Intraocular Efficacy and Safety of Small Molecule Proteostasis Modulators in Mice

Abstract

Purpose : Unfolded Protein Response (UPR) and Integrated Stress Response (ISR) are intracellular signal transduction mechanisms that control organismal protein quality. Many eye diseases such as retinitis pigmentosa, achromatopsia, and glaucoma have UPR and ISR as pathomechanisms. Key regulators of UPR and ISR include PERK, ATF6, and IRE-XBP1. In cell culture, various small molecules have been found to modulate UPR but have not been rigorously tested in mice eyes. This study investigates if small molecule proteostasis modulators are safe and effective in the murine eye through intraocular injections, biochemical assays, RNA sequencing, and visual function testing.

Methods : We tested 5 small molecule proteostasis agents – ISRIB (ISR inhibitor), Salubrinal (ISR activator), Ceapin-A7 (CA7, ATF6 inhibitor), AA147 (ATF6 activator), and Tunicamycin (pan-UPR/ISR activator, glycosylation inhibitor) – in C57BL/6J mice, injecting them intravitreally (IV) into both eyes. Saline served as a control, and Tunicamycin as a positive control. Eyes were collected 24 hours and 3 days post-injection for immunohistochemistry and RNA-sequencing, assessing UPR/ISR modulation and retinal histology. Additionally, electroretinogram (ERG) tests were conducted one-week post-injection to assess effects on vision.

Results : GFAP staining revealed gliosis in retinas treated with ISRIB for three days. TUNEL staining indicated cell death in retinas exposed to tunicamycin but not with the small molecule proteostasis agents. RNA-seq data analysis at the 24-hour mark highlighted significant pathway terms associated with visual function and endoplasmic reticulum in CA7, AA147, Salubrinal, and ISRIB. UPR/ISR modulators significantly modulated multiple UPR/ISR-regulated genes evidenced by a p ≤ 0.05 in DESeq2 analysis. ERG results indicated that both scotopic and photopic vision remained unimpaired after seven days of injecting CA7, AA147, and Salubrinal, but ISRIB injection caused lower a- and b-wave amplitudes.

Conclusions : We identify experimental conditions where Ceapin-A7, AA147, and Salubrinal safely modulate proteostasis mechanisms in the retina. These results provide proof-of-principle for the safety and efficacy of some small molecule proteostasis agents for use in the mouse eye. Our data provides foundational parameters to optimize the use of these agents in retinal degeneration, ocular disease models linked to ER stress, and UPR/ISR signaling.

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