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Rachayata Dharmat, Sangbae Kim, Yumei Li, Aiden Eblimit, Rui Chen; Single cell RNA-Seq to elucidate the mechanism of delayed cell death in retinal dystrophy.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1009.
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© ARVO (1962-2015); The Authors (2016-present)
It has been observed that apoptosis of photoreceptor cells in patients with inherited retinal degeneration (IRD) diseases often occurs over an extended period of time that take over several decades. A similar phenomenon has also been observed in animal models of IRD.For example, in Spata7 mutant mice, an LCA5 model, although a large proportion (60%) of photoreceptors (PRs) die in the first 3 months, some PRs can survive up to 1.5 years of age. The goal of the project is to investigate this mechanism of prolonged survival in a subset of PR cells, which can form the basis for developing a new therapeutic approach to delay or prevent PR degeneration.
To address the heterogeneity of PR survival in the Spata7 mutant retina, single-cell RNA-Seq (scRNA-Seq) was performed at multiple time points: postnatal day-14 (P14) (onset of degeneration), P30 (peak cell death), P95, and P180 (surviving/ delayed cell death). Single cell suspensions were prepared by papain-based enzymatic dissociation of the retinae, and were captured on the Wafergen ICELL8 nanowell system. Single-cell RNA-Seq was performed on successfully captured live cells. Individual PRs were identified based on gene expression profile of individual cells. Genes/pathways that are differentially expressed between mutant and wild-type PRs were identified and further analyzed.
Analysis of scRNA-Seq data led to the identification of PRs and other retinal cell types in both wild-type and Spata7 mutant retina. Interestingly, mature Spata7 mutant PRs exhibit distinct transcriptome profile compared to age-matched WT PRs. These distinct Spata7 mutant PRs display a significant reduction of expression in a number of photoreceptor lineage-specific genes, such as Rho. Pathways such as visual perception and phototransduction are the most significantly altered in the PRs exhibiting distinct transcriptome profile.
Single cell RNA-Seq of retinal dystrophy model suggests that down-regulation of PR lineage-specific gene expression might prolong its survival. This is consistent with previous observation that reduction of RHO can partially rescue the Spata7 mutant phenotype. Mechanisms of how PRs can themselves modulate PR gene expression in response to stress is currently underway.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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