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

Extracellular vesicles can mediate communication between tissues, affecting the physiological conditions of recipient cells. They are increasingly investigated in Amyotrophic Lateral Sclerosis, the most common form of Motor Neurone Disease, as transporters of misfolded proteins including SOD1, FUS, TDP43, or other neurotoxic elements, such as the dipeptide repeats resulting from C9orf72 expansions. EVs are classified based on their biogenesis and size and can be separated by differential centrifugation. They include exosomes, released by the fusion of multivesicular bodies with the plasma membrane, and ectosomes, also known as microvesicles or microparticles, resulting from budding or pinching of the plasma membrane. In the current study, EVs were obtained from the myotube cell culture medium of ALS patients or healthy controls. EVs of two different sizes, separating at 20,000 or 100,000 g, were then compared in terms of their effects on recipient motor neurons, astrocytes, and myotubes. Compared to untreated cells, the smaller, exosome-like vesicles of ALS patients reduced the survival of motor neurons by 31% and of myotubes by 18%, decreased neurite length and branching, and increased the proportion of stellate astrocytes, whereas neither those of healthy subjects, nor larger EVs of ALS or healthy subjects, had such effects.

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.

Expression of ALS-linked SOD1 Mutation in Motoneurons or Myotubes Induces Differential Effects on Neuromuscular Function In vitro.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that selectively affects upper and lower motoneurons. Dismantlement of the neuromuscular junction (NMJ) is an early pathological hallmark of the disease whose cellular origin remains still debated. We developed an in vitro NMJ model to investigate the differential contribution of motoneurons and muscle cells expressing ALS-causing mutation in the superoxide dismutase 1 (SOD1) to neuromuscular dysfunction. The primary co-culture system allows the formation of functional NMJs and fosters the expression of the ALS-sensitive fast fatigable type II-b myosin heavy chain (MHC) isoform. Expression of SOD1G93A in myotubes does not prevent the formation of a functional NMJ but leads to decreased contraction frequency and lowers the slow type I MHC isoform transcript levels. Expression of SOD1G93A in both motoneurons and myotubes or in motoneurons alone however alters the formation of a functional NMJ. Our results strongly suggest that motoneurons are a major factor involved in the process of NMJ dismantlement in an experimental model of ALS.

A Systematic Review of Suggested Molecular Strata, Biomarkers and Their Tissue Sources in ALS.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, is an incurable neurodegenerative condition, characterized by the loss of upper and lower motor neurons. It affects 1-1.8/100,000 individuals worldwide, and the number of cases is projected to increase as the population ages. Thus, there is an urgent need to identify both therapeutic targets and disease-specific biomarkers-biomarkers that would be useful to diagnose and stratify patients into different sub-groups for therapeutic strategies, as well as biomarkers to follow the efficacy of any treatment tested during clinical trials. There is a lack of knowledge about pathogenesis and many hypotheses. Numerous "omics" studies have been conducted on ALS in the past decade to identify a disease-signature in tissues and circulating biomarkers. The first goal of the present review was to group the molecular pathways that have been implicated in monogenic forms of ALS, to enable the description of patient strata corresponding to each pathway grouping. This strategy allowed us to suggest 14 strata, each potentially targetable by different pharmacological strategies. The second goal of this review was to identify diagnostic/prognostic biomarker candidates consistently observed across the literature. For this purpose, we explore previous biomarker-relevant "omics" studies of ALS and summarize their findings, focusing on potential circulating biomarker candidates. We systematically review 118 papers on biomarkers published during the last decade. Several candidate markers were consistently shared across the results of different studies in either cerebrospinal fluid (CSF) or blood (leukocyte or serum/plasma). Although these candidates still need to be validated in a systematic manner, we suggest the use of combinations of biomarkers that would likely reflect the "health status" of different tissues, including motor neuron health (e.g., pNFH and NF-L, cystatin C, Transthyretin), inflammation status (e.g., MCP-1, miR451), muscle health (miR-338-3p, miR-206) and metabolism (homocysteine, glutamate, cholesterol). In light of these studies and because ALS is increasingly perceived as a multi-system disease, the identification of a panel of biomarkers that accurately reflect features of pathology is a priority, not only for diagnostic purposes but also for prognostic or predictive applications.