RESUMO
Friedreich's ataxia (FRDA) is a progressive disorder caused by insufficient expression of frataxin, which plays a critical role in assembly of iron-sulfur centers in mitochondria. Individuals are cognitively normal but display a loss of motor coordination and cardiac abnormalities. Many ultimately develop heart failure. Administration of nicotinamide adenine dinucleotide-positive (NAD+) precursors has shown promise in human mitochondrial myopathy and rodent models of heart failure, including mice lacking frataxin in cardiomyocytes. We studied mice with systemic knockdown of frataxin (shFxn), which display motor deficits and early mortality with cardiac hypertrophy. Hearts in these mice do not "fail" per se but become hyperdynamic with small chamber sizes. Data from an ongoing natural history study indicate that hyperdynamic hearts are observed in young individuals with FRDA, suggesting that the mouse model could reflect early pathology. Administering nicotinamide mononucleotide or riboside to shFxn mice increases survival, modestly improves cardiac hypertrophy, and limits increases in ejection fraction. Mechanistically, most of the transcriptional and metabolic changes induced by frataxin knockdown are insensitive to NAD+ precursor administration, but glutathione levels are increased, suggesting improved antioxidant capacity. Overall, our findings indicate that NAD+ precursors are modestly cardioprotective in this model of FRDA and warrant further investigation.
Assuntos
Modelos Animais de Doenças , Frataxina , Ataxia de Friedreich , Proteínas de Ligação ao Ferro , NAD , Animais , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Camundongos , Humanos , NAD/metabolismo , Fenótipo , Masculino , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Mononucleotídeo de Nicotinamida/farmacologia , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Compostos de Piridínio , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
Trinucleotide repeat (TNR) expansion is the cause of over 40 neurodegenerative diseases, including Huntington's disease and Friedreich's ataxia (FRDA). There are no effective treatments for these diseases due to the poor understanding of molecular mechanisms underlying somatic TNR expansion and contraction in neural systems. We and others have found that DNA base excision repair (BER) actively modulates TNR instability, shedding light on the development of effective treatments for the diseases by contracting expanded repeats through DNA repair. In this study, temozolomide (TMZ) was employed as a model DNA base damaging agent to reveal the mechanisms of the BER pathway in modulating GAA repeat instability at the frataxin (FXN) gene in FRDA neural cells and transgenic mouse mice. We found that TMZ induced large GAA repeat contraction in FRDA mouse brain tissue, neurons, and FRDA iPSC-differentiated neural cells, increasing frataxin protein levels in FRDA mouse brain and neural cells. Surprisingly, we found that TMZ could also inhibit H3K9 methyltransferases, leading to open chromatin and increasing ssDNA breaks and recruitment of the key BER enzyme, pol ß, on the repeats in FRDA neural cells. We further demonstrated that the H3K9 methyltransferase inhibitor BIX01294 also induced the contraction of the expanded repeats and increased frataxin protein in FRDA neural cells by opening the chromatin and increasing the endogenous ssDNA breaks and recruitment of pol ß on the repeats. Our study provides new mechanistic insight illustrating that inhibition of H3K9 methylation can crosstalk with BER to induce GAA repeat contraction in FRDA. Our results will open a new avenue for developing novel gene therapy by targeting histone methylation and the BER pathway for repeat expansion diseases.
