Mitochondrial DNA inheritance in Drosophila

果蝇线粒体DNA遗传

• 批准号: 8939901
• 负责人: Hong Xu
• 金额: $ 128.3万
• 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/8939901
• 关键词: Address; Affect; Age; Aging; Apoptosis; Apoptotic; Architecture; Automobile Driving; Behavioral Genetics; Binding; Binding Proteins; Biogenesis; Biological; Bypass; Cell Death; Cell Differentiation process; Cells; Communication; Complex; Crista ampullaris; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Cytochrome a; Cytochromes; Cytoplasm; DNA Repair; DNA Replication Factor; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Deterioration; Development; Diffuse; Disease; Drosophila genus; Embryonic Development; Evolution; Exhibits; Eye; Female; Fertility; Frequencies; Generations; Genes; Genetic; Genetic Epistasis; Genetic Screening; Germ Lines; Goals; Health; Heat shock proteins; Heme; Homeostasis; Homologous Gene; Human; Inherited; Knock-out; Longevity; Mammals; Manuscripts; Membrane; Membrane Proteins; Messenger RNA; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Matrix; Mitochondrial RNA; Molecular; Morphology; Mus; Mutation; Myopathy; Nature; Nerve Degeneration; Nuclear; Oocytes; Oogenesis; Organelles; Organism; Outer Mitochondrial Membrane; Ovary; Oxidative Phosphorylation; Pathogenesis; Pathway interactions; Pattern; Peptide Hydrolases; Phenotype; Phosphorylation Site; Play; Ploidies; Polymerase; Premature aging syndrome; Prevalence; Proteins; Publishing; RNA-Binding Proteins; Reporting; Retinal Degeneration; Role; Scheme; Signal Transduction; Staging; Surface; Temperature; Testing; Therapeutic; Time; Tissues; Translation Initiation; Variant; Work; age related; aged; alternative oxidase; cytochrome c oxidase; egg; fitness; fly; genome wide association study; human disease; improved; knock-down; mitochondrial DNA mutation; mitochondrial dysfunction; mitochondrial membrane; mtTF1 transcription factor; mutant; novel; novel strategies; oxidative damage; phosphoric diester hydrolase; prevent; restriction enzyme; segregation; stress protein; tool; transmission process; uptake

1. Selective propagation of functional mtDNA during oogenesis restricts the transmission of a deleterious mitochondrial variant Though mitochondrial DNA is prone to mutation and few mtDNA repair mechanisms exist, crippling mitochondrial mutations are exceedingly rare. Recent studies demonstrated strong purifying selection in the mouse female germline. However, the mechanisms underlying the positive selection of healthy mitochondria remain to be elucidated. We visualized mtDNA replication during Drosophila oogenesis. We found that mtDNA replication commenced prior to oocyte determination during the late germarium stage, and was dependent on mitochondrial fitness. We isolated a temperature-sensitive lethal mtDNA mutation, mt:CoIT300I, which displayed reduced mtDNA replication in the germarium at the restrictive temperature. Additionally, the frequency of mt:CoIT300I in heteroplasmic flies was decreased both through oogenesis and over multiple generations at the restrictive temperature. Furthermore, we determined that selection against mt:CoIT300I overlaps with the timing of selective replication of mtDNA in the germarium. These findings establish a previously uncharacterized developmental mechanism for selective amplification of healthy mtDNA, which may be evolutionarily conserved to limit transmission of deleterious mutations. This work was published in Nature Genetics (Hill, J. H., Chen, Z. & Xu, H. Selective replication of functional mtDNA during oogenesis restricts the transmission of a deleterious mutation. Nat. Genetics 46, 389392 (2014). 