Pathophysiogical and therapeutic progress in Friedreich ataxia

revue neurologique 170 (2014) 355–365

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Mitochondrial diseases

Pathophysiogical and therapeutic progress in Friedreich ataxia Ataxie de Friedreich : pathophysiologie et perspectives the´rapeutiques H. Puccio a,b,c,d,e, M. Anheim a,b,c,d,f, C. Tranchant a,b,c,d,f,* a Translational medicine and neurogenetics, institut de ge´ne´tique et de biologie mole´culaire et cellulaire (IGBMC), 1, rue Laurent-Fries, BP 10142, 67404 Illkirch cedex, France b Inserm, U596, 1, rue Laurent-Fries, 67400 Illkirch Graffenstaden, France c CNRS, UMR7104, 1, rue Laurent-Fries, 67400 Illkirch Graffenstaden, France d Universite´ de Strasbourg, 4, rue Blaise-Pascal, 67400 Strasbourg, France e Colle`ge de France, chaire de ge´ne´tique humaine, 1, rue Laurent-Fries, 67400 Illkirch Graffenstaden, France f Service de neurologie, unite´ des pathologies du mouvement, hoˆpital de Hautepierre, hoˆpital universitaire, 1, place de l’Hoˆpital, 67000 Strasbourg, France

info article

abstract

Article history:

Friedreich ataxia (FRDA) is the most common hereditary autosomal recessive ataxia, but is

Received 16 December 2013

also a multisystemic condition with frequent presence of cardiomyopathy or diabetes. It has

Received in revised form

been linked to expansion of a GAA-triplet repeat in the first intron of the FXN gene, leading to a

25 March 2014

reduced level of frataxin, a mitochondrial protein which, by controlling both iron entry and/or

Accepted 26 March 2014

sulfide production, is essential to properly assemble and protect the Fe-S cluster during the

Available online 29 April 2014

initial stage of biogenesis. Several data emphasize the role of oxidative damage in FRDA, but

Keywords:

animal models. Conditional knockout models recapitulate important features of the human

better understanding of pathophysiological consequences of FXN mutations has led to develop Friedreich ataxia

disease but lack the genetic context, GAA repeat expansion-based knock-in and transgenic

Mitochondria

models carry a GAA repeat expansion but they only show a very mild phenotype. Cells derived

Frataxine

from FRDA patients constitute the most relevant frataxin-deficient cell model as they carry the

Physiopathology

complete frataxin locus together with GAA repeat expansions and regulatory sequences.

Therapeutic

Induced pluripotent stem cell (iPSC)-derived neurons present a maturation delay and lower

Mots cle´s :

degeneration, with frequent dark mitochondria and proliferation/accumulation of normal

mitochondrial membrane potential, while cardiomyocytes exhibit progressive mitochondrial Ataxie de Friedreich

mitochondria. Efforts in developing therapeutic strategies can be divided into three categories:

Mitochondries

iron chelators, antioxidants and/or stimulants of mitochondrial biogenesis, and frataxin level

Frataxine

modifiers. A promising therapeutic strategy that is currently the subject of intense research is

Physiopathologie

to directly target the heterochromatin state of the GAA repeat expansion with histone

Approches the´rapeutiques

deacytelase inhibitors (HDACi) to restore frataxin levels. # 2014 Elsevier Masson SAS. All rights reserved.

* Corresponding author. E-mail address: [email protected] (C. Tranchant). http://dx.doi.org/10.1016/j.neurol.2014.03.008 0035-3787/# 2014 Elsevier Masson SAS. All rights reserved.

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revue neurologique 170 (2014) 355–365

