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1
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Dong YN, Mercado-Ayón E, Coulman J, Flatley L, Ngaba LV, Adeshina MW, Lynch DR. The Regulation of the Disease-Causing Gene FXN. Cells 2024; 13:1040. [PMID: 38920668 PMCID: PMC11202134 DOI: 10.3390/cells13121040] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2024] [Revised: 06/10/2024] [Accepted: 06/13/2024] [Indexed: 06/27/2024] Open
Abstract
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease caused in almost all patients by expanded guanine-adenine-adenine (GAA) trinucleotide repeats within intron 1 of the FXN gene. This results in a relative deficiency of frataxin, a small nucleus-encoded mitochondrial protein crucial for iron-sulfur cluster biogenesis. Currently, there is only one medication, omaveloxolone, available for FRDA patients, and it is limited to patients 16 years of age and older. This necessitates the development of new medications. Frataxin restoration is one of the main strategies in potential treatment options as it addresses the root cause of the disease. Comprehending the control of frataxin at the transcriptional, post-transcriptional, and post-translational stages could offer potential therapeutic approaches for addressing the illness. This review aims to provide a general overview of the regulation of frataxin and its implications for a possible therapeutic treatment of FRDA.
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Affiliation(s)
- Yi Na Dong
- Departments of Pediatrics and Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | | | - Jennifer Coulman
- Departments of Pediatrics and Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Liam Flatley
- The Wharton School, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Lucie Vanessa Ngaba
- Departments of Pediatrics and Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Miniat W. Adeshina
- Departments of Pediatrics and Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - David R. Lynch
- Departments of Pediatrics and Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA
- Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
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2
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Kioutchoukova IP, Foster DT, Thakkar RN, Foreman MA, Burgess BJ, Toms RM, Molina Valero EE, Lucke-Wold B. Neurologic orphan diseases: Emerging innovations and role for genetic treatments. World J Exp Med 2023; 13:59-74. [PMID: 37767543 PMCID: PMC10520757 DOI: 10.5493/wjem.v13.i4.59] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2023] [Revised: 07/16/2023] [Accepted: 08/11/2023] [Indexed: 09/15/2023] Open
Abstract
Orphan diseases are rare diseases that affect less than 200000 individuals within the United States. Most orphan diseases are of neurologic and genetic origin. With the current advances in technology, more funding has been devoted to developing therapeutic agents for patients with these conditions. In our review, we highlight emerging options for patients with neurologic orphan diseases, specifically including diseases resulting in muscular deterioration, epilepsy, seizures, neurodegenerative movement disorders, inhibited cognitive development, neuron deterioration, and tumors. After extensive literature review, gene therapy offers a promising route for the treatment of neurologic orphan diseases. The use of clustered regularly interspaced palindromic repeats/Cas9 has demonstrated positive results in experiments investigating its role in several diseases. Additionally, the use of adeno-associated viral vectors has shown improvement in survival, motor function, and developmental milestones, while also demonstrating reversal of sensory ataxia and cardiomyopathy in Friedreich ataxia patients. Antisense oligonucleotides have also been used in some neurologic orphan diseases with positive outcomes. Mammalian target of rapamycin inhibitors are currently being investigated and have reduced abnormal cell growth, proliferation, and angiogenesis. Emerging innovations and the role of genetic treatments open a new window of opportunity for the treatment of neurologic orphan diseases.
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Affiliation(s)
| | - Devon T Foster
- Florida International University Herbert Wertheim College of Medicine, Florida International University Herbert Wertheim College of Medicine, Miami, FL 33199, United States
| | - Rajvi N Thakkar
- College of Medicine, University of Florida, Gainesville, FL 32611, United States
| | - Marco A Foreman
- College of Medicine, University of Florida, Gainesville, FL 32611, United States
| | - Brandon J Burgess
- College of Medicine, University of Florida, Gainesville, FL 32611, United States
| | - Rebecca M Toms
- College of Medicine, University of Florida, Gainesville, FL 32611, United States
| | | | - Brandon Lucke-Wold
- Department of Neurosurgery, University of Florida, Gainesville, FL 32611, United States
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3
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Dionisi C, Chazalon M, Rai M, Keime C, Imbault V, Communi D, Puccio H, Schiffmann SN, Pandolfo M. 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. Brain Commun 2023; 5:fcad007. [PMID: 36865673 PMCID: PMC9972525 DOI: 10.1093/braincomms/fcad007] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2022] [Revised: 09/28/2022] [Accepted: 01/14/2023] [Indexed: 01/19/2023] Open
Abstract
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.
