Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

Laura Le Gall, William Duddy, Cecile Martinat, Virginie Mariot, Owen Connolly, Vanessa Milla, Ekene Anakor, Zamalou G Ouandaogo, Stéphanie Millecamps, Jeanne Lainé, Geetha Vijayakumar, Susan Knoblach, Cedric Raoul, Olivier Lucas, Jean Philippe Loeffler, Peter Bede, Anthony Behin, Helene Blasco, Gaelle Bruneteau, Maria Del Mar AmadorDavid Devos, Alexandre Henriques, Adele Hesters, Lucette Lacomblez, Pascal Laforet, Timothee Langlet, Pascal Le Blanc, Nadine Le Forestier, Thierry Maisonobe, Vincent Meininger, Laura Robelin, Francois Salachas, Tanya Stojkovic, Girogia Querin, Julie Dumonceaux, Gillian Butler-Browne, Jose-Luis Gonzales De Aguilar, Stephanie Duguez, Pierre-Francois Pradat

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Abstract

Background: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle‐secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. Methods: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS‐R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged‐matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT‐qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human‐derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. Results: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy‐derived denervation‐naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome‐like vesicles and disruption of RNA‐processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA‐processing proteins. The RNA‐processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti‐CD63 immuno‐blocking of vesicle uptake. Conclusions: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle‐mediated toxicity in ALS.
Original languageEnglish
Pages (from-to)1385-1402
Number of pages18
JournalJournal of Cachexia, Sarcopenia and Muscle
Volume13
Issue number2
Early online date22 Feb 2022
DOIs
Publication statusPublished - 22 Feb 2022

Bibliographical note

Funding Information:
We thank the Human Cell Culture Platform of The Institute of Myology, and the ‘Plateforme Biopuces et Séquençage de l'IGBMC’. We thank Aswini Panigrahi for help with data upload. This work was financed by Target‐ALS (ViTAL consortium, PI: S Duguez), ARsLA (TEAM consortium, PI: S Duguez), European Union Regional Development Fund (ERDF) EU Sustainable Competitiveness Programme for N. Ireland, Northern Ireland Public Health Agency (HSC R&D), and Ulster University (PI: T Bjourson). L.L.G. is a recipient from ArSLA. It was also partially supported by European Community's Health Seventh Framework Programme under grant agreement No. 259867 (Euro‐MOTOR), INSERM, Sorbonne University, and the AFM. All research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre ‐ the views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The study was sponsored by APHP. Several samples (3) were obtained from an ongoing observational and prospective multicentric cohort (PULSE; Protocol 2013‐A00969‐36) sponsored by the University Hospital of Lille, conducted in 16 ALS expert centres from the clinical research networks in France (FILSLAN, ACT4ALS‐MND), approved from the CPP Nord Ouest‐IV Ethical Committee and registered in the ClinicalTrials.gov website (NCT02360891). We thank all the participants and their families for their cooperation. The authors are grateful for financial support of PULSE from the French ARSLA charity (), support from the French clinical research networks FILSLAN and ACT4ALS‐MND and the . Christine Tabuenca et Marie France Cazalère Fédération de la Recherche Clinique du CHU de Lille

Funding Information:
We thank the Human Cell Culture Platform of The Institute of Myology, and the ?Plateforme Biopuces et S?quen?age de l'IGBMC?. We thank Aswini Panigrahi for help with data upload. This work was financed by Target-ALS (ViTAL consortium, PI: S Duguez), ARsLA (TEAM consortium, PI: S Duguez), European Union Regional Development Fund (ERDF) EU Sustainable Competitiveness Programme for N. Ireland, Northern Ireland Public Health Agency (HSC R&D), and Ulster University (PI: T Bjourson). L.L.G. is a recipient from ArSLA. It was also partially supported by European Community's Health Seventh Framework Programme under grant agreement No. 259867 (Euro-MOTOR), INSERM, Sorbonne University, and the AFM. All research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health is made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre - the views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The study was sponsored by APHP. Several samples (3) were obtained from an ongoing observational and prospective multicentric cohort (PULSE; Protocol 2013-A00969-36) sponsored by the University Hospital of Lille, conducted in 16 ALS expert centres from the clinical research networks in France (FILSLAN, ACT4ALS-MND), approved from the CPP Nord Ouest-IV Ethical Committee and registered in the ClinicalTrials.gov website (NCT02360891). We thank all the participants and their families for their cooperation. The authors are grateful for financial support of PULSE from the French ARSLA charity (Christine Tabuenca et Marie France Cazal?re), support from the French clinical research networks FILSLAN and ACT4ALS-MND and the F?d?ration de la Recherche Clinique du CHU de Lille. The authors of this manuscript certify that they comply with the ethical guidelines for authorship and publishing in the Journal of Cachexia, Sarcopenia and Muscle.48

Publisher Copyright:
© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.

Keywords

  • Secreted vesicles
  • Cell-cell communication
  • MND
  • sporadic ALS
  • Muscle Cells/metabolism
  • Humans
  • Proteomics
  • Induced Pluripotent Stem Cells/metabolism
  • Aged
  • Amyotrophic Lateral Sclerosis/genetics
  • Motor Neurons/metabolism

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