Identification of functional networks associated with cell death in the retina of OXYS rats during the development of retinopathy

Abstract

Age-related macular degeneration (AMD) is a major cause of blindness in developed countries, and the molecular pathogenesis of early events in AMD is poorly understood. Senescence-accelerated OXYS rats develop AMD-like retinopathy. The aim of this study was to explore the differences in retinal gene expression between OXYS and Wistar (control) rats at age 20 d and to identify the pathways of retinal cell death involved in the OXYS retinopathy initiation and progression. Retinal mRNA profiles of 20-day-old OXYS and Wistar rats were generated at the sequencing read depth 40 mln, in triplicate, using Illumina GAIIx. A terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay was performed to measure the apoptosis level. GeneMANIA was used to construct interaction networks for differentially expressed (DE) apoptosis-related genes at ages 20 d and 3 and 18 months. Functional analysis was suggestive of a developmental process, signal transduction, and cell differentiation as the most enriched biological processes among 245 DE genes at age 20 d An increased level of apoptosis was observed in OXYS rats at age 20 d but not at advanced stages. We identified functional clusters in the constructed interaction networks and possible hub genes (Rasa1, cFLAR, Birc3, Cdk1, Hspa1b, Erbb3, and Ntf3). We also demonstrated the significance of the extrinsic apoptotic pathway at preclinical, early, and advanced stages of retinopathy development. Besides the cell death signaling pathways, immune system-related processes and lipid-metabolic processes showed overrepresentation in the clusters of all networks. These characteristics of the expression profile of the genes functionally associated with apoptosis may contribute to the pathogenesis of AMD-like retinopathy in senescence-accelerated OXYS rats.

Keywords: OXYS rats; RNA-Seq; age-related macular degeneration; aging; apoptosis; cell death; retinal transcriptome.

Figure 1.

Apoptotic activity was analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. (A) Positive TUNEL straining (green) was observed under a fluorescence microscope. (B) Quantitative analysis. The number of apoptotic cells was calculated by averaging the number of positive TUNEL signals. *Significant interstrain differences, р < 0.05; ^#significant differences with the preceding age, р < 0.05. Bar 50 mkm.

Figure 2.

Statistically significant (p < 0.05) Gene Ontology terms that are related to the genes whose expression is changed in the retina of OXYS rats in comparison with Wistar rats at age 20 d.

Figure 3.

This Venn diagram shows overlapping sets of genes at the 3 ages.

Figure 4.

Illustration of the association networks of apoptosis in 20-day-old (A), 3-month-old (B), and 18-month-old rats (C). In each of these networks, black circles denote differentially expressed genes (DEG), whereas the GeneMANIA-predicted genes are shown in gray.

Figure 5.

Clusters in the schemes of gene networks for 20-day-old (A), 3-month-old (B), and 18-month-old rats (C). In each of these networks, black circles denote differentially expressed genes (DEG), whereas the GeneMANIA-predicted genes are shown in gray. The hub genes are yellow