−/−.C3−/− mice exhibit worse inflammation than CFH−/− single mutants.

Testable Predictions

1. Mitochondrial ROS as an upstream trigger – In aged wild‑type mice, mitochondrial ROS levels in the sub‑RPE will rise before detectable C3 deposition. Pharmacological scavenging of mitochondria‑targeted ROS (e.g., MitoTEMPO) will delay C3 accumulation and MAC formation.
2. Autophagy flux links C3 to mitochondrial health – Enhancing mitophagy with Urolithin A or overexpressing Parkin in RPE cells will reduce C3‑positive puncta and lower LC3B‑II/I ratios, whereas genetic blockade of autophagy (Atg5 knockdown) will exacerbate C3 deposition even when ROS are scavenged.
3. C3 deficiency redirects microglial activation – In C3−/− retinas, microglia will show increased expression of C5aR1 and pro‑inflammatory cytokines (IL‑1β, TNFα) compared with wild‑type aged controls. Blocking C5aR1 in C3−/− mice will rescue the exacerbated photoreceptor loss seen in CFH−/−.C3−/− double knockouts.
4. MAC formation correlates with mitophagy deficits – Quantification of C5b‑9 deposits will inversely correlate with mitochondrial membrane potential (measured by TMRM) and positively with p62 accumulation across genotypes and treatment conditions.

Experimental Design

• Animal groups: 12‑month‑old wild‑type, C3−/−, CFH−/−, CFH−/−.C3−/−, each subdivided into vehicle, MitoTEMPO, Urolithin A, and C5aR1 antagonist treatments.
• Readouts: (i) sub‑RPE C3 immunostaining and ELISA; (ii) MAC (C5b‑9) immunofluorescence; (iii) mitochondrial ROS (MitoSOX); (iv) autophagic flux (LC3B-II/I, p62) by western blot; (v) microglial phenotype (flow cytometry for CD68, C5aR1, Arg1); (vi) retinal thickness (OCT) and ERG b‑wave amplitude.
• Analysis: Two‑way ANOVA with genotype and treatment as factors; post‑hoc tests for specific comparisons. Significance set at p<0.05.

Falsification Criteria

• If mitochondrial ROS scavenging fails to alter C3 deposition kinetics, the upstream ROS → C3 link is unsupported.
• If mitophagy enhancement does not reduce C3‑positive puncta or MAC formation, the autophagy‑mediated feedback loop is not operative.
• If C5aR1 blockade does not ameliorate the exacerbated phenotype in CFH−/−.C3−/− mice, the alternative inflammatory pathway hypothesis is incorrect.

By integrating complement activation, mitochondrial quality control, and microglial polarization, this hypothesis provides a mechanistic framework that explains the dual protective and detrimental roles of C3 and points to combinatorial targeting of ROS, mitophagy, and C5a signaling as a potential therapeutic strategy to halt age‑related retinal degeneration.