Abstract
Purpose :
Retinyl ester hydrolase (REH) cleaves retinyl esters (RE) to produce retinol. REH has not been identified in the RPE previously. The purpose of this project is to investigate the role of patatin-like phospholipase domain containing 2 (PNPLA2) as REH in the visual cycle.
Methods :
PNPLA2 mRNA and protein levels were examined in retinal tissues and sections by real-time (RT)-PCR, western blot analysis and immunofluorescence staining. PNPLA2 knockout (PNPLA2-/-) mice were used to examine the retinal function and rhodopsin recovery by electroretinography (ERG). Visual chromophore regeneration was determined by photobleaching mice for 30 min at 5k lux light followed by recovering in the dark for 15 and 40 min. Retinoid profiles were analyzed by HPLC. SiRNA-mediated knock-down was used to analyze the REH activity of endogenous PNPLA2 in HEK293A cells.
Results :
Western blot analysis and immunofluorescence staining of the WT mouse retinal tissues and sections confirmed PNPLA2 expression in the RPE and retina. ERG analysis revealed a- and b-wave amplitudes were significantly lower in PNPLA2-/- mice, suggesting impaired retinal function. PNPLA2-/- mice showed lower a-wave recovery after photobleach compared with WT mice. More prominent lipid droplet accumulation along the plasma membrane was observed in RPE cells from PNPLA2-/- mice relative to WT mice. Visual chromophore regeneration was slower in PNPLA2-/- mice compared with WT mice. The content of RE was significantly higher in PNPLA2-/- mouse eyes relative to age-matched WT mice at all tested time points, implying that PNPLA2 hydrolyzes RE from the retinosomes in the RPE, releasing all-trans-retinol (atROL). In cultured cells, siRNA-mediated knock-down increased RE levels, confirming REH activity of PNPLA2.
Conclusions :
REH activity of PNPLA2 was required to mobilize retinol from lipid bodies in RPE cells and is an essential component of the visual cycle. The absence of PNPLA 2 expression in the RPE led to declined retinal function and impaired visual chromophore regeneration along with a significant amount of lipid accumulation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.