Primary fetal RPE cultures resist accumulation and toxicity of lipofuscin-like material, and accumulated material can be further reduced by autophagy induction.

Abstract

Purpose : The accumulation of indigestible autofluorescent material (IAM) in lysosomal-like structures, termed lipofuscin in vivo, is a hallmark of aged retinal pigment epithelium (RPE). However, whether this accumulated waste is toxic is unclear. We therefore investigated the effects of IAM accumulation in highly differentiated human fetal RPE (hfRPE) cultures and evaluated the ability of small molecule induction of autophagy to clear IAM.

Methods : Unmodified and UV-treated purified bovine outer segments (OS) (10 OS/cell) were fed daily to the ARPE-19 RPE cell line or to hfRPE. The emission spectrum, composition, and morphology of resulting IAM was measured and compared to in vivo lipofuscin. Effects of IAM on a range of RPE phenotypes were assessed. A screen for autophagy inducers in hfRPE was performed and top hits were tested for effects on IAM accumulation during UV-OS feedings and after UV-OS feedings.

Results : Although ARPE-19 cells and hfRPE were fed similar amounts of OS material, hfRPE were markedly less susceptible to IAM buildup. The IAM that did accumulate in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. IAM diminished trans-epithelial electrical resistance (TEER) and degradation of phagocytosed OS, while inducing senescence and subtle epithelial-mesenchymal transition (EMT), but had no obvious effects on RPE metabolism, secretion of drusen components or polarity markers, nor overt cell death. Out of more than 25 autophagy inducer candidates that were screened, 4 were found to induce autophagy reliably in our hfRPE culture system, with some reducing accumulation of IAM.

Conclusions : Cultured hfRPE demonstrates a remarkable resistance to IAM build-up, suggesting that young, highly-differentiated RPE has mechanisms for efficient OS processing that are lost with age or proliferation. While IAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may be at least partially adaptable to lipofuscin accumulation, and that autophagy induction may help decrease this modest lipofuscin burden further.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.