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Massimo Pandolfo
The molecular basis of Friedreich ataxia
Neurología 2000; 15: 325-329

Art: 002193

In 1863, Nicholaus Friedreich, Professor of Medicine in Heidelberg, Germany, described a "degenerative atrophy of the posterior columns of the spinal cord" causing progressive ataxia, sensory loss and muscle weakness. The cause of "Friedreich's ataxia" remained mysterious for more than 130 years thereafter. Research on the disease got a new impulse in the late 1970s and early 1980s, when landmark studies carried out established the autosomal recessive pattern of inheritance1-3 and defined rigorous diagnostic criteria1,2. These criteria allowed the collection of clinically homogenous families that were essential for molecular genetic studies. The eventual isolation of the Friedreich ataxia gene (FRDA) in 19964 was a major success of positional cloning applied to the study of inherited neurodegenerative diseases. Thanks to this discovery, genetic testing, genotype-phenotype correlations, pathophysiological studies and new approaches to treatment became possible.

The Friedreich ataxia gene (FRDA, according to the Human Genome Organization nomenclature) is localized on the long arm of chromosome 95,6, just below the pericentromeric heterochromatic region. Its most abundant, and probably only functionally relevant transcript, has a size of 1.3 Kb and contains five exons, 1 to 5a. The encoded protein, predicted to contain 210 aminoacids, was called frataxin4.

The gene is expressed in all cells, but at variable levels in different tissues and during development4,7,8. In adult humans, frataxin mRNA is most abundant in the heart and spinal cord, followed by liver, skeletal muscle, and pancreas. In mouse embryos, expression starts in the neuroepithelium at embryonic day 10.5 (E10.5), then reaches its highest level at E14.5 and into the postnatal period7,8. In developing mice, high levels of frataxin mRNA are found in the spinal cord, particularly at the thoracolumbar level, in the dorsal root ganglia, in the proliferating neural cells in the periventricular zone, in the cortical plates, in the ganglionic eminence, in the heart, in the axial skeleton, and in some epithelial and mesenchymal tissues. In the adult mouse brain the level of frataxin mRNA is reduced and mostly confined to the ependyma, but remains high in the spinal cord and dorsal root ganglia8. Interestingly, protein levels (estimated by western blot analysis) remain high in the adult human and mouse brain and cerebellum9 (and our unpublished observation).

The most common mutation causing Friedreich ataxia (98%) is the hyperexpansion of a GAA triplet repeat in the first intron of the frataxin gene4. Disease-associated repeats contain from ~70 to more than 1,000 triplets, most commonly 600-900. Because of the recessive nature of the disease, affected individuals have expansions in both homologues of chromosome 9, while heterozygous carriers of the expansion are clinically normal. This expanded triplet repeat, like others that cause disease, is unstable during parent-child transmission10-13. Paternal transmission is most often accompanied by a contraction of the repeat. Accordingly, in male carriers smaller repeats are found in sperm than in leukocytes10. Maternal transmission may result in further expansion or in contraction with about equal probability10,14. Instability also occurs during somatic cell divisions, leading to a degree of somatic mosaicism for expansion sizes, which has been observed in leukocytes, fibroblasts and brain of Friedreich ataxia patients11,12.

The Friedreich ataxia-associated GAA repeat expansion is the most common disease-causing triplet repeat expansion identified so far, 1 in 90 Europeans being a carrier13. In normal chromosomes, two classes of alleles of the GAA repeat can be distinguished. Short normal (SN) alleles contain 6-10 GAA triplets and account for 83% of chromosomes in Caucasians, long normal (LN) alleles contain more than 12 triplets and account for 17% of chromosomes in Caucasians13,15. LN alleles constitute a reservoir of "at risk" alleles that may eventually expand into the disease range, as indicated by linkage dysequilibrium analysis13, and by the direct observation of catastrophic expansions of LN alleles to pathological repeats containing hundreds of triplets13,15. A few LN alleles are interrupted by a hexanucleotide repeat (GAGGAA), which probably has a stabilizing role13,15. The GAA triplet repeat expansion that causes Friedreich ataxia is only found in individuals of European, North African, Middle Eastern or Indian origin. LN alleles are found in these populations as well as in Sub-Saharan Africans. Analysis of closely linked markers suggests that expansions arose through a unique two-step process, the first leading to the appearance of LN alleles in Africa, the second to larger LN alleles and pathological expansions in ancestors of Indo-Europeans and North Africans. A major implication of these findings is that Friedreich ataxia may not exist among Sub-Saharan Africans, Amerindians, and peoples from China, Japan, and South-EastAsia16.

