FRDA is the most prevalent form of recessive cerebellar ataxia characterized by neurodegeneration and cardiomyopathy. Patients with FRDA exhibit a progressive degeneration of the dorsal root ganglia, sensory peripheral nerves, and dentate nuclei of the cerebellum. Most individuals develop several neurological symptoms at a juvenile age, but individuals with adult or late onset are occasionally reported (Koeppen, 2011). Mutations in FXN were discovered as the primary cause of FRDA (Campuzano et al., 1996). Studies of the yeast frataxin homolog identified a role for FXN in iron-sulfur cluster biosynthesis and iron homeostasis (Babcock et al., 1997; Mühlenhoff et al., 2002; Rötig et al., 1997). Loss of FXN has been shown to cause mitochondrial dysfunction in several model organisms (Anderson et al., 2005; Puccio et al., 2001; Rötig et al., 1997). Yet, whether iron accumulates in the nervous system is still under debate, as it has been reported that iron levels are not altered in the nervous system of a conditional knockout mouse model and in FRDA patients (Puccio et al., 2001; Simon et al., 2004; Solbach et al., 2014). However, in FRDA patients, iron was shown to accumulate in the dentate nuclei or in glia cells of dorsal root ganglia (Boddaert et al., 2007; Koeppen et al., 2009). In sum, the iron deposition phenotype is still controversial in mouse models and FRDA patients, and the role of iron in the pathophysiology of FRDA has not yet been determined.

It has been proposed that ROS, generated by impaired mitochondrial electron transport chain complexes or iron deposition, mediate the pathogenesis in FRDA (Bayot et al., 2011; Santos et al., 2010). However, emerging evidence suggests that ROS are not elevated in several model organisms, and several clinical trials based on antioxidant therapy have shown no or limited benefit in FRDA patients (Babcock et al., 1997; Llorens et al., 2007; Macevilly and Muller, 1997; Santhera Pharmaceuticals, 2010; Seznec et al., 2005; Shidara and Hollenbeck, 2010). Hence, the pathogenesis of this disease is still poorly understood.

In a recent study, we described the first fly mutant allele of frataxin homolog (fh), the fly homolog of FXN (Chen et al., 2016). We showed that loss of fh in Drosophila leads to iron accumulation in the nervous system, which in turn induces sphingolipid synthesis and ectopically activates Pdk1 and Mef2. Mef2 activation triggers the aberrant transcription of downstream target genes that causes the degeneration of fly photoreceptors (Chen et al., 2016). However, whether this pathway is also affected in vertebrates upon loss of FXN is unknown. Here, we removed the endogenous mouse Fxn gene by using an adeno-associated virus (AAV) and CRISPR/Cas9 strategy (Swiech et al., 2014). We show that the iron/sphingolipid/PDK1/Mef2 pathway is activated in mice upon loss of Fxn. Furthermore, sphingolipids and PDK1 activity are also up-regulated in hearts of FRDA patients, suggesting that this pathway is also affected in FRDA. Hence, down-regulation of this pathway provides possible new targets to interfere with disease progression.

To test if the iron/sphingolipid/PDK1/Mef2 pathway is activated in vertebrates in vivo upon loss of Fxn, we reduced Fxn levels in the mouse nervous system by using the AAV and CRISPR/Cas9 system (Swiech et al., 2014). We designed two different Fxn single guide RNAs (sgRNAs) and tested their efficiency. The Fxn-sgRNAs induce lesions in the Fxn locus and reduce the levels of FXN protein when compared to the LacZ-sgRNA control in Neuro-2a cells (Figure 1—figure supplement 1A and B). To deliver LacZ- or Fxn-sgRNA into the nervous system, we injected AAV that carried sgRNA into the ventricle of Rosa26-Cas9 knock-in newborn mouse brains (Platt et al., 2014), and the efficiency of AAV infection into the cortical neurons was examined. As shown in Figure 1—figure supplement 1C, the majority of cortical neurons are infected with AAV, and the mRNA levels of Fxn in Fxn-sgRNA mice are less than 40% of the LacZ-sgRNA control mice (Figure 1—figure supplement 1D).

