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Vaughan DP, Real R, Jensen MT, Fumi RG, Hodgson M, Jabbari E, Lux D, Wu L, Warner TT, Jaunmuktane Z, Revesz T, Rowe JB, Rohrer J, Morris HR. Analysis of C9orf72 repeat length in progressive supranuclear palsy, corticobasal syndrome, corticobasal degeneration, and atypical parkinsonism. J Neurol 2025; 272:293. [PMID: 40138021 PMCID: PMC11947049 DOI: 10.1007/s00415-025-12990-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2024] [Revised: 02/09/2025] [Accepted: 02/15/2025] [Indexed: 03/29/2025]
Abstract
BACKGROUND Pathogenic hexanucleotide repeat expansions in C9orf72 are the commonest genetic cause of frontotemporal dementia and/or amyotrophic lateral sclerosis. There is growing interest in intermediate repeat expansions in C9orf72 and their relationship to a wide range of neurological presentations, including Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, corticobasal degeneration, and corticobasal syndromes. AIMS To assess the prevalence of intermediate C9orf72 repeat expansions in a large cohort of prospectively-recruited patients clinically diagnosed with progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and atypical parkinsonism (APS), compared with healthy controls. We also sought to replicate the association between C9orf72 repeat length and CBD in neuropathologically confirmed cases. METHODS 626 cases, including PSP (n = 366), CBS (n = 130), and APS (n = 53) from the PROSPECT study, and 77 cases with pathologically confirmed CBD were screened for intermediate repeat expansions in C9orf72 using repeat-primed PCR. These were compared to controls from the PROSPECT-M-UK study and from the 1958 Birth Cohort. RESULTS There was no difference in the mean or largest allele size in any affected patient group compared with controls. A higher proportion of our affected cohort had large C9orf72 repeat expansions compared to controls, but there was no difference when comparing the frequency of intermediate expansions between affected patients and controls. There was no relationship between repeat length and age at onset, level of disability, or survival. CONCLUSIONS Intermediate expansions in C9orf72 do not appear to be a genetic risk factor for PSP, CBS, CBD, or atypical parkinsonism. They are not associated with age at onset, disability, or survival in our study.
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Affiliation(s)
- David P Vaughan
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Raquel Real
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Marte Theilmann Jensen
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Riona G Fumi
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Megan Hodgson
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Edwin Jabbari
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Danielle Lux
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Lesley Wu
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Thomas T Warner
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Zane Jaunmuktane
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK
- Queen Square Brain Bank, Reta Lila Weston Institute of Neurological Studies, UCL Queen Square Institute of Neurology, London, UK
| | - Tamas Revesz
- Queen Square Brain Bank, Reta Lila Weston Institute of Neurological Studies, UCL Queen Square Institute of Neurology, London, UK
- Department of Neurodegenerative Disease, Queen Square Institute of Neurology, University College London, London, UK
| | - James B Rowe
- Department of Clinical Neurosciences, Cambridge University Hospitals NHS Trust, and MRC Cognition and Brain Sciences Unit, University of Cambridge, Cambridge, UK
| | - Jonathan Rohrer
- Dementia Research Centre, UCL Queen Square Institute of Neurology, London, UK
| | - Huw R Morris
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, University College London, London, UK.
- Movement Disorders Centre, UCL Queen Square Institute of Neurology, London, UK.
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Ramesh N, Evans A, Wojta K, Yang Z, Boks MM, Kahn RS, de Boer SCM, van der Lee SJ, Pijnenburg YAL, Reus LM, Ophoff RA. Accurate DNA Methylation Predictor for C9orf72 Repeat Expansion Alleles in the Pathogenic Range. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.03.20.643775. [PMID: 40196659 PMCID: PMC11974722 DOI: 10.1101/2025.03.20.643775] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2025]
Abstract
The hexanucleotide (G 4 C 2 ) repeat expansion in the promoter region of C9orf72 is the most frequent genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this study, we conducted a genome-wide DNA methylation (DNAm) analysis using EPIC version 2 (EPICv2) arrays on an FTD cohort comprising 27 carriers and 250 non-carriers of the pathogenic C9orf72 repeat expansion from the Amsterdam Dementia Cohort. We identified differentially methylated CpGs probes associated with the pathogenic C9orf72 expansion and used these findings to create a DNAm Least Absolute Shrinkage and Selection Operator (LASSO) predictor to identify repeat expansion carriers. Eight CpG sites at the C9orf72 locus were significantly differentially hypermethylated in repeat expansion carriers compared to non-carriers. The LASSO model predicted repeat expansion status with an average accuracy of 98.6%. The LASSO predictor was further validated in an independent cohort of 2,548 subjects with available EPICv2 data, identifying four C9orf72 repeat expansion carriers, subsequently confirmed by repeat-primed PCR. This result not only illustrates the accuracy of the DNAm predictor of C9orf72 repeat expansion carriers but also suggests that repeat expansion carriers may be more prevalent than expected. The identification of a highly accurate DNAm biomarker for a repeat expansion locus associated with neurodegenerative disorders may provide great value for studying this locus. The approach holds significant promise for investigating this and other repeat expansion loci, particularly given the growing interest in epigenetic epidemiological studies involving large cohorts with available DNAm data. Graphical abstract optional
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Calvo B, Schembri-Wismayer P, Durán-Alonso MB. Age-Related Neurodegenerative Diseases: A Stem Cell's Perspective. Cells 2025; 14:347. [PMID: 40072076 PMCID: PMC11898746 DOI: 10.3390/cells14050347] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2025] [Revised: 02/22/2025] [Accepted: 02/24/2025] [Indexed: 03/15/2025] Open
Abstract
Neurodegenerative diseases encompass a number of very heterogeneous disorders, primarily characterized by neuronal loss and a concomitant decline in neurological function. Examples of this type of clinical condition are Alzheimer's Disease, Parkinson's Disease, Huntington's Disease and Amyotrophic Lateral Sclerosis. Age has been identified as a major risk in the etiology of these disorders, which explains their increased incidence in developed countries. Unfortunately, despite continued and intensive efforts, no cure has yet been found for any of these diseases; reliable markers that allow for an early diagnosis of the disease and the identification of key molecular events leading to disease onset and progression are lacking. Altered adult neurogenesis appears to precede the appearance of severe symptoms. Given the scarcity of human samples and the considerable differences with model species, increasingly complex human stem-cell-based models are being developed. These are shedding light on the molecular alterations that contribute to disease development, facilitating the identification of new clinical targets and providing a screening platform for the testing of candidate drugs. Moreover, the secretome and other promising features of these cell types are being explored, to use them as replacement cells of high plasticity or as co-adjuvant therapy in combinatorial treatments.
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Affiliation(s)
- Belén Calvo
- Faculty of Health Sciences, Catholic University of Ávila, 05005 Ávila, Spain;
| | - Pierre Schembri-Wismayer
- Department of Anatomy, Faculty of Medicine and Surgery, University of Malta, MSD 2080 Msida, Malta;
| | - María Beatriz Durán-Alonso
- Department of Biochemistry and Molecular Biology and Physiology, Faculty of Medicine, University of Valladolid, 47005 Valladolid, Spain
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Rambarack N, Fodder K, Murthy M, Toomey C, de Silva R, Heutink P, Humphrey J, Raj T, Lashley T, Bettencourt C. DNA methylation as a contributor to dysregulation of STX6 and other frontotemporal lobar degeneration genetic risk-associated loci. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.21.634065. [PMID: 39975316 PMCID: PMC11838521 DOI: 10.1101/2025.01.21.634065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/21/2025]
Abstract
Frontotemporal Lobar Degeneration (FTLD) represents a spectrum of clinically, genetically, and pathologically heterogeneous neurodegenerative disorders characterised by progressive atrophy of the frontal and temporal lobes of the brain. The two major FTLD pathological subgroups are FTLD-TDP and FTLD-tau. While the majority of FTLD cases are sporadic, heterogeneity also exists within the familial cases, typically involving mutations in MAPT, GRN or C9orf72, which is not fully explained by known genetic mechanisms. We sought to address this gap by investigating the effect of epigenetic modifications, specifically DNA methylation variation, on genes associated with FTLD genetic risk in different FTLD subtypes. We compiled a list of genes associated with genetic risk of FTLD using text-mining databases and literature searches. Frontal cortex DNA methylation profiles were derived from three FTLD datasets containing different subgroups of FTLD-TDP and FTLD-tau: FTLD1m (N = 23) containing FTLD-TDP type A C9orf72 mutation carriers and TDP Type C sporadic cases, FTLD2m (N = 48) containing FTLD-Tau MAPT mutation carriers, FTLD-TDP Type A GRN mutation carriers, and FTLD-TDP Type B C9orf72 mutation carriers and FTLD3m (N = 163) progressive supranuclear palsy (PSP) cases, and corresponding controls. To investigate the downstream effects of DNA methylation further, we then leveraged transcriptomic and proteomic datasets for FTLD cases and controls to examine gene and protein expression levels. Our analysis revealed shared promoter region hypomethylation in STX6 across FTLD-TDP and FTLD-tau subtypes, though the largest effect size was observed in the PSP cases compared to controls (delta-beta = -32%, adjusted-p value=0.002). We also observed dysregulation of the STX6 gene and protein expression across FTLD subtypes. Additionally, we performed a detailed examination of MAPT, GRN and C9orf72 in subtypes with and without the presence of the genetic mutations and observed nominally significant differentially methylated CpGs in variable positions across the genes, often with unique patterns and downstream consequences in gene/protein expression in mutation carriers. We highlight the contribution of DNA methylation at different gene regions in regulating the expression of genes previously associated with genetic risk of FTLD, including STX6. We analysed the relationship of subtypes and presence of mutations with this epigenetic mechanism to increase our understanding of how these mechanisms interact in FTLD.
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Affiliation(s)
- Naiomi Rambarack
- Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK
| | - Katherine Fodder
- Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK
| | - Megha Murthy
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK
| | - Christina Toomey
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK
- The Francis Crick Institute, London, UK
| | - Rohan de Silva
- Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, UK
- Reta Lila Weston Institute, UCL Queen Square Institute of Neurology, London, UK
| | - Peter Heutink
- German Center for Neurodegenerative Diseases, Tübingen, Germany
| | - Jack Humphrey
- Nash Family Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY USA
| | - Towfique Raj
- Nash Family Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY USA
| | - Tammaryn Lashley
- Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK
| | - Conceição Bettencourt
- Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, London, UK
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Tsuruta M, Shil S, Taniguchi S, Kawauchi K, Miyoshi D. The role of cytosine methylation in regulating the topology and liquid-liquid phase separation of DNA G-quadruplexes. Chem Sci 2025:d4sc06959e. [PMID: 39935503 PMCID: PMC11808335 DOI: 10.1039/d4sc06959e] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2024] [Accepted: 01/28/2025] [Indexed: 02/13/2025] Open
Abstract
Aberrant expansion of GGGGCC DNA repeats that form G-quadruplexes (G4) is the main cause of amyotrophic lateral sclerosis (ALS). Expanded GGGGCC repeats induce liquid-liquid phase separation (LLPS) through their interaction with cellular proteins. Furthermore, GGGGCC expansion induces cytosine methylation (mC). Previous studies have shown that even slight chemical modifications of RNAs and proteins can drastically affect their LLPS ability, yet the relationship between LLPS and epigenetic DNA modifications like mC remains unexplored. As a model system, we investigated the effects of mC on LLPS induced by GGGGCC repeat DNAs and show for the first time that mC suppresses LLPS by altering the topology of G4 from being parallel to antiparallel.
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Affiliation(s)
- Mitsuki Tsuruta
- Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University 7-1-20 Minatojima-minamimachi, Chuo-ku Kobe Hyogo 650-0047 Japan
| | - Sumit Shil
- Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University 7-1-20 Minatojima-minamimachi, Chuo-ku Kobe Hyogo 650-0047 Japan
| | - Shinya Taniguchi
- Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University 7-1-20 Minatojima-minamimachi, Chuo-ku Kobe Hyogo 650-0047 Japan
| | - Keiko Kawauchi
- Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University 7-1-20 Minatojima-minamimachi, Chuo-ku Kobe Hyogo 650-0047 Japan
| | - Daisuke Miyoshi
- Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University 7-1-20 Minatojima-minamimachi, Chuo-ku Kobe Hyogo 650-0047 Japan
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McCallister TX, Lim CKW, Singh M, Zhang S, Ahsan NS, Terpstra WM, Xiong AY, Zeballos C MA, Powell JE, Drnevich J, Kang Y, Gaj T. A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD. Nat Commun 2025; 16:460. [PMID: 39779681 PMCID: PMC11711314 DOI: 10.1038/s41467-024-55548-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Accepted: 12/11/2024] [Indexed: 01/11/2025] Open
Abstract
An abnormal expansion of a GGGGCC (G4C2) hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we develop here a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.
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Affiliation(s)
- Tristan X McCallister
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Colin K W Lim
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Mayuri Singh
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Sijia Zhang
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Najah S Ahsan
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - William M Terpstra
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Alisha Y Xiong
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - M Alejandra Zeballos C
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Jackson E Powell
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Jenny Drnevich
- High-Performance Biological Computing, Roy J. Carver Biotechnology Center, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Yifei Kang
- High-Performance Biological Computing, Roy J. Carver Biotechnology Center, University of Illinois Urbana-Champaign, Urbana, IL, USA
| | - Thomas Gaj
- Department of Bioengineering, The Grainger College of Engineering, University of Illinois Urbana-Champaign, Urbana, IL, USA.
- Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA.
