CRISPR/Cas9 targeted deletions in frataxin rescue Friedreich’s ataxia pathology in a human stem cell-based peripheral sensory neural circuitry
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE148212 | GEO | 2020/07/27
REPOSITORIES: GEO
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Project description:The molecular mechanisms of reduced frataxin (FXN) expression in Friedreich's ataxia (FRDA) are linked to epigenetic modification of the FXN locus caused by the disease-associated GAA expansion. Here, we identify that SUV4-20 histone methyltransferases, specifically SUV4-20 H1, play an important role in the regulation of FXN expression and represent a novel therapeutic target. Using a human FXN-GAA-Luciferase repeat expansion genomic DNA reporter model of FRDA, we screened the Structural Genomics Consortium epigenetic probe collection. We found that pharmacological inhibition of the SUV4-20 methyltransferases by the tool compound A-196 increased the expression of FXN by ∼1.5-fold in the reporter cell line. In several FRDA cell lines and patient-derived primary peripheral blood mononuclear cells, A-196 increased FXN expression by up to 2-fold, an effect not seen in WT cells. SUV4-20 inhibition was accompanied by a reduction in H4K20me2 and H4K20me3 and an increase in H4K20me1, but only modest (1.4-7.8%) perturbation in genome-wide expression was observed. Finally, based on the structural activity relationship and crystal structure of A-196, novel small molecule A-196 analogs were synthesized and shown to give a 20-fold increase in potency for increasing FXN expression. Overall, our results suggest that histone methylation is important in the regulation of FXN expression and highlight SUV4-20 H1 as a potential novel therapeutic target for FRDA.
2020-10-08 | GSE145115 | GEO
Project description:Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the frataxin (FXN) gene is silenced due to the expansion of GAA·TTC repeats in intron 1 of the same gene, leading loss of the essential mitochondrial protein frataxin with several impairment in iron metabolism and respiration, primarily affects the sensory DRG neurons. A major limitation in the study of FRDA is the lack of robust animal and cellular models. To fill this gap, this study presents a novel 3D protocol to generate dorsal root ganglia-like organoids (DRGOs) from human iPSCs in which to model Friedreich's ataxia. DRGOs show a robust expression pattern of peripheral markers, are electro-physiologically active and out-perform peripheral sensory neurons generated with a traditional 2D protocol. Furthermore, when co-cultured with human stretch receptor intrafusal muscle fibers, DRGOs contact their peripheral target and form an in vitro muscle spindle. DRGOs generated from FRDA patient-derived iPSCs recapitulate several aspects of FRDA pathology including reduced expression of FXN and TCA-cycle components and mitochondrial fragmentation. We present also the recovery of this pathological signs, achieved by a new CRISPR-Cas9 mediated deletion of the GAA·TTC tract along with the majority of the affected intron. FRDA patient edited DRGOs with the long deletion boast normalized markers of oxidative stress, as well as recovered mitochondrial defect, particularly in the axonal compartment.
2020-07-27 | GSE133755 | GEO
Project description:The inherited neurodegenerative disease Friedreich’s ataxia (FRDA) is caused by hyperexpansion of GAA•TTC trinucleotide repeats within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA•TTC repeats causes heterochromatin-mediated silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts through retroviral transduction of transcription factors. FXN gene repression is maintained in the iPSCs, as are the mRNA and miRNA global expression signatures reflecting the human disease. GAA•TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme Msh2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in the iPSCs, occupies FXN intron 1, and shRNA silencing of Msh2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.
