Frataxin Expression Research Articles

Lipophilic methylene blue analogues enhance mitochondrial function and increase frataxin levels in a cellular model of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder resulting from reduced expression of the protein frataxin (FXN). Although its function is not fully understood, frataxin appears to help assemble iron sulfur clusters; these are critical for the function of many proteins, including those needed for mitochondrial energy production. Finding ways to increase FXN levels has been a major therapeutic strategy for this disease. Previously, we described a novel series of methylene violet analogues and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Presently, a series of methylene blue analogues has been synthesized and characterized for their in vitro biochemical and biological properties in cultured Friedreich’s ataxia lymphocytes. Favorable methylene blue analogues were shown to increase frataxin levels and mitochondrial biogenesis, and to improve aconitase activity. The analogues were found to be good ROS scavengers, and able to protect cultured FRDA lymphocytes from oxidative stress resulting from inhibition of complex I and from glutathione depletion. The analogues also preserved mitochondrial membrane potential and augmented ATP production. Our results suggest that analogue 5, emerging from the initial structure of the parent compound methylene blue (MB), represents a promising lead structure and lacks the cytotoxicity associated with the parent compound MB.

Increased Frataxin Expression Induced in Friedreich Ataxia Cells by Platinum TALE-VP64s or Platinum TALE-SunTag

Frataxin gene (FXN) expression is reduced in Friedreich’s ataxia patients due to an increase in the number of GAA trinucleotides in intron 1. The frataxin protein, encoded by that gene, plays an important role in mitochondria’s iron metabolism. Platinum TALE (plTALE) proteins targeting the regulatory region of the FXN gene, fused with a transcriptional activator (TA) such as VP64 or P300, were used to increase the expression of that gene. Many effectors, plTALEVP64, plTALEp300, and plTALESunTag, targeting 14 sequences of the FXN gene promoter or intron 1 were produced. This permitted selection of 3 plTALEVP64s and 2 plTALESunTag that increased FXN gene expression by up to 19-fold in different Friedreich ataxia (FRDA) primary fibroblasts. Adeno-associated viruses were used to deliver the best effectors to the YG8R mouse model to validate their efficiencies in vivo. Our results showed that these selected plTALEVP64s or plTALESunTag induced transcriptional activity of the endogenous FXN gene as well as expression of the frataxin protein in YG8R mouse heart by 10-fold and in skeletal muscles by up to 35-fold. The aconitase activity was positively modulated by the frataxin level in mitochondria, and it was, thus, increased in vitro and in vivo by the increased frataxin expression.

Identification of p38 MAPK as a novel therapeutic target for Friedreich\u2019s ataxia

Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder caused by decreased expression of frataxin, a protein that localizes to mitochondria and is critical for iron-sulfur-cluster (ISC) assembly. There are no proven effective treatments for FRDA. We previously screened a random shRNA library and identified a synthetic shRNA (gFA11) that reverses the growth defect of FRDA cells in culture. We now report that gFA11 decreases cytokine secretion in primary FRDA fibroblasts and reverts other changes associated with cell senescence. The gene-expression profile induced by gFA11 is remarkably similar to the gene-expression profile induced by the p38 MAPK inhibitor SB203580. We found that p38 phosphorylation, indicating activation of the p38 pathway, is higher in FRDA cells than in normal control cells, and that siRNA knockdown of frataxin in normal fibroblasts also increases p38 phosphorylation. Treatment of FRDA cells with p38 inhibitors recapitulates the reversal of the slow-growth phenotype induced by clone gFA11. These data highlight the involvement of the p38 MAPK pathway in the pathogenesis of FRDA and the potential use of p38 inhibitors as a treatment for FRDA.

Frataxin overexpression in M\xfcller cells protects retinal ganglion cells in a mouse model of ischemia/reperfusion injury in vivo

Müller cells are critical for retinal function and neuronal survival but can become detrimental in response to retinal ischemia and increased oxidative stress. Elevated oxidative stress increases expression of the mitochondrial enzyme frataxin in the retina, and its overexpression is neuroprotective after ischemia. Whether frataxin expression in Müller cells might improve their function and protect neurons after ischemia is unknown. The aim of this study was to evaluate the effect of frataxin overexpression in Müller cells on neuronal survival after retinal ischemia/reperfusion in the mouse in vivo. Retinal ischemia/reperfusion was induced in mice overexpressing frataxin in Müller cells by transient elevation of intraocular pressure. Retinal ganglion cells survival was determined 14 days after lesion. Expression of frataxin, antioxidant enzymes, growth factors and inflammation markers was determined with qRT-PCR, Western blotting and immunohistochemistry 24 hours after lesion. Following lesion, there was a 65% increase in the number of surviving RGCs in frataxin overexpressing mice. Improved survival was associated with increased expression of the antioxidant enzymes Gpx1 and Sod1 as well as the growth factors Cntf and Lif. Additionally, microglial activation was decreased in these mice. Therefore, support of Müller cell function constitutes a feasible approach to reduce neuronal degeneration after ischemia.