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Affiliation(s)
| | | | - Myriam Rai
- Laboratory of Experimental Neurology, Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium
| | - Céline Keime
- Institut de Génétique et de Biologie Moléculaire et Cellulaire UMR 7104 CNRS-UdS / INSERM U1258, Université de Strasbourg, 67404 Illkirch Cedex, Strasbourg, France
| | - Virginie Imbault
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium
| | - David Communi
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium
| | - Hélène Puccio
- Institut de Génétique et de Biologie Moléculaire et Cellulaire UMR 7104 CNRS-UdS / INSERM U1258, Université de Strasbourg, 67404 Illkirch Cedex, Strasbourg, France,Institut NeuroMyoGene (INMG) UMR5310—INSERM U1217, Faculté de Médecine, Université Claude Bernard—Lyon I, 69008 Lyon, France
| | - Serge N Schiffmann
- Laboratory of Neurophysiology, ULB-Neuroscience Institute (UNI), Université Libre de Bruxelles (ULB), 1070 Brussels, Belgium
| | - Massimo Pandolfo
- Correspondence to: Massimo Pandolfo Department of Neurology and Neurosurgery McGill University, Montreal Neurological Institute 3801 University Street, Montreal, Quebec H3A 2B4, Canada E-mail:
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4
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Schreiber AM, Li Y, Chen YH, Napierala JS, Napierala M. Selected Histone Deacetylase Inhibitors Reverse the Frataxin Transcriptional Defect in a Novel Friedreich's Ataxia Induced Pluripotent Stem Cell-Derived Neuronal Reporter System. Front Neurosci 2022; 16:836476. [PMID: 35281493 PMCID: PMC8904878 DOI: 10.3389/fnins.2022.836476] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Accepted: 01/12/2022] [Indexed: 11/23/2022] Open
Abstract
Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by the expansion of guanine-adenine-adenine repeats within the first intron of the frataxin (FXN) gene. The location and nature of the expansion have been proven to contribute to transcriptional repression of FXN by decreasing the rate of polymerase II (RNA polymerase II) progression and increasing the presence of histone modifications associated with a heterochromatin-like state. Targeting impaired FXN transcription appears as a feasible option for therapeutic intervention, while no cure currently exists. We created a novel reporter cell line containing an FXN-Nanoluciferase (FXN-NLuc) fusion in induced pluripotent stem cells (iPSCs) reprogrammed from the fibroblasts of patients with FRDA, thus allowing quantification of endogenous FXN expression. The use of iPSCs provides the opportunity to differentiate these cells into disease-relevant neural progenitor cells (NPCs). NPCs derived from the FXN-NLuc line responded to treatments with a known FXN inducer, RG109. Results were validated by quantitative PCR and Western blot in multiple FRDA NPC lines. We then screened a commercially available library of compounds consisting of molecules targeting various enzymes and pathways critical for silencing or activation of gene expression. Only selected histone deacetylase inhibitors were capable of partial reactivation of FXN expression. This endogenous, FRDA iPSC-derived reporter can be utilized for high-throughput campaigns performed in cells most relevant to disease pathology in search of FXN transcription activators.
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Affiliation(s)
- Anna M. Schreiber
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Yanjie Li
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Yi-Hsien Chen
- Genome Engineering and iPSC Center, Washington University, St. Louis, MO, United States
| | - Jill S. Napierala
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Marek Napierala
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL, United States
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5
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Yang W, Thompson B, Kwa FAA. Molecular approaches for the treatment and prevention of Friedreich's ataxia. Drug Discov Today 2021; 27:866-880. [PMID: 34763067 DOI: 10.1016/j.drudis.2021.11.003] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 10/01/2021] [Accepted: 11/01/2021] [Indexed: 12/11/2022]
Abstract
Friedreich's ataxia (FRDA) is caused by an intronic guanine-adenine-adenine (GAA) trinucleotide expansion in the gene encoding the frataxin protein (FXN). This triggers the transcriptional silencing of the fratxin gene (FXN) and subsequent FXN deficiency in affected cells, which accounts for the multisystemic symptoms of this condition. Current management strategies aim for symptomatic relief and no treatments can prevent disease onset or progression. Thus, research efforts have focused on targeting the molecular pathways that silence FXN and downstream pathological processes. However, progression of potential therapies into clinical use has been hindered by inconclusive clinical trials because of the small patient sample size associated with the low prevalence of this condition. Here, we discuss various molecular approaches and explore their therapeutic potential to alter the course of this progressive condition.
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Affiliation(s)
- Wenyao Yang
- School of Health Sciences, Swinburne University of Technology, Hawthorn, VIC 3122, Australia
| | - Bruce Thompson
- School of Health Sciences, Swinburne University of Technology, Hawthorn, VIC 3122, Australia
| | - Faith A A Kwa
- School of Health Sciences, Swinburne University of Technology, Hawthorn, VIC 3122, Australia.
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6
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Zimmer-Bensch G, Zempel H. DNA Methylation in Genetic and Sporadic Forms of Neurodegeneration: Lessons from Alzheimer's, Related Tauopathies and Genetic Tauopathies. Cells 2021; 10:3064. [PMID: 34831288 PMCID: PMC8624300 DOI: 10.3390/cells10113064] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2021] [Revised: 11/03/2021] [Accepted: 11/04/2021] [Indexed: 12/14/2022] Open
Abstract
Genetic and sporadic forms of tauopathies, the most prevalent of which is Alzheimer's Disease, are a scourge of the aging society, and in the case of genetic forms, can also affect children and young adults. All tauopathies share ectopic expression, mislocalization, or aggregation of the microtubule associated protein TAU, encoded by the MAPT gene. As TAU is a neuronal protein widely expressed in the CNS, the overwhelming majority of tauopathies are neurological disorders. They are characterized by cognitive dysfunction often leading to dementia, and are frequently accompanied by movement abnormalities such as parkinsonism. Tauopathies can lead to severe neurological deficits and premature death. For some tauopathies there is a clear genetic cause and/or an epigenetic contribution. However, for several others the disease etiology is unclear, with few tauopathies being environmentally triggered. Here, we review current knowledge of tauopathies listing known genetic and important sporadic forms of these disease. Further, we discuss how DNA methylation as a major epigenetic mechanism emerges to be involved in the disease pathophysiology of Alzheimer's, and related genetic and non-genetic tauopathies. Finally, we debate the application of epigenetic signatures in peripheral blood samples as diagnostic tools and usages of epigenetic therapy strategies for these diseases.
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Affiliation(s)
- Geraldine Zimmer-Bensch
- Functional Epigenetics in the Animal Model, Institute for Biology II, RWTH Aachen University, 52074 Aachen, Germany
- Research Training Group 2416 MultiSenses-MultiScales, Institute for Biology II, RWTH Aachen University, 52074 Aachen, Germany
| | - Hans Zempel
- Institute of Human Genetics, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
- Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931 Cologne, Germany
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7
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Vilema-Enríquez G, Quinlan R, Kilfeather P, Mazzone R, Saqlain S, Del Molino Del Barrio I, Donato A, Corda G, Li F, Vedadi M, Németh AH, Brennan PE, Wade-Martins R. Inhibition of the SUV4-20 H1 histone methyltransferase increases frataxin expression in Friedreich's ataxia patient cells. J Biol Chem 2020; 295:17973-17985. [PMID: 33028632 PMCID: PMC7939392 DOI: 10.1074/jbc.ra120.015533] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Indexed: 12/11/2022] Open
Abstract
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.
