Experimental design and treatment responses.

(A) Timeline of intravitreal injections and functional and structural analysis. (B) Scotopic and photopic electroretinogram responses at P68 in DMSO- and RSP-treated P23H-rats. (C) Visual acuity measured by OKT response and contrast threshold, and outer nuclear layer thickness measured by vertical and horizontal scan of OCT in DMSO- and RSP-treated P23H-rats. All parameters were measured in both eyes (8 DMSO-treated and 7 RSP-treated rats). Data were expressed as mean ± SEM, and the Mann-Whitney U test was used to compare DMSO- and RSP-treated groups. RSP: reserpine, OKT: Optokinetic tracking, CT: Contrast threshold, ERG: electroretinogram, OCT: Optical coherence tomography, ONL: outer nuclear layer, ns: not significant, *p<0.05.

Sex-specific treatment responses in ERG analysis.

(A) Sex-associated subgroup analysis for scotopic b-wave amplitude at P68. (B) Individual changes of scotopic b-wave amplitude between P54 and P68 in female rats. (C) Scotopic and photopic electroretinogram responses and contrast threshold at P68 in DMSO- and RSP-treated female P23H-rats. Data were expressed as mean ± SEM, and the Mann- Whitney U test was used to compare DMSO- and RSP-treated groups. RSP: reserpine, *p<0.05; **p<0.01; ***p<0.001.

Effect of reserpine treatment on outer nuclear layer thickness of female P23H rats.

(A) Outer nuclear layer thickness evaluated by vertical scan of OCT at P70. (B) Outer nuclear layer thickness evaluated by horizontal scan of OCT at P70. The scale bar represents 100 µm. (C) Outer nuclear layer thickness evaluated by DAPI-stained retinal section at P70. The representative images were taken from dorsal retina, 1000 µm away from the optic nerve. Data were expressed as mean ± SEM, and the Mann-Whitney U test was used to compare DMSO- and RSP-treated groups. Images are representative of four female rats. The scale bar represents 20 µm. RSP: reserpine, ONL: outer nuclear layer,, INL: inner nuclear layer,, GCL: ganglion cell layer, *p<0.05.

Effect of reserpine treatment on retinal photoreceptors of female P23H rats.

(A,B) Rod photoreceptors were evaluated by immunostaining of REEP6 and rhodopsin (Rho) at P70. The representative images were taken from dorsal retina, 1000 µm away from the optic nerve. The scale bar represents 20 µm. (C) Cone photoreceptors were evaluated by immunostaining of cone arrestin at P70. Number of cone photoreceptors were counted every 500 μm throughout the retina. Data were expressed as mean ± SEM, and the Mann-Whitney U test was used to compare DMSO- and RSP-treated groups. Images are representative of four female rats. The scale bar represents 20 µm. RSP: reserpine, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, *p<0.05.

Pathology of RhoP23H/+ retina impacts key retinal pathways and is influenced by biological sex.

(A) Principal Component Analysis (PCA) plot of retinal transcriptomes from WT, P23H and P23H reserpine-treated rats at P70. Female (.F) and male (.M) animals are represented by circles and triangles, respectively. (B) Heatmap of significantly differentially expressing genes between WT and P23H retinas. Male and female retinas have common as well as unique transcriptomic trends in P23H rodents. Colors are z-scores of row-scaled log2 CPM values of genes. (C) Volcano plot of significantly differential genes between WT and P23H retinal transcriptomes. Top over- and under-expressing genes labeled on the plot. (D) KEGG pathways enriched in mutant retinal transcriptomes of female and male rats. Over and under-enriched pathways are shown in red and blue, while non-significant enrichment is depicted in transparent fill. (E) Phototransduction genes express differently in P23H mutant retinas and have a sex-biased trend. (F) Boxplots showing expression of key phototransduction genes – rhodopsin (Rho), transducin (Gnat1), and rhodopsin kinase (Grk1) in female and male retinas. (G) Heatmap of significantly differential genes in KEGG pathways of transcription, translation and cellular transport that constitute the genetic information processing supergroup. Heatmap colors in panels (E) and (G) are z-scores of row-scaled log2 CPM values of genes and similar to the plot in (B). WT: wild-type, P23H: DMSO treated P23H retinas, RSP: reserpine treated P23H retinas.

Reserpine treatment leads to improved phototransduction and key signaling processes in retinas of female P23H rats.

(A) Heatmap of reserpine associated differentially expressing genes showing clustering of transcriptomic profiles of treated retinas with that of WT rats. Colors are z-scores of row-scaled log2 CPM values of genes and same as in Figure 5B. (B) KEGG pathway enrichment network of reserpine linked over-expressing genes highlighting clusters of pathways impacted by treatment. Analysis performed using ClueGo on Cytoscape. Notable pathways include phototransduction, proteostasis network and p53 signaling among others. (C) Reserpine-treated female P23H retinas have higher gene expression of Rho than that of DMSO administered samples. (D) Gene expression of several key photoreceptor transcription factors – Nrl, Nr2e3 and Crx – recover after reserpine treatment. (E) Transcription of proteostasis network genes in female mutant retinas is reversed by reserpine. (F) Reserpine leads to transcriptomic upregulation of p53 signaling pathway genes. (G) P62 (Sqstm1) and (F) Akt1 genes are transcribed higher in response to reserpine administration. WT: wild-type, P23H and DMSO: DMSO treated P23H retinas, RSP: reserpine treated P23H retinas.