Abstract
Purpose :
Neural Retina Leucine zipper protein (NRL) is a critical transcription factor (TF) for rod photoreceptor differentiation. Loss of NRL results in complete absence of rod photoreceptors in mouse retina with concomitant increase in S-cones. We hypothesize that multiple proteins interact with NRL as part of transcriptional complexes and modulate rod gene expression. The aim of the present project is to understand the mechanisms of photoreceptor (rod) differentiation and to reveal new possible drug targets by altering NRL function and/or its targets.
Methods :
We have identified candidate interactors by performing yeast two-hybrid assays in conjunction with Blast analysis for their identification. We then selected candidates for validation based on criteria defined before the start of this experiment. Our search is for candidates that appear multiple times across independent screens and would be novel interactors of NRL. We are validating these interactions by proximity ligation assay (PLA) and co-immunoprecipitation. We will perform luciferase reporter assays with different rod promoters to investigate synergy between NRL and the candidate interactor protein. We will also investigate a chosen novel interactor’s impact on the retina by performing immunohistochemistry & OCT comparing wildtype mice to knockdown mice generated by AAV injection.
Results :
We have identified over 30 candidate interactors of NRL and used selection criteria to move forward, identifying Activating Transcription Factor 4 (ATF4). We validated interaction by performing PLA in HEK293 cells transfected with NRL-Xpress. We are moving on to preform PLA on human sections to provide support for physiological interaction.
Conclusions :
We have elected to work with Activating Transcription Factor 4 (ATF4) as it is also a leucine zipper transcription factor. ATF4 is associated with oxidative stress response; therefore, We hypothesize that NRL and ATF4 work together in response to stress conditions within rod photoreceptors.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.