Mitochondrial dysfunction in friedreich's ataxia

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disorder caused in the vast majority of cases by a GAA triplet expansion in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein which is severely reduced in FRDA patients. Loss of the homologue of frataxin in yeast is associated with mitochondrial iron overload, increased sensitivity to oxidative stress and profound deficit of oxidative phosphorylation. The demonstration that the human pathology of FRDA is also characterised by mitochondrial iron accumulation, deficit of respiratory chain complex activities and in vivo deficit of tissue energy metabolism establishes FRDA as a 'new' nuclear encoded mitochondrial disease.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ∆yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

Friedreich ataxia: a paradigm for mitochondrial diseases

Friedreich ataxia (FRDA), a progressive neurodegenerative disease, is due to the partial loss of function of frataxin, a mitochondrial protein of unknown function. Loss of frataxin causes mitochondrial iron accumulation, deficiency in the activities of iron-sulfur (Fe-S) proteins, and increased oxidative stress. Mouse models for FRDA demonstrate that the Fe-S deficit precedes iron accumulation, suggesting that iron accumulation is a secondary event. Furthermore, increased oxidative stress in FRDA patients has been demonstrated, and in vitro experiments imply that the frataxin defect impairs early antioxidant defenses. These results taken together suggest that frataxin may function either in mitochondrial iron homeostasis, in Fe-S cluster biogenesis, or directly in the response to oxidative stress. It is clear, however, that the pathogenic mechanism in FRDA involves free-radical production and oxidative stress, a process that appears to be sensitive to antioxidant therapies.     

Mitochondrial Dysfunction in Friedreich's Ataxia: From Pathogenesis to Treatment Perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Mitochondrial dysfunction in Friedreich's ataxia: from pathogenesis to treatment perspectives

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q 10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.     

Deferiprone and idebenone rescue frataxin depletion phenotypes in a Drosophila model of Friedreich's ataxia

Friedreich's ataxia (FRDA), the most common inherited ataxia, is a neurodegenerative disease caused by a reduction in the levels of the mitochondrial protein frataxin, the function of which remains a controversial matter. Several therapeutic approaches are being developed to increase frataxin expression and reduce the intramitochondrial iron aggregates and oxidative damage found in this disease. In this study, we tested separately the response of a Drosophila RNAi model of FRDA ( Llorens et al., 2007) to treatment with the iron chelator deferiprone (DFP) and the antioxidant idebenone (IDE), which are both in clinical trials. The FRDA flies have a shortened life span and impaired motor coordination, and these phenotypes are more pronounced in oxidative stress conditions. In addition, under hyperoxia, the activity of the mitochondrial enzyme aconitase is strongly reduced in the FRDA flies. This study reports that DFP and IDE improve the life span and motor ability of frataxin-depleted flies. We show that DFP eliminates the excess of labile iron in the mitochondria and thus prevents the toxicity induced by iron accumulation. IDE treatment rescues aconitase activity in hyperoxic conditions. These results validate the use of our Drosophila model of FRDA to screen for therapeutic molecules to treat this disease.     

Blood cells from Friedreich ataxia patients harbor frataxin deficiency without a loss of mitochondrial function

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.     

Changes in mitochondrial glutathione levels and protein thiol oxidation in ?yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient ?yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in α-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.     

Rapamycin reduces oxidative stress in frataxin-deficient yeast cells

Friedreich ataxia (FRDA) is a common form of ataxia caused by decreased expression of the mitochondrial protein frataxin. Oxidative damage of mitochondria is thought to play a key role in the pathogenesis of the disease. Therefore, a possible therapeutic strategy should be directed to an antioxidant protection against mitochondrial damage. Indeed, treatment of FRDA patients with the antioxidant idebenone has been shown to improve neurological functions. The yeast frataxin knock-out model of the disease shows mitochondrial iron accumulation, iron-sulfur cluster defects and high sensitivity to oxidative stress. By flow cytometry analysis we studied reactive oxygen species (ROS) production of yeast frataxin mutant cells treated with two antioxidants, N-acetyl-L-cysteine and a mitochondrially-targeted analog of vitamin E, confirming that mitochondria are the main site of ROS production in this model. Furthermore we found a significant reduction of ROS production and a decrease in the mitochondrial mass in mutant cells treated with rapamycin, an inhibitor of TOR kinases, most likely due to autophagy of damaged mitochondria.     

