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Omar OMF, Kimble AL, Cheemala A, Tyburski JD, Pandey S, Wu Q, Reese B, Jellison ER, Hao B, Li Y, Yan R, Murphy PA. Endothelial TDP-43 depletion disrupts core blood-brain barrier pathways in neurodegeneration. Nat Neurosci 2025; 28:973-984. [PMID: 40087396 DOI: 10.1038/s41593-025-01914-5] [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: 01/03/2024] [Accepted: 02/05/2025] [Indexed: 03/17/2025]
Abstract
Endothelial cells (ECs) help maintain the blood-brain barrier but deteriorate in many neurodegenerative disorders. Here we show, using a specialized method to isolate EC and microglial nuclei from postmortem human cortex (92 donors, 50 male and 42 female, aged 20-98 years), that intranuclear cellular indexing of transcriptomes and epitopes enables simultaneous profiling of nuclear proteins and RNA transcripts at a single-nucleus resolution. We identify a disease-associated subset of capillary ECs in Alzheimer's disease, amyotrophic lateral sclerosis and frontotemporal degeneration. These capillaries exhibit reduced nuclear β-catenin and β-catenin-downstream genes, along with elevated TNF/NF-κB markers. Notably, these transcriptional changes correlate with the loss of nuclear TDP-43, an RNA-binding protein also depleted in neuronal nuclei. TDP-43 disruption in human and mouse ECs replicates these alterations, suggesting that TDP-43 deficiency in ECs is an important factor contributing to blood-brain barrier breakdown in neurodegenerative diseases.
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Affiliation(s)
- Omar M F Omar
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT, USA
| | - Amy L Kimble
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT, USA
| | - Ashok Cheemala
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT, USA
| | - Jordan D Tyburski
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT, USA
| | - Swati Pandey
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT, USA
| | - Qian Wu
- Department of Pathology, University of Connecticut Medical School, Farmington, CT, USA
| | - Bo Reese
- Center for Genome Innovation, University of Connecticut, Storrs, CT, USA
| | - Evan R Jellison
- Department of Immunology, University of Connecticut Medical School, Farmington, CT, USA
| | - Bing Hao
- Department of Molecular Biology and Biophysics, University of Connecticut Medical School, Farmington, CT, USA
| | - Yunfeng Li
- Department of Molecular Biology and Biophysics, University of Connecticut Medical School, Farmington, CT, USA
| | - Riqiang Yan
- Department of Neuroscience, University of Connecticut Medical School, Farmington, CT, USA
| | - Patrick A Murphy
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT, USA.
- Department of Immunology, University of Connecticut Medical School, Farmington, CT, USA.
- Department of Neuroscience, University of Connecticut Medical School, Farmington, CT, USA.
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2
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Cheng F, Chapman T, Venturato J, Davidson JM, Polido SA, Rosa‐Fernandes L, San Gil R, Suddull HJ, Zhang S, Macaslam CY, Szwaja P, Chung R, Walker AK, Rayner SL, Morsch M, Lee A. Proteomics Analysis of the TDP-43 Interactome in Cellular Models of ALS Pathogenesis. J Neurochem 2025; 169:e70079. [PMID: 40365763 PMCID: PMC12076276 DOI: 10.1111/jnc.70079] [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: 09/24/2024] [Revised: 04/14/2025] [Accepted: 04/15/2025] [Indexed: 05/15/2025]
Abstract
Cytoplasmic aggregation and nuclear depletion of TAR DNA-binding protein 43 (TDP-43) is a hallmark pathology of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). However, the protein interactome of TDP-43 remains incompletely defined. In this study, we aimed to identify putative TDP-43 protein partners within the nucleus and the cytoplasm and with different disease models of TDP-43 by comparing TDP-43 interaction partners in three different cell lines. We verified the levels of interaction of protein partners under stress conditions as well as after introducing TDP-43 variants containing ALS missense mutations (G294V and A315T). Overall, we identified 58 putative wild-type TDP-43 interactors, including novel binding partners responsible for RNA metabolism and splicing. Oxidative stress exposure broadly led to changes in TDP-43WT interactions with proteins involved in mRNA metabolism, suggesting a dysregulation of the transcriptional machinery early in disease. Conversely, although G294V and A315T mutations are both located in the C-terminal domain of TDP-43, both mutants presented different interactome profiles with most interaction partners involved in translational and transcriptional machinery. Overall, by correlating different cell lines and disease-simulating interventions, we provide a list of high-confidence TDP-43 interaction partners, including novel and previously reported proteins. Understanding pathological changes to TDP-43 and its specific interaction partners in different models of stress is critical to better understand TDP-43 proteinopathies and provide novel potential therapeutic targets and biomarkers.
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Affiliation(s)
- Flora Cheng
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Tyler Chapman
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Juliana Venturato
- Neurodegeneration Pathobiology LaboratoryClem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, the University of QueenslandSt. LuciaAustralia
| | - Jennilee M. Davidson
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Stella A. Polido
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Livia Rosa‐Fernandes
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Rebecca San Gil
- School of Medical Sciences, Faculty of Medicine and HealthThe University of SydneySydneyNew South WalesAustralia
| | - Hannah J. Suddull
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Selina Zhang
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Chiara Y. Macaslam
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Paulina Szwaja
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Roger Chung
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Adam K. Walker
- Neurodegeneration Pathobiology LaboratoryClem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, the University of QueenslandSt. LuciaAustralia
- Sydney Pharmacy School, Faculty of Medicine and HealthThe University of SydneySydneyNew South WalesAustralia
| | - Stephanie L. Rayner
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Marco Morsch
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
| | - Albert Lee
- Motor Neuron Disease Research Centre, Macquarie Medical School, Faculty of Medicine Health, and Human SciencesMacquarie UniversityNorth RydeNew South WalesAustralia
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3
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Cheemala A, Kimble AL, Burrage EN, Helming SB, Tyburski JD, Leclair NK, Omar OM, Zuberi AR, Murphy M, Jellison ER, Reese B, Hu X, Lutz CM, Yan R, Murphy PA. Amyotrophic lateral sclerosis and frontotemporal dementia mutation reduces endothelial TDP-43 and causes blood-brain barrier defects. SCIENCE ADVANCES 2025; 11:eads0505. [PMID: 40238886 PMCID: PMC12002129 DOI: 10.1126/sciadv.ads0505] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/26/2024] [Accepted: 03/12/2025] [Indexed: 04/18/2025]
Abstract
Mutations in the TARDBP gene encoding TDP-43 protein are linked to loss of function in neurons and familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We recently identified reduced nuclear TDP-43 in capillary endothelial cells (ECs) of donors with ALS-FTD. Because blood-brain barrier (BBB) permeability increases in ALS-FTD, we postulated that reduced nuclear TDP-43 in ECs might contribute. Here, we show that nuclear TDP-43 is reduced in ECs of mice with an ALS-FTD-associated mutation in TDP-43 (TardbpG348C) and that this leads to cell-autonomous loss of junctional complexes and BBB integrity. Targeted excision of TDP-43 in brain ECs recapitulates BBB defects and loss of junctional complexes and ultimately leads to fibrin deposition, gliosis, phospho-Tau accumulation, and impaired memory and social interaction. Transcriptional changes in TDP-43-deficient ECs resemble diseased brain ECs. These data show that nuclear loss of TDP-43 in brain ECs disrupts the BBB and causes hallmarks of FTD.
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Affiliation(s)
- Ashok Cheemala
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Amy L. Kimble
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Emily N. Burrage
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Stephen B. Helming
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Jordan D. Tyburski
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Nathan K. Leclair
- MD/PhD Program, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Omar M. Omar
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Aamir R. Zuberi
- Rare Disease Translational Center and Technology Evaluation and Development Laboratory, The Jackson Laboratory, Bar Harbor, ME, USA
| | - Melissa Murphy
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Evan R. Jellison
- Department of Immunology, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Bo Reese
- Center for Genome Innovation, University of Connecticut, Storrs, CT, USA
| | - Xiangyou Hu
- Department of Neuroscience, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Cathleen M. Lutz
- Rare Disease Translational Center and Technology Evaluation and Development Laboratory, The Jackson Laboratory, Bar Harbor, ME, USA
| | - Riqiang Yan
- Department of Neuroscience, University of Connecticut School of Medicine, Farmington, CT, USA
| | - Patrick A. Murphy
- Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, CT, USA
- Department of Immunology, University of Connecticut School of Medicine, Farmington, CT, USA
- Department of Neuroscience, University of Connecticut School of Medicine, Farmington, CT, USA
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4
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Wasielewska JM, Chaves JCS, Cabral-da-Silva MC, Pecoraro M, Viljoen SJ, Nguyen TH, Bella VL, Oikari LE, Ooi L, White AR. A patient-derived amyotrophic lateral sclerosis blood-brain barrier model for focused ultrasound-mediated anti-TDP-43 antibody delivery. Fluids Barriers CNS 2024; 21:65. [PMID: 39138578 PMCID: PMC11323367 DOI: 10.1186/s12987-024-00565-1] [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: 02/28/2024] [Accepted: 08/06/2024] [Indexed: 08/15/2024] Open
Abstract
BACKGROUND Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored. METHODS Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro. RESULTS Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. CONCLUSIONS Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.
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Affiliation(s)
- Joanna M Wasielewska
- Brain and Mental Health Program, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
- Faculty of Medicine, University of Queensland, St. Lucia, QLD, Australia
| | - Juliana C S Chaves
- Brain and Mental Health Program, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
| | - Mauricio Castro Cabral-da-Silva
- Molecular Horizons, School of Chemistry and Molecular Bioscience, University of Wollongong, Northfields Avenue, Wollongong, NSW, Australia
- The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Murdoch Children's Research Institute (MCRI), Parkville, VIC, Australia
| | - Martina Pecoraro
- ALS Clinical Research Centre and Laboratory of Neurochemistry, Department of Biomedicine, Neurosciences and Advanced Diagnosis, University of Palermo, Palermo, Italy
| | - Stephani J Viljoen
- Brain and Mental Health Program, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
- School of Biomedical Science, University of Queensland, St. Lucia, QLD, Australia
| | - Tam Hong Nguyen
- Flow Cytometry and Imaging Facility, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
| | - Vincenzo La Bella
- ALS Clinical Research Centre and Laboratory of Neurochemistry, Department of Biomedicine, Neurosciences and Advanced Diagnosis, University of Palermo, Palermo, Italy
| | - Lotta E Oikari
- Brain and Mental Health Program, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia
| | - Lezanne Ooi
- Molecular Horizons, School of Chemistry and Molecular Bioscience, University of Wollongong, Northfields Avenue, Wollongong, NSW, Australia
| | - Anthony R White
- Brain and Mental Health Program, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia.
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia.
- School of Biomedical Science, University of Queensland, St. Lucia, QLD, Australia.
- A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.
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5
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Scherer NM, Maurel C, Graus M, McAlary L, Richter G, Radford RW, Hogan A, Don E, Lee A, Yerbury J, Francois M, Chung R, Morsch M. RNA-binding properties orchestrate TDP-43 homeostasis through condensate formation in vivo. Nucleic Acids Res 2024; 52:5301-5319. [PMID: 38381071 PMCID: PMC11109982 DOI: 10.1093/nar/gkae112] [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: 06/23/2023] [Revised: 01/12/2024] [Accepted: 02/06/2024] [Indexed: 02/22/2024] Open
Abstract
Insoluble cytoplasmic aggregate formation of the RNA-binding protein TDP-43 is a major hallmark of neurodegenerative diseases including Amyotrophic Lateral Sclerosis. TDP-43 localizes predominantly in the nucleus, arranging itself into dynamic condensates through liquid-liquid phase separation (LLPS). Mutations and post-translational modifications can alter the condensation properties of TDP-43, contributing to the transition of liquid-like biomolecular condensates into solid-like aggregates. However, to date it has been a challenge to study the dynamics of this process in vivo. We demonstrate through live imaging that human TDP-43 undergoes nuclear condensation in spinal motor neurons in a living animal. RNA-binding deficiencies as well as post-translational modifications can lead to aberrant condensation and altered TDP-43 compartmentalization. Single-molecule tracking revealed an altered mobility profile for RNA-binding deficient TDP-43. Overall, these results provide a critically needed in vivo characterization of TDP-43 condensation, demonstrate phase separation as an important regulatory mechanism of TDP-43 accessibility, and identify a molecular mechanism of how functional TDP-43 can be regulated.
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Affiliation(s)
- Natalie M Scherer
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Cindy Maurel
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Matthew S Graus
- The David Richmond Laboratory for Cardio-Vascular Development: gene regulation and editing, Centenary Institute, The University of Sydney, School of Medical Sciences, Sydney, NSW 2006, Australia
- Genome Imaging Centre, Centenary Institute, The University of Sydney, Sydney, NSW 2006, Australia
| | - Luke McAlary
- Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia
| | - Grant Richter
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Rowan A W Radford
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Alison Hogan
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Emily K Don
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Albert Lee
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Justin Yerbury
- Molecular Horizons and School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia
| | - Mathias Francois
- The David Richmond Laboratory for Cardio-Vascular Development: gene regulation and editing, Centenary Institute, The University of Sydney, School of Medical Sciences, Sydney, NSW 2006, Australia
- Genome Imaging Centre, Centenary Institute, The University of Sydney, Sydney, NSW 2006, Australia
| | - Roger S Chung
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
| | - Marco Morsch
- Faculty of Medicine, Health & Human Sciences, Macquarie Medical School, MND Research Centre, Macquarie University, Sydney, NSW 2109, Australia
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6
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Milani M, Della Valle I, Rossi S, Fabbrizio P, Margotta C, Nardo G, Cozzolino M, D'Ambrosi N, Apolloni S. Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models. Neurotherapeutics 2024; 21:e00346. [PMID: 38493058 PMCID: PMC11070272 DOI: 10.1016/j.neurot.2024.e00346] [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: 12/31/2023] [Revised: 02/28/2024] [Accepted: 03/06/2024] [Indexed: 03/18/2024] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease influenced by genetic, epigenetic, and environmental factors, resulting in dysfunction in cellular and molecular pathways. The limited efficacy of current treatments highlights the need for combination therapies targeting multiple aspects of the disease. Niclosamide, an anthelminthic drug listed as an essential medicine, has been repurposed in clinical trials for different diseases due to its anti-inflammatory and anti-fibrotic properties. Niclosamide can inhibit various molecular pathways (e.g., STAT3, mTOR) that are dysregulated in ALS, suggesting its potential to disrupt these altered mechanisms associated with the pathology. We administered niclosamide intraperitoneally to two transgenic murine models, SOD1-G93A and FUS mice, mimicking key pathological processes of ALS. The treatment was initiated at the onset of symptoms, and we assessed disease progression by neurological scores, rotarod and wire tests, and monitored survival. Furthermore, we investigated cellular and molecular mechanisms affected by niclosamide in the spinal cord and muscle of ALS mice. In both models, the administration of niclosamide resulted in a slowdown of disease progression, an increase in survival rates, and an improvement in tissue pathology. This was characterised by reduced gliosis, motor neuron loss, muscle atrophy, and inflammatory pathways. Based on these results, our findings demonstrate that niclosamide can impact multiple pathways involved in ALS. This multi-targeted approach leads to a slowdown in the progression of the disease, positioning niclosamide as a promising candidate for repurposing in the treatment of ALS.