At approximately four months of age the Fxn-sgRNA mice (mice with Fxn-sgRNA AAV injection) develop an abnormal appearance when compared to LacZ-sgRNA control mice. These include a smaller body size and a hunchback phenotype at P130 (Figure 1A). Interestingly, these features are similar to the neuronal conditional knockout mice that were previously reported (Puccio et al., 2001). In addition, the Fxn-sgRNA mice become much less mobile than their LacZ-sgRNA littermates (Videos 1 and 2). The Fxn-sgRNA mice also display an aberrant reflex when lifted by the tail as they fail to spread their hind limbs (Figure 1B). To evaluate the sensorimotor coordination, we performed a rotarod test at two different ages. At P50, we observe no difference between LacZ-sgRNA and Fxn-sgRNA mice. However, Fxn-sgRNA mice show a significant reduction of latency to fall from the rod at age of P130 (Figure 1C). Besides the deficit on the rotarod test, Fxn-sgRNA mice also show a severe impairment in the wire hang test at P130 (Figure 1D). In summary, a removal of Fxn in a substantial neuronal population in the newborn mouse brain causes a severe impairment of neuronal function.

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We further characterized the neuronal phenotype by examining brain sections at P130. We observed that the activating transcription factor 3 (ATF3), a marker that is typically associated with cellular stress response and neuronal damage (Tsujino et al., 2000), is up-regulated in cortical neurons of Fxn-sgRNA mice (Figure 2A). However, no ATF3 positive cells were observed in LacZ-sgRNA mice (Figure 2A). In addition, the axon initial segments, specialized membrane regions where Na⁺ channels cluster to initiate action potentials (Szu-Yu Ho and Rasband, 2011), are lost in cortical neurons of Fxn-sgRNA mice. As shown in Figure 2B, the scaffolding protein required for the formation and maintenance of the axon initial segment, ankyrin-G (AnkG), is severely reduced or lost in cortical neurons in Fxn-sgRNA mice, especially in ATF3 positive neurons (Figure 2B). These data suggest that loss of Fxn in cortical neurons leads to severe neuronal injury and damage.

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To assess if the iron/sphingolipid/PDK1/Mef2 pathway is activated in the brains of Fxn-sgRNA mice, we assessed if iron levels are increased upon loss of Fxn. To examine Fe³⁺ levels, we performed Perls’ staining and used 3,3'-diaminobenzidine (DAB) to enhance the signal. At P130 the DAB signal is strongly increased in brain sections of Fxn-sgRNA mice when compared to control mice (Figure 3A). To further characterize the iron distribution, we co-stain iron with neuronal and glia markers in the same brain sections. Since strong DAB signal interferes with the fluorescence, we chose the cortex region with relatively weak DAB deposits of Fxn-sgRNA mice. Interestingly, the majority of DAB signals are co-localized with a neuronal marker but not glial markers, suggesting that Fe³⁺ accumulates in neurons, although there are some weak diffuse signals distributed outside of the cell body (Figure 3 and Figure 3—figure supplement 1). These data show that Fe³⁺ accumulates in the nervous system upon loss of Fxn. The fact that we observed an obvious Fe³⁺ accumulation, whereas previous reports did not, is likely due to the use of DAB to enhance the Perls' Prussian blue staining, as we did not observe any signal with Perls' Prussian blue staining alone (Figure 3—figure supplement 2A).

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We next assessed Fe²⁺ levels by using RPA (Petrat et al., 2002). RPA is a cell-permeable fluorescent dye that selectively accumulates in mitochondria, and its fluorescence is stoichiometrically quenched by Fe²⁺ (Petrat et al., 2002). Interestingly, we observed Fe²⁺ accumulation in the brain of Fxn-sgRNA mice at P60, a time point at which we did not observe behavioral defects in the Fxn-sgRNA mice. As shown in Figure 3B, the RPA fluorescence is quenched in cells of cortical layer IV and V of Fxn-sgRNA mice when compared to LacZ-sgRNA controls, indicating an accumulation of Fe²⁺ in the nervous system upon loss of Fxn. As a negative control, we used RPAC, a dye that is structurally very similar to RPA but is insensitive to Fe²⁺ mediated fluorescence quenching. RPAC is present at similar levels in both Fxn-sgRNA and LacZ-sgRNA brains (Figure 3B), suggesting that the decreased fluorescence of RPA in Fxn-sgRNA mice is not due to impaired dye uptake but due to Fe²⁺ quenching.