r e´ s u m e´ L’ataxie de Friedreich (FRDA) est la plus fre´quente des ataxies re´cessives, mais est e´galement une maladie pluri syste´mique souvent complique´e de la survenue d’un diabe`te ou d’une cardiomyopathie. Elle est lie´e a` une expansion de triplets GAA dans le ge`ne FXN, responsable d’une baisse du taux de frataxine, une prote´ine mitochondriale implique´e dans le transport du fer, la formation des noyaux Fe-S et la biogene`se des mitochondries. Diffe´rents mode`les animaux ont e´te´ de´veloppe´s pour tenter de pre´ciser la physiopathologie de la FRDA, domine´e par le stress oxydatif. Les mode`les knock-out pour la frataxine reproduisent les signes cliniques de la maladie, alors que les mode`les reproduisant l’extension des triplets GAA n’ont qu’un phe´notype incomplet. Les mode`les les plus performants reposent actuellement sur les cultures de cellules pluripotentes (iPSC) issues de patients FRDA. Les neurones de´rive´s de ces iPSC pre´sentent une maturation retarde´e et un potentiel de membrane mitochondrial bas, alors que les iPSC cardiomyocytes de´veloppent des le´sions mitochondriales. Ces mode`les sont indispensables pour tester les diffe´rentes approches the´rapeutiques : che´lateurs du fer, antioxydants et/ou stimulants de la biogene`se mitochondriale, modificateurs du taux de frataxine. Une strate´gie prometteuse est l’utilisation d’inhibiteurs des histone–de´acyte´lases agissant sur les expansions GAA pour restaurer le taux de frataxine. # 2014 Elsevier Masson SAS. Tous droits re´serve´s.

First described by N. Friedreich in 1863, Friedreich ataxia is the most common hereditary autosomal recessive cerebellar ataxia [1,2]. It has been linked to mutations in the frataxin gene (FXN), leading to a reduced level of frataxin, a mitochondrial protein which is implicated in iron-sulfur cluster biogenesis. Since the discovery of the gene in 1996 [3], the function of frataxin has been extensively studied, multiple models have been generated and analysed in the goal of understanding the pathophysiological process and there have been a number of clinical trials. In this review, we will describe the clinical and genetic features of FA, recent pathophysiological and animal models data, as well as therapeutic perspectives.

1.

Clinical description

Friedreich ataxia (FRDA) is a multisystemic condition with presence of neurological and frequent non-neurological signs (in particular diabetes and cardiomyopathy). Harding’ s neurological clinical criteria (1981) with primary necessary criteria (onset before 25 years, progressive ataxia of limbs and gait, absence of knee and ankles jerks) and secondary criteria (dysarthria, extensor plantar responses or if absent an affected sibling and median motor nerve velocities greater than 40 m/s) [4], keep a good specificity, but since the discovery of the FXN gene [3], this phenotype has been extended to several atypical presentations. In the typical cases [5–9], age of onset is before 25 and in most of the cases, between 10 and 15 years of age. Mixed (cerebellar and proprioceptive) limb and gait ataxia, due to spinocerebellar tracts and dorsal root ganglia degeneration [10,11], is present in all cases and usually begins the disease. Dysarthria is present in more than 90% of cases, but can occur later than 5 years after the onset. Absent lower limbs reflexes and proprioception deficit, due to a peripheral axonal sensory

neuropathy and to spinal posterior degeneration, are usual. Extensor plantar responses are common, whereas lower limbs spasticity occur only in advanced cases. Oculo-motor abnormalities are frequent with square wave jerks (69% of cases), gaze evoked nystagmus, or abnormalities of saccades, pursuits or of vestibulo-ocular reflexes [9,12]. Optic atrophy is found in only 30% of cases, but when performed OCT demonstrates abnormalities in all cases [13]. Decreased visual acuity is described in 20% of cases but can deeply alter quality of life. A few patients can develop sudden bilateral loss of vision (pseudo Leber syndrome). Hearing loss is rare (8 to 39%). Mild cognitive impairment (slowed motor processing speed, impaired conceptual thinking and verbal fluency) has been described [14]. Extraneurological signs include scoliosis or pes cavus (present in more than 50% of cases), hypertrophic cardiomyopathy with abnormal electrocardiogram or echography (in 65% of cases), and diabetes mellitus (10 to 30% of cases) [9]. Walking difficulties increase slowly, but in most of cases wheelchair-bound occurs 15.5 years (3–44 years) after disease onset. Upper limb ataxia increased more slowly but participates to loss of dependence. Dyasrthria in all cases, and dysphagia in numerous cases are present. Arythmia or dilated cardiomyopathy are the most frequent cause of death. MRI rarely shows cerebellar atrophy, but always demonstrates cervical cord atrophy. Four limbs electromyography confirms axonal sensory neuropathy. Molecular biology allows the diagnosis, after exclusion of vitamin E deficiency. All FRDA patients carry an expansion of a GAA-triplet repeat in the first intron of the FXN gene. Most patients are homozygous for this mutation, but a few patients (4%) are compound heterozygous for the GAA expansion and a different mutation (nonsense, missense, deletions, insertions) leading to loss of FXN function [3,15,16]. Phenotype-genotype correlations have found that age of onset and rate of progression is correlated with the size of the

