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Peng Shang, Meysam Yazdankhah, Nadezda Stepicheva, Stacey L Hose, Sayan Ghosh, Imran Ahmed Bhutto, J Samuel Zigler, Debasish Sinha; βA3/A1-crystallin is necessary for intracellular protein trafficking in mature RPE cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3994.
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We have previously shown that βA3/A1-crystallin plays a pivotal role in the lysosomal-mediated clearance process in RPE cells. Our recent studies suggest a novel function of this protein in intracellular trafficking. This study was undertaken to determine how βA3/A1-crystallin modulates protein trafficking in mature RPE cells.
RPE-Choroid complex was isolated from Cryba1fl/fl (control) and Cryba1 (encoding βA3/A1-crystallin) KO mice. Extracts were prepared and used to perform RNAseq and Proteomic analysis. Western blotting and quantitative PCR techniques were used to detect target gene expression at either protein or mRNA level. Immunostaining of RPE flatmounts were used to investigate RPE phenotype and protein expression. Mouse primary RPE cells were cultured and differentiated in vitro into mature RPE cells for intracellular trafficking studies.
Proteomics and RNAseq data revealed that many extracellular matrix proteins are altered in the RPE cells of Cryba1 KO mice compared to that of control mice, including genes involved in focal adhesions and tight junctions, which are decreased in Cryba1 KO mice at 5 months old. These data also indicated abnormal cytoskeleton reorganization, endocytosis and cellular trafficking in RPE cells of KO mice. Primary RPE cells isolated from floxed mice lose βA3/A1-crystallin immediately after plating, but regain its expression after differentiating into a highly polarized RPE monolayer. Western blot and QPCR experiments indicated that expression of the epithelial cell marker E-Cadherin is reduced and the mRNA levels of snail1 and snail2 are up-regulated in Cryba1 conditional KO (cKO) RPE cells. We also found EGFR expression in Cryba1 KO RPE cells to be higher than that of control mice. RNAseq data indicated the up-regulation of the MAPK signaling pathway in the RPE of 3-month old Cryba1 cKO mice compared to control mice. Ezrin and Radixin, two proteins known to interact with the plasma membrane and the cytoskeleton, were decreased in the RPE of Cryba1 cKO mice. Immunostaining and RPE flatmount showed that RPE cells in Cryba1 KO mice start losing normal hexagonal shape by 9 months of age.
βA3/A1-crystallin may regulate polarized endocytic routes in RPE cells. Loss of βA3/A1-crystallin results in EMT-like process in RPE cells, which may lead to subretinal fibrosis and further contribute to the AMD-like phenotype seen in our mouse models.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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