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Affiliation(s)
- Martina Milani
- Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy; Program in Cellular and Molecular Biology, University of Rome Tor Vergata, 00133 Rome, Italy
| | - Ilaria Della Valle
- Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy; Program in Cellular and Molecular Biology, University of Rome Tor Vergata, 00133 Rome, Italy
| | - Simona Rossi
- Institute of Translational Pharmacology, CNR, 00133 Rome, Italy
| | - Paola Fabbrizio
- Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milan, Italy
| | - Cassandra Margotta
- Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milan, Italy
| | - Giovanni Nardo
- Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milan, Italy
| | - Mauro Cozzolino
- Institute of Translational Pharmacology, CNR, 00133 Rome, Italy
| | - Nadia D'Ambrosi
- Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy
| | - Savina Apolloni
- Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy; UniCamillus-Saint Camillus International University of Health Sciences, Rome, Italy.
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7
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Cheemala A, Kimble AL, Tyburski JD, Leclair NK, Zuberi AR, Murphy M, Jellison ER, Reese B, Hu X, Lutz CM, Yan R, Murphy PA. Loss of Endothelial TDP-43 Leads to Blood Brain Barrier Defects in Mouse Models of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.13.571184. [PMID: 38168388 PMCID: PMC10760101 DOI: 10.1101/2023.12.13.571184] [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
Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the TARDBP gene that encodes the protein are linked to familial Frontal Temporal Lobar Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Although the focus has been on neuronal cell dysfunction caused by TDP-43 variants, TARDBP mRNA transcripts are expressed at similar levels in brain endothelial cells (ECs). Since increased permeability across the blood brain barrier (BBB) precedes cognitive decline, we postulated that altered functions of TDP-43 in ECs contributes to BBB dysfunction in neurodegenerative disease. To test this hypothesis, we examined EC function and BBB properties in mice with either knock-in mutations found in ALS/FTLD patients (TARDBP G348C and GRN R493X ) or EC-specific deletion of TDP-43 throughout the endothelium (Cdh5(PAC)CreERT2; Tardbp ff ) or restricted to brain endothelium (Slco1c1(BAC)CreERT2; Tardbp ff ). We found that TARDBP G348C mice exhibited increased permeability to 3kDa Texas Red dextran and NHS-biotin, relative to their littermate controls, which could be recapitulated in cultured brain ECs from these mice. Nuclear levels of TDP-43 were reduced in vitro and in vivo in ECs from TARDBP G348C mice. This coincided with a reduction in junctional proteins VE-cadherin, claudin-5 and ZO-1 in isolated ECs, supporting a cell autonomous effect on barrier function through a loss of nuclear TDP-43. We further examined two models of Tardbp deletion in ECs, and found that the loss of TDP-43 throughout the endothelium led to systemic endothelial activation and permeability. Deletion specifically within the brain endothelium acutely increased BBB permeability, and eventually led to hallmarks of FTD, including fibrin deposition, microglial and astrocyte activation, and behavioral defects. Together, these data show that TDP-43 dysfunction specifically within brain ECs would contribute to the BBB defects observed early in the progression of ALS/FTLD.
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Affiliation(s)
- Ashok Cheemala
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT
| | - Amy L Kimble
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT
| | - Jordan D Tyburski
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT
| | - Nathan K Leclair
- MD/PhD Program, University of Connecticut School of Medicine, Farmington, CT
| | - Aamir R Zuberi
- Rare Disease Translational Center and Technology Evaluation and Development Laboratory, The Jackson Laboratory, Bar Harbor, ME
| | - Melissa Murphy
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT
| | - Evan R Jellison
- Department of Immunology, University of Connecticut Medical School, Farmington, CT
| | - Bo Reese
- Center for Genome Innovation, University of Connecticut, Storrs, CT
| | - Xiangyou Hu
- Department of Neuroscience, University of Connecticut Medical School, Farmington, CT
| | - Cathleen M Lutz
- Rare Disease Translational Center and Technology Evaluation and Development Laboratory, The Jackson Laboratory, Bar Harbor, ME
| | - Riqiang Yan
- Department of Neuroscience, University of Connecticut Medical School, Farmington, CT
| | - Patrick A Murphy
- Center for Vascular Biology, University of Connecticut Medical School, Farmington, CT
- Department of Immunology, University of Connecticut Medical School, Farmington, CT
- Department of Neuroscience, University of Connecticut Medical School, Farmington, CT
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8
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Woo E, Bredvik K, Liu B, Fuchs TJ, Manfredi G, Konrad C. Machine learning approaches based on fibroblast morphometry do not predict ALS. Neurobiol Aging 2023; 130:80-83. [PMID: 37473581 DOI: 10.1016/j.neurobiolaging.2023.06.010] [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: 01/02/2023] [Revised: 06/08/2023] [Accepted: 06/13/2023] [Indexed: 07/22/2023]
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease with limited therapeutic options. Biomarkers are needed for early disease detection, clinical trial design, and personalized medicine. Early evidence suggests that specific morphometric features in ALS primary skin fibroblasts may be used as biomarkers; however, this hypothesis has not been rigorously tested in conclusively large fibroblast populations. Here, we imaged ALS-relevant organelles (mitochondria, endoplasmic reticulum, lysosomes) and proteins (TAR DNA-binding protein 43, Ras GTPase-activating protein-binding protein 1, heat-shock protein 60) at baseline and under stress perturbations and tested their predictive power on a total set of 443 human fibroblast lines from ALS and healthy individuals. Machine learning approaches were able to confidently predict stress perturbation states (ROC-AUC ∼0.99) but not disease groups or clinical features (ROC-AUC 0.58-0.64). Our findings indicate that multivariate models using patient-derived fibroblast morphometry can accurately predict different stressors but are insufficient to develop viable ALS biomarkers.
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Affiliation(s)
- Evan Woo
- Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Kirsten Bredvik
- Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Bangyan Liu
- Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Thomas J Fuchs
- Hasso Plattner Institute for Digital Health, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Giovanni Manfredi
- Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA
| | - Csaba Konrad
- Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY, USA.
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9
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Guerra M, Meola L, Lattante S, Conte A, Sabatelli M, Sette C, Bernardini C. Characterization of SOD1-DT, a Divergent Long Non-Coding RNA in the Locus of the SOD1 Human Gene. Cells 2023; 12:2058. [PMID: 37626868 PMCID: PMC10453398 DOI: 10.3390/cells12162058] [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: 06/29/2023] [Revised: 08/03/2023] [Accepted: 08/06/2023] [Indexed: 08/27/2023] Open
Abstract
Researchers studying Amyotrophic Lateral Sclerosis (ALS) have made significant efforts to find a unique mechanism to explain the etiopathology of the different forms of the disease. However, despite several mutations associated with ALS having been discovered in recent years, the link between the mutated genes and its onset has not yet been fully elucidated. Among the genes associated with ALS, superoxide dismutase 1 (SOD1) was the first to be identified, but its role in the etiopathogenesis of the disease is still unclear. In recent years, research has been focused on the non-coding part of the genome to fully understand the mechanisms underlying gene regulation. Non-coding RNAs are conserved molecules and are not usually translated in protein. A total of 98% of the human genome is composed of non-protein coding sequences with roles in the transcriptional and post-transcriptional regulation of gene expression. In this study, we characterized a divergent nuclear lncRNA (SOD1-DT) transcribed in the antisense direction from the 5' region of the SOD1 coding gene in both the SH-SY5Y cell line and fibroblasts derived from ALS patients. Interestingly, this lncRNA seems to regulate gene expression, since its inhibition leads to the upregulation of surrounding genes including SOD1. SOD1-DT represents a very complex molecule, displaying allelic and transcriptional variability concerning transposable elements (TEs) included in its sequence, widening the scenario of gene expression regulation in ALS disease.
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Affiliation(s)
- Marika Guerra
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Hearth, 00168 Rome, Italy; (L.M.); (C.S.)
- GSTeP-Organoids Research Core Facility, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy
| | - Lucia Meola
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Hearth, 00168 Rome, Italy; (L.M.); (C.S.)
| | - Serena Lattante
- Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy;
| | - Amelia Conte
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy; (A.C.); (M.S.)
| | - Mario Sabatelli
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy; (A.C.); (M.S.)
- Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
| | - Claudio Sette
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Hearth, 00168 Rome, Italy; (L.M.); (C.S.)
- GSTeP-Organoids Research Core Facility, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy
| | - Camilla Bernardini
- Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Hearth, 00168 Rome, Italy; (L.M.); (C.S.)
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10
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Gianferrari G, Martinelli I, Simonini C, Zucchi E, Fini N, Carra S, Moglia C, Mandrioli J. Case report: p.Glu134del SOD1 mutation in two apparently unrelated ALS patients with mirrored phenotype. Front Neurol 2023; 13:1052341. [PMID: 36686515 PMCID: PMC9846158 DOI: 10.3389/fneur.2022.1052341] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2022] [Accepted: 12/05/2022] [Indexed: 01/05/2023] Open
Abstract
With upcoming personalized approaches based on genetics, it is important to report new mutations in amyotrophic lateral sclerosis (ALS) genes in order to understand their pathogenicity and possible patient responses to specific therapies. SOD1 mutations are the second most frequent genetic cause of ALS in European populations. Here, we describe two seemingly unrelated Italian patients with ALS carrying the same SOD1 heterozygous c.400_402 deletion (p.Glu134del). Both patients had spinal onset in their lower limbs, progressive muscular weakness with respiratory involvement, and sparing bulbar function. In addition to the clinical picture, we discuss the possible pathogenic role of this unfamiliar SOD1 mutation.
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Affiliation(s)
- Giulia Gianferrari
- Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Ilaria Martinelli
- Department of Neurosciences, Azienda Ospedaliero-Universitaria di Modena, Modena, Italy,Clinical and Experimental PhD Program, University of Modena and Reggio Emilia, Modena, Italy,*Correspondence: Ilaria Martinelli ✉
| | - Cecilia Simonini
- Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Elisabetta Zucchi
- Department of Neurosciences, Azienda Ospedaliero-Universitaria di Modena, Modena, Italy,Neurosciences PhD Program, University of Modena and Reggio Emilia, Modena, Italy
| | - Nicola Fini
- Department of Neurosciences, Azienda Ospedaliero-Universitaria di Modena, Modena, Italy
| | - Serena Carra
- Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Cristina Moglia
- S.C Neurology 1U, Azienda Ospedaliero-Universitaria Città della Salute e della Scienza Torino, Torino, Italy,“Rita Levi Montalcini” Department of Neuroscience, University of Turin, Torino, Italy
| | - Jessica Mandrioli
- Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy,Department of Neurosciences, Azienda Ospedaliero-Universitaria di Modena, Modena, Italy
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11
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Romano R, De Luca M, Del Fiore VS, Pecoraro M, Lattante S, Sabatelli M, La Bella V, Bucci C. Allele-specific silencing as therapy for familial amyotrophic lateral sclerosis caused by the p.G376D TARDBP mutation. Brain Commun 2022; 4:fcac315. [PMID: 36751500 PMCID: PMC9897181 DOI: 10.1093/braincomms/fcac315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Revised: 08/25/2022] [Accepted: 11/28/2022] [Indexed: 12/23/2022] Open
Abstract
Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by the degeneration of motor neurons. There is no treatment for this disease that affects the ability to move, eat, speak and finally breathe, causing death. In an Italian family, a heterozygous pathogenic missense variant has been previously discovered in Exon 6 of the gene TARDBP encoding the TAR DNA-binding protein 43 protein. Here, we developed a potential therapeutic tool based on allele-specific small interfering RNAs for familial amyotrophic lateral sclerosis with the heterozygous missense mutation c.1127G>A. We designed a small interfering RNA that was able to diminish specifically the expression of the exogenous Green Fluorescent Protein (TAR DNA-binding protein 43G376D mutant protein) in HEK-293T cells but not that of the Green Fluorescent Protein (TAR DNA-binding protein 43 wild-type). Similarly, this small interfering RNA silenced the mutated allele in fibroblasts derived from patients with amyotrophic lateral sclerosis but did not silence the wild-type gene in control fibroblasts. In addition, we established that silencing the mutated allele was able to strongly reduce the pathological cellular phenotypes induced by TAR DNA-binding protein 43G376D expression, such as the presence of cytoplasmic aggregates. Thus, we have identified a small interfering RNA that could be used to silence specifically the mutated allele to try a targeted therapy for patients carrying the p.G376D TAR DNA-binding protein 43 mutation.
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Affiliation(s)
- Roberta Romano
- Department of Biological and Environmental Sciences and Technologies (DiSTeBA), Via Provinciale Lecce-Monteroni n.165, 73100 Lecce, Italy
| | - Maria De Luca
- Department of Biological and Environmental Sciences and Technologies (DiSTeBA), Via Provinciale Lecce-Monteroni n.165, 73100 Lecce, Italy
| | - Victoria Stefania Del Fiore
- Department of Biological and Environmental Sciences and Technologies (DiSTeBA), Via Provinciale Lecce-Monteroni n.165, 73100 Lecce, Italy
| | - Martina Pecoraro
- ALS Clinical Research Center, P Giaccone University Hospital and Department of Biomedicine, Neuroscience and advanced Diagnostic (BIND), University of Palermo, via Gaetano La Loggia n° 1, 90129 Palermo, Italy
| | - Serena Lattante
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy,Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Mario Sabatelli
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy,Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy
| | - Vincenzo La Bella
- ALS Clinical Research Center, P Giaccone University Hospital and Department of Biomedicine, Neuroscience and advanced Diagnostic (BIND), University of Palermo, via Gaetano La Loggia n° 1, 90129 Palermo, Italy
| | - Cecilia Bucci
- Correspondence to: Cecilia Bucci Department of Biological and Environmental Sciences and Technologies (DiSTeBA) Via Provinciale Lecce-Monteroni n.165 73100 Lecce, Italy E-mail:
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12
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Martello F, Lattante S, Doronzio PN, Conte A, Bisogni G, Orteschi D, Luigetti M, Marrucci MA, Zollino M, Sabatelli M, Marangi G. Generation of an induced pluripotent stem cell line (UCSCi002-A) from a patient with a variant in TARDBP gene associated with familial amyotrophic lateral sclerosis and frontotemporal dementia. Stem Cell Res 2022; 62:102825. [PMID: 35667216 DOI: 10.1016/j.scr.2022.102825] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Revised: 04/21/2022] [Accepted: 05/29/2022] [Indexed: 11/18/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects motor neurons. In 20% of cases, ALS appears in comorbidity with frontotemporal dementia (FTD). We generated patient-derived-induced Pluripotent Stem Cells (iPSCs), from an ALS/FTD patient. The patient had a familial form of the disease and a missense variant in TARDBP gene. We used an established protocol based on Sendai virus to reprogram fibroblasts. We confirmed the stemness and the pluripotency of the iPSC clones, thus generating embryoid bodies. We believe that the iPSC line carrying a TARDBP mutation could be a valuable tool to investigate TDP-43 proteinopathy linked to ALS.