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smaller GAA expansion [6]. Furthermore, cardiomyopathy, extensor plantar, weakness and skeletal deformities frequencies increase with the size of GAA expansion. In patients with smaller expansions (GAA < 500), onset of the disease, onset of dysarthria and loss of deambulation is delayed compared with patients with larger repeats [9]. Most frequent atypical forms are early onset cerebellar ataxia with retained reflexes, late onset and very late onset Friedreich ataxia. Late onset FA (LOFA) and very late onset FA (VLOFA) are defined by age of onset after 25 or 40 respectively. Clinical signs are similar to those of patients with typical FA [17] with gait and limb ataxia, dysarthria, loss of vibration sense and abnormal eyes movement. However spasticity, lower limb ataxia and retained reflexes are more frequent, whereas non-neurological signs and cardiomyopathy are rarer. MRI cerebellar atrophy (especially for vermis) is described in some cases [17]. Some patients with minimal GAA expansion have been presented with adult onset spastic paraplegia [18]. Earlier (mean age of onset: 7) and more severe cases have been described in patients with exonic deletions of FXN [19].

2.

Genetics

The mutated gene in FRDA, which is localized on 9q21.11, encodes a small mitochondrial protein called frataxin (FXN) [20,21]. While normal alleles contain up to 40 GAA repeats, FRDA patients exhibit larger expansions, ranging from 70 to 1700 GAA repeats, most commonly 600 to 900 [3]. This GAA expansion leads to a partial transcriptional silencing of FXN through a mechanism involving epigenetic changes including histone hypoacetylation and trimethylation leading to heterochromatin formation, thereby impairing gene transcription [22,23]. This results in a strongly reduced frataxin protein expression in all tissues. Indeed, FRDA patients show significantly lower amounts of frataxin protein compared to controls, with 21.1% in buccal cells and 32.2% in whole blood [24]. There is an inverse correlation between the length of the smaller of the two alleles and the disease severity [25]. The other rare mutations in FXN that have been associated with FRDA lead to production of nonfunctional or partially functional proteins [26]. In most cases, compound heterozygous patients are undistinguishable from patients that are homozygous for GAA expansions, although a few missense mutations (i.e. G130V, D122Y, R165P, L106S) cause atypical or milder clinical presentations [15,16]. No patient carrying a double point mutation with normal GAA repeat length has ever been reported, suggesting that a minimal level of normal frataxin expression is required. This is supported by work in multicellular eukaryotes, demonstrating that frataxin is essential for embryonic development, as complete frataxin deletion leads to early embryonic lethality in plants and mice [27–29] and to L2/L3 larval stage arrest in C. elegans [30].

3.

Frataxin function

Frataxin is a small globular protein, highly conserved in most organisms from bacteria to mammalians [31,32]. Human

