ALBERT

Description: Various human disorders are associated with misdistribution of iron within or across cells. Friedreich ataxia (FRDA), a deficiency in the mitochondrial iron-chaperone frataxin, results in defective use of iron and its misdistribution between mitochondria and cytosol. We assessed the possibility of functionally correcting the cellular properties affected by frataxin deficiency with a siderophore capable of relocating iron and facilitating its metabolic use. Adding the chelator deferiprone at clinical concentrations to inducibly frataxin-deficient HEK-293 cells resulted in chelation of mitochondrial labile iron involved in oxidative stress and in reactivation of iron-depleted aconitase. These led to (1) restoration of impaired mitochondrial membrane and redox potentials, (2) increased adenosine triphosphate production and oxygen consumption, and (3) attenuation of mitochondrial DNA damage and reversal of hypersensitivity to staurosporine-induced apoptosis. Permeant chelators of higher affinity than deferiprone were not as efficient in restoring affected functions. Thus, although iron chelation might protect cells from iron toxicity, rendering the chelated iron bioavailable might underlie the capacity of deferiprone to restore cell functions affected by frataxin deficiency, as also observed in FRDA patients. The siderophore-like properties of deferiprone provide a rational basis for treating diseases of iron misdistribution, such as FRDA, anemia of chronic disease, and X-linked sideroblastic anemia with ataxia.

Description: Various pathologies are characterized by the accumulation of toxic iron in cell compartments. In anemia of chronic disease, iron is withheld by macrophages, leaving extracellular fluids iron-depleted. In Friedreich ataxia, iron levels rise in the mitochondria of excitable cells but decrease in the cytosol. We explored the possibility of using deferiprone, a membrane-permeant iron chelator in clinical use, to capture labile iron accumulated in specific organelles of cardiomyocytes and macrophages and convey it to other locations for physiologic reuse. Deferiprone's capacity for shuttling iron between cellular organelles was assessed with organelle-targeted fluorescent iron sensors in conjunction with time-lapse fluorescence microscopy imaging. Deferiprone facilitated transfer of iron from extracellular media into nuclei and mitochondria, from nuclei to mitochondria, from endosomes to nuclei, and from intracellular compartments to extracellular apotransferrin. Furthermore, it mobilized iron from iron-loaded cells and donated it to preerythroid cells for hemoglobin synthesis, both in the presence and in the absence of transferrin. These unique properties of deferiprone underlie mechanistically its capacity to alleviate iron accumulation in dentate nuclei of Friedreich ataxia patients and to donate tissue-chelated iron to plasma transferrin in thalassemia intermedia patients. Deferiprone's shuttling properties could be exploited clinically for treating diseases involving regional iron accumulation.

Description: Iron raises to toxic levels in mitochondria of excitable cells in some forms of neuro-degeneration with brain accumulation (NBIA), in sideroblastic anemia and in Friedreich’s ataxia (FA), often leaving the cytosol iron-depleted. In anemia of chronic disease (ACD) iron is withheld by macrophages, while iron levels in extracellular fluids (e.g. plasma) are drastically reduced. Although excessive iron deposition occurring in organs of iron overloaded (IO) patients can be reduced with iron chelators, it is uncertain whether this is applicable to conditions where iron accumulates within selected tissues/cells in the absence of systemic IO. Objective. We assessed whether deferiprone (DFP), a membrane-permeant bidentate chelator in clinical use for treating systemic IO, might serve as an iron relocating agent in settings of regional iron accumulation by a. capturing labile iron accumulated in cell compartments and b. conveying the chelated iron either to other cell locations for metabolic integration or to transferrin for systemic reutilization. Methods. DFP capacity to shuttle iron intracellularly and transcellularly was assessed in macrophages (J774) and heart (H9c2) cell models using organelle-targeted fluorescent iron-sensors in conjunction with fluorescence microscopy imaging. We employed pairs of sensors targeted to different cell compartments and iron-evoked quenching and chelator-evoked dequenching as means to trace DFP mediated iron transfer. Mitochondrial iron accumulation was generated with succinylacetone or by silencing genes affecting mitochondrial iron metabolism. Results. DFP facilitated iron transfer: a. from iron-laden cell organelles to other cell compartments or to medium (and vice versa) and b. from iron loaded macrophages to pre-erythroid MEL cells (for chemically induced hemoglobin synthesis) either directly or via transfer to extracellular transferrin. Discussion The results of this study indicate that relocation of cell accumulated iron can be used as a modality of chelation for treating conditions of regional iron accumulation by applying chelators able to permeate into cell compartments but also to transfer the chelated iron to cell acceptors or to the extracellular iron carrier transferrin.

