Hypothesis

We propose that in aging retinal pigment epithelium (RPE), sublytic membrane attack complex (MAC) formation triggers lysosomal calcium efflux, which activates calcium‑dependent proteases (calpains) that cleave the autophagy essential protein ATG5. This cleavage uncouples the protective opsonocytic functions of C3 from the detrimental terminal complement pathway, leading to impaired autophagy, oxidative stress, and retinal dysfunction. Restricting MAC assembly downstream of C5 while preserving upstream C3 activity will rescue autophagy flux and retinal homeostasis without compromising clearance of apoptotic debris.

Rationale

• Aged mice show C3‑dependent accumulation in the sub‑RPE space, correlating with retinal thinning and reduced electroretinographic responses (1).
• C3 deficiency preserves retinal thickness and enhances autophagy (LC3B‑II/I ratio ↑) but complete C3 loss exacerbates photoreceptor loss, Bruch’s membrane thickening and inflammation (4), indicating a protective upstream role for C3 in debris clearance.
• MAC deposition in RPE lysosomes causes lysosomal membrane permeabilization (3), a known source of calcium leakage.
• Calcium overload activates calpains, which can cleave ATG5 and other autophagy proteins, thereby blocking autophagosome formation.
• Genetic variants in CFH modulate MAC formation in human AMD lesions (6), suggesting that individual susceptibility to MAC‑mediated lysosomal damage may underlie variable disease progression.

Predictions

1. In aged wild‑type RPE, MAC deposition will colocalize with lysosomal LAMP1 and be accompanied by a measurable rise in lysosomal calcium (detectable with Fluo‑4 AM).
2. Pharmacological inhibition of calpain (e.g., with MDL‑28170) or genetic ablation of calpain‑1 in RPE will prevent ATG5 cleavage, restore LC3B‑II/I ratios, and reduce oxidative markers (4‑HNE, nitrotyrosine) despite persistent C3 deposition.
3. RPE‑specific overexpression of CD59 (a MAC inhibitor) will attenuate lysosomal calcium spikes, preserve ATG5 integrity, and maintain autophagic flux, resulting in thicker outer nuclear layers and improved scotopic ERG responses relative to aged controls.
4. Global C3 knockout mice will exhibit exacerbated lysosomal calcium dysregulation and ATG5 cleavage when MAC formation is driven by alternative pathways (e.g., via C5aR agonism), confirming that loss of C3’s opsonocytic function unmasks MAC‑driven pathology.
5. Human RPE cultures derived from CFH risk‑allele carriers will show heightened MAC‑induced lysosomal calcium release and greater sensitivity to calpain‑mediated ATG5 cleavage compared with non‑risk alleles.

Experimental Design

• Model: Aged (18‑month) C57BL/6J mice, RPE‑specific CD59 transgenic line, RPE‑specific calpain‑1 knockout, and C3‑null mice.
• Readouts: Lysosomal calcium imaging (live‑cell Fluo‑4 AT), western blot for full‑length vs cleaved ATG5, LC3B‑II/I ratios, immunostaining for C3d and MAC (C5b‑9), oxidative stress markers, OCT retinal thickness, scotopic photopic ERG.
• Interventions: Intravitreal delivery of calpain inhibitor, CD59‑AAV vector, or C5aR antagonist; controls receive vehicle.
• Human validation: iPSC‑derived RPE from donors genotyped for CFH Y402H; treat with sublytic complement serum; measure lysosomal calcium and ATG5 integrity.

Potential Outcomes and Falsifiability

If the hypothesis is correct, inhibiting calpain or blocking MAC formation will rescue autophagy and retinal function without altering C3 deposition levels. Conversely, if MAC‑induced lysosomal calcium does not affect ATG5 cleavage (e.g., ATG5 remains intact despite calcium flux) or if rescuing autophagy fails to improve retinal outcomes, the hypothesis would be falsified. Additionally, demonstrating that C3 loss exacerbates pathology only when MAC formation is chemically induced would confirm the dual role of upstream complement protection versus terminal pathway damage.