Vitamin A dimers trigger the protracted death of retinal pigment epithelium cells

Cellular events responsible for the initiation of major neurodegenerative disorders of the eye leading to blindness, including age-related macular degeneration, Stargardt and Best diseases, are poorly understood. Accumulation of vitamin A dimers, such as N-retinylidene-N-retinylethanolamine (A2E) in the retinal pigment epithelium (RPE), is one of the earliest measurable events preceding retinal degeneration. However, the extent to which these dimers contribute to tissue degeneration is not clear. To determine if A2E could trigger morphological changes associated with the degenerating RPE and subsequent cell death, we evaluated its toxicity to cultured human RPE cells (ARPE-19). We show that A2E triggered the accumulation of debris followed by a protracted death. A2E was up to≈14-fold more toxic than its precursor, retinaldehyde. Measurements reveal that the concentration of A2E in the aged human eye could exceed the concentration of all other retinoids, opening the possibility of A2E-triggered cell death by several reported mechanisms. Findings suggest that accumulation of vitamin A dimers such as A2E in the human eye might be responsible for the formation of ubiquitous RPE debris, an early indication of retinal degeneration, and that preventing or reducing the accumulation of vitamin A dimers is a prudent strategy to prevent blindness.The elucidation of environmental and genetic drivers of RPE senescence has been a persistent goal toward understanding and preventing degenerative disease of the retina. Since their structural elucidation in the 1990s, dimers of dietary vitamin A, in particular N-retinylidene-N-retinylethanolamine (A2E), have been postulated as chemical triggers, driving retinal senescence and associated degeneration. The eye uses vitamin A as a cofactor to sense light. A striking chemical signature of the aging and degenerating retina is the accumulation of vitamin A dimers in the retinal pigment epithelium (RPE)^{1, 2} and underlying Bruch''s membrane.³ In rodent models of macular degeneration,^{4, 5, 6, 7, 8} high levels of vitamin A dimers correlate with poor retinal health and a variety of mechanisms have been proposed by which dimers of vitamin A may induce retinal toxicity ranging from non specific to direct antagonistic/protagonistic mechanisms.^{9, 10, 11, 12, 13, 14, 15, 16} As a cationic ambiphilic pyridinium, A2E has been shown to solubilize lipid membranes, inactivate lysosomes by increasing lysosomal pH, and accumulates in the negatively charged mitochondrial compartment. Once dimerized, the special orientation of the polyene chains make them especially susceptible to oxidative degradation¹⁶ leading to secondary reactive aldehyde and epoxide toxicants.¹⁷ Direct reported mechanisms of A2E toxicity include, acting as an agonist for retinal pigment epithelium-specific 65-kDa protein,¹⁸ retinoic acid receptors,¹⁰ cyclooxygenase-2,¹⁹ and covalent modification of biomolecules,^{20, 21} among others.Despite data demonstrating that dimers of vitamin A can disrupt cellular hemostasis, there is less direct evidence supporting their role as primary drivers of retinal senescence. More recently, based on studies in ARPE-19 cells, it has been proposed that A2E''s chemical precursor, vitamin A aldehyde (retinaldehyde), might play a role in the degenerative process and that A2E might be a benign biomarker of increased levels of retinaldehyde.^{16, 17, 18} To determine if A2E could chemically trigger the degenerative process in the retina, we explored the acute and long-term toxicity of A2E to human retinal pigment epithelium (RPE) cells in vitro. ARPE-19 cells treated with A2E showed degraded mitochondria, accumulated glycogen and lipofuscin debris, and underwent a protracted, dose-dependent death over several days to months. These data suggest that A2E can trigger the accumulation of lipofuscin-like debris in the in vivo RPE and can be detrimental to the retina''s health. Data further reveal that increasing concentrations of A2E in the RPE potentially plays a larger role in retinal senescence than previously thought.