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Affiliation(s)
- Francesco Martello
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Serena Lattante
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.
| | - Paolo Niccolò Doronzio
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Amelia Conte
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy; Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy
| | - Giulia Bisogni
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy; Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy
| | - Daniela Orteschi
- Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Marco Luigetti
- Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Maria Alessandra Marrucci
- Section of Legal Medicine, Department of Healthcare Surveillance and Bioethics, Università Cattolica del Sacro Cuore, Rome, Italy
| | - Marcella Zollino
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Mario Sabatelli
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy; Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy
| | - Giuseppe Marangi
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
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13
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Perciballi E, Bovio F, Rosati J, Arrigoni F, D’Anzi A, Lattante S, Gelati M, De Marchi F, Lombardi I, Ruotolo G, Forcella M, Mazzini L, D’Alfonso S, Corrado L, Sabatelli M, Conte A, De Gioia L, Martino S, Vescovi AL, Fusi P, Ferrari D. Characterization of the p.L145F and p.S135N Mutations in SOD1: Impact on the Metabolism of Fibroblasts Derived from Amyotrophic Lateral Sclerosis Patients. Antioxidants (Basel) 2022; 11:antiox11050815. [PMID: 35624679 PMCID: PMC9137766 DOI: 10.3390/antiox11050815] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2022] [Revised: 04/13/2022] [Accepted: 04/20/2022] [Indexed: 12/24/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1L145F and SOD1S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.
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Affiliation(s)
- Elisa Perciballi
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
| | - Federica Bovio
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
| | - Jessica Rosati
- Cellular Reprogramming Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini 1, 71013 San Giovanni Rotondo, Italy; (J.R.); (A.D.); (G.R.)
| | - Federica Arrigoni
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
| | - Angela D’Anzi
- Cellular Reprogramming Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini 1, 71013 San Giovanni Rotondo, Italy; (J.R.); (A.D.); (G.R.)
| | - Serena Lattante
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Largo Francesco Vito, 1, 00168 Rome, Italy;
- Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, Italy
| | - Maurizio Gelati
- UPTA Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini 1, 71013 San Giovanni Rotondo, Italy;
| | - Fabiola De Marchi
- ALS Centre Maggiore della Carità Hospital and Università del Piemonte Orientale, 28100 Novara, Italy; (F.D.M.); (L.M.)
| | - Ivan Lombardi
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
| | - Giorgia Ruotolo
- Cellular Reprogramming Unit, Fondazione IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini 1, 71013 San Giovanni Rotondo, Italy; (J.R.); (A.D.); (G.R.)
| | - Matilde Forcella
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
| | - Letizia Mazzini
- ALS Centre Maggiore della Carità Hospital and Università del Piemonte Orientale, 28100 Novara, Italy; (F.D.M.); (L.M.)
| | - Sandra D’Alfonso
- Department of Health Sciences, Center on Autoimmune and Allergic Diseases (CAAD), UPO, University of Eastern Piedmont, 28100 Novara, Italy; (S.D.); (L.C.)
| | - Lucia Corrado
- Department of Health Sciences, Center on Autoimmune and Allergic Diseases (CAAD), UPO, University of Eastern Piedmont, 28100 Novara, Italy; (S.D.); (L.C.)
| | - Mario Sabatelli
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, Italy; (M.S.); (A.C.)
- Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Largo Francesco Vito, 1, 00168 Rome, Italy
| | - Amelia Conte
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Largo A. Gemelli 8, 00168 Rome, Italy; (M.S.); (A.C.)
- Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Largo Francesco Vito, 1, 00168 Rome, Italy
| | - Luca De Gioia
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
| | - Sabata Martino
- Department of Chemistry, Biology and Biotechnology, University of Perugia, Via del Giochetto, 06123 Perugia, Italy;
| | - Angelo Luigi Vescovi
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
- Fondazione IRCCS Casa Sollievo della Sofferenza, Viale dei Cappuccini 1, 71013 San Giovanni Rotondo, Italy
| | - Paola Fusi
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
- Correspondence: (P.F.); (D.F.); Tel.: +39-348-004-6641 (D.F.)
| | - Daniela Ferrari
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza, 2, 20126 Milan, Italy; (E.P.); (F.B.); (F.A.); (I.L.); (M.F.); (L.D.G.); (A.L.V.)
- Correspondence: (P.F.); (D.F.); Tel.: +39-348-004-6641 (D.F.)
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14
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Sahu R, Mehan S, Kumar S, Prajapati A, Alshammari A, Alharbi M, Assiri MA, Narula AS. Effect of alpha-mangostin in the prevention of behavioural and neurochemical defects in methylmercury-induced neurotoxicity in experimental rats. Toxicol Rep 2022; 9:977-998. [PMID: 35783250 PMCID: PMC9247835 DOI: 10.1016/j.toxrep.2022.04.023] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Revised: 04/17/2022] [Accepted: 04/20/2022] [Indexed: 12/11/2022] Open
Abstract
Methylmercury (MeHg+) is a known neurotoxin that causes progressive motor neuron degeneration in the central nervous system. Axonal degeneration, oligodendrocyte degeneration, and myelin basic protein (MBP) deficits are among the neuropathological abnormalities caused by MeHg+ in amyotrophic lateral sclerosis (ALS). This results in demyelination and motor neuron death in both humans and animals. Previous experimental studies have confirmed that overexpression of the extracellular signalling regulated kinase (ERK1/2) signalling contributes to glutamate excitotoxicity, inflammatory response of microglial cells, and oligodendrocyte (OL) dysfunction that promotes myelin loss. Alpha-mangostin (AMG), an active ingredient obtained from the tree "Garcinia mangostana Linn," has been used in experimental animals to treat a variety of brain disorders, including Parkinson's and Huntington's disease memory impairment, Alzheimer's disease, and schizophrenia, including Parkinson's disease and Huntington's disease memory impairment, Alzheimer's disease, and schizophrenia. AMG has traditionally been used as an antioxidant, anti-inflammatory, and neuroprotective agent.Accordingly, we investigated the therapeutic potential of AMG (100 and 200 mg/kg) in experimental rats with methylmercury (MeHg+)-induced neurotoxicity. The neuroprotective effect of AMG on behavioural, cellular, molecular, and other gross pathological changes, such as histopathological alterations in MeHg+ -treated rat brains, is presented. The neurological behaviour of experimental rats was evaluated using a Morris water maze (MWM), open field test (OFT), grip strength test (GST), and force swim test (FST). In addition, we investigate AMG's neuroprotective effect by restoring MBP levels in cerebral spinal fluid and whole rat brain homogenate. The apoptotic, pro-inflammatory, and oxidative stress markers were measured in rat blood plasma samples and brain homogenate. According to the findings of this study, AMG decreases ERK-1/2 levels and modulates neurochemical alterations in rat brains, minimising MeHg+ -induced neurotoxicity.
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Affiliation(s)
- Rakesh Sahu
- Neuropharmacology Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India
| | - Sidharth Mehan
- Neuropharmacology Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India
| | - Sumit Kumar
- Neuropharmacology Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India
| | - Aradhana Prajapati
- Neuropharmacology Division, Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India
| | - Abdulrahman Alshammari
- Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Post Box 2455, Riyadh 11451, Saudi Arabia
| | - Metab Alharbi
- Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Post Box 2455, Riyadh 11451, Saudi Arabia
| | - Mohammed A. Assiri
- Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Post Box 2455, Riyadh 11451, Saudi Arabia
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15
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Rubio MA, Herrando-Grabulosa M, Velasco R, Blasco I, Povedano M, Navarro X. TDP-43 Cytoplasmic Translocation in the Skin Fibroblasts of ALS Patients. Cells 2022; 11:209. [PMID: 35053327 PMCID: PMC8773870 DOI: 10.3390/cells11020209] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 12/23/2021] [Accepted: 01/04/2022] [Indexed: 12/10/2022] Open
Abstract
Diagnosis of ALS is based on clinical symptoms when motoneuron degeneration is significant. Therefore, new approaches for early diagnosis are needed. We aimed to assess if alterations in appearance and cellular localization of cutaneous TDP-43 may represent a biomarker for ALS. Skin biopsies from 64 subjects were analyzed: 44 ALS patients, 10 healthy controls (HC) and 10 neurological controls (NC) (Parkinson's disease and multiple sclerosis). TDP-43 immunoreactivity in epidermis and dermis was analyzed, as well as the percentage of cells with TDP-43 cytoplasmic localization. We detected a higher amount of TDP-43 in epidermis (p < 0.001) and in both layers of dermis (p < 0.001), as well as a higher percentage of TDP-43 cytoplasmic positive cells (p < 0.001) in the ALS group compared to HC and NC groups. Dermal cells containing TDP-43 were fibroblasts as identified by co-labeling against vimentin. ROC analyses (AUC 0.867, p < 0.001; CI 95% 0.800-0.935) showed that detection of 24.1% cells with cytoplasmic TDP-43 positivity in the dermis had 85% sensitivity and 80% specificity for detecting ALS. We have identified significantly increased TDP-43 levels in epidermis and in the cytoplasm of dermal cells of ALS patients. Our findings provide support for the use of TDP-43 in skin biopsies as a potential biomarker.
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Affiliation(s)
- Miguel A. Rubio
- Neuromuscular Unit, Department of Neurology, Hospital del Mar, 08003 Barcelona, Spain;
- Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences and CIBERNED, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; (M.H.-G.); (R.V.); (I.B.)
| | - Mireia Herrando-Grabulosa
- Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences and CIBERNED, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; (M.H.-G.); (R.V.); (I.B.)
| | - Roser Velasco
- Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences and CIBERNED, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; (M.H.-G.); (R.V.); (I.B.)
- Neuro-Oncology Unit, Department of Neurology, Hospital Universitari de Bellvitge-ICO and IDIBELL, 08907 L’Hospitalet, Spain
| | - Israel Blasco
- Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences and CIBERNED, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; (M.H.-G.); (R.V.); (I.B.)
| | - Monica Povedano
- Department of Neurology, Hospital Universitari de Bellvitge, 08907 L’Hospitalet, Spain;
| | - Xavier Navarro
- Department of Cell Biology, Physiology and Immunology, Institute of Neurosciences and CIBERNED, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; (M.H.-G.); (R.V.); (I.B.)
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16
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Leskelä S, Hoffmann D, Rostalski H, Huber N, Wittrahm R, Hartikainen P, Korhonen V, Leinonen V, Hiltunen M, Solje E, Remes AM, Haapasalo A. FTLD Patient-Derived Fibroblasts Show Defective Mitochondrial Function and Accumulation of p62. Mol Neurobiol 2021; 58:5438-5458. [PMID: 34328616 PMCID: PMC8599259 DOI: 10.1007/s12035-021-02475-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Accepted: 06/25/2021] [Indexed: 11/25/2022]
Abstract
Frontotemporal lobar degeneration (FTLD) is a clinically, genetically, and neuropathologically heterogeneous group of neurodegenerative syndromes, leading to progressive cognitive dysfunction and frontal and temporal atrophy. C9orf72 hexanucleotide repeat expansion (C9-HRE) is the most common genetic cause of FTLD, but pathogenic mechanisms underlying FTLD are not fully understood. Here, we compared cellular features and functional properties, especially related to protein degradation pathways and mitochondrial function, of FTLD patient–derived skin fibroblasts from C9-HRE carriers and non-carriers and healthy donors. Fibroblasts from C9-HRE carriers were found to produce RNA foci, but no dipeptide repeat proteins, and they showed unchanged levels of C9orf72 mRNA transcripts. The main protein degradation pathways, the ubiquitin–proteasome system and autophagy, did not show alterations between the fibroblasts from C9-HRE-carrying and non-carrying FTLD patients and compared to healthy controls. An increase in the number and size of p62-positive puncta was evident in fibroblasts from both C9-HRE carriers and non-carriers. In addition, several parameters of mitochondrial function, namely, basal and maximal respiration and respiration linked to ATP production, were significantly reduced in the FTLD patient–derived fibroblasts from both C9-HRE carriers and non-carriers. Our findings suggest that FTLD patient–derived fibroblasts, regardless of whether they carry the C9-HRE expansion, show unchanged proteasomal and autophagic function, but significantly impaired mitochondrial function and increased accumulation of p62 when compared to control fibroblasts. These findings suggest the possibility of utilizing FTLD patient–derived fibroblasts as a platform for biomarker discovery and testing of drugs targeted to specific cellular functions, such as mitochondrial respiration.
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Affiliation(s)
- Stina Leskelä
- A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211, Kuopio, Finland
| | - Dorit Hoffmann
- A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211, Kuopio, Finland
| | - Hannah Rostalski
- A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211, Kuopio, Finland
| | - Nadine Huber
- A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211, Kuopio, Finland
| | - Rebekka Wittrahm
- Institute of Biomedicine, University of Eastern Finland, Yliopistonranta 1E, 70211, Kuopio, Finland
| | - Päivi Hartikainen
- Neuro Center, Neurology, Kuopio University Hospital, 70029, Kuopio, Finland
| | - Ville Korhonen
- Neuro Center, Neurosurgery, Kuopio University Hospital, 70029, Kuopio, Finland
- Institute of Clinical Medicine - Neurosurgery, University of Eastern Finland, Yliopistonranta 1C, 70211, Kuopio, Finland
| | - Ville Leinonen
- Neuro Center, Neurosurgery, Kuopio University Hospital, 70029, Kuopio, Finland
- Institute of Clinical Medicine - Neurosurgery, University of Eastern Finland, Yliopistonranta 1C, 70211, Kuopio, Finland
| | - Mikko Hiltunen
- Institute of Biomedicine, University of Eastern Finland, Yliopistonranta 1E, 70211, Kuopio, Finland
| | - Eino Solje
- Neuro Center, Neurology, Kuopio University Hospital, 70029, Kuopio, Finland
- Institute of Clinical Medicine - Neurology, University of Eastern Finland, Yliopistonranta 1C, 70211, Kuopio, Finland
| | - Anne M Remes
- Unit of Clinical Neuroscience, Neurology, University of Oulu, P.O. Box 8000, 90014, Oulu, Finland
- MRC Oulu, Oulu University Hospital, P.O. Box 8000, 90014, Oulu, Finland
| | - Annakaisa Haapasalo
- A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Neulaniementie 2, 70211, Kuopio, Finland.
