Ke Yao

Involvement of endoplasmic reticulum stress in all-trans-retinal-induced retinal pigment epithelium degeneration.

Excess accumulation of endogenous all-trans-retinal (atRAL) contributes to degeneration of the retinal pigment epithelium (RPE) and photoreceptor cells, and plays a role in the etiologies of age-related macular degeneration (AMD) and Stargardts disease. In this study, we reveal that human RPE cells tolerate exposure of up to 5u2009µM atRAL without deleterious effects, but higher concentrations are detrimental and induce cell apoptosis. atRAL treatment significantly increased production of intracellular reactive oxygen species (ROS) and up-regulated mRNA expression of Nrf2, HO-1, and γ-GCSh within RPE cells, thereby causing oxidative stress. ROS localized to 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-eIF2α-ATF4 signaling pathway was activated. Expression levels of ATF4, CHOP, and GADD34 in RPE cells increased in a concentration-dependent manner after incubation with atRAL. Salubrinal, a selective inhibitor of ER stress, alleviated atRAL-induced cell death. The antioxidant N-acetylcysteine (NAC) effectively blocked RPE cell loss 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 demonstrate that atRAL-driven ROS overproduction-induced ER stress is involved in cellular mitochondrial dysfunction and apoptosis of RPE cells.

Structures and biogenetic analysis of lipofuscin bis-retinoids

Age-related macular degeneration (AMD) is still an incurable blinding eye disease because of complex pathogenic mechanisms and unusual diseased regions. With the use of chemical biology tools, great progress has been achieved in improving the understanding of AMD pathogenesis. The severity of AMD is, at least in part, linked to the non-degradable lipofuscin bis-retinoids in retinal pigment epithelial (RPE). This material is thought to result from the lifelong accumulation of lysosomal residual bodies containing the end products derived from the daily phagocytosis of rod outer segments by RPE cells. Here, we present previously recognized bis-retinoids with focus on structures and biosynthetic pathways. In addition to a brief discussion on the mutual conversion relationships of bis-retinoids, future perspectives and the medical relevance of such studies on these lipofuscin constituents are also highlighted.

Identification of a Novel Lipofuscin Pigment (iisoA2E) in Retina and Its Effects in the Retinal Pigment Epithelial Cells

Background: Macular degeneration implicates lipofuscin deposition in the retina. Results: Bisretinoid iisoA2E in the retina was characterized; excessive accumulation of iisoA2E was cytotoxic to retinal pigment epithelial cells. Conclusion: Pyridinium iisoA2E is a unique diretinal adduct and serves as a fluorescent biomarker of aberrant all-trans-retinal metabolism. Significance: Characterization of iisoA2E gives a more complete understanding of the biosynthesis of retinal bisretinoid lipofuscin. Lipofuscin accumulation in retinal pigment epithelial (RPE) cells of the eye implicates the etiologies of Stargardt disease and age-related macular degeneration, a leading cause of blindness in the elderly. 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 is a new type of pyridinium bisretinoid presenting an unusual structure, in which two polyenic side chains are 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 leads to a significant loss of cell viability and caused membrane damage. Phospholipase D-mediated phosphodiester cleavage of the A2PE series generated isoA2E and iisoA2E, in addition to A2E, thus corroborating the presence of isoA2PE and iisoA2PE that may serve as biosynthetic precursors of isoA2E and iisoA2E.

Retinal metabolism in humans induces the formation of an unprecedented lipofuscin fluorophore 'pdA2E'.

Toxic lipofuscin in the RPE (retinal pigment epithelium) is implicated in blindness in AMD (age-related macular degeneration) or recessive Stargardts disease patients. In the present study, we identified a novel fluorescent lipofuscin component in human and bovine RPEs. Using 1D and 2D NMR and MS, we confirmed the structure of this pigment and called it pdA2E. It exhibits absorbance maxima at 492 and 342 nm, and is susceptible to photocatalytic isomerization and oxidation. This fluorophore was also detected in the eyecup extracts of Abca4(-/-)Rdh8(-/-) (Abca4 encodes ATP-binding cassette transporter 4 and Rdh8 encodes retinol dehydrogenase 8) mice, an AMD/recessive Stargardts disease model. Excess amassing of pdA2E within RPE cells caused significant cell viability loss and membrane damage. The formation of pdA2E occurred when atRAL (all-trans-retinal) reacted with excess ethanolamine in the absence of acetic acid, and the process is likely to involve the participation of three atRAL molecules. Our findings suggest that endogenous pdA2E may serve as a sensitizer for yielding singlet oxygen and a singlet oxygen quencher, as well as a by-product of retinal metabolism, and its complete characterization facilitates the understanding of biosynthetic pathways by which adverse RPE lipofuscin constituents form.

Potential Therapeutic Agents Against Retinal Diseases Caused by Aberrant Metabolism of Retinoids.

The retinoid (visual) cycle is a complex enzymatic pathway that operates in the retina for the regeneration of 11-cis-retinal (11-cis-Ral), the inherent visual chromophore indispensable for vision. Deficiencies in the retinoid metabolism are involved in pathologic mechanisms of several forms of retinal diseases including age-related macular degeneration, Stargardts disease, and Lebers congenital amaurosis, for which no effective cures presently exist. Nevertheless, the interference of abnormal retinoid metabolism with chemicals has been considered to be a promising strategy aimed at alleviating these retinal dysfunctions. Moreover, since gene therapy is gaining increasing importance in clinical practice, the modulation of key enzymes implicated with the retinoid cycle at a genetic level will hold great promise for the treatment of patients with degenerative diseases of the retina.

Preparative and Biosynthetic Insights Into pdA2E and isopdA2E, Retinal-Derived Fluorophores of Retinal Pigment Epithelial Lipofuscin

PURPOSEnRetinal-derived fluorophores that accumulate as RPE lipofuscin are implicated in pathological mechanisms of AMD. One component of RPE lipofuscin has been characterized as pdA2E, a pyridinium adduct derived from all-trans-retinal and excess ethanolamine. One-step preparation and biosynthetic studies of pdA2E and its novel isomer called isopdA2E are reported.nnnMETHODSnBiosynthetic reaction mixtures, RPE/choroids and neural retinas dissected from bovines, eyes harvested from Abca4(-/-)Rdh8(-/-) mice, irradiated samples, and enzyme-treated solutions were analyzed by HPLC, mass spectrometry, nuclear magnetic resonance spectroscopy, fluorescence spectrophotometry, and density functional theory (DFT).nnnRESULTSnOptimization of the in vitro synthesis of pdA2E resulted in a biomimetic preparation of this pigment in a yield of 15%; this protocol also allowed the identification of isopdA2E, a double-bond isomer of pdA2E at the C13C14 position in bovine RPE lipofuscin. Interconversion between these two molecules occurs when either pdA2E or isopdA2E is exposed to light. A phospholipase D-based assay demonstrated the possibility of pdA2-PE being formed in neural retina and served as a precursor of pdA2E in the biosynthetic pathway. DFT calculations revealed that the 492-nm absorbance was assigned to the long arm of pdA2E/isopdA2E and the 340/342-nm absorbance to the short arm. Fluorescence efficiency of pdA2E and isopdA2E is very similar, but is much weaker in comparison with A2E, isoA2E, and iisoA2E.nnnCONCLUSIONSnOur results facilitate the understanding of compositions and biosynthetic pathways of adverse RPE lipofuscin.

Effects of organic solvents on two retinal pigment epithelial lipofuscin fluorophores, A2E and all-trans-retinal dimer

Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt’s disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinyl-ethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II–IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II–IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.