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frataxin is synthetised as a precursor of 210 amino acids that is imported in the mitochondrion where it is maturated by the mitochondrial processing peptidase (MMP) through a two-step process, leading to the successive generation of an intermediate form of 19 kDa form (FXN42-210) and a mature form of 14 kDa (FXN81-210) [33–35]. The FXN81-210 mature form is fully functional for cell survival and is the most suitable for further functional studies [36]. The structure of frataxin is conserved and consist of two helices packing against a contiguous antiparallel beta sheet assembled in the sequence alpha-(beta)5-7-alpha [37–41]. The N-terminal eukaryotic tail of the human protein was shown to be intrinsically unfolded and highly flexible [40,42]. Interestingly, all clinically important missense mutations observed in heterozygous FRDA patients affect conserved residues [15]. In vitro studies demonstrated that these mutants retain a native fold under physiological conditions but have reduced thermodynamic stabilities that, in the order, follow the trend: wild-type > W155R > I154F > D122Y > G130 V [43]. Unfolding is reversible ruling out the possibility of formation of insoluble aggregates. These results suggest that the point mutation lead to loss of function of frataxin. Frataxin is a ubiquitous mitochondrial protein, whose exact function was long controversial. Although frataxin has been proposed to be a multifunctional protein involved in different iron-dependent mitochondrial pathways, phylogenetic, genetic and biochemical studies point to the essential role of frataxin in Fe-S cluster metabolism [31,44–48]. Early in vitro studies of bacterial, yeast or human frataxin showed a medium to low affinity for ferrous iron (3–55 mM range), through interaction with a conserved acidic ridge of the mature protein[41,49,50]. The proposed function of frataxin as an iron donor was suggested both by iron-dependent interaction of yeast frataxin with Nfs1 and Isu1 [51] and by in vitro studies demonstrating the capacity of iron-loaded mature human frataxin to provide iron for Fe-S cluster formation on human ISCU [50]. Frataxin has also been shown to form oligomers in the presence of iron in vitro, initially suggesting an iron detoxification and storage function for this protein [39,52–54]. However, this hypothesis has been questioned by in vivo experiments. Indeed, the deletion of yeast frataxin can be complemented by a mutant frataxin defective in ironinduced oligomerization [55]. Furthermore, the mature form of human frataxin (FXN81-210), which has been shown not to oligomerize [56], rescued cellular viability of murine fibroblasts deleted for frataxin [36]. Both results indicate that the formation of oligomers is not a requisite for frataxin to be functional. Conversely, in vitro reconstitution experiments showed that oligomeric iron-loaded bacterial and yeast frataxins could provide iron for Fe-S cluster formation on IscU-Isu1. The possibility that frataxin might act as a regulator of the cysteine desulfurase activity has recently emerged [44,45,48], thereby opening new venues on how frataxin might be involved in Fe-S cluster formation. De novo Fe-S cluster biogenesis is a highly conserved but still poorly characterized process that occurs in mitochondria in eukaryotes [57]. The first step of Fe-S cluster biogenesis involves the assembly of a Fe-S cluster on a scaffold protein ISCU (Isu1 in yeast) from inorganic iron and sulfur. In this process, the sulfur is provided through a persulfide

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intermediate by a cysteine desulfurase (comprising NFS1 and ISD11). Although controversial data regarding the direct frataxin protein partner were subsequently reported [51,53,58,59], very recent independent work using mammalian recombinant proteins reconciled the different results by showing that frataxin interacts with a preformed complex composed of NFS1, ISCU and ISD11 [36,48]. It was subsequently shown that frataxin stabilizes the complex and controls iron entry through activation of the cysteine desulfurase activity [45,48]. Recently, a point mutation in the Isu1 gene (M107I) was found to partially rescue the DYfh1 yeast model, pointing again to the role of frataxin in early steps of Fe-S cluster biogenesis [60]. An elegant recent biochemical characterization of the separate steps of the cysteine desulfurase activity points to the role of frataxin in positioning the substratebinding site of the cysteine desulfurase, a conformational change that can be bypassed by the Isu1M107I mutation [61]. Altogether, these results suggest that frataxin, by controlling both iron entry and/or sulfide production through a yet undefined conformational change, is essential to properly assemble and protect the Fe-S cluster during the initial stage of biogenesis.

4.

Pathogenesis

After the identification of the disease-causing mutation and its gene product, much effort has been made to elucidate both the pathophysiology and the genetic basis of the disease. Studies performed on biological material from FRDA patients revealed:  the presence of iron deposits in cardiomyocytes;  decreased ISC-containing enzyme activities in heart homogenates (aconitase, respiratory chain complex I, II and III) [63] and;  presence of markers of oxidative damage in urine and blood [64–66]. These three biochemical features are tightly interconnected, and have been alternately proposed in the literature to be the primary event in the disease, leading to the hypothesis that frataxin deficiency results in a vicious circle of cellular defects [67]. In correlation with the clinical signs, the cellular defects associated with frataxin deficiency are reported only in specific cells of certain tissues (i.e. sensory neurons, cardiomyocytes). As the affected cells are known to have a high mitochondrial content and high metabolic rate, these postmitotic cells could be more sensitive to frataxin deficiency. Furthermore, this tissue specificity partially correlates with the distribution of the FXN transcript in the organism: its expression is strong in DRG and heart, mild in cerebellum, pancreas, liver and skeletal muscle, and low in other tissues, such as cortex [21]. However, differential FXN expression levels cannot be the only explanation. It is thought that part of the tissue-specificity as well as the progressive nature of the disease might be a direct consequence of the genetic basis of the disease. Indeed, analysis of mutation load in patients and their families has revealed that GAA repeat expansions are highly dynamic, exhibiting both intergenerational and somat-

