Protective effect of autophagy on human retinal pigment epithelial cells against lipofuscin fluorophore A2E: implications for age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the secretion of inflammatory cytokines and VEGFA.Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among elderly people and is becoming a major public health issue.^{1, 2, 3} The pathological change in AMD is located in the macula, which is the central and posterior portion of the retina containing the retinal pigment epithelium (RPE) and photoreceptors. Central visual impairment caused by AMD results from the loss or damage of RPE cells and the photoreceptors.⁴ Currently, the etiology and pathogenesis of AMD is not fully understood and there is no effective treatment.^{5, 6} A chronic aberrant inflammatory response in RPE cells is considered to be one of the major factors contributing to the pathogenesis of AMD.^{7, 8}Lipofuscin is a complex aggregate of fluorescent material, formed in a variety of tissues but best studied in the eye.⁹ The buildup of lipofuscin in RPE cells has been identified as a byproduct of the visual cycle, and is derived from the ingestion of photoreceptor outer segments, which has been implicated in several retinal degenerations, including AMD.^{10, 11} As revealed by spectroscopic analyses, the bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is the first isolated, major fluorophore from RPE lipofuscin. Numerous in vitro and in vivo studies have found that toxicity effects associated with this compound, and A2E is involved in the pathological pathways of AMD, especially the inflammatory response.^{12, 13} Although several studies have suggested that A2E may induce cytokine production, activate inflammasomes or the complement system in RPE cells, and contribute to chronic inflammation in AMD,^{14, 15, 16} the exact mechanisms by which A2E exerts an effect on RPE cells remains unclear.Autophagy is an evolutionarily conserved cellular housekeeping process that removes damaged organelles and protein aggregates that are unnecessary or dysfunctional to the cells by delivering cytoplasmic substrates to lysosomes for degeneration.¹⁷ In addition to turnover of cellular components, autophagy is involved in development, differentiation, and tissue remodeling in various organisms.¹⁸ The failure of autophagy in aged postmitotic cells, including RPE cells, can result in the accumulation of aggregation-prone proteins, cellular degeneration, and finally the induction of cell death.^{19, 20} Currently, a large amount of evidence indicates that autophagy is associated with RPE damage and AMD pathology.^{21, 22, 23} In RPE cells, the preservation of autophagic activity, together with functional lysosomal enzymes, is a prerequisite to prevent detrimental intracellular accumulation of damaged molecules.²¹ A well-functioning proteolytic machine guarantees that there is sufficient capacity to handle damaged proteins and organelles.²⁴ In addition, Saadat KA et al.²⁵ have shown that RPE cell death is induced in the presence of A2E and the autophagic inhibitor 3-methyladenine (3-MA). Nevertheless, whether the autophagic pathway has effects on A2E-induced cell damage through the production of chemokines and cytokines remains unclear. Furthermore, the relationship between A2E and autophagy and how this interaction influences RPE cells'' inflammatory response requires further clarification.Therefore, the protective effect of autophagy on human RPE cells against lipofuscin fluorophore A2E-induced cell death and the inflammatory response were studied in the present article. This work facilitates our understanding of the role of autophagy in the survival and death of RPE cells accumulating excess lipofuscin and provides a new strategy in the treatment of AMD.