Frataxin Research Articles

Retinal hypoplasia and degeneration result in vision loss in Friedreich ataxia.

Friedreich ataxia (FRDA) is an inherited condition caused by a GAA triplet repeat (GAA-TR) expansion in the FXN gene. Clinical features of FRDA include ataxia, cardiomyopathy, and in some, vision loss. In this study, we characterize features of vision loss in a large cohort of adults and children with FRDA. Using optical coherence tomography (OCT), we measured peripapillary retinal nerve fiber layer (RNFL) thickness in 198 people with FRDA, and 77 controls. Sloan letter charts were used to determine visual acuity. RNFL thickness and visual acuity were compared to measures of disease severity obtained from the Friedreich Ataxia Clinical Outcomes Measures Study (FACOMS). The majority of patients, including children, had pathologically thin RNFLs (mean = 73 ± 13 μm in FRDA; 98 ± 9 μm in controls) and low-contrast vision deficits early in the disease course. Variability in RNFL thickness in FRDA (range: 36 to 107 μm) was best predicted by disease burden (GAA-TR length X disease duration). Significant deficits in high-contrast visual acuity were apparent in patients with an RNFL thickness of ≤68 μm. RNFL thickness decreased at a rate of -1.2 ± 1.4 μm/year and reached 68 μm at a disease burden of approximately 12,000 GAA years, equivalent to disease duration of 17 years for participants with 700 GAAs. These data suggest that both hypoplasia and subsequent degeneration of the RNFL may be responsible for the optic nerve dysfunction in FRDA and support the development of a vision-directed treatment for selected patients early in the disease to prevent RNFL loss from reaching the critical threshold.

Large-scale expansions of Friedreich's ataxia GAA•TTC repeats in an experimental human system: role of DNA replication and prevention by LNA-DNA oligonucleotides and PNA oligomers.

Friedreich's ataxia (FRDA) is caused by expansions of GAA•TTC repeats in the first intron of the human FXN gene that occurduring both intergenerational transmissions and in somatic cells. Here we describe an experimental system to analyze large-scale repeat expansions in cultured human cells. It employs a shuttle plasmid that can replicate from the SV40 origin in human cells or be stably maintained in S. cerevisiae utilizing ARS4-CEN6. It also contains a selectable cassette allowing us to detect repeat expansions that accumulated in human cells upon plasmid transformation into yeast. We indeed observed massive expansions of GAA•TTC repeats, making it the first genetically tractable experimental system to study large-scale repeat expansions in human cells. Further, GAA•TTC repeats stall replication fork progression, while the frequency of repeat expansions appears to depend on proteins implicated in replication fork stalling, reversal, and restart. Locked nucleic acid (LNA)-DNA mixmer oligonucleotides and peptide nucleic acid (PNA) oligomers, which interfere with triplex formation at GAA•TTC repeats in vitro, prevented the expansion of these repeats in human cells. We hypothesize, therefore, that triplex formation by GAA•TTC repeats stall replication fork progression, ultimately leading to repeat expansions during replication fork restart.

Frataxin deficiency disrupts mitochondrial respiration and pulmonary endothelial cell function

Deficiency of iron‑sulfur (FeS) clusters promotes metabolic rewiring of the endothelium and the development of pulmonary hypertension (PH) in vivo. Joining a growing number of FeS biogenesis proteins critical to pulmonary endothelial function, recent data highlighted that frataxin (FXN) reduction drives Fe-S-dependent genotoxic stress and senescence across multiple types of pulmonary vascular disease. Trinucleotide repeat mutations in the FXN gene cause Friedreich's ataxia, a disease characterized by cardiomyopathy and neurodegeneration. These tissue-specific phenotypes have historically been attributed to mitochondrial reprogramming and oxidative stress. Whether FXN coordinates both nuclear and mitochondrial processes in the endothelium is unknown. Here, we aim to identify the mitochondria-specific effects of FXN deficiency in the endothelium that predispose to pulmonary hypertension. Our data highlight an Fe-S-driven metabolic shift separate from previously described replication stress whereby FXN knockdown diminished mitochondrial respiration and increased glycolysis and oxidative species production. In turn, FXN-deficient endothelial cells had increased vasoconstrictor production (EDN1) and decreased nitric oxide synthase expression (NOS3). These data were observed in primary pulmonary endothelial cells after pharmacologic inhibition of FXN, mice carrying a genetic endothelial deletion of FXN, and inducible pluripotent stem cell-derived endothelial cells from patients with FXN mutations. Altogether, this study indicates FXN is an upstream driver of pathologic aberrations in metabolism and genomic stability. Moreover, our study highlights FXN-specific vasoconstriction in vivo, prompting future studies to investigate available and novel PH therapies in contexts of FXN deficiency.

