Abstract
Purpose :
The RPE is a multifunctional polarized cell layer at the interface of the neural retina and choroid. Serving as a gatekeeper, it stabilizes ions in the subretinal space, participates in water and nutrient transport, photoreceptor phagocytosis, the visual cycle, and secretion of factors critical to the structural integrity of its neighboring tissues. The cytoskeleton is a network of protein fibers supporting cellular function and stability, the dynamics of which varies with age. We investigated the RPE cytoskeleton profile and its associated proteins to establish a framework of the RPE cytoskeletome, and changes in the protein network during aging.
Methods :
Primary RPE cells established from young (45 and 48 yr old; female and male) and old (66, 83, and 93 yr old; male, female and female) donor eyes were grown to post-confluence and their integrity confirmed by immunocytochemistry and qPCR of RPE markers. Cytoskeletal and cytoplasmic-rich fractions were isolated using a mild detergent cocktail followed by a nuclease. The composition of the fractions was confirmed by Western Blot analysis. LC-MS/MS analyses were performed using a MClass UPLC system coupled to a Thermo Orbitrap Fusion Lumos mass spectrometer. Gene ontology and enrichment analyses were performed using ShinyGO and PANTHER analysis tools. Statistical analyses of data (three technical replicates/biological sample; reported as mean ± SEM) involved comparing between groups using t-test and one-way ANOVA. p < 0.05 was considered statistically significant.
Results :
RPE cells demonstrated epithelial morphology, expressing ZO-1, occludin-19, Rpe65, and others. Cytoskeletal enriched fractions expressed vimentin and β-actin, while soluble fractions expressed cytoplasmic proteins (e.g., α-tubulin and GAPDH). After performing label-free quantification of protein samples from cytoskeletal fractions by LC-MS/MS analysis, an abundance of cytoskeleton and associated proteins were detected, with over 288 up- and 207 down-regulated in old versus young samples. Differentially regulated proteins belonged to actin filament based, cytoskeleton organization, extracellular matrix organization, exocytosis, DNA binding, and glucose binding processes among others.
Conclusions :
This is the first report of the RPE cytoskeletal network with age. This framework will assist in further discovery of age-related cellular structural changes that may predispose to disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.