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Affiliation(s)
| | - Robert Quinlan
- Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom; Alzheimer's Research UK Oxford Drug Discovery Institute, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Peter Kilfeather
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
| | - Roberta Mazzone
- Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom; Alzheimer's Research UK Oxford Drug Discovery Institute, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Saba Saqlain
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
| | | | - Annalidia Donato
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
| | - Gabriele Corda
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
| | - Fengling Li
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada
| | - Masoud Vedadi
- Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada
| | - Andrea H Németh
- Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom; Oxford Centre for Genomic Medicine, Oxford University Hospitals National Health Service Trust, Oxford, United Kingdom
| | - Paul E Brennan
- Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom; Alzheimer's Research UK Oxford Drug Discovery Institute, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
| | - Richard Wade-Martins
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.
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8
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Russi M, Martin E, D'Autréaux B, Tixier L, Tricoire H, Monnier V. A Drosophila model of Friedreich ataxia with CRISPR/Cas9 insertion of GAA repeats in the frataxin gene reveals in vivo protection by N-acetyl cysteine. Hum Mol Genet 2020; 29:2831-2844. [PMID: 32744307 DOI: 10.1093/hmg/ddaa170] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2020] [Revised: 07/10/2020] [Accepted: 07/23/2020] [Indexed: 12/11/2022] Open
Abstract
Friedreich ataxia (FA) is caused by GAA repeat expansions in the first intron of FXN, the gene encoding frataxin, which results in decreased gene expression. Thanks to the high degree of frataxin conservation, the Drosophila melanogaster fruitfly appears as an adequate animal model to study this disease and to evaluate therapeutic interventions. Here, we generated a Drosophila model of FA with CRISPR/Cas9 insertion of approximately 200 GAA in the intron of the fly frataxin gene fh. These flies exhibit a developmental delay and lethality associated with decreased frataxin expression. We were able to bypass preadult lethality using genetic tools to overexpress frataxin only during the developmental period. These frataxin-deficient adults are short-lived and present strong locomotor defects. RNA-Seq analysis identified deregulation of genes involved in amino-acid metabolism and transcriptomic signatures of oxidative stress. In particular, we observed a progressive increase of Tspo expression, fully rescued by adult frataxin expression. Thus, Tspo expression constitutes a molecular marker of the disease progression in our fly model and might be of interest in other animal models or in patients. Finally, in a candidate drug screening, we observed that N-acetyl cysteine improved the survival, locomotor function, resistance to oxidative stress and aconitase activity of frataxin-deficient flies. Therefore, our model provides the opportunity to elucidate in vivo, the protective mechanisms of this molecule of therapeutic potential. This study also highlights the strength of the CRISPR/Cas9 technology to introduce human mutations in endogenous orthologous genes, leading to Drosophila models of human diseases with improved physiological relevance.
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Affiliation(s)
- Maria Russi
- Université de Paris, BFA Unit of Functional and Adaptative Biology, UMR 8251, CNRS, Paris F-75013, France
| | - Elodie Martin
- Université de Paris, BFA Unit of Functional and Adaptative Biology, UMR 8251, CNRS, Paris F-75013, France
| | - Benoit D'Autréaux
- Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette cedex 91198, France
| | - Laura Tixier
- Université de Paris, BFA Unit of Functional and Adaptative Biology, UMR 8251, CNRS, Paris F-75013, France
| | - Hervé Tricoire
- Université de Paris, BFA Unit of Functional and Adaptative Biology, UMR 8251, CNRS, Paris F-75013, France
| | - Véronique Monnier
- Université de Paris, BFA Unit of Functional and Adaptative Biology, UMR 8251, CNRS, Paris F-75013, France
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9
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Bizzoca A, Caracciolo M, Corsi P, Magrone T, Jirillo E, Gennarini G. Molecular and Cellular Substrates for the Friedreich Ataxia. Significance of Contactin Expression and of Antioxidant Administration. Molecules 2020; 25:E4085. [PMID: 32906751 PMCID: PMC7570916 DOI: 10.3390/molecules25184085] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Revised: 08/28/2020] [Accepted: 09/02/2020] [Indexed: 11/16/2022] Open
Abstract
In this study, the neural phenotype is explored in rodent models of the spinocerebellar disorder known as the Friedreich Ataxia (FA), which results from mutations within the gene encoding the Frataxin mitochondrial protein. For this, the M12 line, bearing a targeted mutation, which disrupts the Frataxin gene exon 4 was used, together with the M02 line, which, in addition, is hemizygous for the human Frataxin gene mutation (Pook transgene), implying the occurrence of 82-190 GAA repeats within its first intron. The mutant mice phenotype was compared to the one of wild type littermates in regions undergoing differential profiles of neurogenesis, including the cerebellar cortex and the spinal cord by using neuronal (β-tubulin) and glial (Glial Fibrillary Acidic Protein) markers as well as the Contactin 1 axonal glycoprotein, involved in neurite growth control. Morphological/morphometric analyses revealed that while in Frataxin mutant mice the neuronal phenotype was significantly counteracted, a glial upregulation occurred at the same time. Furthermore, Contactin 1 downregulation suggested that changes in the underlying gene contributed to the disorder pathogenesis. Therefore, the FA phenotype implies an alteration of the developmental profile of neuronal and glial precursors. Finally, epigallocatechin gallate polyphenol administration counteracted the disorder, indicating protective effects of antioxidant administration.
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Affiliation(s)
| | | | | | | | | | - Gianfranco Gennarini
- Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Medical School, University of Bari, Piazza Giulio Cesare, 11. I-70124 Bari, Italy; (A.B.); (M.C.); (P.C.); (T.M.); (E.J.)
