Abstract
Purpose :
To identify molecular mechanisms underlying enhanced S-cone sensitivity syndrome in a murine ESCS knock-in model.
Methods :
We generated a ‘knock-in’ mouse line with a targeted modification in the ligand-binding domain (LBD) of the photoreceptor-specific nuclear receptor Nr2e3 at amino acid at position 311 (Nr2e3R311Q/R311Q) . We characterized mice at P0, P3, P6, P10, P14, P21, P28, 6m, and 1y; in vivo, by fundus photography, optical coherence tomography (OCT), electroretinography (ERG) and fluorescein angiography; post mortem by histology, immunohistochemistry, in situ hybridization and ex vivo electroporation. In vitro analyses included RNA expression profiling (RNAseq), Immunoprecipitations, Western blotting and BRET (bioluminescence resonance energy transfer) assays.
Results :
The Nr2e3R311Q/R311Q mouse phenocopies the well-described Nr2e3rd7/rd7 mice by fundus photography, OCT and histology at all time-points. During early postnatal development, Nr2e3 colocalizes with S-Opsin, Rhodopsin and Nrl in wild-type and Nr2e3R311Q/R311Q retinas, as assessed by in situ hybridization. Nr2e3 also colocalized with the nuclear receptor corepressors atrophin-1 and atrophin-2. Co-immunoprecipitation experiments showed a decrease in binding of Nr2e3 to atrophin-1 in presence of the Nr2e3-R311Q mutant protein. By BRET, all tested pathogenic variants located in the LBD showed impaired binding to atrophin-1 and atrophin-2. Knock-down of atrophin-1 by siRNA in ex vivo P0-P2 electroporated wild-type retinas lead to a decrease in rod-specific gene expression and an increase in cone-specific gene expression. Of note, the nuclear receptor corepressor 1 was not interacting with Nr2e3. RNAseq analysis identified about 8700 differentially expressed genes and specific expression profiles were identified in Nr2e3R311Q/R311Q and Nr2e3rd7/rd7 mouse models. This expression data will be available as a resource to the community.
Conclusions :
In vivo, the Nr2e3-R311Q protein shows impaired binding to the corepressors atrophins, and in vitro, all pathogenic variants located in the LBD of Nr2e3 show impaired corepressor binding. These data suggest that impaired corepressor binding is a disease mechanism in ESCS.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.