Omaveloxolone (SkyclarysTM) for patients with Friedreich’s ataxia

STRUCTURE: Omaveloxolone {N-[(4aS,6aR,6bS,8aR,12aS,14aR,14bS)-11-cyano-2,2,6a,6b, 9,9,12a-heptamethyl-10,14-dioxo-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,12a,14,14a,14b-octa-decahydropicen-4a-yl]-2,2-difluoropropanamide} is a semisynthetic triterpenoid based on the natural product oleanolic acid. It contains a highly reactive cyanoenone functionality, which binds covalently and reversibly by Michael addition to cysteines in proteins, such as the cysteine-based sensor protein Kelch-like ECH-associated protein 1 (KEAP1). The molecular formula of omaveloxolone is C33H44F2N2O3 and its molecular weight is 554.7 g/mol. MECHANISM OF ACTION: The pathophysiology of Friedreich’s ataxia is associated with an expansion of GAA repeats in the first intron of FXN encoding the small mitochondrial protein frataxin (FXN), which has a role in mitochondrial homeostasis and iron metabolism. FXN deficiency leads to impaired mitochondrial function and increased production of reactive oxygen species (ROS) (1), in turn causing inflammation with further ROS production, creating a vicious cycle that ultimately results in cellular dysfunction (2). Omaveloxolone is an inducer of a network of endogenous cytoprotective proteins regulated by transcription factor nuclear factor-erythroid 2 p45-related factor 2 (NRF2), the master regulator of cellular redox homeostasis. At basal state, the levels of NRF2 are low due to its continuous proteasomal degradation (3) mediated by KEAP1, a substrate adapter of a Cullin-RING E3 ubiquitin ligase. Omaveloxolone (pink circle), via its cyanoenone functionality, binds to sensor cysteines (primarily C151) in KEAP1 and inactivates it (4). As a result, the newly synthesized NRF2 accumulates, forms a heterodimer with a small musculoaponeurotic fibrosarcoma (sMAF) protein, and induces transcription of its target genes by binding to the antioxidant response element (ARE) sequences in their regulatory regions. The NRF2-transcriptional network encompasses an array of broadly cytoprotective proteins (5), including those responsible for the biosynthesis of glutathione (GSH). Collectively, the NRF2 transcriptional targets counteract oxidative and inflammatory stress, and support proteostasis, mitochondrial function, and bioenergetics. Omaveloxolone also inhibits inflammation, in part via NRF2 (which inhibits transcription of proinflammatory genes) and, in part, due to its potential to bind to cysteines in proteins involved in inflammatory cascades (e.g., IKKβ, shown in beige). Omaveloxolone (also known as RTA-408); brand name is SKYCLARYS. Omaveloxolone is the first and only FDA-approved drug for patients with Friedreich’s ataxia. Omaveloxolone has received Orphan Drug, Fast Track, and Rare Pediatric Disease Designations from the FDA, and Orphan Drug Designation for the treatment of Friedreich’s ataxia from the European Commission. It is currently not approved outside of the USA. SKYCLARYS™ is approved in the USA by the FDA and is indicated for the treatment of Friedreich’s ataxia in adults and adolescents aged 16 years and older. The recommended dosage is 150 mg to be taken orally daily. Reata Pharmaceuticals. Headache (37%), nausea (33%), diarrhea (20%), abdominal pain (29%), fatigue (24%), musculoskeletal pain (20%), vomiting (16%), oropharyngeal pain (18%), influenza (16%), muscle spasms (14%), back pain (13%), decreased appetite (12%), rash (10%). The most common laboratory abnormalities (occurring in 37% of patients) are elevated aspartate/alanine transaminases. In clinical trials, most effects diminished or stopped after 12 weeks of treatment. 2014–2020: Phase 1 trials (NCT02029716, NCT03664453, NCT04008186) 2014–present: Phase 2 trials (NCT02255435, NCT02029729, NCT02142959, NCT02128113, NCT03902002, NCT02255422) February 2023: FDA approval for SKYCLARYS™ (omaveloxolone). We thank the Medical Research Council (MR/W023806/1 and MR/T014644/1), the Biotechnology and Biological Sciences Research Council and GlaxoSmithKline (BB/T508111/1, BB/X00029X/1, and BB/T017546/1), Tenovus Scotland (T19/30 and T22/08), Reata Pharmaceuticals, and Medical Research Scotland (PHD-50477-2021) for supporting our research and the COST Action CA20121, supported by the European Cooperation in Science and Technology (www.cost.eu) (https://benbedphar.org/about-benbedphar/). A.T. D.-K. is a member of the Scientific Advisory Board of Evgen Pharma and collaborates with GlaxoSmithKline and Reata Pharmaceuticals. S.D.N. has no interests to declare.