2. Genetic mosaic analysis of a deleterious mitochondrial DNA mutation in Drosophila Various human diseases are associated with mitochondrial DNA (mtDNA) mutations, but heteroplasmy--the coexistence of mutant and wild-type mtDNA--complicates their study. We have previously isolated a temperature lethal mtDNA mutation in Drosophila, mt:CoIT300I affecting the cytochrome c oxidase subunit I locus. In current study, we found that the decrease in COX activity was ascribable to a temperature dependent destabilization of cytochrome a heme. Consistently, the viability of homoplasmic flies at 29C was fully restored by expressing the alternative oxidase, which selectively bypassing the cytochrome chains. Heteroplasmic flies are fully viable and were used to explore the age-related and tissue-specific phenotypes of mt:CoIT300I. Using a genetic scheme that expresses a mitochondrially targeted restriction enzyme to induce tissue-specific homoplasmy in heteroplasmic flies, we found that mt:CoIT300I homoplasmy in the eye caused severe neurodegeneration at 29C. Degeneration was suppressed by improving mitochondrial Ca2+ uptake, suggesting that Ca2+ mishandling contributed to mt:CoIT300I pathogenesis. Our results demonstrate a novel approach for Drosophila mtDNA genetics, and its application in studying mtDNA diseases and testing therapeutic approaches. The manuscript describing this work is currently under revesion with Nature Communications. 3. Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration Mitochondrial AAA proteases i-AAA and m-AAAs are closely related and play major roles in the inner membranes protein homeostasis. Mutations of m-AAA proteases are associated with neuro-muscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. We generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1Ldel). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1Ldel flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1Ldel flies had reduced Complex I activity, increased level of ROS, severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. Our results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to accumulation of oxidative damage, progressive deterioration of membrane integrity and eventual apoptosis. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency. This work is currently under consideration with Cell death and Differentiation 4. Prune, a Drosophila mitochondrial phosphodiesterase, promotes mitochondrial DNA replication through maintaining TFAM Compartmentalized cAMP signaling regulates mitochondrial dynamics, morphology and oxidative phosphorylation. However, the regulators of mitochondrial cAMP pathway and its broad impacts on organelle function remain to be explored. Here we report that Drosophila Prune is a cyclic nucleotide phosphodiesterase and localizes to mitochondrial matrix. Knocking down prune in cultured cells reduces mitochondrial transcription factor A (TFAM) and mtDNA levels. A TFAM mutant abolishing the conserved PKA phosphorylation site is stable and restores the mtDNA level in prune knockdown cells. Our data suggest that Prune stabilizes TFAM and promotes mtDNA replication through down-regulating the mitochondrial cAMP signaling. Our work demonstrates the prevalence of the mitochondrial cAMP signaling in metazoan and its new role in mitochondrial biogenesis. This work has been submitted. 5. Spoon, a mitochondrial outer membrane RNA binding protein is essential for mtDNA inheritance and selection by promoting mtDNA replication during oogenesis Developmentally regulated mtDNA replication during oogenesis is essential for selection against defective variants, also prepares vast amount of mitochondria for the early embryogenesis. Over a span of 3 days, the mtDNA contents increase over 10,000 fold in the oocytes. However, the molecular mechanism driving this enormous replication remains elusive. We indentified Spoon, a mitochondrial outer membrane protein from a genetic screening for genes required for mtDNA replication in ovary. Spoon mutant flies showed severely disrupted mtDNA replication in ovary. Female mutant flies are sub-fertile, and their eggs