Description: Context: The principle of Sickle Scan (Biomedomics, Inc.) is a rapid, point-of-care qualitative lateral flow immuno-assay kit for the identification of sickle cell conditions of Hb A, S and C. Sickle Scan was specifically developed to allow for the identification of sickle cell trait Hb AS, heterozygotes AC, and Hb SS, Hb SC and Sβ° patients. Other sickle cell conditions as SD, SE, SO-Arab, S Lepore,… cannot be identified using Sickle Scan system. The test must be done using venous blood or capillary blood (fresh or dried blood spots). Patients and methods: Two hundred and fifty patient samples (143 adults and 107 newborns) were analyzed. All tests were performed according the manufacturer's recommendations in one laboratory by 2 observers. The reference tests used for comparison were HPLC (NBS Variant - Bio-Rad) and capillary electrophoresis (Capillarys 3 - Sebia). Results: Comments: In adult patients, the 2 observers concordantly detected the presence of Hb A, Hb S and Hb C. There were 4 differences of interpretation between them (no Hb A in a AS patient and no Hb A in 3 SS transfused patients). The percentage of Hb A in these 3 last patients was respectively 13.6%, 17.7% and 18%. There was no false positivity neither in the patient heterozygous AE nor in the patient SD. No false negativity occurred for Hb S and C. In newborns, the accuracy of the test was excellent for the identification of the phenotypes FA, FAS, FAC, FS (SS / Sβ°). The lowest detected values of Hb S and Hb C in FAS and FAC newborns were respectively 2.4 and 3.4 %. We observed an inconstant cross-reactivity of the antibody anti Hb S with the hemoglobins E and D, in respectively 3/25 FAE phenotype and 6/26 in the FAD phenotype. There was no cross reaction with hemoglobin Bart's and O-Arab. In FAS newborns the mean and extreme values of the percentage of Hb A were m=8.2 (2.6-15.5) and no difficulties occurred for the identification of these low percentages of Hb A. This observation is different from those made in adult patients for which one observer did not find Hb A in transfused patients with highest values of Hb A comprised between 13.6 and 18. Conclusions: In this series of adult and newborn patients, the Sickle Scan appeared as an accurate method for the identification of AS, AC, SS/Sβ° and SC phenotypes. False positive tests were observed in some patients with hemoglobin E or D but no false negative results were found as regards the identification of Hb S and Hb C. Table Table. Disclosures Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding.

Description: Genetic disorders of iron metabolism and chronic inflammation often evoke local iron accumulation. In Friedreich ataxia, decreased iron-sulphur cluster and heme formation leads to mitochondrial iron accumulation and ensuing oxidative damage that primarily affects sensory neurons, the myocardium, and endocrine glands. We assessed the possibility of reducing brain iron accumulation in Friedreich ataxia patients with a membrane-permeant chelator capable of shuttling chelated iron from cells to transferrin, using regimens suitable for patients with no systemic iron overload. Brain magnetic resonance imaging (MRI) of Friedreich ataxia patients compared with age-matched controls revealed smaller and irregularly shaped dentate nuclei with significantly (P 〈 .027) higher H-relaxation rates R2*, indicating regional iron accumulation. A 6-month treatment with 20 to 30 mg/kg/d deferiprone of 9 adolescent patients with no overt cardiomyopathy reduced R2* from 18.3 s−1 (± 1.6 s−1) to 15.7 s−1 (± 0.7 s−1; P 〈 .002), specifically in dentate nuclei and proportionally to the initial R2* (r = 0.90). Chelator treatment caused no apparent hematologic or neurologic side effects while reducing neuropathy and ataxic gait in the youngest patients. To our knowledge, this is the first clinical demonstration of chelation removing labile iron accumulated in a specific brain area implicated in a neurodegenerative disease. The use of moderate chelation for relocating iron from areas of deposition to areas of deprivation has clinical implications for various neurodegenerative and hematologic disorders.

Description: Friedreich's ataxia (FRDA) is a frequent autosomal recessive disease caused by a GAA repeat expansion in the FXN gene encoding frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biogenesis. Resulting frataxin deficiency affects ISC-containing proteins and causes iron to accumulate in the brain and heart of FRDA patients. Here we report on abnormal cellular iron homeostasis in FRDA fibroblasts inducing a massive iron overload in the cytosol and mitochondria. We observe membrane transferrin receptor 1 (TfR1) accumulation, increased TfR1 endocytosis, and delayed transferrin recycling, ascribing this to impaired TfR1 palmitoylation. Frataxin deficiency is shown to reduce coenzyme A (CoA) availability for TfR1 palmitoylation. Finally, we demonstrate that artesunate, CoA, and dichloroacetate improve TfR1 palmitoylation and decrease iron overload, paving the road for evidence-based therapeutic strategies at the actionable level of TfR1 palmitoylation in FRDA.