The P2X7 receptor is an intermediate in the retinoic acid-signaling pathway that induces neuronal remodeling in retinitis pigmentosa

Reviewer #1 (Public review):

In this study, Telias et al. identify the P2X7 receptor as a key component of retinoic acid signaling-mediated remodeling in the degenerating rd1 retina. The authors report increased P2X7R expression in the inner retina following photoreceptor loss and link P2X7R signaling to retinal ganglion cell hyperactivity, membrane hyperpermeability, and altered calcium homeostasis. Genetic deletion of p2rx7 abolishes ganglion cell hyperpermeability and reduces several features of pathological remodeling in the rd1 retina. The study provides valuable mechanistic insight into retinal remodeling following photoreceptor degeneration. However, several issues currently limit the strength of the conclusions. In particular, key comparisons are confounded by differences in genetic background, the cellular source of P2X7R expression is not sufficiently resolved, and several experiments require additional controls and more cautious interpretation.

Major comments:

(1) The Methods state that C57BL/6J mice were used as wild-type controls, whereas rd1 mice were maintained on a C3H/HeJ background, and the rd1-p2rx7 knockout line is on a mixed background. Direct comparisons among these groups are therefore potentially confounded by strain-specific differences. Authors should use littermate rd1 het mice as healthy controls in all their experiments.

(2) The authors do not demonstrate P2X7R expression in RGCs. The P2X7R signal in the rd1 retina shown in Figure 1B appears saturated and is therefore difficult to compare directly with the WT image in Figure 1A. Furthermore, Figure 1E indicates that overall P2X7R fluorescence in the GCL is not significantly different between WT and rd1 retinas, whereas the representative images appear to suggest a marked increase. The authors should provide images acquired and displayed under identical settings and consider including retinal whole-mount staining with an RGC-specific marker. P2X7R abundance should then be quantified specifically within identified RGCs in both healthy and rd1 retinas. As mentioned above, het rd1 mice should be included. As an additional control, the authors also should include the staining of rd1 p2x7r KO retinas.

(3) Does the increase in P2X7R abundance correlate with photoreceptor loss? Please include staining of younger rd1 mice along with het rd1 littermates.

(4) In Figure 1G-H, the description of the reporter is internally inconsistent: the Results refer to an artificial mini-Pax6 promoter, whereas the Figure 1 legend describes the Ple344 neuronal mini-promoter derived from Tubb3. Please clarify which promoter is used and what cell population the ECFP signal labels. Figure 1G should explain the function of each reporter element and how RAR activity is inferred. Figure 1H should include an RGC marker such as RBPMS and provide quantitative analysis of RBPMS-positive, reporter-positive, and Yo-Pro-positive cells. Additional controls are needed to exclude effects of viral transduction or retinal inflammation on Yo-Pro uptake. They should include rd1 retinas without AAV, rd1 het retinas with and without the reporter AAV.

(5) In addition, lines 143-144 state that two experiments were performed, but only one is described in that paragraph; the text should be reorganized or clarified.

(6) Yo-Pro-1-positive cell density in rd1 mice between Figure 1H and Figure 2D is different. Why?

(7) Constitutive deletion of p2rx7 may cause developmental or compensatory changes that could contribute to the observed phenotype in Figures 2 and 3. Additional controls are therefore needed to distinguish acute effects of p2rx7 loss from developmental consequences. The authors should assess whether p2rx7 deletion alters retinal cell-type composition, including RGC density, or affects the timing or extent of photoreceptor degeneration. Perform a rescue experiment to determine whether overexpression of p2rx7 in the knockout background restores the phenotype. For all experiments, het rd1 control mice should be included.

(8) Figure 3D and E experiments should include control AAV expression such as GFP.

(9) Figure 5 experiments should include control rd1 het mice.

Reviewer #2 (Public review):

Summary:

In this study, the authors used genetic, transcriptomic, imaging, and electrophysiological approaches to investigate the role of P2X7R in retinal remodeling in rd1 mice. The authors propose that RA signaling upregulates P2X7R, leading to altered Ca²⁺ signaling, HCN1 expression, and spontaneous RGC hyperactivity.

Strengths:

Multiple lines of experiments were conducted, focusing on an important question in the field.

Weaknesses:

However, some aspects of the experimental design, statistical analysis, and interpretation require clarification. In particular, the genetic controls and causal evidence should be strengthened before the proposed RA-P2X7R-Ca²⁺-HCN1 pathway can be fully supported.

Some major issues:

(1) The Abstract states that P2X7R deletion prevents the upregulation of RA-responsive genes, whereas the Results state that RAR-dependent genes were not consistently changed by P2rx7 deletion. This central statement should be corrected and clarified.

(2) The P2rx7 knockout model requires further discussion. JAX strain 005576 targets exon 13 and has previously been reported to retain truncated P2X7 transcripts with residual activity (Masin et al., 2012). The authors should avoid describing this allele as complete loss of the entire P2rx7 gene unless additional isoform-specific validation is provided.

(3) The evidence that RAR directly regulates P2rx7 transcription remains incomplete. Predicted promoter motifs and reduced P2X7R protein after BMS-493 treatment are supportive, but they do not establish direct transcriptional regulation. Measurement of P2rx7 mRNA, RAR promoter occupancy, or promoter mutagenesis would strengthen this conclusion.

(4) Several statistical results require verification. In Figure 2H, four paired eyes are analyzed using an unpaired Mann-Whitney test, and the reported p<0.001 is difficult to reconcile with n=4. Similarly, the significance levels in Figure 5A-D are not compatible with a two-sided Wilcoxon rank-sum test using n=3 mice per group. The statistical unit, exact P values, and number of biological replicates should be rechecked.

(5) The overall causal pathway remains partially inferential. The study does not directly show that increased Ca²⁺ causes HCN1 upregulation or that HCN1 is required for RGC hyperactivity. These steps should either be experimentally tested or presented as a proposed model rather than an established mechanism.