Abstract
Purpose :
X-linked juvenile retinoschisis is a monogenic disease caused by mutation in the RS1 gene. RS1 encodes retinoschisin, an extracellular binding protein which is secreted from many classes of retinal cells but most abundantly from photoreceptors. However, aside from adhering the retinal cells, the lack of retinoschisin alters the actions of many intracellular processes (e.g., TRIPM1 signaling). We performed a pathway analysis on the retinal proteome of RS1-/y mice, some treated by rAAV2/8 RS1 gene therapy, to identify significantly regulated biological themes and potential targets for novel molecular therapeutics.
Methods :
Intravitreal rAAV2/8 RS1 was administered to a group of RS1-/y mice at postnatal day (P) 20; untreated littermates and wild type animals served as controls. The treated animals were sacrificed at P40 and their retinae extracted. Protein concentration was determined for each sample using a bicinchoninic acid assay (Micro-BCA, Thermo Fisher Scientific). After normalization, peptides were separated, detected, sequenced, and quantified using liquid chromatography mass spectrometry (Thermo Fisher Proteomics Core). Differential abundant protein analysis was performed to normalize percent relative abundance. Pathway enrichment analysis of proteins involved in phototransduction and inflammation was performed in Ingenuity.
Results :
A total of 3,901 proteins were identified in the samples. The amount of proteins analyzed in each sample was similar, evidenced by the similarity of the relative abundances for the majority of proteins across the samples. Pathway analyses of proteins in RS1-/y mice identified significant changes in G-proteins and related biochemical signaling cascades (including TRPM1). Notably, the most heavily regulated biological theme was phototransduction.
Conclusions :
Gene, protein, and pathway analyses suggest that the phototransduction cascade in the RS1-/y mouse is significantly altered, implicating the photoreceptor as an important, little-recognized target of pathology in retinoschisis.
This striking result seems to echo the occasionally reported effect seen in the ERGs of boys with retinoschisis—that the photoreceptors are affected.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.