It was previously proposed that iron deposition triggers free radical production via Fenton’s reaction. We therefore measured oxidative stress by examining the levels of 4-hydroxynonenal (4-HNE), a major product of lipid peroxidation (Ayala et al., 2014). However, we did not observe an increase of 4-HNE in Fxn-sgRNA brain tissues (Figure 3—figure supplement 2B), consistent with previous observations in flies (Chen et al., 2016). We next determined if iron triggers the activation of the PDK1/Mef2 pathway in Fxn-sgRNA mice. Phosphorylation of S241 in the PDK1 activation loop is required for its activity (Casamayor et al., 1999). We observed an increase in phosphorylation levels of S241 in Fxn-sgRNA mice compared to the LacZ-sgRNA mice at P60, prior to the onset of symptoms (Figure 3C and Figure 3—figure supplement 2C). To assess if Mef2 is activated in Fxn-sgRNA mice, we determined the mRNA levels of several validated Mef2 downstream targets (Anderson et al., 2004; Ewen et al., 2011; Potthoff et al., 2007; Woronicz et al., 1995). As shown in Figure 3D, many, but not all, transcripts are up-regulated 1.5–3 fold in Fxn-sgRNA mice at P60. This is consistent with previous findings that only about half of Mef2 targets are up-regulated (to 2–3 fold) when Mef2A is overexpressed (Ewen et al., 2011). In summary, these results provide evidence that the PDK1/Mef2 pathway is activated in the vertebrate nervous system upon loss of Fxn prior to the onset of phenotypes that are observed about two months later.

Last, we tested if the same pathway is also activated in FRDA patients. As we were unable to obtain cerebella and dorsal root ganglia of patients, we used heart samples that were previously shown to accumulate Fe³⁺ (Bradley et al., 2000; Koeppen, 2011). We first investigated the sphingolipid levels in frozen heart tissues of five controls and six FRDA patients. As shown in Figure 4A, a substantial fraction of the sphingolipids are increased in FRDA patients when compared to controls, including short chain ceramides (C14-C18), different dihydroceramides, dihydrosphingosines, dihydrosphingosine-1-phosphate, and sphingosine. Finally, we tested if PDK1 activity is also increased in FRDA patients. The phosphorylation levels of S241 in PDK1 are elevated in FRDA patients when compared to controls (Figure 4B). Note that patient six showed no obvious increase in PDK1 phosphorylation when compared to the other patients. This patient had relatively mild symptoms and a later onset of disease (age 18) than other patients (ages 4 to 9). The age of death was also much later in patient 6 (age 67) compared to the other patients (age 25–37). Similarly, the increased levels of sphingolipids in patient six are also less pronounced than other patients. In sum, these data suggest that the iron/sphingolipid/PDK1/Mef2 pathway is activated in FRDA patients and that it may contribute to the pathogenesis of FRDA.

Figure 4

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Here we show that removal of Fxn in the central nervous system using AAV and CRISPR/Cas9 causes behavioral and neurological phenotypes in mice. This new FXN mouse model exhibits several similar phenotypes to the previous neuronal conditional knockout mouse model, including smaller body size, hunchback phenotype, impaired locomotion, and shortened life span (Puccio et al., 2001). By using RPA and Perls'/DAB stainings, more sensitive iron staining assays than Perls’ Prussian blue staining alone, we found that Fe²⁺ and Fe³⁺ levels are increased in the cerebral cortex of Fxn-sgRNA mice, and the activities of PDK1 and Mef2 are up-regulated, suggesting that the iron/sphingolipid/PDK1/Mef2 pathway, previously identified in fly fh mutants, is also activated in Fxn-sgRNA mice. Importantly, sphingolipids and PDK1 activity are also up-regulated in the hearts of FRDA patients, indicating that a similar pathway is also activated and may contribute to the pathogenic mechanism of FRDA.

