Abstract
Purpose :
Age-dependent processes like extenuated vascular density as well as reduced choroidal blood flow limit oxygen supply to the outer retina. Together with the formation of drusen and thickening of Bruch’s membrane this might lead to hypoxic conditions in the ageing retina, linking age-dependent changes to hypoxia. To understand the cellular response of specific retinal cell types upon hypoxic conditions, we performed droplet-based single-cell RNA sequencing (scRNAseq) of mouse retina tissue after hypoxic exposure.
Methods :
Droplet-based single-cell RNA sequencing of hypoxic (7% O2 for 6h) and normoxic male and female mouse retinas (N=2) was performed using the 10X Genomics platform. Data analysis focused on differential gene expression of hypoxic and normoxic cells in each cluster. In-situ hybridization targeting Kif4 was used to confirm the exclusive expression in cone photoreceptors. qPCR was performed to identify Rbm3 and Cirbp isoforms.
Results :
21 retinal cell types and one RPE cluster were identified among the 9410 normoxic and 9033 hypoxic cells in the analysis. Known hypoxic markers including Bnip3, Higd1a and Egln1 were significantly upregulated in various hypoxic clusters, confirming a general response to hypoxia. Interestingly, RNA-binding proteins Rbm3 and Cirbp were differentially expressed across clusters. Remarkably, both genes underwent an isoform switch in hypoxia leading to the preferential expression of short variants. Focusing on photoreceptor-specific responses, we identified a kinesin motor protein (Kif4) to be induced specifically in cones upon hypoxic exposure. In-situ hybridization confirmed the observed cone specific expression.
Conclusions :
Our results identified an oxygen-dependent isoform switch in RNA-binding proteins Rbm3 and Cirbp, suggesting functional consequences that might contribute to the pathological impact of reduced tissue oxygenation. Additionally, we identified a hypoxia-induced upregulation of a cone-specific motor protein, Kif4, which needs to be further characterized. Possible functions include axonal transport, cargo-delivery to the cone outer segment and enrichment of transmembrane receptors or channels. Identifying the cargo of this cone-specific kinesin might open the possibility for a new therapeutic target.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.