PO-01-126 SEXUAL DIMORPHISM AND CARDIAC ARRHYTHMIAS IN A MOUSE MODEL OF FRIEDREICH'S ATAXIA: WHAT DO WE KNOW?

Friedreich ataxia (FA) is a monogenic recessive disorder caused by the reduction of the mitochondrial protein, frataxin (FXN). Despite neurological deficits place patients with FA in the wheelchairs, most patients die from lethal cardiomyopathy in their 40s. FA patients develop progressive cardiac disease with characteristics similar to primary hypertrophic cardiomyopathy but with more progressive cardiac fibrosis, both diastolic and systolic dysfunction, and arrhythmias. However, the mechanisms leading to cardiomyopathy and arrhythmias in FA patients are poorly understood. To characterize electrical signal propagation and cardiac function in a mouse model which imitates the late-stage cardiomyopathy in FA patients. Cardiac-specific McK-Cre FXN knockout (FXN cKO) mice were used as a model of severe cardiomyopathy which is characteristic for the late-stage of FA. Due to premature death, animals of either sex at 8-weeks of age were used to assess cardiac function in-vivo by echocardiography and surface electrocardiogram (ECG). We found that FXN cKO mice developed idiosyncratic arrhythmias with varying time durations for beat-to-beat (RR) intervals and heart rate variability (HRV) parameters such as root mean squared successive differences (RMSSD, p<0.0358), revealing increased vagal activity. Mice presented extended P-wave (p<0.0113) and QTc intervals (>150 ms, p<0.0015), suggesting impaired atrial and left-ventricular function, resembling human patients. HRV frequency parameters showed increased low-frequency (0.023), high-frequency (p<0.264) and total (p<0.0326) power. Non-linear parameters showed varying short-term variation (SD1, p<0.019), increased long-term variation (SD2, p<0.0017) and ellipse area values (S, p<0.0074). Poincare plots of FXN cKO males included multiple clustering of RR and RR+1 identities while females generally sustained a single clustered ellipse (area, p<0.028). Similarly, QTc intervals were increased mostly in FXN cKO males. Contractile dysfunction was also more severe in FXN cKO males as evident by reduction in left ventricular fractional shortening, ejection fraction, stroke volume and cardiac output. Our study revealed sexual dimorphism and significant impairment in electrical and contractile function in FXN cKO males compared to females. A detailed characterization of impaired electrical signals in FA hearts will build a platform for drug testing to treat lethal cardiomyopathy in FA.

Apparent Opportunities and Hidden Pitfalls: The Conflicting Results of Restoring NRF2-Regulated Redox Metabolism in Friedreich's Ataxia Pre-Clinical Models and Clinical Trials.

Friedreich's ataxia (FRDA) is an autosomal, recessive, inherited neurodegenerative disease caused by the loss of activity of the mitochondrial protein frataxin (FXN), which primarily affects dorsal root ganglia, cerebellum, and spinal cord neurons. The genetic defect consists of the trinucleotide GAA expansion in the first intron of FXN gene, which impedes its transcription. The resulting FXN deficiency perturbs iron homeostasis and metabolism, determining mitochondrial dysfunctions and leading to reduced ATP production, increased reactive oxygen species (ROS) formation, and lipid peroxidation. These alterations are exacerbated by the defective functionality of the nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor acting as a key mediator of the cellular redox signalling and antioxidant response. Because oxidative stress represents a major pathophysiological contributor to FRDA onset and progression, a great effort has been dedicated to the attempt to restore the NRF2 signalling axis. Despite this, the beneficial effects of antioxidant therapies in clinical trials only partly reflect the promising results obtained in preclinical studies conducted in cell cultures and animal models. For these reasons, in this critical review, we overview the outcomes obtained with the administration of various antioxidant compounds and critically analyse the aspects that may have contributed to the conflicting results of preclinical and clinical studies.

