Treatment and prediction of therapeutic response in patients with Friedreich's ataxia

Abstract

To provide methods for treating Friedreich ataxia (FRDA), and methods for predicting whether a patient of FRDA can achieve therapeutic response or not.SOLUTION: A method for treating FRDA comprises a step of administering to a patient a therapeutically effective amount of an agent capable of increasing palmitoylation of transferrin receptor (TfR1). A method for predicting whether a patient suffering from FRDA can achieve therapeutic response or not comprises the steps of: i) determining total intracellular iron content of peripheral blood monocyte (PBMC) obtained from a subject and cultured in a medium containing a predetermined amount of iron; ii) comparing the iron content obtained in step i) with a predetermined reference value; and iii) determining whether the subject will achieve the response or not when the difference between the content value determined in step i) and the predetermined reference value is detected.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of the invention: The present invention relates to methods for treating Friedreich's ataxia (FRDA) as well as methods for predicting whether a patient with FRDA will achieve a therapeutic response.

[0002] Background of the invention: Friedreich's ataxia (FRDA) is a frequent autosomal recessive degenerative disorder (1 in 50,000 births) characterized by progressive gait and limb ataxia, loss of leg tendon reflexes, dysarthria, and weakness of the pyramidal tracts of the lower limbs. Hypertrophic cardiomyopathy is observed in most patients with Friedreich's ataxia. The gene that causes the disease, FXN, encodes the 210-amino acid mitochondrial protein frataxin. FRDA is primarily caused by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles, resulting in reduced steady-state levels of frataxin. Frataxin is involved in the first step of iron-sulfur complex (ISC) assembly, in which the [2Fe-2S] cluster is assembled on the Isu1 scaffold protein. This first step involves five other proteins, including ferredoxin reductase, which enables sulfur reduction. However, the consequences of frataxin deficiency remain incompletely understood. Indeed, although defects in the mitochondrial respiratory chain have been implicated in impaired assembly of complexes I–III iron–sulfur complexes (Rotig et al., 1997), many other features remain unexplained, including intramitochondrial iron accumulation at the expense of the cytoplasm accompanied by increased reactive oxygen species (ROS) (Vaubel and Isaya, 2013), dysregulation of intracellular iron metabolism (Martelli and Puccio, 2014), mitochondrial protein hyperacetylation mediated by SIRT3 inhibition (Wagner et al., 2012), and increased sphingolipid synthesis (Chen et al., 2016a). Recently, a novel mechanism of regulation of transferrin receptor (TfR1) by palmitoylation and its alterations in neurodegenerative disorders with brain iron accumulation (NBIA), a heterogeneous condition resulting from iron accumulation in the basal ganglia, was reported ( Drecourt et al., 2018 ). However, the existence of a similar mechanism in FRDA has not been investigated so far.

[0003] Summary of the Invention: As defined by the claims, the present invention relates to methods for treating Friedreich's ataxia (FRDA) as well as methods for predicting whether a patient with FRDA will achieve a therapeutic response.

[0004] Detailed description of the invention: Friedreich's ataxia (FRDA) is caused by a GAA repeat expansion in the FXN gene, which encodes frataxin, a mitochondrial protein involved in iron-sulfur complex (ISC) assembly. Frataxin deficiency results in abnormal ISC-containing proteins, namely respiratory chain complexes I-III and aconitase, leading to iron accumulation in the brain and heart of affected individuals. Here, we show that FRDA fibroblasts are unable to limit iron uptake, leading to massive cytoplasmic iron accumulation and, to a lesser extent, mitochondrial accumulation. We also observed increased steady-state levels of transferrin receptor (TfR1) and membrane accumulation of TfR1, which we attribute to impaired posttranslational modification by palmitoylation and delayed transferrin recycling. Finally, we have shown that artesunate, dichloroacetic acid, and coenzyme A improve palmitoylation of TfR1 and therefore represent candidate molecules for the treatment of patients with Friedreich's ataxia.

[0005] Treatment: Therefore, a first object of the present invention relates to a method for treating Friedreich's ataxia (FRDA) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent capable of increasing palmitoylation of TfR1.

[0006] As used herein, the term "Friedreich's ataxia" or "FRDA" has its common meaning in the art and refers to a frequent autosomal recessive degenerative disease (1 in 50,000 births) characterized by progressive gait and limb ataxia, loss of leg tendon reflexes, dysarthria, and weakness of the pyramidal tracts of the lower extremities. Hypertrophic cardiomyopathy is observed in most patients with Friedreich's ataxia. The gene causing the disease, FXN, encodes frataxin, a 210-amino acid mitochondrial protein. FRDA is primarily caused by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles, resulting in reduced steady-state levels of frataxin.

[0007] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment, as well as curative or disease-modifying treatment (including treatment of patients at risk of or suspected of having a disease, as well as treatment of patients who are ill or have been diagnosed with a disease or medical condition), including the suppression of clinical recurrence. Treatment may be administered to a subject who has a medical disorder or who may ultimately suffer from a disorder in order to prevent, cure, or delay the onset of the disorder or a recurring disorder, reduce its severity, or ameliorate one or more symptoms thereof, or to prolong the subject's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of disease treatment, e.g., a dosing pattern used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. The induction regimen may (in part or in whole) use a "loading regimen," which may involve administering a higher dose of drug than the physician would use during a maintenance regimen, administering drug more frequently than the physician would use during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used to maintain a patient during disease treatment, e.g., to keep the patient in remission over an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment when certain predetermined criteria (e.g., disease manifestation) are reached).

[0008] In some embodiments, the drug is selected from the group consisting of artesunate, dichloroacetic acid, and coenzyme A.

[0009] In some embodiments, the drug is artesunate. As used herein, the term "artesunate" has its general meaning in the art and refers to (3R,5aS,6R,8aS,9R,10S,12R,12aR)-decahydro-3,6,9-trimethyl-3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10-ol, hydrogen succinate. The term encompasses any individual enantiomer of artesunate. In particular, the term can refer to a single enantiomer, or a racemic or non-racemic mixture of enantiomers. The term also includes polymorphs and hydrates of artesunate. The term also includes salts and esters of artesunate. The term also includes prodrugs of artesunate, as well as enantiomers, racemic mixtures, non-racemic mixtures, polymorphs, hydrates, salts, and esters of the prodrugs.

[0010] As used herein, the term "dichloroacetic acid" has its common meaning in the art and refers to the compound of formula CHClCOOH, which is also known as dichloroethanolic acid, bichloroacetic acid, DCA, BCA, i.e., dichloroacetic acid, bichloroacetic acid.

[0011] As used herein, the term "coenzyme A" has its general meaning in the art and refers to a coenzyme known particularly for its role in the synthesis and oxidation of fatty acids and the oxidation of pyruvate in the citric acid cycle. Its IUPAC name is [[2R,3S,4R,5R]-5-(6-aminopurin-9-yl)-4-hydroxy-3-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl][(3R)-3-hydroxy-2,2-dimethyl-4-oxo-4-[[3-oxo-3-(2-sulfanylethylamino)propyl]amino]butyl] hydrogen phosphate.

[0012] By "therapeutically effective amount" of a drug as defined above, we mean an amount sufficient to produce a therapeutic effect. However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the subject's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concomitantly with the specific polypeptide used; and similar factors well known in the medical field. For example, it is well within the skill of the art to start doses of a compound at levels lower than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of a product can vary over a wide range, from 0.01 mg to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient, with dosage adjustment depending on the symptoms of the subject being treated. Pharmaceutical preparations typically contain from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0013] According to the present invention, the drug is administered to a subject in the form of a pharmaceutical composition. Typically, the drug can be combined with a pharmaceutically acceptable excipient and, optionally, a sustained-release matrix, such as a biodegradable polymer, to form a therapeutic composition. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered appropriately to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or any type of formulation auxiliary. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active ingredient can be administered alone or in combination with another active ingredient in a unit dosage form, in a mixture with a conventional pharmaceutical support, to animals and humans. Suitable unit dosage forms include oral route dosage forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, and intranasal dosage forms, and rectal dosage forms. Typically, pharmaceutical compositions contain a pharmaceutically acceptable vehicle for injectable formulations. These may be, in particular, isotonic sterile saline solutions (such as monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or dried, especially lyophilized, compositions that can be reconstituted for injection by adding sterile water or saline, as appropriate. Pharmaceutical dosage forms suitable for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Solutions containing the compounds of the present invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The drug can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, or calcium, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use of an absorption-delaying agent, for example, gelatin, in the composition. Sterile injectable solutions are prepared by incorporating the required amount of the active compound in the appropriate solvent, along with several other ingredients as enumerated above, as needed, followed by sterile filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredient from a previously sterile-filtered solution thereof.The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, and the use of DMSO as a solvent is envisioned, which allows for extremely rapid penetration and delivery of high concentrations of the active agent. When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, for example, the types of injection solutions described above, but drug-release capsules and the like can also be used. For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dosage for the individual subject.

[0014] Methods for predicting treatment response: A further object of the present invention relates to a method for determining whether a subject will achieve a response using a drug capable of increasing palmitoylation of TfR1, comprising the steps of: i) measuring the total intracellular iron content of peripheral blood mononuclear cells (PBMCs) obtained from the subject cultured in a medium containing an amount of iron; ii) comparing the iron content measured in step i) with a predetermined reference value; and iii) concluding whether the subject will achieve a response if a difference between the measured content in step i) and the predetermined reference value is detected.