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10
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Sherzai M, Valle A, Perry N, Kalef-Ezra E, Al-Mahdawi S, Pook M, Anjomani Virmouni S. HMTase Inhibitors as a Potential Epigenetic-Based Therapeutic Approach for Friedreich's Ataxia. Front Genet 2020; 11:584. [PMID: 32582297 PMCID: PMC7291394 DOI: 10.3389/fgene.2020.00584] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Accepted: 05/14/2020] [Indexed: 12/17/2022] Open
Abstract
Friedreich's ataxia (FRDA) is a progressive neurodegenerative disorder caused by a homozygous GAA repeat expansion mutation in intron 1 of the frataxin gene (FXN), which instigates reduced transcription. As a consequence, reduced levels of frataxin protein lead to mitochondrial iron accumulation, oxidative stress, and ultimately cell death; particularly in dorsal root ganglia (DRG) sensory neurons and the dentate nucleus of the cerebellum. In addition to neurological disability, FRDA is associated with cardiomyopathy, diabetes mellitus, and skeletal deformities. Currently there is no effective treatment for FRDA and patients die prematurely. Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, we investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin protein levels was observed. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies.
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Affiliation(s)
- Mursal Sherzai
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom
| | - Adamo Valle
- Energy Metabolism and Nutrition, Research Institute of Health Sciences (IUNICS) and Health Research Institute of Balearic Islands (IdISBa), University of Balearic Islands, Palma de Mallorca, Spain.,Biomedical Research Networking Center for Physiopathology of Obesity and Nutrition (CIBERobn), Instituto de Salud Carlos III, Madrid, Spain
| | - Nicholas Perry
- Division of Cancer Biology, The Institute of Cancer Research, London, United Kingdom
| | - Ester Kalef-Ezra
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom
| | - Sahar Al-Mahdawi
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom
| | - Mark Pook
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom
| | - Sara Anjomani Virmouni
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom
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11
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Fernández-Frías I, Pérez-Luz S, Díaz-Nido J. Analysis of Putative Epigenetic Regulatory Elements in the FXN Genomic Locus. Int J Mol Sci 2020; 21:E3410. [PMID: 32408537 PMCID: PMC7279236 DOI: 10.3390/ijms21103410] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Revised: 05/05/2020] [Accepted: 05/09/2020] [Indexed: 12/22/2022] Open
Abstract
Friedreich´s ataxia (FRDA) is an autosomal recessive disease caused by an abnormally expanded Guanine-Adenine-Adenine (GAA) repeat sequence within the first intron of the frataxin gene (FXN). The molecular mechanisms associated with FRDA are still poorly understood and most studies on FXN gene regulation have been focused on the region around the minimal promoter and the region in which triplet expansion occurs. Nevertheless, since there could be more epigenetic changes involved in the reduced levels of FXN transcripts, the aim of this study was to obtain a more detailed view of the possible regulatory elements by analyzing data from ENCODE and Roadmap consortia databases. This bioinformatic analysis indicated new putative regulatory regions within the FXN genomic locus, including exons, introns, and upstream and downstream regions. Moreover, the region next to the end of intron 4 is of special interest, since the enhancer signals in FRDA-affected tissues are weak or absent in this region, whilst they are strong in the rest of the analyzed tissues. Therefore, these results suggest that there could be a direct relationship between the absence of enhancer sequences in this specific region and their predisposition to be affected in this pathology.
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Affiliation(s)
- Iván Fernández-Frías
- Departamento Biología Molecular and Centro de Biología Molecular “Severo Ochoa” (UAM-CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain; (I.F.-F.); (J.D.-N.)
- Instituto Investigación Sanitaria Puerta de Hierro-Majadahonda, 28222 Madrid, Spain
| | - Sara Pérez-Luz
- Departamento Biología Molecular and Centro de Biología Molecular “Severo Ochoa” (UAM-CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain; (I.F.-F.); (J.D.-N.)
- Instituto Investigación Sanitaria Puerta de Hierro-Majadahonda, 28222 Madrid, Spain
| | - Javier Díaz-Nido
- Departamento Biología Molecular and Centro de Biología Molecular “Severo Ochoa” (UAM-CSIC), Universidad Autónoma de Madrid, 28049 Madrid, Spain; (I.F.-F.); (J.D.-N.)
- Instituto Investigación Sanitaria Puerta de Hierro-Majadahonda, 28222 Madrid, Spain
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12
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Fedotova E, Abramycheva N, Nuzhny E, Ershova M, Klyushnikov S, Illarioshkin S. Friedreich ataxia: FXN gene expression and its relationship with DNA methylation pattern. BULLETIN OF RUSSIAN STATE MEDICAL UNIVERSITY 2019. [DOI: 10.24075/brsmu.2019.062] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Friedreich ataxia (FRDA) is the most common autosomal recessive ataxia associated with the non-coding GAA tandem repeats expansion in the FXN gene. Transcription impairment and frataxin protein deficiency are the key features of the disease pathogenesis. Our research was aimed to study the FXN gene mRNA expression as well as to carry out the clinical, genetic and epigenetic correlation analysis in a group of patients with homozygous expansion, in a group of their relatives with heterozygous expansion and in a control group. The FXN mRNA level was determined using the real-time polymerase chain reaction. Methylation pattern of CpG sites was evaluated by direct bisulfite sequencing. As a result of the study, the threshold values were obtained between the FRDA patients group, the group of heterozygous carriers and the control group (15 and 79%, respectively). The clinical and genetic features comparison with the FXN expression level revealed no significant correlation. When comparing gene expression with an epigenetic profile, it was found that hypermethylation of a number of CpG sites upstream of the trinucleotide repeats and some non-CpG sites downstream of the region of repeats inhibited expression. Thus, the identified methylated sites may be considered as a target for epigenome editing to increase the FXN transcription and, consequently, for target therapy of the disease.