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17
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Martello F, Lattante S, Doronzio PN, Conte A, Bisogni G, Orteschi D, Pirozzi F, Sabatelli M, Zollino M, Marangi G. Generation of an induced pluripotent stem cell line (UCSCi001-A) from a patient with early-onset amyotrophic lateral sclerosis carrying a FUS variant. Stem Cell Res 2021; 55:102461. [PMID: 34303285 DOI: 10.1016/j.scr.2021.102461] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2021] [Revised: 07/02/2021] [Accepted: 07/09/2021] [Indexed: 11/25/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting upper and lower motor neurons. We generated patient-derived-induced Pluripotent Stem Cells (iPSCs), from an ALS patient affected by an early-onset and aggressive form of the disease, carrying a missense pathogenic variant in FUS gene. We reprogrammed somatic cells using an established Sendai virus protocol and we obtained clones of iPSC. We confirmed their stemness and further generated embryoid bodies, showing their potential of differentiating in all three germ layers. This iPSC line, carrying a pathogenic FUS variant, is a valuable tool to deeply investigate pathogenic mechanisms leading to ALS.
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Affiliation(s)
- Francesco Martello
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Serena Lattante
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.
| | - Paolo Niccolò Doronzio
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Amelia Conte
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Giulia Bisogni
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Daniela Orteschi
- Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Filomena Pirozzi
- Seattle Children's Research Institute, Center for Integrative Brain Research, Seattle, WA, USA
| | - Mario Sabatelli
- Adult NEMO Clinical Center, Unit of Neurology, Department of Aging, Neurological, Orthopedic and Head-Neck Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy; Section of Neurology, Department of Neuroscience, Faculty of Medicine and Surgery, Università Cattolica del Sacro Cuore, Rome, Italy
| | - Marcella Zollino
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
| | - Giuseppe Marangi
- Section of Genomic Medicine, Department of Life Sciences and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy; Unit of Medical Genetics, Department of Laboratory and Infectious Disease Sciences, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy
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18
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Milani M, Mammarella E, Rossi S, Miele C, Lattante S, Sabatelli M, Cozzolino M, D'Ambrosi N, Apolloni S. Targeting S100A4 with niclosamide attenuates inflammatory and profibrotic pathways in models of amyotrophic lateral sclerosis. J Neuroinflammation 2021; 18:132. [PMID: 34118929 PMCID: PMC8196441 DOI: 10.1186/s12974-021-02184-1] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2020] [Accepted: 05/28/2021] [Indexed: 12/23/2022] Open
Abstract
Background An increasing number of studies evidences that amyotrophic lateral sclerosis (ALS) is characterized by extensive alterations in different cell types and in different regions besides the CNS. We previously reported the upregulation in ALS models of a gene called fibroblast-specific protein-1 or S100A4, recognized as a pro-inflammatory and profibrotic factor. Since inflammation and fibrosis are often mutual-sustaining events that contribute to establish a hostile environment for organ functions, the comprehension of the elements responsible for these interconnected pathways is crucial to disclose novel aspects involved in ALS pathology. Methods Here, we employed fibroblasts derived from ALS patients harboring the C9orf72 hexanucleotide repeat expansion and ALS patients with no mutations in known ALS-associated genes and we downregulated S100A4 using siRNA or the S100A4 transcriptional inhibitor niclosamide. Mice overexpressing human FUS were adopted to assess the effects of niclosamide in vivo on ALS pathology. Results We demonstrated that S100A4 underlies impaired autophagy and a profibrotic phenotype, which characterize ALS fibroblasts. Indeed, its inhibition reduces inflammatory, autophagic, and profibrotic pathways in ALS fibroblasts, and interferes with different markers known as pathogenic in the disease, such as mTOR, SQSTM1/p62, STAT3, α-SMA, and NF-κB. Importantly, niclosamide in vivo treatment of ALS-FUS mice reduces the expression of S100A4, α-SMA, and PDGFRβ in the spinal cord, as well as gliosis in central and peripheral nervous tissues, together with axonal impairment and displays beneficial effects on muscle atrophy, by promoting muscle regeneration and reducing fibrosis. Conclusion Our findings show that S100A4 has a role in ALS-related mechanisms, and that drugs such as niclosamide which are able to target inflammatory and fibrotic pathways could represent promising pharmacological tools for ALS. Supplementary Information The online version contains supplementary material available at 10.1186/s12974-021-02184-1.
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Affiliation(s)
- Martina Milani
- Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 1, 00133, Rome, Italy
| | - Eleonora Mammarella
- Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 1, 00133, Rome, Italy
| | - Simona Rossi
- Institute of Translational Pharmacology, CNR, 00133, Rome, Italy
| | - Chiara Miele
- Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 1, 00133, Rome, Italy
| | - Serena Lattante
- Unità Operativa Complessa di Genetica Medica, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168, Rome, Italy.,Sezione di Medicina Genomica, Università Cattolica del Sacro Cuore, 00168, Rome, Italy
| | - Mario Sabatelli
- Unità Operativa Complessa di Neurologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168, Rome, Italy.,Centro Clinico NEMO, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168, Rome, Italy.,Sezione di Neurologia, Università Cattolica del Sacro Cuore, 00168, Rome, Italy
| | - Mauro Cozzolino
- Institute of Translational Pharmacology, CNR, 00133, Rome, Italy
| | - Nadia D'Ambrosi
- Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 1, 00133, Rome, Italy.
| | - Savina Apolloni
- Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 1, 00133, Rome, Italy.
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19
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Gaweda-Walerych K, Walerych D, Berdyński M, Buratti E, Zekanowski C. Parkin Levels Decrease in Fibroblasts With Progranulin (PGRN) Pathogenic Variants and in a Cellular Model of PGRN Deficiency. Front Mol Neurosci 2021; 14:676478. [PMID: 34054428 PMCID: PMC8155584 DOI: 10.3389/fnmol.2021.676478] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Accepted: 04/19/2021] [Indexed: 11/17/2022] Open
Abstract
Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with TDP-43 mislocalization and aggregation. Genetic forms of FTLD and ALS are caused by pathogenic variants in various genes, such as PGRN (progranulin). To date, depletion of parkin E3 ubiquitin protein ligase, a key mitophagy regulator, has been reported in sporadic ALS patients and ALS mice models with TDP-43 proteinopathy. In this work, we show parkin downregulation also in fibroblasts derived from FTLD patients with four different PGRN pathogenic variants. We corroborate this finding in control fibroblasts upon PGRN silencing, demonstrating additionally the decrease of parkin downstream targets, mitofusin 2 (MFN2) and voltage dependent anion channel 1 (VDAC1). Importantly, we show that TDP-43 overexpression rescues PRKN levels upon transient PGRN silencing, but not in FTLD fibroblasts with PGRN pathogenic variants, despite upregulating PGRN levels in both cases. Further observation of PRKN downregulation upon TDP-43 silencing, suggests that TDP-43 loss-of-function contributes to PRKN decrease. Our results provide further evidence that parkin downregulation might be a common and systemic phenomenon in neurodegenerative diseases with TDP- 43 loss-of-function.
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Affiliation(s)
- Katarzyna Gaweda-Walerych
- Laboratory of Neurogenetics, Mossakowski Medical Research Institute, Department of Neurodegenerative Disorders, Polish Academy of Sciences, Warsaw, Poland
| | - Dawid Walerych
- Laboratory of Human Disease Multiomics, Mossakowski Medical Research Institute, Polish Academy of Sciences, Warsaw, Poland
| | - Mariusz Berdyński
- Laboratory of Neurogenetics, Mossakowski Medical Research Institute, Department of Neurodegenerative Disorders, Polish Academy of Sciences, Warsaw, Poland
| | - Emanuele Buratti
- Molecular Pathology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
| | - Cezary Zekanowski
- Laboratory of Neurogenetics, Mossakowski Medical Research Institute, Department of Neurodegenerative Disorders, Polish Academy of Sciences, Warsaw, Poland
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20
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The role of RNA-binding and ribosomal proteins as specific RNA translation regulators in cellular differentiation and carcinogenesis. Biochim Biophys Acta Mol Basis Dis 2020; 1867:166046. [PMID: 33383105 DOI: 10.1016/j.bbadis.2020.166046] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Revised: 12/03/2020] [Accepted: 12/10/2020] [Indexed: 02/07/2023]
Abstract
Tight control of mRNA expression is required for cell differentiation; imbalanced regulation may lead to developmental disorders and cancer. The activity of the translational machinery (including ribosomes and translation factors) regulates the rate (slow or fast) of translation of encoded proteins, and the quality of these proteins highly depends on which mRNAs are available for translation. Specific RNA-binding and ribosomal proteins seem to play a key role in controlling gene expression to determine the differentiation fate of the cell. This demonstrates the important role of RNA-binding proteins, specific ribosome-binding proteins and microRNAs as key molecules in controlling the specific proteins required for the differentiation or dedifferentiation of cells. This delicate balance between specific proteins (in terms of quality and availability) and post-translational modifications occurring in the cytoplasm is crucial for cell differentiation, dedifferentiation and oncogenic potential. In this review, we report how defects in the regulation of mRNA translation can be dependent on specific proteins and can induce an imbalance between differentiation and dedifferentiation in cell fate determination.
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21
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Wang F, Fu S, Lei J, Wu H, Shi S, Chen K, Hu J, Xu X. Identification of novel FUS and TARDBP gene mutations in Chinese amyotrophic lateral sclerosis patients with HRM analysis. Aging (Albany NY) 2020; 12:22859-22868. [PMID: 33159016 PMCID: PMC7746354 DOI: 10.18632/aging.103967] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2020] [Accepted: 07/30/2020] [Indexed: 11/25/2022]
Abstract
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons. More than 30 genes have been linked to ALS to date, including FUS and TARDBP, which exhibit similar roles in RNA metabolism. This study explored the use of high-resolution melting (HRM) analysis to screen for FUS and TARDBP mutation hotspot regions in 146 Chinese ALS patients, which achieved 100% detection. Two FUS mutations were observed in two different familial ALS probands, a missense mutation (p.R521H) and a novel splicing mutation (c.1541+1G>A). Five TARDBP mutations were identified in six ALS patients, including a novel 3'UTR mutation (c.*731A>G) and four missense mutations (p.G294V, p.M337V, p.G348V, and p.I383V). We found that FUS mutations were present in 1.4% of Chinese ALS patients, whereas TARDBP mutations were responsible for 4.1% of Chinese ALS cases. Here, we describe the accuracy of using highly sensitive HRM analysis to identify two novel FUS and TARDBP mutations in Chinese sporadic and familial ALS cases. Our study contributes to the further understanding of the genetic and phenotypic diversity of ALS.
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Affiliation(s)
- Feng Wang
- Department of Clinical Laboratory, Shenzhen Baoan Women's and Children's Hospital, Jinan University, Shenzhen, China
| | - Shengyu Fu
- Faculty of Health Sciences, University of Macau, Taipa, Macau, China
| | - Jiafan Lei
- Department of Clinical Laboratory, Shenzhen Baoan Women's and Children's Hospital, Jinan University, Shenzhen, China
| | - Hongchen Wu
- Department of Neurology, Chunking General Hospital, Chongqing, China
| | - Shugui Shi
- Department of Neurology, Chunking General Hospital, Chongqing, China
| | - Kangning Chen
- Department of Neurology, First Affiliated Hospital of Army Medical University, Army Medical University, Chongqing, China
| | - Jun Hu
- Department of Neurology, First Affiliated Hospital of Army Medical University, Army Medical University, Chongqing, China
| | - Xueqing Xu
- Department of Clinical Laboratory, Shenzhen Baoan Women's and Children's Hospital, Jinan University, Shenzhen, China
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22
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McGurk L, Rifai OM, Bonini NM. TDP-43, a protein central to amyotrophic lateral sclerosis, is destabilized by tankyrase-1 and -2. J Cell Sci 2020; 133:jcs245811. [PMID: 32409565 PMCID: PMC7328137 DOI: 10.1242/jcs.245811] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Accepted: 04/24/2020] [Indexed: 12/12/2022] Open
Abstract
In >95% of cases of amyotrophic lateral sclerosis (ALS) and ∼45% of frontotemporal degeneration (FTD), the RNA/DNA-binding protein TDP-43 is cleared from the nucleus and abnormally accumulates in the cytoplasm of affected brain cells. Although the cellular triggers of disease pathology remain enigmatic, mounting evidence implicates the poly(ADP-ribose) polymerases (PARPs) in TDP-43 neurotoxicity. Here we show that inhibition of the PARP enzymes tankyrase 1 and tankyrase 2 (referred to as Tnks-1/2) protect primary rodent neurons from TDP-43-associated neurotoxicity. We demonstrate that Tnks-1/2 interacts with TDP-43 via a newly defined tankyrase-binding domain. Upon investigating the functional effect, we find that interaction with Tnks-1/2 inhibits the ubiquitination and proteasomal turnover of TDP-43, leading to its stabilization. We further show that proteasomal turnover of TDP-43 occurs preferentially in the nucleus; our data indicate that Tnks-1/2 stabilizes TDP-43 by promoting cytoplasmic accumulation, which sequesters the protein from nuclear proteasome degradation. Thus, Tnks-1/2 activity modulates TDP-43 and is a potential therapeutic target in diseases associated with TDP-43, such as ALS and FTD.This article has an associated First Person interview with the first author of the paper.
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Affiliation(s)
- Leeanne McGurk
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Olivia M Rifai
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Nancy M Bonini
- Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
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23
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Pham J, Keon M, Brennan S, Saksena N. Connecting RNA-Modifying Similarities of TDP-43, FUS, and SOD1 with MicroRNA Dysregulation Amidst A Renewed Network Perspective of Amyotrophic Lateral Sclerosis Proteinopathy. Int J Mol Sci 2020; 21:ijms21103464. [PMID: 32422969 PMCID: PMC7278980 DOI: 10.3390/ijms21103464] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2020] [Revised: 05/10/2020] [Accepted: 05/11/2020] [Indexed: 12/11/2022] Open
Abstract
Beyond traditional approaches in understanding amyotrophic lateral sclerosis (ALS), multiple recent studies in RNA-binding proteins (RBPs)-including transactive response DNA-binding protein (TDP-43) and fused in sarcoma (FUS)-have instigated an interest in their function and prion-like properties. Given their prominence as hallmarks of a highly heterogeneous disease, this prompts a re-examination of the specific functional interrelationships between these proteins, especially as pathological SOD1-a non-RBP commonly associated with familial ALS (fALS)-exhibits similar properties to these RBPs including potential RNA-regulatory capabilities. Moreover, the cytoplasmic mislocalization, aggregation, and co-aggregation of TDP-43, FUS, and SOD1 can be identified as proteinopathies akin to other neurodegenerative diseases (NDs), eliciting strong ties to disrupted RNA splicing, transport, and stability. In recent years, microRNAs (miRNAs) have also been increasingly implicated in the disease, and are of greater significance as they are the master regulators of RNA metabolism in disease pathology. However, little is known about the role of these proteins and how they are regulated by miRNA, which would provide mechanistic insights into ALS pathogenesis. This review seeks to discuss current developments across TDP-43, FUS, and SOD1 to build a detailed snapshot of the network pathophysiology underlying ALS while aiming to highlight possible novel therapeutic targets to guide future research.