Role of Oxidative Damage in Friedreich's Ataxia

Plasma malondialdehyde (MDA) levels were raised in Friedreich's ataxia (FRDA) patients. These levels correlated with increasing age and disease duration, suggesting lipid peroxidation increased with disease progression. Using fibroblasts from FRDA patients we observed that GSH levels and aconitase activities were normal, suggesting their antioxidant status was unchanged. When exposed to various agents to increase free radical generation we observed that intracellular superoxide generation induced by paraquat caused enhanced oxidative damage. This correlated with the size of the GAA1 expansion, suggesting decreased frataxin levels may render the cells more vulnerable to mild oxidative stress. More severe oxidative stress induced by hydrogen peroxide caused increased cell death in FRDA fibroblasts but was not significantly different from control cells. We propose that abnormal respiratory chain function and iron accumulation may lead to a progressive increase in oxidative damage, but increased sensitivity to free radicals may not require detectable respiratory chain dysfunction.     

Actin glutathionylation increases in fibroblasts of patients with Friedreich's ataxia: a potential role in the pathogenesis of the disease

Increasing evidence suggests that iron-mediated oxidative stress might underlie the development of neurodegeneration in Friedreich's ataxia (FRDA), an autosomal recessive ataxia caused by decreased expression of frataxin, a protein implicated in iron metabolism. In this study, we demonstrate that, in fibroblasts of patients with FRDA, the cellular redox equilibrium is shifted toward more protein-bound glutathione. Furthermore, we found that actin is glutathionylated, probably as a result of the accumulation of reactive oxygen species, generated by iron overload in the disease. Indeed, high-pressure liquid chromatography analysis of control fibroblasts in vivo treated with FeSO4 showed a significant increase in the protein-bound/free GSH ratio, and Western blot analysis indicated a relevant rise in glutathionylation. Actin glutathionylation contributes to impaired microfilament organization in FRDA fibroblasts. Rhodamine phalloidin staining revealed a disarray of actin filaments and a reduced signal of F-actin fluorescence. The same hematoxylin/eosin-stained cells showed abnormalities in size and shape. When we treated FRDA fibroblasts with reduced glutathione, we obtained a complete rescue of cytoskeletal abnormalities and cell viability. Thus, we conclude that oxidative stress may induce actin glutathionylation and impairment of cytoskeletal functions in FRDA fibroblasts.     

Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.     

Friedreich's ataxia, no changes in mitochondrial labile iron in human lymphoblasts and fibroblasts: a decrease in antioxidative capacity?

Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with (55)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H(2)O(2) was significantly increased in patient cells, and H(2)O(2) toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2'-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.     

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.     

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

Background

Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)_n expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology

We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN^G130V and hFXN^I154F) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN^I154F mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions

These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.     

Overexpression of frataxin in the mitochondria increases resistance to oxidative stress and extends lifespan in Drosophila

In Friedreich's ataxia, reduction of the mitochondria protein frataxin results in the accumulation of iron and reactive oxygen species, which leads to oxidative damage, neurodegeneration and a diminished lifespan. Recent studies propose that frataxin might play a role in the antioxidative process. Here we show that overexpression of Drosophila frataxin in the mitochondria of female transgenic animals increases antioxidant capability, resistance to oxidative stress insults, and longevity. This suggests that Drosophila frataxin may function to protect the mitochondria from oxidative stresses and the ensuing cellular damage.     