[0015] Therefore, the method is particularly suitable for distinguishing responders from non-responders. As used herein, the term "responder" in the context of the present disclosure refers to a patient who will achieve a response. For example, reduced gait and / or limb ataxia, improved leg tendon reflexes, reduced dysarthria and / or lower limb pyramidal weakness, and improved cardiac function are characteristics of a patient achieving a response. Typically, the characterization of a patient as a responder or non-responder can be performed by reference to a standard or training set. The standard can be a profile of patients known to be responders or non-responders, or alternatively, a numerical value. Such a predetermined standard can be provided in any suitable form, such as a printed list or diagram, a computer software program, or other media. If a patient is concluded to be a non-responder, the physician can decide to discontinue treatment to avoid any further adverse side effects.

[0016] The methods of the present invention are particularly suitable for detecting earlier patient responses related to a decrease in total cellular iron content. Therefore, measurement of total iron content represents a surrogate marker for assessing the probability of treatment success (i.e., response). Thus, the methods of the present invention are performed once a patient has been treated with a drug (e.g., after 1, 2, 3, 4, or 5 cycles of treatment).

[0017] In some embodiments, the drug is selected from the group consisting of artesunate, dichloroacetic acid, and coenzyme A.

[0018] As used herein, the terms "PBMC" or "peripheral blood mononuclear cells" or "unfractionated PBMC" refer to complete PBMCs, i.e., a population of white blood cells with round nuclei that are not enriched for a given subpopulation. Umbilical cord blood mononuclear cells are also included in this definition. Typically, a PBMC sample according to the present invention has not been subjected to a selection process to contain only adherent PBMCs (consisting substantially of more than 90% monocytes) or non-adherent PBMCs (containing T cells, B cells, natural killer (NK) cells, NK T cells, and dendritic progenitor cells). Therefore, a PBMC sample according to the present invention contains lymphocytes (B cells, T cells, NK cells, NK T cells), monocytes, and their progenitors. Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates the blood layers, with the PBMCs forming a cell ring below the plasma layer. Furthermore, PBMCs can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells.

[0019] Any culture medium suitable for the proliferation, survival, and differentiation of PBMCs can be used. Typically, it consists of a basal medium containing nutrients (carbon source, amino acid source), pH buffer, and salts, which may be supplemented with serum and / or growth factors and / or antibiotics of human or other origin. Typically, the basal medium can be RPMI 1640, DMEM, IMDM, X-VIVO, or AIM-V medium, all of which are commercially available standard media.

[0020] Typically, the source of iron in the culture medium is provided by ferric ammonium citrate (FAC), a soluble form of non-transferrin-bound iron (NTBI) that enters cells in a timely manner via endogenous transporters or endocytic pathways.

[0021] In some embodiments, total iron content is measured by any method known in the art. Typically, total iron content is measured using a ferrozine-based iron assay modified from (Barbeito et al., Mol Neurodegener. 2010 Nov 10;5:50).

[0022] Typically, the PMBCs are cultured for 1;2;3;4;5;6;7;8;9;10;11;12;13;14;15;16;17;18;19;20;21;22;23;24;25;26;27;28;29;30;31;32;33;34;35;36;37;38;39;40;41;42;43;44;45;46;47;48;49; or 50 hours before measuring the total iron content, particularly, the PMBCs are cultured for 0, 8, 16, 24, 32, or 40 hours before measuring the total iron content.

[0023] In some embodiments, the predetermined reference value is a threshold value. The threshold value must be determined to obtain optimal sensitivity and specificity according to the test function and the benefit / risk balance (clinical results of false positives and false negatives). Typically, the optimal sensitivity and specificity (and also the threshold value) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the total iron content in a reference group, an algorithmic analysis is used for statistical processing of the levels of immune markers measured in the samples to be tested, thereby obtaining a classification standard with significance for sample classification. The full name of the ROC curve is the receiver operating characteristic curve, which is also known as the receiver operating characteristic curve. It is mainly used for biochemical clinical diagnostic tests. The ROC curve is a comprehensive index reflecting the continuous variables of the true positive rate (sensitivity) and the false positive rate (1-specificity). The ROC curve uses image synthesis to reveal the relationship between sensitivity and specificity. A series of different cutoff values ​​(thresholds or critical values, i.e., boundary values ​​between normal and abnormal results of a diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Sensitivity is then used as the vertical coordinate and specificity is used as the horizontal coordinate to plot the curve. The higher the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the upper left corner of the coordinate diagram is the critical point, which has both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When the AUC is greater than 0.5, the closer the AUC is to 1, the better the diagnostic result. When the AUC is between 0.5 and 0.7, the accuracy is low. When the AUC is between 0.7 and 0.9, the accuracy is moderate. When the AUC is greater than 0.9, the accuracy is very high. This algorithm is preferably implemented using a computer.Existing software or systems in the art can be used to draw ROC curves, such as MedCalc 9.2.0.1 Medical Statistics Software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Silver Spring, MD, USA), etc.

[0024] In some embodiments, the predetermined reference value is the total iron content of PBMCs obtained from the patient before treatment. In such embodiments, if the total iron content is lower than the predetermined reference value (typically by 0.5; 1; 2; 3; 4; 5; 6; 7; 8; 9; or 10 times or more), it is concluded that the patient will achieve a response with the drug. Conversely, if the total iron content is approximately the same as (or higher than) the predetermined reference value, it is concluded that the patient will not achieve a response with the drug.

[0025] In some embodiments, the predetermined reference value is the total iron content of PBMCs obtained from the patient before treatment and cultured in the presence of the drug. In such embodiments, if the total iron content is higher than the predetermined reference value (typically by 0.5; 1; 2; 3; 4; 5; 6; 7; 8; 9; or 10 times or more), it is concluded that the patient will not achieve a response with the drug. Conversely, if the total iron content is approximately the same as (or lower than) the predetermined reference value, it is concluded that the patient will achieve a response with the drug.