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van Zundert B, Montecino M. Epigenetics in Neurodegenerative Diseases. Subcell Biochem 2025; 108:73-109. [PMID: 39820861 DOI: 10.1007/978-3-031-75980-2_3] [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] [Indexed: 01/19/2025]
Abstract
Healthy brain functioning requires a continuous fine-tuning of gene expression, involving changes in the epigenetic landscape and 3D chromatin organization. Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) are three multifactorial neurodegenerative diseases (NDDs) that are partially explained by genetics (gene mutations and genetic risk factors) and influenced by non-genetic factors (i.e., aging, lifestyle, and environmental conditions). Examining comprehensive studies of global and locus-specific (epi)genomic and transcriptomic alterations in human and mouse brain samples at the cell-type resolution has uncovered important phenomena associated with AD. First, DNA methylation and histone marks at promoters contribute to transcriptional dysregulation of genes that are directly implicated in AD pathogenesis (i.e., APP), neuroplasticity and cognition (i.e., PSD95), and microglial activation (i.e., TREM2). Second, the presence of AD genetic risk variants in cell-type-specific distal enhancers (i.e., BIN1 in microglia) alters transcription, presumably by disrupting associated enhancer-promoter interactions and chromatin looping. Third, epigenomic erosion is associated with widespread transcriptional disruption and cell identity loss. And fourth, aging, high cholesterol, air pollution, and pesticides have emerged as potential drivers of AD by inducing locus-specific and global epigenetic modifications that impact key AD-related pathways. Epigenetic studies in ALS/FTD also provide evidence that genetic and non-genetic factors alter gene expression profiles in neurons and astrocytes through aberrant epigenetic mechanisms. We additionally overview the recent development of potential new therapeutic strategies involving (epi)genetic editing and the use of small chromatin-modifying molecules (epidrugs).
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Affiliation(s)
- Brigitte van Zundert
- Faculty of Medicine and Faculty of Life Sciences, Institute of Biomedical Sciences (ICB), Universidad Andres Bello, Santiago, Chile.
- Millennium Nucleus of Neuroepigenetics and Plasticity (EpiNeuro), Santiago, Chile.
- Department of Neurology, University of Massachusetts Chan Medical School (UMMS), Worcester, MA, USA.
| | - Martin Montecino
- Faculty of Medicine and Faculty of Life Sciences, Institute of Biomedical Sciences (ICB), Universidad Andres Bello, Santiago, Chile.
- Millennium Nucleus of Neuroepigenetics and Plasticity (EpiNeuro), Santiago, Chile.
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Udine E, Finch NA, DeJesus-Hernandez M, Jackson JL, Baker MC, Saravanaperumal SA, Wieben E, Ebbert MTW, Shah J, Petrucelli L, Rademakers R, Oskarsson B, van Blitterswijk M. Targeted long-read sequencing to quantify methylation of the C9orf72 repeat expansion. Mol Neurodegener 2024; 19:99. [PMID: 39709476 DOI: 10.1186/s13024-024-00790-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2024] [Accepted: 12/12/2024] [Indexed: 12/23/2024] Open
Abstract
BACKGROUND The gene C9orf72 harbors a non-coding hexanucleotide repeat expansion known to cause amyotrophic lateral sclerosis and frontotemporal dementia. While previous studies have estimated the length of this repeat expansion in multiple tissues, technological limitations have impeded researchers from exploring additional features, such as methylation levels. METHODS We aimed to characterize C9orf72 repeat expansions using a targeted, amplification-free long-read sequencing method. Our primary goal was to determine the presence and subsequent quantification of observed methylation in the C9orf72 repeat expansion. In addition, we measured the repeat length and purity of the expansion. To do this, we sequenced DNA extracted from blood for 27 individuals with an expanded C9orf72 repeat. RESULTS For these individuals, we obtained a total of 7,765 on-target reads, including 1,612 fully covering the expanded allele. Our in-depth analysis revealed that the expansion itself is methylated, with great variability in total methylation levels observed, as represented by the proportion of methylated CpGs (13 to 66%). Interestingly, we demonstrated that the expanded allele is more highly methylated than the wild-type allele (P-Value = 2.76E-05) and that increased methylation levels are observed in longer repeat expansions (P-Value = 1.18E-04). Furthermore, methylation levels correlate with age at collection (P-Value = 3.25E-04) as well as age at disease onset (P-Value = 0.020). Additionally, we detected repeat lengths up to 4,088 repeats (~ 25 kb) and found that the expansion contains few interruptions in the blood. CONCLUSIONS Taken together, our study demonstrates robust ability to quantify methylation of the expanded C9orf72 repeat, capturing differences between individuals harboring this expansion and revealing clinical associations.
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Affiliation(s)
- Evan Udine
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
- Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Jacksonville, FL, USA
| | - NiCole A Finch
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
| | | | - Jazmyne L Jackson
- Fels Cancer Institute for Personalized Medicine, Temple University, Lewis Katz School of Medicine, Philadelphia, PA, USA
| | - Matthew C Baker
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
| | | | - Eric Wieben
- Genome Analysis Core, Mayo Clinic, Rochester, MN, USA
| | - Mark T W Ebbert
- Department of Neuroscience, University of Kentucky Sanders-Brown Center on Aging, Lexington, KY, USA
| | - Jaimin Shah
- Department of Neurology, Mayo Clinic, Jacksonville, FL, USA
| | - Leonard Petrucelli
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
- Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Jacksonville, FL, USA
| | - Rosa Rademakers
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
- VIB Center for Molecular Neurology, Antwerp, Belgium
- Department of Biomedical Science, University of Antwerp, Antwerp, Belgium
| | | | - Marka van Blitterswijk
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.
- Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic, Jacksonville, FL, USA.
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9
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Gilbert JW, Kennedy Z, Godinho BM, Summers A, Weiss A, Echeverria D, Bramato B, McHugh N, Cooper D, Yamada K, Hassler M, Tran H, Gao FB, Brown RH, Khvorova A. Identification of selective and non-selective C9ORF72 targeting in vivo active siRNAs. MOLECULAR THERAPY. NUCLEIC ACIDS 2024; 35:102291. [PMID: 39233852 PMCID: PMC11372813 DOI: 10.1016/j.omtn.2024.102291] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Accepted: 07/29/2024] [Indexed: 09/06/2024]
Abstract
A hexanucleotide (G4C2) repeat expansion (HRE) within intron one of C9ORF72 is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). C9ORF72 haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for C9ORF72 driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different C9ORF72 transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing C9ORF72 mRNA variants are found to preferentially localize to the nucleus, and thus C9ORF72 mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting C9ORF72 mRNA variants specifically or non-selectively reduce the expression of C9ORF72 mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all C9ORF72 mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing C9ORF72 mRNA transcripts. Combined, these data support RNAi-based degradation of C9ORF72 as a potential therapeutic paradigm.
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Affiliation(s)
| | | | | | | | - Alexandra Weiss
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA 01655, USA
| | | | | | | | - David Cooper
- RNA Therapeutic Institute, Worcester, MA 01655, USA
| | - Ken Yamada
- RNA Therapeutic Institute, Worcester, MA 01655, USA
| | | | - Hélène Tran
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA 01655, USA
| | - Fen Biao Gao
- RNA Therapeutic Institute, Worcester, MA 01655, USA
| | - Robert H. Brown
- Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA 01655, USA
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Svikle Z, Paramonova N, Siliņš E, Pahirko L, Zariņa L, Baumane K, Petrovski G, Sokolovska J. DNA Methylation Profiles of PSMA6, PSMB5, KEAP1, and HIF1A Genes in Patients with Type 1 Diabetes and Diabetic Retinopathy. Biomedicines 2024; 12:1354. [PMID: 38927561 PMCID: PMC11202151 DOI: 10.3390/biomedicines12061354] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2024] [Revised: 06/12/2024] [Accepted: 06/14/2024] [Indexed: 06/28/2024] Open
Abstract
We explored differences in the DNA methylation statuses of PSMA6, PSMB5, HIF1A, and KEAP1 gene promoter regions in patients with type 1 diabetes and different diabetic retinopathy (DR) stages. Study subjects included individuals with no DR (NDR, n = 41), those with non-proliferative DR (NPDR, n = 27), and individuals with proliferative DR or those who underwent laser photocoagulation (PDR/LPC, n = 46). DNA methylation was determined by Zymo OneStep qMethyl technique. The methylation of PSMA6 (NDR 5.9 (3.9-8.7) %, NPDR 4.5 (3.8-5.7) %, PDR/LPC 6.6 (4.7-10.7) %, p = 0.003) and PSMB5 (NDR 2.2 (1.9-3.7) %, NPDR 2.2 (1.9-3.0) %, PDR/LPC 3.2 (2.5-7.1) %, p < 0.01) differed across the groups. Consistent correlations were observed between the methylation levels of HIF1A and PSMA6 in all study groups. DNA methylation levels of PSMA6, PSMB5, and HIF1A genes were positively correlated with the duration of diabetes, HbA1c, and albuminuria in certain study groups. Univariate regression models revealed a significant association between the methylation level z-scores of PSMA6, PSMB5, and HIF1A and severe DR (PSMA6: OR = 1.96 (1.15; 3.33), p = 0.013; PSMB5: OR = 1.90 (1.14; 3.16), p = 0.013; HIF1A: OR = 3.19 (1.26; 8.06), p = 0.014). PSMB5 remained significantly associated with DR in multivariate analysis. Our findings suggest significant associations between the severity of DR and the DNA methylation levels of the genes PSMA6, PSMB5, and HIF1A, but not KEAP1 gene.
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Affiliation(s)
- Zane Svikle
- Faculty of Medicine, University of Latvia, Jelgavas Street 3, LV 1004 Riga, Latvia; (Z.S.); (L.Z.); (K.B.)
| | - Natalia Paramonova
- Institute of Biology, University of Latvia, Jelgavas Street 1, LV 1004 Riga, Latvia;
| | - Emīls Siliņš
- Faculty of Physics, Mathematics and Optometry, University of Latvia, Jelgavas Street 3, LV 1004 Riga, Latvia; (E.S.); (L.P.)
| | - Leonora Pahirko
- Faculty of Physics, Mathematics and Optometry, University of Latvia, Jelgavas Street 3, LV 1004 Riga, Latvia; (E.S.); (L.P.)
| | - Līga Zariņa
- Faculty of Medicine, University of Latvia, Jelgavas Street 3, LV 1004 Riga, Latvia; (Z.S.); (L.Z.); (K.B.)
- Ophthalmology Department, Riga East University Hospital, Hipokrata Street 2, LV 1038 Riga, Latvia
| | - Kristīne Baumane
- Faculty of Medicine, University of Latvia, Jelgavas Street 3, LV 1004 Riga, Latvia; (Z.S.); (L.Z.); (K.B.)
- Ophthalmology Department, Riga East University Hospital, Hipokrata Street 2, LV 1038 Riga, Latvia
| | - Goran Petrovski
- Center of Eye Research and Innovative Diagnostics, Department of Ophthalmology, Oslo University Hospital, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0372 Oslo, Norway;
| | - Jelizaveta Sokolovska
- Faculty of Medicine, University of Latvia, Jelgavas Street 3, LV 1004 Riga, Latvia; (Z.S.); (L.Z.); (K.B.)
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11
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Hernan-Godoy M, Rouaux C. From Environment to Gene Expression: Epigenetic Methylations and One-Carbon Metabolism in Amyotrophic Lateral Sclerosis. Cells 2024; 13:967. [PMID: 38891099 PMCID: PMC11171807 DOI: 10.3390/cells13110967] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2024] [Revised: 05/22/2024] [Accepted: 05/28/2024] [Indexed: 06/21/2024] Open
Abstract
The etiology of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) is complex and considered multifactorial. The majority of ALS cases are sporadic, but familial cases also exist. Estimates of heritability range from 8% to 61%, indicating that additional factors beyond genetics likely contribute to ALS. Numerous environmental factors are considered, which may add up and synergize throughout an individual's lifetime building its unique exposome. One level of integration between genetic and environmental factors is epigenetics, which results in alterations in gene expression without modification of the genome sequence. Methylation reactions, targeting DNA or histones, represent a large proportion of epigenetic regulations and strongly depend on the availability of methyl donors provided by the ubiquitous one-carbon (1C) metabolism. Thus, understanding the interplay between exposome, 1C metabolism, and epigenetic modifications will likely contribute to elucidating the mechanisms underlying altered gene expression related to ALS and to developing targeted therapeutic interventions. Here, we review evidence for 1C metabolism alterations and epigenetic methylation dysregulations in ALS, with a focus on the impairments reported in neural tissues, and discuss these environmentally driven mechanisms as the consequences of cumulative exposome or late environmental hits, but also as the possible result of early developmental defects.
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Affiliation(s)
| | - Caroline Rouaux
- Inserm UMR_S 1329, Strasbourg Translational Neuroscience and Psychiatry, Université de Strasbourg, Centre de Recherche en Biomédecine de Strasbourg, 1 Rue Eugène Boeckel, 67 000 Strasbourg, France;
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12
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Ramos-Campoy O, Comas-Albertí A, Hervás D, Borrego-Écija S, Bosch B, Sandoval J, Fort-Aznar L, Moreno-Izco F, Fernández-Villullas G, Molina-Porcel L, Balasa M, Lladó A, Sánchez-Valle R, Antonell A. Genome-Wide DNA Methylation in Early-Onset-Dementia Patients Brain Tissue and Lymphoblastoid Cell Lines. Int J Mol Sci 2024; 25:5445. [PMID: 38791483 PMCID: PMC11121630 DOI: 10.3390/ijms25105445] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Revised: 05/08/2024] [Accepted: 05/13/2024] [Indexed: 05/26/2024] Open
Abstract
Epigenetics, a potential underlying pathogenic mechanism of neurodegenerative diseases, has been in the scope of several studies performed so far. However, there is a gap in regard to analyzing different forms of early-onset dementia and the use of Lymphoblastoid cell lines (LCLs). We performed a genome-wide DNA methylation analysis on sixty-four samples (from the prefrontal cortex and LCLs) including those taken from patients with early-onset forms of Alzheimer's disease (AD) and frontotemporal dementia (FTD) and healthy controls. A beta regression model and adjusted p-values were used to obtain differentially methylated positions (DMPs) via pairwise comparisons. A correlation analysis of DMP levels with Clariom D array gene expression data from the same cohort was also performed. The results showed hypermethylation as the most frequent finding in both tissues studied in the patient groups. Biological significance analysis revealed common pathways altered in AD and FTD patients, affecting neuron development, metabolism, signal transduction, and immune system pathways. These alterations were also found in LCL samples, suggesting the epigenetic changes might not be limited to the central nervous system. In the brain, CpG methylation presented an inverse correlation with gene expression, while in LCLs, we observed mainly a positive correlation. This study enhances our understanding of the biological pathways that are associated with neurodegeneration, describes differential methylation patterns, and suggests LCLs are a potential cell model for studying neurodegenerative diseases in earlier clinical phases than brain tissue.