Assuntos
Cromatina , Reparo do DNA , Frataxina , Ataxia de Friedreich , Proteínas de Ligação ao Ferro , Camundongos Transgênicos , Expansão das Repetições de Trinucleotídeos , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Animais , Camundongos , Expansão das Repetições de Trinucleotídeos/genética , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Cromatina/metabolismo , Cromatina/genética , Humanos , Dano ao DNA , Temozolomida/farmacologia , Neurônios/metabolismo , DNA Polimerase beta/metabolismo , DNA Polimerase beta/genéticaRESUMO
Friedreich's Ataxia (FRDA) stands out as the most prevalent form of hereditary ataxias, marked by progressive movement ataxia, loss of vibratory sensitivity, and skeletal deformities, severely affecting daily functioning. To date, the only medication available for treating FRDA is Omaveloxolone (Skyclarys®), recently approved by the FDA. Missense mutations within the human frataxin (FXN) gene, responsible for intracellular iron homeostasis regulation, are linked to FRDA development. These mutations induce FXN dysfunction, fostering mitochondrial iron accumulation and heightened oxidative stress, ultimately triggering neuronal cell death pathways. This study amalgamated 226 FXN genetic variants from the literature and database searches, with only 18 previously characterized. Predictive analyses revealed a notable prevalence of detrimental and destabilizing predictions for FXN mutations, predominantly impacting conserved residues crucial for protein function. Additionally, an accurate, comprehensive three-dimensional model of human FXN was constructed, serving as the basis for generating genetic variants I154F and W155R. These variants, selected for their severe clinical implications, underwent molecular dynamics (MD) simulations, unveiling flexibility and essential dynamic alterations in their N-terminal segments, encompassing FXN42, FXN56, and FXN78 domains pivotal for protein maturation. Thus, our findings indicate potential interaction profile disturbances in the FXN42, FXN56, and FXN78 domains induced by I154F and W155R mutations, aligning with the existing literature.
Assuntos
Frataxina , Ataxia de Friedreich , Proteínas de Ligação ao Ferro , Simulação de Dinâmica Molecular , Humanos , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Mutação de Sentido Incorreto , Simulação por Computador , Variação GenéticaRESUMO
Friedreich's ataxia (FRDA) is a rare neurodegenerative disease caused by an expansion of a GAA repeat sequence within the Frataxin (FXN) gene. Prominent regions of neurodegeneration include sensory neurons within the dorsal root ganglia. Here we present a set of genetically modified FRDA induced pluripotent stem cell (iPSC) lines that carry an inducible neurogenin-2 (NGN2) expression cassette. Exogenous expression of NGN2 in iPSC derived neural crest progenitors efficiently generates functionally mature sensory neurons. These cell lines will provide a streamlined source of FRDA iPSC sensory neurons for studying both disease mechanism and screening potential therapeutics.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Proteínas do Tecido Nervoso , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Ataxia de Friedreich/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Linhagem Celular , Diferenciação Celular , FrataxinaRESUMO
The translocator protein TSPO is an evolutionary conserved mitochondrial protein overexpressed in various contexts of neurodegeneration. Friedreich Ataxia (FA) is a neurodegenerative disease due to GAA expansions in the FXN gene leading to decreased expression of frataxin, a mitochondrial protein involved in the biosynthesis of iron-sulfur clusters. We previously reported that Tspo was overexpressed in a Drosophila model of this disease generated by CRISPR/Cas9 insertion of approximately 200 GAA in the intron of fh, the fly frataxin gene. Here, we describe a new Drosophila model of FA with 42 GAA repeats, called fh-GAAs. The smaller expansion size allowed to obtain adults exhibiting hallmarks of the FA disease, including short lifespan, locomotory defects and hypersensitivity to oxidative stress. The reduced lifespan was fully rescued by ubiquitous expression of human FXN, confirming that both frataxins share conserved functions. We observed that Tspo was overexpressed in heads and decreased in intestines of these fh-GAAs flies. Then, we further overexpressed Tspo specifically in glial cells and observed improved survival. Finally, we investigated the effects of Tspo overexpression in healthy flies. Increased longevity was conferred by glial-specific overexpression, with opposite effects in neurons. Overall, this study highlights protective effects of glial TSPO in Drosophila both in a neurodegenerative and a healthy context.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Frataxina , Proteínas de Ligação ao Ferro , Longevidade , Neuroglia , Animais , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Neuroglia/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Modelos Animais de Doenças , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Receptores de GABA/genética , Receptores de GABA/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Drosophila/genética , Animais Geneticamente ModificadosRESUMO
