Department of Ophthalmology, Peking University Third Hospital, Beijing, China.
Department of Ophthalmology & Clinical Centre of Optometry, Eye diseases and optometry Institute, Beijing Key Laboratory of Diagnosis and Therapy of Retinal and Choroid Diseases, College of Optometry, Peking University Health Science Center, Peking University People's Hospital, Xizhimen South Street 11, Xi Cheng District, Beijing, 100044, China.
Graefes Arch Clin Exp Ophthalmol. 2020 Oct;258(10):2251-2261. doi: 10.1007/s00417-020-04636-5. Epub 2020 Jun 7.
Best vitelliform macular dystrophy (BVMD) and autosomal recessive bestrophinopathy (ARB) are two kinds of bestrophinopathies which are caused by BEST1 mutations and characterized by accumulation of lipofuscin-like materials on the retinal pigment epithelium cell-photoreceptor interface. In the past two decades, research about the pathogenesis of bestrophinopathies was mainly focused on the anion channel and intracellular Ca signaling, but seldom concentrated on the function of retinal pigment epithelium (RPE) cells. In this study, we explored the possible effect of the three BEST1 mutations p.V143F, p.S142G, and p.A146T on the apoptosis in human fetal RPE cells.
Wild-type plasmid and mutant plasmids BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T were transfected to human fetal RPE cells. The molecules caspase-3, phospho-Bcl-2, BAX, PARP, and AIF associated with apoptosis were determined by quantitative PCR and Western blot. Apoptotic rate and active Caspase-3 staining were analyzed by flow cytometry.
Caspase-3 and PARP expression were significantly increased in BEST1-pcDNA3.1 p.S142G and p.A146T group. Flow cytometry showed that the apoptosis rates were significantly increased in the BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T group compared with the wild-type group.
For the first time, we found that the three mutations promoted RPE cell apoptosis. Furthermore, the results indicated that BEST1 mutations p.S142G and p.A146T may contribute apoptosis of RPE cells by targeting Caspase 3. Our observations suggested that the apoptosis of RPE cells may be one of the mechanisms in bestrophinopathies, which may provide a new potential therapeutic target for the treatment of this disease.
Best 型黄斑营养不良（BVMD）和常染色体隐性 Bestrophinopathy（ARB）是两种由 BEST1 基因突变引起的 Bestrophinopathy，其特征是视网膜色素上皮细胞-光感受器界面上脂褐素样物质的积累。在过去的二十年中，Bestrophinopathy 发病机制的研究主要集中在阴离子通道和细胞内 Ca 信号转导上，但很少集中在视网膜色素上皮（RPE）细胞的功能上。在这项研究中，我们探讨了三种 BEST1 突变 p.V143F、p.S142G 和 p.A146T 对人胎儿 RPE 细胞凋亡的可能影响。
将野生型质粒和突变型质粒 BEST1-pcDNA3.1 p.V143F、p.S142G 和 p.A146T 转染到人胎儿 RPE 细胞中。通过定量 PCR 和 Western blot 测定与凋亡相关的分子 caspase-3、磷酸化 Bcl-2、BAX、PARP 和 AIF。通过流式细胞术分析细胞凋亡率和活性 Caspase-3 染色。
BEST1-pcDNA3.1 p.S142G 和 p.A146T 组 caspase-3 和 PARP 的表达明显增加。流式细胞术显示，与野生型组相比，BEST1-pcDNA3.1 p.V143F、p.S142G 和 p.A146T 组的凋亡率明显增加。
我们首次发现这三种突变促进了 RPE 细胞凋亡。此外，结果表明，BEST1 突变 p.S142G 和 p.A146T 可能通过靶向 Caspase 3 导致 RPE 细胞凋亡。我们的观察结果表明，RPE 细胞凋亡可能是 Bestrophinopathy 的机制之一，这可能为治疗这种疾病提供新的潜在治疗靶点。