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Affiliation(s)
- Oscar Ramos-Campoy
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Aina Comas-Albertí
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - David Hervás
- Department of Applied Statistics and Operations Research and Quality, Universitat Politècnica de València, 46022 Valencia, Spain
| | - Sergi Borrego-Écija
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Beatriz Bosch
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Juan Sandoval
- Epigenomics Core Facility, Health Research Institute La Fe, 46026 Valencia, Spain
| | - Laura Fort-Aznar
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Fermín Moreno-Izco
- Cognitive Disorders Unit, Department of Neurology, Hospital Universitario Donostia, 20014 San Sebastian, Spain
- Instituto de Investigación Sanitaria Biogipuzkoa, Neurosciences Area, Group of Neurodegenerative Diseases, 20014 San Sebastian, Spain
| | - Guadalupe Fernández-Villullas
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Laura Molina-Porcel
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
- Neurological Tissue Bank, Biobank-Hospital Clinic-IDIBAPS, 08036 Barcelona, Spain
| | - Mircea Balasa
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Albert Lladó
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Raquel Sánchez-Valle
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
- Facultat de Medicina i Ciències de la Salut, Institut de Neurociències, Universitat de Barcelona (UB), 08036 Barcelona, Spain
| | - Anna Antonell
- Alzheimer's Disease and Other Cognitive Disorders Unit, Neurology Service, Hospital Clínic de Barcelona, FRCB-IDIBAPS, Universitat de Barcelona (UB), 08036 Barcelona, Spain
- Facultat de Medicina i Ciències de la Salut, Institut de Neurociències, Universitat de Barcelona (UB), 08036 Barcelona, Spain
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13
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Jagaraj CJ, Shadfar S, Kashani SA, Saravanabavan S, Farzana F, Atkin JD. Molecular hallmarks of ageing in amyotrophic lateral sclerosis. Cell Mol Life Sci 2024; 81:111. [PMID: 38430277 PMCID: PMC10908642 DOI: 10.1007/s00018-024-05164-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Revised: 01/21/2024] [Accepted: 02/06/2024] [Indexed: 03/03/2024]
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal, severely debilitating and rapidly progressing disorder affecting motor neurons in the brain, brainstem, and spinal cord. Unfortunately, there are few effective treatments, thus there remains a critical need to find novel interventions that can mitigate against its effects. Whilst the aetiology of ALS remains unclear, ageing is the major risk factor. Ageing is a slowly progressive process marked by functional decline of an organism over its lifespan. However, it remains unclear how ageing promotes the risk of ALS. At the molecular and cellular level there are specific hallmarks characteristic of normal ageing. These hallmarks are highly inter-related and overlap significantly with each other. Moreover, whilst ageing is a normal process, there are striking similarities at the molecular level between these factors and neurodegeneration in ALS. Nine ageing hallmarks were originally proposed: genomic instability, loss of telomeres, senescence, epigenetic modifications, dysregulated nutrient sensing, loss of proteostasis, mitochondrial dysfunction, stem cell exhaustion, and altered inter-cellular communication. However, these were recently (2023) expanded to include dysregulation of autophagy, inflammation and dysbiosis. Hence, given the latest updates to these hallmarks, and their close association to disease processes in ALS, a new examination of their relationship to pathophysiology is warranted. In this review, we describe possible mechanisms by which normal ageing impacts on neurodegenerative mechanisms implicated in ALS, and new therapeutic interventions that may arise from this.
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Affiliation(s)
- Cyril Jones Jagaraj
- MND Research Centre, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW, 2109, Australia
| | - Sina Shadfar
- MND Research Centre, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW, 2109, Australia
| | - Sara Assar Kashani
- MND Research Centre, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW, 2109, Australia
| | - Sayanthooran Saravanabavan
- MND Research Centre, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW, 2109, Australia
| | - Fabiha Farzana
- MND Research Centre, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW, 2109, Australia
| | - Julie D Atkin
- MND Research Centre, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, 75 Talavera Road, Sydney, NSW, 2109, Australia.
- La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Melbourne, VIC, 3086, Australia.
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14
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Giannini LAA, Boers RG, van der Ende EL, Poos JM, Jiskoot LC, Boers JB, van IJcken WFJ, Dopper EG, Pijnenburg YAL, Seelaar H, Meeter LH, van Rooij JGJ, Scheper W, Gribnau J, van Swieten JC. Distinctive cell-free DNA methylation characterizes presymptomatic genetic frontotemporal dementia. Ann Clin Transl Neurol 2024; 11:744-756. [PMID: 38481040 DOI: 10.1002/acn3.51997] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Revised: 12/01/2023] [Accepted: 12/27/2023] [Indexed: 03/27/2024] Open
Abstract
OBJECTIVE Methylation of plasma cell-free DNA (cfDNA) has potential as a marker of brain damage in neurodegenerative diseases such as frontotemporal dementia (FTD). Here, we study methylation of cfDNA in presymptomatic and symptomatic carriers of genetic FTD pathogenic variants, next to healthy controls. METHODS cfDNA was isolated from cross-sectional plasma of 10 presymptomatic carriers (4 C9orf72, 4 GRN, and 2 MAPT), 10 symptomatic carriers (4 C9orf72, 4 GRN, and 2 MAPT), and 9 healthy controls. Genome-wide methylation of cfDNA was determined using a high-resolution sequencing technique (MeD-seq). Cumulative scores based on the identified differentially methylated regions (DMRs) were estimated for presymptomatic carriers (vs. controls and symptomatic carriers), and reevaluated in a validation cohort (8 presymptomatic: 3 C9orf72, 3 GRN, and 2 MAPT; 26 symptomatic: 7 C9orf72, 6 GRN, 12 MAPT, and 1 TARDBP; 13 noncarriers from genetic FTD families). RESULTS Presymptomatic carriers showed a distinctive methylation profile compared to healthy controls and symptomatic carriers. Cumulative DMR scores in presymptomatic carriers enabled to significantly differentiate presymptomatic carriers from healthy controls (p < 0.001) and symptomatic carriers (p < 0.001). In the validation cohort, these scores differentiated presymptomatic carriers from symptomatic carriers (p ≤ 0.007) only. Transcription-start-site methylation in presymptomatic carriers, generally associated with gene downregulation, was enriched for genes involved in ubiquitin-dependent processes, while gene body methylation, generally associated with gene upregulation, was enriched for genes involved in neuronal cell processes. INTERPRETATION A distinctive methylation profile of cfDNA characterizes the presymptomatic stage of genetic FTD, and could reflect neuronal death in this stage.
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Affiliation(s)
- Lucia A A Giannini
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Ruben G Boers
- Department of Developmental Biology, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Emma L van der Ende
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
- Department of Clinical Chemistry, Amsterdam Neuroscience, Amsterdam UMC, Vrije Universiteit, Amsterdam, The Netherlands
| | - Jackie M Poos
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Lize C Jiskoot
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Joachim B Boers
- Department of Developmental Biology, Erasmus Medical Center, Rotterdam, The Netherlands
| | - Wilfred F J van IJcken
- Erasmus Center for Biomics, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Elise G Dopper
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Yolande A L Pijnenburg
- Alzheimer Center Amsterdam, Neurology, Vrije Universiteit, Amsterdam UMC location Vumc, Amsterdam, The Netherlands
- Amsterdam Neuroscience, Neurodegeneration, Amsterdam, The Netherlands
| | - Harro Seelaar
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Lieke H Meeter
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Jeroen G J van Rooij
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Wiep Scheper
- Amsterdam Neuroscience, Neurodegeneration, Amsterdam, The Netherlands
- Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Faculty of Science, Vrije Universiteit, Amsterdam, The Netherlands
- Department of Human Genetics, Vrije Universiteit, Amsterdam UMC location Vumc, Amsterdam, The Netherlands
| | - Joost Gribnau
- Department of Developmental Biology, Erasmus Medical Center, Rotterdam, The Netherlands
| | - John C van Swieten
- Department of Neurology, Alzheimer Center Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
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15
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Rautila OS, Kaivola K, Rautila H, Hokkanen L, Launes J, Strandberg TE, Laaksovirta H, Palmio J, Tienari PJ. The shared ancestry between the C9orf72 hexanucleotide repeat expansion and intermediate-length alleles using haplotype sharing trees and HAPTK. Am J Hum Genet 2024; 111:383-392. [PMID: 38242117 PMCID: PMC10870140 DOI: 10.1016/j.ajhg.2023.12.019] [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: 09/08/2023] [Revised: 12/15/2023] [Accepted: 12/18/2023] [Indexed: 01/21/2024] Open
Abstract
The C9orf72 hexanucleotide repeat expansion (HRE) is a common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The inheritance is autosomal dominant, but a high proportion of subjects with the mutation are simplex cases. One possible explanation is de novo expansions of unstable intermediate-length alleles (IAs). Using haplotype sharing trees (HSTs) with the haplotype analysis tool kit (HAPTK), we derived majority-based ancestral haplotypes of HRE samples and discovered that IAs containing ≥18-20 repeats share large haplotypes in common with the HRE. Using HSTs of HRE and IA samples, we demonstrate that the longer IA haplotypes are largely indistinguishable from HRE haplotypes and that several ≥18-20 IA haplotypes share over 5 Mb (>600 markers) haplotypes in common with the HRE haplotypes. These analysis tools allow physical understanding of the haplotype blocks shared with the majority-based ancestral haplotype. Our results demonstrate that the haplotypes with longer IAs belong to the same pool of haplotypes as the HRE and suggest that longer IAs represent potential premutation alleles.
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Affiliation(s)
- Osma S Rautila
- Translational Immunology, Research Programs Unit, University of Helsinki, Helsinki, Finland; Department of Neurology, Helsinki University Hospital, Helsinki, Finland.
| | - Karri Kaivola
- Translational Immunology, Research Programs Unit, University of Helsinki, Helsinki, Finland; Department of Neurology, Helsinki University Hospital, Helsinki, Finland
| | - Harri Rautila
- Translational Immunology, Research Programs Unit, University of Helsinki, Helsinki, Finland
| | - Laura Hokkanen
- Department of Psychology and Logopedics, University of Helsinki, Helsinki, Finland
| | - Jyrki Launes
- Department of Psychology and Logopedics, University of Helsinki, Helsinki, Finland
| | - Timo E Strandberg
- University of Helsinki and Helsinki University Hospital, Helsinki, Finland
| | - Hannu Laaksovirta
- Department of Neurology, Helsinki University Hospital, Helsinki, Finland
| | - Johanna Palmio
- Neuromuscular Research Center, Tampere University and Tampere University Hospital, Tampere, Finland
| | - Pentti J Tienari
- Translational Immunology, Research Programs Unit, University of Helsinki, Helsinki, Finland; Department of Neurology, Helsinki University Hospital, Helsinki, Finland
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16
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McCallister TX, Lim CKW, Terpstra WM, Alejandra Zeballos C M, Zhang S, Powell JE, Gaj T. A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.12.571328. [PMID: 38168370 PMCID: PMC10760048 DOI: 10.1101/2023.12.12.571328] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.
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Affiliation(s)
- Tristan X. McCallister
- Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
| | - Colin K. W. Lim
- Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
| | - William M. Terpstra
- Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
| | - M. Alejandra Zeballos C
- Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
| | - Sijia Zhang
- Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
| | - Jackson E. Powell
- Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
| | - Thomas Gaj
- Department of Bioengineering, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
- Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801, USA
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Rizzuti M, Sali L, Melzi V, Scarcella S, Costamagna G, Ottoboni L, Quetti L, Brambilla L, Papadimitriou D, Verde F, Ratti A, Ticozzi N, Comi GP, Corti S, Gagliardi D. Genomic and transcriptomic advances in amyotrophic lateral sclerosis. Ageing Res Rev 2023; 92:102126. [PMID: 37972860 DOI: 10.1016/j.arr.2023.102126] [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: 06/01/2023] [Revised: 11/09/2023] [Accepted: 11/10/2023] [Indexed: 11/19/2023]
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder and the most common motor neuron disease. ALS shows substantial clinical and molecular heterogeneity. In vitro and in vivo models coupled with multiomic techniques have provided important contributions to unraveling the pathomechanisms underlying ALS. To date, despite promising results and accumulating knowledge, an effective treatment is still lacking. Here, we provide an overview of the literature on the use of genomics, epigenomics, transcriptomics and microRNAs to deeply investigate the molecular mechanisms developing and sustaining ALS. We report the most relevant genes implicated in ALS pathogenesis, discussing the use of different high-throughput sequencing techniques and the role of epigenomic modifications. Furthermore, we present transcriptomic studies discussing the most recent advances, from microarrays to bulk and single-cell RNA sequencing. Finally, we discuss the use of microRNAs as potential biomarkers and promising tools for molecular intervention. The integration of data from multiple omic approaches may provide new insights into pathogenic pathways in ALS by shedding light on diagnostic and prognostic biomarkers, helping to stratify patients into clinically relevant subgroups, revealing novel therapeutic targets and supporting the development of new effective therapies.
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Affiliation(s)
- Mafalda Rizzuti
- Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Luca Sali
- Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Valentina Melzi
- Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Simone Scarcella
- Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy
| | - Gianluca Costamagna
- Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy
| | - Linda Ottoboni
- Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy
| | - Lorenzo Quetti
- Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Lorenzo Brambilla
- Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | | | - Federico Verde
- Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy; Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan, Italy
| | - Antonia Ratti
- Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan, Italy; Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milan, Italy
| | - Nicola Ticozzi
- Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy; Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan, Italy
| | - Giacomo Pietro Comi
- Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy; Neuromuscular and Rare Diseases Unit, Department of Neuroscience, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
| | - Stefania Corti
- Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy.
| | - Delia Gagliardi
- Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy.
