Abstract
Purpose :
RPE is one of the most phagocytic cells in the body, both autophagy and phagocytosis are highly active in the RPE. The terminal events of both autophagy and phagocytosis involve degradation in the lysosome. Lysosome plays a crucial role in the proper functioning of both autophagy and phagocytosis in the RPE. Imbalances in lysosomal function can lead to the accumulation of undegraded autophagy and phagocytic substrates in the RPE. Transcription factor EB (TFEB) is identified as a master of the regulator of autophagy and lysosomal function. Decrease expression and activity of TFEB is known to increase the pathological accumulation of cellular substrates in different cell types. In this study, we investigated the expression and regulation of TFEB in the ABCA4 knockout (KO) RPE.
Methods :
ABCA4 KO RPE cells were isolated and the expression of TFEB was monitored using immunoblotting analysis and quantitative real-time PCR (qRT-PCR) analysis. The expression of TFEB regulated coordinated lysosomal expression and regulation (CLEAR) network genes was studied by qRT-PCR analysis. The cells were also transfected with a constitutively active TFEBS142AS211A and the expression of TFEB and CLEAR network gene, Cathepsin D (CTSD), Lysosomal-associated membrane protein 1 (LAMP1), Beclin-1(BECN1), UV radiation resistance-associated gene (UVRAG), Sequestosome 1(SQSTM1), Microtubule Associated Protein 1 Light Chain 3 Beta (MAP1LC3B) by qRT-PCR. Cathepsin D activity was also measured using the Cathepsin D activity assay kit.
Results :
Our result shows that both mRNA and protein levels of TFEB were downregulated in the ABCA4 KO RPE compared to age-matched WT RPE. The expression of TFEB regulated CLEAR network genes were rescued upon overexpression of constitutively active TFEB in the ABCA4 KO RPE. Our results also show rescue of Cathepsin D activity in ABCA4 KO RPE upon overexpression of constitutively active TFEB in the ABCA4 KO RPE.
Conclusions :
Our study shows that decreased expression of TFEB in the ABCA4 KO RPE is accompanied by downregulation of TFEB-regulated CLEAR network genes. TFEB-regulated transcriptional program is rescued by overexpression of constitutively active TFEB in the ABCA4 KO RPE. These results suggest that TFEB plays a crucial role in the regulation of cellular clearance in the ABCA4 KO RPE.
This is a 2021 ARVO Annual Meeting abstract.