Abstract
Purpose :
Phosphoinositides (PI) are low-abundant but critical, cellular membrane phospholipids. Mutations in PI-converting enzymes are associated with human diseases including retinal degeneration. In the mammalian genome around 50 genes encode phosphoinositide (PI) kinases and PI phosphatases that generate seven distinct PIs. However, there are no studies describing the distribution of these enzymes in the various retinal cell types. In this study, we investigated the distribution of PI Converting Enzymes between various retinal cells.
Methods :
Using translating ribosome affinity purification (TRAP), we have identified the in vivo distribution of PI-converting enzymes in rods, cones, retinal pigment epithelium (RPE), Müller glia, and retinal ganglion cells. HA-tagging of ribosomal protein Rpl22 was induced with Cre-recombinases under the control of the rod, cone, retinal pigment epithelium (RPE), and Müller cell promoters. AAV2-Cre was used to isolate actively translating mRNAs from retinal ganglion cells. Actively translating mRNAs associated with polyribosomes were then isolated by immunoprecipitation with HA antibody, followed by RNA isolation and qRT-PCR Analysis.
Results :
The retinal neurons: rods, cones, and RGCs are characterized by the enrichment of PI-converting enzymes whereas the Müller glia and RPE are characterized by the depletion of these enzymes. We also identified distinct differences between the expression of PI kinases and PI phosphatases in each retinal cell type. Our studies show a predisposition to active PI metabolism in rod, cone, and RGC, but not in the RPE and MG. In rods and cones, PI3-kinases and phosphatases are enriched. In RGCs, PI3-kinases are enriched but not PI3-phosphatases. However, PI4-phosphatases are enriched in RGCs. In RPE, PI4-and PI5-kinases are enriched.
Conclusions :
Our studies first time demonstrate the expression of PI kinases and PI phosphatases in various retinal cells. Since mutations in PI-converting enzymes are linked to human diseases including retinal diseases, the results of this study will provide a guide for what cell types are likely to be affected by retinal degenerative diseases brought on by changes in PI metabolism. The results reported in this study provide a physiological atlas identifying what retinal cells are likely to detrimentally respond to changes in PI metabolism.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.