Dimorphic frataxin and its gene regulation by sex steroids in hamsters

The role of frataxin (FXN) has been studied extensively in Friedreich ataxia patients, however, the molecular bases underlining the sex steroid-dependent gene expression profiles of FXN in adult tissues are unknown. I describe the molecular characterization of hamster FXN by examining the sexually dimorphic expression and its regulation by sex steroids. Sequence analysis of FXN cDNA showed 630bp-long ORF encoding 209 amino acids. qPCR analysis revealed that FXN is detected in a wide range of tissues, with the highest expression in the heart, liver, and epididymis, and the weakest expression in the lung, spleen, uterus, and gut. In the male Harderian gland (HG), castration decreased FXN expression, while dihydrotestosterone (DHT) administration reestablished levels. FXN expression levels were higher in the male HG than the female HG. Expression levels in endocrine tissues showed a certain degree of sexual dimorphism; the transcript in the testis was significantly higher than those in the ovary. The effects of the estrous cycle on FXN expression remained unchanged in the HG, ovary, and adrenal glands; however, in the pancreas, the FXN mRNA was overexpressed during proestrus and exhibited sexual dimorphism as compared to the male pancreas. The mRNA expression results indicated that Harderian FXN may play a dynamic role in intracellular Fe of heme required for processing cytochromes and other hemeproteins, also suggesting that the moderate sexual dimorphism present in the HG and gonads could be regulated by androgens, while sexually dimorphic expression of FXN in the female pancreas may be controlled by sex steroids.

A Novel Metric for Predicting Severity of Disease Features in Friedreich's Ataxia.

Friedreich's ataxia (FRDA), most commonly caused by a GAA triplet repeat (GAA-TR) expansion in intron 1 of the FXN gene, is characterized by deficiency of frataxin protein and clinical features such as progressive ataxia, dysarthria, impaired proprioception and vibration, abolished deep tendon reflexes, Babinski sign, and vision loss in association with non-neurological features such as skeletal anomalies, hearing loss, cardiomyopathy, and diabetes. Pathogenic GAA-TRs range in size from 60 to 1500 triplets and negatively correlate with age of onset. Clinical severity is predicted by a combination of GAA-TR length and disease duration (DD) via multivariable regressions, which cannot typically be used for the small sample sizes in most studies on this rare disease. We aimed to develop a single metric, which we call "disease burden" (DB), that encompasses both GAA-TR length and DD for predicting disease features of FRDA in small sample sizes. Linear regression and multivariable regression analysis was used to determine correlation coefficients between different disease features of FRDA. Using large datasets for validation, we found that DB predicts measures of neurological dysfunction in FRDA better than GAA-TR length or DD. Analogous results were found using small datasets. FRDA DB is a novel metric of disease severity that has utility in small datasets to demonstrate correlations that would not otherwise be evident with either GAA-TR or DD alone. This is important for discovering new biomarkers, as well as improving the prediction of severity of disease features in FRDA. © 2023 International Parkinson and Movement Disorder Society.

Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango.

Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe-4S]2+ prosthetic group which yields an inactive [3Fe-4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe-S clusters. A high yield of pure ACO2 (≥99%, 22±2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102M-1s-1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1±0.5 and 43.6±0.2°C for [4Fe-4S]2+ and [3Fe-4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe-S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe-4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe-4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.

Insights from yeast: Transcriptional reprogramming following metformin treatment is similar to that of deferiprone in a yeast Friedreich's ataxia model.