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18
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Reis AHDEO, Figalo LB, Orsini M, Lemos B. The implications of DNA methylation for amyotrophic lateral sclerosis. AN ACAD BRAS CIENC 2023; 95:e20230277. [PMID: 37909610 DOI: 10.1590/0001-3765202320230277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2023] [Accepted: 07/11/2023] [Indexed: 11/03/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a complex and serious neurodegenerative disorder that develops in consequence of the progressive loss of the upper and lower motor neurons. Cases of ALS are classified as sporadic (sALS), or familial (fALS). Over 90% of cases are sALS, while roughly 10% are related to inherited genetic mutations (fALS). Approximately 70% of the genetic mutations that contribute to fALS have been identified. On the other hand, the majority of the sALS cases have an undetermined genetic contributor and few mutations have been described, despite the advanced genetic analysis methods. Also, several factors contribute to the onset and progression of ALS. Numerous lines of evidence indicate that epigenetic changes are linked to aging, as well as neurodegenerative disorders, such as ALS. In most cases, they act as the heritable regulation of transcription by DNA methylation, histone modification and expression of noncoding RNAs. Mechanisms involving aberrant DNA methylation could be relevant to human ALS pathobiology and therapeutic targeting. Despite advances in research to find factors associated with ALS and more effective treatments, this disease remains complex and has low patient survival. Here, we provide a narrative review of the role of DNA methylation for this complex neurodegenerative disorder.
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Affiliation(s)
- Adriana Helena DE Oliveira Reis
- Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Genética, Pavilhão Haroldo Lisboa da Cunha, Rua São Francisco Xavier, 524, Sala 501F, 20550-900 Rio de Janeiro, RJ, Brazil
| | - Luna B Figalo
- Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Genética, Pavilhão Haroldo Lisboa da Cunha, Rua São Francisco Xavier, 524, Sala 501F, 20550-900 Rio de Janeiro, RJ, Brazil
| | - Marco Orsini
- Programa de Pós-Graduação em Vigilância em Saúde, Universidade Iguaçu, Av. Abílio Augusto Távora, 2134, 26260-045 Nova Iguaçu, RJ, Brazil
- Universidade Federal do Rio de Janeiro, Departamento de Psiquiatria, Av. Venceslau Brás, 71, Botafogo, 22290-140 Rio de Janeiro, RJ, Brazil
| | - Bernardo Lemos
- Coit Center for longevity and Neurotheraéutics, Departament of pharmacology and toxicology, R Ken Coit College of Pharmacy, University of Arizona, 1703 E. Mabel St. PO Box 210207 Tucson, Arizona, USA
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19
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Liu Y, Huang Z, Liu H, Ji Z, Arora A, Cai D, Wang H, Liu M, Simko EAJ, Zhang Y, Periz G, Liu Z, Wang J. DNA-initiated epigenetic cascades driven by C9orf72 hexanucleotide repeat. Neuron 2023; 111:1205-1221.e9. [PMID: 36822200 PMCID: PMC10121948 DOI: 10.1016/j.neuron.2023.01.022] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2022] [Revised: 12/08/2022] [Accepted: 01/27/2023] [Indexed: 02/24/2023]
Abstract
The C9orf72 hexanucleotide repeat expansion (HRE) is the most frequent genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we describe the pathogenic cascades that are initiated by the C9orf72 HRE DNA. The HRE DNA binds to its protein partner DAXX and promotes its liquid-liquid phase separation, which is capable of reorganizing genomic structures. An HRE-dependent nuclear accumulation of DAXX drives chromatin remodeling and epigenetic changes such as histone hypermethylation and hypoacetylation in patient cells. While regulating global gene expression, DAXX plays a key role in the suppression of basal and stress-inducible expression of C9orf72 via chromatin remodeling and epigenetic modifications of the promoter of the major C9orf72 transcript. Downregulation of DAXX or rebalancing the epigenetic modifications mitigates the stress-induced sensitivity of C9orf72-patient-derived motor neurons. These studies reveal a C9orf72 HRE DNA-dependent regulatory mechanism for both local and genomic architectural changes in the relevant diseases.
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Affiliation(s)
- Yang Liu
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Zhiyuan Huang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Honghe Liu
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Zhicheng Ji
- Department of Biostatistics and Bioinformatics, Duke University School of Medicine, Duke University, Durham, NC 27710, USA
| | - Amit Arora
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Danfeng Cai
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA; Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Hongjin Wang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Mingming Liu
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Eric A J Simko
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Yanjun Zhang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Goran Periz
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Zhe Liu
- Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA
| | - Jiou Wang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
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20
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Boeynaems S, Ma XR, Yeong V, Ginell GM, Chen JH, Blum JA, Nakayama L, Sanyal A, Briner A, Haver DV, Pauwels J, Ekman A, Schmidt HB, Sundararajan K, Porta L, Lasker K, Larabell C, Hayashi MAF, Kundaje A, Impens F, Obermeyer A, Holehouse AS, Gitler AD. Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.09.531820. [PMID: 36945394 PMCID: PMC10028949 DOI: 10.1101/2023.03.09.531820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/11/2023]
Abstract
Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name 'polycation poisoning'. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.
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Affiliation(s)
- Steven Boeynaems
- Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
- Jan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX 77030, USA
- Therapeutic Innovation Center (THINC), Baylor College of Medicine, Houston, TX 77030, USA
- Center for Alzheimer’s and Neurodegenerative Diseases (CAND), Texas Children’s Hospital, Houston, TX 77030, USA
- Dan L Duncan Comprehensive Cancer Center (DLDCCC), Baylor College of Medicine, Houston, TX 77030, USA
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - X. Rosa Ma
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Vivian Yeong
- Department of Chemical Engineering, Columbia University, New York, NY, 10027, USA
| | - Garrett M. Ginell
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA
- Center for Biomolecular Condensates, Washington University in St Louis, St. Louis, MO 63130, USA
| | - Jian-Hua Chen
- Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
- Department of Anatomy, University of California, San Francisco, CA 94143, USA
| | - Jacob A. Blum
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Lisa Nakayama
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Anushka Sanyal
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Adam Briner
- Clem Jones Centre for Ageing Dementia Research (CJCADR), Queensland Brain Institute (QBI), The University of Queensland, Brisbane, QLD 4072, Australia
| | - Delphi Van Haver
- VIB-UGent Center for Medical Biotechnology, 9000 Gent, Belgium
- VIB Proteomics Core, 9000 Gent, Belgium
- Department of Biochemistry, Ghent University, 9000 Gent, Belgium
| | - Jarne Pauwels
- VIB-UGent Center for Medical Biotechnology, 9000 Gent, Belgium
- VIB Proteomics Core, 9000 Gent, Belgium
- Department of Biochemistry, Ghent University, 9000 Gent, Belgium
| | - Axel Ekman
- Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
- Department of Anatomy, University of California, San Francisco, CA 94143, USA
| | - H. Broder Schmidt
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Kousik Sundararajan
- Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Lucas Porta
- Department of Pharmacology, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), Sao Paulo, Brazil
| | - Keren Lasker
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Carolyn Larabell
- Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
- Department of Anatomy, University of California, San Francisco, CA 94143, USA
| | - Mirian A. F. Hayashi
- Department of Pharmacology, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), Sao Paulo, Brazil
| | - Anshul Kundaje
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
- Department of Computer Science, Stanford University, Stanford, CA 94305, USA
| | - Francis Impens
- VIB-UGent Center for Medical Biotechnology, 9000 Gent, Belgium
- VIB Proteomics Core, 9000 Gent, Belgium
- Department of Biochemistry, Ghent University, 9000 Gent, Belgium
| | - Allie Obermeyer
- Department of Chemical Engineering, Columbia University, New York, NY, 10027, USA
| | - Alex S. Holehouse
- Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110, USA
- Center for Biomolecular Condensates, Washington University in St Louis, St. Louis, MO 63130, USA
| | - Aaron D. Gitler
- Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
- Chan Zuckerberg Biohub, San Francisco, CA 94158, USA
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21
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Younesian S, Yousefi AM, Momeny M, Ghaffari SH, Bashash D. The DNA Methylation in Neurological Diseases. Cells 2022; 11:3439. [PMID: 36359835 PMCID: PMC9657829 DOI: 10.3390/cells11213439] [Citation(s) in RCA: 55] [Impact Index Per Article: 18.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Accepted: 10/25/2022] [Indexed: 07/30/2023] Open
Abstract
DNA methylation is critical for the normal development and functioning of the human brain, such as the proliferation and differentiation of neural stem cells, synaptic plasticity, neuronal reparation, learning, and memory. Despite the physical stability of DNA and methylated DNA compared to other epigenetic modifications, some DNA methylation-based biomarkers have translated into clinical practice. Increasing reports indicate a strong association between DNA methylation profiles and various clinical outcomes in neurological diseases, making DNA methylation profiles valuable as novel clinical markers. In this review, we aim to discuss the latest evidence concerning DNA methylation alterations in the development of neurodegenerative, neurodevelopmental, and neuropsychiatric diseases. We also highlighted the relationship of DNA methylation alterations with the disease progression and outcome in many neurological diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, and autism.
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Affiliation(s)
- Samareh Younesian
- Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran 1971653313, Iran
| | - Amir-Mohammad Yousefi
- Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran 1971653313, Iran
| | - Majid Momeny
- The Brown Foundation Institute of Molecular Medicine, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA
| | - Seyed H. Ghaffari
- Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran 1411713135, Iran
| | - Davood Bashash
- Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences, Tehran 1971653313, Iran
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22
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Cao Y, Tian W, Wu J, Song X, Cao L, Luan X. DNA hypermethylation of NOTCH2NLC in neuronal intranuclear inclusion disease: a case-control study. J Neurol 2022; 269:6049-6057. [PMID: 35857137 DOI: 10.1007/s00415-022-11272-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2022] [Revised: 05/27/2022] [Accepted: 07/04/2022] [Indexed: 11/27/2022]
Abstract
BACKGROUND GGC repeat expansions in NOTCH2NLC gene have been recently proposed to cause neuronal intranuclear inclusion disease (NIID) via prevailing gain-of-function mechanism (protein and RNA toxicity). Nevertheless, increasing evidences suggest that epigenetics can also play a role in the pathogenesis of repeat-mediated disorders. METHODS In this study, using MethylTarget sequencing, we performed a quantitative analysis of the methylation status of 68 CpG sites located around the NOTCH2NLC promoter in 25 NIID patients and 25 age- and gender-matched healthy controls. We further explored the correlation of DNA methylation (DNAm) status with disease features and performed receiver operating characteristic (ROC) analysis. RESULTS DNAm levels of GGC repeats and adjacent CpG islands were higher in the NIID patients than in controls, independent of gender and family history. DNAm levels at 4 CpG sites (CpG_207, CpG_421, GpG_473 and CpG_523) were negatively correlated with age at onset, and DNAm levels at 7 CpG sites (CpG_25, CpG_298, CpG_336, CpG_374, CpG_411, CpG_421 and CpG_473) were positively correlated with GGC repeats. NIID patients had concomitant system symptoms besides nervous system symptoms, and negative correlations between NOTCH2NLC DNAm levels and the number of multi-systemic involvement were observed in the study. The area under the ROC curve at NOTCH2NLC DNAm level reached to 0.733 for the best cutoff point of 0.012. CONCLUSIONS Our findings suggested the aberrant DNAm status of the NOTCH2NLC promoter in NIID, and we explored the link between DNAm levels and disease features quantitatively for the first time, which may help to further explore pathogenic mechanism.
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Affiliation(s)
- Yuwen Cao
- Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
| | - Wotu Tian
- Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
| | - Jingying Wu
- Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China
| | - Xingwang Song
- Institute of Neuroscience and Department of Neurology, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
| | - Li Cao
- Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
| | - Xinghua Luan
- Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
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23
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Czuppa M, Dhingra A, Zhou Q, Schludi C, König L, Scharf E, Farny D, Dalmia A, Täger J, Castillo-Lizardo M, Katona E, Mori K, Aumer T, Schelter F, Müller M, Carell T, Kalliokoski T, Messinger J, Rizzu P, Heutink P, Edbauer D. Drug screen in iPSC-Neurons identifies nucleoside analogs as inhibitors of (G 4C 2) n expression in C9orf72 ALS/FTD. Cell Rep 2022; 39:110913. [PMID: 35675776 DOI: 10.1016/j.celrep.2022.110913] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2021] [Revised: 12/22/2021] [Accepted: 05/12/2022] [Indexed: 11/03/2022] Open
Abstract
An intronic (G4C2)n expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent. Hit compounds nearly abolish sense and antisense RNA foci and reduce expression of the repeat-containing nascent C9orf72 RNA transcript and its mature mRNA with minimal effects on global gene expression, suggesting that they specifically act on repeat transcription. Importantly, decitabine treatment reduces (G4C2)n foci and DPRs in C9orf72 BAC transgenic mice. Our findings suggest that nucleoside analogs are a promising compound class for therapeutic development in C9orf72 repeat-expansion-associated disorders.
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Affiliation(s)
- Mareike Czuppa
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
| | - Ashutosh Dhingra
- German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
| | - Qihui Zhou
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
| | - Carina Schludi
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
| | - Laura König
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
| | - Elisabeth Scharf
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
| | - Daniel Farny
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
| | - Anupriya Dalmia
- German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany
| | - Joachim Täger
- German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany
| | | | - Eszter Katona
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany
| | - Kohji Mori
- Psychiatry, Graduate School of Medicine, Osaka University, Suita, Japan
| | - Tina Aumer
- Ludwig-Maximilians-University Munich, Faculty of Chemistry and Pharmacy, Munich, Germany
| | - Florian Schelter
- Ludwig-Maximilians-University Munich, Faculty of Chemistry and Pharmacy, Munich, Germany
| | - Markus Müller
- Ludwig-Maximilians-University Munich, Faculty of Chemistry and Pharmacy, Munich, Germany
| | - Thomas Carell
- Ludwig-Maximilians-University Munich, Faculty of Chemistry and Pharmacy, Munich, Germany
| | | | - Josef Messinger
- Orion Corporation Orion Pharma, Medicine Design, Espoo, Finland
| | - Patrizia Rizzu
- German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany
| | - Peter Heutink
- German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany; Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany
| | - Dieter Edbauer
- German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Ludwig-Maximilians-University Munich, Graduate School of Systemic Neurosciences (GSN), Munich, Germany.
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24
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Loveland AB, Svidritskiy E, Susorov D, Lee S, Park A, Zvornicanin S, Demo G, Gao FB, Korostelev AA. Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM. Nat Commun 2022; 13:2776. [PMID: 35589706 PMCID: PMC9120013 DOI: 10.1038/s41467-022-30418-0] [Citation(s) in RCA: 40] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2020] [Accepted: 04/29/2022] [Indexed: 12/15/2022] Open
Abstract
Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome's peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.
