In vivo overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency

Huichalaf, Claudia; Perfitt, Tyler L.; Kuperman, Anna; Gooch, Renea; Kovi, Ramesh C.; Brenneman, Karrie A.; Chen, Xian; Hirenallur-Shanthappa, Dinesh; Ma, Tiffany; Assaf, Basel T.; Pardo, Ingrid; Franks, Tania; Monarski, Laura; Cheng, Teng-Hu; Le, Kevin X.; Su, Chunyan; Somanathan, Suryanarayan; Whiteley, Laurence O.; Bulawa, Christine E.; Pregel, Marko J.; Martelli, Alain

doi:10.1016/j.omtm.2022.02.002

Cited by 28 publications

(31 citation statements)

References 24 publications

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“…Platelets and reticulocytes with half-lives of 2 and 10 days, respectively, contain some 96.3% of the mitochondria that are present in a whole blood sample (Figure ). Therefore, it should be possible to quantify modulation of dysregulated protein expression in these blood cells during relatively short-term phase I clinical trials that could serve as a proof of concept that the therapeutic approach is effective and nontoxic, an important consideration given the toxicity of elevated levels of the frataxin protein . For extra-mitochondrial proteins, the situation is more complex as it will depend on whether the protein is present in all the blood cells, just the erythrocytes, or just the reticulocytes, platelets, granulocytes, monocytes, and lymphocytes (Figure ).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, it should be possible to quantify modulation of dysregulated protein expression in these blood cells during relatively short-term phase I clinical trials that could serve as a proof of concept that the therapeutic approach is effective and nontoxic, an important consideration given the toxicity of elevated levels of the frataxin protein. 11 For extra-mitochondrial proteins, the situation is more complex as it will depend on whether the protein is present in all the blood cells, just the erythrocytes, or just the reticulocytes, platelets, granulocytes, monocytes, and lymphocytes ( Figure 2 ). Frataxin-E is an unusual protein where it is mainly found in erythrocytes so that most of the protein can be quantified by analyzing a whole blood sample.…”

Section: Discussionmentioning

confidence: 99%

“… 10 Thus, there is a compelling need to monitor frataxin protein levels as FRDA disease biomarkers so that the efficacy of various therapeutic regimens can be assessed and the potential over-expression of toxic levels of frataxin can be monitored. 11 Full-length (FL) frataxin is a protein of 210 amino acids ( M W = 23,135 Da), which contains a mitochondrial targeting sequence at its amino terminus. Therefore, when FL frataxin is expressed in the cytosol, it rapidly translocates into the mitochondria where it undergoes a two-step cleavage process mediated by mitochondrial processing peptidase (MPP), which removes the mitochondrial targeting sequence and yields mature mitochondrial frataxin (frataxin-M) as a 130-amino acid protein ( M W = 14,268 Da, Figure 1 ).…”

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Liquid Chromatography–Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich’s Ataxia

Rojsajjakul

Grady

et al. 2023

Anal. Chem.

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Friedreich’s ataxia (FRDA) is caused primarily by expanded GAA repeats in intron 1 of both alleles of the FXN gene, which causes transcriptional silencing and reduced expression of frataxin mRNA and protein. FRDA is characterized by slowly progressive ataxia and cardiomyopathy. Symptoms generally appear during adolescence, and patients slowly progress to wheelchair dependency usually in the late teens or early twenties with death on average in the 4th decade. There are two known mature proteoforms of frataxin. Mitochondrial frataxin (frataxin-M) is a 130-amino acid protein with a molecular weight of 14,268 Da, and there is an alternatively spliced N-terminally acetylated 135-amino acid form (frataxin-E) with a molecular weight of 14,953 Da found in erythrocytes. There is reduced expression of frataxin in the heart and brain, but frataxin is not secreted into the systemic circulation, so it cannot be analyzed in serum or plasma. Blood is a readily accessible biofluid that contains numerous different cell types that express frataxin. We have found that pig blood can serve as an excellent surrogate matrix to validate an assay for frataxin proteoforms because pig frataxin is lost during the immunoprecipitation step used to isolate human frataxin. Frataxin-M is expressed in blood cells that contain mitochondria, whereas extra-mitochondrial frataxin-E is found in erythrocytes. This means that the analysis of frataxin in whole blood provides information on the concentration of both proteoforms without having to isolate the individual cell types. In the current study, we observed that the distributions of frataxin levels for a sample of 25 healthy controls and 50 FRDA patients were completely separated from each other, suggesting 100% specificity and 100% sensitivity for distinguishing healthy controls from FRDA cases, a very unusual finding for a biomarker assay. Additionally, frataxin levels were significantly correlated with the GAA repeat length and age of onset with higher correlations for extra-mitochondrial frataxin-E than those for mitochondrial frataxin-M. These findings auger well for using frataxin levels measured by the validated stable isotope dilution ultrahigh-performance liquid chromatography–multiple reaction monitoring/mass spectrometry assay to monitor therapeutic interventions and the natural history of FRDA. Our study also illustrates the utility of using whole blood for protein disease biomarker discovery and validation.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Liquid Chromatography–Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich’s Ataxia