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.Supplementary key words: frataxin, ceramides, fatty acids oxidation, triglycerides, phospholipids, lipidomics, lipid remodeling, neurodegenerative disorders, triplet repeat expansion, stable isotope tracing

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder with an incidence of 1 in 29,000 (1). Currently it has no approved treatment (1). The main clinical features in FRDA include gait and limb ataxia, dysarthria, sensory loss, and cardiomyopathy (2). Heart failure from cardiomyopathy is the primary cause of death in the majority of patients with FRDA (3). FRDA is caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene that leads to gene silencing and decreased levels of the mitochondrial protein frataxin (4). The number of GAA repeats inversely correlates with frataxin protein level and age of disease onset, both of which determine disease severity (5, 6). The tissues most affected are the heart, dorsal root ganglia, posterior columns of the spinal cord, dentate nucleus, and corticospinal tracts. The exact mechanism by which frataxin deficiency leads to neuro- and cardiodegeneration is not completely understood.One function of frataxin is in the formation of the iron-sulfur (Fe-S) cluster prosthetic groups that are critical for enzymes in the Krebs cycle (aconitase), oxidative phosphorylation (electron transport chain components of complexes I–III), and fatty acid breakdown (β-oxidation) (7, 8). Frataxin localization in the mitochondria (9) further suggests that mitochondrial dysfunction plays a role in FRDA. Decreased conversion of labeled glucose to acetyl-CoA in platelets from patients with FRDA (10) is consistent with studies that show diminished pyruvate oxidation in FRDA (10). Increased incorporation of labeled palmitate into HMG-CoA, an important intermediate in ketogenesis and sterol synthesis, in patients with FRDA suggests increased fatty acid metabolism through β-oxidation (11). Increased β-oxidation produces FADH₂ and NADH that can be utilized to maintain the electrochemical gradient across the inner mitochondrial membrane needed for ATP synthesis. Therefore, increased lipid metabolism observed in FRDA could be important to maintain cellular homeostasis during mitochondrial dysfunction.A recent study found reactive oxygen species-independent accumulation of iron in the nervous system of an FRDA fly model with a mutant frataxin homolog, associated with enhanced sphingolipid synthesis (12). Sphingolipids are linked to increased inflammation (13) and activate 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2) to trigger neurodegeneration (12). The findings in the fly model were replicated in a frataxin knockdown mouse model suggesting that the mechanism is evolutionarily conserved (14). PDK1 activity and sphingolipid levels were also elevated in heart tissues of patients with FRDA compared with healthy controls suggesting that a similar pathway may be activated in humans with FRDA (14).Ceramides are central intermediates in sphingolipid metabolism and have been implicated in several cellular processes including apoptosis (15). Dysregulated ceramides have been the focus of study in a variety of cardiac diseases. High ceramide ratios of Cer 16:0 and 18:0 to Cer 24:0 in plasma are strongly associated with increased risk for major adverse cardiac events (16). Furthermore, increased ceramide levels have been associated with diabetic cardiomyopathy (17) and increased de novo ceramide synthesis has been linked to advanced heart failure (18). The observation of elevated ceramides in FRDA heart tissue raises the question of whether sphingolipids will be dysregulated in other affected and nonaffected tissues.Ideally, metabolic and lipidomic abnormalities should be studied in the most affected tissues, but frataxin deficiency is present in all tissues to different extents (19). Since it is difficult to sample human cardiac tissue from living individuals, peripheral tissues, such as fibroblasts, can be used as models to study metabolic profiles of FRDA. Fibroblasts in culture have the additional advantage of not being influenced by diet or environment, thus providing a stable system for comparing metabolic flux between patients and controls. Recently, RNA sequencing and gene ontology analysis was used to identify differentially expressed genes between FRDA and healthy control fibroblasts and indicated that fibroblasts are an accessible system to study dysregulated pathways in FRDA (20). In the present study, we used highly sensitive and specific liquid chromatography-high resolution mass spectrometry (LC-HRMS) assays to perform metabolomic and lipidomic profiles in fibroblast cells from patients with FRDA with different disease severities. This study complements the RNA sequencing data and gives new insights into the disease mechanism.     

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.