The expanded GAA repeat inhibits the expression of the frataxin gene. Severely reduced levels of frataxin mRNA and protein have been demonstrated in tissue samples and cultured cells from Friedreich ataxia patients. The reduction is proportional to the size of the expanded GAA repeats, particularly of the smaller one9. Lengths of GAA triplet repeats as those associated with Friedreich ataxia can form triple helical structures in physiological conditions, which can associate in bimolecular complexes (sticky DNA)17. Triplexes are known to block transcription18, providing a mechanism for inhibited gene expression in Friedreich ataxia19.

As expected by the experimental finding that smaller expansions allow a higher residual gene expression9,19,20, expansion sizes have an influence on the severity of the phenotype. A direct correlation has been firmly established between the size of GAA repeats and earlier age of onset, earlier age when confined in wheelchair, more rapid rate of disease progression, and presence of non-obligatory disease manifestations indicative of more widespread degeneration11,14,21-24. However, differences in GAA expansions account for only about 50% of the variability in age of onset, indicating that other factors influence the phenotype. These may include somatic mosaicism for expansion sizes, variations in the frataxin gene itself, modifier genes and environmental factors.

About 2% of the Friedreich ataxia chromosomes have a normal GAA repeat but carry a missense, nonsense, or splice site mutations ultimately affecting the frataxin coding sequence4,25,26. All affected individuals with a point mutation so far identified are heterozygous for an expanded GAA repeat on the other homologue of chromosome 9. It is possible that homozygotes for point mutations have not yet been found just because point mutations are rare, but it is more likely that homozygosity for frataxin point mutations would cause a lethal phenotype, as suggested by the recent observation that frataxin knock-out mice and mice homozygous for a frataxin missense mutation die during embryonic development27 (P. Ioannou, personal communication).

A few missense mutations are associated with milder atypical phenotypes with slow progression, suggesting that the mutated proteins preserve some residual function. Patients carrying the G130V mutation have early onset but slow progression, no dysarthria, mild limb ataxia, and retained reflexes25,26. A similar phenotype occurs in individuals with the mutations D122Y25 and R165P28. For unclear reasons, optic atrophy is more frequent in patients with point mutations of any kind (50%)25.

Frataxin does not resemble any protein of known function. It is highly conserved during evolution4, with homologs in mammals, invertebrates, yeast and plants. The protein is localized in the mitochondrial matrix9,29,30. Genes can be easily disrupted (knocked out) in yeast by homologous recombination, providing a powerful tool to study their function. This was accomplished for the yeast frataxin homolog gene (YFH1). Most YFH1 knock-out yeast strains, called ? YFH1, lose the ability to carry out oxidative phosphorylation, forming petite colonies with defects or loss of mitochondrial DNA that cannot grow on non-fermentable substrates29,31. In ? YFH1, iron accumulates in mitochondria, more then 10-fold in excess of wild type yeast, at the expense of cytosolic iron. Loss of respiratory competence requires the presence of iron in the culture medium, and occurs more rapidly as iron concentration in the medium is increased, suggesting that permanent mitochondrial damage is the consequence of iron toxicity. Iron in mitochondria can react with reactive oxygen species (ROS) that form in these organelles. Even in normal mitochondria a few electrons prematurely leak from the respiratory chain, mostly from reduced ubiquinone (or probably its semiquinone form), directly reducing molecular oxygen to superoxide (O2-). Mitochondrial Mn-dependent superoxide dismutase (SOD2) generates hydrogen peroxide (H2O2) from O2-, then glutathione peroxidase oxidizes glutathione to transforms H2O2 into H2O. Iron may intervene in this process and be engaged in a cycle with O2- and H2O2 as follows:

Fe(III) + O2- + 2H+ * Fe(II) + H2O2

Fe(II) + H2O2 * Fe(III) + OHo + OH- (Fenton reaction)

The hydroxyl radical (OH*) produced by the Fenton reaction is highly toxic and causes lipid peroxidation, protein and nucleic acid damage. Occurrence of the Fenton reaction in ? YFH1 yeast cells is suggested by their highly enhanced sensitivity to H2O229.

Several mitochondrial enzymes are impaired in ? YFH1 yeast cells, including respiratory chain complexes I, II, and III and aconitase32. These enzymes have in common that they contain iron-sulfur (Fe-S) clusters in their active sites. Fe-S proteins are remarkably sensitive to free radicals33. Whether iron-sulfur clusters are damaged by free radicals, or their synthesis is also directly affected by lack of frataxin homolog is not known.

Disruption of frataxin causes a general dysregulation of iron metabolism in yeast cells. The reason for mitochondrial iron accumulation in ? YFH1 cells may in principle involve increased iron uptake, altered utilization or decreased export from these organelles. Experiments involving induction of frataxin expression from a plasmid transformed into ? YFH1 yeast cells indicate that the protein stimulates a flux of non-heme iron out of mitochondria34, but this can again be compatible with different functional roles for frataxin. As iron is trapped in the mitochondrial fraction, cytosolic iron decreases, resulting in a marked induction (10- to 50-fold) of the high-affinity iron transport system of the cell membrane, normally not expressed in yeast cells that are iron replete29. As a consequence, iron crosses the plasma membrane in large amounts and further accumulates in mitochondria, engaging the cell in a vicious cycle. Interestingly, heme synthesis is normal in ? YFH1 yeast, suggesting that ferrochelatase function and the transport of heme out of mitochondria are not affected by frataxin deficiency. The yeast frataxin homolog, yfh1p (the protein product of the YFH1 gene), may be an iron-binding protein35. Monomers of yfh1p are not capable of binding iron, but experiments using gel filtration and analytical ultracentrifugation have suggested that a high molecular weight yfh1p-iron complex may form when ferrous iron is added to the protein at a 40:1 molar ratio. Small amount of intermediates containing 2,3 or more molecules of yfh1p complexed with iron form at lower iron:protein ratios. The high molecular weight complex is estimated to contain about 60 molecules of frataxin and 4,000 atoms of iron. Western blot analysis of gel filtration fractions of yeast extracts suggest that high molecular weight complexes containing yfh1p may exist in vivo. According to these data, yfh1p may protect iron in mitochondria from contacts with free radicals. Since iron in the complexes seems to be readily accessible to chelators, so probably bioavailable, yfh1p could be a sort of mitochondrial iron chaperone, in the absence of which several biosyntheses and transport processes are impaired and iron accumulates in a toxic, redox-active form. The structure of frataxin is the object of intensive analysis. Preliminary data indicate that mature frataxin is a globular protein containing an N-terminal * helix, a middle ß sheet region, and a C-terminal * helix. Hydrophobic aminoacids are buried inside the structure, with several charged residues on the surface. Hopefully, a correlation between structural data and biochemical findings will soon be available. Normal human frataxin is able to complement the defect in ? YFH1 cells, while human frataxin carrying a point mutation found in Friedreich ataxia patients is unable to do so31, strongly suggesting that the function of yfh1p is conserved in human frataxin. Several observation reinforce the hypothesis that altered iron metabolism, free radical damage, and mitochondrial dysfunction all occur in Friedreich ataxia. Involvement of iron was suggested twenty years ago by the finding of deposits of this metal in myocardial cells from Friedreich ataxia patients36. Iron accumulation has been demonstrated by magnetic resonance imaging (MRI) in the dentate nucleus, a severely affected structure in the central nervous system37. We have confirmed an increase in dentate nucleus iron by atomic absorption spectroscopy analysis of pathological samples from three