BACKGROUND: Early studies in cellular models suggested an iron accumulation in Friedreich's ataxia (FA), yet findings from patients are lacking. OBJECTIVES: The objective is to characterize systemic iron metabolism, body iron storages, and intracellular iron regulation in FA patients. METHODS: In FA patients and matched healthy controls, we assessed serum iron parameters, regulatory hormones as well as the expression of regulatory proteins and iron distribution in peripheral blood mononuclear cells (PBMCs). We applied magnetic resonance imaging with R2*-relaxometry to quantify iron storages in the liver, spleen, and pancreas. Across all evaluations, we assessed the influence of the genetic severity as expressed by the length of the shorter GAA-expansion (GAA1). RESULTS: We recruited 40 FA patients (19 women). Compared to controls, FA patients displayed lower serum iron and transferrin saturation. Serum ferritin, hepcidin, mean corpuscular hemoglobin and mean corpuscular volume in FA inversely correlated with the GAA1-repeat length, indicating iron deficiency and restricted availability for erythropoiesis with increasing genetic severity. R2*-relaxometry revealed a reduction of splenic and hepatic iron stores in FA. Liver and spleen R2* values inversely correlated with the GAA1-repeat length. FA PBMCs displayed downregulation of ferritin and upregulation of transferrin receptor and divalent metal transporter-1 mRNA, particularly in patients with >500 GAA1-repeats. In FA PBMCs, intracellular iron was not increased, but shifted toward mitochondria. CONCLUSIONS: We provide evidence for a previously unrecognized iron starvation signature at systemic and cellular levels in FA patients, which is related to the underlying genetic severity. These findings challenge the use of systemic iron lowering therapies in FA. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Ataxia de Friedreich , Ferro , Humanos , Ataxia de Friedreich/genética , Ataxia de Friedreich/sangue , Ataxia de Friedreich/metabolismo , Feminino , Masculino , Adulto , Ferro/metabolismo , Fígado/metabolismo , Fígado/patologia , Pessoa de Meia-Idade , Imageamento por Ressonância Magnética , Adulto Jovem , Baço/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Ferritinas/sangue , Ferritinas/metabolismo , Hepcidinas/genética , Hepcidinas/sangue , Hepcidinas/metabolismo , Pâncreas/metabolismo , Pâncreas/patologiaRESUMO
Immunometabolism investigates the intricate relationship between the immune system and cellular metabolism. This study delves into the consequences of mitochondrial frataxin (FXN) depletion, the primary cause of Friedreich's ataxia (FRDA), a debilitating neurodegenerative condition characterized by impaired coordination and muscle control. By using single-cell RNA sequencing, we have identified distinct cellular clusters within the cerebellum of an FRDA mouse model, emphasizing a significant loss in the homeostatic response of microglial cells lacking FXN. Remarkably, these microglia deficient in FXN display heightened reactive responses to inflammatory stimuli. Furthermore, our metabolomic analyses reveal a shift towards glycolysis and itaconate production in these cells. Remarkably, treatment with butyrate counteracts these immunometabolic changes, triggering an antioxidant response via the itaconate-Nrf2-GSH pathways and suppressing the expression of inflammatory genes. Furthermore, we identify Hcar2 (GPR109A) as a mediator involved in restoring the homeostasis of microglia without FXN. Motor function tests conducted on FRDA mice underscore the neuroprotective attributes of butyrate supplementation, enhancing neuromotor performance. In conclusion, our findings elucidate the role of disrupted homeostatic function in cerebellar microglia in the pathogenesis of FRDA. Moreover, they underscore the potential of butyrate to mitigate inflammatory gene expression, correct metabolic imbalances, and improve neuromotor capabilities in FRDA.
