Mitochondrial DNA inheritance in Drosophila

果蝇线粒体DNA遗传

• 批准号: 8558085
• 负责人: Hong Xu
• 金额: $ 144.77万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/8558085
• 关键词: Amino Acid Substitution; Animal Model; Autophagocytosis; Bacteria; Bacterial Proteins; Biolistics; Biological; Biological Assay; Cell Survival; Cells; Chloramphenicol Resistance; Cytochromes; DNA; DNA Restriction Enzymes; DNA biosynthesis; Defect; Disease; Drosophila genus; Engineering; Ensure; Etiology; Female; Fibroblasts; Frequencies; Functional disorder; Future; Galactose; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Goals; Hereditary Disease; High temperature of physical object; Human; Image; In Vitro; Individual; Length; Life; Longevity; Lysosomes; Mammalian Cell; Mammals; Measures; Methods; Mitochondria; Mitochondrial DNA; Modeling; Monitor; Mothers; Mus; Muscle; Mutagenesis; Mutation; Neurologic; Nucleic Acids; Oocytes; Oogenesis; Organelles; Organism; Ovary; Oxidases; Oxidation-Reduction; Pathogenesis; Pathway interactions; Phenotype; Physiological; Plants; Plasmids; Process; Protein Denaturation; Proteins; Pupa; Quality Control; Reactive Oxygen Species; Recruitment Activity; Resistance; Scheme; Staging; Staining method; Stains; Stress-Induced Protein; Structure; Temperature; Testing; Time; Tissues; Variant; Work; Yeasts; age related; animal tissue; base; cold temperature; cytochrome c oxidase; fitness; fly; glutathione peroxidase; mitochondrial DNA mutation; mitochondrial genome; mutant; novel; parkin gene/protein; porin; prevent; purge; relating to nervous system; restriction enzyme; rho; segregation; tool; transmission process

Project 1: Genetic method for selective elimination of damaged mitochondria Mitochondrial turnover has been postulated as a mechanism for mitochondrial quality control. However, it remains a question whether cells are indeed able to eliminate defective mitochondria selectively. Quantitative and live imaging assays are required to measure selective mitochondrial degradation and visualize this process in real time, while a genetic approach is essential to probe mitochondrial turnover in a physiological context. We expressed a toxic bacterial protein, PorB, to damage a subpopulation of total cellular mitochondria in cultured Drosophila cells and tissues. Damaged mitochondria concentrated with PorB were segregated from the mitochondrial network through a fission/fusion process and selectively removed by lysosomes through the autophagy pathway in otherwise healthy cells. We demonstrated for the first time the Parkin-dependent degradation of damaged mitochondria in an animal tissue, the Drosophila flight muscle. Our work proves in principle that defective mitochondria are selectively removed in healthy cells, and also provides a novel genetic approach to monitor mitochondrial turnover and dissect the underlying mechanisms. Project 2: Selective transmission of healthy mtDNA during Drosophila oogenesis One of the most prominent questions regarding mtDNA inheritance is how mothers ensure the transmission of healthy mitochondria to their progeny. Recent studies of mtDNA mutator mice show that mtDNA mutations are purged from germline cells, even though their levels in the cells are too low to impair overall cellular fitness. The most plausible explanation for this phenomenon is that mtDNA mutations can be selected against on an organelles level based on the functionality of an individual mitochondrion. To examine this question, we turned to Drosophila oogenesis to explore the mechanisms of mitochondrial DNA transmission. We generated heteroplasmic fly lines that contain both wild type and mt:Co1T300I mtDNAs. mt:Co1T300I is a temperature sensitive mutation in the mtDNA gene cytochrome c oxidase 1 (mt:Co1). Homoplasmic mt:Co1T300I flies cannot survive at high temperature, while is mostly healthy at low temperature. We found that the frequency of mutant mtDNA in the heteroplasmic flies increased in the progeny of female flies shifted from 18C to 29C, indicating a direct selection against defective mitochondria. Cell biological analysis revealed that mtDNA replication occurs at a specialized structure in the germarium, known as the fusome, and disruption of this structure leads to a decrease in mtDNA replication early