In the absence of YFH1, the yeast orthologof the human FXN gene, budding yeast Saccharomyces cerevisiae experience similar problems to those of cells with Friedreich's ataxia (FRDA). The comparable phenotypic traits consist of impaired respiration, problems in iron homeostasis, decreased oxidative stress tolerance, and diminished iron-sulfur cluster synthesis, rendering yeast of potential use in FRDA modeling and drug trials. Deferiprone, an iron chelator, is one of the long-term studied potential drugs for FRDA, whereas metformin is a biguanide prescribed to treat type 2 diabetes. In the present study, the effects of deferiprone and metformin treatment on the yeast FRDA model are explored via RNA-sequencing analyses. The comparative inquiry of transcriptome data reveals new promising roles for metformin in FRDA treatment since deferiprone and metformin treatments produce overlapping transcriptional and phenotypic responses in YFH1Δ cells. The results revealed that both deferiprone and metformin treatment does not rescue aerobic respiration in YFH1Δ cells, but they alleviate the FRDA phenotype probably by triggering the retrograde mitochondria-to-nucleus signaling.

Liquid Chromatography-Mass Spectrometry Analysis of Frataxin Proteoforms in Whole Blood as Biomarkers of the Genetic Disease Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is caused primarily by expanded GAA repeats in intron 1 of both alleles of the FXN gene, which causes transcriptional silencing and reduced expression of frataxin mRNA and protein. FRDA is characterized by slowly progressive ataxia and cardiomyopathy. Symptoms generally appear during adolescence, and patients slowly progress to wheelchair dependency usually in the late teens or early twenties with death on average in the 4th decade. There are two known mature proteoforms of frataxin. Mitochondrial frataxin (frataxin-M) is a 130-amino acid protein with a molecular weight of 14,268 Da, and there is an alternatively spliced N-terminally acetylated 135-amino acid form (frataxin-E) with a molecular weight of 14,953 Da found in erythrocytes. There is reduced expression of frataxin in the heart and brain, but frataxin is not secreted into the systemic circulation, so it cannot be analyzed in serum or plasma. Blood is a readily accessible biofluid that contains numerous different cell types that express frataxin. We have found that pig blood can serve as an excellent surrogate matrix to validate an assay for frataxin proteoforms because pig frataxin is lost during the immunoprecipitation step used to isolate human frataxin. Frataxin-M is expressed in blood cells that contain mitochondria, whereas extra-mitochondrial frataxin-E is found in erythrocytes. This means that the analysis of frataxin in whole blood provides information on the concentration of both proteoforms without having to isolate the individual cell types. In the current study, we observed that the distributions of frataxin levels for a sample of 25 healthy controls and 50 FRDA patients were completely separated from each other, suggesting 100% specificity and 100% sensitivity for distinguishing healthy controls from FRDA cases, a very unusual finding for a biomarker assay. Additionally, frataxin levels were significantly correlated with the GAA repeat length and age of onset with higher correlations for extra-mitochondrial frataxin-E than those for mitochondrial frataxin-M. These findings auger well for using frataxin levels measured by the validated stable isotope dilution ultrahigh-performance liquid chromatography-multiple reaction monitoring/mass spectrometry assay to monitor therapeutic interventions and the natural history of FRDA. Our study also illustrates the utility of using whole blood for protein disease biomarker discovery and validation.

Removal of the GAA repeat in the heart of a Friedreich's ataxia mouse model using CjCas9.

Most Friedreich ataxia (FRDA) cases are caused by the elongation of the GAA repeat (GAAr) sequence in the first intron of the FXN gene, leading to a decrease of the frataxin protein expression. Deletion of this GAAr with CRISPR/Cas9 technology leads to an increase in frataxin expression in vitro. We are therefore aiming to develop FRDA treatment based on the deletion of GAAr with CRISPR/Cas9 technology using a single AAV expressing a small Cas9 (CjCas9) and two single guide RNAs (sgRNAs) targeting the FXN gene. This AAV was intraperitoneally administrated to YG8sR (250-300 GAAr) and to YG8-800 (800 GAAr) mice. DNA and RNA were extracted from different organs a month later. PCR amplification of part of intron 1 of the FXN gene detected some GAAr deletion in some cells in heart and liver of both mouse models, but the editing rate was not sufficient to cause an increase in frataxin mRNA in the heart. However, the correlation observed between the editing rate and the distribution of AAV suggests a possible therapy based on the removal of the GAAr with a better delivery tool of the CRISPR/Cas9 system.

