RNA-Seq Analysis for Exploring the Pathogenesis of Retinitis Pigmentosa in P23H Knock-In Mice

Abstract

<b><i>Introduction:</i></b> Mechanisms contributing to the progression of autosomal dominant retinitis pigmentosa (adRP) due to the P23H rhodopsin mutation are complex and diverse. Previous studies showed that mechanisms like endoplasmic reticulum (ER) stress, pyroptosis, and oxidative stress were involved in the pathogenesis of the disease. However, the roles and relationships of different mechanisms are not precisely known. In this study, we aimed to evaluate certain mechanisms and find novel genes involved in P23H-related adRP. <b><i>Methods:</i></b> Total RNA extracted at postnatal day (PN) 14, PN21, and PN35 was used for RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses were conducted for RNA-seq data. Additionally, data from the clustered regularly interspaced short palindromic repeats (CRISPR) screening library and the RNA-seq data of several mechanisms were used for generating custom gene sets for gene set enrichment analysis (GSEA). Next, we obtained the intersection of the aforementioned gene sets and our RNA-seq data to identify candidate genes, which were verified using real-time quantitative PCR (qPCR). <b><i>Results:</i></b> Functional enrichment analyses were consistent with disease phenotypes. All time points observed pyroptosis. In the results of GSEA, ER stress, pyroptosis, and oxidative stress were observed at PN14. ER stress and pyroptosis were shown on PN35. A total of 22 candidate genes were identified. The expression levels of selected genes verified by qPCR were concordant with the RNA-seq data. <b><i>Conclusions:</i></b> In our study, we conclude that pyroptosis and ER stress might play a central role in RP progression. We also identified differentially expressed gene clusters related to ER stress and pyroptosis, which deserve further study. These findings provide a novel perspective for the investigation of P23H-related adRP.

Network biology analysis of P23H rhodopsin interactome identifies protein and mRNA quality control mechanisms

“…With respect to P23H rhodopsin, RQC could contribute to the elimination of P23H rhodopsin during translation and target P23H rhodopsin mRNA for nucleolytic cleavage to prevent synthesis of further misfolded protein. Interestingly, previous studies found that mRNA levels of P23H rhodopsin are lower than WT rhodopsin in the same mice although the copy numbers of the two mice are not significantly different 14 , 70 .…”

“…With respect to P23H rhodopsin, RQC could contribute to the elimination of P23H rhodopsin during translation and target P23H rhodopsin mRNA for nucleolytic cleavage to prevent synthesis of further misfolded protein. Interestingly, previous studies found that mRNA levels of P23H rhodopsin are lower than WT rhodopsin in the same mice although the copy numbers of the two mice are not significantly different 14,70 . www.nature.com/scientificreports/ Aside from translational and post-translational quality control mechanisms, we also found significant and selective association of P23H rhodopsin interactome with synaptic processes.…”

Inherited Retinal Dystrophies: Role of Oxidative Stress and Inflammation in Their Physiopathology and Therapeutic Implications

“…This inflammatory-induced cell death mechanism has been described in different neurodegenerative diseases, such as Alzheimer’s disease [ 68 ]. Moreover, there is evidence of its possible involvement in photoreceptors and RPE cell death in experimental IRDs models [ 69 ]. However, the cellular mechanisms by which it occurs are unknown and further investigation is needed.…”

Protective mechanism of TCF7L1 against retinal photoreceptor cell injury following retinitis pigmentosa based on the GEO database

“…10 More recently, an RNA-Seq analysis in P23H knock-in mice identified differentially expressed gene (DEG) clusters associated with the endoplasmic reticulum (ER) stress and pyroptosis. 11 Donato et al also identified genetic variations in RP punctata albescens in two affected Egyptian children along with healthy consanguineous parents through Sanger sequencing; the PRPH2, RHO, and RLBP1 genes were suggested to be coexpressed and colocalized in RP tissues, sharing several common pathways in the etiopathogenesis of this disease. 12 A recent study illustrated that ferroptosis gave rise to RPE degeneration evoked by the adduct N-retinylidene-N-retinylethanolamine and blue light and that dysregulated genes relevant to retinal degeneration driven by oxidative damage and inflammation held promise as potential targets for the treatment of related disorders.…”

“…In an existing study, 58 genes, most of which are located in photoreceptors and the retinal pigment epithelium (RPE), have been implicated in the pathology of RP 10 . More recently, an RNA‐Seq analysis in P23H knock‐in mice identified differentially expressed gene (DEG) clusters associated with the endoplasmic reticulum (ER) stress and pyroptosis 11 . Donato et al also identified genetic variations in RP punctata albescens in two affected Egyptian children along with healthy consanguineous parents through Sanger sequencing; the PRPH2, RHO, and RLBP1 genes were suggested to be coexpressed and colocalized in RP tissues, sharing several common pathways in the etiopathogenesis of this disease 12 .…”