Analysis of Secretory Lipidomics and Proteomics of Late-Onset Retinal Degeneration iPSC-derived RPE

Abstract

Purpose : Late-Onset Retinal Degeneration (L-ORD) is a rare autosomal dominant disorder caused by a single S163R amino acid substitution in a secreted protein known as CTRP5. The disease phenotype (delayed dark adaptation, sub-RPE deposits) is reminiscent of more prevalent retinal degenerations, thus providing valuable insight into the shared mechanisms underlying their pathogenesis.

Methods : Fibroblasts from two L-ORD affected and two unaffected siblings were reprogrammed into induced pluripotent stem cells (iPSCs) using sendai viral vectors containing the four Yamanaka factors. iPSCs were fully characterized via immunocytochemistry, karyotyping, and spontaneous differentiation of embryoid bodies into all three germ layers. iPSCs were then differentiated into RPE using a developmentally guided protocol. Cell shapemetric analysis was performed using custom software on iPSC-derived RPE stained with ZO-1. Apical media was analyzed for protein and lipid composition.

Results : Although iPSC-derived RPE from patients and unaffected siblings have similar hexagonality (score, side ratio, area ratio), L-ORD patient iPSC-derived RPE tended be more variable in size and on average possessed greater cell areas. Mass Spect analysis revealed elevated sequence coverage of glutathione, cystatin C, keratins, vimentin, actin, and tubulin secreted by patient iPSC-RPE. Lipidomic analysis of patient iPSC-RPE showed higher secretion levels of very long chain- polyunsaturated fatty acids (VLC-PUFAs) corresponding with higher DHA secretion into the apical media compared to unaffected siblings.

Conclusions : The more variable and larger RPE cell sizes in L-ORD patient iPSC-RPE are indicative of cytoskeletal rearrangement perhaps resulting from enhanced uncompensated oxidative stress. In agreement, proteomic analysis of the apical media revealed proteins identified as biomarkers for oxidative stress. Lastly, lipidomic analysis revealed an over-abundance of DHA and VLC-PUFAs in L-ORD patient iPSC-RPE suggesting that elevated levels of DHA may reflect an attempt to set in motion protective instructive signals from DHA and when overproduced may contribute to lipid peroxidation and susceptibility of these cells to oxidative stress-mediated damage.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.