Abstract
Purpose :
Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries. There are currently no treatments for the dry version of AMD (DAMD), driving a need to understand molecular systems to create treatments. DAMD patients demonstrate decreased mitochondrial function, suggesting that mitochondrial metabolism may be an avenue for future therapies. Our lab generated a cytoplasmic hybrid (cybrid) disease model to interrogate mitochondria's specific role in DAMD. This model involves fusing a mitochondrially depleted immortalized ARPE-19 cell line with mitochondria-rich platelets from DAMD or normal patients. However, it is still necessary to validate that it accurately replicates in vivo mitochondrial and metabolic hallmarks of AMD. Fisher et al (Scientific Reports, 2022) analyzed mitochondrial protein alterations in response to mitochondrial uncoupling in primary cultured RPE cells, providing insight into the mitochondrial dynamics of DAMD. These tests were replicated using our cybrid model to determine if our cybrid model replicates the trends seen in primary RPE cells.
Methods :
Normal control (n=5) and DAMD (n=5) age-matched, sex-matched, and mtDNA haplogroup-matched cybrids were treated with 2.5 μM FCCP (a protonophore that disrupts ATP synthesis) for 4 hours and then allowed to recover for 0, 6, 24, and 72 hours before collecting protein. Mitochondrial protein content was assessed using western blotting techniques.
Results :
DAMD cybrids demonstrated decreased MFN1 expression compared to normal controls at baseline (P<0.001) and after a 24-hour recovery (P<0.01). DRP1 was significantly increased in DAMD cybrids at baseline and 72 hours of recovery (P<0.001 for both). OPA1 showed a slight decrease in DAMD cybrids after 6 hours of recovery (P<0.05).
Conclusions :
My preliminary data indicates a preference for mitochondrial fission in DAMD cybrids compared to normal cybrids. This is consistent with previously seen mitochondrial dysfunction in patients with DAMD. However, these data are not entirely in agreement with the primary RPE culture study, suggesting differences between the two models. Further analysis of mitochondrial fission, fusion, and biogenesis markers will elucidate the role of our lab’s cybrid model in interrogating the metabolic hallmarks of DAMD in vitro.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.