Single-nucleus multi-modal analysis of photoreceptor alterations during cellular stress in MFRP-associated retinal degeneration

Abstract

Purpose : Membrane frizzled-related protein (MFRP) is an RPE expressed gene of unknown function. The Mfrp gene knock-out (KO) model develops early-onset retinal degeneration. In Mfrp gene ablated models, rods and cones can survive cellular stress during later stages of disease despite RPE abnormalities, prompting investigation into the cellular state of photoreceptors upon the loss of Mfrp.

Methods : Retina and RPE tissue from 28 day (d) (n=3) and 2.5 mo (n=3) Wt and Mfrp KO mice were isolated and analyzed using single-nucleus RNA-sequencing (snRNA-seq). Differentially expressed genes (DEGs) and Enrichr pathway analyses were analyzed followed by validation.

Results : Cluster analysis of the snRNA-seq data (~60,000 nuclei) revealed 18 clusters of known retinal cell types in Mfrp KO mice. Enrichr analysis revealed shared and divergent genes and pathways in 28d (early-stage disease) and 2.5mo (mid-stage disease) rod and cone clusters. Shared pathways in Mfrp KO vs. Wt rods and cones include extracellular matrix organization and filipodium assembly. In 28d and 2.5mo Mfrp KO vs. Wt rods, Wnt signaling and cholesterol metabolism pathways were downregulated, and the JAK-STAT cascade was upregulated. Although visual perception pathways were downregulated in 28d Mfrp KO vs. Wt cones, we observed that 2.5mo cones upregulate transforming growth factor beta (TGFβ) signaling and apoptotic clearance, which were not detected in rods. However, a few select pathways related to survival (e.g., protein kinase B signaling) were upregulated in 2.5mo rods, but not in cones. Differential expression analysis revealed DEGs (p<0.05) involved in Wnt signaling (Fzd1, Sfrp1 and Sox4), cholesterol metabolism (Abca1, Abcg1and Apoe) and JAK-STAT cascade (Socs3 and Stat3) in rods and apoptotic clearance (C4b) and TGFβ signaling (Col1a2, Fmod, Ltbp4 and Thbs1) in cones. Validation of select DEGs via IHC, Western blotting, and RT-PCR in Mfrp KO mice showed altered expression in the neural retina compared to Wt mice.

Conclusions : Mfrp KO rods and cones display unique alterations in pathways and DEGs during cellular stress as revealed by snRNA-seq. TGFβ signaling and apoptotic clearance pathways may play a key role in photoreceptor changes upon loss of Mfrp. Additional pathways and epigenetic changes (via ATAC-seq) are being studied to gain further insight into Mfrp KO photoreceptor alterations during disease.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.