2010-07-02 | GSE22651 | GEO
Project description:Friedreich's Ataxia (FRDA), a rare, inherited, progressive degenerative disease, is caused by a defective expression of mitochondrial protein frataxin (FXN), due to homozygous hyper-expansion of GAA triplets in the gene which severely reduce its transcription. FXN is crucial for cell survival and its deficiency in humans critically affects viability of neurons, cardiomyocytes and pancreatic beta cells. Around two thirds of individuals with FRDA show typical manifestation of hypertrophic cardiomyopathy, that can progress in heart failure and death. Except for FXN, very few genes have been correlated with ataxia and cardiac disease in FRDA. To identify other genes involved in pathogenesis of FRDA, a microarray analysis on FRDA patient's lymphoblastoid cells stably reconstituted with FXN was performed. HS-1 associated protein X-1 (HAX-1), a family of proteins playing important roles in the protection of cardiomyocytes from apoptosis, is the highest up-regulated transcript (FC=+2, p<0.0006). HAX-1 is highly expressed in heart and HAX-1 heterozygous-deficient hearts exhibit increases in infarct size. The microarray result was further assessed with qRT-PCR and western blot analysis performed on HEK293 stably transfected with empty vector or FXN-vector and lymphoblasts from FRDA patients compared to clinically unaffected heterozygous parents, confirming a strong co-expression of HAX-1 and FXN both at mRNA and protein level. Moreover, analysis of FXN and HAX-1 in peripheral blood mononuclear cells (PBMCs) of FRDA patients (n=13), heterozygous parents (n=6) and non-correlated healthy controls (n=13) revealed that their expressions are correlated. The positive correlation is more significant in FRDA patients showing a cardiac disease (10/13). The positive correlation between frataxin and HAX-1 expression level suggests a potential link of HAX-1 expression to pro-survival or protective cellular pathways related to frataxin. Thus, HAX-1 may be considered a specific biomarker of cardiomyopathy in FRDA and may represent a prognostic implement in these patients. Moreover, the discovery of novel genetic "risk" or "protective" factors in cardiomyopathy is crucial to improve risk stratification strategies and to identify new therapeutic targets.
2019-12-31 | GSE108200 | GEO
Project description:Friedreich's ataxia (FRDA), the most common inherited ataxia in humans, is caused by recessive mutations that lead to a substantial reduction in the levels of frataxin (FXN), a mitochondrial iron binding protein. FRDA is a multi-system disease, involving multiple neurological, cardiac, and metabolic manifestations whose study is hindered by a paucity of animal models that faithfully recapitulate human disease features. We developed an inducible (doxycycline) mouse model of Fxn deficiency (FRDAkd) that enabled us to control the onset, progression and potential rescue of disease phenotypes by the modulation of Fxn levels using RNA interference. We found that systemic knockdown of Fxn in adult mice led to multiple features paralleling those observed in human patients, including electrophysiological, cellular, biochemical and structural phenotypes associated with cardiomyopathy, as well as dorsal root ganglion and retinal neuronal degeneration and reduced axonal size and myelin sheath thickness in the spinal cord. Fxn knockdown mice also exhibited other abnormalities similar to patients, including weight loss, reduced locomotor activity, ataxia, reduced muscular strength, and reduced survival, as well as genome-wide transcriptome changes. We performed microarray analysis of heart, cerebellum and dorsal root ganglia in FRDAkd mouse model of frataxin deficiency, and found several molecular pathway dysfunction via genome-wide transcriptome analyses. We also found that, upon restoration of near wild-type FXN levels, we observed significant recovery of function, pathology and associated transcriptomic changes, even after significant motor dysfunction was observed. The rapidity of Fxn expression due to doxycycline removal and its robust correction of various parameters, even when restored after the onset of motor dysfunction, makes this FRDAkd mouse model an appealing potential preclinical tool for testing various therapeutics for FRDA.
2017-12-19 | GSE98790 | GEO
Project description:Friedreich’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 have low FXN mRNA and frataxin protein levels when compared with heterozygous carriers or healthy controls. 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. 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. Transcriptome profiling of FRDA skin fibroblasts revealed significantly upregulated expression of genes encoding plasma membrane solute carrier proteins. Conversely, the expression of genes encoding accessory factors and enzymes involved in cytoplasmic and mitochondrial protein synthesis was consistently decreased in the FRDA cells. Finally, comparison of genes differentially expressed in FRDA fibroblasts to 3 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.
