Hypothesis In aged retinal tissue, low‑level formation of the membrane attack complex (MAC, C5b‑9) in the sub‑RPE domain produces a biphasic effect on lysosomal function: sublytic MAC insertion triggers Ca2+‑dependent activation of TFEB and enhances lysosomal biogenesis and autophagic flux, thereby providing a protective clearance mechanism; when MAC density exceeds a threshold, lysosomal membranes become permeabilized, releasing cathepsins that activate the NLRP3 inflammasome and drive RPE apoptosis. This model explains why both excessive C3 activation and complete C3 loss are detrimental, and predicts a narrow therapeutic window where MAC formation is modulated rather than abolished.

Rationale

• Complement C3 deposition is localized to the sub‑RPE region in aged retinas [2].
• The terminal complement pathway (C5‑9) has not been systematically characterized in this locale [5].
• Sublytic MAC is known to cause Ca2+ influx and can stimulate TFEB‑mediated lysosomal biogenesis in other cell types.
• Lysosomal dysfunction and inflammasome activation are hallmarks of age‑related RPE decline [4].
• Complete C3 loss impairs debris clearance and antioxidant signaling, while uncontrolled C3 activation drives pathology [1][2].

Mechanistic Model

1. Age‑related oxidative stress and alternative pathway dysregulation increase C3 convertase activity in the Bruch's membrane.
2. Elevated C5 convertase generates C5a (chemotactic) and C5b, which initiates MAC assembly on the basal lamina of RPE cells.
3. At low MAC density, C5b‑9 pores permit limited Ca2+ entry, activating calmodulin‑dependent kinases that phosphorylate TFEB, promoting its nuclear translocation and upregulation of lysosomal genes (LAMP1, CATB, etc.). Enhanced lysosomal activity improves phagocytosis of photoreceptor outer segments and mitigates amyloid‑β accumulation.
4. When MAC density rises—due to sustained complement activation or impaired regulatory proteins (e.g., CD59)—pores enlarge, causing lysosomal membrane permeabilization (LMP). Cathepsin B release into the cytosol triggers NLRP3 inflammasome assembly, caspase‑1 activation, and IL‑1β maturation, culminating in RPE pyroptosis/apoptosis.
5. The shift from protective to destructive outcomes defines a complement‑dependent "lysosomal tipping point" that varies with age and genetic background (e.g., CFH Y402H variant).

Testable Predictions

• In aged WT mice, sub‑RPE tissue will show detectable C5b‑9 immunoreactivity that colocalizes with LAMP1 and phosphorylated TFEB; the magnitude of this signal will correlate positively with lysosomal enzyme activity and negatively with markers of LMP (e.g., cytosolic cathepsin B).
• Mice deficient in C5 (C5‑/-) or treated with a sublytic MAC inhibitor (e.g., CD59‑Fc fusion) will exhibit reduced TFEB activation, impaired lysosomal biogenesis, and accelerated accumulation of undegraded material despite lower overall complement activation.
• Conversely, aged mice with heterozygous loss of CD59 (showing higher MAC density) will display increased LMP, cathepsin B release, NLRP3 inflammasome activation, and RPE loss, even when total C3 levels are unchanged.
• In primary human RPE cultures carrying the CFH Y402H risk allele, exposure to physiological levels of C5b‑9 will increase TFEB nuclear translocation and lysosomal flux at low doses, but induce LMP and inflammasome activation at higher doses; blockade of MAC formation with anti‑C5 antibody will abolish both responses.
• Therapeutic administration of a MAC‑modulating agent (not a full C3 inhibitor) in aged mice will improve ERT b‑wave amplitudes and reduce drusen‑like deposits only when given after the onset of sublytic MAC deposition, confirming an age‑dependent therapeutic window.

Experimental Approach

• Use immunofluorescence and immunoelectron microscopy to quantify C5b‑9, LAMP1, p‑TFEB, and cytosolic cathepsin B in retinal sections from young, middle‑aged, and aged WT, C3-/-, C5-/-, and CD59+/- mice.
• Measure lysosomal activity (Magic Red cathepsin assays) and autophagic flux (LC3-II turnover with bafilomycin) in isolated RPE/choroid complexes.
• Assess inflammasome activation via Western blot for cleaved caspase‑1 and IL‑1β, and pyroptosis via LDH release.
• Functional outcomes: ERG a‑ and b‑wave amplitudes, OCT‑based retinal thickness, and histologic scoring of photoreceptor loss and Bruch's membrane thickening.
• Intervention: intravitreal delivery of CD59‑Fc or anti‑C5 antibody at defined ages; compare to C3 knockout and wild‑type controls.

Falsification If sublytic MAC deposition does not correlate with TFEB activation or lysosomal enhancement, or if MAC modulation fails to alter lysosomal function or inflammasome activation in aged RPE, the hypothesis would be refuted. Conversely, demonstration that MAC density directly governs a switch from protective lysosomal biogenesis to destructive lysosomal permeabilization would strongly support the model.