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Affiliation(s)
- Anna B Loveland
- RNA Therapeutics Institute, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA
| | - Egor Svidritskiy
- RNA Therapeutics Institute, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA
| | - Denis Susorov
- RNA Therapeutics Institute, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA
| | - Soojin Lee
- Department of Neurology, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA
| | - Alexander Park
- RNA Therapeutics Institute, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA
| | - Sarah Zvornicanin
- RNA Therapeutics Institute, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA
| | - Gabriel Demo
- RNA Therapeutics Institute, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA
- Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, 625 00, Czech Republic
| | - Fen-Biao Gao
- Department of Neurology, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA.
| | - Andrei A Korostelev
- RNA Therapeutics Institute, UMass Chan Medical School, 368 Plantation Street, Worcester, MA, 01605, USA.
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25
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Barbé L, Finkbeiner S. Genetic and Epigenetic Interplay Define Disease Onset and Severity in Repeat Diseases. Front Aging Neurosci 2022; 14:750629. [PMID: 35592702 PMCID: PMC9110800 DOI: 10.3389/fnagi.2022.750629] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2021] [Accepted: 03/01/2022] [Indexed: 11/13/2022] Open
Abstract
Repeat diseases, such as fragile X syndrome, myotonic dystrophy, Friedreich ataxia, Huntington disease, spinocerebellar ataxias, and some forms of amyotrophic lateral sclerosis, are caused by repetitive DNA sequences that are expanded in affected individuals. The age at which an individual begins to experience symptoms, and the severity of disease, are partially determined by the size of the repeat. However, the epigenetic state of the area in and around the repeat also plays an important role in determining the age of disease onset and the rate of disease progression. Many repeat diseases share a common epigenetic pattern of increased methylation at CpG islands near the repeat region. CpG islands are CG-rich sequences that are tightly regulated by methylation and are often found at gene enhancer or insulator elements in the genome. Methylation of CpG islands can inhibit binding of the transcriptional regulator CTCF, resulting in a closed chromatin state and gene down regulation. The downregulation of these genes leads to some disease-specific symptoms. Additionally, a genetic and epigenetic interplay is suggested by an effect of methylation on repeat instability, a hallmark of large repeat expansions that leads to increasing disease severity in successive generations. In this review, we will discuss the common epigenetic patterns shared across repeat diseases, how the genetics and epigenetics interact, and how this could be involved in disease manifestation. We also discuss the currently available stem cell and mouse models, which frequently do not recapitulate epigenetic patterns observed in human disease, and propose alternative strategies to study the role of epigenetics in repeat diseases.
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Affiliation(s)
- Lise Barbé
- Center for Systems and Therapeutics, Gladstone Institutes, San Francisco, CA, United States
- Department of Neurology, University of California, San Francisco, San Francisco, CA, United States
- Department of Physiology, University of California, San Francisco, San Francisco, CA, United States
| | - Steve Finkbeiner
- Center for Systems and Therapeutics, Gladstone Institutes, San Francisco, CA, United States
- Department of Neurology, University of California, San Francisco, San Francisco, CA, United States
- Department of Physiology, University of California, San Francisco, San Francisco, CA, United States
- *Correspondence: Steve Finkbeiner,
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26
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Hartung T, Rhein M, Kalmbach N, Thau-Habermann N, Naujock M, Müschen L, Frieling H, Sterneckert J, Hermann A, Wegner F, Petri S. Methylation and Expression of Mutant FUS in Motor Neurons Differentiated From Induced Pluripotent Stem Cells From ALS Patients. Front Cell Dev Biol 2021; 9:774751. [PMID: 34869374 PMCID: PMC8640347 DOI: 10.3389/fcell.2021.774751] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Accepted: 10/20/2021] [Indexed: 11/25/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive disease leading to degeneration of motor neurons (MNs). Epigenetic modification of gene expression is increasingly recognized as potential disease mechanism. In the present study we generated motor neurons from induced pluripotent stem cells from ALS patients carrying a mutation in the fused in sarcoma gene (FUS) and analyzed expression and promoter methylation of the FUS gene and expression of DNA methyltransferases (DNMTs) compared to healthy control cell lines. While mutant FUS neural progenitor cells (NPCs) did not show a difference in FUS and DNMT expression compared to healthy controls, differentiated mutant FUS motor neurons showed significantly lower FUS expression, higher DNMT expression and higher methylation of the proximal FUS gene promoter. Immunofluorescence revealed perceived proximity of cytoplasmic FUS aggregates in ALS MNs together with 5-methylcytosin (5-mC). Targeting disturbed methylation in ALS may therefore restore transcriptional alterations and represent a novel therapeutic strategy.
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Affiliation(s)
- T Hartung
- Department of Neurology, Hannover Medical School, Hannover, Germany.,Department of Neurology, Charité-Universitätsmedizin Berlin, Berlin, Germany
| | - M Rhein
- Department of Psychiatry, Social Psychiatry and Psychotherapy, Hanover Medical School, Hanover, Germany
| | - N Kalmbach
- Department of Neurology, Hannover Medical School, Hannover, Germany
| | - N Thau-Habermann
- Department of Neurology, Hannover Medical School, Hannover, Germany
| | - M Naujock
- Department of Neurology, Hannover Medical School, Hannover, Germany.,Evotec International GmbH, Göttingen, Germany
| | - L Müschen
- Department of Neurology, Hannover Medical School, Hannover, Germany
| | - H Frieling
- Department of Psychiatry, Social Psychiatry and Psychotherapy, Hanover Medical School, Hanover, Germany
| | - J Sterneckert
- Center for Regenerative Therapies TU Dresden (CRTD), Technische Universität Dresden, Dresden, Germany
| | - A Hermann
- Translational Neurodegeneration Section "Albrecht Kossel", Department of Neurology and Center for Transdisciplinary Neuroscience (CTNR), University Medical Center Rostock, University of Rostock, Rostock, Germany.,German Center for Neurodegenerative Diseases (DZNE) Rostock/Greifswald, Rostock, Germany
| | - F Wegner
- Department of Neurology, Hannover Medical School, Hannover, Germany
| | - S Petri
- Department of Neurology, Hannover Medical School, Hannover, Germany
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27
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Ozcan KA, Ghaffari LT, Haeusler AR. The effects of molecular crowding and CpG hypermethylation on DNA G-quadruplexes formed by the C9orf72 nucleotide repeat expansion. Sci Rep 2021; 11:23213. [PMID: 34853325 PMCID: PMC8636472 DOI: 10.1038/s41598-021-02041-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Accepted: 11/09/2021] [Indexed: 01/09/2023] Open
Abstract
A nucleotide repeat expansion (NRE), (G4C2)n, located in a classically noncoding region of C9orf72 (C9), is the most common genetic mutation associated with ALS/FTD. There is increasing evidence that nucleic acid structures formed by the C9-NRE may both contribute to ALS/FTD, and serve as therapeutic targets, but there is limited characterization of these nucleic acid structures under physiologically and disease relevant conditions. Here we show in vitro that the C9-NRE DNA can form both parallel and antiparallel DNA G-quadruplex (GQ) topological structures and that the structural preference of these DNA GQs can be dependent on the molecular crowding conditions. Additionally, 5-methylcytosine DNA hypermethylation, which is observed in the C9-NRE locus in some patients, has minimal effects on GQ topological preferences. Finally, molecular dynamic simulations of methylated and nonmethylated GQ structures support in vitro data showing that DNA GQ structures formed by the C9-NRE DNA are stable, with structural fluctuations limited to the cytosine-containing loop regions. These findings provide new insight into the structural polymorphic preferences and stability of DNA GQs formed by the C9-NRE in both the methylated and nonmethylated states, as well as reveal important features to guide the development of upstream therapeutic approaches to potentially attenuate C9-NRE-linked diseases.
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Affiliation(s)
- Kadir A Ozcan
- Department of Neuroscience, Jefferson Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - Layla T Ghaffari
- Department of Neuroscience, Jefferson Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA
| | - Aaron R Haeusler
- Department of Neuroscience, Jefferson Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA. .,Department of Neuroscience, Jefferson Weinberg ALS Center, Vickie and Jack Farber Institute for Neuroscience, Thomas Jefferson University, 900 Walnut Street, JHN suite 410, Philadelphia, PA, 19107, USA.
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28
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Ramic M, Andrade NS, Rybin MJ, Esanov R, Wahlestedt C, Benatar M, Zeier Z. Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD. Brain Sci 2021; 11:brainsci11111543. [PMID: 34827542 PMCID: PMC8616043 DOI: 10.3390/brainsci11111543] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Revised: 11/11/2021] [Accepted: 11/18/2021] [Indexed: 01/04/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport “biosensors”, composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype—persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects—resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.
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Affiliation(s)
- Melina Ramic
- Center for Therapeutic Innovation, Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136, USA; (M.R.); (N.S.A.); (M.J.R.); (R.E.); (C.W.)
| | - Nadja S. Andrade
- Center for Therapeutic Innovation, Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136, USA; (M.R.); (N.S.A.); (M.J.R.); (R.E.); (C.W.)
| | - Matthew J. Rybin
- Center for Therapeutic Innovation, Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136, USA; (M.R.); (N.S.A.); (M.J.R.); (R.E.); (C.W.)
| | - Rustam Esanov
- Center for Therapeutic Innovation, Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136, USA; (M.R.); (N.S.A.); (M.J.R.); (R.E.); (C.W.)
| | - Claes Wahlestedt
- Center for Therapeutic Innovation, Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136, USA; (M.R.); (N.S.A.); (M.J.R.); (R.E.); (C.W.)
| | - Michael Benatar
- Department of Neurology, University of Miami Miller School of Medicine, 1120 NW 14th St., Miami, FL 33136, USA;
| | - Zane Zeier
- Center for Therapeutic Innovation, Department of Psychiatry & Behavioral Sciences, University of Miami Miller School of Medicine, 1501 NW 10th Ave, Miami, FL 33136, USA; (M.R.); (N.S.A.); (M.J.R.); (R.E.); (C.W.)
- Correspondence: ; Tel.: +1-305-243-1367
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29
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Malik I, Kelley CP, Wang ET, Todd PK. Molecular mechanisms underlying nucleotide repeat expansion disorders. Nat Rev Mol Cell Biol 2021; 22:589-607. [PMID: 34140671 PMCID: PMC9612635 DOI: 10.1038/s41580-021-00382-6] [Citation(s) in RCA: 200] [Impact Index Per Article: 50.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 04/30/2021] [Indexed: 02/05/2023]
Abstract
The human genome contains over one million short tandem repeats. Expansion of a subset of these repeat tracts underlies over fifty human disorders, including common genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72), polyglutamine-associated ataxias and Huntington disease, myotonic dystrophy, and intellectual disability disorders such as Fragile X syndrome. In this Review, we discuss the four major mechanisms by which expansion of short tandem repeats causes disease: loss of function through transcription repression, RNA-mediated gain of function through gelation and sequestration of RNA-binding proteins, gain of function of canonically translated repeat-harbouring proteins, and repeat-associated non-AUG translation of toxic repeat peptides. Somatic repeat instability amplifies these mechanisms and influences both disease age of onset and tissue specificity of pathogenic features. We focus on the crosstalk between these disease mechanisms, and argue that they often synergize to drive pathogenesis. We also discuss the emerging native functions of repeat elements and how their dynamics might contribute to disease at a larger scale than currently appreciated. Lastly, we propose that lynchpins tying these disease mechanisms and native functions together offer promising therapeutic targets with potential shared applications across this class of human disorders.
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Affiliation(s)
- Indranil Malik
- Department of Neurology, University of Michigan, Ann Arbor, MI, USA
| | - Chase P Kelley
- Department of Molecular Genetics and Microbiology, Center for NeuroGenetics, Genetics Institute, University of Florida, Gainesville, FL, USA
- Genetics and Genomics Graduate Program, University of Florida, Gainesville, FL, USA
| | - Eric T Wang
- Department of Molecular Genetics and Microbiology, Center for NeuroGenetics, Genetics Institute, University of Florida, Gainesville, FL, USA.
| | - Peter K Todd
- Department of Neurology, University of Michigan, Ann Arbor, MI, USA.
- VA Ann Arbor Healthcare System, Ann Arbor, MI, USA.
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30
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Xu M, Zhu J, Liu XD, Luo MY, Xu NJ. Roles of physical exercise in neurodegeneration: reversal of epigenetic clock. Transl Neurodegener 2021; 10:30. [PMID: 34389067 PMCID: PMC8361623 DOI: 10.1186/s40035-021-00254-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2021] [Accepted: 07/29/2021] [Indexed: 12/17/2022] Open
Abstract
The epigenetic clock is defined by the DNA methylation (DNAm) level and has been extensively applied to distinguish biological age from chronological age. Aging-related neurodegeneration is associated with epigenetic alteration, which determines the status of diseases. In recent years, extensive research has shown that physical exercise (PE) can affect the DNAm level, implying a reversal of the epigenetic clock in neurodegeneration. PE also regulates brain plasticity, neuroinflammation, and molecular signaling cascades associated with epigenetics. This review summarizes the effects of PE on neurodegenerative diseases via both general and disease-specific DNAm mechanisms, and discusses epigenetic modifications that alleviate the pathological symptoms of these diseases. This may lead to probing of the underpinnings of neurodegenerative disorders and provide valuable therapeutic references for cognitive and motor dysfunction.
