Mechanism Of All-trans-retinal-induced Cytotoxicity In Retinal Pigment Epithelial Cells And Identification Of A Novel Lipofuscin Pigment (iisoA2E) In Retina

Age-related macular degeneration (AMD) is a major cause of vision loss worldwide. It is a progressive chronic disease of the central retina that mostly affects old adults. The pathogenesis of this disease is currently unclear. Thus, elucidating the mechanism of AMD is of increasing importance. A large number of recent studies have indicated that aberrant ocular retinoid metabolism is involved in the development of macular degeneration. Excess accumulation of all-trans-retinal (atRAL) induces severe degeneration in the retina, but the molecular mechanism remains to be further clarified. Given that the retinal pigment epithelial (RPE) cells are important to maintain the normal function of the retina, we employed a human RPE cell line, the ARPE-19 cell, to clarify the mechanism of cytotoxicity of atRAL and to explore its metabolic pathway in RPE cells.The role of cumulative fundus lipofuscin pigments in AMD pathogenesis is controversial. A large number of studies indicated that these products have certain biological activities, and they may be critical in the pathogenesis of retinopathy. The lipofuscin pigment compositions are extremely complex. Thus our understanding of these pigments depends largely on their isolation, biomimetic synthesis, structural analysis and bioactivity test. In the last part of this study, we identified a previously unknown RPE lipofuscin component. We also studied its properties, bioactivities and biosynthetic pathways.1. Involvement of endoplasmic reticulum stress in all-trans-retinal-induced retinal pigment epithelium degenerationExcess accumulation of endogenous all-trans-retinal (atRAL) contributes to the degeneration of the RPE and photoreceptor cells, and plays a role in the etiologies of AMD and Stargardt’s Disease. In this study, we revealed that human RPE cells tolerate exposure of up to 5μM of atRAL without deleterious effects, but higher concentrations were detrimental and induced cell apoptosis. atRAL treatment significantly increase production of intracellular reactive oxygen species (ROS) and up-regulated the mRNA expression of Nrf2, HO-1, and y-GCSh within RPE cells, thereby causing oxidative stress. ROS was localized in mitochondria and endoplasmic reticulum (ER). ER resident molecular chaperone BiP, a marker of ER stress, was up-regulated at the translational level, and meanwhile, the PERK-eIF2a-ATF4 signaling pathway was activated. Expression levels of ATF4, CHOP and GADD34 were increased in a concentration-dependent manner after incubation with atRAL. Salubrinal,a selective inhibitor of ER stress, alleviated the atRAL-related cell death. The antioxidant N-acetylcysteine (NAC) effectively blocked RPE cell losses and ER stress activation, suggesting that atRAL-induced ROS generation is responsible for RPE degeneration and is an early trigger of ER stress. Furthermore, the mitochondrial transmembrane potential was lost after atRAL exposure, and was followed by caspase-3 activation and poly-(ADP-ribose) polymerase cleavage. The results demonstrated that ER stress induced by atRAL-driven ROS overproduction was involved in cellular mitochondrial dysfunction and apoptosis of RPE cells.2. Preliminary studies on the metabolism of all-trans-retinal in human retinal pigment epithelial cells and role of all-trans-retinal dimer formationEffective clearance of atRAL from RPE cells is important for avoiding its cytotoxicity. However, the metabolism of atRAL in RPE cells is poorly clarified. In this section, we found that after incubation with RPE cells for 6 h, atRAL could be metabolized into all-trans-retinol (atROL) and atRAL condensation products, such as all-trans-retinal dimer (atRAL-dimer) and A2E. Moreover, we found that the content of atRAL-dimer was more than that of A2E in RPE cells. In RDH8-/-ABCA4-/- mice, the model of AMD, atRAL-dimer was increased relative to wild type and was far more abundant than the build-up of A2E. For comparing the cytotoxicity of atRAL and atRAL-dimer in RPE cells, we assayed their impacts on RPE cellular viability, ΔΨm, intracellular ROS production and the expression levels of oxidative stress related genes (Nrf2, HO-1, and y-GCSh). We found that atRAL-dimer was safer than atRAL. Our work indicated that the formation of atRAL-dimer could ameliorate the cytotoxicity induced by aberrant accumulation of atRAL in retina.3. Identification of a novel lipofuscin pigment iisoA2E in retina and its effects in the retinal pigment epithelial cellsLipofuscin accumulation in RPE cells of the eye may implicate the etiologies of Stargardt’s Disease and AMD. Here we have identified a previously unknown RPE lipofuscin component. By one-and two-dimensional NMR techniques and mass spectrometry, we confirmed that this compound was a new type of pyridinium bisretinoid presenting an unusual structure, in which two polyenic side-chains were attached to adjacent carbons of a pyridinium ring. This pigment is a light-induced isomer of isoA2E, rather than A2E, referred to as iisoA2E. This pigment is a fluorescent lipofuscin compound with absorbance maxima at-430 and 352 nm detected in human, pig, mouse and bovine eyes. Formation of iisoA2E was found in reaction mixtures of all-trans-retinal and ethanolamine. Excess intracellular accumulation of this adduct in RPE cells in vitro led to a significant loss of cell viability and caused membrane damage. Phospholipase D-mediated phosphodiester cleavage of A2PE series generated isoA2E and iisoA2E, in addition to A2E, thus corroborating the presence of isoA2PE and iisoA2PE that may serve as the precursors of isoA2E and iisoA2E.