Therapeutic Prospects for Friedreich’s Ataxia Zhang, Siyuan; Napierala, Marek; Napierala, Jill S.
Trends in pharmacological sciences (Regular ed.),
April 2019, 2019-04-00, 20190401, Letnik:
40, Številka:
4
Journal Article
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Friedreich’s ataxia (FRDA) is a progressive disease affecting multiple organs that is caused by systemic insufficiency of the mitochondrial protein frataxin. Current therapeutic strategies aim to ...elevate frataxin levels and/or alleviate the consequences of frataxin deficiency. Recent significant advances in the FRDA therapeutic pipeline are bringing patients closer to a cure.
Friedreich's ataxia (FRDA) is a genetic neurodegenerative disorder caused by transcriptional silencing of the frataxin gene (FXN) due to expansions of GAA repeats in intron 1. FRDA manifests with ...multiple symptoms, which may include ataxia, cardiomyopathy and diabetes mellitus. Expanded GAA tracts are genetically unstable, exhibiting both expansions and contractions. GAA length correlates with severity of FRDA symptoms and inversely with age of onset. Thus, tissue-specific somatic instability of long GAA repeats may be implicated in the development of symptoms and disease progression. Herein, we determined the extent of somatic instability of the GAA repeats in heart, cerebral cortex, spinal cord, cerebellar cortex, and pancreatic tissues from 15 FRDA patients. Results demonstrate differences in the lengths of the expanded GAAs among different tissues, with significantly longer GAA tracts detected in heart and pancreas than in other tissues. The expansion bias detected in heart and pancreas may contribute to disease onset and progression, making the mechanism of somatic instability an important target for therapy. Additionally, we detected significant differences in GAA tract lengths between lymphocytes and fibroblast pairs derived from 16 FRDA patients, with longer GAA tracts present in the lymphocytes. This result urges caution in direct comparisons of data obtained in these frequently used FRDA models. Furthermore, we conducted a longitudinal analysis of the GAA repeat length in lymphocytes collected over a span of 7-9 years and demonstrated progressive expansions of the GAAs with maximum gain of approximately 9 repeats per year. Continuous GAA expansions throughout the patient's lifespan, as observed in FRDA lymphocytes, should be considered in clinical trial designs and data interpretation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Friedreich’s ataxia (FRDA) is a rare neurodegenerative disease caused by an expansion of a GAA repeat sequence within the Frataxin (FXN) gene. Prominent regions of neurodegeneration include sensory ...neurons within the dorsal root ganglia. Here we present a set of genetically modified FRDA induced pluripotent stem cell (iPSC) lines that carry an inducible neurogenin-2 (NGN2) expression cassette. Exogenous expression of NGN2 in iPSC derived neural crest progenitors efficiently generates functionally mature sensory neurons. These cell lines will provide a streamlined source of FRDA iPSC sensory neurons for studying both disease mechanism and screening potential therapeutics.
Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to ...decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.
Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin (FXN). Most FRDA patients are homozygous for large expansions of GAA ...repeats in intron 1 of FXN, while some are compound heterozygotes with an expanded GAA tract in one allele and a missense or nonsense mutation in the other. A missense mutation, changing a glycine to valine at position 130 (G130V), is prevalent among the clinical variants. We and others have demonstrated that levels of mature FXN protein in FRDA G130V samples are reduced below those detected in samples harboring homozygous repeat expansions. Little is known regarding expression and function of endogenous FXN-G130V protein due to lack of reagents and models that can distinguish the mutant FXN protein from the wild-type FXN produced from the GAA-expanded allele. We aimed to determine the effect of the G130V (murine G127V) mutation on Fxn expression and to define its multi-system impact in vivo. We used CRISPR/Cas9 to introduce the G127V missense mutation in the Fxn coding sequence and generated homozygous mice (FxnG127V/G127V). We also introduced the G127V mutation into a GAA repeat expansion FRDA mouse model (FxnGAA230/KO; KIKO) to generate a compound heterozygous strain (FxnG127V/GAA230). We performed neurobehavioral tests on cohorts of WT and Fxn mutant animals at three-month intervals for one year, and collected tissue samples to analyze molecular changes during that time. The endogenous Fxn G127V protein is detected at much lower levels in all tissues analyzed from FxnG127V/G127V mice compared to age and sex-matched WT mice without differences in Fxn transcript levels. FxnG127V/G127V mice are significantly smaller than WT counterparts, but perform similarly in most neurobehavioral tasks. RNA sequencing analysis revealed reduced expression of genes in oxidative phosphorylation and protein synthesis, underscoring the metabolic consequences in our mouse model expressing extremely low levels of Fxn. Results of these studies provide insight into the unique pathogenic mechanism of the FXN G130V mechanism and the tolerable limit of Fxn/FXN expression in vivo.
•Extremely low Fxn levels are detected in CNS and heart tissues of Fxn G127V mice.•Fxn G127V mice are small and develop a hunched posture.•Endurance and activity are reduced in adult Fxn G127V mice.•Motor coordination is not affected in adult Fxn G127V mice.