ic instability [68,69]. The latter is both tissue-specific and agerelated, and has therefore been hypothesized to be important for the disease progression [70]. While the molecular mechanisms underlying GAA repeat instability remain unknown, major progress has been made in understanding the mechanism of GAA repeat expansion-induced FXN gene silencing using artificial constructs in transfected human cells, together with FRDA patient fibroblasts, lymphoblasts, autopsy tissues and FRDA models [71]. Although these ISC enzyme deficits were initially thought to be a consequence of increased oxidative stress generated through the Fenton reaction by mitochondrial iron accumulation, studies using conditional mouse models with tissuespecific frataxin deletions demonstrate that the primary deficit in the disease is the ISC protein deficiency followed by secondary mitochondrial iron accumulation, with no overt sign of oxidative stress damage [72,73]. It is now commonly accepted that frataxin deficiency leads to primary mitochondrial and extramitochondrial ISC deficits [63,72,74]. Whether iron dysregulation has a causal role in the disease pathogenesis remains to be demonstrated and further cellular and animal studies will be crucial to determine whether a subtle increase in redox-active mitochondrial iron plays a role in the disease pathogenesis. The role of oxidative stress in the pathogenesis of FRDA was discovered very early with the demonstration that frataxin-deficient yeast and cultured cells from FRDA patients exhibit increased sensitivity to oxidative stress reagents [75–77]. Numerous studies have demonstrated an impaired response of antioxidant enzymes in cell lines and model organisms [73,78–80]. This disabled response was recently proposed to be the consequence of a disorganization of the Nrf2-dependent phase II antioxidant-signaling pathway [81]. Finally, reports of increased levels of oxidative stress markers are not consistent, with initial studies showing low but significant increases of oxidative stress markers in blood and urine samples from FRDA patients [64–66], while more recent studies have failed to replicate these data [82–85]. Interestingly, a recent study presented data suggesting increases in nuclear and mitochondrial DNA damage in peripheral blood samples from FRDA patients [86]. Whether this increase in DNA damage is a consequence of an impaired antioxidant defense or directly linked to an ISC deficit remains to be determined, as several damage recognition and DNA repair proteins are ISC proteins [74,87]. Considering these data, although a net increase in the level of oxidative damage is difficult to measure in humans and animal models, it is clear that oxidative damage is an important player in FRDA, and warrants further studies to understand the pathophysiological consequences and therapeutic approaches.

5.

Models of FRDA

The genetic basis of FRDA raises challenges for modelling the disease in other species. However, in the past 15 years, many models of FRDA have been generated that have enabled advances in understanding the function of frataxin, the pathophysiology of the disease and some of the mechanisms implicated in GAA-based silencing and instability. Due to its high evolutionary conservation, the effect of FXN depletion

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has been modelled in diverse organisms, including yeast [88,89], invertebrates (C. elegans [30,90,91] and Drosophila [79,92]) and mice [72,93,94]. However, due to the complexity of the clinical phenotype of FRDA patients and the species specificity of certain fundamental pathways, mouse or mammalian cells are probably better suited to answer pathophysiological questions. It was first shown that complete deletion of frataxin in mice is not viable, resulting in lethality at embryonic day 6.5 [28]. Similarly, mouse fibroblasts cannot be sustained in culture after frataxin deletion [78]. This lethal phenotype either in cells or in the whole organism can be rescued by exogenous expression of human or murine frataxin [78,95]. These results are in agreement with the absence of FRDA patients identified with a complete loss of frataxin. The subsequent discovery of the essential role of frataxin in ISC biosynthesis, and the recent identification of cytosolic and nuclear ISC proteins with indispensable function for cell viability, including ABCE1 involved in translation, or DNA helicases, polymerases and primases involved in replication and repair [96] provide a rational explanation for the lethality associated with complete frataxin deficiency during embryogenesis and cell division. To study the consequences of frataxin depletion after early embryonic stages and in a tissue specific manner, several conditional knockout mouse models corresponding to tissues affected in FRDA patients were generated using the robust CreloxP system. Together, these models reproduce hypertrophic cardiomyopathy, progressive spinocerebellar and sensory ataxia, and some aspects of the diabetes. The cardiac-specific model, expressing Cre recombinase under the muscle creatine kinase (MCK) promoter, reproduces the chronological development of the cardiomyopathy observed in patients [72]. In addition, the MCK mutants reproduce the biochemical features observed in FRDA patients, with defects in ISCenzyme activities (i.e. aconitase, succinate dehydrogenase) occurring early, prior to cardiac dysfunction and iron accumulation [72]. Mitochondrial iron accumulation is observed from 7 weeks of life, and is tightly associated with dysregulation of proteins involved in iron uptake (transferrin receptor 1), iron storage (ferritins H and L) and iron release (ferroportin 1), probably as a consequence of activation of IRP1, one of the main cellular regulators of iron metabolism, directly due to ISC deficit [73,97]. Interestingly, no detectable oxidative damage was observed in the MCK mutants, suggesting that oxidative stress might not be involved in the early onset of the disease. Recently, Wagner and colleagues reported that a defective mitochondrial respiratory chain is followed by hyperacetylation of cardiac proteins for MCK mice due to the inhibition of SIRT3 [98]. As SIRT3 is involved in regulation of oxidative metabolism, its inhibition might play a role in the cardiomyopathy observed in FRDA patients, as well as in the increased sensitivity to oxidative stress reported in FRDA cells. An inducible conditional mouse model was generated with the Cre recombinase under the control of the mouse prion protein (PRP) promoter. The inducible approach allows spatial and temporal control of Fxn deletion, mediated by Cre recombinase translocation to the nucleus upon tamoxifen injection. In the PRP model, Fxn deletion is restricted to DRGs, spinal cord and cerebellum [99]. After tamoxifen injection at four weeks of age, mice develop a progressive mixed cerebellar