in oogenesis. Homoplasmic mt:Co1T300I flies contain an intact fusome, but display disruption of mtDNA replication in the germarium. Further, we expressed a bacterial porin protein to damage a subset of mitochondria in ovary. We observed segregation of damaged mitochondria away from the future oocyte during early and mid-stage oogenesis. Our results demonstrate that healthy mitochondria are selectively recruited to the fusome, where mtDNAs are preferentially replicated. The selective amplification of healthy mitochondria that containing wild type mtDNA will reciprocally reduce the frequency of mtDNA in ovary and their opportunity of transmission. Future studies will seek to determine the machinery involved in recognition and selection of healthy mitochondria during oogenesis. Project 3: A Drosophila model reveals novel pathogenic mechanism of mtDNA mutation. Applying a selection scheme based on mitochondrially targeted restriction enzymes, we isolated a homoplasmic mitochondria DNA mutant, mt:CoIT300I that carrying a single amino acid substitution on cytochrome C oxidase (COX) subunit I (CoI) locus. The mt:CoIT300I flies had reduced COX activity and decreased ATP levels. The mt:CoIT300I flies are temperature sensitivities. Very few survived through the pupa stage at 29 C. In addition, mt:CoIT300I flies displayed greatly reduced life span as well as impaired mobility and neural activities at permissive temperature. The defects are exacerbated in old flies, which indicates an age-dependent neurological and muscular dysfunction. Most of the phenotypes resembled typical features of human mtDNA diseases, validating mt:CoIT300I as a Drosophila model to understand the conserved features of mtDNA mutations. Expression analyses revealed handful genes that are involved in maintaining cellular redox potential and protecting against stress induced protein denaturation, are up regulated in the mutant background. Over expression of glutathione peroxidase in mt:CoIT300I flies can partially suppress the phenotype, further confirming the idea that deregulation of cellular redox potential is one of the mitochondria dysfunctions and contribute to cellular deficiencies downstream. In contrast to the common hypothesis that reactive oxygen species are one of the major players in pathogenesis induced by mitochondrial deficiencies, we found not evidence of involvement of ROS in the process in mt:CoIT300I flies. We results suggest a novel pathway of mitochondrial etiology, and provide a genetic handle to further delineate the whole process. Since most mtDNA diseases shows tissue-to-tissue variation in extent and phenotypes, we established a genetic scheme to make homoplasmic mtDNA mutation in tissue specific manner in Drosophila for a better modeling of human mtDNA diseases in future. Project 4: Targeted mutagenesis of mammalian mtDNA through direct transformation of engineered mtDNA The ability to induce specific mutations into the mammalian mitochondrial genome would facilitate studies into human mitochondrial genetics and genetic disorders. Until now, the delivery to and expression of exogenous nucleic acids in mitochondria has been limited to yeast, plants, and algal species owing to the highly active recombination of native mitochondrial genomes. In certain mammalian tissues, however, recombination of the mitochondrial DNA (mtDNA) is markedly minimal to absent. Full-length mtDNA was cloned into a plasmid and stably amplified and mutagenized in bacteria. This Engineered mtDNA plasmid was delivered to mouse fibroblast mitochondria via biolistic bombardment. The constructs were selected for using an inducible mitochondrial-targeted restriction endonuclease to which our constructs are resistant, and the 2433 T-to-C chloramphenicol resistance polymorphism. Galactose treatment assured that rho-zero cells were eliminated. Simple cell survival, along with restriction digest analysis and mtDNA fluorescent staining, indicated that our constructs had been stably integrated into mitochondria. Our preliminary evidences showed the promise of direct transformation of mammalian cell with in-vitro modified mtDNA, which could enable the study of human mtDNA disorders in animal models as well as accelerate the understanding of mtDNA genetics in mammals.