Iron toxicity has been long considered as a possible pathogenic contributor of FRDA. However, the iron accumulation phenotypes have never been reported in the nervous system in an inducible conditional knockout mouse model (Martelli and Puccio, 2014; Puccio et al., 2001; Simon et al., 2004). We provide the first direct evidence that both Fe²⁺ and Fe³⁺ accumulate in the brain upon loss of Fxn. In addition, we noticed that irons are not only deposited inside the neuronal cell body but also present in the extracellular space. For example, DAB staining showed a diffuse pattern in and surrounding the neuronal cell body (Figure 3A and Figure 3—figure supplement 1), and the RPA fluorescence is similarly reduced in the extracellular space (Figure 3B), suggesting that iron accumulation occurs both intracellularly and extracellularly.

It is worth noting that the iron/sphingolipid/PDK1/Mef2 pathway is conserved from yeast to fly (Chen et al., 2016; Lee et al., 2012), and that this pathway is also activated in Fxn-sgRNA mice and the hearts of FRDA patients. Interestingly, genetic variations at the 5’UTR of Mef2C that promote Mef2C transcription are associated with hypertrophic cardiomyopathy (Alonso-Montes et al., 2012). In addition, overexpression of Mef2A or Mef2C causes cell sarcomeric disorganization and elongation in cultured cardiomyocytes (Xu et al., 2006). These data suggest that an activation of iron/sphingolipid/PDK1/Mef2 pathway may play a role in the induction of cardiomyopathy in FRDA patients. Inhibition of this pathway may not only suppress neurodegeneration, but also prevent or delay cardiomyopathy, the major cause of lethality in FRDA. In sum, this pathway may provide us with therapeutic targets based on antisense oligonucleotide strategies (Ackermann et al., 2012), an option that should be explored.

1. 1. Ackermann EJ
   2. Guo S
   3. Booten S
   4. Alvarado L
   5. Benson M
   6. Hughes S
   7. Monia BP

   (2012) Clinical development of an antisense therapy for the treatment of transthyretin-associated polyneuropathy

   Amyloid 19 (Suppl 1):43–44.

   https://doi.org/10.3109/13506129.2012.673140

   • PubMed
   • Google Scholar
2. 1. Alonso-Montes C
   2. Naves-Diaz M
   3. Fernandez-Martin JL
   4. Rodriguez-Reguero J
   5. Moris C
   6. Coto E
   7. Cannata-Andia JB
   8. Rodriguez I

   (2012) New polymorphisms in human MEF2C gene as potential modifier of hypertrophic cardiomyopathy

   Molecular Biology Reports 39:8777–8785.

   https://doi.org/10.1007/s11033-012-1740-7

   • PubMed
   • Google Scholar
3. 1. Anderson JP
   2. Dodou E
   3. Heidt AB
   4. De Val SJ
   5. Jaehnig EJ
   6. Greene SB
   7. Olson EN
   8. Black BL

   (2004) HRC is a direct transcriptional target of MEF2 during cardiac, skeletal, and arterial smooth muscle development in vivo

   Molecular and Cellular Biology 24:3757–3768.

   https://doi.org/10.1128/MCB.24.9.3757-3768.2004

   • PubMed
   • Google Scholar
4. 1. Anderson PR
   2. Kirby K
   3. Hilliker AJ
   4. Phillips JP

   (2005) RNAi-mediated suppression of the mitochondrial iron chaperone, frataxin, in Drosophila

   Human Molecular Genetics 14:3397–3405.

   https://doi.org/10.1093/hmg/ddi367

   • PubMed
   • Google Scholar
5. 1. Ayala A
   2. Muñoz MF
   3. Argüelles S

   (2014) Lipid peroxidation: production, metabolism, and signaling mechanisms of Malondialdehyde and 4-hydroxy-2-nonenal

   Oxidative Medicine and Cellular Longevity 2014:1–31.

