Abstract
Purpose :
Autophagy is a cellular process for the removal of damaged or abnormal cellular components. We have previously reported that age-related alterations in Bruch's membrane decreases autophagic activity in retinal pigment epithelial (RPE) cells. Herein we investigate the molecular mechanism of the effects of aged basement membrane on autophagic activity in RPE cells.
Methods :
Extracellular matrix (ECM) was treated with sodium nitrite to mimic the ECM aging process as described previously. ARPE-19 cells were cultured on nitrite-modified or PBS treated- (control) ECM for three weeks. Tandem Sensor RFP-GFP-LC3B (Invitrogen, Carlsbad, CA) was used to monitor autophogic autophagosome and autolysosome formation in RPE cells. Autophogic activities are measured as the intensity of LC-3 GFP or RFP fluorescence. MEK1/2, an autophagy inducer, was measured in the nitrite-modified ECM culture system. Rapamycin and U0126 were used to observe autophagic effects on this experimental system. Autophagy and other metabolic pathway related gene expression (mRNA) were quantitively measured by real-time RT-PCR to elucidate the molecular mechanism.
Results :
RPE cells display reduced confluency on nitrite-modified ECM. 72 hours after transduction LC3 fluorescence protein can be visualized with fluorescence microscope in RPE cells surrounding the cell nucleus, suggesting autophagic activity. At day 21, LC3-protein fluorescence intensity is lower in RPE cells on nitrite ECM compared with those on PBS-treated ECM (control group). MEK1/2 expression was correlated with autophagy activity. Rapamycin enhanced autophagy activity on nitrite-modified ECM and U0126 could reverse this. The expression levels of other autophagy related genes such as (ATG) like ATG 5 and CLN3 (Battenin) were altered in RPE cells cultured on nitrite-modified ECM.
Conclusions :
Reduced autophagic activity in RPE on nitrite-modified ECM may be through MAPK/ERK signal transduction pathway. Aged human Bruch's membrane, which is associated with AMD pathology, may adversely affect the ability of the RPE cells to maintain healthy intracellular homeostasis. The significance of these changes to AMD pathophysiology remain to be elucidated.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.