项目1：选择性消除受损线粒体的遗传方法 线粒体周转率已被认为是线粒体质量控制的一种机制。但是，仍然是一个问题，是否确实能够选择性地消除有缺陷的线粒体。需要进行定量和实时成像测定，以测量选择性的线粒体降解并实时可视化此过程，而遗传方法对于在生理环境中探测线粒体转换至关重要。我们表达了一种有毒的细菌蛋白PORB，以损害培养的果蝇细胞和组织中总细胞线粒体的亚群。通过裂变/融合过程从线粒体网络中分离浓缩的线粒体受损的线粒体被分离，并通过溶酶体通过自噬途径在其他健康的细胞中选择性去除。我们首次证明了在动物组织（果蝇飞行肌肉）中帕金依赖性降解的降解。我们的工作原则上证明了有缺陷的线粒体在健康细胞中有选择地去除，还提供了一种新型的遗传方法来监测线粒体周转并剖析潜在的机制。 项目2：果蝇期间健康mtDNA的选择性传播 关于mtDNA遗传的最突出的问题之一是，母亲如何确保健康的线粒体传播到其后代。最近对mtDNA突变器小鼠的研究表明，MTDNA突变是从种系细胞中清除的，即使细胞中的水平太低而无法损害整体细胞适应性。对此现象的最合理的解释是，可以根据单个线粒体的功能在细胞器水平上选择mtDNA突变。为了研究这个问题，我们转向果蝇卵子来探索线粒体DNA传播的机制。我们产生了含有野生型和MT：CO1T300I MTDNA的异质飞行线。 MT：CO1T300I是mtDNA基因细胞色素C氧化酶1（MT：CO1）中的温度敏感突变。同质MT：CO1T300I苍蝇在高温下无法生存，而在低温下大多是健康的。我们发现，在雌性苍蝇的后代中，突变mtDNA的频率从18c转移到29c，表明针对有缺陷的线粒体进行了直接选择。细胞生物学分析表明，mtDNA复制发生在胚芽中的专业结构（称为熔炉）中，这种结构的破坏会导致卵子早期的mtDNA复制减少。同质MT：Co1t300i蝇包含完整的圆锥体，但在胚芽中显示了mtDNA复制的破坏。此外，我们表达了一种细菌孔蛋白蛋白，以损害卵巢中线粒体的一部分。我们观察到在早期和中期卵子发生期间，损坏的线粒体从未来的卵母细胞中分离出来。我们的结果表明，健康的线粒体被选择性地募集到Fusome，在该木材中优先复制mtdnas。含有野生型mtDNA的健康线粒体的选择性扩增将相互减少卵巢中mtDNA的频率及其传播机会。未来的研究将寻求确定卵子发生过程中识别和选择健康线粒体的机械。 项目3：果蝇模型揭示了mtDNA突变的新型致病机制。 应用基于线粒体靶向限制酶的选择方案，我们分离了同型线粒体DNA突变体，MT：COIT300I，该coit300i在细胞色素c氧化酶（COX）亚基I（COI）基因座上携带单个氨基酸取代。 MT：COIT300I苍蝇降低了COX活性并降低了ATP水平。 MT：COIT300I蝇是温度敏感性。在29 C的PUPA阶段中，很少有MT：COIT300I苍蝇大大降低了寿命，并且在宽松温度下的迁移率和神经活动受损。这些缺陷在旧苍蝇中加剧，这表明依赖年龄的神经系统和肌肉功能障碍。大多数表型类似于人类mtDNA疾病的典型特征，将MT：COIT300I作为果蝇模型验证，以了解mtDNA突变的保守特征。表达分析表明，少数基因在维持细胞氧化还原电位和预防应激诱发蛋白质变性的基因中受到突变背景中的调节。谷胱甘肽过氧化物酶在MT：COIT300I苍蝇中的表达可以部分抑制表型，进一步证实了这样一种观念，即细胞氧化还原电位的管制是线粒体功能障碍之一，并有助于下游的细胞缺陷。与普遍的假设相反，即活性氧是线粒体缺陷引起的发病机理的主要参与者之一，我们没有发现ROS参与MT：COIT300I苍蝇的过程。我们的结果提出了一种新的线粒体病因途径，并提供了进一步描述整个过程的遗传处理方法。由于大多数mtDNA疾病都显示了组织到组织的范围和表型的组织变化，因此我们建立了一种遗传方案，以在果蝇中以组织特异性方式使同性恋MTDNA突变在未来的人类mtDNA疾病中进行更好的建模。 项目4：通过直接转化工程mtDNA的哺乳动物mtDNA的靶向诱变 将特定突变诱导到哺乳动物线粒体基因组中的能力将促进对人线粒体遗传学和遗传疾病的研究。到目前为止，由于天然线粒体基因组的高度活性重组，线粒体中外源核酸的递送和表达一直限于酵母，植物和藻类。然而，在某些哺乳动物组织中，线粒体DNA（mtDNA）的重组显然很少。将全长mtDNA克隆到质粒中，并稳定地放大并在细菌中诱变。该工程的mtDNA质粒通过生物轰击传递到小鼠成纤维细胞线粒体。选择构建体用于使用诱导的线粒体靶向限制性核酸内切酶，并使用其抗抗性，并使用2433 T-TO-C氯霉素耐药性多态性。半乳糖处理确保消除了Rho-Zero细胞。简单的细胞存活以及限制性消化分析和mtDNA荧光染色，表明我们的构建体稳定地整合到了线粒体中。我们的初步证据表明，哺乳动物细胞与体外修饰的mtDNA直接转化，这可以使动物模型中人类mtDNA疾病的研究以及对哺乳动物中mtDNA遗传学的理解。