   https://doi.org/10.1155/2014/360438

   • Google Scholar
6. 1. Ayuso E
   2. Mingozzi F
   3. Montane J
   4. Leon X
   5. Anguela XM
   6. Haurigot V
   7. Edmonson SA
   8. Africa L
   9. Zhou S
   10. High KA
   11. Bosch F
   12. Wright JF

   (2010) High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

   Gene Therapy 17:503–510.

   https://doi.org/10.1038/gt.2009.157

   • PubMed
   • Google Scholar
7. 1. Babcock M
   2. de Silva D
   3. Oaks R
   4. Davis-Kaplan S
   5. Jiralerspong S
   6. Montermini L
   7. Pandolfo M
   8. Kaplan J

   (1997) Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin

   Science 276:1709–1712.

   https://doi.org/10.1126/science.276.5319.1709

   • PubMed
   • Google Scholar
8. 1. Bayot A
   2. Santos R
   3. Camadro JM
   4. Rustin P

   (2011) Friedreich's ataxia: the vicious circle hypothesis revisited

   BMC Medicine 9:112.

   https://doi.org/10.1186/1741-7015-9-112

   • PubMed
   • Google Scholar
9. 1. Boddaert N
   2. Le Quan Sang KH
   3. Rötig A
   4. Leroy-Willig A
   5. Gallet S
   6. Brunelle F
   7. Sidi D
   8. Thalabard JC
   9. Munnich A
   10. Cabantchik ZI

   (2007) Selective iron chelation in Friedreich ataxia: biologic and clinical implications

   Blood 110:401–408.

   https://doi.org/10.1182/blood-2006-12-065433

   • PubMed
   • Google Scholar
10. 1. Bradley JL
    2. Blake JC
    3. Chamberlain S
    4. Thomas PK
    5. Cooper JM
    6. Schapira AH

    (2000) Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia

    Human Molecular Genetics 9:275–282.

    https://doi.org/10.1093/hmg/9.2.275

    • PubMed
    • Google Scholar
11. 1. Campuzano V
    2. Montermini L
    3. Moltò MD
    4. Pianese L
    5. Cossée M
    6. Cavalcanti F
    7. Monros E
    8. Rodius F
    9. Duclos F
    10. Monticelli A
    11. Zara F
    12. Cañizares J
    13. Koutnikova H
    14. Bidichandani SI
    15. Gellera C
    16. Brice A
    17. Trouillas P
    18. De Michele G
    19. Filla A
    20. De Frutos R
    21. Palau F
    22. Patel PI
    23. Di Donato S
    24. Mandel JL
    25. Cocozza S
    26. Koenig M
    27. Pandolfo M

    (1996) Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion

    Science 271:1423–1427.

    https://doi.org/10.1126/science.271.5254.1423

    • PubMed
    • Google Scholar
12. 1. Casamayor A
    2. Morrice NA
    3. Alessi DR

    (1999) Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo

    Biochemical Journal 342:287–292.

    https://doi.org/10.1042/bj3420287

    • PubMed
    • Google Scholar
13. 1. Chen K
    2. Lin G
    3. Haelterman NA
    4. Ho TS
    5. Li T
    6. Li Z
    7. Duraine L
    8. Graham BH
    9. Jaiswal M
    10. Yamamoto S
    11. Rasband MN
    12. Bellen HJ

    (2016) Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration

    eLife 5:e16043.

    https://doi.org/10.7554/eLife.16043

    • PubMed
    • Google Scholar
14. 1. Ewen EP
    2. Snyder CM
    3. Wilson M
    4. Desjardins D
    5. Naya FJ

    (2011) The Mef2A transcription factor coordinately regulates a costamere gene program in cardiac muscle

    Journal of Biological Chemistry 286:29644–29653.

    https://doi.org/10.1074/jbc.M111.268094

    • PubMed
    • Google Scholar
15. 1. Grimm D
    2. Lee JS
    3. Wang L
    4. Desai T
    5. Akache B
    6. Storm TA
    7. Kay MA

    (2008) In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses

    Journal of Virology 82:5887–5911.