Friedreich ataxia patients (our unpublished data). The observation of a moderate, but significant increase in iron concentration in the mitochondrial fraction from Friedreich ataxia fibroblasts has been reported38. Oxidative stress is suggested by the observation that Friedreich ataxia fibroblasts are sensitive to low doses of H2O2, that induce cell shrinkage, nuclear condensation and apoptotic cell death at lower doses than in control fibroblasts39 (and our unpublished observations). This finding suggests that even non-affected cells are in an "at risk" status for oxidative stress as a consequence of the primary genetic defect. A further hint of a possible role of free radicals comes from the observation that vitamin E deficiency produces a phenotype resembling Friedreich ataxia40. Vitamin E localizes in mitochondrial membranes where it acts as a free radical scavenger41. Mitochondrial dysfunction has been proven to occur in vivo in Friedreich ataxia patients. Magnetic resonance spectroscopy analysis of skeletal muscle shows a reduced rate of ATP synthesis after exercise, which is inversely correlated to GAA expansion sizes42. In addition, Rötig et al32 demonstrated the same multiple enzyme dysfunctions found in ? YFH1 yeast (deficit of respiratory complexes I, II and III, and of aconitase) in endomyocardial biopsies from two Friedreich ataxia patients32. A general abnormality of iron metabolism may also be occurring in Friedreich ataxia patients, as suggested by the high level of circulating transferrin receptor, the principal carrier of iron into human cells43. As ? YFH1 yeast increases cellular iron uptake because of low cytosolic iron, a similar process may occur in human patients. In higher eukaryotes, cytosolic iron is sensed by two iron responsive element binding proteins (IRP-1 and IRP-2), that regulate the expression of several genes at the post-transcriptional level. When activated by low iron, they bind to specific sequence elements (iron responsive elements, IREs) present in some mRNAs. IRP binding stabilizes mRNAs encoding proteins that enhance iron uptake, as the transferrin receptor (TfR), while blocking the translation of mRNAs encoding proteins that utilize or store iron, as ferritin44. IRP-1 is a cytosolic aconitase containing an Fe-S cluster. It is activated not only in response to low cytosolic iron, but also to oxidative radicals and to signaling molecules as nitric oxide (NO) and carbon monoxide (CO)44. If the loss of aconitase activity observed by Rötig et al32 involves the cytosolic enzyme, it might result in changes in the abundance of IRP-1-regulated proteins32, including the observed increase in transferrin receptor. It should be noted that the expression of frataxin does not seem to be regulated by iron (our unpublished observation) and its mRNA does not contain an IRE. Removal of excess mitochondrial iron and/or anti-oxidant treatment may in principle be attempted. However, removal of excess mitochondrial iron is problematic with the currently available drugs. Desferioxamine (DFO) is effective in chelating iron in the extracellular fluid and cytosol, not directly in mitochondria. Furthermore, DFO toxicity may be higher when there is no overall iron overload. Thus, chelation therapy has a number of unknowns: it is probably better tested in pilot trials involving a small number of closely monitored patients. Iron depletion by phlebotomy, though less risky, presents the same uncertainties concerning possible efficacy. As far as antioxidants are concerned, these include a long list of molecules with specific mechanisms of action and pharmacokinetic properties. To have the potential to be effective in FRDA, an antioxidant must protect against the damage caused by the free radicals involved in this disease, in particular OH*, act in the mitochondrial compartment and be able to cross the blood-brain barrier. At this time, CoQ derivatives, like its short chain analog idebenone, appear to be interesting molecules and are object of pilot studies45. However, new knowledge on frataxin function and pathogenesis is needed to progress towards an effective treatment of the disease. In the long term, gene replacement, protein replacement, or reactivation of the expression of endogenous frataxin could be cures and are all worth exploring.