Abstract
Purpose :
Retinal detachment (RD) is a severe vision-threatening condition despite high surgical success rate, mainly because of cone photoreceptor death. Identifying novel therapeutic avenues for maintaining cones health has the potential to improve vision and the quality of life for patients suffering from retinal detachment. This work was conducted to explore the new targets with potential to reduce retinal detachment-induced cone degeneration in mice by single-cell RNA sequencing (scRNAseq) analysis.
Methods :
24 hours after retinal detachment established in eight-week-old C57BL/6J mice, retinal cells were dissociated and subjected to 10x Genomics scRNAseq. The data were analyzed using the Seurat package and visualized by Uniform Manifold Approximation and Projection (UMAP). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on the differential genes of cone cluster. The Single-cell regulatory network inference and clustering (SCENIC) analysis was conducted through R packages SCENIC (v1.1.3) with default parameters. Immunohistochemistry (IHC) staining was used to verify the expression of key genes.
Results :
Four to six individual retinas of control or retinal detached mice were sequenced and analyzed. Among 54,491 single cells, we identified 22 clusters of retinal cells by specific marker genes. We identified 3302 genes that significantly changed in cones after retinal detachment. Through KEGG and GO analysis, we noticed top three upregulated pathways about cell death and growth, including apoptosis, specifically, endoplasmic reticulum (ER) stress-induced apoptosis, necroptosis and ferroptosis, in cones after retinal detachment. We also found several upregulated genes that involved in the above pathways are also related to neuronal degeneration, Atf4 (activating transcription factor 4), Ddit3 (DNA-damage inducible transcript 3), Tfrc (transferrin receptor) and so on. The cell-enriched transcription factors (TF) analysis by SCENIC showed Atf4 was one of significantly upregulated TFs in cones after retinal detachment. And high expression of Atf4 and its down-stream target, Ddit3 in cones of detached retinas was verified by IHC.
Conclusions :
In this work, scRNAseq analysis reveals that cone photoreceptors can die through mainly three ways in detached retina. Moreover, Atf4 mediated ER stress-induced apoptosis may play an important role in cones survival.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.