Assuntos
Frataxina , Ataxia de Friedreich , Succinatos , Animais , Camundongos , Butiratos , Frataxina/genética , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Glucose , Microglia/metabolismoRESUMO
BACKGROUND: Friedreich's Ataxia (FRDA) is a rare hereditary neurodegenerative disorder characterized by progressive ataxia, cardiomyopathy, and diabetes. The disease is caused by a deficiency of frataxin, a mitochondrial protein involved in iron-sulfur cluster synthesis and iron metabolism. OBJECTIVE: This review aims to summarize recent advances in the development of treatment strategies for FRDA, with a focus on potential drug candidates and their mechanisms of action. METHODS: A comprehensive literature search was conducted using various authentic scientific databases to identify studies published in the last decade that investigated potential treatment strategies for FRDA. The search terms used included "Friedreich's ataxia", "treatment", "drug candidates", and "mechanisms of action". RESULTS: To date, only one drug got approval from US-FDA in the year 2023; however, significant developments were achieved in FRDA-related research focusing on diverse therapeutic interventions that could potentially alleviate the symptoms of this disease. Several promising drug candidates have been identified for the treatment of FRDA, which target various aspects of frataxin deficiency and aim to restore frataxin levels, reduce oxidative stress, and improve mitochondrial function. Clinical trials have shown varying degrees of success, with some drugs demonstrating significant improvements in neurological function and quality of life in FRDA patients. CONCLUSION: While there has been significant progress in the development of treatment strategies for FRDA, further research is needed to optimize these approaches and identify the most effective and safe treatment options for patients. The integration of multiple therapeutic strategies may be necessary to achieve the best outcomes in FRDA management.
Assuntos
Ataxia de Friedreich , Proteínas de Ligação ao Ferro , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Frataxina , AnimaisRESUMO
Friedreich's ataxia is a neurodegenerative disorder caused by the hyper expansion of (GAA-TTC)n triplet repeats in the first intron of the FXN gene. Here, we generated iPSC lines from two individuals with FRDA, both of whom have homozygous GAA repeat expansion in the first intron of FXN gene. Both iPSC lines demonstrated characteristics of pluripotency, including expression of pluripotency markers, stable karyotypes and ability to develop into all three germ layers, and presence of GAA repeat expansion with reduced FXN mRNA expression. These iPSC lines will serve as invaluable tools for investigating the pathophysiology and phenotypes of FRDA.
Assuntos
Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , ÍntronsRESUMO
Nqo15 is a subunit of respiratory complex I of the bacterium Thermus thermophilus, with strong structural similarity to human frataxin (FXN), a protein involved in the mitochondrial disease Friedreich's ataxia (FRDA). Recently, we showed that the expression of recombinant Nqo15 can ameliorate the respiratory phenotype of FRDA patients' cells, and this prompted us to further characterize both the Nqo15 solution's behavior and its potential functional overlap with FXN, using a combination of in silico and in vitro techniques. We studied the analogy of Nqo15 and FXN by performing extensive database searches based on sequence and structure. Nqo15's folding and flexibility were investigated by combining nuclear magnetic resonance (NMR), circular dichroism, and coarse-grained molecular dynamics simulations. Nqo15's iron-binding properties were studied using NMR, fluorescence, and specific assays and its desulfurase activation by biochemical assays. We found that the recombinant Nqo15 isolated from complex I is monomeric, stable, folded in solution, and highly dynamic. Nqo15 does not share the iron-binding properties of FXN or its desulfurase activation function.
Assuntos
Frataxina , Ataxia de Friedreich , Humanos , Complexo I de Transporte de Elétrons/metabolismo , Thermus thermophilus/metabolismo , Simulação de Dinâmica Molecular , Ferro/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Ataxia de Friedreich/metabolismoRESUMO
Friedreich's ataxia (FA) is a debilitating, multisystemic disease caused by the depletion of frataxin (FXN), a mitochondrial iron-sulfur (Fe-S) cluster biogenesis factor. To understand the cellular pathogenesis of FA, we performed quantitative proteomics in FXN-deficient human cells. Nearly every annotated Fe-S cluster-containing protein was depleted, indicating that as a rule, cluster binding confers stability to Fe-S proteins. We also observed depletion of a small mitoribosomal assembly factor METTL17 and evidence of impaired mitochondrial translation. Using comparative sequence analysis, mutagenesis, biochemistry, and cryoelectron microscopy, we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability. METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN-depleted cells. These findings suggest that METTL17 acts as an Fe-S cluster checkpoint, promoting translation of Fe-S cluster-rich oxidative phosphorylation (OXPHOS) proteins only when Fe-S cofactors are replete.
