Aberrant buildup of all-Trans-retinal dimer, a nonpyridinium bisretinoid lipofuscin fluorophore, contributes to the degeneration of the retinal pigment epithelium

Abstract

PURPOSE. Nondegradable fluorophores that accumulate as deleterious lipofuscin of RPE are involved in pathological mechanisms leading to the degeneration of RPE in AMD. A2E, a major component of RPE lipofuscin, could cause damage to RPE cells. Nevertheless, all-trans-retinal dimer (atRAL dimer) was found to be much more abundant than that of A2E in eyes of Abca4-/- Rdh8-/- double-knockout (DKO) mice, a rodent model showing the typical characteristics of retinopathies in AMD patients. Our aim was to elucidate the effect and mechanism of atRAL dimer–induced RPE degeneration. METHODS. Eyes harvested from C57BL/6J wild-type (WT) and Abca4-/- Rdh8-/- DKO mice were examined by HPLC. Cellular uptake, subcellular localization, 5-bromo-2-deoxyuridine (BrdU), Cdc25C, DNA strand breaks, mitochondrial membrane potential (DWm), and cytochrome c were analyzed by fluorescence microscopy. Cellular toxicity was assayed by lactate dehydrogenase (LDH) assay and dead cell staining. Apoptosis and cell-cycle stages were detected by flow cytometry. Furthermore, in vitro and in vivo expression of proteins associated with cell cycle and apoptosis was measured by immunoblot assays. RESULTS. All-trans-retinal dimer clearly could damage RPE cell membrane and inhibit the proliferation of RPE cells as well as induce DNA damage and cell-cycle arrest at the G2/M phase via activating the ATM/ATR-Chk2-p53 signaling pathway. Moreover, this di-retinal adduct triggered mitochondrion-associated apoptosis in RPE cells. Evidence from the cell-based experiments was also corroborated by a remarkable abnormality in expression of proteins associated with cell cycle (Cyclin B1 and Cdc2) and apoptosis (p53, Bcl-2 and Bax) in the RPE of Abca4-/- Rdh8-/- DKO mice. CONCLUSIONS. These findings suggest that atRAL dimer that accumulates beyond a critical level, facilitates age-dependent RPE degeneration.