Degeneration- and RA-dependent upregulation of p2rx7 in the rd1 inner retina.

A) Immunofluorescence labeling of cell nuclei (DAPI, blue), P2X7R protein (green) and the retinal ganglion cell marker RBPMS (red) in a retinal slice from an adult WT mouse. Yellow arrows show two non-specific bands stained by the secondary antibody (see also Fig. S1) and yellow regions of interest illustrating the method employed to quantify fluorescence. INL – inner nuclear layer; IPL – inner plexiform layer; GCL – ganglion cell layer. B) Similar to (A), but in the retina of an adult rd1 mouse, fully degenerated. C-E) Quantification of P2X7R green fluorescence in all three regions of interest detailed above. Images here had their brightness and contrast artificially enhanced for graphic purposes, but analysis was conducted on raw images without any manipulation. Individual data points represent the average of 3-5 fields of view in a single retina from a single mouse (WT n=7 mice; rd1 n=10 mice, P60-90). Data are shown as mean ± SEM. **p<0.01, ***p<0.001, n.s. – non-significant; 1-tailed t-test after Shapiro-Wilk normality test. No outliners detected (Thompson Tau test). F) Illustration of the type, number and position of Retinoic Acid Response Element (RARE) sequences for all three RAR subtypes (α, β, γ), as well as for RXRα, in the first 1500 bp immediately upstream to the 5’ untranslated region of the P2RX7 promoter, depicting the mouse gene (Mus musculus) on the top, and the human homolog region on the bottom (Homo sapiens). G) Schematic sequence map for the RAR-reporter construct, including enhanced cyan fluorescent protein (ECFP) under the neuronal artificial mini-promoter Ple344 derived from Tubb3, the expression of red fluorescent protein (mCherry) under the regulation of the weak SV40 promoter and three repetitions of a RARE sequence. Fluorescent proteins express the degradation peptide PEST, to prevent overexpression. H) Images of a flat-mounted retinal sample, with the GCL facing up, dissected from a fully degenerated rd1 mouse after intravitreal delivery of the RAR-reporter and acute incubation with the P2X7R-dependent nuclear dye Yo-Pro-1.

Genomic deletion of p2rx7 in rd1 mice rescues membrane hyperpermeability.

A) Illustration of breeding strategy to obtain double mutant rd1-p2rx7ko mice (for strain details see STAR Methods). B) Gel electrophoresis of PCR amplicons corresponding to the wild-type (top) and mutant (bottom) alleles of the mouse p2rx7 gene in the original knock-out strain. Mice with no retinal degeneration (WT) were compared to rd1 and rd1-p2rx7ko (P21, n=2/condition). The single-nucleotide mutation in both Pde6b alleles in rd1 and rd1-p2rx7ko mice was confirmed by DNA sequencing. C) Gel electrophoresis of reverse transcription PCR amplicons for p2xr7 and b-actin demonstrate lack of p2xr7 mRNA expression in rd1-p2rx7ko. RNA was purified from total retinal lysates in P60 mice (n=2/condition). D) Confocal images of flat-mounted retinas from WT, rd1 and rd1-p2rx7ko mice, incubated with Yo-Pro-1 dye, showing regions of interest corresponding to the ganglion cell layer (GCL, top) and the inner nuclear layer (INL). The GCL of WT and rd1-p2rx7ko retinas show Yo-Pro-positive pericytes in blood vessels. E) Quantification of the percentage of cells positively stained with Yo-Pro-1 out of all cells in each region of interest (DAPI, not shown). Data points correspond to a 200 µm x 200 µm area, 2 – 4 regions of interest/retina, 1-2 retinas/mouse, and 5-7 mice/strain, all at P60-90. Data are shown as mean ± SEM. *p<0.05, ***p<0.001, 2-tailed t-test after Shapiro-Wilk normality test. F) Illustration of intravitreal drug delivery of 2 µl solution containing 100 µM BMS-493 (BMS) in one eye, and contralateral vehicle control (PBS), repeated in 4 individual rd1 mice. G-H) Western blot gel bands for loading control GAPDH (red) and P2X7R (green) individual retinas from the left (OS) or right (OD) eye. Yellow boxes correspond to regions of interest used for quantitative analysis. Data are shown as mean ± SEM.***p<0.001; Mann-Whitney U test.

