Aberrant Buildup of All-Trans-Retinal Dimer, a Nonpyridinium Bisretinoid Lipofuscin Fluorophore, Contributes to the Degeneration of the Retinal Pigment Epithelium.
Aberrant Buildup of All-Trans-Retinal Dimer, a Nonpyridinium Bisretinoid Lipofuscin Fluorophore, Contributes to the Degeneration of the Retinal Pigment Epithelium.
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DOI：
10.1167/iovs.16-20734
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发表时间：
2017-02
影响因子：
4.4
通讯作者：
Junli Zhao;Yi Liao;Jing-meng Chen;Xinran Dong;Zhan Gao;Houjian Zhang;Xiaodan Wu;Zuguo Liu;Yalin Wu
中科院分区：
文献类型：
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作者：
Junli Zhao;Yi Liao;Jing-meng Chen;Xinran Dong;Zhan Gao;Houjian Zhang;Xiaodan Wu;Zuguo Liu;Yalin Wu
不可降解的荧光团作为有害的脂褐素在RPE中积累，参与了导致AMD RPE变性的病理机制。A2E是RPE脂褐素的主要成分，可引起RPE细胞损伤。然而，在Abca4-/- rdh8 -/-双敲除（DKO）小鼠的眼睛中，发现全反式视网膜二聚体（atRAL二聚体）比A2E丰富得多，这是一种显示AMD患者视网膜病变典型特征的啮齿动物模型。我们的目的是阐明天然二聚体诱导的RPE变性的作用和机制。方法采用高效液相色谱法检测C57BL/6J野生型（WT）和Abca4-/- rdh8 -/- DKO小鼠的眼睛。荧光显微镜分析细胞摄取、亚细胞定位、5-溴-2-脱氧尿苷（BrdU）、Cdc25C、DNA链断裂、线粒体膜电位（ΔΨm）和细胞色素c。采用乳酸脱氢酶（LDH）法和死细胞染色法检测细胞毒性。流式细胞术检测细胞凋亡和细胞周期分期。此外，通过免疫印迹法检测与细胞周期和凋亡相关的蛋白在体外和体内的表达。结果全反式视网膜二聚体通过激活ATM/ATR-Chk2-p53信号通路，明显损伤RPE细胞膜，抑制RPE细胞增殖，诱导DNA损伤和细胞周期阻滞在G2/M期。此外，这种双视网膜加合物引发了RPE细胞中线粒体相关的凋亡。基于细胞的实验也证实了Abca4-/- rdh8 -/- DKO小鼠RPE中与细胞周期相关的蛋白（Cyclin B1和Cdc2）和凋亡相关的蛋白（p53， Bcl-2和Bax）表达的显著异常。这些发现表明，atRAL二聚体积累超过临界水平，促进年龄依赖性RPE变性。
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 (ΔΨm), 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.