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Affiliation(s)
| | | | - E.P. Nuzhny
- Research Center of Neurology, Moscow, Russia
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13
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Gottesfeld JM. Molecular Mechanisms and Therapeutics for the GAA·TTC Expansion Disease Friedreich Ataxia. Neurotherapeutics 2019; 16:1032-1049. [PMID: 31317428 PMCID: PMC6985418 DOI: 10.1007/s13311-019-00764-x] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Friedreich ataxia (FRDA), the most common inherited ataxia, is caused by transcriptional silencing of the nuclear FXN gene, encoding the essential mitochondrial protein frataxin. Currently, there is no approved therapy for this fatal disorder. Gene silencing in FRDA is due to hyperexpansion of the triplet repeat sequence GAA·TTC in the first intron of the FXN gene, which results in chromatin histone modifications consistent with heterochromatin formation. Frataxin is involved in mitochondrial iron homeostasis and the assembly and transfer of iron-sulfur clusters to various mitochondrial enzymes and components of the electron transport chain. Frataxin insufficiency leads to progressive spinocerebellar neurodegeneration, causing symptoms of gait and limb ataxia, slurred speech, muscle weakness, sensory loss, and cardiomyopathy in many patients, resulting in death in early adulthood. Numerous approaches are being taken to find a treatment for FRDA, including excision or correction of the repeats by genome engineering methods, gene activation with small molecules or artificial transcription factors, delivery of frataxin to affected cells by protein replacement therapy, gene therapy, or small molecules to increase frataxin protein levels, and therapies aimed at countering the cellular consequences of reduced frataxin. This review will summarize the mechanisms involved in repeat-mediated gene silencing and recent efforts aimed at development of therapeutics.
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Affiliation(s)
- Joel M Gottesfeld
- Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, 92037, USA.
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14
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Qadir MI, Bukhat S, Rasul S, Manzoor H, Manzoor M. RNA therapeutics: Identification of novel targets leading to drug discovery. J Cell Biochem 2019; 121:898-929. [DOI: 10.1002/jcb.29364] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Accepted: 08/20/2019] [Indexed: 12/23/2022]
Affiliation(s)
- Muhammad Imran Qadir
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Sherien Bukhat
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Sumaira Rasul
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Hamid Manzoor
- Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
| | - Majid Manzoor
- College of Pharmaceutical Sciences Zhejiang University Hangzhou China
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15
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Eimer H, Sureshkumar S, Singh Yadav A, Kraupner-Taylor C, Bandaranayake C, Seleznev A, Thomason T, Fletcher SJ, Gordon SF, Carroll BJ, Balasubramanian S. RNA-Dependent Epigenetic Silencing Directs Transcriptional Downregulation Caused by Intronic Repeat Expansions. Cell 2018; 174:1095-1105.e11. [PMID: 30057112 DOI: 10.1016/j.cell.2018.06.044] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2017] [Revised: 04/20/2018] [Accepted: 06/22/2018] [Indexed: 01/07/2023]
Abstract
Transcriptional downregulation caused by intronic triplet repeat expansions underlies diseases such as Friedreich's ataxia. This downregulation of gene expression is coupled with epigenetic changes, but the underlying mechanisms are unknown. Here, we show that an intronic GAA/TTC triplet expansion within the IIL1 gene of Arabidopsis thaliana results in accumulation of 24-nt short interfering RNAs (siRNAs) and repressive histone marks at the IIL1 locus, which in turn causes its transcriptional downregulation and an associated phenotype. Knocking down DICER LIKE-3 (DCL3), which produces 24-nt siRNAs, suppressed transcriptional downregulation of IIL1 and the triplet expansion-associated phenotype. Furthermore, knocking down additional components of the RNA-dependent DNA methylation (RdDM) pathway also suppressed both transcriptional downregulation of IIL1 and the repeat expansion-associated phenotype. Thus, our results show that triplet repeat expansions can lead to local siRNA biogenesis, which in turn downregulates transcription through an RdDM-dependent epigenetic modification.
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Affiliation(s)
- Hannes Eimer
- School of Biological Sciences, Monash University, Clayton Campus, VIC 3800, Australia
| | - Sridevi Sureshkumar
- School of Biological Sciences, Monash University, Clayton Campus, VIC 3800, Australia
| | - Avilash Singh Yadav
- School of Biological Sciences, Monash University, Clayton Campus, VIC 3800, Australia
| | | | - Champa Bandaranayake
- School of Biological Sciences, Monash University, Clayton Campus, VIC 3800, Australia
| | - Andrei Seleznev
- School of Biological Sciences, Monash University, Clayton Campus, VIC 3800, Australia
| | - Tamblyn Thomason
- School of Biological Sciences, Monash University, Clayton Campus, VIC 3800, Australia
| | - Stephen J Fletcher
- School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia QLD 4072, Australia
| | | | - Bernard J Carroll
- School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia QLD 4072, Australia
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16
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Monnier V, Llorens JV, Navarro JA. Impact of Drosophila Models in the Study and Treatment of Friedreich's Ataxia. Int J Mol Sci 2018; 19:E1989. [PMID: 29986523 PMCID: PMC6073496 DOI: 10.3390/ijms19071989] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2018] [Revised: 06/26/2018] [Accepted: 07/03/2018] [Indexed: 02/07/2023] Open
Abstract
Drosophila melanogaster has been for over a century the model of choice of several neurobiologists to decipher the formation and development of the nervous system as well as to mirror the pathophysiological conditions of many human neurodegenerative diseases. The rare disease Friedreich’s ataxia (FRDA) is not an exception. Since the isolation of the responsible gene more than two decades ago, the analysis of the fly orthologue has proven to be an excellent avenue to understand the development and progression of the disease, to unravel pivotal mechanisms underpinning the pathology and to identify genes and molecules that might well be either disease biomarkers or promising targets for therapeutic interventions. In this review, we aim to summarize the collection of findings provided by the Drosophila models but also to go one step beyond and propose the implications of these discoveries for the study and cure of this disorder. We will present the physiological, cellular and molecular phenotypes described in the fly, highlighting those that have given insight into the pathology and we will show how the ability of Drosophila to perform genetic and pharmacological screens has provided valuable information that is not easily within reach of other cellular or mammalian models.
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Affiliation(s)
- Véronique Monnier
- Unité de Biologie Fonctionnelle et Adaptative (BFA), Sorbonne Paris Cité, Université Paris Diderot, UMR8251 CNRS, 75013 Paris, France.
| | - Jose Vicente Llorens
- Department of Genetics, University of Valencia, Campus of Burjassot, 96100 Valencia, Spain.
| | - Juan Antonio Navarro
- Lehrstuhl für Entwicklungsbiologie, Universität Regensburg, 93040 Regensburg, Germany.
