New Retinal Pigment Epithelial Cell Model to Unravel Neuroprotection Sensors of Neurodegeneration in Retinal Disease

Abstract

Retinal pigment epithelial (RPE) cells sustain photoreceptor integrity, and when this function is disrupted, retinal degenerations ensue. Herein, we characterize a new cell line from human RPE that we termed ABC. These cells remarkably recapitulate human eye native cells. Distinctive from other epithelia, RPE cells originate from the neural crest and follow a neural development but are terminally differentiated into “epithelial” type, thus sharing characteristics with their neuronal lineages counterparts. Additionally, they form microvilli, tight junctions, and honeycomb packing and express distinctive markers. In these cells, outer segment phagocytosis, phagolysosome fate, phospholipid metabolism, and lipid mediator release can be studied. ABC cells display higher resistance to oxidative stress and are protected from senescence through mTOR inhibition, making them more stable in culture. The cells are responsive to Neuroprotectin D1 (NPD1), which downregulates inflammasomes and upregulates antioxidant and anti-inflammatory genes. ABC gene expression profile displays close proximity to native RPE lineage, making them a reliable cell system to unravel signaling in uncompensated oxidative stress (UOS) and retinal degenerative disease to define neuroprotection sites.

Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels

“…Cellular regulation of RPE phagocytosis can be studied in cell cultures by challenging RPE cells (primary culture as well as ARPE-19 cells) with isolated POS [ 9 , 29 , 30 , 31 ]. In addition, it has been reported that RPE cells can mediate non-specific phagocytosis by engulfing latex beads, for example [ 9 , 29 , 32 ].…”

“…Cellular regulation of RPE phagocytosis can be studied in cell cultures by challenging RPE cells (primary culture as well as ARPE-19 cells) with isolated POS [ 9 , 29 , 30 , 31 ]. In addition, it has been reported that RPE cells can mediate non-specific phagocytosis by engulfing latex beads, for example [ 9 , 29 , 32 ]. To study the effect of HG on ARPE-19 cells phagocytic function, they were seeded in 96-well plates and exposed to HG (33 mM) for 24, 48, or 72 h. Then, non-specific phagocytosis was measured using pHrodo™ green E. coli BioParticles ® , as described in the Materials and Methods section.…”

“…In addition, we measured POS phagocytosis by ARPE-19 cells exposed to HG for 48 h and incubated or not with PLD inhibitors. For this purpose, cells were exposed to the experimental conditions, then incubated with bovine POS for 16 h. Although it was previously shown that internalized POS are detected in ARPE-19 cells after a 6 h incubation time, POS phagocytosis was shown to be maximal after a 16 h incubation [ 29 ]. Then, internalized POS were identified by immunocytochemistry followed by fluorescence and confocal microscopy or by western blot (WB) using an anti-rhodopsin antibody.…”

“…They form microvilli, tight junctions, and honeycomb packing. In addition, ABC cells express the right markers compared to ARPE-19 cells and phagocyte POS [ 29 ].…”

“…Although we were not able to achieve full silencing of PLDs, the partial reduction in PLD1 and PLD2 expression induced by the combination of both siRNAs was able to reduce by 25% the phosphorylation (activation) of the mTOR downstream effector p70 S6K ( Figure 3 D), suggesting reduced PA-mediated mTOR activation. RNAseq analysis was performed as previously described [ 29 ] in ABC, ARPE-19, and hRPE49 cells. Figure 3 C shows that the three RPE cell lines expressed both classical PLDs and that PLD1 and PLD2 gene expression was lower in ARPE-19 and hRPE49 cells with respect to ABC cells ( Figure 3 C).…”