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Affiliation(s)
- Jade Pham
- Faculty of Medicine, The University of New South Wales, Kensington, Sydney, NSW 2033, Australia;
| | - Matt Keon
- Iggy Get Out, Neurodegenerative Disease Section, Darlinghurst, Sydney, NSW 2010, Australia; (M.K.); (S.B.)
| | - Samuel Brennan
- Iggy Get Out, Neurodegenerative Disease Section, Darlinghurst, Sydney, NSW 2010, Australia; (M.K.); (S.B.)
| | - Nitin Saksena
- Iggy Get Out, Neurodegenerative Disease Section, Darlinghurst, Sydney, NSW 2010, Australia; (M.K.); (S.B.)
- Correspondence:
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24
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Hergesheimer RC, Chami AA, de Assis DR, Vourc'h P, Andres CR, Corcia P, Lanznaster D, Blasco H. The debated toxic role of aggregated TDP-43 in amyotrophic lateral sclerosis: a resolution in sight? Brain 2020; 142:1176-1194. [PMID: 30938443 PMCID: PMC6487324 DOI: 10.1093/brain/awz078] [Citation(s) in RCA: 123] [Impact Index Per Article: 24.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2018] [Revised: 02/11/2019] [Accepted: 02/16/2019] [Indexed: 12/11/2022] Open
Abstract
Transactive response DNA-binding protein-43 (TDP-43) is an RNA/DNA binding protein that forms phosphorylated and ubiquitinated aggregates in the cytoplasm of motor neurons in amyotrophic lateral sclerosis, which is a hallmark of this disease. Amyotrophic lateral sclerosis is a neurodegenerative condition affecting the upper and lower motor neurons. Even though the aggregative property of TDP-43 is considered a cornerstone of amyotrophic lateral sclerosis, there has been major controversy regarding the functional link between TDP-43 aggregates and cell death. In this review, we attempt to reconcile the current literature surrounding this debate by discussing the results and limitations of the published data relating TDP-43 aggregates to cytotoxicity, as well as therapeutic perspectives of TDP-43 aggregate clearance. We point out key data suggesting that the formation of TDP-43 aggregates and the capacity to self-template and propagate among cells as a 'prion-like' protein, another pathological property of TDP-43 aggregates, are a significant cause of motor neuronal death. We discuss the disparities among the various studies, particularly with respect to the type of models and the different forms of TDP-43 used to evaluate cellular toxicity. We also examine how these disparities can interfere with the interpretation of the results pertaining to a direct toxic effect of TDP-43 aggregates. Furthermore, we present perspectives for improving models in order to better uncover the toxic role of aggregated TDP-43. Finally, we review the recent studies on the enhancement of the cellular clearance mechanisms of autophagy, the ubiquitin proteasome system, and endocytosis in an attempt to counteract TDP-43 aggregation-induced toxicity. Altogether, the data available so far encourage us to suggest that the cytoplasmic aggregation of TDP-43 is key for the neurodegeneration observed in motor neurons in patients with amyotrophic lateral sclerosis. The corresponding findings provide novel avenues toward early therapeutic interventions and clinical outcomes for amyotrophic lateral sclerosis management.
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Affiliation(s)
| | - Anna A Chami
- UMR 1253, iBRAIN, Université de Tours, INSERM, Tours, France
| | | | - Patrick Vourc'h
- UMR 1253, iBRAIN, Université de Tours, INSERM, Tours, France.,CHU de Tours, Service de Biochimie et Biologie Moléculaire, Tours, France
| | - Christian R Andres
- UMR 1253, iBRAIN, Université de Tours, INSERM, Tours, France.,CHU de Tours, Service de Biochimie et Biologie Moléculaire, Tours, France
| | - Philippe Corcia
- UMR 1253, iBRAIN, Université de Tours, INSERM, Tours, France.,CHU de Tours, Service de Neurologie, Tours, France
| | | | - Hélène Blasco
- UMR 1253, iBRAIN, Université de Tours, INSERM, Tours, France.,CHU de Tours, Service de Biochimie et Biologie Moléculaire, Tours, France
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25
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Romano N, Catalani A, Lattante S, Belardo A, Proietti S, Bertini L, Silvestri F, Catalani E, Cervia D, Zolla L, Sabatelli M, Welshhans K, Ceci M. ALS skin fibroblasts reveal oxidative stress and ERK1/2-mediated cytoplasmic localization of TDP-43. Cell Signal 2020; 70:109591. [PMID: 32126264 DOI: 10.1016/j.cellsig.2020.109591] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2019] [Revised: 02/14/2020] [Accepted: 02/26/2020] [Indexed: 12/20/2022]
Abstract
The main hallmark of many forms of familiar and sporadic amyotrophic lateral sclerosis (ALS) is a reduction in nuclear TDP-43 protein and its inclusion in cytoplasmic aggregates in motor neurons. In order to understand which cellular and molecular mechanisms underlie the mislocalization of TDP-43, we examined human skin fibroblasts from two individuals with familial ALS, both with mutations in TDP-43, and two individuals with sporadic ALS, both without TDP-43 mutations or mutations in other ALS related genes. We found that all ALS fibroblasts had a partially cytoplasmic localization of TDP-43 and had reduced cell metabolism as compared to fibroblasts from apparently healthy individuals. ALS fibroblasts showed an increase in global protein synthesis and an increase in 4E-BP1 and rpS6 phosphorylation, which is indicative of mTORC1 activity. We also observed a decrease in glutathione (GSH), which suggests that oxidative stress is elevated in ALS. ERK1/2 activity regulated the extent of oxidative stress and the localization of TDP-43 in the cytoplasm in all ALS fibroblasts. Lastly, ALS fibroblasts showed reduced stress granule formation in response to H2O2 stress. In conclusion, these findings identify specific cellular and molecular defects in ALS fibroblasts, thus providing insight into potential mechanisms that may also occur in degenerating motor neurons.
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Affiliation(s)
- Nicla Romano
- Department of Ecological and Biological Science (DEB), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy
| | - Alessia Catalani
- Department of Molecular Sciences, University of Urbino "Carlo Bo", Via Santa Chiara, 27 61029 Urbino, PU, Italy
| | - Serena Lattante
- Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Genetica Medica, 00168 Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Medicina Genomica, 00168 Roma, Italy
| | - Antonio Belardo
- Department of Ecological and Biological Science (DEB), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy
| | - Silvia Proietti
- Department of Ecological and Biological Science (DEB), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy
| | - Laura Bertini
- Department of Ecological and Biological Science (DEB), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy
| | - Federica Silvestri
- Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy
| | - Elisabetta Catalani
- Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy
| | - Davide Cervia
- Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy
| | - Lello Zolla
- Department of Science and Technology for Agriculture, Forestry, Nature and Energy, University of Tuscia (DAFNE), 01100 Viterbo, Italy
| | - Mario Sabatelli
- Fondazione Policlinico Universitario A. Gemelli IRCCS, UOC Neurologia, Dipartimento Scienze dell'invecchiamento, neurologiche, ortopediche e della testa-collo, 00168 Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Neurologia, Centro Clinico NEMO, 00168 Roma, Italy
| | - Kristy Welshhans
- Department of Biological Sciences, School of Biomedical Sciences and Brain Health Research Institute, Kent State University, Kent, OH 44236, USA
| | - Marcello Ceci
- Department of Ecological and Biological Science (DEB), University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy.
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26
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Riancho J, Castanedo-Vázquez D, Gil-Bea F, Tapia O, Arozamena J, Durán-Vían C, Sedano MJ, Berciano MT, Lopez de Munain A, Lafarga M. ALS-derived fibroblasts exhibit reduced proliferation rate, cytoplasmic TDP-43 aggregation and a higher susceptibility to DNA damage. J Neurol 2020; 267:1291-1299. [PMID: 31938860 DOI: 10.1007/s00415-020-09704-8] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2019] [Revised: 01/07/2020] [Accepted: 01/09/2020] [Indexed: 11/28/2022]
Abstract
BACKGROUND Dermic fibroblasts have been proposed as a potential genetic-ALS cellular model. This study aimed to explore whether dermic fibroblasts from patients with sporadic-ALS (sALS) recapitulate alterations typical of ALS motor neurons and exhibit abnormal DNA-damage response. METHODS Dermic fibroblasts were obtained from eight sALS patients and four control subjects. Cellular characterization included proliferation rate analysis, cytoarchitecture studies and confocal immunofluorescence assessment for TDP-43. Additionally, basal and irradiation-induced DNA damage was evaluated by confocal immunofluorescence and biochemical techniques. RESULTS sALS-fibroblasts showed decreased proliferation rates compared to controls. Additionally, whereas control fibroblasts exhibited the expected normal spindle-shaped morphology, ALS fibroblasts were thinner, with reduced cell size and enlarged nucleoli, with frequent cytoplasmic TDP-43aggregates. Also, baseline signs of DNA damage were evidenced more frequently in ALS-derived fibroblasts (11 versus 4% in control-fibroblasts). Assays for evaluating the irradiation-induced DNA damage demonstrated that DNA repair was defective in ALS-fibroblasts, accumulating more than double of γH2AX-positive DNA damage foci than controls. Very intriguingly, the proportion of fibroblasts particularly vulnerable to irradiation (with more than 15 DNA damage foci per nucleus) was seven times higher in ALS-derived fibroblasts than in controls. CONCLUSIONS Dermic-derived ALS fibroblasts recapitulate relevant cellular features of sALS and show a higher susceptibility to DNA damage and defective DNA repair responses. Altogether, these results support that dermic fibroblasts may represent a convenient and accessible ALS cellular model to study pathogenetic mechanisms, particularly those related to DNA damage response, as well as the eventual response to disease-modifying therapies.
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Affiliation(s)
- Javier Riancho
- Service of Neurology, Hospital Sierrallana-IDIVAL, Barrio Ganzo s/n, 39300, Torrelavega, Spain. .,Department of Medicine and Psychiatry, University of Cantabria, Santander, Spain. .,Centro de Investigación en Red de Enfermedades Neurodegenerativas, CIBERNED. Instituto Carlos III, Madrid, Spain.
| | | | - Francisco Gil-Bea
- Centro de Investigación en Red de Enfermedades Neurodegenerativas, CIBERNED. Instituto Carlos III, Madrid, Spain.,Neurosciences Area. Biodonostia Research Institute, San Sebastián, Spain
| | - Olga Tapia
- Centro de Investigación en Red de Enfermedades Neurodegenerativas, CIBERNED. Instituto Carlos III, Madrid, Spain.,Department of Anatomy and Cell Biology, University of Cantabria-IDIVAL, Santander, Spain
| | - Jana Arozamena
- Department of Anatomy and Cell Biology, University of Cantabria-IDIVAL, Santander, Spain
| | - Carlos Durán-Vían
- Service of Dermatology, Hospital Universitario Marqués de Valdecilla, Santander, Spain
| | - María José Sedano
- Centro de Investigación en Red de Enfermedades Neurodegenerativas, CIBERNED. Instituto Carlos III, Madrid, Spain.,Service of Neurology, Hospital Universitario Marqués de Valdecilla-IDIVAL, Santander, Spain
| | - Maria Teresa Berciano
- Centro de Investigación en Red de Enfermedades Neurodegenerativas, CIBERNED. Instituto Carlos III, Madrid, Spain.,Department of Anatomy and Cell Biology, University of Cantabria-IDIVAL, Santander, Spain
| | - Adolfo Lopez de Munain
- Centro de Investigación en Red de Enfermedades Neurodegenerativas, CIBERNED. Instituto Carlos III, Madrid, Spain.,Neurosciences Area. Biodonostia Research Institute, San Sebastián, Spain.,Service of Neurology, Hospital Universitario Donostia, San Sebastián, Spain.,Department of Neurosciences. School of Medicine and Nursery, University of the Basque Country, San Sebastián, Spain
| | - Miguel Lafarga
- Centro de Investigación en Red de Enfermedades Neurodegenerativas, CIBERNED. Instituto Carlos III, Madrid, Spain.,Department of Anatomy and Cell Biology, University of Cantabria-IDIVAL, Santander, Spain
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27
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Riancho J, Arozamena S, López de Munaín A. Dermic-derived fibroblasts for the study of amyotrophic lateral sclerosis. Neural Regen Res 2020; 15:2043-2044. [PMID: 32394958 PMCID: PMC7716046 DOI: 10.4103/1673-5374.282257] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Affiliation(s)
- Javier Riancho
- Service of Neurology, Hospital Sierrallana-IDIVAL, Torrelavega; Department of Medicine and Psychiatry, University of Cantabria, Santander; Centro de Investigación en Red de Enfermedades Neurodegenerativas, Instituto Carlos III, Madrid, Spain
| | - Sara Arozamena
- Service of Neurology, Hospital Sierrallana-IDIVAL, Torrelavega, Spain
| | - Adolfo López de Munaín
- Centro de Investigación en Red de Enfermedades Neurodegenerativas, Instituto Carlos III, Madrid; Neurosciences Area, Biodonostia Research Institute; Service of Neurology, Hospital Universitario Donostia; Department of Neurosciences, School of Medicine and Nursery, University of the Basque Country, San Sebastián, Spain
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28
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Lattante S, Doronzio PN, Marangi G, Conte A, Bisogni G, Bernardo D, Russo T, Lamberti D, Patrizi S, Apollo FP, Lunetta C, Scarlino S, Pozzi L, Zollino M, Riva N, Sabatelli M. Coexistence of variants in TBK1 and in other ALS-related genes elucidates an oligogenic model of pathogenesis in sporadic ALS. Neurobiol Aging 2019; 84:239.e9-239.e14. [PMID: 31000212 DOI: 10.1016/j.neurobiolaging.2019.03.010] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2018] [Revised: 03/04/2019] [Accepted: 03/19/2019] [Indexed: 11/19/2022]
Abstract
Variants in tank-binding kinase 1 (TBK1) are responsible for a significant proportion of amyotrophic lateral sclerosis (ALS) cases. In the present study, we analyzed variants in TBK1 extracted by targeted sequencing of 32 genes in a group of 406 Italian patients with ALS. We identified 7 different TBK1 variants in 7 sporadic cases, resulting in a frequency of 1.7%. Three patients had missense variants (p.R357Q, p.R358H, and p.R724C), one patient had a small deletion (p.E618del), and 3 had truncating variants (p.Y482*, p.R229*, and p.N681*). Notably, we found that 4 patients had an additional variant in ALS-related genes: 2 in OPTN and 2 in the 3'UTR region of FUS. By studying an independent group of 7 TBK1-mutated patients previously reported, we found another variant in the 3'UTR region of FUS in one patient. The presence of a second variant in TBK1 variant carriers is an interesting finding that needs to be investigated in larger cohorts of patients. These findings suggest that TBK1 belongs to the category of genes conferring a significantly increased risk but not sufficient to cause disease.