[0026] The present invention will be further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0027] [Figure 1A]Characterization of FRDA fibroblasts. A. Western blot analysis of frataxin and proteins involved in iron homeostasis and antioxidant defense in fibroblasts from five patients (P1-5) with GAA repeat expansions and controls (C1-3). Frataxin, ferritin, IRP1 (iron regulatory protein 1), IRP2, SOD1, and FBXL5 were tested under reducing conditions, while TfR1, SOD2, and ferredoxin reductase (FDXR) were tested under nonreducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation). GAPDH was used as a loading control. B. Measurement of mitochondrial reactive oxygen species (ROS) by flow cytometry using MitoSox in control (C1-3) and FRDA fibroblasts (P1-5) grown in normal medium (+ fetal bovine serum (FBS), - ferric ammonium citrate (FAC)) or high-iron medium (- FBS, + 100 μM FAC). Data show the percentage of MitoSox-positive cells in controls and patients. C. Iron quantification using a ferrozine-based colorimetric assay in complete fibroblasts or mitochondrial extracts (D) from control (C1-3) and FRDA patients (P1-5) grown in normal medium (+ FBS, - FAC). E. Ratio of intramitochondrial iron content to total intracellular iron content. Patient values ​​were compared to the mean control values ​​in B, C, D, and E using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. [Figure 1B]Characterization of FRDA fibroblasts. A. Western blot analysis of frataxin and proteins involved in iron homeostasis and antioxidant defense in fibroblasts from five patients (P1-5) with GAA repeat expansions and controls (C1-3). Frataxin, ferritin, IRP1 (iron regulatory protein 1), IRP2, SOD1, and FBXL5 were tested under reducing conditions, while TfR1, SOD2, and ferredoxin reductase (FDXR) were tested under nonreducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation). GAPDH was used as a loading control. B. Measurement of mitochondrial reactive oxygen species (ROS) by flow cytometry using MitoSox in control (C1-3) and FRDA fibroblasts (P1-5) grown in normal medium (+ fetal bovine serum (FBS), - ferric ammonium citrate (FAC)) or high-iron medium (- FBS, + 100 μM FAC). Data show the percentage of MitoSox-positive cells in controls and patients. C. Iron quantification using a ferrozine-based colorimetric assay in complete fibroblasts or mitochondrial extracts (D) from control (C1-3) and FRDA patients (P1-5) grown in normal medium (+ FBS, - FAC). E. Ratio of intramitochondrial iron content to total intracellular iron content. Patient values ​​were compared to the mean control values ​​in B, C, D, and E using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. [Figure 1C]Characterization of FRDA fibroblasts. A. Western blot analysis of frataxin and proteins involved in iron homeostasis and antioxidant defense in fibroblasts from five patients (P1-5) with GAA repeat expansions and controls (C1-3). Frataxin, ferritin, IRP1 (iron regulatory protein 1), IRP2, SOD1, and FBXL5 were tested under reducing conditions, while TfR1, SOD2, and ferredoxin reductase (FDXR) were tested under nonreducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation). GAPDH was used as a loading control. B. Measurement of mitochondrial reactive oxygen species (ROS) by flow cytometry using MitoSox in control (C1-3) and FRDA fibroblasts (P1-5) grown in normal medium (+ fetal bovine serum (FBS), - ferric ammonium citrate (FAC)) or high-iron medium (- FBS, + 100 μM FAC). Data show the percentage of MitoSox-positive cells in controls and patients. C. Iron quantification using a ferrozine-based colorimetric assay in complete fibroblasts or mitochondrial extracts (D) from control (C1-3) and FRDA patients (P1-5) grown in normal medium (+ FBS, - FAC). E. Ratio of intramitochondrial iron content to total intracellular iron content. Patient values ​​were compared to the mean control values ​​in B, C, D, and E using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. [Figure 1D]Characterization of FRDA fibroblasts. A. Western blot analysis of frataxin and proteins involved in iron homeostasis and antioxidant defense in fibroblasts from five patients (P1-5) with GAA repeat expansions and controls (C1-3). Frataxin, ferritin, IRP1 (iron regulatory protein 1), IRP2, SOD1, and FBXL5 were tested under reducing conditions, while TfR1, SOD2, and ferredoxin reductase (FDXR) were tested under nonreducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation). GAPDH was used as a loading control. B. Measurement of mitochondrial reactive oxygen species (ROS) by flow cytometry using MitoSox in control (C1-3) and FRDA fibroblasts (P1-5) grown in normal medium (+ fetal bovine serum (FBS), - ferric ammonium citrate (FAC)) or high-iron medium (- FBS, + 100 μM FAC). Data show the percentage of MitoSox-positive cells in controls and patients. C. Iron quantification using a ferrozine-based colorimetric assay in complete fibroblasts or mitochondrial extracts (D) from control (C1-3) and FRDA patients (P1-5) grown in normal medium (+ FBS, - FAC). E. Ratio of intramitochondrial iron content to total intracellular iron content. Patient values ​​were compared to the mean control values ​​in B, C, D, and E using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. [Figure 1E]Characterization of FRDA fibroblasts. A. Western blot analysis of frataxin and proteins involved in iron homeostasis and antioxidant defense in fibroblasts from five patients (P1-5) with GAA repeat expansions and controls (C1-3). Frataxin, ferritin, IRP1 (iron regulatory protein 1), IRP2, SOD1, and FBXL5 were tested under reducing conditions, while TfR1, SOD2, and ferredoxin reductase (FDXR) were tested under nonreducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation). GAPDH was used as a loading control. B. Measurement of mitochondrial reactive oxygen species (ROS) by flow cytometry using MitoSox in control (C1-3) and FRDA fibroblasts (P1-5) grown in normal medium (+ fetal bovine serum (FBS), - ferric ammonium citrate (FAC)) or high-iron medium (- FBS, + 100 μM FAC). Data show the percentage of MitoSox-positive cells in controls and patients. C. Iron quantification using a ferrozine-based colorimetric assay in complete fibroblasts or mitochondrial extracts (D) from control (C1-3) and FRDA patients (P1-5) grown in normal medium (+ FBS, - FAC). E. Ratio of intramitochondrial iron content to total intracellular iron content. Patient values ​​were compared to the mean control values ​​in B, C, D, and E using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. [Figure 2A]Regulation of iron homeostasis in FRDA fibroblasts. Posttranscriptional regulation of iron homeostasis in control and FRDA fibroblasts grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium. TfR1 (TFRC, A) and ferritin (FTH, B) mRNA were quantified by ddPCR and expressed as a ratio to GUSB mRNA. Data are means ± standard error of three independent experiments. No significant variation between samples and controls was estimated using a two-way analysis of variance for multiple comparisons using the Holm-Sydak method. Patient values ​​were compared to the mean control values ​​using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. C. Iron quantification using a ferrozine-based colorimetric assay in control (C1-3) and FRDA patient (P1-5) fibroblasts grown in low iron (-FAC) or high iron (+FAC) conditions in DMEM medium without fetal bovine serum. Error bars indicate standard error (n=3). Patient values ​​were compared to the mean control values ​​using an unpaired t-test. ** and *** correspond to p-values ​​of <0.01 and <0.001, respectively. ns: not significant. D. Steady-state levels of proteins involved in iron homeostasis. TfR1, SOD2, and FBXL5 were assayed under non-reducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation) in control (C1-3) and FRDA fibroblasts (P1-5) grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium; ferritin and SOD2 were assayed under reducing conditions. GAPDH was used as a loading control. [Figure 2B]Regulation of iron homeostasis in FRDA fibroblasts. Posttranscriptional regulation of iron homeostasis in control and FRDA fibroblasts grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium. TfR1 (TFRC, A) and ferritin (FTH, B) mRNA were quantified by ddPCR and expressed as a ratio to GUSB mRNA. Data are means ± standard error of three independent experiments. No significant variation between samples and controls was estimated using a two-way analysis of variance for multiple comparisons using the Holm-Sydak method. Patient values ​​were compared to the mean control values ​​using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. C. Iron quantification using a ferrozine-based colorimetric assay in control (C1-3) and FRDA patient (P1-5) fibroblasts grown in low iron (-FAC) or high iron (+FAC) conditions in DMEM medium without fetal bovine serum. Error bars indicate standard error (n=3). Patient values ​​were compared to the mean control values ​​using an unpaired t-test. ** and *** correspond to p-values ​​of <0.01 and <0.001, respectively. ns: not significant. D. Steady-state levels of proteins involved in iron homeostasis. TfR1, SOD2, and FBXL5 were assayed under non-reducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation) in control (C1-3) and FRDA fibroblasts (P1-5) grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium; ferritin and SOD2 were assayed under reducing conditions. GAPDH was used as a loading control. [Figure 2C]Regulation of iron homeostasis in FRDA fibroblasts. Posttranscriptional regulation of iron homeostasis in control and FRDA fibroblasts grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium. TfR1 (TFRC, A) and ferritin (FTH, B) mRNA were quantified by ddPCR and expressed as a ratio to GUSB mRNA. Data are means ± standard error of three independent experiments. No significant variation between samples and controls was estimated using a two-way analysis of variance for multiple comparisons using the Holm-Sydak method. Patient values ​​were compared to the mean control values ​​using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. C. Iron quantification using a ferrozine-based colorimetric assay in control (C1-3) and FRDA patient (P1-5) fibroblasts grown in low iron (-FAC) or high iron (+FAC) conditions in DMEM medium without fetal bovine serum. Error bars indicate standard error (n=3). Patient values ​​were compared to the mean control values ​​using an unpaired t-test. ** and *** correspond to p-values ​​of <0.01 and <0.001, respectively. ns: not significant. D. Steady-state levels of proteins involved in iron homeostasis. TfR1, SOD2, and FBXL5 were assayed under non-reducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation) in control (C1-3) and FRDA fibroblasts (P1-5) grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium; ferritin and SOD2 were assayed under reducing conditions. GAPDH was used as a loading control. [Figure 2D]Regulation of iron homeostasis in FRDA fibroblasts. Posttranscriptional regulation of iron homeostasis in control and FRDA fibroblasts grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium. TfR1 (TFRC, A) and ferritin (FTH, B) mRNA were quantified by ddPCR and expressed as a ratio to GUSB mRNA. Data are means ± standard error of three independent experiments. No significant variation between samples and controls was estimated using a two-way analysis of variance for multiple comparisons using the Holm-Sydak method. Patient values ​​were compared to the mean control values ​​using an unpaired samples t-test. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. C. Iron quantification using a ferrozine-based colorimetric assay in control (C1-3) and FRDA patient (P1-5) fibroblasts grown in low iron (-FAC) or high iron (+FAC) conditions in DMEM medium without fetal bovine serum. Error bars indicate standard error (n=3). Patient values ​​were compared to the mean control values ​​using an unpaired t-test. ** and *** correspond to p-values ​​of <0.01 and <0.001, respectively. ns: not significant. D. Steady-state levels of proteins involved in iron homeostasis. TfR1, SOD2, and FBXL5 were assayed under non-reducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation) in control (C1-3) and FRDA fibroblasts (P1-5) grown in low-iron (-FAC) or high-iron (+FAC) conditions in fetal bovine serum-free DMEM medium; ferritin and SOD2 were assayed under reducing conditions. GAPDH was used as a loading control. [Figure 3A]Accumulation of TfR1 and incomplete Tf recycling in FRDA fibroblasts. A. Example of TfR1 labeling in control fibroblasts (C1 is representative of three controls) and FRDA fibroblasts (P1-5). Cell analysis was based on Hoechst-positive signals. Scale bar, 10 μm. B. Quantification of membrane-bound TfR1 signals on at least 20,000 fibroblasts using IDEAS software (Amnis). Data are means ± standard error of three independent experiments. Error bars indicate standard error (n = 3). Patient values ​​were compared to the mean control values ​​using a Student's t-test. ** and *** correspond to p-values ​​of <0.01 and <0.001, respectively. C. Relative mean fluorescence intensity of Tf-RED signals tracked for 40 min in control (C1-3) and FRDA patient (P1-5) fibroblasts as a percentage of initial Tf-RED staining. The number of analyzed cells was greater than 20 in three independent experiments. Data are the mean ± standard error of three independent experiments. Patient values ​​were compared to the mean control values ​​using the Holm-Sydak multiple comparison test. *** corresponds to a p-value of <0.001. [Figure 3B]Accumulation of TfR1 and incomplete Tf recycling in FRDA fibroblasts. A. Example of TfR1 labeling in control fibroblasts (C1 is representative of three controls) and FRDA fibroblasts (P1-5). Cell analysis was based on Hoechst-positive signals. Scale bar, 10 μm. B. Quantification of membrane-bound TfR1 signals on at least 20,000 fibroblasts using IDEAS software (Amnis). Data are means ± standard error of three independent experiments. Error bars indicate standard error (n = 3). Patient values ​​were compared to the mean control values ​​using a Student's t-test. ** and *** correspond to p-values ​​of <0.01 and <0.001, respectively. C. Relative mean fluorescence intensity of Tf-RED signals tracked for 40 min in control (C1-3) and FRDA patient (P1-5) fibroblasts as a percentage of initial Tf-RED staining. The number of analyzed cells was greater than 20 in three independent experiments. Data are the mean ± standard error of three independent experiments. Patient values ​​were compared to the mean control values ​​using the Holm-Sydak multiple comparison test. *** corresponds to a p-value of <0.001. [Figure 3C]Accumulation of TfR1 and incomplete Tf recycling in FRDA fibroblasts. A. Example of TfR1 labeling in control fibroblasts (C1 is representative of three controls) and FRDA fibroblasts (P1-5). Cell analysis was based on Hoechst-positive signals. Scale bar, 10 μm. B. Quantification of membrane-bound TfR1 signals on at least 20,000 fibroblasts using IDEAS software (Amnis). Data are means ± standard error of three independent experiments. Error bars indicate standard error (n = 3). Patient values ​​were compared to the mean control values ​​using a Student's t-test. ** and *** correspond to p-values ​​of <0.01 and <0.001, respectively. C. Relative mean fluorescence intensity of Tf-RED signals tracked for 40 min in control (C1-3) and FRDA patient (P1-5) fibroblasts as a percentage of initial Tf-RED staining. The number of analyzed cells was greater than 20 in three independent experiments. Data are the mean ± standard error of three independent experiments. Patient values ​​were compared to the mean control values ​​using the Holm-Sydak multiple comparison test. *** corresponds to a p-value of <0.001. [Figure 4A]Palmitoylation of TfR1 in FRDA fibroblasts. A. Control (C1-3) and FRDA patient (P1-5) fibroblasts were treated with DMSO alone (upper left panel), 25 μM CoA for 72 hours (upper right panel), 5 mM dichloroacetic acid (DCA) for 72 hours (lower left panel), or 25 μM artesunate for 48 hours (lower right panel). TfR1 protein was immunoprecipitated with a mouse anti-TfR1 antibody and a palmitoylation assay was performed. For each condition, the upper panel shows the palmitoylated TfR1 level (IB: biotin) and the amount of immunoprecipitated TfR1 (IB: TfR1). Input TfR1 was used as a loading control for each condition. IP: immunoprecipitation; IB: immunoblot. B. Iron quantification using a ferrozine-based colorimetric assay in control (C1-3) and FRDA patient (P1-5) fibroblasts grown in normal DMEM medium for 72 hours in the presence or absence of 25 μM CoA. Error bars indicate standard error (n=3). Patient values ​​were compared to the mean control values ​​using a two-way analysis of variance for multiple comparisons with the Holm-Sidak multiple comparison test. *** corresponds to a p-value of <0.001. ns: not significant. C. Steady-state levels of PDH-E2, lipoylated PDH-E2 (PDH-E2-LA), and lipoylated α-KGDH (α-KGDH-LA) in mitochondria of control (C1-3) and FRDA patient (P1-5) fibroblasts untreated (-DCA) or treated with 5 mM DCA (+DCA) for 72 hours. Fibroblast mitochondria were analyzed by immunoblotting using an antibody against lipoic acid (which detects lipoic acid binding to PDH-E2 and α-KGDH). Porin was used as a loading control. [Figure 4B]Palmitoylation of TfR1 in FRDA fibroblasts. A. Control (C1-3) and FRDA patient (P1-5) fibroblasts were treated with DMSO alone (upper left panel), 25 μM CoA for 72 hours (upper right panel), 5 mM dichloroacetic acid (DCA) for 72 hours (lower left panel), or 25 μM artesunate for 48 hours (lower right panel). TfR1 protein was immunoprecipitated with a mouse anti-TfR1 antibody and a palmitoylation assay was performed. For each condition, the upper panel shows the palmitoylated TfR1 level (IB: biotin) and the amount of immunoprecipitated TfR1 (IB: TfR1). Input TfR1 was used as a loading control for each condition. IP: immunoprecipitation; IB: immunoblot. B. Iron quantification using a ferrozine-based colorimetric assay in control (C1-3) and FRDA patient (P1-5) fibroblasts grown in normal DMEM medium for 72 hours in the presence or absence of 25 μM CoA. Error bars indicate standard error (n=3). Patient values ​​were compared to the mean control values ​​using a two-way analysis of variance for multiple comparisons with the Holm-Sidak multiple comparison test. *** corresponds to a p-value of <0.001. ns: not significant. C. Steady-state levels of PDH-E2, lipoylated PDH-E2 (PDH-E2-LA), and lipoylated α-KGDH (α-KGDH-LA) in mitochondria of control (C1-3) and FRDA patient (P1-5) fibroblasts untreated (-DCA) or treated with 5 mM DCA (+DCA) for 72 hours. Fibroblast mitochondria were analyzed by immunoblotting using an antibody against lipoic acid (which detects lipoic acid binding to PDH-E2 and α-KGDH). Porin was used as a loading control. [Figure 4C]Palmitoylation of TfR1 in FRDA fibroblasts. A. Control (C1-3) and FRDA patient (P1-5) fibroblasts were treated with DMSO alone (upper left panel), 25 μM CoA for 72 hours (upper right panel), 5 mM dichloroacetic acid (DCA) for 72 hours (lower left panel), or 25 μM artesunate for 48 hours (lower right panel). TfR1 protein was immunoprecipitated with a mouse anti-TfR1 antibody and a palmitoylation assay was performed. For each condition, the upper panel shows the palmitoylated TfR1 level (IB: biotin) and the amount of immunoprecipitated TfR1 (IB: TfR1). Input TfR1 was used as a loading control for each condition. IP: immunoprecipitation; IB: immunoblot. B. Iron quantification using a ferrozine-based colorimetric assay in control (C1-3) and FRDA patient (P1-5) fibroblasts grown in normal DMEM medium for 72 hours in the presence or absence of 25 μM CoA. Error bars indicate standard error (n=3). Patient values ​​were compared to the mean control values ​​using a two-way analysis of variance for multiple comparisons with the Holm-Sidak multiple comparison test. *** corresponds to a p-value of <0.001. ns: not significant. C. Steady-state levels of PDH-E2, lipoylated PDH-E2 (PDH-E2-LA), and lipoylated α-KGDH (α-KGDH-LA) in mitochondria of control (C1-3) and FRDA patient (P1-5) fibroblasts untreated (-DCA) or treated with 5 mM DCA (+DCA) for 72 hours. Fibroblast mitochondria were analyzed by immunoblotting using an antibody against lipoic acid (which detects lipoic acid binding to PDH-E2 and α-KGDH). Porin was used as a loading control. [Figure 5A]Treatment with artesunate reduces TfR1 accumulation and improves Tf recycling in FRDA fibroblasts. A. Examples of TfR1 labeling in control fibroblasts (C1 is representative of three controls) and FRDA fibroblasts (P1-5) supplemented with or without 25 μM artesunate for 48 hours. Cell analysis was based on Hoechst-positive signals. Scale bar, 10 μm. B. Quantification of membrane-bound TfR1 signals on at least 20,000 fibroblasts from control (C1-3) and FRDA patients (P1-5) using IDEAS software (Amnis). Error bars indicate standard error (n=3). C. Transferrin recycling. Relative mean fluorescence intensity of Tf-RED signals tracked for 40 minutes in control (C1-3) and FRDA patient (P1-5) fibroblasts as a percentage of Tf-RED initial staining. The number of analyzed cells exceeds 20 in three independent experiments. Data are the mean ± standard error of three independent experiments. D. Iron quantification using a ferrozine-based colorimetric assay in fibroblasts grown in normal DMEM medium with or without 25 μM artesunate for 48 hours. Error bars indicate standard error (n=3). Two-way ANOVA with Holm-Sidak multiple comparison test was used in B, C, and D. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. ns: not significant. [Figure 5B]Treatment with artesunate reduces TfR1 accumulation and improves Tf recycling in FRDA fibroblasts. A. Examples of TfR1 labeling in control fibroblasts (C1 is representative of three controls) and FRDA fibroblasts (P1-5) supplemented with or without 25 μM artesunate for 48 hours. Cell analysis was based on Hoechst-positive signals. Scale bar, 10 μm. B. Quantification of membrane-bound TfR1 signals on at least 20,000 fibroblasts from control (C1-3) and FRDA patients (P1-5) using IDEAS software (Amnis). Error bars indicate standard error (n=3). C. Transferrin recycling. Relative mean fluorescence intensity of Tf-RED signals tracked for 40 minutes in control (C1-3) and FRDA patient (P1-5) fibroblasts as a percentage of Tf-RED initial staining. The number of analyzed cells exceeds 20 in three independent experiments. Data are the mean ± standard error of three independent experiments. D. Iron quantification using a ferrozine-based colorimetric assay in fibroblasts grown in normal DMEM medium with or without 25 μM artesunate for 48 hours. Error bars indicate standard error (n=3). Two-way ANOVA with Holm-Sidak multiple comparison test was used in B, C, and D. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. ns: not significant. [Figure 5C]Treatment with artesunate reduces TfR1 accumulation and improves Tf recycling in FRDA fibroblasts. A. Examples of TfR1 labeling in control fibroblasts (C1 is representative of three controls) and FRDA fibroblasts (P1-5) supplemented with or without 25 μM artesunate for 48 hours. Cell analysis was based on Hoechst-positive signals. Scale bar, 10 μm. B. Quantification of membrane-bound TfR1 signals on at least 20,000 fibroblasts from control (C1-3) and FRDA patients (P1-5) using IDEAS software (Amnis). Error bars indicate standard error (n=3). C. Transferrin recycling. Relative mean fluorescence intensity of Tf-RED signals tracked for 40 minutes in control (C1-3) and FRDA patient (P1-5) fibroblasts as a percentage of Tf-RED initial staining. The number of analyzed cells exceeds 20 in three independent experiments. Data are the mean ± standard error of three independent experiments. D. Iron quantification using a ferrozine-based colorimetric assay in fibroblasts grown in normal DMEM medium with or without 25 μM artesunate for 48 hours. Error bars indicate standard error (n=3). Two-way ANOVA with Holm-Sidak multiple comparison test was used in B, C, and D. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. ns: not significant. [Figure 5D]Treatment with artesunate reduces TfR1 accumulation and improves Tf recycling in FRDA fibroblasts. A. Examples of TfR1 labeling in control fibroblasts (C1 is representative of three controls) and FRDA fibroblasts (P1-5) supplemented with or without 25 μM artesunate for 48 hours. Cell analysis was based on Hoechst-positive signals. Scale bar, 10 μm. B. Quantification of membrane-bound TfR1 signals on at least 20,000 fibroblasts from control (C1-3) and FRDA patients (P1-5) using IDEAS software (Amnis). Error bars indicate standard error (n=3). C. Transferrin recycling. Relative mean fluorescence intensity of Tf-RED signals tracked for 40 minutes in control (C1-3) and FRDA patient (P1-5) fibroblasts as a percentage of Tf-RED initial staining. The number of analyzed cells exceeds 20 in three independent experiments. Data are the mean ± standard error of three independent experiments. D. Iron quantification using a ferrozine-based colorimetric assay in fibroblasts grown in normal DMEM medium with or without 25 μM artesunate for 48 hours. Error bars indicate standard error (n=3). Two-way ANOVA with Holm-Sidak multiple comparison test was used in B, C, and D. *, **, and *** correspond to p-values ​​of <0.5, <0.01, and <0.001, respectively. ns: not significant. [Figure 6A]Artesunate treatment improves iron homeostasis in FRDA fibroblasts. A. Iron quantification using a ferrozine-based colorimetric assay in fibroblasts grown in high-iron conditions (100 μM FAC) with or without 25 μM artesunate for 48 h. Error bars indicate standard error (n=3). Two-way ANOVA with Holm-Sidak multiple comparison test was used. *** corresponds to p<0.001. B. Steady-state levels of proteins involved in iron homeostasis. TfR1, SOD2, and FBXL5 were assayed under nonreducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation) in control (C1-3) and FRDA fibroblasts (P1-5) grown in normal DMEM medium with or without 25 μM artesunate for 48 h. IRP1, IPR2, and ferritin were assayed under reducing conditions. [Figure 6B] Artesunate treatment improves iron homeostasis in FRDA fibroblasts. A. Iron quantification using a ferrozine-based colorimetric assay in fibroblasts grown in high-iron conditions (100 μM FAC) with or without 25 μM artesunate for 48 h. Error bars indicate standard error (n=3). Two-way ANOVA with Holm-Sidak multiple comparison test was used. *** corresponds to p<0.001. B. Steady-state levels of proteins involved in iron homeostasis. TfR1, SOD2, and FBXL5 were assayed under nonreducing conditions (12% acrylamide, no dithiothreitol (DTT), no heat denaturation) in control (C1-3) and FRDA fibroblasts (P1-5) grown in normal DMEM medium with or without 25 μM artesunate for 48 h. IRP1, IPR2, and ferritin were assayed under reducing conditions. [Figure 7A]Iron content in FRDA PBMCs. A. Measurement of iron content in PBMCs grown in high-iron medium (100 μM FAC). Iron content was measured every 8 hours for 40 hours using a ferrozine-based colorimetric assay in control (C4-7), heterozygous carriers of the FXN GAA expansion (carriers 1-3), and FRDA PBMCs (P1, P6-P14). B. Iron content in PBMCs grown in high-iron medium (100 μM FAC) with or without 25 μM artesunate for 40 hours. C8-9 are controls, carriers 4-5 are heterozygous carriers of the FXN GAA expansion, and P15-16 is an FRDA patient with a compound heterozygous FXN GAA expansion. [Figure 7B] Iron content in FRDA PBMCs. A. Measurement of iron content in PBMCs grown in high-iron medium (100 μM FAC). Iron content was measured every 8 hours for 40 hours using a ferrozine-based colorimetric assay in control (C4-7), heterozygous carriers of the FXN GAA expansion (carriers 1-3), and FRDA PBMCs (P1, P6-P14). B. Iron content in PBMCs grown in high-iron medium (100 μM FAC) with or without 25 μM artesunate for 40 hours. C8-9 are controls, carriers 4-5 are heterozygous carriers of the FXN GAA expansion, and P15-16 is an FRDA patient with a compound heterozygous FXN GAA expansion.

