The role of frataxin, the deficient protein in Friedreich's ataxia (FRDA), in iron metabolism is now firmly established. The first clues came from the analysis of yeast mutants with a deleted frataxin homolog gene (Δyfh1), which exhibit mitochondrial iron accumulation and dysregulation of iron uptake (Babcock et al. 1997). Δyfh1 yeast shows the apparent paradox of activating the high-affinity iron uptake system, which is normally induced by cellular iron depletion, while having high intracellular iron content. A closer analysis of Δyfh1 cells reveals that the excess iron content is all concentrated in mitochondria, with concomitant cytosolic iron depletion. In yeast, the high-affinity iron uptake system is regulated at the transcriptional level by the iron sensor and transcription factor Aft1, which is localized in the cytosol. Hence, loss of frataxin results in the induction of iron uptake because of the unbalanced intracellular distribution of the metal, and implies that iron entering the cell is directly channeled into mitochondria, where it accumulates. Experimental evidence indeed supported that mitochondrial iron accumulation may be prevented by diverting the flow of iron coming into the cell into a different vesicular compartment, which may be obtained by over-expressing the vesicular metal transporter Ccc1(Chen and Kaplan 2000). Furthermore, restoring frataxin expression in Δyfh1 yeast, by transfection with a frataxin-expressing plasmid, induces a rapid flow of iron out of mitochondria, re-equilibrating the intracellular iron distribution (Radisky et al. 1999). Important clues to the underlying mechanism came with the discovery that frataxin deficiency leads to a depletion of iron–sulfur (Fe–S) clusters. Fe–S clusters are important cofactors for many proteins with different functions and subcellular localizations, including the Krebs cycle enzyme aconitase and several subunits of respiratory complexes I, II, and III in the mitochondria, enzymes of aminoacid and purine metabolism in the cytosol, and DNA replication and repair factors in the nucleus(Sheftel et al. 2010). Deficiencies of Fe–S dependent enzymes were first observed in endomyocardial biopsies from FRDA patients and in Δyfh1 yeast (Rötig et al. 1997), then in conditional knock-out mice with tissue-specific deletion of the Fxn gene (Puccio et al. 2001; Puccio 2007). Subsequent observations suggested that these deficiencies are the consequence of impaired Fe–S cluster synthesis. An initial clue came by the observation that deletion in yeast of any gene encoding a protein involved in Fe–S cluster biogenesis leads to the same impairment of iron metabolism observed in Δyfh1 cells (Chen et al. 2002). Then, a series of studies, first in yeast, then in mammalian systems, confirmed the role of frataxin as an activator of mitochondrial Fe–S cluster biogenesis (Mühlenhoff et al. 2002; Stehling et al. 2004). The most recent findings revealed that frataxin is part of a multiprotein complex responsible for Fe–S cluster biogenesis in the mitochondrial matrix (Schmucker et al. 2011), in which it acts as an allosteric activator of the cysteine desulfurase enzyme that provides the sulfur moiety for Fe–S cluster assembly (Tsai and Barondeau 2010; Colin et al. 2013). Other postulated functions of frataxin oligomers and multimers in mitochondrial iron storage and detoxification (Vaubel and Isaya 2012) cannot be excluded, but remain controversial, and in any case do not contradict its major role in Fe–S synthesis. Mammalian cells contain Fe–S assembly factors also in the cytosol, but an essential precursor for cytosolic Fe–S synthesis has to be exported from mitochondria, where its synthesis depends on mitochondrial Fe–S cluster biogenesis (Lill et al. 2012; Rouault 2012). Accordingly, frataxin-deficient cells have impaired activities of Fe–S proteins in all cellular compartments (Martelli et al. 2007). This is important to understand the consequences of frataxin deficiency on iron metabolism in mammalian cells, because a key regulator of iron metabolism in higher eukaryotes, iron responsive element binding protein 1 (IRP1), which is localized in the cytosol, is a Fe–S protein. IRP1 and a structurally similar protein, IRP2, regulate iron metabolism in mammalian cells by binding to sequence motifs (iron responsive elements, IREs) that are present in the mRNAs for proteins involved in iron metabolism (Aisen et al. 2001). RNA binding of IRP1 and IRP2 normally occurs when cytosolic iron levels are low. IRP2, which has no Fe–S cluster, undergoes iron-dependent degradation, so its level increases as cytosolic iron decreases, whereas IRP1 functions as a cytosolic aconitase when its Fe-S cluster is fully assembled, and becomes an IRE-binding protein when its Fe-S cluster is partially or fully disassembled. IRE-binding by IRP1 and 2 protects mRNAs of iron import proteins from degradation, like the transferrin receptor, and prevents translation of mRNAs for proteins that need iron for their function, like aconitase, or store iron, like ferritin. A possible scenario when frataxin is deficient, is the activation of IRP1 as IRE-binding protein because of impaired Fe–S cluster biogenesis, resulting in increased cellular iron uptake. IRP1 activation has indeed been detected in frataxin-deficient cells and animal models (Lobmayr et al. 2005). Iron then is funneled into mitochondria where it should be utilized for Fe–S cluster (and heme) synthesis. Up-regulation of mitochondrial iron uptake via mitochondrial iron transporter (Richardson et al. 2010) also occurs when frataxin is deficient, contributing to iron accumulation in these organelles. Overall, these changes can be interpreted as a homeostatic response to restore Fe–S cluster biogenesis when it occurs at low levels, normally the consequence of iron deficiency. However, when frataxin is low, the extra iron imported into mitochondria cannot be efficiently utilized, it is oxidized, and progressively accumulates. Iron oxidation is in part the result of the Fenton reaction between ferrous iron, the normal substrate for Fe–S synthesis, and H2O2, which is produced in excess in mitochondria because of respiratory chain dysfunction, generating the highly toxic hydroxyl radical (OH˙) (Fig. 1).
The use of iron chelators in FRDA was first proposed as a mean to eliminate the excess iron accumulating in mitochondria, thus decreasing free radical generation through the Fenton reaction. However, iron chelators that poorly penetrate biological membranes, as desferoxamine, can only do that indirectly, by profoundly depleting iron in the extracellular medium and therefore in the whole cell, a clearly unwanted outcome that could even worsen Fe–S protein deficiencies. To target the iron imbalance in FRDA, an iron chelator should instead be able to reach into mitochondria and redistribute iron to other cellular compartments, without causing overall depletion. Such an iron chelator should therefore be membrane-permeable and have relatively low affinity for iron. Deferiprone, an orally administered, lipid-soluble iron chelator used in several countries as an alternative to desferoxamine to treat iron overload in polytransfused individuals with hemoglobinopathies, can easily cross the blood–brain barrier and cellular membranes. In addition, because of its relatively low affinity for iron, lower than that of transferrin, deferiprone is less likely to cause iron depletion than desferoxamine. These properties made deferiprone an interesting candidate as FRDA therapeutic.