Hypothesis

Age-dependent microglial C3 synthesis in the outer retina likely creates a localized complement "priming" state. When combined with pre-existing RPE lysosomal vulnerability, this drives terminal pathway activation—specifically C5b-9/MAC deposition—in the sub-RPE space. The key insight is that neither microglial C3 overexpression nor RPE lysosomal stress alone appears sufficient for pathological MAC formation. Rather, their convergence in the aging retina represents the critical threshold for complement-mediated RPE degeneration.

Mechanistic Reasoning

The research shows that aged microglia become autonomous C3 sources, while RPE cells with complement risk variants expose galectin-8 following lysosomal damage, triggering complement activation. These likely represent temporally distinct hits: microglial C3 accumulates progressively from around 12 months, while RPE lysosomal integrity degrades with age-related oxidative stress.

The mechanism proposed here is that microglial-derived C3 localizes to the sub-RPE space through microglial processes that infiltrate this region with age, creating a high-concentration C3 reservoir. When RPE lysosomal damage occurs—the second hit—the local C3 pool enables rapid alternative pathway amplification and terminal pathway completion. This could explain why direct MAC quantification has been elusive; it may be spatially restricted to regions where both hits converge.

This model addresses the paradoxical finding that complete C3 absence worsens some disease models: systemic C3 depletion removes both pathological microglial C3 and protective baseline complement activity. Selective inhibition of microglial C3 synthesis—through CSF1R modulation or astrocyte-targeted siRNA, for example—would preserve systemic complement function while blocking the pathogenic local amplification.

Testable Predictions

1. Spatial co-localization: In aged (18-month) wild-type mice, C5b-9 immunostaining will co-localize with both Iba1+ microglial processes in the sub-RPE space and regions of RPE galectin-8 exposure, but not with distant microglia.

2. Two-hit requirement: Young mice with enforced microglial C3 overexpression (via AAV-C3 in outer retina) will not develop sub-RPE MAC deposition unless concurrent RPE lysosomal stress is induced (e.g., sublucent phototoxicity or sodium iodate).

3. Therapeutic window: Selective microglial C3 knockdown in aged mice (using Cx3cr1-CreERT2 conditional knockout) will reduce sub-RPE MAC deposition while preserving systemic complement activity, unlike complete C3 knockout.

4. Age-specificity: Cross-sectional human donor retina analysis will reveal MAC deposition predominantly in aged eyes (>70 years) with drusen, co-localizing with microglial markers (Iba1, TMEM119) in the sub-RPE region.

Falsifiability

This hypothesis is falsifiable if: (a) aged mouse retinas show abundant sub-RPE MAC without detectable RPE lysosomal damage; (b) young mice with microglial C3 overexpression develop MAC without additional stress; or (c) human aged retinas show MAC deposition independent of microglial presence. Negative results in any prediction would require revision of the two-hit framework.