Müller glial responses compensate for degenerating photoreceptors in retinitis pigmentosa

Abstract

Photoreceptor degeneration caused by genetic defects leads to retinitis pigmentosa, a rare disease typically diagnosed in adolescents and young adults. In most cases, rod loss occurs first, followed by cone loss as well as altered function in cells connected to photoreceptors directly or indirectly. There remains a gap in our understanding of retinal cellular responses to photoreceptor abnormalities. Here, we utilized single-cell transcriptomics to investigate cellular responses in each major retinal cell type in retinitis pigmentosa model (P23H) mice vs. wild-type littermate mice. We found a significant decrease in the expression of genes associated with phototransduction, the inner/outer segment, photoreceptor cell cilium, and photoreceptor development in both rod and cone clusters, in line with the structural changes seen with immunohistochemistry. Accompanying this loss was a significant decrease in the expression of genes involved in metabolic pathways and energy production in both rods and cones. We found that in the Müller glia/astrocyte cluster, there was a significant increase in gene expression in pathways involving photoreceptor maintenance, while concomitant decreases were observed in rods and cones. Additionally, the expression of genes involved in mitochondrial localization and transport was increased in the Müller glia/astrocyte cluster. The Müller glial compensatory increase in the expression of genes downregulated in photoreceptors suggests that Müller glia adapt their transcriptome to support photoreceptors and could be thought of as general therapeutic targets to protect against retinal degeneration.

Fig. 1. Decreased photoreceptor layer thickness in P23H mice at PW4.

a Schematic of retinal structure and representative OCT images from the nasal to temporal sides of WT and P23H mouse retinas at PW4. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer (amacrine, bipolar, horizontal, and Müller glial cell nuclear layer); ONL, outer nuclear layer (photoreceptor nuclear layer); IS/OS, photoreceptor inner/outer segment; RPE, retinal pigment epithelium. The edge of each layer on the OCT images is labeled with a black line. b Decreased thickness of the ONL and IS/OS in P23H vs. WT retinas. The fold change in thickness compared to that of the WT mice was calculated. n = 11 eyes (WT), n = 27 eyes (P23H). The data are presented as mean ± SEM. Unpaired t test. ^***P < 0.001, n.s., not significant.

Fig. 2. Retinal neuronal degeneration and Müller glia/astrocyte gliosis occurred in P23H mice.

Rod degeneration (rhodopsin) was observed at PW4 along with decreased thickness of the ONL and IS/OS, which was further reduced at PW7. Rhodopsin was also observed in the INL at PW7 in WT retinas but not in P23H retinas. Cone degeneration (cone arrestin) was also observed at PW4 with shortened outer segments, with further disruption at PW7. Retinal gliosis (GFAP) was observed at PW4 and to a greater extent at PW7. No significant changes were observed for amacrine cells (calretinin) or rod bipolar cells (PKCα). DAPI staining was used to show cell nuclei. ILM, inner limiting membrane; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Images were taken at 200X magnification. Scale bar, 50 μm.

Fig. 3. Single-cell transcriptomics of retinal cells from P23H vs. WT mice at PW7.

a tSNE projection of different color-coded retinal cell types from P23H vs. WT littermate controls. n = 4 mice per group, littermates. b Violin plots of normalized marker gene expression for different cell types, as follows: Rho and Pde6b (rods), Opn1mw and Opn1sw (cones), Rlbp1 and Slc3a1 (Müller glia and astrocytes), Vsx2 and Pcp2 (bipolar cells), Slc6a9 and Gad1 (amacrine cells), Calb2 and Slc17a6 (retinal ganglion cells, RGCs), Cldn5 and Pecam1 (endothelial cells), and Cx3cr1 and Ccl4 (macrophages and microglia). c Validation of Rho, Pde6b, and Pde6g expression in P23H vs. WT mouse retinas at PW7 with qPCR. n = 5 mice for WT, n = 3 mice for P23H. The data are presented as mean ± SEM. Unpaired t test, ^*P < 0.05. d Validation of RHO levels in P23H vs. WT mouse retinas at PW7 with western blot analysis. n = 4 mice per group. The data are presented as mean ± SEM. Unpaired t test, ^**P < 0.01.

Fig. 4. Downregulated pathways in rods in P23H mice at PW7.

a The most downregulated genes in the rod cluster of P23H retinas were associated with photoreceptor maintenance, and light-response Gene Ontology terms (phototransduction, IS/OS segment, photoreceptor cell cilium, and photoreceptor development) and ATP metabolic process-related Gene Ontology terms. The P values for the enriched Gene Ontology (GO) terms are shown in bar graphs (P < 0.001). The gene ratio for each pathway is shown in the heatmap. b Heatmap of genes involved in photoreceptor maintenance, light responses, and ATP metabolic processes in the rod population of P23H mouse retinas.

Fig. 5. Downregulated pathways in cones in P23H mice at PW7.

a The most downregulated genes in the cone population of P23H retinas were associated with photoreceptor maintenance- and light response-related Gene Ontology terms (phototransduction, IS/OS segment, photoreceptor cell cilium, and photoreceptor development) and metabolism-related Gene Ontology terms. The P values for the enriched Gene Ontology (GO) terms are shown in bar graphs (P < 0.001). The gene ratio for each pathway is shown in the heatmap. b Heatmap of genes involved in photoreceptor maintenance, light responses, and metabolism in the cone population of P23H mouse retinas.

Fig. 6. Upregulated pathways of Müller glia/astrocytes in P23H mice at PW7.

a The most upregulated genes in the Müller glia/astrocyte population of P23H retinas were associated with photoreceptor maintenance-related Gene Ontology terms (phototransduction, IS/OS segment, photoreceptor cell cilium, and photoreceptor development) and mitochondrial location-related Gene Ontology terms. The P values for the enriched Gene Ontology (GO) terms are shown in bar graphs (P < 0.001). The gene ratio for each pathway is shown in the heatmap. b Heatmap of genes involved in photoreceptor maintenance, light responses, and mitochondrial location in the Müller glia/astrocyte population of P23H mouse retinas. Repeated genes observed in both categories are labeled in red.

Fig. 7. Analysis of the gene profile in Cluster 12 in P23H vs. WT mice.

a Violin plots of normalized marker gene expression in Cluster 12. Cluster 12 was positive for Rho and Pde6b (rods), Opn1mw and Opn1sw (cones), and Rlbp1 and Slc3a1 (Müller glia and astrocytes). b Fraction of Cluster 12 cells in P23H het vs. WT littermate mice. n = 4 mice per group. The data are presented as mean ± SEM. n.s., not significant, two-tailed unpaired t test. c Heatscatter plot of normalized expression for the indicated genes in individual cells in Cluster 12. The cells are presented in the tSNE projection and color-coded by the normalized expression from gray (low) to blue (high). d Comparison of the fraction of cells in major retinal clusters in P23H vs. WT mice (with Cluster 12 excluded). ^***P < 0.001, ^**P < 0.01, unpaired t test.

Fig. 8. Gene-set enrichment analysis of retinal transcription factors in P23H vs. WT mice.

Gene-set enrichment analysis was performed using Encode ChIP seq gene sets. Gene-set enrichment analysis revealed significant enrichment of STAT1 and STAT3 binding motifs in rods (a) and JUND in Müller glia/astrocytes (b) in the differentially expressed genes induced in P23H mice (compared to littermate WT controls).