were devoid of mtDNA and displayed various mitochondrial dysfunctions including mitochondria hyper-fusion and reduced ATP level. Spoon was highly expressed in the female germ line, and its expression was up regulated at the late germarium stage. This spatial pattern is coincided with the timing of commencement of mtDNA replication. We found that the protein levels of several key mtDNA replication factors including mtDNA polymerase, mtRNA polymerase were significantly reduced in the spoon mutant background. Additionally, Spoon physically interacted with Larp1, a mRNA binding protein stabilizing mRNA and promoting translation initiation. Larp1 localized to mitochondria surface in wild type, but diffused into cytoplasm in the Spoon mutant ovary. Furthermore, Spoon bound to a subset of mRNAs encoding mitochondrial replication factor. Taken together, we propose that Spoon promotes the local synthesis of these factors on mitochondrial surface, and facilitates their translocation into mitochondria subsequently, through which drives the mtDNA replication. Similarly, the knock out of spoon homolog in mice also reduces female fertility. Thus, spoon dependent mtDNA replication may represent an evolutionary conserved mechanism of mtDNA replication during oogenesis, and plays essential roles in two aspects of mtDNA inheritance: selection against deleterious variants and providing high amount of mtDNA for early embryogenesis. Currently we are preparing a manuscript describing this work.

1。卵子发生过程中功能mtDNA的选择性传播限制了有害线粒体变体的传播 尽管线粒体DNA容易突变，并且很少有mtDNA修复机制，但线粒体突变的残废非常罕见。最近的研究表明，在小鼠雌性种系中进行了强烈的纯化选择。但是，健康线粒体阳性选择的基础机制仍有待阐明。我们在果蝇中观察了mtDNA复制。我们发现，在胚芽晚期的卵母细胞测定之前，mtDNA复制开始，并依赖于线粒体适应性。我们分离了对温度敏感的致命mtDNA突变，MT：COIT300I，该突变在限制性温度下显示出降低的菌群中mtDNA复制。 此外，在限制性温度下，通过卵子发生和多代杂质中的MT：COIT300I的频率降低了。此外，我们确定针对MT：COIT300I的选择与胚芽中mtDNA选择性复制的时间重叠。这些发现建立了一种先前未表征的发育机制，用于选择性扩增健康mtDNA，这可能是进化性的，以限制有害突变的传播。 这项工作发表在自然遗传学中（Hill，J。H.，Chen，Z。＆Xu，H。卵子发生过程中功能mtDNA的选择性复制限制了有害突变的传播。NAT。遗传学46，389392（2014）。 2。果蝇中有害线粒体DNA突变的遗传镶嵌分析 各种人类疾病与线粒体DNA（mtDNA）突变有关，但是异质性 - 突变体和野生型mtDNA的共存 - 使他们的研究复杂化。以前，我们已经在果蝇中分离了果蝇中的温度致命mtDNA突变：coit300i影响了细胞色素C氧化酶亚基I基因座。在当前的研究中，我们发现Cox活性的降低可归因于细胞色素a血红素的依赖性不稳定。一致地，通过表达替代氧化酶的替代氧化酶，可以选择性地绕过细胞色素链，从而完全恢复同质果蝇在29c处的生存能力。杂质蝇完全可行，用于探索MT：COIT300I的年龄相关和组织特异性表型。使用表达线粒体靶向限制酶以诱导组织特异性同质质的遗传方案，我们发现眼睛中的MT：COIT300I同质质在29c时引起严重的神经变性。通过改善线粒体Ca2+摄取来抑制退化，这表明Ca2+不当行为有助于MT：COIT300I发病机理。我们的结果证明了果蝇mtDNA遗传学的一种新方法，及其在研究mtDNA疾病和测试治疗方法中的应用。 描述这项工作的手稿目前正在与自然通信有关。 3。果蝇I-AAA蛋白酶的丢失，Dyme1l，导致线粒体异常和凋亡变性 线粒体AAA蛋白酶I-AAA和M-AAAS密切相关，并在内膜蛋白稳态中起主要作用。 M-AAA蛋白酶的突变与人类的神经肌肉疾病有关。但是，I-AAA在后生动物中的作用知之甚少。我们产生了影响果蝇I-AAA，dyme1l（dyme1ldel）的缺失。突变果蝇表现出过早衰老，进行性运动缺乏和神经变性，类似于M-AAA疾病的某些关键特征。 Dyme1ldel果蝇显示出升高的线粒体展开的蛋白质胁迫和不规则的cr。老化的dyme1ldel果蝇降低了复杂的I活性，ROS的水平升高，线粒体膜严重混乱并增加了凋亡。 此外，通过靶向DOMI或DIAP1抑制凋亡，或者减少ROS积累抑制视网膜变性。我们的结果表明，I-AAA对于去除展开的蛋白质和维持线粒体膜结构至关重要。 I-AAA的丧失会导致氧化损伤的积累，膜完整性的进行性恶化和最终凋亡。含有ROS水平可能是管理线粒体AAA蛋白酶缺乏症的潜在策略。 目前正在考虑这项工作与细胞死亡和分化 4。修剪，一种果蝇线粒体磷酸二酯酶，通过维持TFAM来促进线粒体DNA复制 分隔的cAMP信号传导调节线粒体动力学，形态和氧化磷酸化。但是，线粒体cAMP通路的调节剂及其对细胞器功能的广泛影响仍有待探索。在这里，我们报告果蝇是一种环状核苷酸磷酸二酯酶，并定位于线粒体基质。在培养的细胞中敲击修剪可降低线粒体转录因子A（TFAM）和mtDNA水平。废除保守PKA磷酸化位点的TFAM突变体是稳定的，并恢复了修剪的麦芽磷酸化细胞中的mtDNA水平。我们的数据表明，修剪稳定TFAM并通过下调线粒体cAMP信号传导来促进mtDNA复制。我们的工作证明了后生动物中线粒体cAMP信号的普遍性及其在线粒体生物发生中的新作用。 这项工作已提交。 5。Spoon，线粒体外膜RNA结合蛋白对于MTDNA的遗传和选择至关重要。 在卵子发生过程中，在发育中调节的mtDNA复制对于针对有缺陷的变体的选择至关重要，还为早期胚胎发生准备了大量的线粒体。在3天的时间里，卵母细胞中的mtDNA含量增加了10,000倍以上。但是，驱动这种巨大复制的分子机制仍然难以捉摸。我们将勺子倒下，这是一种从卵巢中mtDNA复制所需的基因的遗传筛选的线粒体外膜蛋白。勺子突变体在卵巢中显示出严重破坏的mtDNA复制。雌性突变果蝇是亚肥胖的，其卵没有mtDNA，并显示出各种线粒体功能障碍，包括线粒体超融合和ATP水平降低。汤匙在女性系中高度表达，其表达在胚芽晚期阶段受到调节。这种空间模式与mtDNA复制的开始时间相吻合。我们发现，在勺子突变体背景下，几种关键mtDNA复制因子（包括mtDNA聚合酶，mTRNA聚合酶）的蛋白水平显着降低。另外，勺子与LARP1（mRNA结合蛋白稳定mRNA并促进翻译起始）物理相互作用。 LARP1位于野生型中的线粒体表面，但在勺子突变体卵巢中扩散成细胞质。此外，勺子与编码线粒体复制因子的mRNA子集结合。综上所述，我们提出勺子促进了线粒体表面上这些因素的局部合成，并促进了它们的易位，然后将其转移到线粒体中，然后通过该迁移驱动mtDNA复制。同样，在小鼠中脱落的勺子同源物也降低了女性的生育能力。因此，依赖勺子的mtDNA复制可以代表卵子发生过程中mtDNA复制的进化保守机制，并且在mtDNA遗传的两个方面扮演着重要的作用：针对有害变体的选择，并为早期胚胎发生提供了大量的mtDNA。 目前，我们正在准备一个描述这项工作的手稿。