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17
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Chandran V, Gao K, Swarup V, Versano R, Dong H, Jordan MC, Geschwind DH. Inducible and reversible phenotypes in a novel mouse model of Friedreich's Ataxia. eLife 2017; 6:e30054. [PMID: 29257745 PMCID: PMC5736353 DOI: 10.7554/elife.30054] [Citation(s) in RCA: 65] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Accepted: 11/20/2017] [Indexed: 12/13/2022] Open
Abstract
Friedreich's ataxia (FRDA), the most common inherited ataxia, is caused by recessive mutations that reduce the levels of frataxin (FXN), a mitochondrial iron binding protein. We developed an inducible mouse model of Fxn deficiency that enabled us to control the onset and progression of disease phenotypes by the modulation of Fxn levels. Systemic knockdown of Fxn in adult mice led to multiple phenotypes paralleling those observed in human patients across multiple organ systems. By reversing knockdown after clinical features appear, we were able to determine to what extent observed phenotypes represent reversible cellular dysfunction. Remarkably, upon restoration of near wild-type FXN levels, we observed significant recovery of function, associated pathology and transcriptomic dysregulation even after substantial motor dysfunction and pathology were observed. This model will be of broad utility in therapeutic development and in refining our understanding of the relative contribution of reversible cellular dysfunction at different stages in disease.
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Affiliation(s)
- Vijayendran Chandran
- Program in Neurogenetics, Department of Neurology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
| | - Kun Gao
- Program in Neurogenetics, Department of Neurology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
| | - Vivek Swarup
- Program in Neurogenetics, Department of Neurology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
| | - Revital Versano
- Program in Neurogenetics, Department of Neurology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
| | - Hongmei Dong
- Program in Neurogenetics, Department of Neurology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
| | - Maria C Jordan
- Department of Physiology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
| | - Daniel H Geschwind
- Program in Neurogenetics, Department of Neurology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
- Department of Human Genetics, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUnited States
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18
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Schenkel LC, Rodenhiser D, Siu V, McCready E, Ainsworth P, Sadikovic B. Constitutional Epi/Genetic Conditions: Genetic, Epigenetic, and Environmental Factors. J Pediatr Genet 2017; 6:30-41. [PMID: 28180025 PMCID: PMC5288004 DOI: 10.1055/s-0036-1593849] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Accepted: 04/14/2016] [Indexed: 12/12/2022]
Abstract
There are more than 4,000 phenotypes for which the molecular basis is at least partly known. Though defects in primary DNA structure constitute a major cause of these disorders, epigenetic disruption is emerging as an important alternative mechanism in the etiology of a broad range of congenital and developmental conditions. These include epigenetic defects caused by either localized (in cis) genetic alterations or more distant (in trans) genetic events but can also include environmental effects. Emerging evidence suggests interplay between genetic and environmental factors in the epigenetic etiology of several constitutional "epi/genetic" conditions. This review summarizes our broadening understanding of how epigenetics contributes to pediatric disease by exploring different classes of epigenomic disorders. It further challenges the simplistic dogma of "DNA encodes RNA encodes protein" to best understand the spectrum of factors that can influence genetic traits in a pediatric population.
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Affiliation(s)
- Laila C. Schenkel
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada
- Children's Health Research Institute, London, Ontario, Canada
| | - David Rodenhiser
- Children's Health Research Institute, London, Ontario, Canada
- Department of Biochemistry, Western University, London, Ontario, Canada
- Department of Pediatrics, Western University, London, Ontario, Canada
- London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada
- Department of Oncology, Western University, London, Ontario, Canada
| | - Victoria Siu
- Children's Health Research Institute, London, Ontario, Canada
- Department of Pediatrics, Western University, London, Ontario, Canada
- London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada
| | - Elizabeth McCready
- Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
| | - Peter Ainsworth
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada
- Children's Health Research Institute, London, Ontario, Canada
- Department of Biochemistry, Western University, London, Ontario, Canada
- Department of Pediatrics, Western University, London, Ontario, Canada
- London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada
- Department of Oncology, Western University, London, Ontario, Canada
| | - Bekim Sadikovic
- Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada
- Children's Health Research Institute, London, Ontario, Canada
- London Regional Cancer Program, London Health Sciences Centre, London, Ontario, Canada
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19
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Abstract
Most of the human genome encodes RNAs that do not code for proteins. These non-coding RNAs (ncRNAs) may affect normal gene expression and disease progression, making them a new class of targets for drug discovery. Because their mechanisms of action are often novel, developing drugs to target ncRNAs will involve equally novel challenges. However, many potential problems may already have been solved during the development of technologies to target mRNA. Here, we discuss the growing field of ncRNA - including microRNA, intronic RNA, repetitive RNA and long non-coding RNA - and assess the potential and challenges in their therapeutic exploitation.
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Affiliation(s)
- Masayuki Matsui
- Departments of Pharmacology and Biochemistry, UT Southwestern, Dallas, Texas 75390-9041, USA
| | - David R Corey
- Departments of Pharmacology and Biochemistry, UT Southwestern, Dallas, Texas 75390-9041, USA
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20
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Viswambharan V, Thanseem I, Vasu MM, Poovathinal SA, Anitha A. miRNAs as biomarkers of neurodegenerative disorders. Biomark Med 2017; 11:151-167. [PMID: 28125293 DOI: 10.2217/bmm-2016-0242] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Neurodegenerative diseases (NDDs) are the result of progressive deterioration of neurons, ultimately leading to disabilities. There is no effective cure for NDDs at present; ongoing therapies are mainly aimed at treating the most bothersome symptoms. Since early treatment is crucial in NDDs, there is an urgent need for specific and sensitive biomarkers that can aid in early diagnosis of these disorders. Recently, altered expression of miRNAs has been implicated in several neurological disorders, including NDDs. miRNA expression has been extensively investigated in the cells, tissues and body fluids of patients with different types of NDDs. The aim of this review is to provide a comprehensive overview of miRNAs as biomarkers and therapeutic targets for NDDs.