Low apolipoprotein A-I levels in Friedreich's ataxia and in frataxin-deficient cells: Implications for therapy.

Friedreich’s ataxia (FA) is an autosomal recessive neurodegenerative disorder, which results primarily from reduced expression of the mitochondrial protein frataxin. FA has an estimated prevalence of one in 50,000 in the population, making it the most common hereditary ataxia. Paradoxically, mortality arises most frequently from cardiomyopathy and cardiac failure rather than from neurological effects. Decreased high-density lipoprotein (HDL) and apolipoprotein A-I (ApoA-l) levels in the general population are associated with an increased risk of mortality from cardiomyopathy and heart failure. However, the pathophysiology of heart disease in FA is non-vascular and there are conflicting data on HDL-cholesterol in FA. Two studies have shown a decrease in HDL-cholesterol compared with controls and two have shown there was no difference between FA and controls. One also showed that there was no difference in serum Apo-A-I levels in FA when compared with controls. Using a highly specific stable isotope dilution mass spectrometry-based assay, we demonstrated a 21.6% decrease in serum ApoA-I in FA patients (134.8 mg/dL, n = 95) compared with non-affected controls (172.1 mg/dL, n = 95). This is similar to the difference in serum ApoA-I levels between non-smokers and tobacco smokers. Knockdown of frataxin by > 70% in human hepatoma HepG2 cells caused a 20% reduction in secreted ApoA-I. Simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor caused a 200% increase in HMG-CoA in the control HepG2 cells with a similar increase in the frataxin knockdown HepG2 cells, back to levels found in the control cells. There was a concomitant 20% increase in secreted ApoA-I to levels found in the control cells that were treated with simvastatin. This study provides compelling evidence that ApoA-I levels are reduced in FA patients compared with controls and suggest that statin treatment would normalize the ApoA-I levels.

Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

Liquid Chromatography-High Resolution Mass Spectrometry Analysis of Platelet Frataxin as a Protein Biomarker for the Rare Disease Friedreich's Ataxia.

Friedreich's ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50 000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood cannot be used because it is present in long-lived erythrocytes. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of 10 days. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/mass spectrometry. The lower limit of quantification was 0.078 pg frataxin/μg protein, and the assay had 100% sensitivity and specificity for discriminating between controls and FA cases. The mean levels of control and FA platelet frataxin were 9.4 ± 2.6 and 2.4 ± 0.6 pg/μg protein, respectively. The assay should make it possible to rigorously monitor the effects of therapeutic interventions on frataxin expression in this devastating disease.

Effect of diazoxide on Friedreich ataxia models.

Friedreich ataxia (FRDA) is an inherited recessive disorder caused by a deficiency in the mitochondrial protein frataxin. There is currently no effective treatment for FRDA available, especially for neurological deficits. In this study, we tested diazoxide, a drug commonly used as vasodilator in the treatment of acute hypertension, on cellular and animal models of FRDA. We first showed that diazoxide increases frataxin protein levels in FRDA lymphoblastoid cell lines, via the mammalian target of rapamycin (mTOR) pathway. We then explored the potential therapeutic effect of diazoxide in frataxin-deficient transgenic YG8sR mice and we found that prolonged oral administration of 3 mpk/d diazoxide was found to be safe, but produced variable effects concerning efficacy. YG8sR mice showed improved beam walk coordination abilities and footprint stride patterns, but a generally reduced locomotor activity. Moreover, they showed significantly increased frataxin expression, improved aconitase activity, and decreased protein oxidation in cerebellum and brain mitochondrial tissue extracts. Further studies are needed before this drug should be considered for FRDA clinical trials.

Sustained FXN expression in dorsal root ganglia from a nonreplicative genomic HSV-1 vector.