Friedreich's ataxia (FA) is a neurodegenerative disease with no approved therapy that is the result of frataxin deficiency. The identification of human FA blood biomarkers related to disease severity ...and neuro-pathomechanism could support clinical trials of drug efficacy. To try to identify human biomarkers of neuro-pathomechanistic relevance, we compared the overlapping gene expression changes of primary blood and skin cells of FA patients with changes in the Dorsal Root Ganglion (DRG) of the KIKO FA mouse model. As DRG is the primary site of neurodegeneration in FA, our goal was to identify which changes in blood and skin of FA patients provide a 'window' into the FA neuropathomechanism inside the nervous system. In addition, gene expression in frataxin-deficient neuroglial cells and FA mouse hearts were compared for a total of 5 data sets. The overlap of these changes strongly supports mitochondrial changes, apoptosis and alterations of selenium metabolism. Consistent biomarkers were observed, including three genes of mitochondrial stress (MTIF2, ENO2), apoptosis (DDIT3/CHOP), oxidative stress (PREX1), and selenometabolism (SEPW1). These results prompted our investigation of the GPX1 activity as a marker of selenium and oxidative stress, in which we observed a significant change in FA patients. We believe these lead biomarkers that could be assayed in FA patient blood as indicators of disease severity and progression, and also support the involvement of mitochondria, apoptosis and selenium in the neurodegenerative process.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Friedreich's ataxia is caused by large homozygous, intronic expansions of GAA repeats in the frataxin (FXN) gene, resulting in severe downregulation of its expression. Pathogenic repeats are located ...in intron one, hence patients express unaffected FXN protein, albeit in low quantities. Although FRDA symptoms typically afflict the nervous system, hypertrophic cardiomyopathy is the predominant cause of death. Our studies were conducted using cardiomyocytes differentiated from induced pluripotent stem cells derived from control individuals, FRDA patients, and isogenic cells corrected by zinc finger nucleases-mediated excision of pathogenic expanded GAA repeats. This correction of the FXN gene removed the primary trigger of the transcription defect, upregulated frataxin expression, reduced pathological lipid accumulation observed in patient cardiomyocytes, and reversed gene expression signatures of FRDA cardiomyocytes. Transcriptome analyses revealed hypertrophy-specific expression signatures unique to FRDA cardiomyocytes, and emphasized similarities between unaffected and ZFN-corrected FRDA cardiomyocytes. Thus, the iPSC-derived FRDA cardiomyocytes exhibit various molecular defects characteristic for cellular models of cardiomyopathy that can be corrected by genome editing of the expanded GAA repeats. These results underscore the utility of genome editing in generating isogenic cellular models of FRDA and the potential of this approach as a future therapy for this disease.
•Decreased frataxin expression in FRDA iPSC derived cardiomyocytes•Lipid droplet accumulation in FRDA cardiomyocytets•Cardiac hypertrophy expression signature FRDA cardiomyocytets•Frataxin deficiency alleviated by excision of expanded GAA repeats.•Reduced lipid droplet accumulation and reversal of gene expression changes upon correction of frataxin gene.
Abstract
Friedreich’s ataxia (FRDA) is a severe multisystem disease caused by transcriptional repression induced by expanded GAA repeats located in intron 1 of the Frataxin (FXN) gene encoding ...frataxin. FRDA results from decreased levels of frataxin; thus, stabilization of the FXN mRNA already present in patient cells represents an attractive and unexplored therapeutic avenue. In this work, we pursued a novel approach based on oligonucleotide-mediated targeting of FXN mRNA ends to extend its half-life and availability as a template for translation. We demonstrated that oligonucleotides designed to bind to FXN 5′ or 3′ noncoding regions can increase FXN mRNA and protein levels. Simultaneous delivery of oligonucleotides targeting both ends increases efficacy of the treatment. The approach was confirmed in several FRDA fibroblast and induced pluripotent stem cell-derived neuronal progenitor lines. RNA sequencing and single-cell expression analyses confirmed oligonucleotide-mediated FXN mRNA upregulation. Mechanistically, a significant elongation of the FXN mRNA half-life without any changes in chromatin status at the FXN gene was observed upon treatment with end-targeting oligonucleotides, indicating that transcript stabilization is responsible for frataxin upregulation. These results identify a novel approach toward upregulation of steady-state mRNA levels via oligonucleotide-mediated end targeting that may be of significance to any condition resulting from transcription downregulation.
Frataxin deficiency in Friedreich's ataxia results from transcriptional downregulation of the FXN gene caused by expansion of the intronic trinucleotide guanine-adenine-adenine (GAA) repeats. We used ...multiple transcriptomic approaches to determine the molecular mechanism of transcription inhibition caused by long GAAs. We uncovered that transcription of FXN in patient cells is prematurely terminated upstream of the expanded repeats leading to the formation of a novel, truncated and stable RNA. This FXN early terminated transcript (FXN-ett) undergoes alternative, non-productive splicing and does not contribute to the synthesis of functional frataxin. The level the FXN-ett RNA directly correlates with the length of the longer of the two expanded GAA tracts. Targeting GAAs with antisense oligonucleotides or excision of the repeats eliminates the transcription impediment, diminishes expression of the aberrant FXN-ett, while increasing levels of FXN mRNA and frataxin. Non-productive transcription may represent a common phenomenon and attractive therapeutic target in diseases caused by repeat-mediated transcription aberrations.
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