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and sensory ataxia associated with loss of proprioception and absence of motor defects. Histologically, the first event is a degeneration of large sensory neurons in DRGs, with the presence of vacuoles, signs of autophagy and lipofuscin accumulation. Chromatolysis in sensory neurons of the posterior horn of the spinal cord is then observed, followed later by degeneration of motor neurons in the anterior horn. The observations made in the PRP mutants suggest that impaired autophagy might be a key process in the neurodegeneration. In parallel to the loss of the sensory neurons, degeneration of the granule cells of the cerebellum is observed. While non-specific to FRDA, this feature contributes also to the cerebellar ataxia observed in the PRP mutants and reveals the sensitivity of these particular cells to frataxin loss. Diabetes mellitus and glucose intolerance are identified in 10% and 20% of FRDA patients, respectively, but to date few studies have been performed to address the mechanism underlying this phenotype. Conditional models lacking frataxin primarily in the pancreatic b cells were generated using an Ins2-Cre transgene [100]. These mice develop impaired glucose tolerance followed by slowly progressive diabetes probably as a consequence of decreased b cell mass, due to increased apoptosis and a reduced proliferation rate. However, more investigations need to be done in FRDA patients and mouse models to unravel the specific pathways which link frataxin deficiency and impairment of glucose homeostasis. All together, these different Fxn deletion mouse models reproduce most of the symptoms associated with FRDA, and have allowed better understanding of the pathophysiology of the disease. Moreover, they are relevant for therapeutic tests, as they have been used for testing antioxidants, iron chelators and protein therapy. However, frataxin deletion in the Fxn knockout mouse models occurs at a specific time in development, whereas FRDA is characterized by partial frataxin deficiency in all cells throughout life. As a consequence, these models cannot be used to study the tissue specific aspects of the disease. Therefore, in parallel to the conditional Fxn knockout models, mice with residual frataxin levels are essential to further investigate the pathophysiological mechanisms involved in FRDA. The generation of GAA repeat expansion-based mouse models is appealing. Indeed, the presence of a pathological GAA expansion in mouse is expected to lead to a residual amount of frataxin in all tissues, as observed in patients. However, due to the specific genomic context and the length of the repeats, it is not easy to generate mice with large GAA repeat expansions. For this purpose, two strategies have thus far been used. Firstly, a model has been generated using a knock-in approach by introducing a (GAA)230 repeat expansion into the first intron of the mouse Fxn locus, with the homozygous knock-in mice designated as KIKI mice [94]. The KIKI mice were then crossed with frataxin knockout mice to obtained compound heterozygous mice, designated as KIKO mice. The KIKI and KIKO mice have 66–83% and 25–36% residual frataxin protein expression compared to WT, respectively. The KIKO mice show no iron deposits and only mild signs of fibrosis (collagen) in the heart and no coordination defects assessed by rotarod. This absence of cardiac and neurological phenotypes argues for a pathologic threshold of frataxin expression below