[0028] Working Example: method: Informed consent for diagnostic and research studies was obtained from all subjects according to the Declaration of Helsinki protocol and approved by the Paris Regional Institutional Review Board.

[0029] patient The patient carried a GAA repeat expansion within the first intron of the frataxin gene.

[0030] [Table 1]

[0031] cell culture solution Dermal fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 2.5 mM pyruvate, 100 μg / ml streptomycin, and 100 U / ml penicillin at 37° C. For treatment with ferric ammonium citrate (FAC), cells at 80% confluence were incubated in serum-free DMEM (i.e., without transferrin) with or without 100 μM FAC for 72 hours. For drug treatment, cells at 80% confluence were incubated for 48 h in DMEM + 10% FBS supplemented with 25 μM artesunate (Sigma) in DMSO, 72 h in DMEM + 10% FBS supplemented with 25 μM CoA (Sigma), or 72 h in DMEM + 10% FBS supplemented with 5 mM DCA (Sigma).

[0032] PBMC isolation and culture PBMCs were isolated from 8 ml blood samples by density centrifugation using Ficoll (GE Healthcare) and grown in regular DMEM for 2 h before treatment with 100 μM FAC for various times.

[0033] Western blot Dermal fibroblasts cultured in reducing lysis buffer (2 μM dithiothreitol (DTT), denatured at 95°C for 5 min) or non-reducing lysis buffer (no DTT, no heat denaturation) were harvested by scraping on ice. Western blot analysis was performed on 12% acrylamide gels or 4-15% gradient gels using 20 μg of whole cell protein extracts or mitochondrial-enriched fractions. Immunodetection was performed in PBS containing 5% or 1% milk and 0.05% Tween 20 (Sigma) using the following antibodies: rabbit anti-SOD1 antibody (Abcam, ab16831), rabbit anti-SOD2 antibody (Abcam, ab13533), mouse anti-TfR1 antibody (Invitrogen, 13-6800), rabbit anti-TfR1 antibody (Abcam, 108985), rabbit anti-ferritin antibody (Abcam, ab75973), rabbit anti-IRP1 antibody (Abcam, ab126595), rabbit anti-IRP2 antibody (Abcam, ab80339), rabbit anti-FBXL5 antibody (Abcam, ab140175), rabbit anti-FDXR antibody (Abcam, 204310), rabbit anti-FXN antibody (Proteintech, 14147-1-AP), and rabbit anti-PDH. E2 subunit antibody (Abcam, ab172617), rabbit anti-lipoic acid antibody (Abcam, ab58724), goat anti-biotin antibody (Thermo Fisher Scientific, 31852), and mouse anti-VDAC / porin antibody (Abcam, ab14734).

[0034] The blots were incubated with mouse anti-ATP5a (Abcam, ab14748), mouse anti-vinculin (Abcam, ab130007), rabbit anti-TFAM (Proteintech, 19998-1-AP), rabbit ATP8 (Proteintech, 26723-1-AP), and mouse anti-GAPDH (ab8245). The blots were incubated with fluorescent secondary antibodies (IRDye800CW / 680LT goat anti-rabbit or anti-mouse IgG (LI-COR)) or horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG HRP (Abcam), goat anti-mouse IgG HRP (Abcam), or donkey anti-goat IgG HRP (Santa Cruz)) before electrochemiluminescence-based detection (SuperSignal West Dura, Thermo Fisher Scientific). Signals were acquired using either an Odyssey near-infrared fluorescence imaging system using Image Studio Lite v5.2 (LI-COR Biosciences) or a CDD camera using Image Lab v3.0 (Bio-Rad).

[0035] MitoSOX Mitochondrial superoxide was quantified by a flow cytometry assay adapted from (Mukhopadhyay et al., 2007). Fibroblasts were supplemented with 10 μM MitoSox Red for 20 min, trypsinized, and neutralized with fresh medium (regular medium or DMEM with FACS). Flow cytometry was performed using a Gallios (Beckman Coulter). MitoSOX Red was excited at 488 nm. Data were collected in the forward scatter, side scatter, and 580 nm (FL2) channels for at least 20,000 cells using Calusa software (Beckman Coulter). Cell debris was excluded for analysis. Histograms of the mean intensity of MitoSOX fluorescence were displayed in the FL2 channel.

[0036] Quantification of TFRC and FTH transcripts Total RNA was extracted using the RNeasyMini Kit (Qiagen) and DNase-free RNase kit (Qiagen) treated with RNase-free DNase kit according to the manufacturer's protocol. The concentration and purity of total RNA were assessed using a Nanodrop-8000 spectrophotometer (Thermo Fisher Scientific) before storage at -80°C. mRNA was then reverse transcribed from 2 μg of intact RNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) according to the manufacturer's instructions, using random priming. Quantitative RT-PCR (qRT-PCR) was performed using digital droplet PCR (ddPCR) with a QX200 Droplet Digital PCR System (Bio-Rad). TFRC and FTH cDNA were amplified using specific primers. β-Glucuronidase (GUSB, NM_000181.3) was used for normalization. Data were analyzed on a QX200 droplet reader using Quantasoft analysis software (Bio-Rad). Expression levels of TFRC and FTH were normalized to the average copy number of the GUSB housekeeping gene.