Substitution to hydrophobic linker and formation of host–guest complex enhanced the effect of synthetic transcription factor made of pyrrole−imidazole polyamide

GAA repeat expansion in the first intron of the frataxin (FXN) gene represses the transcription of FXN, and that induces Friedreich’s ataxia (FRDA). Pyrrole−imidazole polyamides (PIPs) are the class of oligopeptide that targets double-stranded DNA with sequence selectivity. Previously, bromodomain inhibitors such as JQ1 conjugated with PIPs were reported to selectively increase transcription. Here, we report the synthesis of a compound that increases the transcription of FXN in cells derived from an FRDA patient. The compound was effective in lower (one tenth) concentration than the compound that previously reported. High concentration of the compound is toxic, but toxicity was reduced with a host–guest complex.

Bioactive fumarate improves cardiac function and expands lifespan in Friedrech's ataxia.

Friedreich's ataxia (FA) is a recessive ataxia caused by reduction of mitochondrial protein, frataxin (FXN). Cardiomyopathy is the leading cause of death in FA patients due to deficient FXN expression in the heart. We have developed a novel monomethyl fumarate prodrug, IMF, which has improved pharmacokinetic profile and compared its effect to fumarate prodrug dimethyl fumarate (DMF, Tecfidera).

A new FRDA mouse model [Fxn null:YG8s(GAA) > 800] with more than 800 GAA repeats.

Friedreich's ataxia (FRDA) is an inherited recessive neurodegenerative disorder caused by a homozygous guanine-adenine-adenine (GAA) repeat expansion within intron 1 of the FXN gene, which encodes the essential mitochondrial protein frataxin. There is still no effective therapy for FRDA, therefore the development of optimal cell and animal models of the disease is one of the priorities for preclinical therapeutic testing. We obtained the latest FRDA humanized mouse model that was generated on the basis of our previous YG8sR, by Jackson laboratory [YG8JR, Fxn null:YG8s(GAA) > 800]. We characterized the behavioral, cellular, molecular and epigenetics properties of the YG8JR model, which has the largest GAA repeat sizes compared to all the current FRDA mouse models. We found statistically significant behavioral deficits, together with reduced levels of frataxin mRNA and protein, and aconitase activity in YG8JR mice compared with control Y47JR mice. YG8JR mice exhibit intergenerational GAA repeat instability by the analysis of parent and offspring tissue samples. Somatic GAA repeat instability was also detected in individual brain and cerebellum tissue samples. In addition, increased DNA methylation of CpG U13 was identified in FXN GAA repeat region in the brain, cerebellum, and heart tissues. Furthermore, we show decreased histone H3K9 acetylation and increased H3K9 methylation of YG8JR cerebellum tissues within the FXN gene, upstream and downstream of the GAA repeat region compared to Y47JR controls. These studies provide a detailed characterization of the GAA repeat expansion-based YG8JR transgenic mouse models that will help investigations of FRDA disease mechanisms and therapy.

Small-molecule inhibitors of proteasome increase CjCas9 protein stability.

The small size of CjCas9 can make easier its vectorization for in vivo gene therapy. However, compared to the SpCas9, the CjCas9 is, in general, less efficient to generate indels in target genes. The factors that affect its efficacity are not yet determined. We observed that the CjCas9 protein expressed in HEK293T cells after transfection of this transgene under a CMV promoter was much lower than the SpCas9 protein in the same conditions. We thus evaluated the effect of proteasome inhibitors on CjCas9 protein stability and its efficiency on FXN gene editing. Western blotting showed that the addition of MG132 or bortezomib, significantly increased CjCas9 protein levels in HEK293T and HeLa cells. Moreover, bortezomib increased the level of CjCas9 protein expressed under promoters weaker than CMV such as CBH or EFS but which are specific for certain tissues. Finally, ddPCR quantification showed that bortezomib treatment enhanced CjCas9 efficiency to delete GAA repeat region of FXN gene in HEK293T cells. The improvement of CjCas9 protein stability would facilitate its used in CRISPR/Cas system.

Neurobehavioral deficits of mice expressing a low level of G127V mutant frataxin

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.