    https://doi.org/10.1128/JVI.00254-08

    • PubMed
    • Google Scholar
16. 1. Ho TS
    2. Zollinger DR
    3. Chang KJ
    4. Xu M
    5. Cooper EC
    6. Stankewich MC
    7. Bennett V
    8. Rasband MN

    (2014) A hierarchy of ankyrin-spectrin complexes clusters sodium channels at nodes of Ranvier

    Nature Neuroscience 17:1664–1672.

    https://doi.org/10.1038/nn.3859

    • PubMed
    • Google Scholar
17. 1. Koeppen AH
    2. Morral JA
    3. Davis AN
    4. Qian J
    5. Petrocine SV
    6. Knutson MD
    7. Gibson WM
    8. Cusack MJ
    9. Li D

    (2009) The dorsal root ganglion in Friedreich's ataxia

    Acta Neuropathologica 118:763–776.

    https://doi.org/10.1007/s00401-009-0589-x

    • PubMed
    • Google Scholar
18. 1. Koeppen AH

    (2011) Friedreich's ataxia: pathology, pathogenesis, and molecular genetics

    Journal of the Neurological Sciences 303:1–12.

    https://doi.org/10.1016/j.jns.2011.01.010

    • PubMed
    • Google Scholar
19. 1. Lee YJ
    2. Huang X
    3. Kropat J
    4. Henras A
    5. Merchant SS
    6. Dickson RC
    7. Chanfreau GF

    (2012) Sphingolipid signaling mediates iron toxicity

    Cell Metabolism 16:90–96.

    https://doi.org/10.1016/j.cmet.2012.06.004

    • PubMed
    • Google Scholar
20. 1. Ljungberg MC
    2. Ali YO
    3. Zhu J
    4. Wu CS
    5. Oka K
    6. Zhai RG
    7. Lu HC

    (2012) CREB-activity and nmnat2 transcription are down-regulated prior to neurodegeneration, while NMNAT2 over-expression is neuroprotective, in a mouse model of human tauopathy

    Human Molecular Genetics 21:251–267.

    https://doi.org/10.1093/hmg/ddr492

    • PubMed
    • Google Scholar
21. 1. Llorens JV
    2. Navarro JA
    3. Martínez-Sebastián MJ
    4. Baylies MK
    5. Schneuwly S
    6. Botella JA
    7. Moltó MD

    (2007) Causative role of oxidative stress in a Drosophila model of Friedreich ataxia

    The FASEB Journal 21:333–344.

    https://doi.org/10.1096/fj.05-5709com

    • PubMed
    • Google Scholar
22. 1. Macevilly CJ
    2. Muller DP

    (1997) Oxidative stress does not appear to be involved in the aetiology of Friedreich's ataxia

    Restorative Neurology and Neuroscience 11:131–137.

    https://doi.org/10.3233/RNN-1997-11303

    • PubMed
    • Google Scholar
23. 1. Martelli A
    2. Puccio H

    (2014) Dysregulation of cellular iron metabolism in Friedreich ataxia: from primary iron-sulfur cluster deficit to mitochondrial iron accumulation

    Frontiers in Pharmacology 5:130.

    https://doi.org/10.3389/fphar.2014.00130

    • PubMed
    • Google Scholar
24. 1. Mühlenhoff U
    2. Richhardt N
    3. Ristow M
    4. Kispal G
    5. Lill R

    (2002) The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins

    Human Molecular Genetics 11:2025–2036.

    https://doi.org/10.1093/hmg/11.17.2025

    • PubMed
    • Google Scholar
25. 1. Petrat F
    2. Weisheit D
    3. Lensen M
    4. de Groot H
    5. Sustmann R
    6. Rauen U

    (2002) Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor

    Biochemical Journal 362:137–147.

    https://doi.org/10.1042/bj3620137

    • PubMed
    • Google Scholar
26. 1. Platt RJ
    2. Chen S
    3. Zhou Y
    4. Yim MJ
    5. Swiech L
    6. Kempton HR
    7. Dahlman JE
    8. Parnas O
    9. Eisenhaure TM
    10. Jovanovic M
    11. Graham DB
    12. Jhunjhunwala S
    13. Heidenreich M
    14. Xavier RJ
    15. Langer R
    16. Anderson DG
    17. Hacohen N
    18. Regev A
    19. Feng G
    20. Sharp PA
    21. Zhang F

