Donaldson Kevin J, Chrenek Micah A, Boatright Jeffrey H, Nickerson John M
Department of Ophthalmology, Emory University, Atlanta, GA 30322, USA.
Atlanta Veterans Administration Center for Visual and Neurocognitive Rehabilitation, Decatur, GA 30033, USA.
Biomolecules. 2025 Jul 26;15(8):1084. doi: 10.3390/biom15081084.
The retinal pigment epithelium (RPE), a monolayer of pigmented cells, is critical for visual function through its interaction with the neural retina. In healthy eyes, RPE cells exhibit a uniform hexagonal arrangement, but under stress or disease, such as age-related macular degeneration (AMD), dysmorphic traits like cell enlargement and apparent multinucleation emerge. Multinucleation has been hypothesized to result from cellular fusion, a compensatory mechanism to maintain cell-to-cell contact and barrier function, as well as conserve resources in unhealthy tissue. However, traditional two-dimensional (2D) imaging using apical border markers alone may misrepresent multinucleation due to the lack of lateral markers. We present high-resolution confocal images enabling three-dimensional (3D) visualization of apical (ZO-1) and lateral (α-catenin) markers alongside nuclei. In two RPE damage models, we find that seemingly multinucleated cells are often single cells with displaced neighboring nuclei and lateral membranes. This emphasizes the need for 3D analyses to avoid misidentifying multinucleation and underlying fusion mechanisms. Lastly, images from the NaIO oxidative damage model reveal variability in RPE damage, with elongated, dysmorphic cells showing increased ZsGreen reporter protein expression driven by EMT-linked CAG promoter activity, while more regular RPE cells displayed somewhat reduced green signal more typical of epithelial phenotypes.
视网膜色素上皮（RPE）是一层色素细胞，通过与神经视网膜相互作用对视觉功能至关重要。在健康眼睛中，RPE细胞呈现均匀的六边形排列，但在应激或疾病状态下，如年龄相关性黄斑变性（AMD），会出现细胞增大和明显多核等畸形特征。多核现象被认为是细胞融合的结果，这是一种维持细胞间接触和屏障功能以及在不健康组织中保存资源的补偿机制。然而，仅使用顶端边界标记的传统二维（2D）成像可能会因缺乏侧向标记而误判多核现象。我们展示了高分辨率共聚焦图像，能够对顶端（ZO-1）和侧向（α-连环蛋白）标记以及细胞核进行三维（3D）可视化。在两个RPE损伤模型中，我们发现看似多核的细胞通常是单个细胞，其相邻细胞核和侧向膜发生了移位。这强调了进行3D分析以避免误判多核现象和潜在融合机制的必要性。最后，来自NaIO氧化损伤模型的图像显示RPE损伤存在变异性，形态异常的细长细胞显示由EMT相关CAG启动子活性驱动的ZsGreen报告蛋白表达增加，而更规则的RPE细胞显示出更典型上皮表型的绿色信号略有降低。
