A non-retinoid chaperone of rhodopsin protects the Rho^P23H/+ mouse retina in ex vivo culture.

Abstract

Purpose : RHODOPSIN (RHO) mutations accounts for ~25-30% of autosomal dominant retinitis pigmentosa (adRP). Structural instability of mutant rhodopsin (RHO) causes dominant negative effect leads to the death of rod photoreceptors and vision loss. We hypothesize to restore the RHO homeostasis and rescue rods from RHO-associated adRP using previously discovered non-retinoid chaperone of RHO. The goal of this study is to test this hypothesis in ex vivo culture of the Rho^P23H/+ knock-in mouse retina.

Methods : We established retinal explant culture with RPE from the Rho^P23H/+ and Rho^+/+ mouse. Retinal morphology was analyzed by immunohistochemistry (IHC) of retinal explants at different days in vitro (DIV). To test the effect of small molecule chaperone on RHO homeostasis and retinal degeneration, Rho^P23H/+ retinal explants were treated with the compound and DMSO for 10 DIV. Retinae were collected for IHC, immunoblots and RNA-seq. The safety of compound was examined by TUNEL assay using Rho^+/+ retinal explants.

Results : IHC of RHO showed a gradual decrease of RHO level and outer segment (OS) length in both the Rho^P23H/+ and Rho^+/+ retinae along with time. The Rho^P23H/+, but not Rho^+/+ retinal explants showed a time-dependent decrease of outer nuclear layer (ONL), confirming a progressive degeneration in dish as seen in vivo. Importantly, treated with the compound, the Rho^P23H/+ retinae showed higher RHO level, improved RHO glycosylation, less ubiquitinated RHO, longer OS length, and thicker ONL, suggesting the compound indeed improved RHO homeostasis and supported photoreceptor survival. Interestingly, this small molecule increased the ratio of RHO in the OS/IS layer to that in the ONL of Rho^P23H/P23H retinal explants, consolidating its chaperone activity. The RNA-seq data suggests that the compound mitigated the primary immune response and reduced microglia activation. Additionally, protein homeostasis pathways were upregulated.

Conclusions : We showed that the non-retinoid chaperone improved RHO homeostasis and protected photoreceptors in the Rho^P23H/+ knock-in mouse retinae in ex vivo culture. The efficacious dose of the compound showed no significant toxicity. Our results strongly support the notion that improving RHO homeostasis is sufficient to protect retina from RHO-associated adRP. Future development of a continuous ocular drug delivery system will allow us to test the efficacy of this compound in vivo.

This is a 2021 ARVO Annual Meeting abstract.