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25% and probably for a role of the genomic context in the disease. A second GAA repeat expansion-based mouse model has been obtained by initially crossing transgenic mice that contain the entire FXN gene within a human YAC clone onto a mouse Fxn null background, showing that transgenic human frataxin can substitute for endogenous mouse frataxin [95]. Then similar YAC FXN transgenic mice were generated, but this time with a GAA repeat expansion appropriately inserted into the FXN locus, producing the YG8 line with two GAA sequences of 90 and 190 repeats and the YG22 line with one sequence of 190 repeats [101]. Both FXN transgenic mice were then crossed with heterozygous frataxin knockout mice to obtain the YG8R and YG22R (R = ‘rescue’) FRDA mouse models that contain GAA repeat expansions and express only human frataxin [93]. A decrease in transgenic human frataxin mRNA was observed in all tissues, with the greatest decrease in cerebellum of YG22R and YG8R (62% and 57%, respectively) and skeletal muscle of YG8R (57%). Both models showed a mild progressive phenotype, with slight coordination impairment, together with locomotor defects. Similarly to PRP mutants, giant vacuoles were observed in large sensory neurons of DRGs as well as chromatolysis and lipofuscin accumulation. No overt cardiac phenotype was observed in YG22R. However, a decrease in heart aconitase activity was measured in 6 months old YG8R mice compared to WT mice. Contrary to the conditional knockout mouse models, slight signs of oxidative stress were reported in YG8R and YG22R tissues. The differences observed between YG8R and YG22R lines might be due to different integration sites of the YAC. In conclusion, the different results obtained with the various mouse models highlight two main points when considering the pathophysiology of FRDA disease. On the one hand, only the conditional knockout models recapitulate important features of the human disease but they lack the genetic context. On the other hand, the GAA repeat expansionbased knock-in and transgenic models carry a GAA repeat expansion, which is the primary pathological mutation for humans, but they only show a very mild phenotype. As a consequence, efforts to generate mouse models with longer GAA repeats, either through novel constructs or through various genetic crosses and selecting for GAA expansion, are ongoing. Cells derived from FRDA patients constitute the most relevant frataxin-deficient cell model as they carry the complete frataxin locus together with GAA repeat expansions and regulatory sequences. Some cell types, such as primary fibroblasts and lymphocytes, are easily accessible, but these cells do not spontaneously exhibit the complex biochemical phenotype associated with FRDA, despite having reduced levels of frataxin [63,102]. However, viability of these cells is affected under stress conditions (i.e. treatment with hydrogen peroxide or buthionine sulfoximine), suggesting defects in cellular defense against oxidative stress [80,103]. With the recent advances in generating induced pluripotent stem cell (iPSC) from somatic cell and their potential differentiation in all cell types [104], several groups have derived iPSC from FRDA patients which carry large GAA repeat [105–108]. These cells show reduced frataxin levels, epigenetic changes associated to GAA repeat and repeat instability [105–108]. iPSC-derived

neurons present a maturation delay and lower mitochondrial membrane potential [105,106], while cardiomyocytes exhibit progressive mitochondrial degeneration, with frequent dark mitochondria and proliferation/accumulation of normal mitochondria [106]. Further studies using iPSC-derived cells are warranted to further elucidate the pathophysiological consequences of frataxin deficiency in a human cell model.

6.

