Iron homeostasis alterations in mutant CHCHD10 mitochondria

突变体 CHCHD10 线粒体中铁稳态的改变

• 批准号: 10615913
• 负责人: Nicole Sayles
• 金额: $ 4.77万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-27 至 2024-04-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10615913
• 关键词: AA Spectrophotometry; Aconitate Hydratase; Address; Affect; Antioxidants; Binding; Binding Proteins; Biochemical; Bioenergetics; Biogenesis; Biological Assay; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cell Nucleus; Cells; Cytosol; Defect; Disease; Disease Progression; Down-Regulation; Enzymes; Event; Ferritin; Foundations; Friedreich Ataxia; Frontotemporal Dementia; Gene Expression Profiling; Genes; Heart; Heart Mitochondria; Heart failure; Heme; Homeostasis; Impairment; In Vitro; Iron; Iron Overload; Knock-in Mouse; Knock-out; Knowledge; Lead; Link; Liver; Measures; Metabolic; Mitochondria; Mitochondrial Diseases; Mitochondrial Matrix; Mitochondrial Proteins; Molecular; Molecular Chaperones; Morphology; Motor Neuron Disease; Mus; Mutation; Myocardial dysfunction; Myopathy; Nervous System; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Peptide Hydrolases; Peptides; Phenocopy; Process; Production; Protein Import; Proteins; Proteomics; Reactive Oxygen Species; Role; SOD2 gene; Signal Transduction; Succinate Dehydrogenase; Sulfur; Testing; Tissues; Transferrin; autosome; combat; effective therapy; frataxin; functional group; gain of function; heart damage; heart function; heme biosynthesis; human disease; macromolecule; mitochondrial cardiomyopathies; mitochondrial dysfunction; motor deficit; mouse model; mutant; novel; oxidative damage; paralogous gene; response; targeted treatment; transcriptomics

Project Summary Mutations in the mitochondrial protein coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10, D10) have recently been linked to autosomal dominant mitochondrial diseases, characterized by cardiomyopathy, myopathy, motor neuron disease, and frontotemporal dementia. However, the pathogenic mechanisms of these mutations remain unclear. This application seeks to address this gap in knowledge by investigating a S55L mutant D10 (D10S55L, equivalent to the human disease mutation S59L) mouse model. My group previously showed that the D10S55L knock in mouse develops a fatal mitochondrial disease associated with D10 aggregation in heart mitochondria, ultimately leading to mitochondrial dysfunction and fatal cardiomyopathy. While little is known about the normal function of D10, analyses of mitochondrial protein interactome indicate an interaction with mitochondrial import components, including peptide proteases MPP and PITRM1, suggesting that D10 may play a role in the processing of matrix-bound proteins imported from the cytosol. Notably, MPP and PITRM1 process frataxin (FXN), a protein required for the assembly of iron- sulfur clusters (ISCs), which is defective in Friedreich's ataxia (FRDA), a fatal mitochondrial disease, affecting the heart and the nervous system. The D10S55L mouse model phenocopies the FXN loss seen in FRDA, with a loss of processed FXN in the heart. Similar to FRDA, gene expression analyses indicate iron dysregulation in the D10S55L mouse heart, with increased ferritin and transferrin and decreased mitoferrin. I hypothesize that altered ISC biogenesis due to impairment of FXN processing could underlie iron dysregulation leading to oxidative stress and mitochondrial damage in heart. In accord, I find a marked activation of the antioxidant response Nrf2-ARE regulated genes, such Hmox-1 and Nqo1, and downregulation of heme biosynthesis. I propose that ISC assembly defects, iron accumulation, and heme biosynthesis defects cause mitochondrial dysfunction and ultimately cardiomyocyte damage. I will test this hypothesis in aim 1, by assessing protein import efficiency in heart mitochondria of D10S55L mice, focusing on FXN maturation, and compare it with other imported mitochondrial matrix proteins, including MnSOD and TFAM. To this end, I will use established in vitro import assays comparing mitochondria isolated from affected (heart) and unaffected (liver) tissues at different disease stages. In aim 2, I will investigate iron homeostasis in D10S55L mitochondria, by measuring total, cytosolic, and mitochondrial iron, and by assessing the activity of ISC-dependent enzymes, such as succinate dehydrogenase and aconitase. I will also measure ROS production and cardiac oxidative damage. Importantly, I will evaluate longitudinally histopathological and functional cardiac alterations in mutant D10 heart. These studies will elucidate the role of D10 in mitochondrial import and processing of key metabolic proteins, such as frataxin, and in iron homeostasis, and will illuminate the pathogenic mechanisms of mutant D10 in the heart, offering novel pathogenic pathways to be targeted for therapy in cardiomyopathies.

项目摘要 线粒体蛋白10螺旋结构域突变(CHCHD10，D10) 最近被认为与常染色体显性遗传性线粒体疾病有关，以心肌病为特征， 肌病、运动神经元病和额颞叶痴呆。然而，其致病机制尚不清楚。 这些突变仍不清楚。此应用程序试图通过调查一个 S55L突变D10(D10S55L，相当于人类疾病突变S59L)小鼠模型。我的小组 此前研究表明，D10S55L敲击小鼠会患上一种致命的线粒体疾病 D10聚集在心脏线粒体，最终导致线粒体功能障碍并致死 心肌病。虽然人们对D10的正常功能知之甚少，但对线粒体蛋白的分析 相互作用组表明与线粒体输入成分相互作用，包括多肽蛋白酶MPP 和PITRM1，提示D10可能在加工从国外进口的基质结合蛋白中发挥作用 细胞质。值得注意的是，MPP和PITRM1处理Frataxin(FXN)，这是一种组装铁所需的蛋白质-- 硫簇(ISCs)，这是一种致命的线粒体疾病，在Friedreich‘s共济失调(FRDA)中存在缺陷，影响 心脏和神经系统。D10S55L小鼠模型表现出FXN在FRDA中的丢失，具有 心脏中失去加工的FXN。与FRDA相似，基因表达分析表明铁代谢紊乱 D10S55L小鼠心脏铁蛋白和转铁蛋白升高，丝裂原铁蛋白降低。我假设 FXN加工障碍引起的ISC生物发生改变可能是导致铁失调的原因 心脏的氧化应激和线粒体损伤。总之，我发现抗氧化剂有明显的激活作用 反应Nrf2-是调节基因，如Hmox-1和NQO1，并下调血红素的生物合成。我 提出ISC组装缺陷、铁积累和血红素生物合成缺陷导致线粒体 功能障碍，最终导致心肌细胞损伤。我将在目标1中通过评估蛋白质来验证这一假设 D10S55L小鼠心肌线粒体的导入效率，以FXN成熟为重点，并与其他 进口线粒体基质蛋白，包括MnSOD和TFAM。为此，我将使用在体外建立的 进口分析比较从病变(心脏)和未病变(肝脏)组织中分离的线粒体在不同时间 疾病分期。在目标2，我将研究D10S55L线粒体铁的动态平衡，通过测量总的， 通过评估依赖于ISC的酶的活性，如琥珀酸 脱氢酶和乌头酸酶。我还将测量ROS的产生和心脏的氧化损伤。重要的是 我将对D10突变心脏的纵向组织病理学和功能改变进行评估。这些 研究将阐明D10在线粒体输入和处理关键代谢蛋白中的作用，如 Frataxin，并在铁稳态中，并将阐明心脏中突变D10的致病机制， 为心肌病的治疗提供新的致病途径。