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

项目摘要/摘要 老年性黄斑变性(AMD)是发达国家致盲的一个主要原因， 已有大量研究致力于探索其原因和可能的治疗方法。而线粒体 已发现对AMD的发展至关重要，但对其确切途径知之甚少 线粒体通过它影响核基因的表达和疾病的进展。我们已经开发出一种 一种新的传递线粒体胞质模型，允许我们产生具有相同基因的视网膜色素上皮细胞系 细胞核，但线粒体来自不同AMD和年龄匹配的正常受试者。使用这些网络病毒，我们的 实验室发现，AMD线粒体的引入会导致细胞凋亡、氧化应激和 细胞存活率下降。目前，我们正在探索AMD患者的线粒体如何导致这些 胞质细胞系的戏剧性变化，以及这种影响如何被调节以改善细胞健康。一 我们研究的潜在途径之一是对microRNA的调节。我们初步的赛博德数据 表明AMD线粒体的引入会导致七个具有功能的microRNA的表达发生变化 与AMD病理相关。随后的工作重点是有针对性地下调两个过度表达的基因 在AMD胞质中存在microRNA(miRNA135B-5P和miRNA148A-3P)。下调miRNA 135B- 5P导致细胞凋亡和血管生成相关基因表达减少，同时下调 MiRNA148A-3P的表达导致与线粒体生物发生相关的基因表达水平增加 减少了活性氧的产生。这些发现证明了microRNA的有效性。 调制作为一种改善细胞健康和潜在治疗方法的方法。建立在这些基础上 初步数据，我们对这项资助的中心假设是，带有AMD线粒体(A)的细胞表现出改变 MicroRNA表达和(B)通过靶向抑制调节这些失调的microRNA水平 或过度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 了解microRNA在AMD模型中的作用对其他多种疾病具有重要的潜在影响 线粒体功能障碍的疾病，包括阿尔茨海默氏症和帕金森氏症。

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.