Autophagy inhibition-induced alterations of energy metabolism and fluorescence lifetime of the retinal pigment epithelium ex vivo

Abstract

Purpose : Reduced autophagy function of retinal pigment epithelial (RPE) cells is suggested as one of the key factors in the pathogenesis of degenerative chorioretinal disorders, such as age-related macular degeneration. The purpose of this study is to investigate the energy metabolism of ex vivo RPE under the inhibitory condition of autophagy, and its correlation with its fluorescence lifetime (FLT) using fluorescence lifetime imaging ophthalmoscopy (FLIO).

Methods : Porcine ex-vivo RPE/choroid-explants were exposed to the autophagy inhibitor Bafilomycin A1 (1x10^-8 M) for 24 h. The viability of the RPE cells was examined with a calcein test. The inhibitory effect of Bafilomycin A1 on autophagy was verified by measuring intracellular autophagosomes. To understand the influence of the autophagy inhibition on the energy metabolism of the RPE, the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured by a Seahorse-XF-Analyzer after 24 h of Bafilomycin A1 incubation. The FLT of the RPE was examined by FLIO (excitation: 473 nm, detection in the short spectral channel (SSC): 498-560 nm and in the long spectral channel (LSC): 560-700 nm). In order to enable the FLIO on the tissues placed in a culture plate with culture medium, the horizontal stage and a 45-degree-angled mirror were built in front of the FLIO camera.

Results : Incubation of the explants with 1x10^-8 M Bafilomycin A1 for 24 h did not cause increased cell death compared with the non-treated control group, whereas Bafilomycin A1 was proven to inhibit autophagy. The OCR of the treated tissue was significantly reduced (e.g., basal respiration from 220 pmol/min ± 43 pmol/min to 147 pmol/min ± 62 pmol/min, p<0.05). The ECAR, an indicator of the glycolytic activity was not significantly altered. In FLIO, Bafilomycin A1 significantly extended the FLT (mean FLT: τ_m) of the RPE by about 350 ps in both detection channels.

Conclusions : The results of the metabolic analysis clearly showed that the autophagy inhibition may induce mitochondrial dysfunction in RPE cells. In this study, it was experimentally demonstrated that FLIO may indicate alteration of mitochondrial function in RPE cells and could be utilized to assess it in clinical practice.

This is a 2021 ARVO Annual Meeting abstract.