P2X7 receptors mediate degeneration-dependent RGC hyperactivity.

A) Representative multielectrode array recording of spontaneous extracellular retinal activity in rd1 and rd1-p2rx7ko excised retinas, during 1 minute, under continuous perfusion with oxygenated saline at 34°C. Raster plots are shown on the top, and the combined firing activity (in Hz) of all detected units, shown on the bottom. B) Quantification of mean firing frequency for rd1 retinas in saline (Ctrl) or treated with a pan-P2X antagonist (TNP-ATP, 100 µM), and rd1-p2rx7ko retinas in saline. C) In a second experimental set, we repeated the same conditions while perfusing saline containing a cocktail of synaptic blockers (see STAR Methods). Data are shown as mean ± SEM. *p<0.05, ***p<0.001, n.s. – non-significant, 2-tailed t-test after Shapiro-Wilk normality test. D) Flat-mount fluorescent-light imaging of the GCL in a WT mouse infected intravitreally with an adeno-associated virus (AAV) delivering overexpression of p2rx7 and green fluorescent protein (GFP) under the chicken beta actin (CBA) constitutive promoter. E) Quantification of spontaneous activity in the dark from the GCL of WT retinas treated with pAAV_CBA_P2RX7_GFP or not, under saline perfusion with or without synaptic blockers. Data are shown as mean ± SEM. *p<0.05, ***p<0.001, 2-tailed t-test after Shapiro-Wilk normality test.

Degeneration-enhanced expression of ion channels is p2rx7-dependent.

A) Heatmap of transcriptional regulation patterns. The image visually represents the differential expression of the top 1000 genes in each retinal sample from rd1-p2rx7ko and rd1 mice. Three strains were tested (n=4 per strain), each column represents a sample, each row corresponds to a gene, and each cell displays normalized gene expression values. B) KEGG enrichment analysis of significant down-regulated pathways. The figure presents the top 32 significant pathways based on p-adj <0.05 and abs(log2FoldChange) > 0, identified by RNA-seq. The y-axis indicates the pathways identified by their ID (see the readable term name table in supplement materials). The left x-axis demonstrates the percentage of genes, the expressed genes (left number) out of the total genes number (right number) in each GO term. The right x-axis demonstrates the number of genes per term, the bubble size indicates the proportion of expressed genes. The color bar indicates the p- adjusted value, the blue represents a higher value, the red represents lower value. C) Gene set enrichment analysis (GSEA) of the top 48 down-regulated genes associated with the 32 significant down-regulated pathways. Pathways are shown horizontally and genes vertically. The colored cells indicate the frequency of gene expression association within each pathway, aligning with its p-adjusted value. D) Distribution of top down-expressed genes according to their cellular localization. E) Molecular functions and interactions network visualization. The terms and their corresponding connections to the associated genes are colored labeled. Nodes represent genes, blue for nuclear, gray for plasma membrane and orange for cytoplasmic gene localization. The black lines represent the type of protein-protein interaction network analyzed by STRING.

P2RX7-dependent expression of HCN1 in the degenerated retina.

A-D) Relative transcript levels of HCN1 (A), HCN2 (B), HCN3 (C) and HCN4 (D) as assessed by qRT-PCR using the ΔΔCt-quantification method and the housekeeping gene GAPDH, in retinal lysates from 3 rd1 and 3 rd1-p2rx7ko mice at P60 and at P120 (total n=12). E-G) Immunolabeling of WT (E), rd1 (F) and rd1-p2×7ko (G) retinal slices with a primary antibody against HCN1 revealed with a red-fluorescent secondary antibody and counterstained with the nuclear blue dye DAPI. The staining profile for blue and red fluorescence across the retina, from the outer limit of the INL to the anterior edge of the GCL (∼100 µm wide) is displayed next to each image. H-I) Quantification of red fluorescence in the INL (H) and IPL (I) in the retinas of 8 WT, 8 rd1 and 6 rd1-p2rx7ko mice. A-D, H-I) Values are shown for individual datapoints (WT – square; circle – rd1; triangle – rd1-p2×7ko), and as mean ± SEM. Wilcoxon test, *p<0.05, **p<0.01, ***p<0.001.