[0037] Iron content and imaging flow cytometry (ImageStream) Fibroblasts were starved for 1 hour in fetal bovine serum-free DMEM medium (i.e., transferrin-free), treated with 5 mM EDTA to harvest them without disrupting the cell surface-located TfR1, as with trypsin, and then labeled with anti-TfR1 antibodies at 4°C to avoid TfR1 internalization. Under these conditions, only membrane-bound TfR1 was quantified. Cell sorting was based on a Hoechst-positive signal, which allowed for the selection of live cells.

[0038] Total iron content was measured using a Ferrozine-based iron assay modified from (Barbeito et al., 2010). For imaging flow cytometry, fibroblasts were starved in fetal bovine serum-free DMEM medium for 1 hour, treated with 5 mM EDTA to harvest them without disrupting cell surface-located TfR1, as with trypsin, washed three times with cold PBS, and then labeled with anti-TfR1 antibody (A24) (Moura et al., 2004) for 1 hour on ice to avoid TfR1 internalization. Under these conditions, only membrane-bound TfR1 was quantified. Secondary staining was performed on ice for 30 minutes using Alexa fluor 488 goat anti-mouse antibody (Life Technologies). Cells were washed and stained with Hoechst for 5 minutes in a total volume of 50 μl, followed by direct acquisition. Cell analysis was based on the Hoechst-positive signal, which allows for the selection of live cells. Samples were subjected to flow cytometry on an ImageStream ISX mkII (Amnis, Millipore, Seattle, WA), which combines detailed cell imaging and functional studies. A 40x magnification was used for all acquisitions. Data were acquired using INSPIRE software (Amnis) and analyzed using IDEAS™ software (version 6.2, Amnis) for at least 20,000 events. Spectral correction was performed using single-stained samples. A special mask was designed for the analysis of TfR1 membrane localization. This mask was the result of a 5-pixel subtraction from the full brightfield mask and a 1-pixel dilation from the brightfield mask, resulting in a donut-like mask. Results were expressed as mean pixel intensity values, which are intensity normalized to surface area.

[0039] Confocal microscope For Tf recirculation, fibroblasts were plated at 30% confluence on glass slides (IBIDI) 24 h before the experiment. Cells were then starved in fetal bovine serum-free DMEM medium for 1 h and Tf-RED (12.5 μg / mL) was added for 30 min at 37°C. Cells were washed with PBS and then incubated in normal medium for live imaging using a Zeiss spinning disk confocal microscope. Slides were placed in an incubation chamber at 37°C under 5% CO2. At least 20 cells were captured per minute for 40 min using Zen software with a 63x oil immersion objective. Nuclear regions (NRs) were defined using Icy software v1.9. Each NR was magnified 2x to create perinuclear regions of interest (PNROIs). The PNROI mask was then applied to the RED channel using the Spot Detector plugin to obtain quantitative mean fluorescent particle intensity. Only particles with a size of at least 8 pixels were considered. Mean intensity values ​​were normalized to surface area. Data were expressed as a ratio to the initial Tf signal at 0 min.

[0040] Palmitoylation assay TfR1 palmitoylation in cultured skin fibroblasts was modified from (Ba et al., 2012). Briefly, cells were lysed in DTT-free cell lysis buffer on ice, and endogenous TfR1 was immunoprecipitated overnight using a mouse anti-TfR1 antibody (Life Technologies, 136890) with Protein G magnetic beads (Bio-Rad). After washing with PBS, the beads were sequentially incubated with 50 mM N-ethylmaleimide (NEM) for 2 h at room temperature and 1 M hydroxylamine and 50 mM HPDP-biotin (Thermo Fisher Scientific) for 2 h in the dark. Samples were run on a 12% acrylamide gel, and biotin-labeled TfR1 levels were determined by immunoblotting under non-reducing conditions using Chemidoc technology with a CCD camera (Bio-Rad) and ImageLab software v3.0 (Bio-Rad).

[0041] Mitochondrial isolation Mitochondria were isolated by differential centrifugation in Mito isolation buffer (320 mM sucrose, 10 mM Tris-HCl (pH 7.5), EDTA with a protease inhibitor cocktail) as described (Metodiev et al., 2009). Mitochondria were either resuspended in Mito isolation buffer for protein analysis or preserved as a dry pellet for iron quantification.

[0042] statistics All statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software) using two-tailed unpaired t-tests or one-way analysis of variance for multiple comparisons using the Holm-Sidak method. * , ** and *** corresponds to P values ​​of <0.05, <0.01, and <0.001, respectively. ns: non-significant.

[0043] result Characterization of frataxin, TfR1, and IRPs in FRDA fibroblasts Frataxin steady-state levels in cultured skin fibroblasts from FRDA patients were reduced to 30-68% of control values ​​(GAA repeat expansions >2.1 kb, Figure 1A). Ferredoxin reductase (FDXR) steady-state levels were also reduced, suggesting co-regulation of the two proteins in FRDA fibroblasts (Figure 1A).

[0044] Intracellular iron is primarily imported via transferrin-bound iron uptake via TfR1-mediated endocytosis. Intracellular iron homeostasis is regulated by a post-transcriptional mechanism involving iron regulatory proteins 1-2 (IRP1-2). IRPs regulate several iron-related genes, particularly TfR1 and ferritin, at the post-transcriptional level. Western blot analysis of FRDA fibroblasts showed increased steady-state levels of TfR1 (1.6-fold increase), IRP1, and IRP2 (2.2- and 2.4-fold, respectively; Figure 1A), mimicking the iron-deficiency state previously reported after disruption of the mitochondrial iron-sulfur complex machinery (Muhlenhoff et al., 2015). Paradoxically, steady-state levels of ferritin were also increased (2.1-fold) in FRDA fibroblasts, which contradicts the elevated TfR1 and IRP1-2 levels and suggests intracellular iron overload. F-box / leucine-rich repeat protein 5 (FBXL5), an iron sensor protein that reflects the labile iron pool, was also increased, again supporting cytoplasmic iron overload. Consistently, cytoplasmic superoxide dismutase SOD1 was increased (average increase of 232%), suggesting cytoplasmic stress likely associated with cytoplasmic iron overload (Figure 1C). Mitochondrial superoxide dismutase SOD2 showed a two-fold increase, reflecting severe oxidative stress likely triggered by mitochondrial iron overload in FRDA fibroblasts (Figure 1A). Consistently, a 2.5-fold increase in mitochondrial reactive oxygen species was detected by flow cytometry in FRDA fibroblasts using MitoSOX (Figure 1B).

[0045] FRDA fibroblasts were unable to regulate iron uptake. The elevated steady-state levels of ferritin and FBXL5 prompted us to assess iron content in FRDA fibroblasts using a Ferrozine-based iron assay modified from (Barbeito et al., 2010). Under basal conditions, total intracellular iron was 3-4 fold higher in FRDA fibroblasts compared to controls (Figure 1C), whereas iron content in mitochondrial extracts was only 2-fold higher in FRDA fibroblasts compared to controls (Figure 1D). Intramitochondrial iron accounted for primarily 1-1.5% of total iron in controls, but only 0.6-0.8% in FRDA fibroblasts (Figure 1E). Thus, FRDA fibroblasts accumulate iron primarily in the cytoplasm and, to a lesser extent, in mitochondria, suggesting that total intracellular iron does not reflect the actual intramitochondrial iron pool in FRDA fibroblasts.

[0046] Intracellular iron was quantified under either low-iron conditions (without fetal bovine serum (FBS)—i.e., no transferrin-bound iron) or high-iron conditions provided by ferric ammonium citrate (FAC). FAC is a soluble form of non-transferrin-bound iron (NTBI) that enters cells in a timely manner via endogenous transporters or endocytic pathways. Under low-iron conditions (without FAC or FBS), FRDA fibroblasts exhibited 2- to 4-fold higher iron content compared with controls (Figure 2A). However, after 3 days of incubation with FAC, FRDA fibroblasts exhibited a large intracellular iron increase (32- to 42-fold change), whereas control fibroblasts exhibited a 10-fold increase (Figure 2A). This was not due to a respiratory chain defect, as intracellular iron levels in patients with biallelic SURF1 mutations were similar to those in controls grown under the same conditions (Figure 2A). These data suggested that cultured FRDA fibroblasts were unable to regulate iron uptake and exhibited significant iron overload when grown in high iron conditions.

[0047] Iron regulatory proteins (IRPs) are sensors of intracellular iron content. When cytoplasmic iron increases, IRP1 is converted to aconitase, while IRP2 is targeted for ubiquitination and proteasomal degradation by the iron-binding protein FBXL5. Reduced IRP1-2 downregulates TfR1, limiting iron uptake, while ferritin is upregulated, allowing iron storage in the cytoplasm. Given the significant iron overload in FRDA fibroblasts, we investigated the posttranscriptional regulation of iron homeostasis. TfR1 (TFRC) and H-ferritin (FTH) mRNAs were quantified by digital droplet PCR (ddPCR) in fibroblasts grown for 3 days under either low-iron (-FAC) or high-iron (+FAC) conditions. TFRC mRNA levels were similar in control and FRDA fibroblasts grown under low iron conditions (-FAC) and decreased under high iron conditions, suggesting normal downregulation of TFRC transcripts (Figure 2B). Ferritin mRNA was concomitantly increased in control and FRDA fibroblasts grown under high iron conditions (Figure 2C). These results suggest efficient post-transcriptional regulation of TfR1 and ferritin in FRDA fibroblasts.