Friedreich's ataxia (FA) is an autosomal recessive neurodegenerative disease caused by mutations in the frataxin gene (FXN), which lead to reduced levels of the essential mitochondrial protein frataxin. Currently, there is no effective cure. With the aim of developing a gene therapy for FA neuropathology, we describe the construction and preliminary characterization of a high-capacity nonreplicative genomic herpes simplex virus type 1 vector (H24B-FXNlac vector) carrying a reduced version of the human FXN genomic locus, comprising the 5-kb promoter and the FXN cDNA with the inclusion of intron 1. We show that the transgene cassette contains the elements necessary to preserve physiological neuronal regulation of human FXN expression. Transduction of cultured fetal rat dorsal root ganglia neurons with the H24B-FXNlac vector results in sustained expression of human FXN transcripts and frataxin protein. Rat footpad inoculation with the H24B-FXNlac vector results in human FXN transgene delivery to the dorsal root ganglia, with expression persisting for at least 1month. The results of the present study support the feasibility of using this vector for sustained neuronal expression of human frataxin for FA gene therapy.

Comprehensive analysis of gene expression patterns in Friedreich's ataxia fibroblasts by RNA sequencing reveals altered levels of protein synthesis factors and solute carriers.

ABSTRACTFriedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disease usually caused by large homozygous expansions of GAA repeat sequences in intron 1 of the frataxin (FXN) gene. FRDA patients homozygous for GAA expansions have low FXN mRNA and protein levels when compared with heterozygous carriers or healthy controls. Frataxin is a mitochondrial protein involved in iron–sulfur cluster synthesis, and many FRDA phenotypes result from deficiencies in cellular metabolism due to lowered expression of FXN. Presently, there is no effective treatment for FRDA, and biomarkers to measure therapeutic trial outcomes and/or to gauge disease progression are lacking. Peripheral tissues, including blood cells, buccal cells and skin fibroblasts, can readily be isolated from FRDA patients and used to define molecular hallmarks of disease pathogenesis. For instance, FXN mRNA and protein levels as well as FXN GAA-repeat tract lengths are routinely determined using all of these cell types. However, because these tissues are not directly involved in disease pathogenesis, their relevance as models of the molecular aspects of the disease is yet to be decided. Herein, we conducted unbiased RNA sequencing to profile the transcriptomes of fibroblast cell lines derived from 18 FRDA patients and 17 unaffected control individuals. Bioinformatic analyses revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins in FRDA fibroblasts. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in FRDA fibroblasts. Finally, comparison of genes differentially expressed in FRDA fibroblasts to three previously published gene expression signatures defined for FRDA blood cells showed substantial overlap between the independent datasets, including correspondingly deficient expression of antioxidant defense genes. Together, these results indicate that gene expression profiling of cells derived from peripheral tissues can, in fact, consistently reveal novel molecular pathways of the disease. When performed on statistically meaningful sample group sizes, unbiased global profiling analyses utilizing peripheral tissues are critical for the discovery and validation of FRDA disease biomarkers.

Peptide SS-31 upregulates frataxin expression and improves the quality of mitochondria: implications in the treatment of Friedreich ataxia

Friedreich ataxia is a progressive neurodegenerative disease caused by the expansion of GAA trinucleotide repeats within the first intron of the FXN gene, which encodes frataxin. The pathophysiology of the disease is thought to be derived from the decrease of Fe-S cluster biogenesis due to frataxin deficiency. There is currently no effective treatment for the disease. In our study, we demonstrated that treatment with the mitochondrion-targeted peptide SS-31 reduced frataxin deficiency-induced oxidative stress in lymphoblasts and fibroblasts derived from patients. Interestingly, SS-31 treatment translationally upregulated the protein level of frataxin in a dose-dependent manner. Furthermore, SS-31 treatment increased the enzymatic activities of the iron-sulphur enzymes, including aconitase and complex II and III of the respiratory chain. Further evaluation of the quality of mitochondria showed that mitochondrial membrane potential, ATP content, NAD+/NADH, and the morphology of mitochondria all improved. Our results suggest that SS-31 might potentially be a new drug for the early treatment of Friedreich ataxia.

