Abstract
Purpose :
Age-related macular degeneration (AMD) is a progressive retinal degenerative disease that is the leading cause of vision loss in the elderly. The progression of AMD has been linked to lipid metabolism and autophagy. Accumulation of lipids in the retinal pigment epithelium (RPE) is one of the pathological characteristics of AMD. Lipid droplet accumulation in the RPE has been observed in aged mice and an AMD mouse model. Here, we aimed to explore the mechanism underlying the accumulation of lipid-rich deposits in the RPE. To achieve this purpose, we focus on optineurin (OPTN), a selective autophagy receptor, and investigate its role in the subcellular distributions of a lipid droplet marker, perilipin 2 (PLIN2).
Methods :
The targeting sequence against human OPTN gene (OPTN-sh) was inserted into a pU6-promoter-driven vector. The cDNA containing either human OPTN or PLIN2 was transferred into the pCAG-IRES-GFP vector. ARPE-19 was cultured and transfected using the Neon NxT Electroporation System following manufacturer protocols. Lipid droplets were detected by staining with Lipi-Green (Dojindo, Kumamoto, Japan). Embryonic transfection in the RPE was performed in Slc:ICR mice (Japan SLC, Hamamatsu, Japan) at gestational day 14.5. Animals were maintained in a 12-h light/12-h dark cycle until postnatal 21 days, and the harvested retinas were subjected to immunostaining. The images were obtained by Olympus confocal microscope FV3000.
Results :
To monitor the subcellular distribution of lipid droplets, we generated the construct expressing PLIN2 double-tagged with mCherry and pHluorin. In the control ARPE-19 cells, typical round structures for lipid droplets appeared in the perinuclear region with both pHluorin and mCherry fluorescence (yellow), whereas OPTN-suppressed APRE19 cells had enlarged amorphous debris with only mCherry fluorescence (red), suggesting that the accumulation of PLIN2 in acidic compartments such as autolysosomes. The suppression of OPTN also resulted in the accumulation of lipid droplets. Co-immunoprecipitation assay revealed the interaction between expressed OPTN and PLIN2 in ARPE19 cells. In the tranfected murine RPE, the signals of expressed OPTN were partially overlapped with PLIN2-positive vacuoles.
Conclusions :
OPTN plays a key role in the morphology of lipid droplets. The selective autophagy regulated by OPTN would contribute to RPE lipid homeostasis and its functions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.