Iron homeostasis alterations in mutant CHCHD10 mitochondria

项目摘要 线粒体蛋白卷曲螺旋-螺旋-卷曲螺旋-螺旋结构域10（CHCHD 10，D10）突变 最近与常染色体显性线粒体疾病有关，其特征是心肌病， 肌病、运动神经元疾病和额颞叶痴呆。然而， 这些突变仍不清楚。本申请旨在通过调查 S55 L突变体D10（D10 S55 L，相当于人类疾病突变S59 L）小鼠模型。我的小组 先前的研究表明，小鼠中的D10 S55 L敲除会发展出一种致命的线粒体疾病， D10在心脏线粒体中聚集，最终导致线粒体功能障碍和致命的 心肌病虽然对D10的正常功能知之甚少，但对线粒体蛋白的分析表明， 相互作用组表明与线粒体输入组分相互作用，包括肽蛋白酶MPP 和PITRM 1，这表明D10可能在处理从血管内皮细胞输入的基质结合蛋白质中发挥作用。 细胞质值得注意的是，MPP和PITRM 1处理共济失调蛋白（FXN），这是一种组装铁- 硫簇（ISC），这是有缺陷的弗里德赖希共济失调（FRDA），一种致命的线粒体疾病，影响 心脏和神经系统。D10 S55 L小鼠模型表型模仿FRDA中观察到的FXN损失， 心脏中的FXN加工损失。与FRDA相似，基因表达分析表明， D10 S55 L小鼠心脏，铁蛋白和转铁蛋白增加，线粒体铁蛋白减少。我假设 由于FXN加工受损导致的ISC生物发生的改变可能是铁失调的基础， 氧化应激和线粒体损伤。在雅阁中，我发现抗氧化剂有明显的活性 响应Nrf 2-ARE调节的基因，如Hmox-1和Nqo 1，以及血红素生物合成的下调。我 提出ISC装配缺陷、铁积累和血红素生物合成缺陷导致线粒体 功能障碍并最终导致心肌细胞损伤。我将在aim 1中通过评估蛋白质来验证这一假设。 D10 S55 L小鼠心脏线粒体中的输入效率，重点是FXN成熟，并将其与其他 输入线粒体基质蛋白，包括MnSOD和TFAM。为此，我将使用建立在体外 比较在不同温度下从受影响的（心脏）和未受影响的（肝脏）组织分离的线粒体的输入测定 疾病阶段。在目标2中，我将研究D10 S55 L线粒体中的铁稳态， 细胞溶质和线粒体铁，并通过评估ISC依赖性酶的活性，如琥珀酸 脱氢酶和乌头酸酶。我还会测量ROS的产生和心脏氧化损伤。重要的是， 我将纵向评价突变D10心脏的组织病理学和功能性心脏改变。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和铁稳态，并将阐明突变体D10在心脏中的致病机制， 为心肌病的治疗提供了新的靶向致病途径。

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.