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Affiliation(s)
- Serena Lattante
- Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Genetica Medica, Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Medicina Genomica, Roma, Italy
| | - Paolo Niccolò Doronzio
- Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Genetica Medica, Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Medicina Genomica, Roma, Italy
| | - Giuseppe Marangi
- Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Genetica Medica, Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Medicina Genomica, Roma, Italy
| | | | | | | | - Tommaso Russo
- Dipartimento Scienze dell'invecchiamento, Neurologiche, Ortopediche e della testa-collo, Fondazione Policlinico Universitario A. Gemelli IRCCS, UOC Neurologia, Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Neurologia, Roma, Italy
| | - Dante Lamberti
- Unità di Oncogenomica ed Epigenetica, IRCCS Istituto Nazionale Tumori Regina Elena, Roma, Italy
| | - Sara Patrizi
- Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Genetica Medica, Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Medicina Genomica, Roma, Italy
| | - Francesco Paolo Apollo
- Divisione di Neurologia, Ospedale Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo, Italy
| | | | - Stefania Scarlino
- Divisione di Neuroscienze, Dipartimento di Neurologia, Istituto di Neurologia Sperimentale (INSPE), Istituto Scientifico San Raffaele, Milano, Italy
| | - Laura Pozzi
- Divisione di Neuroscienze, Dipartimento di Neurologia, Istituto di Neurologia Sperimentale (INSPE), Istituto Scientifico San Raffaele, Milano, Italy
| | - Marcella Zollino
- Fondazione Policlinico Universitario A. Gemelli IRCCS, Unità Operativa Complessa di Genetica Medica, Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Medicina Genomica, Roma, Italy
| | - Nilo Riva
- Divisione di Neuroscienze, Dipartimento di Neurologia, Istituto di Neurologia Sperimentale (INSPE), Istituto Scientifico San Raffaele, Milano, Italy
| | - Mario Sabatelli
- Centro Clinico NEMO, Roma, Italy; Dipartimento Scienze dell'invecchiamento, Neurologiche, Ortopediche e della testa-collo, Fondazione Policlinico Universitario A. Gemelli IRCCS, UOC Neurologia, Roma, Italy; Università Cattolica del Sacro Cuore, Istituto di Neurologia, Roma, Italy.
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29
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Serrano A, Apolloni S, Rossi S, Lattante S, Sabatelli M, Peric M, Andjus P, Michetti F, Carrì MT, Cozzolino M, D'Ambrosi N. The S100A4 Transcriptional Inhibitor Niclosamide Reduces Pro-Inflammatory and Migratory Phenotypes of Microglia: Implications for Amyotrophic Lateral Sclerosis. Cells 2019; 8:cells8101261. [PMID: 31623154 PMCID: PMC6829868 DOI: 10.3390/cells8101261] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2019] [Revised: 10/11/2019] [Accepted: 10/14/2019] [Indexed: 02/07/2023] Open
Abstract
S100A4, belonging to a large multifunctional S100 protein family, is a Ca2+-binding protein with a significant role in stimulating the motility of cancer and immune cells, as well as in promoting pro-inflammatory properties in different cell types. In the CNS, there is limited information concerning S100A4 presence and function. In this study, we analyzed the expression of S100A4 and the effect of the S100A4 transcriptional inhibitor niclosamide in murine activated primary microglia. We found that S100A4 was strongly up-regulated in reactive microglia and that niclosamide prevented NADPH oxidase 2, mTOR (mammalian target of rapamycin), and NF-κB (nuclear factor-kappa B) increase, cytoskeletal rearrangements, migration, and phagocytosis. Furthermore, we found that S100A4 was significantly up-regulated in astrocytes and microglia in the spinal cord of a transgenic rat SOD1-G93A model of amyotrophic lateral sclerosis. Finally, we demonstrated the increased expression of S100A4 also in fibroblasts derived from amyotrophic lateral sclerosis (ALS) patients carrying SOD1 pathogenic variants. These results ascribe S100A4 as a marker of microglial reactivity, suggesting the contribution of S100A4-regulated pathways to neuroinflammation, and identify niclosamide as a possible drug in the control and attenuation of reactive phenotypes of microglia, thus opening the way to further investigation for a new application in neurodegenerative conditions.
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Affiliation(s)
- Alessia Serrano
- Institute of Anatomy and Cell Biology, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.
| | - Savina Apolloni
- Department of Biology, University of Rome "Tor Vergata", 00133 Rome, Italy.
| | - Simona Rossi
- Department of Biology, University of Rome "Tor Vergata", 00133 Rome, Italy.
- Institute of Translational Pharmacology, CNR, 00133 Rome, Italy.
| | - Serena Lattante
- Unità Operativa Complessa di Genetica Medica, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy.
- Istituto di Medicina Genomica, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.
| | - Mario Sabatelli
- Unità Operativa Complessa di Neurologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy.
- Centro Clinico NEMO, 00168 Rome, Italy.
- Istituto di Neurologia, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.
| | - Mina Peric
- Institute of Physiology and Biochemistry "Ivan Djaja", Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia.
| | - Pavle Andjus
- Institute of Physiology and Biochemistry "Ivan Djaja", Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia.
| | - Fabrizio Michetti
- Institute of Anatomy and Cell Biology, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.
| | - Maria Teresa Carrì
- Department of Biology, University of Rome "Tor Vergata", 00133 Rome, Italy.
| | - Mauro Cozzolino
- Institute of Translational Pharmacology, CNR, 00133 Rome, Italy.
| | - Nadia D'Ambrosi
- Department of Biology, University of Rome "Tor Vergata", 00133 Rome, Italy.
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30
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Lee SM, Asress S, Hales CM, Gearing M, Vizcarra JC, Fournier CN, Gutman DA, Chin LS, Li L, Glass JD. TDP-43 cytoplasmic inclusion formation is disrupted in C9orf72-associated amyotrophic lateral sclerosis/frontotemporal lobar degeneration. Brain Commun 2019; 1:fcz014. [PMID: 31633109 PMCID: PMC6788139 DOI: 10.1093/braincomms/fcz014] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Revised: 08/10/2019] [Accepted: 08/19/2019] [Indexed: 12/12/2022] Open
Abstract
The G4C2 hexanucleotide repeat expansion mutation in the C9orf72 gene is the most common genetic cause underlying both amyotrophic lateral sclerosis and frontotemporal dementia. Pathologically, these two neurodegenerative disorders are linked by the common presence of abnormal phosphorylated TDP-43 neuronal cytoplasmic inclusions. We compared the number and size of phosphorylated TDP-43 inclusions and their morphology in hippocampi from patients dying with sporadic versus C9orf72-related amyotrophic lateral sclerosis with pathologically defined frontotemporal lobar degeneration with phosphorylated TDP-43 inclusions, the pathological substrate of clinical frontotemporal dementia in patients with amyotrophic lateral sclerosis. In sporadic cases, there were numerous consolidated phosphorylated TDP-43 inclusions that were variable in size, whereas inclusions in C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration were quantitatively smaller than those in sporadic cases. Also, C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration homogenized brain contained soluble cytoplasmic TDP-43 that was largely absent in sporadic cases. To better understand these pathological differences, we modelled TDP-43 inclusion formation in fibroblasts derived from sporadic or C9orf72-related amyotrophic lateral sclerosis/frontotemporal dementia patients. We found that both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia patient fibroblasts showed impairment in TDP-43 degradation by the proteasome, which may explain increased TDP-43 protein levels found in both sporadic and C9orf72 amyotrophic lateral sclerosis/frontotemporal lobar degeneration frontal cortex and hippocampus. Fibroblasts derived from sporadic patients, but not C9orf72 patients, demonstrated the ability to sequester cytoplasmic TDP-43 into aggresomes via microtubule-dependent mechanisms. TDP-43 aggresomes in vitro and TDP-43 neuronal inclusions in vivo were both tightly localized with autophagy markers and, therefore, were likely to function similarly as sites for autophagic degradation. The inability for C9orf72 fibroblasts to form TDP-43 aggresomes, together with the observations that TDP-43 protein was soluble in the cytoplasm and formed smaller inclusions in the C9orf72 brain compared with sporadic disease, suggests a loss of protein quality control response to sequester and degrade TDP-43 in C9orf72-related diseases.
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Affiliation(s)
- Samuel M Lee
- Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA.,Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
| | - Seneshaw Asress
- Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA.,Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
| | - Chadwick M Hales
- Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA.,Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
| | - Marla Gearing
- Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA.,Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA.,Department of Pathology & Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
| | - Juan C Vizcarra
- Department of Biomedical Engineering, Emory University School of Medicine, Atlanta, GA, USA.,Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA
| | - Christina N Fournier
- Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA.,Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
| | - David A Gutman
- Department of Biomedical Engineering, Emory University School of Medicine, Atlanta, GA, USA.,Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA
| | - Lih-Shen Chin
- Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA.,Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA
| | - Lian Li
- Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA.,Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, USA
| | - Jonathan D Glass
- Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA.,Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA.,Department of Pathology & Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
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31
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Mandrioli J, Crippa V, Cereda C, Bonetto V, Zucchi E, Gessani A, Ceroni M, Chio A, D’Amico R, Monsurrò MR, Riva N, Sabatelli M, Silani V, Simone IL, Sorarù G, Provenzani A, D’Agostino VG, Carra S, Poletti A. Proteostasis and ALS: protocol for a phase II, randomised, double-blind, placebo-controlled, multicentre clinical trial for colchicine in ALS (Co-ALS). BMJ Open 2019; 9:e028486. [PMID: 31152038 PMCID: PMC6549675 DOI: 10.1136/bmjopen-2018-028486] [Citation(s) in RCA: 31] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Revised: 02/25/2019] [Accepted: 04/10/2019] [Indexed: 12/17/2022] Open
Abstract
INTRODUCTION Disruptions of proteasome and autophagy systems are central events in amyotrophic lateral sclerosis (ALS) and support the urgent need to find therapeutic compounds targeting these processes. The heat shock protein B8 (HSPB8) recognises and promotes the autophagy-mediated removal of misfolded mutant SOD1 and TDP-43 fragments from ALS motor neurons (MNs), as well as aggregating species of dipeptides produced in C9ORF72-related diseases. In ALS-SOD1 mice and in human ALS autopsy specimens, HSPB8 is highly expressed in spinal cord MNs that survive at the end stage of disease. Moreover, the HSPB8-BAG3-HSP70 complex maintains granulostasis, which avoids conversion of dynamic stress granules (SGs) into aggregation-prone assemblies. We will perform a randomised clinical trial (RCT) with colchicine, which enhances the expression of HSPB8 and of several autophagy players, blocking TDP-43 accumulation and exerting crucial activities for MNs function. METHODS AND ANALYSIS Colchicine in amyotrophic lateral sclerosis (Co-ALS) is a double-blind, placebo-controlled, multicentre, phase II RCT. ALS patients will be enrolled in three groups (placebo, colchicine 0.01 mg/day and colchicine 0.005 mg/day) of 18 subjects treated with riluzole; treatment will last 30 weeks, and follow-up will last 24 weeks. The primary aim is to assess whether colchicine decreases disease progression as measured by ALS Functional Rating Scale - Revised (ALSFRS-R) at baseline and at treatment end. Secondary aims include assessment of (1) safety and tolerability of Colchicine in patiets with ALS; (2) changes in cellular activity (autophagy, protein aggregation, and SG and exosome secretion) and in biomarkers of disease progression (neurofilaments); (3) survival and respiratory function and (4) quality of life. Preclinical studies with a full assessment of autophagy and neuroinflammation biomarkers in fibroblasts, peripheral blood mononuclear cells and lymphoblasts will be conducted in parallel with clinic assessment to optimise time and resources. ETHICS AND DISSEMINATION The study protocol was approved by the Ethics Committee of Area Vasta Emilia Nord and by Agenzia Italiana del Farmaco (EUDRACT N.2017-004459-21) based on the Declaration of Helsinki. This research protocol was written without patient involvement. Patients' association will be involved in disseminating the study design and results. Results will be presented during scientific symposia or published in scientific journals. TRIAL REGISTRATION NUMBER EUDRACT 2017-004459-21; NCT03693781; Pre-results.
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Affiliation(s)
- Jessica Mandrioli
- Department of Neurosciences, St. Agostino Estense Hospital, Azienda Ospedaliero Universitaria di Modena, Modena, Italy
| | - Valeria Crippa
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milano, Italy
| | - Cristina Cereda
- Genomics and Post-Genomics Center, IRCCS Mondino Foundation, Pavia, Italy
| | - Valentina Bonetto
- Laboratory of Translational Biomarkers, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano, Italy
| | - Elisabetta Zucchi
- Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Annalisa Gessani
- Department of Neurosciences, St. Agostino Estense Hospital, Azienda Ospedaliero Universitaria di Modena, Modena, Italy
| | - Mauro Ceroni
- Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy
- Department of General Neurology, Rare Diseases Unit, IRCCS Mondino Foundation, Pavia, Italy
| | - Adriano Chio
- “Rita Levi Montalcini” Departmentof Neurosciences, ALS Centre, University of Turin and Azienda Ospedaliero Universitaria Città della Salute e della Scienza, Turin, Italy
| | - Roberto D’Amico
- Department of Diagnostic, Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy
| | - Maria Rosaria Monsurrò
- Dipartimento ad attività integratedi Medicina Interna e Specialistica, Azienda Ospedaliero Universitaria “L. Vanvitelli”, Napoli, Italy
| | - Nilo Riva
- Department of Neurology, Institute of Experimental Neurology (INSPE), Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Milan, Italy
| | - Mario Sabatelli
- Neuromuscular Omni Centre (NEMO), Fondazione Serena Onlus, Policlinico A. Gemelli IRCCS, Roma, Italy
- Istituto di Neurologia, Università Cattolica del Sacro Cuore, Roma, Italy
- UOC di Neurologia, Dipartimento di Scienze dell’invecchiamento, Neurologiche, ortopediche e della testa collo, Fondazione Policlinico Universitario A. Gemelli IRCCS, Roma, Italy
| | - Vincenzo Silani
- Department of Neurology-Stroke Unitand Laboratory of Neuroscience, Istituto Auxologico Italiano IRCCS, Milan, Italy
- Department of Pathophysiology and Transplantation, "Dino Ferrari" Center, Università degli Studi di Milano, Milano, Italy
| | - Isabella Laura Simone
- Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari, Bari, Italy
| | - Gianni Sorarù
- Department of Neurosciences, University of Padua, Padua, Italy
| | | | | | - Serena Carra
- Centre for Neuroscience and Nanotechnology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy
| | - Angelo Poletti
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Centro di Eccellenza sulle Malattie Neurodegenerative, Università degli Studi di Milano, Milano, Italy
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32
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Riancho J, Gil-Bea FJ, Castanedo-Vazquez D, Sedano MJ, Zufiría M, de Eulate GFG, Poza JJ, Lopez de Munain A. Clinical evidences supporting the Src/c-Abl pathway as potential therapeutic target in amyotrophic lateral sclerosis. J Neurol Sci 2018; 393:80-82. [DOI: 10.1016/j.jns.2018.08.013] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2018] [Revised: 07/20/2018] [Accepted: 08/10/2018] [Indexed: 12/12/2022]
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Jeon GS, Shim YM, Lee DY, Kim JS, Kang M, Ahn SH, Shin JY, Geum D, Hong YH, Sung JJ. Pathological Modification of TDP-43 in Amyotrophic Lateral Sclerosis with SOD1 Mutations. Mol Neurobiol 2018; 56:2007-2021. [PMID: 29982983 PMCID: PMC6394608 DOI: 10.1007/s12035-018-1218-2] [Citation(s) in RCA: 54] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2018] [Accepted: 06/29/2018] [Indexed: 12/12/2022]
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset, progressive neurodegenerative disorder with no known cure. Cu/Zn-superoxide dismutase (SOD1) was the first identified protein associated with familial ALS (fALS). Recently, TAR DNA-binding protein 43 (TDP-43) has been found to be a principal component of ubiquitinated cytoplasmic inclusions in neurons and glia in ALS. However, it remains unclear whether these ALS-linked proteins partly have a shared pathogenesis. Here, we determine the association between mutant SOD1 and the modification of TDP-43 and the relationship of pathologic TDP-43 to neuronal cytotoxicity in SOD1 ALS. In this work, using animal model, human tissue, and cell models, we provide the evidence that the association between the TDP-43 modification and the pathogenesis of SOD1 fALS. We demonstrated an age-dependent increase in TDP-43 C-terminal fragments and phosphorylation in motor neurons and glia of SOD1 mice and SOD1G85S ALS patient. Cytoplasmic TDP-43 was also observed in iPSC-derived motor neurons from SOD1G17S ALS patient. Moreover, we observed that mutant SOD1 interacts with TDP-43 in co-immunoprecipitation assays with G93A hSOD1-transfected cell lines. Mutant SOD1 overexpression led to an increase in TDP-43 modification in the detergent-insoluble fraction in the spinal cord of SOD1 mice and fALS patient. Additionally, we showed cellular apoptosis in response to the interaction of mutant SOD1 and fragment forms of TDP-43. These findings suggest that mutant SOD1 could affect the solubility/insolubility of TDP-43 through physical interactions and the resulting pathological modifications of TDP-43 may be involved in motor neuron death in SOD1 fALS.