P2rx7-dependent increase in HCN currents in the Off-RGCs of degenerated retinas.

A-B) Current-voltage (IV) curves in putative On- (A) and Off-RGCs (B), in WT (triangles), rd1 (circles), and rd1-p2rx7ko (squares) mouse retinas. C-E) Average density of Ih at -110 mV for each recorded RGC in WT (C), rd1 (D) and rd1-p2rx7ko (E), plotted against the depth of dendritic penetration in the inner plexiform layer (IPL). F) Summary quantification of C-E, where Ih density is shown as mean ± SEM in putative On- (white bars) and Off-RGCs (grey bars). Wilcoxon test, ***p<0.001. A-F) Recordings from a total of 16 WT-RGCs (On = 7, Off = 9); 19 rd1-RGCs (On = 10, Off = 9); and 13 rd1-p2rx7ko-RGCs (On = 5, Off = 8).

P2X7 receptor signaling sustains elevated steady-state Ca²⁺ levels in rd1 RGCs.

(A) Confocal image of GCaMP6f fluorescence in the ganglion cell layer (GCL) following intravitreal delivery of pAAV-hSyn1-GCaMP6f. Robust neuronal expression enables measurement of steady-state intracellular Ca²⁺ dynamics in situ. (B) Quantification of mean steady-state Ca²⁺ levels (F/F₀, arbitrary units) in WT, rd1, and rd1-p2rx7ko retinas under baseline conditions (left) and during synaptic blockade (right). rd1 RGCs exhibit significantly elevated Ca²⁺ compared to WT, which is reduced in rd1-p2rx7ko mice. Synaptic blockade does not abolish the Ca²⁺ elevation in rd1 but markedly reduces Ca²⁺ in rd1-p2rx7ko. Values are shown as mean ± SEM, *p<0.05, **p<0.01; one-way ANOVA with post hoc Tukey’s HSD; n = 275 - 328 cells from 6 mice and 12 retinas per genotype. (C) Wide-field image of rd1-Thy1-GCaMP6s retina illustrating sparse RGC labeling and large-scale imaging field. Inset shows higher magnification of RGCs with axons projecting to the optic disk. (D) Paired Ca²⁺ imaging in identified rd1 RGCs before and after pharmacological manipulation. Left, synaptic blockade significantly reduces steady-state Ca²⁺ in a subset of rd1 RGCs. Right, addition of TNP-ATP under synaptic blockade does not further reduce Ca²⁺ levels. Grey lines connect values in individual mice; black circles indicate mean ± SEM. *p<0.05; ns, not significant; paired t-test after Shapiro Wilk normality test; n = 25-40 cells per retina in 10 retinas from 5 mice, per condition. (E) Dual imaging of rd1-Thy1-GCaMP6s (green) with Yo-Pro-3 (red) labeling to identify P2X7R-permeable pore formation. Green arrows indicate GCaMP6s⁺/Yo-Pro-3⁻ RGCs; yellow arrows indicate double-positive cells (GCaMP6s⁺/Yo-Pro-3⁺), consistent with P2X7R activation. (F) Steady-state Ca²⁺ levels in Yo-Pro-3⁻ and Yo-Pro-3⁺ rd1 RGCs under baseline conditions (left) and during synaptic blockade (right). Yo-Pro-3⁺ RGCs display significantly elevated Ca²⁺ compared to Yo-Pro-3⁻ cells, an effect that persists under synaptic blockade. Values are shown as mean ± SEM with individual recorded cells are shown as circles. *p<0.05, **p<0.01; Mann–Whitney test.