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Mitochondrial DNA inheritance in Drosophila

果蝇线粒体DNA遗传

基本信息

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
项目类别：
财政年份：
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

项目摘要

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.