[0048] Consistently, Western blot analysis showed that in control cells grown under high iron conditions (+FAC), low levels of TfR1 prevented iron accumulation, and H-ferritin and L-ferritin levels were also increased, allowing iron storage (Figure 2D). Increased intracellular iron induced strong cytoplasmic and mitochondrial reactive oxygen species overproduction, as indicated by high levels of SOD1-2. Under these conditions (+FAC), FRDA fibroblasts failed to downregulate, and even increased, TfR1 content despite high steady-state ferritin levels. Similar results were observed with FBXL5 (Figure 2D). In the context of iron overload, the concomitant increase in TfR1 and ferritin is paradoxical, since stored iron should downregulate TfR1 levels and metal import. This result suggests that TfR1 appears to circumvent IRP regulation in FRDA fibroblasts. Recalling that post-transcriptional regulation of TfR1 is unaffected, these results suggest aberrant post-translational regulation of TfR1 in FRDA, as previously described in NBIA ( Drecourt et al., 2018 ).

[0049] TfR1 accumulates on the cell surface of FRDA fibroblasts. We hypothesized that the increased steady-state levels of TfR1 might be related to the accumulation of TfR1 at the membrane in FRDA, as observed in NBIA. TfR1 abundance was quantified by immunofluorescence using next-generation imaging flow cytometry with an Amnis ImageStream (X) Mark II, combining flow cytometry with detailed cellular imaging and functional studies. This analysis demonstrated increased amounts of TfR1 on the cell surface of FRDA fibroblasts compared with controls (Figure 3A). Quantification of TfR1 in over 20,000 fibroblasts grown under basal conditions using IDEAS software (Amnis) revealed significantly increased TfR1 signal in patients (Figure 3B). These results indicate that despite iron overload and correct posttranscriptional downregulation, FRDA fibroblasts accumulate TfR1 at the cell membrane, preventing them from regulating iron uptake.

[0050] Delayed transferrin recycling in FRDA fibroblasts. Spinning-disk confocal microscopy was used to assess the perinuclear immunofluorescence intensity of transferrin (Tf)-Alexa555 staining in patient and control fibroblasts. At the TO time point of the Tf-Alexa555 pulse-chase, Tf staining was similar in patient and control cells. Control fibroblasts showed a rapid decrease in Tf staining due to Tf recycling (Figure 3C). In contrast, Tf recycling was significantly delayed in FRDA fibroblasts, as specific signals concentrated around the nucleus failed to decrease after a 10-minute Tf-Alexa555 incubation and were delayed even later (Figure 3C).

[0051] Palmitoylation of TfR1 in FRDA fibroblasts TfR1 binds Cys via a thioester bond. 62 and Cys 67Frataxin is post-translationally modified by the covalent attachment of an S-acyl group to TfR1, with palmitate being the primary fatty acid donor. Decreased palmitoylation has previously been shown to increase TfR1 endocytosis and iron uptake (Alvarez et al., 1990). Furthermore, defective TfR1 palmitoylation was recently reported in NBIA fibroblasts, where iron homeostasis is also altered (Drecourt et al., 2018). Studying cultured cells from FRDA patients revealed a dramatic reduction in TfR1 palmitoylation to only 16–22% of control values, suggesting that frataxin deficiency severely affects TfR1 palmitoylation for reasons that remain unclear (Figure 4A). Acetyl-coenzyme A (CoA) is the sole donor of the acetyl group for palmitoyltransferase. We previously reported that adding CoA to cultured fibroblasts carrying biallelic mutations in two NBIA genes (PANK2 and CRAT) involved in CoA biosynthesis increased palmitoylation of TfR1, suggesting that impaired CoA biosynthesis secondary to altered palmitoylation of TfR1 (Drecourt et al., 2018). Cells can obtain CoA from extracellular sources because CoA can be hydrolyzed extracellularly by ectonucleotide pyrophosphatase, producing membrane-permeable 4'-phosphopantetheine, which can be converted to CoA intracellularly (Srinivasan et al., 2015). Supplementation of cultured cells with 25 μM CoA for 72 hours increased TfR1 palmitoylation in FRDA fibroblasts (2.1- to 3.2-fold increase, Figure 4A), suggesting that frataxin deficiency limits the CoA pool and secondarily affects TfR1 palmitoylation. CoA supplementation also reduced the steady-state levels of TfR1 in FRDA fibroblasts (Figure 4A). Furthermore, unlike controls, FRDA fibroblasts grown under high iron conditions (+FAC) and supplemented with 25 μM CoA for 72 hours showed a 1.6- to 2.4-fold decrease in intracellular iron content (Figure 4B).This suggests a direct link between frataxin deficiency, CoA availability, TfR1 palmitoylation, and iron homeostasis.

[0052] Impaired TfR1 palmitoylation in FRDA fibroblasts is associated with defective pyruvate dehydrogenase (PDH) lipoylation Lipoic acid synthase (LIAS) is a [4Fes-4S] cluster-containing protein and a key enzyme in lipoic acid (LA) synthesis. LA is a cofactor for several mitochondrial proteins, including dihydrolipoamide acetyltransferase (DLAT or PDH-E2), one of the three pyruvate dehydrogenase (PDH) subunits. Cultured fibroblasts from patients with biallelic mutations in various genes involved in iron-sulfur complex biosynthesis (NFU1, IBA57, ISCA2, and FDX1L) show impaired lipoylation of DLAT and other mitochondrial proteins, as well as reduced PDH activity (Lebigot et al., 2017).

[0053] Furthermore, FXN deficiency in mice (Martelli et al., 2015) and knockdown in HeLa cells resulted in severely defective lipoylation of PDH and α-ketoglutarate dehydrogenase (α-KGDH, Tong et al., 2018). We also observed altered lipoylation of PDH-E2 and α-KGDH, as well as reduced steady-state levels of the PDH-E2 subunit, in FRDA fibroblasts (Figure 4C). Given that CoA supplementation improved TfR1 palmitoylation, we hypothesized that frataxin deficiency might affect TfR1 palmitoylation via defective PDH lipoylation. This is because acetyl-CoA is primarily produced by oxidative decarboxylation of pyruvate by the PDH complex in mitochondria. Dichloroacetic acid (DCA), an inhibitor of PDH kinase (PDHK) that inactivates the PDH complex, is known to increase pyruvate oxidation and the pool of acetyl-CoA. Supplementing FRDA fibroblasts with 5 mM DCA for 72 hours significantly increased palmitoylation of TfR1, just as it did CoA (Figure 4A), demonstrating that reduced PDH activity indeed affects the pool of CoA available for palmitoylation of TfR1. DCA also significantly reduced the steady-state levels of TfR1 in FRDA fibroblasts (Figure 4A). Furthermore, DCA supplementation completely or partially rescued the steady-state levels of PDH-E2 and altered the lipoylation of PDH and α-KGDH in FRDA fibroblasts. This is likely because octanoic acid, the precursor of lipoic acid, is synthesized through fatty acid oxidation.

[0054] Artesunate rescues TfR1 palmitoylation, Tf recycling, and iron overload in FRDA fibroblasts. Artesunate is known to alter intracellular iron homeostasis by palmitoylating TfR1, resulting in a decrease in membrane TfR1 (Ba et al., 2012). Addition of 25 μM artesunate to the culture medium for 48 hours enhanced TfR1 palmitoylation to 80% of control levels, and significantly reduced steady-state TfR1 levels in FRDA fibroblasts (Figure 4A). Imaging flow cytometry demonstrated that artesunate significantly reduced membrane TfR1 in FRDA fibroblasts (15–30% reduction), whereas it only slightly increased it in control cells (1.1–1.3-fold, Figures 5A and 5B).

[0055] To examine the effect of artesunate on transferrin recycling, we recorded the immunofluorescence intensity of Tf-Alexa555 staining in FRDA and control cells. Artesunate rapidly reduced the Tf signal to control values ​​in FRDA fibroblasts, completely rescued the delayed Tf recycling, and perinuclear Tf staining disappeared (Figure 5C). Consistently, iron overload decreased to 62–79% of the initial intracellular iron content 48 h after artesunate supplementation in FRDA fibroblasts grown under basal conditions (Figure 5D). It should be noted that artesunate-treated control cells showed a slight delay in Tf recycling and a mild, but not significant, increase in iron content (Figures 5C and 5D). Addition of 25 μM artesunate to FRDA cultured fibroblasts grown in high iron conditions (100 μM FAC) reduced intracellular iron content by 71-79%, demonstrating a dramatic rescue of the patient's cells' ability to regulate iron uptake and handling (Figure 6A). Notably, artesunate also reduced the iron content of control cells grown in high iron conditions (Figure 6A).

[0056] Western blot analysis confirmed that artesunate supplementation of FRDA fibroblasts reduced TfR1, reaching 136% of control levels in artesunate-free medium (Figure 6B). Furthermore, steady-state levels of ferritin and FbXL5 returned to control levels, paralleling the decreased iron content, which likely reduced reactive oxygen species production, as suggested by the decreased SOD2. IRP1-2 were not modified by artesunate treatment. In control cells, the slight increase in TfR1 by artesunate treatment likely results from a slight, but not significant, decrease in TfR1 palmitoylation. Nevertheless, ferritin and FbXL5 were not modified.

[0057] Artesunate reduces iron overload in peripheral blood mononuclear cells from patients with FRDA We quantified the total intracellular iron content of peripheral blood mononuclear cells (PBMCs) grown in high-iron conditions for 40 hours in patients and controls. After 24 hours, the iron content of PBMCs from FRDA patients was much higher than that of controls, eventually doubling after 40 hours (Figure 7A). This suggested that PBMCs also were unable to regulate iron uptake under high-iron conditions. The iron content of PBMCs from three heterozygous carriers was similar to control values, even after 40 hours of incubation in high-iron medium. Addition of 25 μM artesunate to the PBMC culture medium reduced iron content twofold in both FRDA and control PBMCs (Figure 7B).