Assuntos
Ataxia de Friedreich , Proteínas Ferro-Enxofre , Humanos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Microscopia Crioeletrônica , Frataxina , Biossíntese de Proteínas , Mitocôndrias/genética , Mitocôndrias/metabolismo , Ataxia de Friedreich/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismoRESUMO
Heart, dentate nucleus, and dorsal root ganglia (DRG) are targets of tissue damage in Friedreich ataxia (FA). This report summarizes the histology and histopathology of iron in the main tissues affected by FA. None of the affected anatomical sites reveals an elevation of total iron levels. In the myocardium, a small percentage of fibers shows iron-reactive granular inclusions. The accumulation of larger iron aggregates and fiber invasion cause necrosis and damage to the contractile apparatus. In the dentate nucleus, the principal FA-caused tissue injury is neuronal atrophy and grumose reaction. X-ray fluorescence mapping of iron in the dentate nucleus in FA shows retention of the metal in the center of the collapsed structure. Immunohistochemistry of ferritin, a surrogate marker of tissue iron, confirms strong expression in oligodendrocytes of the efferent white matter of the dentate nucleus and abundance of ferritin-positive microglia in the atrophic gray matter. Iron dysmetabolism in DRG is complex and consists of prominent expression of ferritin in hyperplastic satellite cells and residual nodules, also a loss of the iron export protein ferroportin from the cytoplasm of the remaining DRG nerve cells.
Assuntos
Ataxia de Friedreich , Ferro , Humanos , Ferro/metabolismo , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Ferritinas/metabolismo , Neurônios/metabolismo , Citoplasma/metabolismoRESUMO
Friedreich ataxia (FA) is an inherited autosomal recessive neurodegenerative disease (NDD) characterized primarily by progressive sensory and spinocerebellar ataxia associated with hypertrophic cardiomyopathy. FA is due to an intronic GAA repeat expansion within the frataxin gene (FXN) leading to reduced levels of frataxin (FXN) which causes mitochondrial dysfunction, production of reactive oxygen species (ROS), and altered iron metabolism. To date there is no resolutive cure for FA; however, the FDA has recently approved omaveloxolone - a potent activator of nuclear factor erythroid 2-related factor 2 (NRF2) - as the first treatment for FA. We discuss herein the urgency to find a resolutive cure for NDDs that will most probably be achieved via combinatorial therapy targeting multiple disease pathways, and how omavaloxolone serves as an example for future treatments.
Assuntos
Cardiomiopatia Hipertrófica , Ataxia de Friedreich , Doenças Neurodegenerativas , Triterpenos , Humanos , Ataxia de Friedreich/tratamento farmacológico , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Triterpenos/uso terapêutico , FrataxinaRESUMO
Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional Fxn allele and one floxed-out/null allele. These mice quickly develop cardiomyopathy that becomes fatal by 9-11 wk of age. Here, we generated a cardiac-specific model with floxed Fxn allele homozygosity (MCK-Fxnflox/flox). MCK-Fxnflox/flox mice were phenotypically normal at 9 wk of age, despite no detectable FXN protein expression. Between 13 and 15 wk of age, these mice began to display progressive cardiomyopathy, including decreased ejection fraction and fractional shortening and increased left ventricular mass. MCK-Fxnflox/flox mice began to lose weight around 16 wk of age, characteristically associated with heart failure in other cardiac-specific FA models. By 18 wk of age, MCK-Fxnflox/flox mice displayed elevated markers of Fe-S deficiency, cardiac stress and injury, and cardiac fibrosis. This modified model reproduced important pathophysiological and biochemical features of FA over a longer timescale than previous cardiac-specific mouse models, offering a larger window for studying potential therapeutics.NEW & NOTEWORTHY Previous cardiac-specific frataxin knockout models exhibit rapid and fatal cardiomyopathy by 9 wk of age. This severe phenotype poses challenges for the design and execution of intervention studies. We introduce an alternative cardiac-specific model, MCK-Fxnflox/flox, with increased longevity and delayed onset of all major phenotypes. These phenotypes develop to the same severity as previous models. Thus, this new model provides the same cardiomyopathy-associated mortality with a larger window for potential studies.