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Affiliation(s)
- Miao Xu
- Department of Anatomy, Histology and Embryology, Kunming Medical University, Kunming, 650500, China.,Collaborative Innovation Center for Brain Science, Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
| | - JiaYi Zhu
- Collaborative Innovation Center for Brain Science, Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.,Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
| | - Xian-Dong Liu
- Collaborative Innovation Center for Brain Science, Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.,Department of Neurology and Institute of Neurology, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
| | - Ming-Ying Luo
- Department of Anatomy, Histology and Embryology, Kunming Medical University, Kunming, 650500, China
| | - Nan-Jie Xu
- Collaborative Innovation Center for Brain Science, Department of Anatomy and Physiology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. .,Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. .,Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
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31
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Ratti A, Peverelli S, D'Adda E, Colombrita C, Gennuso M, Prelle A, Silani V. Genetic and epigenetic disease modifiers in an Italian C9orf72 family expressing ALS, FTD or PD clinical phenotypes. Amyotroph Lateral Scler Frontotemporal Degener 2021; 23:292-298. [PMID: 34382491 DOI: 10.1080/21678421.2021.1962355] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Objective: The presence of the hexanucleotide repeat expansion (HRE) in C9orf72 gene is associated to the ALS/FTD spectrum, but also to parkinsonisms. We here describe an Italian family with the father diagnosed with Parkinson disease (PD) at the age of 67 and the two daughters developing FTD and ALS at 45 years of age. We searched for C9orf72 HRE with possible genetic and epigenetic modifiers to account for the intrafamilial phenotypic variability. Methods: C9orf72 mutational analysis was performed by fragment length analysis, Repeat-primed PCR and Southern blot. Targeted next generation sequencing was used to analyze 48 genes associated to neurodegenerative diseases. Promoter methylation was analyzed by bisulfite sequencing. Results: Genetic analysis identified C9orf72 HRE in all the affected members with a similar repeat expansion size. Both the father and the FTD daughter also carried the heterozygous p.Ile946Phe variant in ATP13A2 gene, associated to PD. In addition, the father also showed a heterozygous EIF4G1 variant (p.Ala13Pro), that might increase his susceptibility to develop PD. The DNA methylation analysis showed that all the 26 CpG sites within C9orf72 promoter were unmethylated in all family members. Conclusions: Neither C9orf72 HRE size nor promoter methylation act as disease modifiers within this family, at least in blood, not excluding HRE mosaicism and a different methylation pattern in the brain. However, the presence of rare genetic variants in PD genes suggests that they may influence the clinical manifestation in the father. Other genetic and/or epigenetic modifiers must be responsible for disease variability in this C9orf72 family case.
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Affiliation(s)
- Antonia Ratti
- Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milano, Italy.,Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Milano, Italy
| | - Silvia Peverelli
- Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milano, Italy
| | | | - Claudia Colombrita
- Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milano, Italy
| | | | - Alessandro Prelle
- U.O.C. of Neurology - Stroke Unit, ASST Ovest milanese, Legnano, Italy
| | - Vincenzo Silani
- Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milano, Italy.,Department of Pathophysiology and Transplantation, "Dino Ferrari" Center, Università degli Studi di Milano, Milano, Italy
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32
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Wang E, Thombre R, Shah Y, Latanich R, Wang J. G-Quadruplexes as pathogenic drivers in neurodegenerative disorders. Nucleic Acids Res 2021; 49:4816-4830. [PMID: 33784396 PMCID: PMC8136783 DOI: 10.1093/nar/gkab164] [Citation(s) in RCA: 90] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2019] [Revised: 02/20/2021] [Accepted: 03/29/2021] [Indexed: 12/12/2022] Open
Abstract
G-quadruplexes (G4s), higher-order DNA and RNA secondary structures featuring guanine-rich nucleic acid sequences with various conformations, are widely distributed in the human genome. These structural motifs are known to participate in basic cellular processes, including transcription, splicing, and translation, and their functions related to health and disease are becoming increasingly recognized. In this review, we summarize the landscape of G4s involved in major neurodegenerative disorders, describing the genes that contain G4-forming sequences and proteins that have high affinity for G4-containing elements. The functions of G4s are diverse, with potentially protective or deleterious effects in the pathogenic cascades of various neurological diseases. While the studies of the functions of G4s in vivo, including those involved in pathophysiology, are still in their early stages, we will nevertheless discuss the evidence pointing to their biological relevance. A better understanding of this unique structural element in the biological context is important for unveiling its potential roles in the pathogenesis of diseases such as neurodegeneration and for designing new diagnostic and therapeutic strategies.
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Affiliation(s)
- Ernest Wang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore MD, 21205, USA.,Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Ravi Thombre
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore MD, 21205, USA.,Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Yajas Shah
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore MD, 21205, USA.,Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Rachel Latanich
- Department of Medicine, Division of Gastroenterology and Hepatology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
| | - Jiou Wang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore MD, 21205, USA.,Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA
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Smeyers J, Banchi EG, Latouche M. C9ORF72: What It Is, What It Does, and Why It Matters. Front Cell Neurosci 2021; 15:661447. [PMID: 34025358 PMCID: PMC8131521 DOI: 10.3389/fncel.2021.661447] [Citation(s) in RCA: 87] [Impact Index Per Article: 21.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Accepted: 03/17/2021] [Indexed: 12/11/2022] Open
Abstract
When the non-coding repeat expansion in the C9ORF72 gene was discovered to be the most frequent cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) in 2011, this gene and its derived protein, C9ORF72, were completely unknown. The mutation appeared to produce both haploinsufficiency and gain-of-function effects in the form of aggregating expanded RNAs and dipeptide repeat proteins (DPRs). An unprecedented effort was then unleashed to decipher the pathogenic mechanisms and the functions of C9ORF72 in order to design therapies. A decade later, while the toxicity of accumulating gain-of-function products has been established and therapeutic strategies are being developed to target it, the contribution of the loss of function starts to appear more clearly. This article reviews the current knowledge about the C9ORF72 protein, how it is affected by the repeat expansion in models and patients, and what could be the contribution of its haploinsufficiency to the disease in light of the most recent findings. We suggest that these elements should be taken into consideration to refine future therapeutic strategies, compensating for the decrease of C9ORF72 or at least preventing a further reduction.
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Affiliation(s)
- Julie Smeyers
- Sorbonne Université, Institut du Cerveau - Paris Brain Institute - ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière, DMU Neuroscience 6, Paris, France
- PSL Research university, EPHE, Neurogenetics team, Paris, France
| | - Elena-Gaia Banchi
- Sorbonne Université, Institut du Cerveau - Paris Brain Institute - ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière, DMU Neuroscience 6, Paris, France
| | - Morwena Latouche
- Sorbonne Université, Institut du Cerveau - Paris Brain Institute - ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière, DMU Neuroscience 6, Paris, France
- PSL Research university, EPHE, Neurogenetics team, Paris, France
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34
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Handal T, Eiges R. Correction of Heritable Epigenetic Defects Using Editing Tools. Int J Mol Sci 2021; 22:ijms22083966. [PMID: 33921346 PMCID: PMC8070094 DOI: 10.3390/ijms22083966] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 04/02/2021] [Accepted: 04/07/2021] [Indexed: 11/21/2022] Open
Abstract
Epimutations refer to mistakes in the setting or maintenance of epigenetic marks in the chromatin. They lead to mis-expression of genes and are often secondary to germline transmitted mutations. As such, they are the cause for a considerable number of genetically inherited conditions in humans. The correction of these types of epigenetic defects constitutes a good paradigm to probe the fundamental mechanisms underlying the development of these diseases, and the molecular basis for the establishment, maintenance and regulation of epigenetic modifications in general. Here, we review the data to date, which is limited to repetitive elements, that relates to the applications of key editing tools for addressing the epigenetic aspects of various epigenetically regulated diseases. For each approach we summarize the efforts conducted to date, highlight their contribution to a better understanding of the molecular basis of epigenetic mechanisms, describe the limitations of each approach and suggest perspectives for further exploration in this field.
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Affiliation(s)
- Tayma Handal
- Stem Cell Research Laboratory, Medical Genetics Institute Shaare Zedek Medical Center, Jerusalem 91031, Israel;
- School of Medicine, The Hebrew University, Campus Ein Kerem, Jerusalem 91120, Israel
| | - Rachel Eiges
- Stem Cell Research Laboratory, Medical Genetics Institute Shaare Zedek Medical Center, Jerusalem 91031, Israel;
- School of Medicine, The Hebrew University, Campus Ein Kerem, Jerusalem 91120, Israel
- Correspondence:
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35
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Characterization of C9orf72 haplotypes to evaluate the effects of normal and pathological variations on its expression and splicing. PLoS Genet 2021; 17:e1009445. [PMID: 33780440 PMCID: PMC8031855 DOI: 10.1371/journal.pgen.1009445] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 04/08/2021] [Accepted: 02/25/2021] [Indexed: 11/19/2022] Open
Abstract
Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients’ cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative diseases, whose most frequent genetic cause is hexanucleotide repeat (HR) expansion from normal 2 to 20 repeats to pathological hundreds of repeats within a non-coding region of the C9orf72 gene. Haplotype is a specific combination of multiple polymorphic sites along a chromosome that are inherited together in block. We characterized the single nucleotide polymorphism (SNP) signature and HR length of the major C9orf72 haplotypes in Caucasians to identify the allelic origin of C9orf72 transcripts per patient and determine the effects of expanded HR on C9orf72 gene expression and splicing. In C9orf72 ALS patients’ cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of downstream initiated transcript variant, increased levels of upstream initiated transcripts, accumulation of introns 1 and 2, and abnormal splicing at cryptic splice sites along intron 1. The C9orf72 haplotypes DNA signatures described here are valuable for studying C9-ALS/FTD pathogenesis and for developing allele-specific therapeutic strategies.
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36
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Quezada E, Cappelli C, Diaz I, Jury N, Wightman N, Brown RH, Montecino M, van Zundert B. BET bromodomain inhibitors PFI-1 and JQ1 are identified in an epigenetic compound screen to enhance C9ORF72 gene expression and shown to ameliorate C9ORF72-associated pathological and behavioral abnormalities in a C9ALS/FTD model. Clin Epigenetics 2021; 13:56. [PMID: 33726839 PMCID: PMC7962347 DOI: 10.1186/s13148-021-01039-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2020] [Accepted: 02/23/2021] [Indexed: 12/25/2022] Open
Abstract
BACKGROUND An intronic GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), referred to as C9ALS/FTD. No cure or effective treatment exist for C9ALS/FTD. Three major molecular mechanisms have emerged to explain C9ALS/FTD disease mechanisms: (1) C9ORF72 loss-of-function through haploinsufficiency, (2) dipeptide repeat (DPR) proteins mediated toxicity by the translation of the repeat RNAs, and more controversial, (3) RNA-mediated toxicity by bidirectional transcription of the repeats that form intranuclear RNA foci. Recent studies indicate a double-hit pathogenic mechanism in C9ALS/FTD, where reduced C9ORF72 protein levels lead to impaired clearance of toxic DPRs. Here we explored whether pharmacological compounds can revert these pathological hallmarks in vitro and cognitive impairment in a C9ALS/FTD mouse model (C9BAC). We specifically focused our study on small molecule inhibitors targeting chromatin-regulating proteins (epidrugs) with the goal of increasing C9ORF72 gene expression and reduce toxic DPRs. RESULTS We generated luciferase reporter cell lines containing 10 (control) or ≥ 90 (mutant) G4C2 HRE located between exon 1a and 1b of the human C9ORF72 gene. In a screen of 14 different epidrugs targeting bromodomains, chromodomains and histone-modifying enzymes, we found that several bromodomain and extra-terminal domain (BET) inhibitors (BETi), including PFI-1 and JQ1, increased luciferase reporter activity. Using primary cortical cultures from C9BAC mice, we further found that PFI-1 treatment increased the expression of V1-V3 transcripts of the human mutant C9ORF72 gene, reduced poly(GP)-DPR inclusions but enhanced intranuclear RNA foci. We also tested whether JQ1, an BETi previously shown to reach the mouse brain by intraperitoneal (i.p.) injection, can revert behavioral abnormalities in C9BAC mice. Interestingly, it was found that JQ1 administration (daily i.p. administration for 7 days) rescued hippocampal-dependent cognitive deficits in C9BAC mice. CONCLUSIONS Our findings place BET bromodomain inhibitors as a potential therapy for C9ALS/FTD by ameliorating C9ORF72-associated pathological and behavioral abnormalities. Our finding that PFI-1 increases accumulation of intranuclear RNA foci is in agreement with recent data in flies suggesting that nuclear RNA foci can be neuroprotective by sequestering repeat transcripts that result in toxic DPRs.
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Affiliation(s)
- Esteban Quezada
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile
| | - Claudio Cappelli
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile
| | - Iván Diaz
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile
| | - Nur Jury
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile
| | - Nicholas Wightman
- Department of Neurology, University of Massachusetts Medical School (UMMS), Worcester, MA, USA
| | - Robert H Brown
- Department of Neurology, University of Massachusetts Medical School (UMMS), Worcester, MA, USA
| | - Martín Montecino
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.
- FONDAP Center for Genome Regulation, Santiago, Chile.
| | - Brigitte van Zundert
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.
- Department of Neurology, University of Massachusetts Medical School (UMMS), Worcester, MA, USA.
- CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile.
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Beckers J, Tharkeshwar AK, Van Damme P. C9orf72 ALS-FTD: recent evidence for dysregulation of the autophagy-lysosome pathway at multiple levels. Autophagy 2021; 17:3306-3322. [PMID: 33632058 PMCID: PMC8632097 DOI: 10.1080/15548627.2021.1872189] [Citation(s) in RCA: 64] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two clinically distinct classes of neurodegenerative disorders. Yet, they share a range of genetic, cellular, and molecular features. Hexanucleotide repeat expansions (HREs) in the C9orf72 gene and the accumulation of toxic protein aggregates in the nervous systems of the affected individuals are among such common features. Though the mechanisms by which HREs cause toxicity is not clear, the toxic gain of function due to transcribed HRE RNA or dipeptide repeat proteins (DPRs) produced by repeat-associated non-AUG translation together with a reduction in C9orf72 expression are proposed as the contributing factors for disease pathogenesis in ALS and FTD. In addition, several recent studies point toward alterations in protein homeostasis as one of the root causes of the disease pathogenesis. In this review, we discuss the effects of the C9orf72 HRE in the autophagy-lysosome pathway based on various recent findings. We suggest that dysfunction of the autophagy-lysosome pathway synergizes with toxicity from C9orf72 repeat RNA and DPRs to drive disease pathogenesis. Abbreviation: ALP: autophagy-lysosome pathway; ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related; ASO: antisense oligonucleotide; C9orf72: C9orf72-SMCR8 complex subunit; DENN: differentially expressed in normal and neoplastic cells; DPR: dipeptide repeat protein; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; ER: endoplasmic reticulum; FTD: frontotemporal dementia; GAP: GTPase-activating protein; GEF: guanine nucleotide exchange factor; HRE: hexanucleotide repeat expansion; iPSC: induced pluripotent stem cell; ISR: integrated stress response; M6PR: mannose-6-phosphate receptor, cation dependent; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; ND: neurodegenerative disorder; RAN: repeat-associated non-ATG; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SLC66A1/PQLC2: solute carrier family 66 member 1; SMCR8: SMCR8-C9orf72 complex subunit; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; WDR41: WD repeat domain 41.