Overexpression of Drosophila frataxin triggers cell death in an iron-dependent manner

Friedreich ataxia (FRDA) is the most important autosomal recessive ataxia in the Caucasian population. FRDA patients display severe neurological and cardiac symptoms that reflect a strong cellular and axonal degeneration. FRDA is caused by a loss of function of the mitochondrial protein frataxin which impairs the biosynthesis of iron-sulfur clusters and in turn the catalytic activity of several enzymes in the Krebs cycle and the respiratory chain leading to a diminished energy production. Although FRDA is due to frataxin depletion, overexpression might also be very helpful to better understand cellular functions of frataxin. In this work, we have increased frataxin expression in neurons to elucidate specific roles that frataxin might play in these tissues. Using molecular, biochemical, histological and behavioral methods, we report that frataxin overexpression is sufficient to increase oxidative phosphorylation, modify mitochondrial morphology, alter iron homeostasis and trigger oxidative stress-dependent cell death. Interestingly, genetic manipulation of mitochondrial iron metabolism by silencing mitoferrin successfully improves cell survival under oxidative-attack conditions, although enhancing antioxidant defenses or mitochondrial fusion failed to ameliorate frataxin overexpression phenotypes. This result suggests that cell degeneration is directly related to enhanced incorporation of iron into the mitochondria. Drosophila frataxin overexpression might also provide an alternative approach to identify processes that are important in FRDA such as changes in mitochondrial morphology and oxidative stress induced cell death.

Erythropoietin and small molecule agonists of the tissue-protective erythropoietin receptor increase FXN expression in neuronal cells in vitro and in Fxn-deficient KIKO mice in vivo

Friedreich's ataxia (FA) is a progressive neurodegenerative disease caused by reduced levels of the mitochondrial protein frataxin (FXN). Recombinant human erythropoietin (rhEPO) increased FXN protein in vitro and in early clinical studies, while no published reports evaluate rhEPO in animal models of FA. STS-E412 and STS-E424 are novel small molecule agonists of the tissue-protective, but not the erythropoietic EPO receptor. We find that rhEPO, STS-E412 and STS-E424 increase FXN expression in vitro and in vivo. RhEPO, STS-E412 and STS-E424 increase FXN by up to 2-fold in primary human cortical cells and in retinoic-acid differentiated murine P19 cells. In primary human cortical cells, the increase in FXN protein was accompanied by an increase in FXN mRNA, detectable within 4 h. RhEPO and low nanomolar concentrations of STS-E412 and STS-E424 also increase FXN in normal and FA patient-derived PBMC by 20%–40% within 24 h, an effect that was comparable to that by HDAC inhibitor 4b. In vivo, STS-E412 increased Fxn mRNA and protein in wild-type C57BL6/j mice. RhEPO, STS-E412, and STS-E424 increase FXN expression in the heart of FXN-deficient KIKO mice. In contrast, FXN expression in the brains of KIKO mice increased following treatment with STS-E412 and STS-E424, but not following treatment with rhEPO. Unexpectedly, rhEPO-treated KIKO mice developed severe splenomegaly, while no splenomegaly was observed in STS-E412- or STS-E424-treated mice. RhEPO, STS-E412 and STS-E424 upregulate FXN expression in vitro at equal efficacy, however, the effects of the small molecules on FXN expression in the CNS are superior to rhEPO in vivo.

Frataxin deficiency impairs mitochondrial biogenesis in cells, mice and humans.

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by inherited deficiency of the mitochondrial protein Frataxin (FXN), which has no approved therapy and is an area in which biomarkers are needed for clinical development. Here, we investigated the consequences of FXN deficiency in patient-derived FRDA fibroblast cell models, the FRDA mouse model KIKO, and in whole blood collected from patients with FRDA. We observed decreased mitochondrial copy number in all the three FRDA models tested: cells, mice and patient blood. In addition, we observed 40% residual mitochondrial gene expression in FRDA patient blood. These deficiencies of mitochondrial biogenesis in FRDA cells and patient blood are significantly correlated with FXN expression, consistent with the idea that the decreased mitochondrial biogenesis is a consequence of FXN deficiency. The observations appear relevant to the FRDA pathophysiological mechanism, as FXN-dependent deficiency in mitochondrial biogenesis and consequent mitochondrial bioenergetic defect could contribute to the neurodegenerative process. The observations may also have translational potential, as mitochondrial biogenesis could now be followed as a clinical biomarker of FRDA as a correlate of disease severity, progression, and therapeutic effect. Also, mitochondrial copy number in blood is objective, scalar and more investigator-independent than clinical-neurological patient rating scales. Thus, FXN deficiency causes mitochondrial deficiency in FRDA cells, the KIKO mouse model, and in whole blood of patients with FRDA, and this deficiency could potentially be used in clinical trial design.

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis.We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An “IronChip” microarray analysis followed by a protein-protein interaction analysis was performed.FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis.The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported.

Cytokine therapy-mediated neuroprotection in a Friedreich's ataxia mouse model.