[0058] Consideration: Here, we report disturbed intracellular iron homeostasis and defective transferrin receptor (TfR1) palmitoylation in cultured fibroblasts from FRDA patients. Both the cytosolic and mitochondrial compartments were found to abnormally accumulate large amounts of iron. We also observed defective lipoylation of the PDH complex due to impaired assembly of the iron-sulfur complex in lipoic acid synthase. Defective lipoylation of the PDH complex, in turn, dramatically reduced the acetyl-CoA pool and caused secondary defects in TfR1 palmitoylation. This resulted in membrane accumulation of TfR1, preventing the regulation of iron uptake and Tf recycling in FRDA fibroblasts. Finally, we demonstrate that artesunate improved TfR1 palmitoylation, reduced membrane TfR1, and rescued Tf recycling and iron overload in FRDA fibroblasts. Similarly, dichloroacetate and CoA also increased palmitoylation of TfR1.

[0059] Iron dysregulation in Friedreich's ataxia has long been recognized and is generally thought to be characterized by mitochondrial iron accumulation, along with cytoplasmic iron deficiency. Here, we show that iron accumulates extensively in the cytoplasm and, to a lesser extent, in mitochondria. Increased steady-state levels of ferritin and FBXL5 paralleled iron accumulation, and elevated SOD1-2 suggested increased reactive oxygen species production, possibly associated with iron overload. Cytoplasmic iron content has rarely been assessed previously and, to our knowledge, has not been formally quantified in various cell and animal models of frataxin deficiency. Although the previously reported iron depletion in the cytoplasm at the expense of mitochondria has been primarily based on mitochondrial iron overload without cytoplasmic quantification (Babcock et al., 1997; Puccio et al., 2001), increased TfR1 and decreased steady-state levels of ferritin have been associated with activation of IRP1, which binds to the iron-responsive element (Martelli et al., 2015; Telot et al., 2018; Whitnall et al., 2012). Surprisingly, in our study, we found that iron accumulation was relatively higher in the cytoplasm than in mitochondria. It should be noted that increased levels of ferritin were observed in the hearts of FRDA patients (Ramirez et al., 2012) and in the muscles of MCK conditional frataxin knockout mice (Whitnall et al., 2012), suggesting that iron accumulation varies between tissues.

[0060] Intracellular iron homeostasis is primarily regulated by post-transcriptional mechanisms that allow the reduction of TfR1 mRNA, thereby limiting iron uptake under high iron conditions. This post-transcriptional regulation by iron regulatory proteins / iron response elements (IRPs) functioned normally in FRDA fibroblasts, as TFRC transcripts were efficiently downregulated to limit iron uptake under high iron conditions. Therefore, the increased steady-state levels of TfR1 and its accumulation at the membrane in FRDA fibroblasts pointed to another level of regulation, i.e., post-translational regulation of TfR1, as previously reported in NBIA (Drecourt et al., 2018).

[0061] TfR1 is post-translationally modified by S-acylation, particularly palmitoylation, because palmitate (C16:0) is the major lipid donor for S-acylated proteins. The palmitoylation level of TfR1 is known to regulate intracellular iron because Cys, the major site of palmitoylation of TfR1, is located at the cytosine endonuclearise (C16:0). 62 and Cys 67Mutations in TfR1 caused increased TfR1 internalization and iron overload. Here, we provide evidence of defective TfR1 palmitoylation in FRDA fibroblasts and subsequent accumulation of TfR1 at the membrane and cytoplasm. Because post-translational regulation of TfR1 by palmitoylation rapidly regulates intracellular iron content, we hypothesize that this regulatory system is impaired in FRDA and contributes, at least in part, to the disease mechanism. We previously attributed defective TfR1 palmitoylation to impaired CoA synthesis associated with mutations in PANK2 and CRAT in NBIA (Drecourt et al., 2018). Because PANK2 and CRAT are directly involved in CoA synthesis, the increased TfR1 palmitoylation and decreased iron content after CoA supplementation in FRDA cultured fibroblasts strongly suggest that frataxin deficiency induces a secondary decrease in the CoA / acetyl-CoA pool. As with other defects in the iron-sulfur complex biosynthesis machinery, frataxin deficiency affects various intracellular proteins, including mitochondrial lipoic acid synthase, resulting in decreased lipoylation of at least the dihydrolipoyl transacetylase (DLAT) subunit of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (Lebigot et al., 2017; Martelli et al., 2015; Tong et al., 2018). Acetyl-CoA is primarily produced in mitochondria by decarboxylation of pyruvate, and the CoA pool is expected to be reduced in FRDA fibroblasts due to incomplete DLAT lipoylation. Increased palmitoylation of TfR1 after inhibition of pyruvate dehydrokinase by dichloroacetate supports this hypothesis.

[0062] FRDA fibroblasts also exhibited defective endosomal circuitry, as indicated by delayed Tf recycling. Due to impaired endosomal recycling, we hypothesize that iron overload may result not only from increased membrane abundance of TfR1 but also from impaired release of cytoplasmic iron stored in uncoated vesicles. Palmitoylation is known to increase protein lipophilicity and regulate its trafficking, stability, and intracellular distribution. Because many endosomal recycling proteins are palmitoylated, it is possible that altered palmitoylation of other, as yet undetermined, endosomal proteins may alter TfR1 recycling and contribute to iron overload.

[0063] The identification of abnormal palmitoylation of TfR1 in FRDA fibroblasts adds another level of complexity to the pathophysiology of the disease. It also helps elucidate the diverse clinical outcomes of frataxin deficiency. Because the time course and / or tissue-specific expression of iron-sulfur complex-containing proteins may actually control organ onset, explaining why respiratory chain defects are observed in the hearts of FRDA patients but not in muscle or fibroblasts (Rotig et al., 1997). The different tissue requirements for acetyl-CoA or SIRT3 inhibition (Wagner et al., 2012), which are known to affect intracellular iron content (Jeong et al., 2015), may also be responsible for the tissue-specific expression of FRDA. In support of this, respiratory chain defects were observed in the hearts of 7-week-old frataxin-deficient mice, whereas iron overload appeared after 3 weeks (Puccio et al., 2001).

[0064] Other consequences of frataxin deficiency may also cause a secondary decrease in the mitochondrial acetyl-CoA pool. As shown for ISCU deficiency (Tong et al., 2018), tissues with reduced mitochondrial aconitase activity accumulate citrate, which activates acetyl-CoA carboxylase and thus reduces the acetyl-CoA pool by inducing the formation of malonyl-CoA. Suppression of SIRT3 deacetylase in mice has been reported to induce hyperacetylation of several mitochondrial proteins, which is known to reduce their activity (Wagner et al., 2012). Hyperacetylation of mitochondrial acetyl-CoA synthetase 2, one of the primary targets of SIRT3, may reduce acetyl-CoA synthesis. Along the same lines, iron accumulation can increase sphingolipid and palmitoyl-CoA synthesis and acyl-CoA consumption, which in turn reduces the palmitoyl-CoA and acyl-CoA pools required for TfR1 palmitoylation (Chen et al., 2016a). Indeed, as in the case of impaired CoA biosynthesis associated with PANK2 mutations (Siudeja et al., 2011), frataxin deficiency reduces global histone acetylation (Tong et al., 2018).

[0065] Finally, we also show that artesunate, CoA, and dichloroacetic acid significantly induced palmitoylation of TfR1 and reduced steady-state levels and membrane accumulation of TfR1. Artesunate rescued Tf recycling and restored the ability of FRDA fibroblasts to regulate iron uptake. Artesunate has potent anticancer properties because it induces iron deficiency, which is toxic to cancer cells (Lai et al., 2013). It is also used to treat malaria caused by Plasmodium falciparum. Although its safety profile and pharmacokinetics have not been evaluated in neurodegenerative diseases, millions of subjects have received artemisinin with very few side effects (Efferth and Kaina, 2010). Our data suggest that this compound and other drugs that increase TfR1 palmitoylation should be considered as potential therapeutic approaches in FRDA. Iron-mediated toxicity in particular has been shown to be a cause of neurodegeneration in a Drosophila model of FRDA (Chen et al., 2016b). Because PBMCs from FRDA patients were unable to regulate iron uptake under high iron conditions, as observed in FRDA-iPSC cardiomyocytes (Lee et al., 2014), we believe that in vivo monitoring of iron homeostasis and TfR1 immunofluorescence may be useful early endpoints for monitoring future clinical trials in FRDA patients.

[0066] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into the present disclosure. [Table 2] TIFF0007822411000003.tif241165 TIFF0007822411000004.tif250165 TIFF0007822411000005.tif147165

Claims

1. 1. A method for testing the likelihood of achieving a therapeutic response in a patient with Friedreich's ataxia (FRDA) with an agent capable of increasing palmitoylation of transferrin receptor 1 (TfR1), comprising: i) measuring the total intracellular iron content of peripheral blood mononuclear cells (PBMCs) obtained from the patient cultured in a medium containing an amount of iron, with or without the agent; wherein the agent is selected from the group consisting of artesunate, dichloroacetic acid, and coenzyme A; and The iron content in the cells cultured in the medium without the drug and the iron content in the cells cultured in the medium with the drug are The method is compared to a criterion that when the iron content in the cells cultured in medium with the drug is lower than the iron content in the cells cultured in medium without the drug, the drug will treat FRDA in the patient.

2. 2. The method of claim 1, wherein the PBMCs are cultured for 8 hours, 16 hours, 24 hours, 32 hours, or 40 hours before measuring the total iron content.

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