Abstract
Purpose :
In the retinal pigment epithelium (RPE), the delivery of lipids to target organelles following phagocytosis of photoreceptor outer segments (POS) by lysosomes is tightly regulated by a network of vesicular and non-vesicular transport mechanisms. Our previous data show that RPE cells lacking ELOVL2 (Elongation of Very Long Chain Fatty Acids-Like 2) and its subsequent products, docosahexaenoic acid (DHA) (22:6n-3) and very long-chain polyunsaturated fatty acids (VLC-PUFAs), exhibit impaired phagocytosis. Here, we investigated whether lack of ELOVL2 results in altered lysosomal lipid trafficking in RPE cells.
Methods :
We used small interfering RNAs (siRNAs) directed against ELOVL2 in ARPE19 cells and performed transcriptomic and lipidomic analyses. We then used live-cell imaging and immunofluorescence to study the movement and distribution of vesicles in the endosomal-lysosomal system. To investigate lipid trafficking between cell compartments, we separated nuclei, organelles and plasma membranes from cells, extracted lipids, and compared changes in the lipidome among the fractions. Quantification of lysosomal marker LAMP1 in plasma membrane fractions was assessed by Western blot.
Results :
Transcriptome analysis in ELOVL2 KD cells revealed downregulation of genes in cellular membranes, such as basolateral plasma membrane, microvillus membrane, basement membrane and apicolateral plasma membrane. Interestingly, ATP6V1F, a vacuolar ATPase component crucial for acidifying endomembrane organelles, was significantly downregulated (log2FC = -1.52, padj = 1.61E-20), suggesting alkalinization of lysosomes. Live-cell imaging showed greater vesicular trafficking to and from the plasma membrane in KD cells. Western blot demonstrated increased plasma membrane LAMP1 normalized to total LAMP1, indicating a greater proportion of lysosomes localized on ELOVL2 KD plasma membranes. Finally, lipidomics of plasma membrane fractions demonstrated accumulation of ceramides, which are known to be abundant in lysosomal membranes, with depletion of glucosylceramides.
Conclusions :
Our data suggest that age-related impaired polyunsaturated fatty acid synthesis perturbs cellular membrane lipids and results in a redistribution of lysosomes to the cell periphery. Future experiments will be aimed at elucidating whether these lysosomes are involved in plasma membrane repair, rather than processing phagocytosed material.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.