Assuntos
Cardiomiopatias , Ataxia de Friedreich , Humanos , Camundongos , Animais , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Alelos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Modelos Animais de Doenças , Frataxina , Miócitos Cardíacos/metabolismoRESUMO
Friedreich's ataxia (FRDA) is a rare neurodegenerativedisorder caused by over expansion of GAA repeats in thefirstintron ofFXN gene. Here, we generated two iPSC lines from FRDA patients with biallelic expansion of GAA repeats in the first intron ofFXNgene.IGIBi014-A and IGIBi015-Aboth iPSC lines demonstrated characteristics of pluripotency, normal karyotypes (46, XY),the capacity to differentiate into all three germ layers, and the ability to sustain the GAA repeat expansion with decreased FXN mRNA expression. These cell lines will be utilized to comprehend the pathophysiology of the illness and the FRDA's predictive phenotypes.
Assuntos
Ataxia de Friedreich , Células-Tronco Pluripotentes Induzidas , Humanos , Ataxia de Friedreich/metabolismo , Íntrons/genética , Frataxina , Células-Tronco Pluripotentes Induzidas/metabolismo , Linhagem Celular , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
BACKGROUND: Friedreich ataxia is the most common inherited ataxia in Europe and is mainly caused by biallelic pathogenic expansions of the GAA trinucleotide repeat in intron 1 of the FXN gene that lead to a decrease in frataxin protein levels. Rarely, affected individuals carry either a large intragenic deletion or whole-gene deletion of FXN on one allele and a full-penetrance expanded GAA repeat on the other allele. CASE PRESENTATION: We report here a patient that presented the typical clinical features of FRDA and genetic analysis of FXN intron 1 led to the assumption that the patient carried the common biallelic expansion. Subsequently, parental sample testing led to the identification of a novel intragenic deletion involving the 5'UTR upstream region and exons 1 and 2 of the FXN gene by MLPA. CONCLUSIONS: With this case, we want to raise awareness about the potentially higher prevalence of intragenic deletions and underline the essential role of parental sample testing in providing accurate genetic counselling.
Assuntos
Ataxia de Friedreich , Humanos , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Expansão das Repetições de Trinucleotídeos , Fenótipo , Éxons , ÍntronsRESUMO
Friedreich ataxia (FRDA) is a rare, inherited neurodegenerative disease caused by an expanded GAA repeat in the first intron of the FXN gene, leading to transcriptional silencing and reduced expression of frataxin. Frataxin participates in the mitochondrial assembly of FeS clusters, redox cofactors of the respiratory complexes I, II and III. To date it is still unclear how frataxin deficiency culminates in the decrease of bioenergetics efficiency in FRDA patients' cells. We previously demonstrated that in healthy cells frataxin is closely attached to the mitochondrial cristae, which contain both the FeS cluster assembly machinery and the respiratory chain complexes, whereas in FRDA patients' cells with impaired respiration the residual frataxin is largely displaced in the matrix. To gain novel insights into the function of frataxin in the mitochondrial pathophysiology, and in the upstream metabolic defects leading to FRDA disease onset and progression, here we explored the potential interaction of frataxin with the FeS cluster-containing respiratory complexes I, II and III. Using healthy cells and different FRDA cellular models we found that frataxin interacts with these three respiratory complexes. Furthermore, by EPR spectroscopy, we observed that in mitochondria from FRDA patients' cells the decreased level of frataxin specifically affects the FeS cluster content of complex I. Remarkably, we also found that the frataxin-like protein Nqo15 from T. thermophilus complex I ameliorates the mitochondrial respiratory phenotype when expressed in FRDA patient's cells. Our data point to a structural and functional interaction of frataxin with complex I and open a perspective to explore therapeutic rationales for FRDA targeted to this respiratory complex.
