Manipulating the Mitochondrial Genome in PD

操纵帕金森病中的线粒体基因组

• 批准号: 7312789
• 负责人: JAMES PEPPER BENNETT
• 金额: $ 36.91万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7312789
• 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.

帕金森氏病（PD）的大多数发生，没有任何常染色体遗传模式。在第一个资金期间，UVA Udall Center组积累了大量的间接证据，即线粒体！基因组对PD发病机理有显着贡献。该证据包括自发产生来自PD血小板mtDNA的PD cybrid中的真正的Lewy体内夹杂物。尽管先前的项目已经确定了mtDNA突变 缺乏在驱动PD发病机理中PD脑MTDNA突变因果关系的直接证明，可以直接证明PD脑的直接证明。此外，有关血小板mtDNA与脑病理学相关性的问题继续困扰着cybrid研究的解释。这些问题在此项目中直接解决。该项目利用了最近在Bennett实验室开发的新技术，该技术允许快速删除和插入 整个人线粒体基因组的表达在活细胞的线粒体中。结合蛋白质转导结构域（“原子能”）的工程线粒体DNA结合蛋白的开发允许从人后大脑中产生表达凝胶纯化的mtDNA的细胞系。表征这些细胞系的表型将允许直接检验脑mtDNA因果关系，以驱动帕金森氏病的发病机理。贝内特组还成功地设计了mtDNA，以在线粒体区室中专门纳入了点突变和外源基因的表达。该成就与原始技术结合使用，将允许创建与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物种的病原体。这个项目有四个目标。 AIM 1将优化原始技术，以创建从根据Braak分期分类的PD和CTL大脑纯化的mtDNA表达的神经细胞系。生物能活性，氧化应激标志物和蛋白酶体功能/蛋白质水平将在起源的大脑样本之间进行比较 以及由这些大脑样品得出的线粒体基因组表达产生的细胞系。 AIM 2将确定由原子型产生的神经细胞的表型，以表达同质的线粒体基因组，而线粒体基因组改变了，以包含项目1中针对PD脑的突变。 AIM 3将研究在神经细胞系中产生的异质，并将检验以下假设：PD致病mtDNA突变具有复制性优势。 AIM 4将开发用于致病性PD MTDNA的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，以支持或反驳脑MtDNA参与PD发病机理，将定义特定mtDNA复合物I基因突变的病原体，并为线粒体基因置换疗法的发展奠定了基础。