Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is caused in almost all cases by homozygous intronic expansions resulting in the loss of frataxin, a mitochondrial protein conserved through evolution, and involved in mitochondrial iron homeostasis. Yeast knockout models, and histological and biochemical data from patient heart biopsies or autopsies indicate that the frataxin defect causes a specific iron–sulfur protein deficiency and mitochondrial iron accumulation leading to the pathological changes. Affected human tissues are rarely available to further examine this hypothesis. To study the mechanism of the disease, we generated a mouse model by deletion of exon 4 leading to inactivation of the Frda gene product. We show that homozygous deletions cause embryonic lethality a few days after implantation, demonstrating an important role for frataxin during early development. These results suggest that the milder phenotype in humans is due to residual frataxin expression associated with the expansion mutations. Surprisingly, in the frataxin knockout mouse, no iron accumulation was observed during embryonic resorption, suggesting that cell death could be due to a mechanism independent of iron accumulation.

Ex vivo manufacture of red blood cells from stem cells is a potential means to ensure an adequate and safe supply of blood cell products. Advances in somatic cell reprogramming of human induced pluripotent stem cells have opened the door to generating specific cells for cell therapy. Human induced pluripotent stem cells represent a potentially unlimited source of stem cells for erythroid generation for transfusion medicine. We characterized the erythroid differentiation and maturation of human induced pluripotent stem cell lines obtained from human fetal (IMR90) and adult fibroblasts (FD-136) compared to those of a human embryonic stem cell line (H1). Our protocol comprises two steps: (i) differentiation of human induced pluripotent stem cells by formation of embryoid bodies with indispensable conditioning in the presence of cytokines and human plasma to obtain early erythroid commitment, and (ii) differentiation/maturation to the stage of cultured red blood cells in the presence of cytokines. The protocol dispenses with major constraints such as an obligatory passage through a hematopoietic progenitor, co-culture on a cellular stroma and use of proteins of animal origin. We report for the first time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells containing fetal hemoglobin in a functional tetrameric form. Red blood cells generated from human induced pluripotent stem cells pave the way for future development of allogeneic transfusion products. This could be done by banking a very limited number of red cell phenotype combinations enabling the safe transfusion of a great number of immunized patients.

Frataxin, the mitochondrial protein deficient in Friedreich ataxia, a rare autosomal recessive neurodegenerative disorder, is thought to be involved in multiple iron-dependent mitochondrial pathways. In particular, frataxin plays an important role in the formation of iron-sulfur (Fe-S) clusters biogenesis. We present data providing new insights into the interactions of mammalian frataxin with the Fe-S assembly complex by combining in vitro and in vivo approaches. Through immunoprecipitation experiments, we show that the main endogenous interactors of a recombinant mature human frataxin are ISCU, NFS1 and ISD11, the components of the core Fe-S assembly complex. Furthemore, using a heterologous expression system, we demonstrate that mammalian frataxin interacts with the preformed core complex, rather than with the individual components. The quaternary complex can be isolated in a stable form and has a molecular mass of ≈190 kDa. Finally, we demonstrate that the mature human FXN_81–210 form of frataxin is the essential functional form in vivo. Our results suggest that the interaction of frataxin with the core ISCU/NFS1/ISD11 complex most likely defines the essential function of frataxin. Our results provide new elements important for further understanding the early steps of de novo Fe-S cluster biosynthesis.

Friedreich ataxia (FRDA) results from a generalized deficiency of mitochondrial and cytosolic iron–sulfur protein activity initially ascribed to mitochondrial iron overload. Recent in vitro data suggest that frataxin is necessary for iron incorporation in Fe–S cluster (ISC) and heme biosynthesis. In addition, several reports suggest that continuous oxidative damage resulting from hampered superoxide dismutases (SODs) signaling participates in the mitochondrial deficiency and ultimately the neuronal and cardiac cell death. This has led to the use of antioxidants such as idebenone for FRDA therapy. To further discern the role of oxidative stress in FRDA pathophysiology, we have tested the potential effect of increased antioxidant defense using an MnSOD mimetic (MnTBAP) and Cu,ZnSOD overexpression on the murine FRDA cardiomyopathy. Surprisingly, no positive effect was observed, suggesting that increased superoxide production could not explain by itself the FRDA cardiac pathophysiology. Moreover, we demonstrate that complete frataxin-deficiency neither induces oxidative stress in neuronal tissues nor alters the MnSOD expression and induction in the early step of the pathology (neuronal and cardiac) as previously suggested. We show that cytosolic ISC aconitase activity of iron regulatory protein-1 progressively decreases, whereas its apo-RNA binding form increases despite the absence of oxidative stress, suggesting that in a mammalian system the mitochondrial ISC assembly machinery is essential for cytosolic ISC biogenesis. In conclusion, our data demonstrate that in FRDA, mitochondrial iron accumulation does not induce oxidative stress and we propose that, contrary to the general assumption, FRDA is a neurodegenerative disease not associated with oxidative damage.