An RPE subpopulation is resistant to oxidative-induced inflammation through SERPINE3-Caspase1 pathway

Abstract

Purpose : RPE heterogeneity and heterogenous RPE degeneration along the peripheral to central axis have been reported. However, the relationship between RPE heterogeneity and regional degeneration remains unknown. This study aims to explore a new subpopulation of RPE cells and determine their role during regional degeneration.

Methods : For searching the new subpopulation of RPE cells, bulk and single cell RNA seq data of human and macaque RPE cells were analyzed. To tracing the subpopulation, a genetic mouse model with a GFP reporter was generated by CRISPR/CAS9 gene editing. To characterize the subpopulation, cell division and oxidative stress response were analyzed by Edu and sodium iodate (SI) injection, respectively. To understand the function of the candidate genes in the subpopulation, gene knockout mice and AAV gene delivery system were used for the loss and gain of function analysis. Afterwards, a proteinase screen, protein structure prediction and protein-protein interactions were analyzed using in vivo or in vitro assays for studing the molecular mechanism.

Results : The RNA seq data shows that SERPINE3 is highly expressed in a population of peripheral RPE cells. Similar patterns of expression are evident in SERPINE3-GFP knock-in mice which the GFP reporter was inserted in the exon 1 of Serpine3 gene. Moreover, GFP+ cells show a high level of cell division during neonatal development. After SI injection, GFP expression increases significantly in the peripheral RPE cells, and these GFP+ RPE cells are preserved relative to GFP- central RPE cells. Furthermore, deletion of SERPINE3 accelerates the SI-induced RPE degeneration as well as aging inflammation. Conversely, overexpression of SERPINE3 prevents the SI-induced RPE degeneration. Moreover, proteinase screen and co-IP revealed that SERPINE3 inhibits caspase 1 and binds Caspase 1 directly. SERPINE3 protein was predicted to have 9 helix domains, 3 sheets domains and a reactive center loop (RCL). Domain mapping showed that RCL is crucial for the function of SERPINE3 to inhibit Caspase 1 activity.

Conclusions : We have identified a new SERPINE3-expressing RPE subpopulation residing in the peripheral RPE. This subpopulation is resistant to oxidative stress and aging-associated inflammation through upregulation of SERPINE3 and subsequent caspase 1 inhibition, providing a new target to widen the current therapeutic strategies for retinal diseases.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.