2017-09-27 | GSE104288 | GEO
Project description:Immunometabolism investigates the complex interplay between the immune system and cellular metabolism. This study highlights the effects of mitochondrial frataxin (FXN) depletion, which causes Friedreich's ataxia (FRDA), a neurodegenerative condition characterized by coordination and muscle control deficiencies. Using single-cell RNA sequencing, we identified specific cell groups in the cerebellum of a FRDA mouse model, emphasizing a notable inflammatory microglial response. These FXN-deficient microglia cells exhibited enhanced inflammatory reactions. Furthermore, our metabolomic analyses revealed increased glycolysis and itaconate production in these cells, possibly driving the inflammation. Remarkably, butyrate treatment counteracted these immunometabolic changes, triggered an antioxidant response via the itaconate-Nrf2-GSH pathways, and dampened inflammation. The study also pinpointed Hcar2 (GPR109A) as a potential agent for butyrate anti-inflammatory impact on microglia. Tests on FRDA mice highlighted the neuroprotective attributes of butyrate intake, bolstering neuromotor performance. In essence, our findings shed light on how cerebellar microglia activation contributes to FRDA and highlight butyrate potential to alleviate neuroinflammation, rectify metabolic imbalances, and boost neuromotor capabilities in FRDA and similar conditions.
2024-04-16 | GSE261655 | GEO
Project description:Immunometabolism investigates the complex interplay between the immune system and cellular metabolism. This study highlights the effects of mitochondrial frataxin (FXN) depletion, which causes Friedreich's ataxia (FRDA), a neurodegenerative condition characterized by coordination and muscle control deficiencies. Using single-cell RNA sequencing, we identified specific cell groups in the cerebellum of a FRDA mouse model, emphasizing a notable inflammatory microglial response. These FXN-deficient microglia cells exhibited enhanced inflammatory reactions. Furthermore, our metabolomic analyses revealed increased glycolysis and itaconate production in these cells, possibly driving the inflammation. Remarkably, butyrate treatment counteracted these immunometabolic changes, triggered an antioxidant response via the itaconate-Nrf2-GSH pathways, and dampened inflammation. The study also pinpointed Hcar2 (GPR109A) as a potential agent for butyrate anti-inflammatory impact on microglia. Tests on FRDA mice highlighted the neuroprotective attributes of butyrate intake, bolstering neuromotor performance. In essence, our findings shed light on how cerebellar microglia activation contributes to FRDA and highlight butyrate potential to alleviate neuroinflammation, rectify metabolic imbalances, and boost neuromotor capabilities in FRDA and similar conditions.
2024-04-16 | GSE261654 | GEO
Project description:Immunometabolism investigates the complex interplay between the immune system and cellular metabolism. This study highlights the effects of mitochondrial frataxin (FXN) depletion, which causes Friedreich's ataxia (FRDA), a neurodegenerative condition characterized by coordination and muscle control deficiencies. Using single-cell RNA sequencing, we identified specific cell groups in the cerebellum of a FRDA mouse model, emphasizing a notable inflammatory microglial response. These FXN-deficient microglia cells exhibited enhanced inflammatory reactions. Furthermore, our metabolomic analyses revealed increased glycolysis and itaconate production in these cells, possibly driving the inflammation. Remarkably, butyrate treatment counteracted these immunometabolic changes, triggered an antioxidant response via the itaconate-Nrf2-GSH pathways, and dampened inflammation. The study also pinpointed Hcar2 (GPR109A) as a potential agent for butyrate anti-inflammatory impact on microglia. Tests on FRDA mice highlighted the neuroprotective attributes of butyrate intake, bolstering neuromotor performance. In essence, our findings shed light on how cerebellar microglia activation contributes to FRDA and highlight butyrate potential to alleviate neuroinflammation, rectify metabolic imbalances, and boost neuromotor capabilities in FRDA and similar conditions.
2024-04-16 | GSE261653 | GEO
Project description:Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by a homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that a single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in the prevention of the neurologic and cardiac complications of FRDA in YG8R mice, and the rescue was mediated by FXN transfer from tissue engrafted HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation of this approach, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients’ CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions in the cells. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. To this end, we generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. FRDA neurons generated from these iPSCs displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. These results were validated by molecular and functional studies, and gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe structural damage to the ER in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage and function in FRDA. In addition, these results validate the efficacy profile of our FXN gene editing approach, in a disease relevant model improving mitochondrial and cellular, and supporting our approach as an effective strategy for therapeutic intervention for Friedreich’s ataxia.
2024-01-29 | GSE244886 | GEO