Abstract
Purpose :
Usher syndrome (USH) is the leading genetic cause of deaf-blindness. Currently, there are four USH types (USH1-4) that differ in severity and age of onset. Mutations in the USH1C gene, which encodes the protein harmonin, cause the most severe form, USH1. Harmonin is a scaffolding protein expressed in ciliary cells. In hair cells, Harmonin has been shown to play a role in development and function; however, its role in the retina is not well understood. The USH1C c.216G>A (216A) founder mutation is the most common cause of Type 1 Usher in Acadian populations of the United States and Canada. Mice engineered to contain this mutation (USH1C) are deaf and have balance and visual deficits similar to patients. To understand the effect of the 216A mutation in the retina, we assessed protein expression in retinal extracts from USH1C, wild-type controls (WT), and 216A-targeted antisense oligonucleotide (ASO)-treated USH1C mice using quantitative discovery-based proteomics. We hypothesize USH1C mice have abnormal protein expression compared to WT controls and that ASO treatment, which improves Ush1c gene expression, rescues differentially expressed proteins in the retina.
Methods :
Liquid chromatography and mass spectrometry were used to process retinal tissue harvested from USH1C, ASO-USH1C, and WT mice at various ages for proteomic analysis. Proteins were then analyzed using Proteome Discoverer 2.3 software to identify common proteins. Immunohistochemistry (IHC) and western blot analysis were used to confirm expression.
Results :
A total of 3500-4000 proteins were identified in USH1C, ASO-USH1C, and WT retinas at 1, 3, 6 and 12 months of age. Four proteins (Acyl, Impg1, Pygm and Sfxn5) were found to be differentially expressed at all ages. Of these, proteomic analysis found Impg1 expression was rescued with ASO treatment in USH1C mice, which IHC and western blot confirmed.
Conclusions :
This data demonstrates that abnormal protein expression in the USH1C retina, which may directly or indirectly depend on Ush1c expression, can be corrected with ASO treatment targeting the Ush1c gene. Additionally, these data suggest a new role for harmonin in the healthy and USH1C retina.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.