Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

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

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

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

Isolement et etude structurale de glucanes de Phytophtora parasitica

National audienc

Toxicité vis-à-vis du tabac des fractions purifiées d'un filtrat de culture de Phytophthora cryptogea Pethyb. & Laff

International audienc

Isolement composition et activite elicitrice chez l'oeillet de fractions extraites de la paroi de Phytophthora parasitica Dastur

National audienceWater-soluble extracts were released from the cell walls of the phytopathogenic fungus Phytophthora parasitica by various treatments. Applied to carnation cuttings, they elicited the accumulation of phytoalexins : the N-benzoylanthranilic derivatives, dianthalexin and dianthramide A. Extract S1, obtained by hot water treatment of the cell walls, had the highest specific activity. It contained proteins (54 %), polysaccharides and trace amounts of lipids. The lipids in S1 were represented by free and esterified fatty acids and, among the latter, a high proportion of unsaturated C20 acids. They were devoid of eliciting ability on carnation (as well as the lipids directly extracted from the cell walls). Using anion exchange chromatography, S1 was separated into fractions A (non retained) and B (retained). beta-D-glucans were the major components of A and were purified. The glucan of lowest molecular weight faintly elicited carnation ; the others did not. Fraction B had the greatest eliciting activity and was composed of proteins and polysaccharides containing a high proportion of mannose. Its activity, as well as that of extract S1, was destroyed by pronase and by periodate oxidation. Thus, the eliciting factor could be a glycoprotein.Divers traitements de la paroi du champignon phytopathogène Phytophthora parasitica fournissent des extraits hydrosolubles capables d’éliciter chez l’oeillet l’accumulation de phytoalexines dérivées de l’acide N-benzoylanthranilique : la dianthalexine et la dianthramide A. L’extrait S1 obtenu par autoclavage de la paroi dans l’eau est quantitativement le plus actif. Il contient des protéines (54 p. 100), des polysaccharides et des traces de lipides. Les lipides de S1 sont représentés par des acides gras libres et des acides gras estérifiés, ces derniers contenant une forte proportion d’acides insaturés en C20. Ils sont dépourvus d’activité élicitrice chez l’oeillet (ainsi que les lipides directement extraits de la paroi). La chromatographie d’échange anionique permet de séparer S1 en une fraction non liée (A) et une fraction retenue (B). Des beta-D-glucanes, composants majoritaires de A, ont été purifiés. Le composé de plus faible masse moléculaire a un faible pouvoir éliciteur, les autres aucun. La fraction la plus élicitrice (B) est composée de protéines et de polysaccharides contenant une forte proportion de mannose. Son activité, comme celle de l’extrait S1, est détruite par action de la pronase et par oxydation periodique, ce qui incite à penser que le principe éliciteur pourrait être de nature glycoprotéique

Caracterisation chimique des inducteurs de phytoalexines chez l'oeillet

National audienc