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Affiliation(s)
- Jimmy Beckers
- Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, Leuven, Belgium.,VIB, Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium
| | - Arun Kumar Tharkeshwar
- Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, Leuven, Belgium.,VIB, Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium
| | - Philip Van Damme
- Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, Leuven, Belgium.,VIB, Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium.,University Hospitals Leuven, Department of Neurology, Leuven, Belgium
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Braems E, Swinnen B, Van Den Bosch L. C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD? Acta Neuropathol 2020; 140:625-643. [PMID: 32876811 PMCID: PMC7547039 DOI: 10.1007/s00401-020-02214-x] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2020] [Revised: 07/28/2020] [Accepted: 08/13/2020] [Indexed: 12/11/2022]
Abstract
A repeat expansion in C9orf72 is responsible for the characteristic neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in a still unresolved manner. Proposed mechanisms involve gain-of-functions, comprising RNA and protein toxicity, and loss-of-function of the C9orf72 gene. Their exact contribution is still inconclusive and reports regarding loss-of-function are rather inconsistent. Here, we review the function of the C9orf72 protein and its relevance in disease. We explore the potential link between reduced C9orf72 levels and disease phenotypes in postmortem, in vitro, and in vivo models. Moreover, the significance of loss-of-function in other non-coding repeat expansion diseases is used to clarify its contribution in C9orf72 ALS/FTD. In conclusion, with evidence pointing to a multiple-hit model, loss-of-function on itself seems to be insufficient to cause neurodegeneration in C9orf72 ALS/FTD.
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Affiliation(s)
- Elke Braems
- Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, 3000, Leuven, Belgium
- Laboratory of Neurobiology, Experimental Neurology, Center for Brain and Disease Research, VIB, Campus Gasthuisberg, O&N4, Herestraat 49, PB 602, 3000, Leuven, Belgium
| | - Bart Swinnen
- Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, 3000, Leuven, Belgium
- Laboratory of Neurobiology, Experimental Neurology, Center for Brain and Disease Research, VIB, Campus Gasthuisberg, O&N4, Herestraat 49, PB 602, 3000, Leuven, Belgium
- Department of Neurology, University Hospitals Leuven, 3000, Leuven, Belgium
| | - Ludo Van Den Bosch
- Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, 3000, Leuven, Belgium.
- Laboratory of Neurobiology, Experimental Neurology, Center for Brain and Disease Research, VIB, Campus Gasthuisberg, O&N4, Herestraat 49, PB 602, 3000, Leuven, Belgium.
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Dong W, Zhang L, Sun C, Gao X, Guan F, Li J, Chen W, Ma Y, Zhang L. Knock in of a hexanucleotide repeat expansion in the C9orf72 gene induces ALS in rats. Animal Model Exp Med 2020; 3:237-244. [PMID: 33024945 PMCID: PMC7529333 DOI: 10.1002/ame2.12129] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Accepted: 07/07/2020] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND The GGGGCC (G4C2) repeat expansion in the human open reading frame 72 on chromosome 9, C9orf72, is the most common cause of amyotrophic lateral sclerosis (ALS). Studies in transgenic mouse models have linked the pathogenic mechanism of G4C2 repeat expansion to RNA foci or the accumulation of unnatural dipeptide repeats in neurons. However, only one of the existing transgenic mouse lines developed typical ALS. METHODS C9orf72 knockin rats were generated by knockin of 80 G4C2 repeats with human flanking fragments within exon1a and exon1b at the rat C9orf72 locus. Protein expression was detected by western blot. Motor coordination and grip force were measured using a Rotarod test and a grip strength test. Neurodegeneration was assessed by Nissl staining with cresyl violet. RESULTS C9orf72 haploinsufficiency reduced C9orf72 protein expression 40% in the cerebrum, cerebellum and spinal cords from knockin rats (P < .05). The knockin (KI) rats developed motor deficits from 4 months of age. Their falling latencies and grip force were decreased by 67% (P < .01) and 44% (P < .01), respectively, at 12 months of age compared to wild-type (WT) mice. The knockin of the hexanucleotide repeat expansion (HRE) caused a 47% loss of motor neurons in the spinal cord (P < .001) and 25% (5/20) of female KI rats developed hind limb paralysis at 13 to 24 months. CONCLUSION Motor defects in KI rats may result from neurotoxicity caused by HRE and the resulting reduction in C9orf72 protein due to haploinsufficiency. These KI rats could be a useful model for investigating the contributions of loss-of-function to neurotoxicity in C9orf72-related ALS.
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Affiliation(s)
- Wei Dong
- Key Laboratory of Human Disease Comparative MedicineNational Health Commission of China (NHC)Institute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
- Neuroscience CenterChinese Academy of Medical SciencesBeijingChina
| | - Li Zhang
- Neuroscience CenterChinese Academy of Medical SciencesBeijingChina
- Beijing Engineering Research Center for Experimental Animal Models of Human DiseasesInstitute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
| | - Caixian Sun
- Key Laboratory of Human Disease Comparative MedicineNational Health Commission of China (NHC)Institute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
| | - Xiang Gao
- Key Laboratory of Human Disease Comparative MedicineNational Health Commission of China (NHC)Institute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
| | - Feifei Guan
- Key Laboratory of Human Disease Comparative MedicineNational Health Commission of China (NHC)Institute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
| | - Jing Li
- Beijing Engineering Research Center for Experimental Animal Models of Human DiseasesInstitute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
| | - Wei Chen
- Key Laboratory of Human Disease Comparative MedicineNational Health Commission of China (NHC)Institute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
| | - Yuanwu Ma
- Beijing Engineering Research Center for Experimental Animal Models of Human DiseasesInstitute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
| | - Lianfeng Zhang
- Key Laboratory of Human Disease Comparative MedicineNational Health Commission of China (NHC)Institute of Laboratory Animal SciencePeking Union Medicine CollegeChinese Academy of Medical SciencesBeijingChina
- Neuroscience CenterChinese Academy of Medical SciencesBeijingChina
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40
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Hao Z, Wang R, Ren H, Wang G. Role of the C9ORF72 Gene in the Pathogenesis of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Neurosci Bull 2020; 36:1057-1070. [PMID: 32860626 DOI: 10.1007/s12264-020-00567-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2020] [Accepted: 04/30/2020] [Indexed: 12/12/2022] Open
Abstract
Since the discovery of the C9ORF72 gene in 2011, great advances have been achieved in its genetics and in identifying its role in disease models and pathological mechanisms; it is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS patients with C9ORF72 expansion show heterogeneous symptoms. Those who are C9ORF72 expansion carriers have shorter survival after disease onset than non-C9ORF72 expansion patients. Pathological and clinical features of C9ORF72 patients have been well mimicked via several models, including induced pluripotent stem cell-derived neurons and transgenic mice that were embedded with bacterial artificial chromosome construct and that overexpressing dipeptide repeat proteins. The mechanisms implicated in C9ORF72 pathology include DNA damage, changes of RNA metabolism, alteration of phase separation, and impairment of nucleocytoplasmic transport, which may underlie C9ORF72 expansion-related ALS/FTD and provide insight into non-C9ORF72 expansion-related ALS, FTD, and other neurodegenerative diseases.
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Affiliation(s)
- Zongbing Hao
- Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Neuropsychiatric Diseases and Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China
| | - Rui Wang
- Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Neuropsychiatric Diseases and Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China
| | - Haigang Ren
- Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Neuropsychiatric Diseases and Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China
| | - Guanghui Wang
- Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Neuropsychiatric Diseases and Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China.
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41
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Dong W, Ma Y, Guan F, Zhang X, Chen W, Zhang L, Zhang L. Ablation of C9orf72 together with excitotoxicity induces ALS in rats. FEBS J 2020; 288:1712-1723. [PMID: 32745320 DOI: 10.1111/febs.15501] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2019] [Revised: 05/19/2020] [Accepted: 07/28/2020] [Indexed: 12/12/2022]
Abstract
Pathogenesis of familial amyotrophic lateral sclerosis (ALS) linked to expansion of the chromosome 9 open reading frame 72 (C9orf72) hexanucleotide repeat that impairs C9orf72 expression. Loss of function of the C9orf72 protein is one of the three main proposed C9orf72-related ALS mechanisms. However, C9orf72 loss of function, by itself, is insufficient to cause severe phenotypes in mice. Excitotoxicity is another major disease mechanism of ALS. We speculate that loss of C9orf72 protein might cause ALS in combination with excitotoxicity. To date, the effect of C9orf72 deficiency in the background of SD rat has not been examined. To test our hypothesis, we generated a line of rat with a deletion of part of the C9orf72 gene ablating the encoded protein. These animals did not develop any ALS phenotypes; however, when they were treated with kainic acid, an excitotoxicity inducer, the rats developed motor deficits and showed loss of motor neurons (MNs), Golgi complex fragmentation, and abnormal vesicle trafficking. RNA sequencing revealed profound changes in the gene profiles that were primarily associated with neural activity. Our results demonstrated that C9orf72 ablation alone was not enough to cause ALS pathogenesis in rat; but the ablation sensitized MNs to other risk factors that synergistically caused the ALS. These results support a loss of function of C9orf72 mechanism of ALS.
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Affiliation(s)
- Wei Dong
- Key Laboratory of Human Disease Comparative Medicine, National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China.,Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, China
| | - Yuanwu Ma
- Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, China.,Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China
| | - Feifei Guan
- Key Laboratory of Human Disease Comparative Medicine, National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China
| | - Xu Zhang
- Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China
| | - Wei Chen
- Key Laboratory of Human Disease Comparative Medicine, National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China
| | - Li Zhang
- Beijing Engineering Research Center for Experimental Animal Models of Human Diseases, Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China
| | - Lianfeng Zhang
- Key Laboratory of Human Disease Comparative Medicine, National Health Commission of China (NHC), Institute of Laboratory Animal Science, Peking Union Medicine College, Chinese Academy of Medical Sciences, Beijing, China.,Neuroscience Center, Chinese Academy of Medical Sciences, Beijing, China
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42
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Liu EY, Russ J, Lee EB. Neuronal Transcriptome from C9orf72 Repeat Expanded Human Tissue is Associated with Loss of C9orf72 Function. FREE NEUROPATHOLOGY 2020; 1:23. [PMID: 32905541 PMCID: PMC10240940 DOI: 10.17879/freeneuropathology-2020-2911] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Accepted: 08/13/2020] [Indexed: 04/02/2025]
Abstract
A hexanucleotide G4C2 repeat expansion in C9orf72 is the most common genetic cause of familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). The mutation is associated with a reduction of C9orf72 protein and accumulation of toxic RNA and dipeptide repeat aggregates. The accumulation of toxic RNA has been proposed to sequester RNA binding proteins thereby altering RNA processing, consistent with previous transcriptome studies that have shown that the C9orf72 repeat expansion is linked to abundant splicing alterations and transcriptome changes. Here, we used a subcellular fractionation method and FACS to enrich for neuronal nuclei from C9orf72 repeat expanded post-mortem human ALS/FTD brains, and to remove neuronal nuclei with TDP-43 pathology which are observed in nearly all symptomatic C9orf72 repeat expanded cases. We show that the C9orf72 expansion is associated with relatively mild gene expression changes. Dysregulated genes were enriched for vesicle transport pathways, which is consistent with the known functions of C9orf72 protein. Further analysis suggests that the C9orf72 transcriptome is not driven by toxic RNA but is rather shaped by the depletion of pathologic TDP-43 nuclei and the loss of C9orf72 expression. These findings argue against RNA binding protein sequestration in neurons as a major contributor to C9orf72 mediated toxicity.
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Affiliation(s)
- Elaine Y. Liu
- Translational Neuropathology Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA
| | - Jenny Russ
- Translational Neuropathology Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA
| | - Edward B. Lee
- Translational Neuropathology Research Laboratory, University of Pennsylvania, Philadelphia, PA, USA
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43
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He F, Flores BN, Krans A, Frazer M, Natla S, Niraula S, Adefioye O, Barmada SJ, Todd PK. The carboxyl termini of RAN translated GGGGCC nucleotide repeat expansions modulate toxicity in models of ALS/FTD. Acta Neuropathol Commun 2020; 8:122. [PMID: 32753055 PMCID: PMC7401224 DOI: 10.1186/s40478-020-01002-8] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2020] [Accepted: 07/22/2020] [Indexed: 12/13/2022] Open
Abstract
An intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G4C2) repeats either inside of an artificial intron within a GFP reporter or within the 5' untranslated region (UTR) of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5' UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus (C-terminus) in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR C-terminus was observed when repeats were expressed in rodent neurons. The presence of the native C-termini across all three reading frames was partly protective. Taken together, these findings suggest that C-terminal sequences outside of the repeat region may alter the behavior and toxicity of dipeptide repeat proteins derived from GGGGCC repeats.
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44
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Ababneh NA, Scaber J, Flynn R, Douglas A, Barbagallo P, Candalija A, Turner MR, Sims D, Dafinca R, Cowley SA, Talbot K. Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair. Hum Mol Genet 2020; 29:2200-2217. [PMID: 32504093 PMCID: PMC7399532 DOI: 10.1093/hmg/ddaa106] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2020] [Revised: 05/04/2020] [Accepted: 06/01/2020] [Indexed: 12/13/2022] Open
Abstract
The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.
