AMD Mitochondria Modulate Expression of microRNA 135b-5p and 148a-3p in RPE Cybrids: Implications for Age-related Macular Degeneration

项目总结/摘要 视网膜相关性黄斑变性（AMD）是发达国家失明的主要原因， 大量研究致力于探索病因和潜在的治疗方法。而线粒体 已经发现，对于AMD的发展至关重要，但对确切的途径知之甚少 线粒体通过其影响核基因表达和疾病进展。我们已经开发出一种 一种新的线粒体胞质杂交模型，使我们能够产生视网膜色素上皮细胞系， 细胞核，但线粒体来自不同的AMD和年龄匹配的正常受试者。利用这些赛伯人， 实验室已经发现AMD线粒体的引入引起细胞凋亡、氧化应激 降低细胞活力。目前，我们正在探索AMD患者的线粒体是如何引起这些变化的。 胞质杂种细胞系的巨大变化，以及如何调节这种影响以改善细胞健康。一 我们研究的潜在途径之一是microRNA的调节。我们初步的赛伯数据 显示AMD线粒体的引入导致7种具有功能的microRNA的表达改变， 与AMD病理学相关。随后的工作集中在靶向下调两个过度表达的 存在于AMD胞质杂交体中的microRNA（miRNA 135 b-5 p和miRNA 148 a-3 p）。下调miRNA 135 b- 5 p导致与凋亡和血管生成相关的基因表达降低，而下调 miRNA 148 a-3 p的表达导致与线粒体生物发生相关的基因表达水平增加， 减少活性氧的产生。这些发现证明了microRNA的有效性。 调节作为改善细胞健康和潜在治疗的方法。在此基础上， 根据初步数据，我们对这项资助的中心假设是，AMD线粒体细胞（a）表现出改变， microRNA表达和（B）通过靶向抑制调节这些失调的microRNA水平 或过表达将影响基因表达、细胞健康和血管生成的体外模型。进一步 理解microRNA在AMD模型中的作用对各种其他研究具有重大的潜在影响。 线粒体功能障碍的疾病，包括阿尔茨海默病和帕金森病。

Project Summary/Abstract Age-related Macular Degeneration (AMD) represents a major cause of blindness in the developed world, and significant research has been devoted to exploring the causes and potential treatments. While the mitochondria have been found to be of critical importance to the development of AMD, less is known about the exact pathways through which mitochondria influence nuclear gene expression and disease progression. We have developed a novel transmitochondrial cybrid model that allows us to generate retinal pigment epithelial cell lines with identical nuclei, but with mitochondria from different AMD and age-matched normal subjects. Using these cybrids, our laboratory has discovered that introduction of AMD mitochondria causes cellular apoptosis, oxidative stress and decreased cell viability. Presently, we are exploring how the mitochondria of AMD patients could cause these dramatic changes in cybrid cell lines, and how this influence could be modulated to improve cellular health. One of the potential pathways that we have investigated is the regulation of microRNA. Our preliminary cybrid data show that introduction of AMD mitochondria causes altered expression of seven microRNA with functions relevant to AMD pathology. Subsequent work has focused on the targeted downregulation of two overexpressed microRNA present in the AMD cybrids (miRNA 135b-5p and miRNA 148a-3p). Downregulation of miRNA 135b- 5p leads to decreased expression of genes associated with apoptosis and angiogenesis, while downregulation of miRNA 148a-3p results in increased expression levels of genes associated with mitochondrial biogenesis and decreased reactive oxygen species production. These findings demonstrate the effectiveness of microRNA modulation as a method for improvement of cellular health and potential treatment. Building upon these preliminary data, our central hypotheses for this grant are that cells with AMD mitochondria (a) exhibit altered microRNA expression and that (b) modulation of these dysregulated microRNA levels through targeted inhibition or overexpression will influence gene expression, cellular health and in vitro models of angiogenesis. Further understanding of the role of microRNA in the AMD model has a significant potential impact for a variety of other diseases with mitochondrial dysfunction, including Alzheimer’s and Parkinson’s diseases.