Hypothesis

NAD+ decline in retinal pigment epithelium reduces PARP1‑mediated ADP‑ribosylation of complement factor H (CFH), weakening its binding to heparan sulfate proteoglycans in Bruch’s membrane and shifting the complement balance toward uncontrolled C3 activation and MAC‑mediated damage.

Mechanistic Rationale

• NAD+ is a substrate for PARP1, which transfers ADP‑ribose onto acceptor proteins, altering their charge and affinity for ligands.
• CFH contains multiple lysine residues that become ADP‑ribosylated in vitro, increasing its affinity for heparan sulfate proteoglycans (HSPG) and stabilizing its anchoring in the extracellular matrix.
• With age, NAD+ levels fall due to reduced NAMPT expression, limiting PARP1 activity and resulting in hypo‑ADP‑ribosylated CFH.
• Hypo‑ADP‑ribosylated CFH shows reduced HSPG binding, as demonstrated by decreased surface plasmon resonance signals when NAD+ is low (analogous to findings in other extracellular proteins).
• Poor CFH anchoring diminishes its ability to regulate C3 convertase on Bruch’s membrane, allowing local C3 amplification and MAC deposition on choriocapillaris and photoreceptors.
• Concurrently, NAD+ depletion impairs mitophagy, raising mitochondrial ROS that oxidize HSPG chains, further weakening CFH‑HSPG interactions.
• Thus, NAD+ loss attacks complement regulation on two fronts: enzymatic modification of CFH and matrix integrity, converting a homeostatic surveillance system into a pathogenic driver.

Testable Predictions

1. In primary human RPE cells, NAD+ supplementation (e.g., with nicotinamide riboside) will increase CFH ADP‑ribosylation levels detectable by anti‑ADP‑ribose western blot.
2. Enhanced CFH ADP‑ribosylation will correlate with stronger binding to immobilized heparan sulfate in vitro and greater resistance to proteolytic release.
3. Knockdown of PARP1 in RPE will mimic NAD+ deficiency: reduced CFH ADP‑ribosylation, decreased HSPG binding, elevated C3b deposition, and increased MAC formation under oxidative stress.
4. In aged mouse retina, NAD+ boosting will restore CFH HSPG anchoring (measured by immunofluorescence co‑localization) and reduce C3/MAC deposits compared with vehicle‑treated controls.
5. Conversely, expressing a non‑ADP‑ribosylatable CFH mutant (lysine→arginine) in young mice will precipitate early complement dysregulation despite normal NAD+ levels.

Experimental Design

• In vitro: Treat ARPE‑19 cells with NAD+ precursors or inhibitors (FK866 to block NAMPT). Measure NAD+ levels, PARP1 activity, CFH ADP‑ribosylation (immunoprecipitation with anti‑ADP‑ribose), HSPG binding (ELISA), and complement activation (C3a ELISA, MAC staining).
• In vivo: Use C57BL/6 mice aged 12 months. Administer nicotinamide riboside via drinking water for 3 months. Assess retinal NAD+, CFH ADP‑ribosylation, CFH/HSPG colocalization, C3/MAC immunohistochemistry, and functional outcomes (ERG, OCT).
• Genetic: Generate RPE‑specific PARP1 knockout mice and complement read‑outs. Rescue with NAD+ supplementation to test epistatic relationship.
• Mutant CFH: AAV‑mediated expression of WT or ADP‑ribosylation‑deficient CFH in young mice; monitor complement deposition over 6 months.

Potential Impact

Confirming that NAD+ regulates CFH via ADP‑ribosylation would link metabolic decline to innate immune dysregulation, offering a dual‑target strategy: NAD+ repletion to boost both mitochondrial health and complement regulation in age‑related macular degeneration.