Frataxin: Its Role in Iron Metabolism and the Pathogenesis of Friedreich's Ataxia

Erika Becker a,b and Des R. Richardson

The Heart Research Institute, 
145 Missenden Rd, 
Camperdown Sydney, 
New South Wales 2050 AUSTRALIA; 
The Department of Medicine, 
Royal Brisbane Hospital, 
Herston, Brisbane, Queensland 4029, AUSTRALIA

Author for Correspondence:  
Dr. D.R. Richardson,
The Heart Research Institute,
145 Missenden Rd, Camperdown, Sydney, NSW 2050 AUSTRALIA.
Ph: +61-2-9550-3560; FAX: +61-2-9550-7502; email:
Idebenone - 150 mg - 30 caps - SALE - Jan 2015


Friedreich's ataxia (FA) is a severe neurodegenerative condition.  In 97% of patients this disease is due to an intronic GAA triplet repeat expansion in the FRDA gene resulting in a marked decrease in its mRNA levels. The protein encoded by this gene is known as frataxin that is localised within the mitochondrion. When the gene homologous to FRDA in the yeast (YFH1) was deleted, it resulted in an accumulation of iron (Fe) within the mitochondrion. When the YFH1 gene was reintroduced back into the yeast cell Fe was exported out of the mitochondrion and into the cytosol.  Evidence that human frataxin is also involved in nitochondrial Fe overload comes from studies in FA patients that have shown there is an accumulation of Fe within the heart. While the precise role of human frataxin remains to be determined, the molecule appears to be involved in regulating the export and/or inport of mitochondrial Fe. These findings of mitochondrial Fe overload suggests that the use of chelators that can permeate the mitochondrion may have potential in the treatment of this disease.

1. General Introduction
All living cells require iron (Fe) for growth, replication, respiration, and DNA synthesis [1,2]. Iron is required for critical proteins within the mitochondrion, including haem and iron-sulfur proteins that involved in electron transport and energy production. On the one hand, while Fe is essential for growth and replication, excess Fe is highly toxic. For many years a number of Fe-loading diseases have been known, the most common being haemochromatosis and b-thalassemia. More recently, studies have indicated that the severe neurodegenerative disease, Friedreich's ataxia, could be caused by Fe overload in the mitochondrion and this is discussed below. Before these latter investigations are described, it is important to describe how cells take up and utilise Fe.

2. Cellular Iron Metabolism
The transport of Fe in the serum is achieved by the Fe-binding protein transferrin (Tf). Serum Tf belongs to an important class of Fe-binding proteins [3,4] that include lactoferrin, ovotransferrin and melanotransferrin. Cells can obtain Fe from serum Tf by a number of mechanisms. The most well characterised process of Fe uptake is via receptor-mediated endocytosis of Tf [5-7]. This latter process involves the binding of Tf to specific Tf receptors (TfR) present on the cell surface followed by internalisation of the molecule into endosomes. The Fe is then released from Tf by a reduction of endosomal pH. Once released the Fe is transported through the endosomal membrane and into the cell via Nramp2 or the divalent metal transporter 1 (DMT1) [8-10]. From this point Fe can be used for a variety of metabolic processes or stored within the protein ferritin. In terms of mitochondrial Fe utilisation, the best characterised function in this organelle involves the use of Fe for the production of haem [11]. Haem is an important component of many proteins involved in respiration and metabolism. These studies, provide a framework for trying to understand the role of many new molecules that have been reported to play a role in Fe metabolism.

3. What is the Function of Frataxin and How Does it Result in Friedreich's Ataxia?
The gene FRDA is defective in FA (Campuzano et al., 1996) and encodes a protein known as frataxin [12,13]. In 97% of cases the defect in the gene is due to an intronic GAA triplet repeat expansion that results in a marked decrease in gene expression [12,13]. Over the last few years, evidence has accumulated to suggest that frataxin could play a role in mitochondrial Fe metabolism [18-19]. However, frataxin has no appreciable sequence homology to other enzymes involved in Fe or haem metabolism and its function remains unclear (see below).

Studies in the baker's yeast (Saccharomyces cerevisiae) have led to an interesting model that may explain the pathogenesis of FA [18,20]. The yeast gene YFH1 is very similar to the human gene FRDA that produces frataxin [20]. In addition, YFH1 encodes a mitochondrial protein (Yfh1p) in the yeast involved in Fe homeostasis and respiration [17,18,20]. When the YFH1 gene was deleted in the yeast there was a marked accumulation of Fe in the mitochondria resulting in the loss of mitochondrial DNA and respiration [14,15,20,21]. Thus, this led to the hypothesis that the accumulated Fe resulted in the production of free radicals that are known to damage lipids, proteins and the mitochondrial genome. When YFH1 was reintroduced back into the yeast, mitochondrial Fe was exported back out into the cytosol [18,20]. The frataxin homologue Yfh1p therefore seemed to regulate Fe export from the mitochondrion, suggesting a mitochondrial Fe cycle in the yeast [18]. It is unlikely that Yfh1p is the actual Fe transporter, because sequence analysis shows that it has no transmembrane sequences necessary for a transport role [18]. Another molecule involved in Fe metabolism in the yeast mitochondrion is the protein Atm1p, which is an ABC transporter situated in the inner mitochondrial membrane [18]. Further studies are necessary to assess whether there are any functional relationships between Yfh1p and Atm1p.

