Iron homeostasis alterations in mutant CHCHD10 mitochondria

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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.