Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States of America.
Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.
Biochim Biophys Acta Gen Subj. 2021 Apr;1865(4):129598. doi: 10.1016/j.bbagen.2020.129598. Epub 2020 Mar 30.
Mitochondrial function in retinal pigmented epithelial (RPE) cells and extracellular vesicle (EV) formation/release are related through the lysosomal and exocytotic pathways that process and eliminate intracellular material, including mitochondrial fragments. We propose that RPE cells with impaired mitochondria will release EVs containing mitochondrial miRNAs that reflect the diminished capacity of mitochondria within these cells.
We screened ARPE-19 cells for miRNAs that localize to the mitochondria, exhibit biological activity, and are present in EVs released by both untreated cells and cells treated with rotenone to induce mitochondrial injury. EVs were characterized by vesicle size, size distribution, presence of EV biomarkers: CD81, CD63, and syntenin-1, miRNA cargo, and number concentration of EVs released per cell.
We found that miR-494-3p was enriched in ARPE-19 mitochondria. Knockdown of miR-494-3p in ARPE-19 cells decreased ATP production and mitochondrial membrane potential in a dose-dependent manner, and decreased basal oxygen consumption rate and maximal respiratory capacity. Increased number of EVs released per cell and elevated levels of miR-494-3p in EVs released from ARPE-19 cells treated with rotenone were also measured.
ARPE-19 mitochondrial function is regulated by miR-494-3p. Elevated levels of miR-494-3p in EVs released by ARPE-19 cells indicate diminished capacity of the mitochondria within these cells.
EV miR-494-3p is a potential biomarker for RPE mitochondrial dysfunction, which plays a central role in non-neovascular age-related macular degeneration, and may be a diagnostic biomarker for monitoring the spread of degeneration to neighboring RPE cells in the retina.
视网膜色素上皮 (RPE) 细胞中的线粒体功能和细胞外囊泡 (EV) 的形成/释放是通过溶酶体和胞吐途径相关的，这些途径处理和消除包括线粒体片段在内的细胞内物质。我们假设线粒体功能受损的 RPE 细胞将释放含有线粒体 miRNA 的 EV，这些 miRNA 反映了这些细胞中线粒体功能的降低。
我们筛选了 ARPE-19 细胞中的 miRNA，这些 miRNA 定位于线粒体，具有生物活性，并且存在于未处理细胞和用鱼藤酮处理以诱导线粒体损伤的细胞释放的 EV 中。EV 通过囊泡大小、大小分布、EV 标志物：CD81、CD63 和 syntenin-1 的存在、miRNA 货物以及每个细胞释放的 EV 数量浓度来表征。
我们发现 miR-494-3p 在 ARPE-19 线粒体中富集。ARPE-19 细胞中 miR-494-3p 的敲低以剂量依赖的方式降低了 ATP 产生和线粒体膜电位，并降低了基础耗氧率和最大呼吸能力。还测量了从用鱼藤酮处理的 ARPE-19 细胞释放的 EV 中每个细胞释放的 EV 数量增加和 miR-494-3p 水平升高。
ARPE-19 线粒体功能受 miR-494-3p 调节。ARPE-19 细胞释放的 EV 中 miR-494-3p 水平升高表明这些细胞中线粒体功能降低。
EV miR-494-3p 是 RPE 线粒体功能障碍的潜在生物标志物，在非新生血管性年龄相关性黄斑变性中起核心作用，并且可能是监测变性向视网膜中相邻 RPE 细胞扩散的诊断生物标志物。
