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）是偶发性的，没有任何常染色体遗传模式。在第一个资助期间，UVA Udall中心小组积累了大量的间接证据，证明线粒体！基因组对PD发病机制有重要作用。这些证据包括在源自PD血小板线粒体DNA的PD胞质杂交体中自发产生真正的路易体内含物。虽然之前的一个项目已经确定了线粒体DNA突变， 尽管复合物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发病机制中的因果关系的直接证据。此外，关于血小板mtDNA与脑病理学的相关性的问题继续困扰着对胞质杂种研究的解释。这些问题在本项目中得到了直接解决。该项目利用班尼特实验室最近开发的新技术，可快速取出和插入 以及将整个人线粒体基因组表达到活细胞的线粒体中。结合蛋白质转导结构域的工程化线粒体DNA结合蛋白（“原转染”）的开发允许产生表达来自人死后脑的凝胶纯化的mtDNA的细胞系。表征这些细胞系的表型将允许直接测试脑mtDNA在驱动帕金森病发病机制中的因果关系。班尼特小组还成功地改造了线粒体DNA，使其在线粒体区室中特异性地掺入点突变和外源基因的表达。这一成就，与protofection技术相结合，将允许创建突变的mtDNA物种纳入与PD脑相关的复合物I基因突变。这些突变mtDNA种类的致病性现在可以通过在细胞系中表达这些mtDNA种类来直接测试。这个项目有四个目标。目的1优化原核转染技术，以建立从PD和CTL脑中纯化的mtDNA表达的神经细胞系。将在来源的脑样品之间比较生物能量活性、氧化应激标志物和蛋白酶体功能/蛋白质水平 以及由来自那些脑样品的线粒体基因组的表达产生的细胞系。目的2将确定通过原生转染产生的神经细胞的表型，以表达同质线粒体基因组，该基因组被改变以包含项目1中发现的突变，从而特异于PD脑。目的3将研究在神经细胞系中产生的异质性，并将检验PD致病性mtDNA突变具有复制优势的假设。目的4建立线粒体基因替代治疗帕金森病的细胞模型 突变。这些目标将提供高度可解释的结果，以支持或反驳脑mtDNA参与PD发病机制，将定义特定mtDNA复合物I基因突变的致病性，并将为线粒体基因替代疗法的发展奠定基础。