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 线粒体周转被认为是线粒体质量控制的机制。然而，细胞是否确实能够选择性地消除有缺陷的线粒体仍然是一个问题。需要定量和实时成像测定来测量选择性线粒体降解并实时可视化该过程，而遗传方法对于探测生理背景下的线粒体周转至关重要。我们表达了一种有毒细菌蛋白 PorB，以损害培养的果蝇细胞和组织中总细胞线粒体的亚群。用 PorB 浓缩的受损线粒体通过裂变/融合过程从线粒体网络中分离出来，并通过其他健康细胞中的自噬途径被溶酶体选择性去除。我们首次证明了动物组织（果蝇飞行肌）中受损线粒体的帕金依赖性降解。我们的工作原则上证明有缺陷的线粒体在健康细胞中被选择性去除，并且还提供了一种新的遗传方法来监测线粒体周转并剖析潜在机制。 项目2：果蝇卵子发生过程中健康线粒体DNA的选择性传递 关于线粒体DNA遗传的最突出的问题之一是母亲如何确保将健康的线粒体传递给后代。最近对 mtDNA 突变小鼠的研究表明，mtDNA 突变已从种系细胞中清除，尽管它们在细胞中的水平太低，不足以损害整体细胞健康。对于这种现象最合理的解释是，可以根据单个线粒体的功能在细胞器水平上选择线粒体DNA突变。为了研究这个问题，我们转向果蝇卵子发生来探索线粒体 DNA 传输的机制。我们生成了同时含有野生型和 mt:Co1T300I mtDNA 的异质蝇系。 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果蝇在高温下不能生存，而在低温下大多健康。我们发现，在雌性果蝇的后代中，异质果蝇中突变线粒体DNA的频率从18°C转移到29°C，这表明对有缺陷的线粒体的直接选择。细胞生物学分析表明，mtDNA 复制发生在生殖细胞的特殊结构（称为纺锤体）处，该结构的破坏会导致卵子发生早期 mtDNA 复制减少。同质 mt:Co1T300I 果蝇含有完整的融合体，但在生殖细胞中表现出 mtDNA 复制的破坏。此外，我们表达了一种细菌孔蛋白来损伤卵巢中的线粒体子集。我们观察到在早期和中期卵子发生过程中受损的线粒体与未来的卵母细胞分离。我们的结果表明，健康的线粒体被选择性地招募到融合体中，其中线粒体 DNA 优先被复制。含有野生型mtDNA的健康线粒体的选择性扩增将反过来减少卵巢中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参与识别和选择健康线粒体的机制。 项目3：果蝇模型揭示线粒体DNA突变的新致病机制。 应用基于线粒体靶向限制性酶的选择方案，我们分离出同质线粒体 DNA 突变体 mt:CoIT300I，其在细胞色素 C 氧化酶 (COX) 亚基 I (CoI) 基因座上携带单个氨基酸取代。 mt:CoIT300I 果蝇降低了 COX 活性并降低了 ATP 水平。 mt:CoIT300I 苍蝇对温度敏感。在 29°C 的条件下，很少有果蝇能在蛹阶段存活下来。此外，mt:CoIT300I 果蝇在允许的温度下表现出寿命大大缩短以及活动能力和神经活动受损。这些缺陷在年老的果蝇中更为严重，这表明存在年龄依赖性的神经和肌肉功能障碍。大多数表型类似于人类 mtDNA 疾病的典型特征，验证 mt:CoIT300I 作为果蝇模型来了解 mtDNA 突变的保守特征。表达分析显示，少数参与维持细胞氧化还原电位和防止应激诱导的蛋白质变性的基因在突变背景中上调。 mt:CoIT300I 果蝇中谷胱甘肽过氧化物酶的过度表达可以部分抑制表型，进一步证实了细胞氧化还原电位失调是线粒体功能障碍之一并导致下游细胞缺陷的观点。与活性氧是线粒体缺陷引起的发病机制的主要参与者之一的常见假设相反，我们在 mt:CoIT300I 果蝇中没有发现 ROS 参与该过程的证据。我们的结果提出了线粒体病因学的一条新途径，并提供了进一步描述整个过程的遗传手柄。由于大多数 mtDNA 疾病在程度和表型上表现出组织间的差异，我们建立了一种遗传方案，在果蝇中以组织特异性方式产生同质 mtDNA 突变，以便将来更好地模拟人类 mtDNA 疾病。 项目4：通过工程mtDNA的直接转化对哺乳动物mtDNA进行定向诱变 诱导哺乳动物线粒体基因组特定突变的能力将有助于对人类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跃重组，外源核酸在线粒体中的递送和表达仅限于酵母、植物和藻类物种。然而，在某些哺乳动物组织中，线粒体 DNA (mtDNA) 的重组明显很少甚至不存在。全长线粒体DNA被克隆到质粒中，并在细菌中稳定扩增和诱变。该工程化 mtDNA 质粒通过基因枪轰击递送至小鼠成纤维细胞线粒体。选择使用我们的构建体具有抗性的诱导型线粒体靶向限制性内切核酸酶和2433 T-to-C氯霉素抗性多态性来选择构建体。半乳糖处理可确保消除 rho-0 细胞。简单的细胞存活以及限制性消化分析和线粒体DNA荧光染色表明我们的构建体已稳定地整合到线粒体中。我们的初步证据显示了利用体外修饰的线粒体DNA直接转化哺乳动物细胞的前景，这可以使我们能够在动物模型中研究人类线粒体DNA疾病，并加速对哺乳动物线粒体DNA遗传学的理解。