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Affiliation(s)
- Gye Sun Jeon
- Department of Neurology, Seoul National University Hospital, Seoul, South Korea.,Biomedical Research Institute, Seoul National University Hospital, Seoul, South Korea
| | - Yu-Mi Shim
- Department of Neurology, Seoul National University Hospital, Seoul, South Korea
| | - Do-Yeon Lee
- Department of Neurology, Seoul National University Hospital, Seoul, South Korea
| | - Jun-Soon Kim
- Department of Neurology, Seoul National University Hospital, Seoul, South Korea
| | - MinJin Kang
- Department of Biomedical Sciences, Korea University College of Medicine, Seoul, South Korea
| | - So Hyun Ahn
- Department of Neurology, Seoul National University Hospital, Seoul, South Korea
| | - Je-Young Shin
- Department of Neurology, Seoul National University Hospital, Seoul, South Korea
| | - Dongho Geum
- Department of Biomedical Sciences, Korea University College of Medicine, Seoul, South Korea
| | - Yoon Ho Hong
- Department of Neurology, Seoul National University Seoul Metropolitan Government Boramae Medical Center, Seoul, South Korea
| | - Jung-Joon Sung
- Department of Neurology, Seoul National University Hospital, Seoul, South Korea. .,Neuroscience Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
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Brancia C, Noli B, Boido M, Pilleri R, Boi A, Puddu R, Marrosu F, Vercelli A, Bongioanni P, Ferri GL, Cocco C. TLQP Peptides in Amyotrophic Lateral Sclerosis: Possible Blood Biomarkers with a Neuroprotective Role. Neuroscience 2018; 380:152-163. [PMID: 29588252 DOI: 10.1016/j.neuroscience.2018.03.023] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2017] [Revised: 03/15/2018] [Accepted: 03/16/2018] [Indexed: 12/12/2022]
Abstract
While the VGF-derived TLQP peptides have been shown to prevent neuronal apoptosis, and to act on synaptic strengthening, their involvement in Amyotrophic Lateral Sclerosis (ALS) remains unclarified. We studied human ALS patients' plasma (taken at early to late disease stages) and primary fibroblast cultures (patients vs controls), in parallel with SOD1-G93A transgenic mice (taken at pre-, early- and late symptomatic stages) and the mouse motor neuron cell line (NSC-34) treated with Sodium Arsenite (SA) to induce oxidative stress. TLQP peptides were measured by enzyme-linked immunosorbent assay, in parallel with gel chromatography characterization, while their localization was studied by immunohistochemistry. In controls, TLQP peptides, including forms compatible with TLQP-21 and 62, were revealed in plasma and spinal cord motor neurons, as well as in fibroblasts and NSC-34 cells. TLQP peptides were reduced in ALS patients' plasma starting in the early disease stage (14% of controls) and remaining so at the late stage (16% of controls). In mice, a comparable pattern of reduction was shown (vs wild type), in both plasma and spinal cord already in the pre-symptomatic phase (about 26% and 70%, respectively). Similarly, the levels of TLQP peptides were reduced in ALS fibroblasts (31% of controls) and in the NSC-34 treated with Sodium Arsenite (53% of decrease), however, the exogeneous TLQP-21 improved cell viability (SA-treated cells with TLQP-21, vs SA-treated cells only: about 83% vs. 75%). Hence, TLQP peptides, reduced upon oxidative stress, are suggested as blood biomarkers, while TLQP-21 exerts a neuroprotective activity.
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Affiliation(s)
- Carla Brancia
- Dept. Biomedical Sciences, University of Cagliari, Monserrato, Italy.
| | - Barbara Noli
- Dept. Biomedical Sciences, University of Cagliari, Monserrato, Italy
| | - Marina Boido
- Neuroscience Institute Cavalieri Ottolenghi, Dept. Neuroscience, University of Turin, Turin, Italy
| | - Roberta Pilleri
- Dept. Biomedical Sciences, University of Cagliari, Monserrato, Italy
| | - Andrea Boi
- Dept. Biomedical Sciences, University of Cagliari, Monserrato, Italy
| | - Roberta Puddu
- Dept. Neurology, Azienda Universitario Ospedaliera di Cagliari & University of Cagliari, Cagliari, Italy
| | - Francesco Marrosu
- Dept. Neurology, Azienda Universitario Ospedaliera di Cagliari & University of Cagliari, Cagliari, Italy
| | - Alessandro Vercelli
- Neuroscience Institute Cavalieri Ottolenghi, Dept. Neuroscience, University of Turin, Turin, Italy
| | - Paolo Bongioanni
- Neurorehabilitation Unit, Dept. Neuroscience, University of Pisa, Pisa, Italy
| | - Gian-Luca Ferri
- Dept. Biomedical Sciences, University of Cagliari, Monserrato, Italy
| | - Cristina Cocco
- Dept. Biomedical Sciences, University of Cagliari, Monserrato, Italy
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35
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Orrù S, Coni P, Floris A, Littera R, Carcassi C, Sogos V, Brancia C. Reduced stress granule formation and cell death in fibroblasts with the A382T mutation of TARDBP gene: evidence for loss of TDP-43 nuclear function. Hum Mol Genet 2018; 25:4473-4483. [PMID: 28172957 DOI: 10.1093/hmg/ddw276] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2016] [Revised: 08/11/2016] [Accepted: 08/13/2016] [Indexed: 12/12/2022] Open
Abstract
TAR deoxyribonucleic acid-binding protein 43 (TDP-43) is a key protein in the pathogenesis of amyoptrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Recent studies suggest that mutations in the TDP-43 coding gene, TARDBP, as well as variations in TDP-43 protein expression levels may disrupt the dynamics of stress granules (SGs). However, it remains unclear whether the pathogenetic effect of the TDP-43 protein is exerted at the cytoplasmic level, through direct participation to SG composition, or at nuclear level, through control of proteins essential to SG assembly. To clarify this point, we investigated the dynamics of SG formation in primary skin fibroblast cultures from the patients with ALS together with the A382T mutation and the patients with ALS and healthy controls with wild-type TDP-43. Under stress conditions induced by sodium arsenite, we found that in human fibroblasts TDP-43 did not translocate to the SGs but instead contributed to the SG formation through a regulatory effect on the G3BP1 core protein. We found that the A382T mutation caused a significant reduction in the number of SGs per cell (P < 0.01) as well as the percentage of cells that form SGs (P < 0.00001). Following stress stimuli, a significant decrease of viability was observed for cells with the TDP-43 A382T mutation (P < 0.0005).
We can therefore conclude that the A382T mutation caused a reduction in the ability of cells to respond to stress through loss of TDP-43 function in SG nucleation. The pathogenetic action revealed in our study model does not seem to be mediated by changes in the localization of the TDP-43 protein.
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Affiliation(s)
- Sandro Orrù
- Medical Genetics, Department of Medical Sciences, University of Cagliari, R. Binaghi Hospital, Cagliari, Italy
| | - Paola Coni
- Paola Coni, Department of Biomedical Sciences, University of Cagliari, Monserrato, Italy
| | - Andrea Floris
- Medical Genetics, Department of Medical Sciences, University of Cagliari, R. Binaghi Hospital, Cagliari, Italy
| | - Roberto Littera
- Regional Transplant Center, R. Binaghi Hospital, ASL Cagliari, Cagliari, Italy
| | - Carlo Carcassi
- Medical Genetics, Department of Medical Sciences, University of Cagliari, R. Binaghi Hospital, Cagliari, Italy
| | - Valeria Sogos
- Paola Coni, Department of Biomedical Sciences, University of Cagliari, Monserrato, Italy
| | - Carla Brancia
- Paola Coni, Department of Biomedical Sciences, University of Cagliari, Monserrato, Italy
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Codron P, Cassereau J, Vourc’h P, Veyrat-Durebex C, Blasco H, Kane S, Procaccio V, Letournel F, Verny C, Lenaers G, Reynier P, Chevrollier A. Primary fibroblasts derived from sporadic amyotrophic lateral sclerosis patients do not show ALS cytological lesions. Amyotroph Lateral Scler Frontotemporal Degener 2018; 19:446-456. [DOI: 10.1080/21678421.2018.1431787] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
- Philippe Codron
- Department of Neurology, ALS Center, University Hospital of Angers, Angers Cedex 9, France,
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
- Department of Neurobiology and Neuropathology, University Hospital of Angers, Angers Cedex 9, France,
| | - Julien Cassereau
- Department of Neurology, ALS Center, University Hospital of Angers, Angers Cedex 9, France,
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
| | - Patrick Vourc’h
- Department of Biochemistry and Molecular Biology, University Hospital of Tours, Tours Cedex 1, France, and
- School of Medicine, INSERM U930, François-Rabelais University, Tours Cedex 1, France
| | - Charlotte Veyrat-Durebex
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
| | - Hélène Blasco
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
- Department of Biochemistry and Molecular Biology, University Hospital of Tours, Tours Cedex 1, France, and
- School of Medicine, INSERM U930, François-Rabelais University, Tours Cedex 1, France
| | - Selma Kane
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
| | - Vincent Procaccio
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
| | - Franck Letournel
- Department of Neurobiology and Neuropathology, University Hospital of Angers, Angers Cedex 9, France,
| | - Christophe Verny
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
| | - Guy Lenaers
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
| | - Pascal Reynier
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
| | - Arnaud Chevrollier
- MitoLab Unit, MITOVASC Institute, CNRS 6015, INSERM U1083, University of Angers, Angers Cedex 9, France,
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37
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Maguire G. Amyotrophic lateral sclerosis as a protein level, non-genomic disease: Therapy with S2RM exosome released molecules. World J Stem Cells 2017; 9:187-202. [PMID: 29312526 PMCID: PMC5745587 DOI: 10.4252/wjsc.v9.i11.187] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/29/2017] [Revised: 08/10/2017] [Accepted: 09/04/2017] [Indexed: 02/06/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that leads to death. No effective treatments are currently available. Based on data from epidemiological, etiological, laboratory, and clinical studies, I offer a new way of thinking about ALS and its treatment. This paper describes a host of extrinsic factors, including the exposome, that disrupt the extracellular matrix and protein function such that a spreading, prion-like disease leads to neurodegeneration in the motor tracts. A treatment regimen is described using the stem cell released molecules from a number of types of adult stem cells to provide tissue dependent molecules that restore homeostasis, including proteostasis, in the ALS patient. Because stem cells themselves as a therapeutic are cumbersome and expensive, and when implanted in a host cause aging of the host tissue and often fail to engraft or remain viable, only the S2RM molecules are used. Rebuilding of the extracellular matrix and repair of the dysfunctional proteins in the ALS patient ensues.
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Affiliation(s)
- Greg Maguire
- BioRegenerative Sciences, Inc., La Jolla, CA 92037, United States
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38
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Penndorf D, Tadić V, Witte OW, Grosskreutz J, Kretz A. DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of amyotrophic lateral sclerosis in vivo and in vitro. PLoS One 2017; 12:e0183684. [PMID: 28832631 PMCID: PMC5568271 DOI: 10.1371/journal.pone.0183684] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2017] [Accepted: 08/09/2017] [Indexed: 12/12/2022] Open
Abstract
Mutations in the human Cu/Zn superoxide dismutase type-1 (hSOD1) gene are common in familial amyotrophic lateral sclerosis (fALS). The pathophysiology has been linked to, e.g., organelle dysfunction, RNA metabolism and oxidative DNA damage conferred by SOD1 malfunction. However, apart from metabolically evoked DNA oxidation, it is unclear whether severe genotoxicity including DNA single-strand breaks (SSBs) and double-strand breaks (DSBs), originates from loss of function of nuclear SOD1 enzyme. Factors that endogenously interfere with DNA integrity and repair complexes in hSOD1-mediated fALS remain similarly unexplored. In this regard, uncontrolled activation of transposable elements (TEs) might contribute to DNA disintegration and neurodegeneration. The aim of this study was to elucidate the role of the fALS-causing hSOD1G93A mutation in the generation of severe DNA damage beyond well-characterized DNA base oxidation. Therefore, DNA damage was assessed in spinal tissue of hSOD1G93A-overexpressing mice and in corresponding motor neuron-enriched cell cultures in vitro. Overexpression of the hSOD1G93A locus did not change the threshold for severe DNA damage per se. We found that levels of SSBs and DSBs were unaltered between hSOD1G93A and control conditions, as demonstrated in post-mitotic motor neurons and in astrocytes susceptible to replication-dependent DNA breakage. Analogously, parameters indicative of DNA damage response processes were not activated in vivo or in vitro. Evidence for a mutation-related elevation in TE activation was not detected, in accordance with the absence of TAR DNA binding protein 43 (TDP-43) proteinopathy in terms of cytoplasmic mislocation or nuclear loss, as nuclear TDP-43 is supposed to silence TEs physiologically. Conclusively, the superoxide dismutase function of SOD1 might not be required to preserve DNA integrity in motor neurons, at least when the function of TDP-43 is unaltered. Our data establish a foundation for further investigations addressing functional TDP-43 interaction with ALS-relevant genetic mutations.
