Abstract
Purpose :
Incompetent proteostasis resulting from protein misfolding is a leading cause of neurodegeneration, including autosomal dominant retinitis pigmentosa (adRP). Unfortunately, effective treatments for adRP are currently lacking, highlighting the need for novel therapeutic strategies that enhance photoreceptor proteostasis. We recently found the dynamics of liquid-liquid phase separation (LLPS) in nuclear speckles play a pivotal role in determining the transcription capacity of proteostasis genes. Enhancing the activity of the nuclear speckle scaffolding protein SON increased fluidity, chromatin recruitment, proteostasis gene expression, and reduced protein aggregation. We aim to testify the proof-of-principle that by pharmacologically invigorating nuclear speckle LLPS dynamics, the entire proteostasis pathway can be amplified and the proteinopathy-related adRP can be treated.
Methods :
Using an image-based assay, we performed an exploratory drug screening using an FDA-approved drug repurposing library.
We performed a series of hit confirmation assays including the reporter assay of a critical regulator of proteostasis, Perk, RNA-seq, and a SON thermoshift assay.
We treated NIH3T3 (RHOP23H) cells with PP and measured RHOP23H level by immunoblots.
We tested the efficacy of PP in the retina explants isolated from the RhoP23H/+ knock-in mouse model of RP by a 10-day treatment. Retinal total thickness was measured at day 0 and day 10 of treatment by a vis-OCT system, and analyzed by histology imaging.
Results :
We identified PP as a potent nuclear speckle rejuvenator. By binding to SON with an EC50 at about 50 nM, PP increased the LLPS by reducing the surface tension of the nuclear speckles. The PP treatment led to the induction of genes involved in the ER-associated protein degradation pathway, autophagy, and vesicle transport in MEFs. Importantly, PP treatment effectively cleared the misfolded RHOP23H mutant in the NIH3T3 cells. PP treatment led to an immediate transcriptomic boost of proteostasis in the eye, resulting in near-complete protection to the RhoP23H/+ mouse retina explants, evidenced by the preserved retina thickness from D0-D10 measured by OCT, and preserved outer nuclear layer cell number similar to the wild-type control by retinal histological analysis.
Conclusions :
Our findings suggest SON stabilization can be a viable therapeutic strategy for ocular proteinopathies including RHO-associated adRP.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.