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Stacey L Hose, Sayan Ghosh, Nadezda A. Stepicheva, Sara Jabalameli, Imran Ahmed Bhutto, Peng Shang, Meysam Yazdankhah, Joseph Weiss, Gerard A Lutty, J Samuel Zigler, Jr, Leah Byrne, Debasish Sinha; Targeting TFEB (transcription factor EB) as a novel approach for AMD therapy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1238.
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© ARVO (1962-2015); The Authors (2016-present)
This study was undertaken to determine whether activation of TFEB in diseased RPE is a viable therapeutic strategy for preventing or delaying the progression of early/dry AMD, by modulating autophagy-lysosomal clearance.
We engineered adeno-associated viral vector (AAV2 and AAV8-tyrosine mutant)-mediated WT-TFEB (will not translocate to the nucleus since it binds to 14-3-3 proteins and gets phosphorylated and, therefore, is retained in the cytoplasm) or constitutively active TFEB-S210A (a mutant form of TFEB that does not bind to 14-3-3 proteins and, therefore, will translocate to the nucleus) constructs for gene therapy studies. Primary cultures of RPE cells from 3-week-old Cryba1 KO mice were prepared following previously published methods. The cells, which have impaired lysosomal clearance, were infected with TFEB-WT-AAV vector or TFEB-S210A-AAV vector for 24 h at a dose of 1 X 109 infective units/ml. Western blotting (WB) and quantitative PCR (QPCR) were used to quantify protein and mRNA levels, respectively, for target genes from the CLEAR (Coordinated Lysosomal Expression and Regulation) network. Cryo-sections from the posterior poles of human AMD and age-matched control eyes were immuno-labeled with antibodies to TFEB and co-stained with DAPI.
Our data showed reduced nuclear TFEB staining in early AMD patient tissues (with small drusen and pigmentary changes at fovea; average age 79) compared to aged-matched controls (average age 83). Cultured Cryba1 KO RPE cells infected with TFEB-WT-AAV vector or TFEB-S210A-AAV vector showed that overexpression of constitutively active TFEB-S210A increased mRNA expression of CLEAR network genes like CTSB (Cathepsin B), LAMP2 and ATP6VOA1 (V-ATPase). Moreover, WB data indicate increased expression of CTSD (Cathepsin D), along with decrease in the expression of the autophagosome marker, p62/SQSTM1. AAV by itself had no effect.
These studies support our hypothesis that TFEB can induce lysosomal activity in RPE cells and increase clearance of cellular waste products. Since lysosomal efficiency declines with age and dysfunction in cellular clearance has been implicated in AMD, these findings have potential therapeutic implications.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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