N-retinylidene- N-retinylethanolamine degradation in human retinal pigment epithelial cells via memantine- and ifenprodil-mediated autophagy

Abstract

N-methyl-D-aspartate (NMDA) receptors are ionic glutamine receptors involved in brain development and functions such as learning and memory formation. NMDA receptor inhibition is associated with autophagy activation. In this study, we investigated whether the NMDA receptor antagonists, memantine and ifenprodil, induce autophagy in human retinal pigment epithelial cells (ARPE-19) to remove Nretinylidene- N-retinylethanolamine (A2E), an intracellular lipofuscin component. Fluorometric analysis using labeled A2E (A2E-BDP) and confocal microscopic examination revealed that low concentrations of NMDA receptor antagonists, which did not induce cytotoxicity, significantly reduced A2E accumulation in ARPE-19 cells. In addition, memantine and ifenprodil activated autophagy in ARPE-19 cells as measured by microtubule-associated protein 1A/1B-light chain3-II formation and phosphorylated p62 protein levels. Further, to understand the correlation between memantine- and ifenprodil-mediated A2E degradation and autophagy, autophagy-related 5 (ATG5) was depleted using RNA interference. Memantine and ifenprodil failed to degrade A2E in ARPE-19 cells lacking ATG5. Taken together, our study indicates that the NMDA receptor antagonists, memantine and ifenprodil, can remove A2E accumulated in cells via autophagy activation in ARPE-19 cells.

Keywords: A2E; ARPE-19; Autophagy; Ifenprodil; Memantine.

Fig. 1. Identification of memantine and ifenprodil as N-retinylidene-N-retinylethanolamine (A2E) degraders.

(A, B) ARPE-19 cells were incubated with 10 μM A2E-BDP for 24 h and then treated with memantine or ifenprodil (10, 25, and 50 μM) for 48 h. A2E-BDP fluorescence intensity was measured using a microplate reader. (C, D) Cell viability of memantine and ifenprodil in ARPE-19 cells. ARPE-19 cells were treated with various concentrations of the compounds. After incubation, cell viability was calculated using an EZ-Cytox assay kit. Results are presented as the mean ± SD. (n = 3). *p < 0.05, **p < 0.01 vs. A2E-BDP.

Fig. 2. Effects of memantine and ifenprodil on N-retinylidene-N-retinylethanolamine (A2E) removal in ARPE-19 cells.

(A) Schematic schedule of compound treatment. A2E accumulated in ARPE-19 cells after 5 μM A2E treatment three times at two-day intervals. A2E-laden ARPE-19 cells were treated with 1 μM memantine, 1 μM ifenprodil, or vehicle (CTR) twice at 24 h intervals. (B) The effects of memantine and ifenprodil on A2E levels in ARPE-19 cells were monitored using confocal microscopy. The fluorescence signals observed are Hoechst 33342 (blue) and A2E (green). Scale bar = 20 μm. (C) The observed A2E fluorescence signals were quantified using Image J software. Representative results are presented as the mean ± SD. (n = 3). ***p < 0.001 vs. A2E.

Fig. 3. Memantine and ifenprodil activate autophagy in ARPE-19 cells.

(A) ARPE-19 cells were treated with 1, 10, and 25 μM memantine for 8 h. LC3-II, p-p62 (S349), p62, and β-actin protein levels were detected using Western blotting. Images were quantified using the Image J software. (B) ARPE-19 cells were treated with 1, 10, and 25 μM ifenprodil for 8 h. Representative results are presented as the mean ± SD. (n = 3). *p < 0.05, **p < 0.01 vs. no-treatment control.

Fig. 4. Effect of autophagy on memantine- and ifenprodil-mediated N-retinylidene-N-retinylethanolamine (A2E) degradation in ARPE-19 cells.

(A) ARPE-19 cells were transfected with non-specific siRNA (siNS) or siRNA targeting ATG5 mRNA (siATG5). The expression level was detected using Western blotting. (B) siATG5-transfected ARPE-19 cells were treated with 5 μM A2E three times at two-day intervals. Then, they were treated with 1 μM memantine, 1 μM ifenprodil, or vehicle (CTR) twice at 24 h intervals. A2E levels in ARPE-19 cells were observed using a confocal microscope. The fluorescence signals observed are Hoechst (blue) and A2E (green). Scale bar = 20 μm. (C) Fluorescence signals on A2E removal were quantified using Image J software. Representative results are presented as the mean ± SD. (n = 3). ***p < 0.001 vs. CTR.