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Shikha Pachauri, Kelsey Dang, Pooja Biswas, Anil Kumar Chekuri, Manisha Dagar, Anne Marie Berry, Berzhan Kurmanov, Ryan Lancione, Donita Garland, Sebastian Preissl, Allen Wang, Radha Ayyagari; Single-nucleus multi-modal analysis of MFRP associated retinal degeneration. Invest. Ophthalmol. Vis. Sci. 2022;63(7):7.
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Mutations in the RPE expressed gene Mfrp (membrane-type frizzled related protein) results in early-onset retinal degeneration (RD) in human and mice. The goal of this study is to identify the cell type-specific transcriptional and gene-regulatory events in the retina that lead to RD using Mfrp gene knock-out (KO) mice.
Retina + RPE tissue from Mfrp-/- and Wild type (WT) mice at 28 days and 2.5 months were analyzed via single nucleus (sn) RNA-seq and ATAC-seq (n=3) followed by experimental validation.
Cluster analysis of the snRNA-seq data (~140,000 nuclei) revealed >30 clusters encompassing known retinal cell-types. To identify the transcriptional changes associated with RD, we performed differential expression analysis (WT vs. Mfrp-/-) with a focus on RPE cells, Rods, and Cones. Notably, more than 50 significantly differentially expressed genes (DEGs) (p < 0.05, Wilcoxon Rank-Sum) in RPE cells, rods, and cones are involved in Mfrp related RD. Among these, ~21 genes are differentially expressed in RPE cells, and about 35 are differentially expressed in rods and cones. In photoreceptors, a majority of DEGs are observed at 28 days, the exception being rhodopsin - which was significantly down-regulated at 2.5 months (P< 0.05) in rods. These DEGs include transcription factors, structural proteins, and members of visual transduction. In RPE cells, we observed more DEGs at 2.5 months than at 28 days. These DEGs with P≤ 0.02 by snRNA-seq are involved in visual transduction and phagocytosis pathways and include Rdh10, Rpe6, Lrp2, Cralbp, Mertk, Igtav which were validated by qRT-PCR (P< 0.05). Genes involved in lipid metabolism (Hmgcs2 P< 0.05 and Slc16a1 P< 0.05) and glucose transport (Slc2a1 P< 0.05) are also downregulated in RPE cells at 2.5 months by qRT-PCR. Lastly, our snRNA-seq analysis revealed downregulation of genes associated with PI3K-AKT pathway in RPE cells at 2.5 months. We further validated that AMPK and mTOR pathways show altered activation in Mfrp-/- mice.
The snRNA-seq data revealed that RPE cells show progressive changes in regulation of key pathways with age while photoreceptors show more prominent alterations in transcriptional regulation at 28 days in Mfrp-/- mice. Analysis of changes in additional cell types as well as analysis of snATAC-seq data is in progress to understand the mechanism underlying Mfrp related RD pathology.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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