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Affiliation(s)
- Vijitha Viswambharan
- Department of Neurogenetics, Institute for Communicative & Cognitive Neurosciences (ICCONS), Shoranur, Palakkad 679 523, Kerala, India
| | - Ismail Thanseem
- Department of Neurogenetics, Institute for Communicative & Cognitive Neurosciences (ICCONS), Shoranur, Palakkad 679 523, Kerala, India
| | - Mahesh M Vasu
- Department of Psychiatry, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka 431 3192, Japan
| | - Suresh A Poovathinal
- Department of Neurology, Institute for Communicative & Cognitive Neurosciences (ICCONS), Shoranur, Palakkad 679 523, Kerala, India
| | - Ayyappan Anitha
- Department of Neurogenetics, Institute for Communicative & Cognitive Neurosciences (ICCONS), Shoranur, Palakkad 679 523, Kerala, India
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21
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Li L, Matsui M, Corey DR. Activating frataxin expression by repeat-targeted nucleic acids. Nat Commun 2016; 7:10606. [PMID: 26842135 PMCID: PMC4742999 DOI: 10.1038/ncomms10606] [Citation(s) in RCA: 59] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2015] [Accepted: 01/04/2016] [Indexed: 12/20/2022] Open
Abstract
Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.
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Affiliation(s)
- Liande Li
- Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, 6001 Forest Park Road, Dallas, Texas 75390-9041, USA
| | - Masayuki Matsui
- Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, 6001 Forest Park Road, Dallas, Texas 75390-9041, USA
| | - David R. Corey
- Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, 6001 Forest Park Road, Dallas, Texas 75390-9041, USA
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22
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Zeier Z, Esanov R, Belle KC, Volmar CH, Johnstone AL, Halley P, DeRosa BA, Khoury N, van Blitterswijk M, Rademakers R, Albert J, Brothers SP, Wuu J, Dykxhoorn DM, Benatar M, Wahlestedt C. Bromodomain inhibitors regulate the C9ORF72 locus in ALS. Exp Neurol 2015; 271:241-50. [PMID: 26099177 DOI: 10.1016/j.expneurol.2015.06.017] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2015] [Revised: 06/05/2015] [Accepted: 06/16/2015] [Indexed: 12/13/2022]
Abstract
A hexanucleotide repeat expansion residing within the C9ORF72 gene represents the most common known cause of amyotrophic lateral sclerosis (ALS) and places the disease among a growing family of repeat expansion disorders. The presence of RNA foci, repeat-associated translation products, and sequestration of RNA binding proteins suggests that toxic RNA gain-of-function contributes to pathology while C9ORF72 haploinsufficiency may be an additional pathological factor. One viable therapeutic strategy for treating expansion diseases is the use of small molecule inhibitors of epigenetic modifier proteins to reactivate expanded genetic loci. Indeed, previous studies have established proof of this principle by increasing the drug-induced expression of expanded (and abnormally heterochromatinized) FMR1, FXN and C9ORF72 genes in respective patient cells. While epigenetic modifier proteins are increasingly recognized as druggable targets, there have been few screening strategies to address this avenue of drug discovery in the context of expansion diseases. Here we utilize a semi-high-throughput gene expression based screen to identify siRNAs and small molecule inhibitors of epigenetic modifier proteins that regulate C9ORF72 RNA in patient fibroblasts, lymphocytes and reprogrammed motor neurons. We found that several bromodomain small molecule inhibitors increase the expression of C9ORF72 mRNA and pre-mRNA without affecting repressive epigenetic signatures of expanded C9ORF72 alleles. These data suggest that bromodomain inhibition increases the expression of unexpanded C9ORF72 alleles and may therefore compensate for haploinsufficiency without increasing the production of toxic RNA and protein products, thereby conferring therapeutic value.
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Affiliation(s)
- Zane Zeier
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Rustam Esanov
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Kinsley C Belle
- John P. Hussman Institute for Human Genomics and The Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, USA
| | - Claude-Henry Volmar
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Andrea L Johnstone
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Paul Halley
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Brooke A DeRosa
- John P. Hussman Institute for Human Genomics and The Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, USA
| | - Nathalie Khoury
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | | | | | | | - Shaun P Brothers
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA
| | - Joanne Wuu
- Department of Neurology, University of Miami Miller School of Medicine, USA
| | - Derek M Dykxhoorn
- John P. Hussman Institute for Human Genomics and The Dr. John T. Macdonald Foundation Department of Human Genetics, University of Miami Miller School of Medicine, USA
| | - Michael Benatar
- Department of Neurology, University of Miami Miller School of Medicine, USA
| | - Claes Wahlestedt
- Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
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23
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Li Y, Polak U, Bhalla AD, Rozwadowska N, Butler JS, Lynch DR, Dent SYR, Napierala M. Excision of Expanded GAA Repeats Alleviates the Molecular Phenotype of Friedreich's Ataxia. Mol Ther 2015; 23:1055-1065. [PMID: 25758173 DOI: 10.1038/mt.2015.41] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2014] [Accepted: 03/03/2015] [Indexed: 12/21/2022] Open
Abstract
Friedreich's ataxia (FRDA) is an autosomal recessive neurological disease caused by expansions of guanine-adenine-adenine (GAA) repeats in intron 1 of the frataxin (FXN) gene. The expansion results in significantly decreased frataxin expression. We report that human FRDA cells can be corrected by zinc finger nuclease-mediated excision of the expanded GAA repeats. Editing of a single expanded GAA allele created heterozygous, FRDA carrier-like cells and significantly increased frataxin expression. This correction persisted during reprogramming of zinc finger nuclease-edited fibroblasts to induced pluripotent stem cells and subsequent differentiation into neurons. The expression of FRDA biomarkers was normalized in corrected patient cells and disease-associated phenotypes, such as decreases in aconitase activity and intracellular ATP levels, were reversed in zinc finger nuclease corrected neuronal cells. Genetically and phenotypically corrected patient cells represent not only a preferred disease-relevant model system to study pathogenic mechanisms, but also a critical step towards development of cell replacement therapy.
