CFH (Y402H) polymorphism sensitizes mitochondria to oxidative stress in RPE cells

Abstract

Purpose : The Y402H polymorphism of Complement Factor H (CFH) is a key genetic risk factor for age-related macular degeneration (AMD). Smoking also significantly contributes to AMD by inducing oxidative stress in cells. The mechanism by which the CFH Y402H variant contributes to AMD pathogenesis in retinal pigment epithelial (RPE) cells remains elusive. This study evaluated the response of iPSC-RPE cells with low-risk (LR, Y402) and high-risk (HR, Y402H) CFH variants to chronic oxidative stress induced by extended exposure to cigarette smoke extract (CSE).

Methods : iPSC-RPE cells, derived from donors with LR or HR CFH variants (N=4-8 per group), were exposed to CSE (0, 50, 100, 150 μg/mL) for two weeks. Mitochondrial function was assessed using Seahorse XFe96. MitoSOX and TMRM assays quantified mitochondrial oxidation status and membrane potential. Proteomic analyses and western blots of cell lysates, along with targeted metabolomics of conditioned media, were conducted.

Results : CSE exposure led to dose-dependent increase in mitochondrial content, mitochondrial superoxide production and a decrease in mitochondrial membrane potential in both LR and HR lines. CSE also caused substantial mitochondrial dysfunction, evidenced by increased proton leak, diminished coupling efficiency, and reduced bioenergetic health index (BHI) in both lines. However, HR lines showed a more pronounced decrease in BHI, with significant reductions in maximal respiration and ATP production starting at 100 μg/mL CSE. Antioxidant responses differed between the LR and HR lines; while Superoxide Dismutase 2 and Catalase levels increased in response to CSE, HR cells exhibited a more diminished response than LR cells. Furthermore, a dose-dependent decrease in Glutathione Peroxidase 1 levels was observed in HR but not LR cells. Metabolomics revealed perturbations in glutathione-related metabolites in both cell types post-CSE. However, HR cells secreted more glutamate and less glutamine compared to LR cells.

Conclusions : Chronic CSE exposure significantly impairs mitochondrial function and health, prompting an upsurge in mitochondrial biosynthesis, which is likely a compensatory attempt to sustain energy production. iPSC-RPE cells with the CFH HR variant display heightened susceptibility to oxidative stress, as reflected in reduced mitochondrial function and increased damage, potentially due to compromised antioxidative defense mechanisms.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.