Rescue of mt DNA-derived defects by mitochondria-tareted mRNA import and translation

通过线粒体靶向 mRNA 导入和翻译来挽救 mt DNA 衍生缺陷

• 批准号: 9180525
• 负责人: Hao Zhu
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9180525
• 关键词: Address; Animals; Anxiety; Back; Base Sequence; Basic Science; Biological Assay; Brain; Catalytic Domain; Cell Line; Cell Nucleus; Cell physiology; Cells; Complex; Control Groups; Coupled; DNA; DNA copy number; Defect; Disease; Disease model; Dyes; Electron Transport; Engineering; Fluorescence Microscopy; Functional disorder; Genes; Genome; Genus Hippocampus; Goals; Health; Holoenzymes; House mice; Human; Immunoblotting; Immunoprecipitation; In Situ; In Vitro; Individual; Intervention; Investigation; Knowledge; Label; Messenger RNA; Metabolic; Methionine; Methods; Mission; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Matrix; Mitochondrial Proteins; Modeling; Mus; Mus musculus domesticus; Mutation; Neurons; Nuclear; Oxidative Stress; Pathology; Pathway interactions; Phenotype; Physiologic pulse; Production; Proteins; Public Health; RNA; Recombinant adeno-associated virus (rAAV); Research; Respiration; Respiratory Chain; Reverse Transcriptase Polymerase Chain Reaction; Role; Single Nucleotide Polymorphism; Staining method; Stains; Transcript; Transgenic Organisms; Translating; Translational Research; Translations; Variant; Western Blotting; aging brain; aging population; behavior test; cytochrome c oxidase; disability; flexibility; gel electrophoresis; human disease; in vivo; inhibitor/antagonist; insight; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial messenger RNA; neuroblastoma cell; non-Native; novel strategies; oligomycin sensitivity-conferring protein; polypeptide; research study; success; targeted delivery; transgene expression; vector

PROJECT ABSTRACT Mitochondrial dysfunction is known to be involved in a number of human diseases. Many mitochondrial proteins are encoded by genes located in nuclear DNA (nDNA), however mitochondria possess their own genome (mtDNA) that encodes 37 genes in humans. Among them, 13 encode polypeptides that are critical subunits for electron transport chain (ETC) complex holoenzyme activity, making them essential components for mitochondrial function. Investigation into nuclear encoded mitochondrial proteins has been simplified due to an abundance of methods available for manipulating the nuclear genome. However, methods for studying the effects of mutations found in Mt-DNA are limited and indirect. Specifically, our ability to generate mtDNA “transgenic” cell lines and animals remains quite limited. This leaves a significant gap in our ability to address fundamental questions about mitochondrial contribution to disease. In an attempt to bridge this gap, our lab recently used an RNA localization sequence previously discovered by the Koehler and Teitell group to deliver mRNA of both native (mtCO2 and mtCO3) and non- native, engineered (eGFP and DNase1) mRNA to mitochondria. Our results indicate successful import and translation of these mRNA's within the mitochondrial matrix of SH-SY5Y human neuroblastoma cells. To our knowledge this is the first successful demonstration of translation of a non-native protein within mitochondria. The objective of this study is to further develop this method as a means of studying mitochondrial dysfunction by targeting nucleus-transcribed mitochondrial mRNA (mt-mRNA) transcripts to mitochondria for translation and incorporation into ETC holoenzymes. Our central hypothesis is that nucleus-transcribed, mitochondria-translated mRNA can produce functional respiratory chain proteins and will facilitate modeling and rescue of mtDNA-derived mitochondrial dysfunction. Our long-term goals are to study the functional consequences of disease-associated single nucleotide polymorphisms on mitochondrial function and brain aging in mice. (Aim 1) We will determine the efficiency and functional effects of mitochondrial-targeted delivery and translation of exogenous transcripts for incorporation into ETC holoenzymes in neuronal cells. (Aim 2) We will investigate whether mitochondrial-targeted delivery and translation of exogenous transcripts rescues mtDNA-derived mitochondrial dysfunction in mouse neurons of a mitochondrial disease model. Our proposed studies are expected to generate a novel approach, both in vitro and in vivo, to analyzing the role of mitochondrial dysfunction in brain aging that can be further utilized for basic and translational research. We believe that our approach will provide insight into mtDNA mutation pathology and offer a pathway-specific method for potential intervention.

项目摘要 已知线粒体功能障碍与许多人类疾病有关。许多线粒体 蛋白质由位于核DNA（nDNA）中的基因编码，然而线粒体具有它们自己的 人类线粒体DNA（mtDNA）编码37个基因。其中，13个编码多肽， 电子传递链（ETC）复合物全酶活性的亚基，使其成为必不可少的组分 线粒体的功能。对核编码线粒体蛋白的研究已经简化， 有大量的方法可以操纵核基因组。然而，研究方法 在Mt-DNA中发现的突变的影响是有限的和间接的。具体来说，我们产生线粒体DNA的能力 “转基因”细胞系和动物仍然相当有限。这就给我们解决以下问题的能力留下了很大的差距： 关于线粒体对疾病的贡献的基本问题。 为了弥补这一差距，我们的实验室最近使用了一个RNA定位序列， 由Koehler和Teitell小组发现，可以传递天然（mtCO 2和mtCO 3）和非天然（mtCO 3）的mRNA。 天然的、工程化的（eGFP和DNase 1）mRNA至线粒体。我们的结果表明成功导入， 这些mRNA在SH-SY 5 Y人神经母细胞瘤细胞的线粒体基质内的翻译。对我们 这是首次成功证明线粒体内非天然蛋白质的翻译。 本研究的目的是进一步发展这种方法作为研究线粒体的一种手段， 通过将核转录的线粒体mRNA（mt-mRNA）转录物靶向线粒体， 翻译和并入ETC全酶中。我们的核心假设是细胞核转录， 翻译的mRNA可以产生功能性呼吸链蛋白， 和拯救线粒体DNA衍生的线粒体功能障碍。我们的长期目标是研究 疾病相关的单核苷酸多态性对线粒体功能和大脑的影响 衰老的老鼠(Aim 1）我们将确定针对性交付的效率和功能效果 以及翻译外源转录物以掺入神经元细胞中的ETC全酶。(Aim 2）我们 将研究是否有针对性的交付和翻译外源性转录拯救 线粒体疾病模型的小鼠神经元中mtDNA衍生的线粒体功能障碍。 我们提出的研究有望产生一种新的方法，在体外和体内，分析 线粒体功能障碍在脑老化中的作用，可进一步用于基础和翻译 research.我们相信，我们的方法将提供深入了解线粒体DNA突变病理学，并提供一个新的研究方向。 潜在干预的具体途径方法。