ObjectivesFriedreich's ataxia is a devastating neurological disease currently lacking any proven treatment. We studied the neuroprotective effects of the cytokines, granulocyte‐colony stimulating factor (G‐CSF) and stem cell factor (SCF) in a humanized murine model of Friedreich's ataxia.MethodsMice received monthly subcutaneous infusions of cytokines while also being assessed at monthly time points using an extensive range of behavioral motor performance tests. After 6 months of treatment, neurophysiological evaluation of both sensory and motor nerve conduction was performed. Subsequently, mice were sacrificed for messenger RNA, protein, and histological analysis of the dorsal root ganglia, spinal cord, and cerebellum.ResultsCytokine administration resulted in significant reversal of biochemical, neuropathological, neurophysiological, and behavioural deficits associated with Friedreich's ataxia. Both G‐CSF and SCF had pronounced effects on frataxin levels (the primary molecular defect in the pathogenesis of the disease) and a regulators of frataxin expression. Sustained improvements in motor coordination and locomotor activity were observed, even after onset of neurological symptoms. Treatment also restored the duration of sensory nerve compound potentials. Improvements in peripheral nerve conduction positively correlated with cytokine‐induced increases in frataxin expression, providing a link between increases in frataxin and neurophysiological function. Abrogation of disease‐related pathology was also evident, with reductions in inflammation/gliosis and increased neural stem cell numbers in areas of tissue injury.InterpretationThese experiments show that cytokines already clinically used in other conditions offer the prospect of a novel, rapidly translatable, disease‐modifying, and neuroprotective treatment for Friedreich's ataxia. Ann Neurol 2017;81:212–226

E3 Ligase RNF126 Directly Ubiquitinates Frataxin, Promoting Its Degradation: Identification of a Potential Therapeutic Target for Friedreich Ataxia

SummaryFriedreich ataxia (FRDA) is a severe genetic neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. To date, there is no therapy to treat this condition. The amount of residual frataxin critically affects the severity of the disease; thus, attempts to restore physiological frataxin levels are considered therapeutically relevant. Frataxin levels are controlled by the ubiquitin-proteasome system; therefore, inhibition of the frataxin E3 ligase may represent a strategy to achieve an increase in frataxin levels. Here, we report the identification of the RING E3 ligase RNF126 as the enzyme that specifically mediates frataxin ubiquitination and targets it for degradation. RNF126 interacts with frataxin and promotes its ubiquitination in a catalytic activity-dependent manner, both in vivo and in vitro. Most importantly, RNF126 depletion results in frataxin accumulation in cells derived from FRDA patients, highlighting the relevance of RNF126 as a new therapeutic target for Friedreich ataxia.

Liver Growth Factor (LGF) Upregulates Frataxin Protein Expression and Reduces Oxidative Stress in Friedreich's Ataxia Transgenic Mice.

Friedreich’s ataxia (FA) is a severe disorder with autosomal recessive inheritance that is caused by the abnormal expansion of GAA repeat in intron 1 of FRDA gen. This alteration leads to a partial silencing of frataxin transcription, causing a multisystem disorder disease that includes neurological and non-neurological damage. Recent studies have proven the effectiveness of neurotrophic factors in a number of neurodegenerative diseases. Therefore, we intend to determine if liver growth factor (LGF), which has a demonstrated antioxidant and neuroprotective capability, could be a useful therapy for FA. To investigate the potential therapeutic activity of LGF we used transgenic mice of the FXNtm1MknTg (FXN)YG8Pook strain. In these mice, intraperitoneal administration of LGF (1.6 μg/mouse) exerted a neuroprotective effect on neurons of the lumbar spinal cord and improved cardiac hypertrophy. Both events could be the consequence of the increment in frataxin expression induced by LGF in spinal cord (1.34-fold) and heart (1.2-fold). LGF also upregulated by 2.6-fold mitochondrial chain complex IV expression in spinal cord, while in skeletal muscle it reduced the relation oxidized glutathione/reduced glutathione. Since LGF partially restores motor coordination, we propose LGF as a novel factor that may be useful in the treatment of FA.

Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)n Repeats by PNA or LNA Targeting.

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.

Alleviating GAA Repeat Induced Transcriptional Silencing of the Friedreich's Ataxia Gene During Somatic Cell Reprogramming.

Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene, which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs, the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming, modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds, sodium butyrate (NaB) and Parnate, led to an increase in FXN expression and correction of repressive marks at the FXN locus, which persisted for several passages. However, prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore, we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together, these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications, thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA.