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Hsuan-Yeh Pan, Kristen Hatton, Abdulla Hadi Alamri, Mallika Valapala; Effects of cyclosporine on lysosomal function and autophagy in the Retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1044.
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RPE is the most active phagocytic cell in the body, phagocytosing 10% of the photoreceptor volume daily. Autophagy, a process involved in recycling of damaged organelles and long-lived proteins is also highly active in the RPE. The terminal events of both phagocytosis and autophagy involve fusion and subsequent degradation in the lysosomes. Dysfunctional lysosomes lead to impaired cellular clearance and subsequent degeneration of the RPE. Transcription Factor EB (TFEB) is identified as a master regulator of lysosomal function and autophagy. A serine/threonine protein phosphatase, calcineurin is known to promote nuclear translocation and activation of TFEB. We investigated the effects of cyclosporine, a calcineurin inhibitor on the TFEB-mediated regulation of lysosomal and autophagy pathway in the RPE.
ARPE-19 and primary mouse RPE cells were treated with 10 µM cyclosporine for 24 hr. Autophagy was induced by culturing the cells in HBSS for 12-24 hr. Quantitative real time PCR (qPCR) analysis was performed to study the expression of the following genes: Lysosome associated membrane protein 1(LAMP-1), Cathepsin D (CTSD), Mucolipin 1 (MCON1), and ATPase, H+ transporting, lysosomal V0 subunit a1 (ATP6V0A1), BECN1 (Beclin 1), UV radiation resistance-associated gene (UVRAG) and Sequestosome 1 (SQSTM1). Immunoblotting and immunostaining with Microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody was used to study the autophagy pathway. LAMP-1 immunostaining was used to estimate lysosome biogenesis
qPCR analysis of ARPE-19 and primary mouse RPE, showed a downregulation of TFEB-target genes in the autophagy and lysosomal pathway upon treatment with cyclosporine. Starvation-induced autophagy was markedly reduced in cells treated with cyclosporine with concomitant reduction in nuclear levels of TFEB. Reduction in lysosomal number and Cathepsin D activity was also observed upon cyclosporine treatment in cells subjected to starvation.
Our studies suggest that pharmacological inhibition of calcineurin by cyclosporine, decreases TFEB localization and expression of TFEB downstream targets in the RPE. Cyclosporine treatment dampens starvation-induced up-regulation of lysosomal biogenesis and autophagy. Our data suggests that TFEB play a crucial role in maintaining cellular homeostasis in RPE.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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