Rojsajjakul

Grady

et al. 2023

Anal. Chem.

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“…To study the role of frataxin deficiency in iPSC‐derived iNs and iCMs from the FRDA‐subject, we sought to restore its expression level by transducing them with frataxin lentivirus (Figure 3a). Prior studies have found that high levels of frataxin can be toxic to cells (PMID: 29794127) (Belbellaa et al, 2020; Huichalaf et al, 2022; Vannocci et al, 2018). Therefore, we modified a transfer plasmid with a weak constitutive promoter (UbC) (113,450, Addgene) (Qin et al, 2010) to better control frataxin overexpression (Figure 3a).…”

Section: Resultsmentioning

confidence: 99%

Frataxin deficiency alters gene expression in Friedreich ataxia derived IPSC‐neurons and cardiomyocytes

Angulo

Bertalovitz

Argenziano

et al. 2022

Molec Gen & Gen Med

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Background Friedreich's ataxia (FRDA) is an autosomal recessive disease, whereby homozygous inheritance of an expanded GAA trinucleotide repeat expansion in the first intron of the FXN gene leads to transcriptional repression of the encoded protein frataxin. FRDA is a progressive neurodegenerative disorder, but the primary cause of death is heart disease which occurs in 60% of the patients. Several functions of frataxin have been proposed, but none of them fully explain why its deficiency causes the FRDA phenotypes nor why the most affected cell types are neurons and cardiomyocytes. Methods To investigate, we generated iPSC‐derived neurons (iNs) and cardiomyocytes (iCMs) from an FRDA patient and upregulated FXN expression via lentivirus without altering genomic GAA repeats at the FXN locus. Results RNA‐seq and differential gene expression enrichment analyses demonstrated that frataxin deficiency affected the expression of glycolytic pathway genes in neurons and extracellular matrix pathway genes in cardiomyocytes. Genes in these pathways were differentially expressed when compared to a control and restored to control levels when FRDA cells were supplemented with frataxin. Conclusions These results offer novel insight into specific roles of frataxin deficiency pathogenesis in neurons and cardiomyocytes.

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“…Early experiments have shown that viral gene therapies to deliver a human FXN gene are a promising approach but may have toxicities. [4][5][6] In a series of interesting and thoughtful experiments published in Molecular Therapy: Methods & Clinical Development, Huichalaf et al 7 have sought to determine expression limits for FXN toxicity and, more importantly, the mechanism(s) of this toxicity. The authors show that overexpression of native human FXN using an AAV9 vector (AAV9-CAG-FXN) in the MCK-Cre conditional ablation FA mouse model (loss of FXN in heart and skeletal muscle) is not only toxic to heart, a key target organ in FA, but is also toxic to liver, which is an off-target organ.…”

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confidence: 99%

Gene therapy for Friedreich ataxia: Too much, too little, or just right?

Payne

2022

Molecular Therapy - Methods & Clinical Development

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In vivo overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency

Cited by 28 publications

References 24 publications

Liquid Chromatography–Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich’s Ataxia

Liquid Chromatography–Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich’s Ataxia

Frataxin deficiency alters gene expression in Friedreich ataxia derived IPSC‐neurons and cardiomyocytes

Gene therapy for Friedreich ataxia: Too much, too little, or just right?

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