A wearable motion capture suit and machine learning predict disease progression in Friedreich’s ataxia

Friedreichʼs ataxia (FA) is caused by a variant of the Frataxin (FXN) gene, leading to its downregulation and progressively impaired cardiac and neurological function. Current gold-standard clinical scales use simplistic behavioral assessments, which require 18- to 24-month-long trials to determine if therapies are beneficial. Here we captured full-body movement kinematics from patients with wearable sensors, enabling us to define digital behavioral features based on the data from nine FA patients (six females and three males) and nine age- and sex-matched controls, who performed the 8-m walk (8-MW) test and 9-hole peg test (9 HPT). We used machine learning to combine these features to longitudinally predict the clinical scores of the FA patients, and compared these with two standard clinical assessments, Spinocerebellar Ataxia Functional Index (SCAFI) and Scale for the Assessment and Rating of Ataxia (SARA). The digital behavioral features enabled longitudinal predictions of personal SARA and SCAFI scores 9 months into the future and were 1.7 and 4 times more precise than longitudinal predictions using only SARA and SCAFI scores, respectively. Unlike the two clinical scales, the digital behavioral features accurately predicted FXN gene expression levels for each FA patient in a cross-sectional manner. Our work demonstrates how data-derived wearable biomarkers can track personal disease trajectories and indicates the potential of such biomarkers for substantially reducing the duration or size of clinical trials testing disease-modifying therapies and for enabling behavioral transcriptomics.

Proprioceptors-enriched neuronal cultures from induced pluripotent stem cells from Friedreich ataxia patients show altered transcriptomic and proteomic profiles, abnormal neurite extension, and impaired electrophysiological properties.

Friedreich ataxia is an autosomal recessive multisystem disorder with prominent neurological manifestations and cardiac involvement. The disease is caused by large GAA expansions in the first intron of the FXN gene, encoding the mitochondrial protein frataxin, resulting in downregulation of gene expression and reduced synthesis of frataxin. The selective loss of proprioceptive neurons is a hallmark of Friedreich ataxia, but the cause of the specific vulnerability of these cells is still unknown. We herein perform an in vitro characterization of human induced pluripotent stem cell-derived sensory neuronal cultures highly enriched for primary proprioceptive neurons. We employ neurons differentiated from healthy donors, Friedreich ataxia patients and Friedreich ataxia sibling isogenic control lines. The analysis of the transcriptomic and proteomic profile suggests an impairment of cytoskeleton organization at the growth cone, neurite extension and, at later stages of maturation, synaptic plasticity. Alterations in the spiking profile of tonic neurons are also observed at the electrophysiological analysis of mature neurons. Despite the reversal of the repressive epigenetic state at the FXN locus and the restoration of FXN expression, isogenic control neurons retain many features of Friedreich ataxia neurons. Our study suggests the existence of abnormalities affecting proprioceptors in Friedreich ataxia, particularly their ability to extend towards their targets and transmit proper synaptic signals. It also highlights the need for further investigations to better understand the mechanistic link between FXN silencing and proprioceptive degeneration in Friedreich ataxia.

Retinal and Visual Pathways Involvement in Carriers of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is a rare autosomal recessive neurodegenerative disorder due to the homozygous pathological expansion of guanine-adenine-adenine (GAA) triplet repeats in the first intron of the FXN gene, which encodes for the mitochondrial protein frataxin. In the visual system, the typical manifestations are ocular motility abnormality, optic neuropathy, and retinopathy. Despite the evidence of ophthalmological impairment in FRDA patients, there is a lack of information about the morpho-functional condition of the retina and of the optic pathways in healthy heterozygous carriers of Friedreich's ataxia (C-FRDA). Ten C-FRDA subjects (providing 20 eyes) and thirty-five Controls (providing 70 eyes) underwent a complete neurological and ophthalmological examination comprehensive of functional (full-field Electroretinogram (ffERG), multifocal Electroretinogram (mfERG), Visual Evoked Potential (VEP), and Pattern Reversal Electroretinogram (PERG)) and morphological assessments (Optical Coherence Tomography, OCT) of the retina, macula, retinal ganglion cells, and visual pathways. The groups' data were compared using a two-sample t-test. Pearson's test was used to investigate the morpho-functional correlations. Statistically significant differences (p &lt; 0.01) between C-FRDA and Control eyes for the values of the following parameters were found: ffERG b-wave amplitude, mfERG Response Amplitude Densities, PERG P50 implicit time and P50-N95 amplitude, VEP P100 implicit time, Retinal Nerve Fiber Layer (RNFL) Overall, and Nasal thickness. The values of the OCT macular volume were not statistically different (p &gt; 0.01) between the two Groups. Therefore, our data suggest that, in C-FRDA, a dysfunction of retinal elements without morphological macular impairment may occur. In addition, a morphological impairment of RNFL associated with an abnormal neural conduction along the visual pathways can be also detected.