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Affiliation(s)
- Yanjie Li
- Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Urszula Polak
- Department of Epigenetics and Molecular Carcinogenesis, Center for Cancer Epigenetics, University of Texas MD Anderson Cancer Center, Science Park, Smithville, Texas, USA; Department of Cell Biology, Poznan University of Medical Sciences, Poznan, Poland
| | - Angela D Bhalla
- Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Natalia Rozwadowska
- Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama, USA; Institute of Human Genetics, Polish Academy of Science, Poznan, Poland
| | - Jill Sergesketter Butler
- Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - David R Lynch
- Division of Neurology and Pediatrics, Children's Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania, USA
| | - Sharon Y R Dent
- Department of Epigenetics and Molecular Carcinogenesis, Center for Cancer Epigenetics, University of Texas MD Anderson Cancer Center, Science Park, Smithville, Texas, USA
| | - Marek Napierala
- Department of Biochemistry and Molecular Genetics, UAB Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama, USA; Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
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24
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Soragni E, Chou CJ, Rusche JR, Gottesfeld JM. Mechanism of Action of 2-Aminobenzamide HDAC Inhibitors in Reversing Gene Silencing in Friedreich's Ataxia. Front Neurol 2015; 6:44. [PMID: 25798128 PMCID: PMC4350406 DOI: 10.3389/fneur.2015.00044] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2014] [Accepted: 02/19/2015] [Indexed: 11/13/2022] Open
Abstract
The genetic defect in Friedreich’s ataxia (FRDA) is the hyperexpansion of a GAA•TTC triplet in the first intron of the FXN gene, encoding the essential mitochondrial protein frataxin. Histone post-translational modifications near the expanded repeats are consistent with heterochromatin formation and consequent FXN gene silencing. Using a newly developed human neuronal cell model, derived from patient-induced pluripotent stem cells, we find that 2-aminobenzamide histone deacetylase (HDAC) inhibitors increase FXN mRNA levels and frataxin protein in FRDA neuronal cells. However, only compounds targeting the class I HDACs 1 and 3 are active in increasing FXN mRNA in these cells. Structural analogs of the active HDAC inhibitors that selectively target either HDAC1 or HDAC3 do not show similar increases in FXN mRNA levels. To understand the mechanism of action of these compounds, we probed the kinetic properties of the active and inactive inhibitors, and found that only compounds that target HDACs 1 and 3 exhibited a slow-on/slow-off mechanism of action for the HDAC enzymes. HDAC1- and HDAC3-selective compounds did not show this activity. Using siRNA methods in the FRDA neuronal cells, we show increases in FXN mRNA upon silencing of either HDACs 1 or 3, suggesting the possibility that inhibition of each of these class I HDACs is necessary for activation of FXN mRNA synthesis, as there appears to be redundancy in the silencing mechanism caused by the GAA•TTC repeats. Moreover, inhibitors must have a long residence time on their target enzymes for this activity. By interrogating microarray data from neuronal cells treated with inhibitors of different specificity, we selected two genes encoding histone macroH2A (H2AFY2) and Polycomb group ring finger 2 (PCGF2) that were specifically down-regulated by the inhibitors targeting HDACs1 and 3 versus the more selective inhibitors for further investigation. Both genes are involved in transcriptional repression and we speculate their involvement in FXN gene silencing. Our results shed light on the mechanism whereby HDAC inhibitors increase FXN mRNA levels in FRDA neuronal cells.
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Affiliation(s)
- Elisabetta Soragni
- Department of Cell and Molecular Biology, The Scripps Research Institute , La Jolla, CA , USA
| | - C James Chou
- Department of Cell and Molecular Biology, The Scripps Research Institute , La Jolla, CA , USA
| | | | - Joel M Gottesfeld
- Department of Cell and Molecular Biology, The Scripps Research Institute , La Jolla, CA , USA
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Al-Mahdawi S, Virmouni SA, Pook MA. The emerging role of 5-hydroxymethylcytosine in neurodegenerative diseases. Front Neurosci 2014; 8:397. [PMID: 25538551 PMCID: PMC4256999 DOI: 10.3389/fnins.2014.00397] [Citation(s) in RCA: 81] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Accepted: 11/18/2014] [Indexed: 12/12/2022] Open
Abstract
DNA methylation primarily occurs within human cells as a 5-methylcytosine (5mC) modification of the cytosine bases in CpG dinucleotides. 5mC has proven to be an important epigenetic mark that is involved in the control of gene transcription for processes such as development and differentiation. However, recent studies have identified an alternative modification, 5-hydroxymethylcytosine (5hmC), which is formed by oxidation of 5mC by ten-eleven translocation (TET) enzymes. The overall levels of 5hmC in the mammalian genome are approximately 10% of 5mC levels, although higher levels have been detected in tissues of the central nervous system (CNS). The functions of 5hmC are not yet fully known, but evidence suggests that 5hmC may be both an intermediate product during the removal of 5mC by passive or active demethylation processes and also an epigenetic modification in its own right, regulating chromatin or transcriptional factors involved in processes such as neurodevelopment or environmental stress response. This review highlights our current understanding of the role that 5hmC plays in neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), fragile X-associated tremor/ataxia syndrome (FXTAS), Friedreich ataxia (FRDA), Huntington's disease (HD), and Parkinson's disease (PD).
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Affiliation(s)
- Sahar Al-Mahdawi
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UK ; Synthetic Biology Theme, Institute of Environment, Health and Societies, Brunel University London Uxbridge, UK
| | - Sara Anjomani Virmouni
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UK ; Synthetic Biology Theme, Institute of Environment, Health and Societies, Brunel University London Uxbridge, UK
| | - Mark A Pook
- Ataxia Research Group, Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, UK ; Synthetic Biology Theme, Institute of Environment, Health and Societies, Brunel University London Uxbridge, UK
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