PKG inhibition reduces rod and cone photoreceptor cell death in the new Rho^I255del mouse model for autosomal dominant retinitis pigmentosa.

Abstract

Purpose : Retinitis Pigmentosa (RP) is a blinding disease typically caused by a loss of photoreceptor cells. High levels of cGMP have been linked to photoreceptor cell death in models for autosomal recessive RP (ARRP; rd1 mice), and autosomal dominant RP (ADRP; Rho^S334ter rats). cGMP activates protein kinase G (PKG), with downstream effectors including calpain-type proteases and poly-ADP-ribose polymerase (PARP). Previously, PKG inhibition was found to prevent primary rod photoreceptor degeneration in rd1 mice. Here, we utilized a new mouse model for ADRP, carrying the Rho^I255del (Rho^I255d) mutation found in a subset of RP patients.

Methods : Retinal tissue sections from wild-type (WT), heterozygous Rho^I255d/+, and homozygous Rho^I255d/I255d mice were immunostained for cGMP accumulation and caspase-3 activation at post-natal day (P) 20 on 4% PFA fixed tissue. Unfixed tissue was used for in situ detection of calpain and PARP activity at timepoints ranging from P11 to P60. We then used organotypic retinal explant cultures derived from P12 WT and Rho^I255d/+ mice to test the neuroprotective efficacy of the PKG inhibitors CN003 and CN238. Cultures were stopped at P18, P20, P24, and P28, readouts included the TUNEL assay and immunostaining of cone arrestin.

Results : Compared to WT, cGMP accumulation was evident in photoreceptor segments in Rho^I255d/+ and, more strongly, in Rho^I255d/I255d retina, but the degeneration progressed more slowly in the Rho^I255d/+. Caspase-3 positive cells were detected in the outer nuclear layer (ONL) of both mutants, concomitant with a strong activation of calpain and PARP. CN003 (-37.14% ± 3.29%, n=8, p<0.05) and CN238 (-51.31% ± 0.97%, n=6, p<0.01) decreased the numbers of dying cells in the ONL. Remarkably, CN238 effectively protected cone photoreceptors even in long-term treatments lasting until P28.

Conclusions : The newly generated homologous Rho^I255d/+ ADRP model mouse displayed a rapid photoreceptor degeneration. The underlying mechanisms may include non-apoptotic cGMP-dependent cell death and classical apoptosis. Still, inhibition of PKG showed strong protection of both rod and cone photoreceptors, highlighting this as a therapeutic approach suitable for both ARRP and ADRP. Future studies in the Rho^I255del mouse may study an effective long-term delivery of PKG inhibitors to the retina.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.