PNPLA2 is required for the degradation of Photoreceptor Outer Segments by the RPE

Abstract

Purpose : We have previously reported that the retinal pigment epithelium (RPE) expresses the PNPLA2 gene coding for a phospholipase A2. We aim to study the contribution of PNPLA2 in phagocytosis of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE).

Methods : A mouse line harboring the cyclization recombinase (Cre)-mediated deletion of Pnpla2 in the RPE was generated (Pnpla2flox/flox; BEST-Cre hereby referred to as cKO). PNPLA2 was silenced in ARPE-19 cells using the RNAi technology with either siPNPLA2 or shPNPLA2. The phospholipase A2 inhibitor bromoenol lactone (BEL) was used. Pnpla2 expression was assessed by quantitative PCR. POS were isolated from bovine retinas. Rhodopsin levels were determined by western blotting. The release of β-hydroxybutyrate (β-HB) to the media was measured using the enzymatic activity of β-HB Dehydrogenase in a colorimetric assay. Fatty acids released to the media were quantified using the enzymatic activity of Acyl-CoA Synthetase in a colorimetric assay. Gene expression analysis was performed using the RT² Profiler PCR Array of fatty acid metabolism-related genes in ARPE-19 cells stably transfected with shPNPLA2 relative to those with the control empty plasmid.

Results : Suppression of PNPLA2 expression was confirmed in cKO RPE/choroid eyecups and in ARPE-19[siPNPLA2] cells. The relative β-HB release from RPE in cKO eyecups challenged with POS was lower than that of littermate controls. PNPLA2 silencing did not affect the uptake of rhodopsin as detected at 30 min, 60 min, and 150 min after POS addition, and was similar to that of RPE cells with siScramble or untransfected. However, rhodopsin levels were higher in BEL-treated ARPE-19 cells as well as in ARPE-19[siPNPLA2] cells when compared to cells treated with the vehicle DMSO and siScramble transfected cells during POS degradation at 16h and 24h. Moreover, siPNPLA2 lowered the release of fatty acids and β-HB from RPE cells at 30 min following POS addition. Gene expression analysis revealed that the silencing of PNPLA2 in ARPE-19 cells affected several genes of the fatty acid metabolism pathway with ~2-fold decrease of genes such as ACA9 and CPT1C.

Conclusions : These findings imply that RPE phagocytosis depends on PNPLA2 activity for the release of fatty acids from POS, identifying a novel contribution of this enzyme in POS degradation.

This is a 2020 ARVO Annual Meeting abstract.