Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles

Abstract

BackgroundThe 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,

The Neurotoxicity of Vesicles Secreted by ALS Patient Myotubes Is Specific to Exosome-Like and Not Larger Subtypes

“…Primary human myoblasts were obtained from human deltoid muscle biopsies from a previous study ( n = 7 ALS and n = 7 healthy) [ 44 ]. The protocol (NCT01984957) was approved by the local Ethical Committee and all subjects signed an informed consent in accordance with institutional guidelines.…”

“…The protocol (NCT01984957) was approved by the local Ethical Committee and all subjects signed an informed consent in accordance with institutional guidelines. ALS gene mutations had been assessed previously [ 44 ], and included testing for the C9orf72 hexanucleotide repeat expansion, ATXN2 repeat length, and the coding regions of SOD1 , TARDBP , FUS , UBQLN2 and TBK1 . Subject characteristics are given in Table 1 .…”

“…EVs were extracted as previously described, at myoblast cell division counts of less than 20 in order to avoid potential artefacts due to pre-senescence [ 13 , 44 , 45 ]. Briefly, the conditioned cell culture media from differentiated myoblasts was centrifuged at 260 g for 10 min and the resulting supernatant centrifuged at 4000 g for 20 min.…”

“…After 72 h of treatment, MN were fixed using 4% formaldehyde, permeabilized, blocked and stained for Tuj1, Islet 1/2 as described [ 44 ]. Fifteen non-overlapping images were acquired across wells using an Olympus UPlan FI 10x/0.30 Ph1 objective.…”

“…Exosomes originating from the astrocytes of ALS murine models [ 36 , 37 ], or from ALS patient iPSC-derived astrocytes [ 38 ] and motor neurons [ 36 ], have been implicated in pathogenesis [ 37 , 39 , 40 , 41 ], carrying misfolded proteins such as SOD1, FUS, TDP43, or toxic elements such as the dipeptide repeats (DPRs) resulting from C9orf72 expansions [ 36 , 37 , 40 , 42 , 43 ]. In alignment with these studies, we recently observed that the skeletal muscle cells of ALS patients secrete exosome-like vesicles (MuVs) that are toxic towards motor neurons [ 44 ].…”