Assuntos
Ataxia de Friedreich , Doenças Neurodegenerativas , Humanos , Transporte de Elétrons , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Membranas Mitocondriais/metabolismo , Doenças Neurodegenerativas/metabolismoRESUMO
Deficiency in human mature frataxin (hFXN-M) protein is responsible for the devastating neurodegenerative and cardiodegenerative disease of Friedreich's ataxia (FRDA). It results primarily through epigenetic silencing of the FXN gene by GAA triplet repeats on intron 1 of both alleles. GAA repeat lengths are most commonly between 600 and 1200 but can reach 1700. A subset of approximately 3% of FRDA patients have GAA repeats on one allele and a mutation on the other. FRDA patients die most commonly in their 30s from heart disease. Therefore, increasing expression of heart hFXN-M using gene therapy offers a way to prevent early mortality in FRDA. We used rhesus macaque monkeys to test the pharmacology of an adeno-associated virus (AAV)hu68.CB7.hFXN therapy. The advantage of using non-human primates for hFXN-M gene therapy studies is that hFXN-M and monkey FXN-M (mFXN-M) are 98.5% identical, which limits potential immunologic side-effects. However, this presented a formidable bioanalytical challenge in quantification of proteins with almost identical sequences. This could be overcome by the development of a species-specific quantitative mass spectrometry-based method, which has revealed for the first time, robust transgene-specific human protein expression in monkey heart tissue. The dose response is non-linear resulting in a ten-fold increase in monkey heart hFXN-M protein expression with only a three-fold increase in dose of the vector.
Assuntos
Ataxia de Friedreich , Proteínas de Ligação ao Ferro , Animais , Humanos , Macaca mulatta , Proteínas de Ligação ao Ferro/genética , Coração , Ataxia de Friedreich/genética , Ataxia de Friedreich/terapia , Ataxia de Friedreich/metabolismo , Terapia Genética , FrataxinaRESUMO
Cardiomyopathy is often fatal in Friedreich ataxia (FA). However, FA hearts maintain adequate function until advanced disease stages, suggesting initial adaptation to the loss of frataxin (FXN). Conditional cardiac knockout mouse models of FXN show transcriptional and metabolic profiles of the mitochondrial integrated stress response (ISRmt), which could play an adaptive role. However, the ISRmt has not been investigated in models with disease-relevant, partial decrease in FXN. We characterized the heart transcriptomes and metabolomes of three mouse models with varying degrees of FXN depletion: YG8-800, KIKO-700 and FXNG127V. Few metabolites were changed in YG8-800 mice, which did not provide a signature of cardiomyopathy or ISRmt; several metabolites were altered in FXNG127V and KIKO-700 hearts. Transcriptional changes were found in all models, but differentially expressed genes consistent with cardiomyopathy and ISRmt were only identified in FXNG127V hearts. However, these changes were surprisingly mild even at advanced age (18â months), despite a severe decrease in FXN levels to 1% of those of wild type. These findings indicate that the mouse heart has low reliance on FXN, highlighting the difficulty in modeling genetically relevant FA cardiomyopathy.
Assuntos
Cardiomiopatias , Ataxia de Friedreich , Camundongos , Animais , Multiômica , Coração , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Cardiomiopatias/genética , Camundongos Knockout , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , FrataxinaRESUMO
In a currently 13-year-old girl of consanguineous Turkish parents, who developed unsteady gait and polyneuropathy at the ages of 3 and 6 years, respectively, we performed whole genome sequencing and identified a biallelic missense variant c.424C>T, p.R142W in glypican 1 (GPC1) as a putative disease-associated variant. Up to date, GPC1 has not been associated with a neuromuscular disorder, and we hypothesized that this variant, predicted as deleterious, may be causative for the disease. Using mass spectrometry-based proteomics, we investigated the interactome of GPC1 WT and the missense variant. We identified 198 proteins interacting with GPC1, of which 16 were altered for the missense variant. This included CANX as well as vacuolar ATPase (V-ATPase) and the mammalian target of rapamycin complex 1 (mTORC1) complex members, whose dysregulation could have a potential impact on disease severity in the patient. Importantly, these proteins are novel interaction partners of GPC1. At 10.5 years, the patient developed dilated cardiomyopathy and kyphoscoliosis, and Friedreich's ataxia (FRDA) was suspected. Given the unusually severe phenotype in a patient with FRDA carrying only 104 biallelic GAA repeat expansions in FXN, we currently speculate that disturbed GPC1 function may have exacerbated the disease phenotype. LC-MS/MS data are accessible in the ProteomeXchange Consortium (PXD040023).