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Affiliation(s)
- Nidaa A Ababneh
- Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
- Cell Therapy Center, University of Jordan, Queen Rania St, Amman 11942, Jordan
| | - Jakub Scaber
- Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
| | - Rowan Flynn
- James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Andrew Douglas
- Human Development and Health, Faculty of Medicine, University of Southampton, Southampton SO17 1BJ, UK
- Wessex Clinical Genetics Service, Level G, Mailpoint 627, Princess Anne Hospital, Coxford Road, Southampton SO16 5YA, UK
| | - Paola Barbagallo
- Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
| | - Ana Candalija
- Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
| | - Martin R Turner
- Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
| | - David Sims
- Weatherall Institute for Molecular Medicine, Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
| | - Ruxandra Dafinca
- Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
| | - Sally A Cowley
- James Martin Stem Cell Facility, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Kevin Talbot
- Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, UK
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Zhu Q, Jiang J, Gendron TF, McAlonis-Downes M, Jiang L, Taylor A, Diaz Garcia S, Ghosh Dastidar S, Rodriguez MJ, King P, Zhang Y, La Spada AR, Xu H, Petrucelli L, Ravits J, Da Cruz S, Lagier-Tourenne C, Cleveland DW. Reduced C9ORF72 function exacerbates gain of toxicity from ALS/FTD-causing repeat expansion in C9orf72. Nat Neurosci 2020; 23:615-624. [PMID: 32284607 PMCID: PMC7384305 DOI: 10.1038/s41593-020-0619-5] [Citation(s) in RCA: 161] [Impact Index Per Article: 32.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2019] [Accepted: 02/28/2020] [Indexed: 02/08/2023]
Abstract
Hexanucleotide expansions in C9orf72, which encodes a predicted guanine exchange factor, are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although repeat expansion has been established to generate toxic products, mRNAs encoding the C9ORF72 protein are also reduced in affected individuals. In this study, we tested how C9ORF72 protein levels affected repeat-mediated toxicity. In somatic transgenic mice expressing 66 GGGGCC repeats, inactivation of one or both endogenous C9orf72 alleles provoked or accelerated, respectively, early death. In mice expressing a C9orf72 transgene with 450 repeats that did not encode the C9ORF72 protein, inactivation of one or both endogenous C9orf72 alleles exacerbated cognitive deficits, hippocampal neuron loss, glial activation and accumulation of dipeptide-repeat proteins from translation of repeat-containing RNAs. Reduced C9ORF72 was shown to suppress repeat-mediated elevation in autophagy. These efforts support a disease mechanism in ALS/FTD resulting from reduced C9ORF72, which can lead to autophagy deficits, synergizing with repeat-dependent gain of toxicity.
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Affiliation(s)
- Qiang Zhu
- Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA
| | - Jie Jiang
- Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA
- Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, USA
| | - Tania F Gendron
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
| | - Melissa McAlonis-Downes
- Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA
| | - Lulin Jiang
- Neuroscience Initiative, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA
| | - Amy Taylor
- Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA
| | - Sandra Diaz Garcia
- Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA
| | - Somasish Ghosh Dastidar
- Department of Pediatrics, University of California at San Diego, La Jolla, CA, USA
- Molecular Neuroscience; Kasturba Medical College, Manipal Academy of Higher Education, Manipal, India
| | - Maria J Rodriguez
- Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA
| | - Patrick King
- Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA
| | - Yongjie Zhang
- Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA
| | - Albert R La Spada
- Department of Pediatrics, University of California at San Diego, La Jolla, CA, USA
- Departments of Neurology, Neurobiology, and Cell Biology, Duke Center for Neurodegeneration and Neurotherapeutics, Duke University School of Medicine, Durham, NC, USA
| | - Huaxi Xu
- Neuroscience Initiative, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA
| | | | - John Ravits
- Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA
| | - Sandrine Da Cruz
- Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA
- VIB Center for Brain and Disease Research, Leuven, Belgium
- Department of Neurosciences, KU Leuven, Leuven, Belgium
| | - Clotilde Lagier-Tourenne
- Department of Neurology, The Sean M. Healey and AMG Center for ALS at Mass General, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
- Broad Institute of Harvard University and MIT, Cambridge, MA, USA.
| | - Don W Cleveland
- Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA.
- Department of Neurosciences, University of California at San Diego, La Jolla, CA, USA.
- Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA.
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Ji YJ, Ugolino J, Zhang T, Lu J, Kim D, Wang J. C9orf72/ALFA-1 controls TFEB/HLH-30-dependent metabolism through dynamic regulation of Rag GTPases. PLoS Genet 2020; 16:e1008738. [PMID: 32282804 PMCID: PMC7188304 DOI: 10.1371/journal.pgen.1008738] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2019] [Revised: 04/28/2020] [Accepted: 03/25/2020] [Indexed: 12/13/2022] Open
Abstract
Nutrient utilization and energy metabolism are critical for the maintenance of cellular homeostasis. A mutation in the C9orf72 gene has been linked to the most common forms of neurodegenerative diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we have identified an evolutionarily conserved function of C9orf72 in the regulation of the transcription factor EB (TFEB), a master regulator of autophagic and lysosomal genes that is negatively modulated by mTORC1. Loss of the C. elegans orthologue of C9orf72, ALFA-1, causes the nuclear translocation of HLH-30/TFEB, leading to activation of lipolysis and premature lethality during starvation-induced developmental arrest in C. elegans. A similar conserved pathway exists in human cells, in which C9orf72 regulates mTOR and TFEB signaling. C9orf72 interacts with and dynamically regulates the level of Rag GTPases, which are responsible for the recruitment of mTOR and TFEB on the lysosome upon amino acid signals. These results have revealed previously unknown functions of C9orf72 in nutrient sensing and metabolic pathways and suggest that dysregulation of C9orf72 functions could compromise cellular fitness under conditions of nutrient stress. An expansion of repeated nucleotides in the non-coding region of the C9orf72 gene has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeat expansion leads to a reduced expression of the C9orf72 gene and loss of function of the C9orf72 protein may contribute to the pathogenesis. In this study, we identified a new mechanism through which C9orf72 influences nutrient sensing, autophagy, and metabolism. In the multi-cellular organism Caenorhabditis elegans, the C9orf72 orthologue regulates the activity of TFEB, a crucial transcriptional regulator of autophagic and lysosomal genes, through which the lipid metabolism and survival are influenced especially under nutrient stress conditions. The regulatory effect of C9orf72 on TFEB is conserved in mammals, and this is mediated by the dynamic regulation of the Rag GTPases by C9orf72. Given the critical role of the Rag GTPases in nutrient sensing and autophagy, we propose that the C9orf72 function is important for metabolic homeostasis in the cell and its deficiency can lead to compromised fitness under stress conditions.
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Affiliation(s)
- Yon Ju Ji
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States of America
- Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD, United States of America
| | - Janet Ugolino
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States of America
- Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD, United States of America
| | - Tao Zhang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States of America
- Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD, United States of America
| | - Jiayin Lu
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States of America
- Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD, United States of America
| | - Dohoon Kim
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States of America
| | - Jiou Wang
- Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, United States of America
- Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, MD, United States of America
- * E-mail:
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Abstract
DNA methylation at CpG sites is an essential epigenetic mark that regulates gene expression during mammalian development and diseases. Methylome refers to the entire set of methylation modifications present in the whole genome. Over the last several years, an increasing number of reports on brain DNA methylome reported the association between aberrant methylation and the abnormalities in the expression of critical genes known to have critical roles during aging and neurodegenerative diseases. Consequently, the role of methylation in understanding neurodegenerative diseases has been under focus. This review outlines the current knowledge of the human brain DNA methylomes during aging and neurodegenerative diseases. We describe the differentially methylated genes from fetal stage to old age and their biological functions. Additionally, we summarize the key aspects and methylated genes identified from brain methylome studies on neurodegenerative diseases. The brain methylome studies could provide a basis for studying the functional aspects of neurodegenerative diseases.
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Affiliation(s)
- Renuka Prasad
- Department of Life Science, University of Seoul, Seoul 02504, Korea
| | - Eek-Hoon Jho
- Department of Life Science, University of Seoul, Seoul 02504, Korea
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Jury N, Abarzua S, Diaz I, Guerra MV, Ampuero E, Cubillos P, Martinez P, Herrera-Soto A, Arredondo C, Rojas F, Manterola M, Rojas A, Montecino M, Varela-Nallar L, van Zundert B. Widespread loss of the silencing epigenetic mark H3K9me3 in astrocytes and neurons along with hippocampal-dependent cognitive impairment in C9orf72 BAC transgenic mice. Clin Epigenetics 2020; 12:32. [PMID: 32070418 PMCID: PMC7029485 DOI: 10.1186/s13148-020-0816-9] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2019] [Accepted: 01/23/2020] [Indexed: 12/13/2022] Open
Abstract
Background Hexanucleotide repeat expansions of the G4C2 motif in a non-coding region of the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Tissues from C9ALS/FTD patients and from mouse models of ALS show RNA foci, dipeptide-repeat proteins, and notably, widespread alterations in the transcriptome. Epigenetic processes regulate gene expression without changing DNA sequences and therefore could account for the altered transcriptome profiles in C9ALS/FTD; here, we explore whether the critical repressive marks H3K9me2 and H3K9me3 are altered in a recently developed C9ALS/FTD BAC mouse model (C9BAC). Results Chromocenters that constitute pericentric constitutive heterochromatin were visualized as DAPI- or Nucblue-dense foci in nuclei. Cultured C9BAC astrocytes exhibited a reduced staining signal for H3K9me3 (but not for H3K9me2) at chromocenters that was accompanied by a marked decline in the global nuclear level of this mark. Similar depletion of H3K9me3 at chromocenters was detected in astrocytes and neurons of the spinal cord, motor cortex, and hippocampus of C9BAC mice. The alterations of H3K9me3 in the hippocampus of C9BAC mice led us to identify previously undetected neuronal loss in CA1, CA3, and dentate gyrus, as well as hippocampal-dependent cognitive deficits. Conclusions Our data indicate that a loss of the repressive mark H3K9me3 in astrocytes and neurons in the central nervous system of C9BAC mice represents a signature during neurodegeneration and memory deficit of C9ALS/FTD.
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Affiliation(s)
- Nur Jury
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Sebastian Abarzua
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile.,FONDAP Center for Genome Regulation, Santiago, Chile
| | - Ivan Diaz
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Miguel V Guerra
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile
| | - Estibaliz Ampuero
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile.,Current address: Faculty of Health Sciences, Universidad Autónoma de Chile, Santiago, Chile
| | - Paula Cubillos
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Pablo Martinez
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Andrea Herrera-Soto
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile
| | - Cristian Arredondo
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Fabiola Rojas
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile
| | - Marcia Manterola
- Program of Human Genetics, ICBM, Faculty of Medicine, Universidad de Chile, Santiago, Chile
| | - Adriana Rojas
- Instituto de Genética Humana, Pontificia Universidad Javeriana, Bogotá, Colombia
| | - Martín Montecino
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.,FONDAP Center for Genome Regulation, Santiago, Chile
| | - Lorena Varela-Nallar
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.
| | - Brigitte van Zundert
- Institute of Biomedical Sciences (ICB), Faculty of Medicine & Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile. .,CARE Biomedical Research Center, Faculty of Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile.
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49
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Jackson JL, Finch NA, Baker MC, Kachergus JM, DeJesus-Hernandez M, Pereira K, Christopher E, Prudencio M, Heckman MG, Thompson EA, Dickson DW, Shah J, Oskarsson B, Petrucelli L, Rademakers R, van Blitterswijk M. Elevated methylation levels, reduced expression levels, and frequent contractions in a clinical cohort of C9orf72 expansion carriers. Mol Neurodegener 2020; 15:7. [PMID: 32000838 PMCID: PMC6993399 DOI: 10.1186/s13024-020-0359-8] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2019] [Accepted: 01/14/2020] [Indexed: 02/06/2023] Open
Abstract
Background A repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) is the most common genetic cause of two debilitating neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Currently, much remains unknown about which variables may modify these diseases. We sought to investigate associations between C9orf72 promoter methylation, RNA expression levels, and repeat length, their potential effects on disease features, as well as changes over time and within families. Methods All samples were obtained through the ALS Center at Mayo Clinic Florida. Our primary cohort included 75 unrelated patients with an expanded C9orf72 repeat, 33 patients who did not possess this expansion, and 20 control subjects without neurodegenerative diseases. Additionally, 67 members from 17 independent C9orf72 families were selected of whom 33 harbored this expansion. Longitudinally collected samples were available for 35 C9orf72 expansion carriers. To increase our understanding of C9orf72-related diseases, we performed quantitative methylation-sensitive restriction enzyme-based assays, digital molecular barcoding, quantitative real-time PCR, and Southern blotting. Results In our primary cohort, higher methylation levels were observed in patients with a C9orf72 repeat expansion than in patients without this expansion (p = 1.7e-13) or in control subjects (p = 3.3e-07). Moreover, we discovered that an increase in methylation levels was associated with a decrease in total C9orf72 transcript levels (p = 5.5e-05). These findings aligned with our observation that C9orf72 expansion carriers had lower expression levels of total C9orf72 transcripts than patients lacking this expansion (p = 3.7e-07) or control subjects (p = 9.1e-05). We also detected an elevation of transcripts containing intron 1a (upstream of the repeat) in patients carrying a C9orf72 repeat expansion compared to (disease) controls (p ≤ 0.01), an indication of abortive transcripts and/or a switch in transcription start site usage. While methylation and expression levels were relatively stable over time, fluctuations were seen in repeat length. Interestingly, contractions occurred frequently in parent-offspring transmissions (> 50%), especially in paternal transmissions. Furthermore, smaller repeat lengths were detected in currently unaffected individuals than in affected individuals (p = 8.9e-04) and they were associated with an earlier age at collection (p = 0.008). Conclusions In blood from C9orf72 expansion carriers, we found elevated methylation levels, reduced expression levels, and unstable expansions that tend to contract in successive generations, arguing against anticipation.
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Affiliation(s)
- Jazmyne L Jackson
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - NiCole A Finch
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Matthew C Baker
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Jennifer M Kachergus
- Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | | | - Kimberly Pereira
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Elizabeth Christopher
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Mercedes Prudencio
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Michael G Heckman
- Division of Biomedical Statistics and Informatics, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - E Aubrey Thompson
- Department of Cancer Biology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Dennis W Dickson
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Jaimin Shah
- Department of Neurology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Björn Oskarsson
- Department of Neurology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Leonard Petrucelli
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA
| | - Rosa Rademakers
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA.
| | - Marka van Blitterswijk
- Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA.
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50
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Cali CP, Park DS, Lee EB. Targeted DNA methylation of neurodegenerative disease genes via homology directed repair. Nucleic Acids Res 2019; 47:11609-11622. [PMID: 31680172 PMCID: PMC7145628 DOI: 10.1093/nar/gkz979] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Revised: 09/18/2019] [Accepted: 10/11/2019] [Indexed: 12/13/2022] Open
Abstract
DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5' regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.
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Affiliation(s)
- Christopher P Cali
- Translational Neuropathology Research Laboratory, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Daniel S Park
- Translational Neuropathology Research Laboratory, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Edward B Lee
- Translational Neuropathology Research Laboratory, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA
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