Abstract
Purpose :
SLOS is an autosomal recessive disease caused by mutations in the gene encoding 7-dehydrocholesterol (7DHC) reductase (DHCR7), which reduces 7DHC to form cholesterol. Here, we analyzed mitochondrial function in induced pluripotent stem cell (iPSC)-derived RPE cells from normal and SLOS donors.
Methods :
iPSC-derived RPE from “mild”-SLOS (CWI, DHCR7 T93M/IVS8–1G>C splice-site compound heterozygote) and “severe”-SLOS (A2, IVS8–1G>C homozygous) vs. normal (DYS) donors (see Farkas et al., Mol Vis., 2022) were cultured at confluence for 6 mo on XFe24 dishes (Agilent). Mito-stress tests were performed with stepwise addition of electron transfer chain (ETC) modulators (oligomycin, FCCP, rotenone/antimycin A) and glycolysis inhibitor 2-deoxyglucose (2-DG). Extracellular acidification (ECAR) and oxygen consumption (OCR) rates were measured (Agilent Seahorse XFe24 Analyzer; N=20 well comparisons; Students t-test, significance threshold p < 0.05).
Results :
DYS and CWI RPE had similar basal OCRs (~320 vs. ~350 pmol/min, respectively) and ATP-linked OCRs post oligomycin (~262 vs. ~302 pmol/min, respectively). Surprisingly, the spare respiratory capacity of CWI was double that of DYS (~533 vs. ~228 pmol/min, p<0.01), but with similar mitochondrial coupling efficiencies (84 % vs. 81 %, respectively). In contrast, A2 RPE had a low ATP-linked OCR (~78 pmol/min) and poor coupling efficiency (16 %). Basal ECAR was higher in A2 vs. CWI or DYS (~100 vs. ~65 and ~56 mpH/min, ~1.5- and ~1.8-fold, p<0.01). Moreover, ECAR increased robustly post oligomycin in DYS and CWI (by ~54 and ~50 mpH/min, respectively), but modestly in A2 (by ~7 mpH/min). Post-ETC inhibition, 2-DG treatment reduced ECAR comparably among all three RPE cell lines.
Conclusions :
Surprisingly, a “mild”-SLOS derived iPSC-RPE line (CWI) exhibited greater respiratory reserve compared to control (DYS) iPSC-RPE. Robust ECAR responses post-oligomycin in DYS and CWI suggest compensatory switches to non-mitochondrial respiration. By contrast, a “severe”-SLOS-derived iPSC-RPE line (A2) maintained a high ECAR under all conditions, indicating greater reliance on non-mitochondrial energy production. Our data show altered energetic profile dependence in donor disease severity, which may be exploited to further develop therapeutic interventions for more effective management of SLOS patients.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.