    (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling

    Cell 159:440–455.

    https://doi.org/10.1016/j.cell.2014.09.014

    • PubMed
    • Google Scholar
27. 1. Potthoff MJ
    2. Arnold MA
    3. McAnally J
    4. Richardson JA
    5. Bassel-Duby R
    6. Olson EN

    (2007) Regulation of skeletal muscle sarcomere integrity and postnatal muscle function by Mef2c

    Molecular and Cellular Biology 27:8143–8151.

    https://doi.org/10.1128/MCB.01187-07

    • PubMed
    • Google Scholar
28. 1. Puccio H
    2. Simon D
    3. Cossée M
    4. Criqui-Filipe P
    5. Tiziano F
    6. Melki J
    7. Hindelang C
    8. Matyas R
    9. Rustin P
    10. Koenig M

    (2001) Mouse models for Friedreich ataxia exhibit cardiomyopathy, sensory nerve defect and Fe-S enzyme deficiency followed by intramitochondrial iron deposits

    Nature Genetics 27:181–186.

    https://doi.org/10.1038/84818

    • PubMed
    • Google Scholar
29. 1. Rötig A
    2. de Lonlay P
    3. Chretien D
    4. Foury F
    5. Koenig M
    6. Sidi D
    7. Munnich A
    8. Rustin P

    (1997) Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia

    Nature Genetics 17:215–217.

    https://doi.org/10.1038/ng1097-215

    • PubMed
    • Google Scholar
30. Clinical Trial

    1. Santhera Pharmaceuticals

    (sponsors) (2010) A Phase III double-blind, randomised, Placebo-controlled study of the efficacy, safety and tolerability of idebenone in the treatment of friedreich's ataxia patients

    https://clinicaltrials.gov/ct2/show/NCT00905268
31. 1. Santos R
    2. Lefevre S
    3. Sliwa D
    4. Seguin A
    5. Camadro JM
    6. Lesuisse E

    (2010) Friedreich ataxia: molecular mechanisms, redox considerations, and therapeutic opportunities

    Antioxidants & Redox Signaling 13:651–690.

    https://doi.org/10.1089/ars.2009.3015

    • PubMed
    • Google Scholar
32. 1. Seznec H
    2. Simon D
    3. Bouton C
    4. Reutenauer L
    5. Hertzog A
    6. Golik P
    7. Procaccio V
    8. Patel M
    9. Drapier JC
    10. Koenig M
    11. Puccio H

    (2005) Friedreich ataxia: the oxidative stress paradox

    Human Molecular Genetics 14:463–474.

    https://doi.org/10.1093/hmg/ddi042

    • PubMed
    • Google Scholar
33. 1. Shidara Y
    2. Hollenbeck PJ

    (2010) Defects in mitochondrial axonal transport and membrane potential without increased reactive oxygen species production in a Drosophila model of Friedreich ataxia

    Journal of Neuroscience 30:11369–11378.

    https://doi.org/10.1523/JNEUROSCI.0529-10.2010

    • PubMed
    • Google Scholar
34. 1. Simon D
    2. Seznec H
    3. Gansmuller A
    4. Carelle N
    5. Weber P
    6. Metzger D
    7. Rustin P
    8. Koenig M
    9. Puccio H

    (2004) Friedreich ataxia mouse models with progressive cerebellar and sensory ataxia reveal autophagic neurodegeneration in dorsal root ganglia

    Journal of Neuroscience 24:1987–1995.

    https://doi.org/10.1523/JNEUROSCI.4549-03.2004

    • PubMed
    • Google Scholar
35. 1. Solbach K
    2. Kraff O
    3. Minnerop M
    4. Beck A
    5. Schöls L
    6. Gizewski ER
    7. Ladd ME
    8. Timmann D

    (2014) Cerebellar pathology in Friedreich's ataxia: atrophied dentate nuclei with normal iron content

