Manipulating the Mitochondrial Genome in PD

操纵帕金森病中的线粒体基因组

• 批准号: 6962406
• 负责人: JAMES PEPPER BENNETT
• 金额: $ 35.82万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6962406
• 关键词: DNA binding protein; Parkinson' s disease; biotechnology; cell line; disease /disorder etiology; disease /disorder model; gene mutation; genetic manipulation; genetic models; genetic susceptibility; genome; human genetic material tag; hybrid cells; mitochondrial DNA; mitochondrial disease /disorder; molecular pathology; pathologic process; phenotype

The majority of Parkinson's disease (PD) occurs sporadically without any autosomal inheritance patterns. During the first funding period, the UVA Udall Center group accumulated substantial circumstantial evidence that mitochondria! genomes contribute significantly to PD pathogenesis. This evidence included spontaneous production of true Lewy body inclusions in PD cybrids derived from PD platelet mtDNA. Although a previous project has identified mtDNA mutations in complex I genes that segregate in and are predictive of PD brain, direct proof of causality of PD brain mtDNA mutations in driving PD pathogenesis is lacking. In addition, questions about the relevance of platelet mtDNA to brain pathology continue to plague interpretation of cybrid studies. These problems are directly addressed in this project. This Project utilizes novel technologies developed recently in the Bennett lab that allow rapid removal of, and insertion and expression of the entire human mitochondrial genome into mitochondria of living cells. The development of an engineered mitochondrial DNA-binding protein incorporating a protein transduction domain ("protofection") allows the creation of cell lines expressing gel-purified mtDNA from human postmortem brain. Characterizing the phenotypes of these cell lines will allow a direct test of causality of brain mtDNA in driving the pathogenesis of Parkinson's disease. The Bennett group has also successfully engineered mtDNA to incorporate point mutations and expression of exogenous genes specifically in the mitochondrial compartment. This achievement, in combination with protofection technology, will allow the creation of mutated mtDNA species incorporating complex I gene mutations associated with PD brain. The pathogenecity of these mutant mtDNA species can now be tested directly by expressing these mtDNA species in cell lines. There are four Aims in this Project. Aim 1 will optimize protofection technology in order to create neural cell lines from expression of mtDNA purified from PD and CTL brains classified according to Braak staging. Bioenergetic activity, oxidative stress markers and proteasomal function/protein levels will be compared between the brain samples of origin and the cell lines resulting from expression of mitochondrial genomes derived from those brain samples. Aim 2 will determine phenotypes of neural cells created by protofection to express homoplasmically mitochondrial genomes altered to contain mutations discovered in Project 1 to be specific to PD brain. Aim 3 will study heteroplasmy that is created in neural cell lines and will test the hypothesis that PD pathogenic mtDNA mutations have a replicative advantage. Aim 4 will develop cell models for mitochondrial gene replacement therapy of pathogenic PD mtDNA mutations. These Aims will provide highly interpretable outcomes to support or refute the involvement of brain mtDNA in PD pathogenesis, will define pathogenecity of specific mtDNA complex I gene mutations and will set the stage for development of mitochondrial gene replacement therapy.

大多数帕金森病（PD）是散发性的，没有任何常染色体遗传模式。在第一个资助期间，UVA 尤德尔中心小组积累了大量间接证据，证明线粒体！基因组对 PD 发病机制有显着贡献。该证据包括源自 PD 血小板 mtDNA 的 PD 细胞杂种中自发产生真正的路易体内含物。尽管之前的一个项目已经鉴定出 mtDNA 突变 虽然复杂 I 基因在 PD 脑中分离并预测 PD 脑，但缺乏 PD 脑 mtDNA 突变在驱动 PD 发病机制中因果关系的直接证据。此外，关于血小板 mtDNA 与脑病理学相关性的问题继续困扰着 cybrid 研究的解释。这些问题都在本项目中得到直接解决。该项目利用贝内特实验室最近开发的新技术，可以快速移除和插入 以及将整个人类线粒体基因组表达到活细胞的线粒体中。包含蛋白质转导结构域（“原转染”）的工程线粒体 DNA 结合蛋白的开发允许创建表达来自人类死后大脑的凝胶纯化 mtDNA 的细胞系。表征这些细胞系的表型将能够直接测试大脑 mtDNA 在驱动帕金森病发病机制中的因果关系。 Bennett 小组还成功地改造了 mtDNA，将点突变和外源基因的表达（特别是在线粒体区室中）结合起来。这一成就与原转染技术相结合，将允许创建包含与 PD 脑相关的复杂 I 基因突变的突变 mtDNA 物种。现在可以通过在细胞系中表达这些 mtDNA 物种来直接测试这些突变 mtDNA 物种的致病性。该项目有四个目标。目标 1 将优化原转染技术，以便通过从根据 Braak 分期分类的 PD 和 CTL 大脑中纯化的 mtDNA 表达来创建神经细胞系。将比较原始脑样本之间的生物能活性、氧化应激标记物和蛋白酶体功能/蛋白质水平 以及源自这些大脑样本的线粒体基因组表达产生的细胞系。目标 2 将确定通过原转染创建的神经细胞的表型，这些神经细胞表达同质线粒体基因组，这些基因组被改变以包含项目 1 中发现的 PD 脑特异性突变。目标 3 将研究神经细胞系中产生的异质性，并检验 PD 致病性 mtDNA 突变具有复制优势的假设。目标 4 将开发用于致病性 PD mtDNA 线粒体基因替代疗法的细胞模型 突变。这些目标将提供高度可解释的结果来支持或反驳大脑 mtDNA 参与 PD 发病机制，将定义特定 mtDNA 复合体 I 基因突变的致病性，并为线粒体基因替代疗法的发展奠定基础。