Therapeutic perspectives

Currently, there is no treatment for FRDA, despite the large efforts in developing therapeutic strategies to intervene in the pathogenetic cascade downstream of frataxin. These therapeutic strategies for FRDA can be divided into three categories: iron chelators, antioxidants and/or stimulants of mitochondrial biogenesis, and frataxin level modifiers. As previously exposed, although the mechanism of iron dysregulation is not fully understood, there is a great deal of evidence that implicate impaired iron homeostasis and mitochondrial iron accumulation as a pathological hallmark of FRDA [109]. Therefore, iron chelation designed to specifically target the mitochondrial iron accumulation is a potential strategy for the treatment of FRDA that was intensively investigated. Several iron chelators that target the mitochondria have been evaluated both in cellulo, in animal models and in clinical trials, including deferoxamine and deferiprone [110– 115]. While deferoxamine showed some promise in cellular models, it was not well-suited for FRDA as it decreased the mRNA level of both aconitase and frataxin [116]. Deferiprone, an orally active, blood-brain barrier permeable iron chelator, can potentially redistribute iron from the mitochondria to other cellular compartments and to blood transferrin [117]. Deferiprone has had variable results, depending on the trial, showing in some instance a modest improvement in neurological function or significant reduction of the cardiac hypertrophy, however, at higher dose, deferiprone was associated with worsening of the neurological function and several reports of serious adverse effects, including agranulocytosis [110]. Overall, results of clinical studies in FRDA suggest a beneficical effect of mild iron chelation on cardiac parameters, but risk of worsening the condition by higher doses of the drugs. At this date, clinical trials with iron chelators have been stopped, until a better understanding of the consequences of iron dysregulation in FRDA is undertaken. FRDA is more a condition of iron deficiency than of iron overload. Inefficient Fe-S cluster biogenesis leads to consequences and homeostatic responses that resemble iron deficiency, including low Fe-S enzyme activities, IRP activation, and increase iron uptake. While the issue is still controversial, a large body of evidence, including pre-clinical and clinical data, support the role of oxidative stress in FRDA, despite the difficulty in measuring net increase in oxidative stress markers in humans and mice. The first drug to reach phase III clinical trials for FRDA was Idebenone, a parabenzoquinone derivative [118– 122]. The rationale was initially based on the antioxidant activity of this molecule, but as it can also stimulate oxidative phosphorylation and ATP production by transferring electrons from complexes I and II to complex III in the electron transport

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chain [123], it may also contribute to counteract the electron transport impairment due to ISC deficits observed in FRDA patients. Idebenone improved intracellular markers of ROS damage and FRDA symptoms in both cellular and murine models [103,121,122,124]. While initial clinical studies were promising with decrease in oxidative stress markers, decrease in cardiac hypertrophy [66,121,125], and even a decrease in neurological symptoms in one study [82], Idebenone failed in phase III studies as it was found not to show significant improvement in neurological function or cardiac outcomes in patients [126,127]. However, these negative results point to the difficulty in evaluating a drug in a very slowly progressive and clinical variable disease such as FRDA, and point to the clear need to stratification of patient for age of onset, disease duration and level of disability. Several efforts at improving and developing similar compounds are currently underway both pre-clinically and in clinical trials. The alternative of restoring frataxin expression in affected cells appears an appealing approach to slow down or stop disease progression. Gene replacement and protein replacement can be used for this purpose. Protein replacement has been investigated for FRDA by using the transactivator of transcription (TAT) protein transduction to deliver human frataxin protein to mitochondria and TAT-FXN resulted in increased lifespan and improvement of cardiac functions as well as increased of aconitase activity in a conditional mouse model [128]. Another therapeutic approach, which is currently under-explored, is gene therapy, which aims to partially or totally restore the levels of frataxin in the affected cells. Indeed, in contrast to the current therapeutic approaches, only a single therapeutic intervention would be needed to correct the cardiac and neurological phenotype. A promising therapeutic strategy that is currently the subject of intense research is to directly target the heterochromatin state of the GAA repeat expansion with histone deacytelase inhibitors (HDACi) to restore frataxin levels [22]. HDACi prevent the deacetylation of histones, making heterochromatin revert to an open active conformation that is conducive for gene expression [129]. A first screening of commercially available HDAC inhibitors was performed on FRDA patient and control lymphoblastoid cells, allowing the identification and the optimization of a promising compound able to increase both frataxin mRNA and protein levels in patient cells [130]. After additional developments, several derivatives were tested on KIKI mice in short-term studies, confirming their ability to temporarily reverse frataxin gene silencing [131,132]. This effect was supported by a five-month study on the mildly affected YG8R mice [133]. A phase I of the therapeutic lead is currently underway to determine whether the drug can safely increase frataxin levels in peripheral blood mononuclear cells in FRDA patients. Development of new more potent HDACi specific to the frataxin locus as well more complete animal studies to determine bioavailability and efficacy are currently underway. In addition to such molecules that directly target the GAA-repeat, a number of additional molecules have also been shown to increase frataxin mRNA or protein levels. In particular, recombinant human erythropoietin (rhuEPO) has been reported to increase frataxin protein in lymphocytes from FRDA patients [134], and clinical trials with rhuEPO are currently in progress. Although, the molecular

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basis of the increase in frataxin protein remains to be determined, it is hypothesized that it is related to increase translation or stabilization of the frataxin protein.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

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