Abstract
Purpose :
A progressive chorioretinopathy is associated with Long-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency (LCHADD). The pathophysiology of LCHADD retinopathy is incompletely understood. We have established a cell model of LCHADD to explore disease mechanisms.
Methods :
Four LCHADD fibroblast lines (HADHA c.1528G>C) were reprogrammed into iPSCs. Two wild type iPSC lines were purchased as controls. iPSCs were differentiated into RPE and assayed for several RPE markers, transepithelial electrical resistance (TEER), and ability to phagocytose labeled outer segments. RPE were fed palmitate (C16) and FAO was measured with the Seahorse bioanalyzer. Acylcarnitines were measured with tandem mass spectrometry. Beta-hydroxybutyrate (BOH) was assayed from transwells. Neutral lipids were detected using LipidTOX and triglycerides were measured. RPE were treated with H2O2 and C16, viability monitored by alamarBlue.
Results :
All RPE lines showed similar expression of RPE markers. Wild type and LCHADD RPE were able to phagocytose outer segments. TEER in wild type (288±13 Ω/cm2) and LCHADD (277±14 Ω/cm2) RPE was similar. The basal oxygen consumption rate (OCR) for wild type RPE increased after C16 compared to BSA alone (41 ± 6.7 BSA to 68 ± 8.5 BSA-C16, p < 0.0001). In contrast, no change was observed in LCHADD (45 ± 7.7 BSA, 51 ± 4.7 BSA-C16). Apical levels of BOH increased in wild type RPE when treated with BSA-C16 (p < 0.002) with no significant change in LCHADD RPE. Increased triglycerides and neutral lipid staining were observed only in LCHADD RPE after C16 exposure. Media from LCHADD RPE accumulated palmitoyl (C16) and 3-hydroxypalmitoyl-carnitine (C16-OH). When treated with H2O2 in the presence of C16, LCHAD RPE showed a decrease in viability compared to wild type RPE (p < 0.0001).
Conclusions :
LCHADD RPE have impaired FAO and ketone body production. Acylcarnitine profiles from LCHADD RPE are similar to those of LCHADD patients. The viability of LCHADD RPE decreased when treated with H2O2 in the presence of long-chain fats. LCHADD RPE may be more susceptible to oxidative stress due to the accumulation of toxic metabolic intermediates such as C16-OH.
This is a 2021 ARVO Annual Meeting abstract.