Interestingly, it was shown that the growth of yeast mutants without the YFH1 gene was highly sensitive to oxidants (eg. H202, Cu) [20,21], probably because of the accumulation of mitochondrial Fe. In addition, disruption of the YFH1 gene resulted in multiple deficiencies in other Fe-S containing enzymes [15]. In accordance with these observations, a loss of aconitase activity has been observed in cardiac biopsies from two FA patients [15]. This decreased enzymatic activity may be due to the sensitivity of Fe-S proteins to free radicals produced by the mitochondrial Fe accumulation.

Fluorescence microscopy has shown that the yeast frataxin homologue is localised to the mitochondrion [13,22]. Frataxin homologues have also been seen in CyaY protein of gram negative bacteria but not their gram positive counterparts [13,22,23]. This latter observation may be significant, since gram negative bacteria share similar characteristics with mitochondria. Human frataxin contains a functional mitochondrial targeting signal, despite low homology with the mitochondrial targeting consensus sequence [13]. The mitochondrial targeting signal is found within the first 20 amino acids and is removed upon protein maturation. Tagging experiments have demonstrated that human frataxin co-localises with the mitochondrial protein cytochrome c oxidase [22].

It has also been found that FRDA was expressed in mouse tissues which are rich in mitochondria, including liver, kidney, skeletal muscle, spleen, thymus, brown fat, heart, and weakly in the brain and lung [22].  In human tissue, FRDA was found to be most highly expressed in the heart, intermediate levels in liver, skeletal muscle and pancreas, and minimal expression in brain [13].  From these data, it is highly likely that the pathology observed in FA is due to mitochondrial Fe overload that leads to the production of toxic free radicals. Consistent with the knockout yeast model, Wong et al. [24], have suggested a pathogenic role for oxidative stress in fibroblasts from FA patients. In addition, Bradley et al. [25] noted that oxidant stress may induce a self-amplifying cycle of oxidative damage. Evidence showing that the pathology of FA in humans is caused by mitochondrial Fe overload includes Fe deposits within the heart myofibrils [26,27], defective myocardial mitochondrial respiration [15], and perturbations in the haem biosynthesis pathway [28]. In summary it can be suggested that eukaryotic frataxins are mitochondrial proteins playing a conserved role in Fe metabolism.

If frataxin plays a role in Fe metabolism, an understanding of the pathogenesis of FA will require the examination of mitochondrial Fe metabolism. Obviously, a mitochondrial function for frataxin has important implications for the understanding of the neurodegenerative process in FA. The best characterised function of mitochondria in Fe metabolism comes from studies in erythroid cells where the mitochondrion is involved in the production of haem for haemoglobin synthesis [11]. Richardson et al. [29] have shown that in erythroid cells Fe is targeted to the mitochondrion where it is incorporated into protoporphyrin to form haem. This latter molecule is then exported out of the mitochondrion to combine with globin chains to form haemoglobin. When the haem biosynthesis pathway is inhibited with succinylacetone (a competitive inhibitor of the enzyme d-aminolevulinic acid dehydratase), Fe accumulates within the mitochondrion [29]. Only when exogenous protoporphyrin is added, does Fe become incorporated into protoporphyrin and form haem which is transported out of the mitochondrion [29]. In the absence of protoporphyrin the Fe accumulates, and under these conditions can result in DNA damage due to the formation of toxic hydroxyl radicals.

Considering the evidence collectively, it is possible to suggest a hypothesis that the decreased levels of frataxin in FA patients may lead to some defect in the process of Fe incorporation into protoporphrin or Fe influx or export which then results in mitochondrial Fe accumulation. In each case, Fe accumulation in the mitochondrion occurs. From the sequence data on the frataxin gene it is already obvious that this protein is not an enzyme of the haem biosynthetic pathway. However, it cannot be excluded that frataxin may play other roles in haem or Fe metabolism that remain unknown at present.

4. Possible Treatment Regimes for Friedreich's Ataxia Patients
Since FA appears to be due to mitochondrial Fe overload, new treatment strategies based upon this new data may provide significant hope for FA patients. One strategy that deserves to be investigated is the use of specific Fe chelators that can permeate the mitochondrion. Already a trial supported by the National Institute of Health (NIH) has begun to investigate the possible utility of the clinically used Fe chelator desferrioxamine (DFO) to treat FA patients [30]. However, DFO cannot efficiently penetrate cell membranes [31], and previous studies have demonstrated that the chelator is not effective at mobilising Fe from Fe-loaded mitochondria in erythroid cells [32].

In contrast to DFO, Ponka and colleagues [33,34] have shown that another chelator known as pyridoxal isonicotinoyl hydrazone (PIH) shows high activity at mobilising mitochondrial Fe. A variety of studies in vitro and in vivo have demonstrated that PIH and its analogues show great potential for the treatment of Fe-overload disease [34-45]. Unfortunately, the development of PIH and its analogues was hindered due to the fact that the chelator was not patented, which meant that pharmaceutical companies had little interest.

To overcome these latter disadvantages we have synthesized a new group of chelators known as the 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH) analogues [46,47]. These latter compounds have been provisionally patented and have been designed so that they share some structural similarities to PIH. Moreover, some of the PCIH analogues are more active than DFO and PIH at mobilising Fe from cells [46], and like PIH can effectively mobilise Fe from the mitochondrion [48]. Further studies in animal models are essential in order to determine the efficiency of these compounds at increasing Fe excretion.

5. Conclusions
The accumulation of Fe within the mitochondrion appears to play a significant role in the development of FA. The build-up of Fe within this organelle is due to a decrease in the levels of frataxin. Further studies are essential to determine the function of frataxin in Fe metabolism, although it can be suggested that the molecule may be involved in regulating either mitochondrial Fe uptake or release. Finally, the development of chelators that can permeate the mitochondrion may provide a novel strategy for the treatment of FA patients.

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