    NeuroImage: Clinical 6:93–99.

    https://doi.org/10.1016/j.nicl.2014.08.018

    • PubMed
    • Google Scholar
36. 1. Swiech L
    2. Heidenreich M
    3. Banerjee A
    4. Habib N
    5. Li Y
    6. Trombetta J
    7. Sur M
    8. Zhang F

    (2015) In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

    Nature Biotechnology 33:102–106.

    https://doi.org/10.1038/nbt.3055

    • PubMed
    • Google Scholar
37. 1. Szu-Yu Ho T
    2. Rasband MN

    (2011) Maintenance of neuronal polarity

    Developmental Neurobiology 71:474–482.

    https://doi.org/10.1002/dneu.20843

    • PubMed
    • Google Scholar
38. 1. Tsujino H
    2. Kondo E
    3. Fukuoka T
    4. Dai Y
    5. Tokunaga A
    6. Miki K
    7. Yonenobu K
    8. Ochi T
    9. Noguchi K

    (2000) Activating transcription factor 3 (ATF3) induction by axotomy in sensory and motoneurons: A novel neuronal marker of nerve injury

    Molecular and Cellular Neuroscience 15:170–182.

    https://doi.org/10.1006/mcne.1999.0814

    • PubMed
    • Google Scholar
39. 1. Woronicz JD
    2. Lina A
    3. Calnan BJ
    4. Szychowski S
    5. Cheng L
    6. Winoto A

    (1995) Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis

    Molecular and Cellular Biology 15:6364–6376.

    https://doi.org/10.1128/MCB.15.11.6364

    • PubMed
    • Google Scholar
40. 1. Xu J
    2. Gong NL
    3. Bodi I
    4. Aronow BJ
    5. Backx PH
    6. Molkentin JD

    (2006) Myocyte enhancer factors 2A and 2C induce dilated cardiomyopathy in transgenic mice

    Journal of Biological Chemistry 281:9152–9162.

    https://doi.org/10.1074/jbc.M510217200

    • PubMed
    • Google Scholar
41. 1. Zhang C
    2. Susuki K
    3. Zollinger DR
    4. Dupree JL
    5. Rasband MN

    (2013) Membrane domain organization of myelinated axons requires βII spectrin

    Journal of Cell Biology 203:437–443.

    https://doi.org/10.1083/jcb.201308116

    • PubMed
    • Google Scholar
42. 1. Zolotukhin S
    2. Byrne BJ
    3. Mason E
    4. Zolotukhin I
    5. Potter M
    6. Chesnut K
    7. Summerford C
    8. Samulski RJ
    9. Muzyczka N

    (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield

    Gene Therapy 6:973–985.

    https://doi.org/10.1038/sj.gt.3300938

    • PubMed
    • Google Scholar

Author details

1. Kuchuan Chen

   Program in Developmental Biology, Baylor College of Medicine, Houston, United States

   Contribution

   KC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Tammy Szu-Yu Ho

   Competing interests

   No competing interests declared.

2. Tammy Szu-Yu Ho

   Department of Neuroscience, Baylor College of Medicine, Houston, United States

   Contribution

   TS-YH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Kuchuan Chen

   Competing interests

   No competing interests declared.

3. Guang Lin

   Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States

   Contribution

   GL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Kai Li Tan

   Program in Developmental Biology, Baylor College of Medicine, Houston, United States

   Contribution

   KLT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

5. Matthew N Rasband

   1. Program in Developmental Biology, Baylor College of Medicine, Houston, United States
   2. Department of Neuroscience, Baylor College of Medicine, Houston, United States

   Contribution

   MNR, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0001-8184-2477

6. Hugo J Bellen

   1. Program in Developmental Biology, Baylor College of Medicine, Houston, United States
   2. Department of Neuroscience, Baylor College of Medicine, Houston, United States
   3. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
   4. Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States
   5. Jan and Dan Duncan Neurological Research Institute, Houston, United States

   Contribution

   HJB, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   hbellen@bcm.edu

   Competing interests

   HJB: Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-5992-5989

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