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Affiliation(s)
- Diane Penndorf
- Hans Berger Department of Neurology, Jena University Hospital, Jena, Thuringia, Germany
| | - Vedrana Tadić
- Hans Berger Department of Neurology, Jena University Hospital, Jena, Thuringia, Germany
| | - Otto W. Witte
- Hans Berger Department of Neurology, Jena University Hospital, Jena, Thuringia, Germany
| | - Julian Grosskreutz
- Hans Berger Department of Neurology, Jena University Hospital, Jena, Thuringia, Germany
| | - Alexandra Kretz
- Hans Berger Department of Neurology, Jena University Hospital, Jena, Thuringia, Germany
- * E-mail:
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Won YH, Lee MY, Choi YC, Ha Y, Kim H, Kim DY, Kim MS, Yu JH, Seo JH, Kim M, Cho SR, Kang SW. Elucidation of Relevant Neuroinflammation Mechanisms Using Gene Expression Profiling in Patients with Amyotrophic Lateral Sclerosis. PLoS One 2016; 11:e0165290. [PMID: 27812125 PMCID: PMC5094695 DOI: 10.1371/journal.pone.0165290] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2016] [Accepted: 10/10/2016] [Indexed: 11/19/2022] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by damage of motor neurons. Recent reports indicate that inflammatory responses occurring within the central nervous system contribute to the pathogenesis of ALS. We aimed to investigate disease-specific gene expression associated with neuroinflammation by conducting transcriptome analysis on fibroblasts from three patients with sporadic ALS and three normal controls. Several pathways were found to be upregulated in patients with ALS, among which the toll-like receptor (TLR) and NOD-like receptor (NLR) signaling pathways are related to the immune response. Genes—toll-interacting protein (TOLLIP), mitogen-activated protein kinase 9 (MAPK9), interleukin-1β (IL-1β), interleukin-8 (IL-8), and chemokine (C-X-C motif) ligand 1 (CXCL1)—related to these two pathways were validated using western blotting. This study validated the genes that are associated with TLR and NLR signaling pathways from different types of patient-derived cells. Not only fibroblasts but also induced pluripotent stem cells (iPSCs) and neural rosettes from the same origins showed similar expression patterns. Furthermore, expression of TOLLIP, a regulator of TLR signaling pathway, decreased with cellular aging as judged by changes in its expression through multiple passages. TOLLIP expression was downregulated in ALS cells under conditions of inflammation induced by lipopolysaccharide. Our data suggest that the TLR and NLR signaling pathways are involved in pathological innate immunity and neuroinflammation associated with ALS and that TOLLIP, MAPK9, IL-1β, IL-8, and CXCL1 play a role in ALS-specific immune responses. Moreover, changes of TOLLIP expression might be associated with progression of ALS.
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Affiliation(s)
- Yu Hui Won
- Department of Physical Medicine and Rehabilitation, Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital, Jeonju, Korea
- Department of Medicine, the Graduate School of Yonsei University, Seoul, Korea
| | - Min-Young Lee
- Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Korea
| | - Young-Chul Choi
- Department of Neurology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea
| | - Yoon Ha
- Department of Neurosurgery, Spine & Spinal Cord Institute, College of Medicine, Yonsei University, Seoul, Korea
| | - Hyongbum Kim
- Department of Pharmacology, Yonsei University College of Medicine, Seoul, Korea
- Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea
| | - Do-Young Kim
- Department of Dermatology, Cutaneous Biology Research Institute, Yonsei University College of Medicine, Seoul, Korea
| | - Myung-Sun Kim
- Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Korea
| | - Ji Hea Yu
- Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Korea
- Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea
| | - Jung Hwa Seo
- Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Korea
- Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea
| | - MinGi Kim
- Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Korea
- Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea
| | - Sung-Rae Cho
- Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine, Seoul, Korea
- Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Korea
- * E-mail: (SWK); (SRC)
| | - Seong-Woong Kang
- Department of Rehabilitation Medicine, Gangnam Severance Hospital, Rehabilitation Institute of Neuromuscular Disease, Yonsei University College of Medicine, Seoul, Korea
- Department of Medicine, the Graduate School of Yonsei University, Seoul, Korea
- * E-mail: (SWK); (SRC)
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40
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VGF Protein and Its C-Terminal Derived Peptides in Amyotrophic Lateral Sclerosis: Human and Animal Model Studies. PLoS One 2016; 11:e0164689. [PMID: 27737014 PMCID: PMC5063282 DOI: 10.1371/journal.pone.0164689] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2016] [Accepted: 09/29/2016] [Indexed: 12/12/2022] Open
Abstract
VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic stage, while showing an earlier down-regulation in the spinal cord. In immunohistochemistry, a large number of gray matter structures were VGF C-terminus immunoreactive in control mice (including nerve terminals, axons and a few perikarya identified as motoneurons), with a striking reduction already in the pre-symptomatic stage. Through gel chromatography and spectrometry analysis, we identified one form likely to be the VGF precursor as well as peptides containing the NAPP- sequence in all tissues studied, while in the mice and fibroblasts, we revealed also AQEE- and TLQP- peptides. Taken together, selective VGF fragment depletion may participate in disease onset and/or progression of ALS.
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41
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De Marco G, Lomartire A, Calvo A, Risso A, De Luca E, Mostert M, Mandrioli J, Caponnetto C, Borghero G, Manera U, Canosa A, Moglia C, Restagno G, Fini N, Tarella C, Giordana MT, Rinaudo MT, Chiò A. Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution. Neuropathol Appl Neurobiol 2016; 43:133-153. [PMID: 27178390 DOI: 10.1111/nan.12328] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2015] [Revised: 04/20/2016] [Accepted: 05/14/2016] [Indexed: 12/12/2022]
Abstract
AIMS Cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 (TDP-43) is an early determinant of motor neuron degeneration in most amyotrophic lateral sclerosis (ALS) cases. We previously disclosed this accumulation in circulating lymphomonocytes (CLM) of ALS patients with mutant TARDBP, the TDP-43-coding gene, as well as of a healthy individual carrying the parental TARDBP mutation. Here, we investigate TDP-43 subcellular localization in CLM and in the constituent cells, lymphocytes and monocytes, of patients with various ALS-linked mutant genes. METHODS TDP-43 subcellular localization was analysed with western immunoblotting and immunocytofluorescence in CLM of healthy controls (n = 10), patients with mutant TARDBP (n = 4, 1 homozygous), valosin-containing protein (VCP; n = 2), fused in sarcoma/translocated in liposarcoma (FUS; n = 2), Cu/Zn superoxide dismutase 1 (SOD1; n = 6), chromosome 9 open reading frame 72 (C9ORF72; n = 4), without mutations (n = 5) and neurologically unaffected subjects with mutant TARDBP (n = 2). RESULTS TDP-43 cytoplasmic accumulation was found (P < 0.05 vs. controls) in CLM of patients with mutant TARDBP or VCP, but not FUS, in line with TDP-43 subcellular localization described for motor neurons of corresponding groups. Accumulation also characterized CLM of the healthy individuals with mutant TARDBP and of some patients with mutant SOD1 or C9ORF72. In 5 patients, belonging to categories described to carry TDP-43 mislocalization in motor neurons (3 C9ORF72, 1 TARDBP and 1 without mutations), TDP-43 cytoplasmic accumulation was not detected in CLM or in lymphocytes but was in monocytes. CONCLUSIONS In ALS forms characterized by TDP-43 mislocalization in motor neurons, monocytes display this alteration, even when not manifest in CLM. Monocytes may be used to support diagnosis, as well as to identify subjects at risk, of ALS and to develop/monitor targeted treatments.
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Affiliation(s)
- G De Marco
- 'Rita Levi Montalcini' Department of Neuroscience, University of Turin, Turin, Italy
| | - A Lomartire
- 'Rita Levi Montalcini' Department of Neuroscience, University of Turin, Turin, Italy
| | - A Calvo
- 'Rita Levi Montalcini' Department of Neuroscience, University of Turin, Turin, Italy.,ALS Center, University of Turin and AOU Città della Salute e della Scienza, Turin, Italy
| | - A Risso
- Molecular Biotechnology Center, University of Turin, Turin, Italy
| | - E De Luca
- Molecular Biotechnology Center, University of Turin, Turin, Italy
| | - M Mostert
- Department of Public Health and Pediatric Sciences, University of Turin, Turin, Italy
| | - J Mandrioli
- Department of Neuroscience, Sant'Agostino Estense Hospital, University of Modena, Modena, Italy
| | - C Caponnetto
- Department of Neurosciences, Ophthalmology, Genetics, Rehabilitation and Child Health, IRCCS AOU San Martino IST, University of Genoa, Genoa, Italy
| | - G Borghero
- Department of Neurology, AOU and University of Cagliari, Cagliari, Italy
| | - U Manera
- 'Rita Levi Montalcini' Department of Neuroscience, University of Turin, Turin, Italy.,ALS Center, University of Turin and AOU Città della Salute e della Scienza, Turin, Italy
| | - A Canosa
- ALS Center, University of Turin and AOU Città della Salute e della Scienza, Turin, Italy.,Department of Neurosciences, Ophthalmology, Genetics, Rehabilitation and Child Health, IRCCS AOU San Martino IST, University of Genoa, Genoa, Italy
| | - C Moglia
- 'Rita Levi Montalcini' Department of Neuroscience, University of Turin, Turin, Italy.,ALS Center, University of Turin and AOU Città della Salute e della Scienza, Turin, Italy
| | - G Restagno
- Molecular Genetics Unit, Department of Clinical Pathology, AOU Città della Salute e della Scienza, University of Turin, Turin, Italy
| | - N Fini
- Department of Neuroscience, Sant'Agostino Estense Hospital, University of Modena, Modena, Italy
| | - C Tarella
- Clinical Hemato-Oncology, European Institute of Oncology (IEO), Milan, Italy
| | - M T Giordana
- 'Rita Levi Montalcini' Department of Neuroscience, University of Turin, Turin, Italy
| | - M T Rinaudo
- Department of Oncology, University of Turin, Turin, Italy
| | - A Chiò
- 'Rita Levi Montalcini' Department of Neuroscience, University of Turin, Turin, Italy.,ALS Center, University of Turin and AOU Città della Salute e della Scienza, Turin, Italy
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Ding Z, Xie H, Huang Y, Lv Y, Yang G, Chen Y, Sun H, Zhou J, Chen F. A simple, rapid, and efficient method for isolating detrusor for the culture of bladder smooth muscle cells. Int Urol Nephrol 2015; 48:47-52. [PMID: 26581565 DOI: 10.1007/s11255-015-1138-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Accepted: 10/10/2015] [Indexed: 12/12/2022]
Abstract
PURPOSE To establish a simple and rapid method to remove serosa and mucosa from detrusor for the culture of bladder smooth muscle cells (SMCs). METHODS Fourteen New Zealand rabbits were randomly allocated to two groups. In the first group, pure bladder detrusor was directly obtained from bladder wall using novel method characterized by subserous injection of normal saline. In the second group, full thickness bladder wall sample was cut down, and then, mucosa and serosa were trimmed off detrusor ex vivo. Twelve detrusor samples from two groups were manually minced and enzymatically digested, respectively, to form dissociated cells whose livability was detected by trypan blue exclusion. Proliferative ability of primary culture cells was detected by CCK-8 kit, and purity of second-passage SMCs was detected by flow cytometric analyses. Another two detrusor samples from two groups were used for histological examination. RESULTS Subserous injection of normal saline combined with blunt dissection can remove mucosa and serosa from detrusor layer easily and quickly. Statistical analysis revealed the first group possessed higher cell livability, shorter primary culture cell doubling time, and higher purity of SMCs than the second group (P < 0.05). Histological examination confirmed no serosa and mucosa existed on the surface of detrusor obtained by novel method, while serosa or mucosa residual can be found on the surface of detrusor obtained by traditional method. CONCLUSION Pure detrusor can be acquired from bladder wall conveniently using novel method. This novel method brought about significantly higher purity and cell livability as compared to traditional method.
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Affiliation(s)
- Zhi Ding
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China
| | - Hua Xie
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China
| | - Yichen Huang
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China
| | - Yiqing Lv
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China
| | - Ganggang Yang
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China
| | - Yan Chen
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China
| | - Huizhen Sun
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China
| | - Junmei Zhou
- Central Laboratory, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China
| | - Fang Chen
- Department of Urology, Shanghai Children's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, 200040, China.
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Evaluation of Skin Fibroblasts from Amyotrophic Lateral Sclerosis Patients for the Rapid Study of Pathological Features. Neurotox Res 2015; 28:138-46. [PMID: 26013250 DOI: 10.1007/s12640-015-9532-1] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2014] [Revised: 05/01/2015] [Accepted: 05/14/2015] [Indexed: 12/20/2022]
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by the progressive degeneration of brain and spinal cord motor neurons. Ubiquitin-proteasome system (UPS) dysfunction and oxidative stress have been implicated in ALS pathogenesis. However, it is unknown whether the defects in these pathways extend to non-neuronal tissues such as fibroblasts. Fibroblasts, unlike neuronal tissue, are readily available and may hold potential for short-term, rapid diagnostic and prognostic purposes. We investigated whether primary skin fibroblasts from ALS patients share, or can be manipulated to develop, functional and pathological abnormalities seen in affected neuronal cells. We inhibited UPS function and induced oxidative stress in the fibroblasts and found that ALS-related cellular changes, such as aggregate formation and ubiquitination of ALS-associated proteins (TDP-43 and ubiquilin 2), can be reproduced in these cells. Higher levels of TDP-43 ubiquitination, as evident by colocalization between TDP-43 and ubiquitin, were found in all six ALS cases compared to controls following extracellular insults. In contrast, colocalization between ubiquilin 2 and ubiquitin was not markedly different between ALS cases and control. A UPS reporter assay revealed UPS abnormalities in patient fibroblasts. Despite the presence of ALS-related cellular changes in the patient fibroblasts, no elevated toxicity was observed. This suggests that aggregate formation and colocalization of ALS-associated proteins may be insufficient alone to confer toxicity in fibroblasts used in the present study. Chronic exposure to ALS-linked stresses and the ALS-linked cellular pathologies may be necessary to breach an unknown threshold that triggers cell death.
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Buratti E. Functional Significance of TDP-43 Mutations in Disease. ADVANCES IN GENETICS 2015; 91:1-53. [DOI: 10.1016/bs.adgen.2015.07.001] [Citation(s) in RCA: 133] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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