Retinal proteome profiling of inherited retinal degeneration across three different mouse models suggests common drug targets in retinitis pigmentosa

Inherited retinal degenerations (IRDs) are a leading cause of blindness among the population of young people in the developed world. Approximately half of IRDs initially manifest as a gradual loss of night vision and visual fields, characteristic of retinitis pigmentosa (RP). Due to challenges in genetic testing and the large heterogeneity of mutations underlying RP, targeted gene therapies are an impractical large-scale solution in the foreseeable future. For this reason, identifying key pathophysiological pathways in IRDs that could be targets for mutation-agnostic and disease-modifying therapies (DMTs) is warranted. In this study, we investigated the retinal proteome of three distinct IRD mouse models, in comparison to sex- and age-matched wild-type mice. Specifically, we used the Pde6βRd10 (rd10) and RhoP23H/WT (P23H) mouse models of autosomal recessive and autosomal dominant RP, respectively, as well as the Rpe65^-/-^ mouse model of Leber's congenital amaurosis type 2 (LCA2). The mice were housed at two distinct institutions and analyzed using LC-MS in three separate facilities/instruments following data-dependent and data-independent acquisition modes. This cross-institutional and multi-methodological approach signifies the reliability and reproducibility of the results. The large-scale profiling of the retinal proteome, coupled with in vivo electroretinography recordings, provided us with a reliable basis for comparing the disease phenotypes and severity. Despite evident inflammation, cellular stress, and downscaled phototransduction observed consistently across all three models, the underlying pathologies of RP and LCA2 displayed many differences, sharing only four general KEGG pathways. The opposite is true for the two RP models in which we identify remarkable convergence in proteomic phenotype, even though the mechanism of primary rod death in rd10 and P23H mice is different. Our data highlights the cAMP and cGMP second-messenger signaling pathways as potential targets for therapeutic intervention. The proteomic data are curated and made publicly available, facilitating the discovery of universal therapeutic targets for RP.

Methods
Animal Models and Study Design

We used three different mouse models of inherited retinal degenerative diseases (IRD) to discover retinal proteomic changes that are common to retinal degeneration (RD). The models used in this study were B6.CXB1-Pde6brd10/J (RRID: IMSR_JAX:004,297, referred to as rd10), B6.129S6(Cg)-Rhotm1.1Kpal/J (RRID: IMSR_JAX:017,628, referred to as P23H), mouse models of recessive and autosomal dominant retinitis pigmentosa (RP), respectively (18, 19), and B6.129-Rpe65tm1Tmr/J (RRID: IMSR_JAX:035,329, referred to as Rpe65−/−) model of Leber congenital amaurosis type 2 (LCA2) which was a kind gift from Dr Michael Redmond (National Institutes of Health) (20). The rd10 colony was kept as a homozygote. Age- and sex-matched C57BL/6J mice (RRID: IMSR_JAX:000,664) were used as controls. P23H heterozygote mice were bred with C57BL/6J mice, yielding P23H heterozygote and wild-type (WT) littermates. To get Rpe65−/− and their WT littermate mice, we bred heterozygote Rpe65+/− mice together. Only WT and homozygote offspring were used in this study. Several cohorts of mice were raised, and their retinal samples were collected at two different institutions: the University of California, Irvine (UCI) and the University of Eastern Finland (UEF) over the years 2019 to 2021 and 2023, respectively (Table 1). Mice were given water and standard feed ad libitum at both institutions. Retinal proteome analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing data-dependent acquisition (DDA) or data-independent acquisition (DIA) modes. Mice reared P0-P29 in DR and in CLR (vivarium) P29-P38.