Amyotrophic lateral sclerosis (ALS) is a terminal late-onset condition characterized by the loss of upper and lower motor neurons. Mutations in more than 30 genes are associated to the disease, but these explain only ~20% of cases. The molecular functions of these genes implicate a wide range of cellular processes in ALS pathology, a cohesive understanding of which may provide clues to common molecular mechanisms across both familial (inherited) and sporadic cases and could be key to the development of effective therapeutic approaches. Here, the different pathways that have been investigated in ALS are summarized, discussing in detail: mitochondrial dysfunction, oxidative stress, axonal transport dysregulation, glutamate excitotoxicity, endosomal and vesicular transport impairment, impaired protein homeostasis, and aberrant RNA metabolism. This review considers the mechanistic roles of ALS-associated genes in pathology, viewed through the prism of shared molecular pathways.

Amyotrophic lateral sclerosis is a rare and fatal neurodegenerative disease characterised by progressive deterioration of upper and lower motor neurons that eventually culminates in severe muscle atrophy, respiratory failure and death. There is a concerning lack of understanding regarding the mechanisms that lead to the onset of ALS and as a result there are no reliable biomarkers that aid in the early detection of the disease nor is there an effective treatment. This review first considers the clinical phenotypes associated with ALS, and discusses the broad categorisation of ALS and ALS-mimic diseases into upper and lower motor neuron diseases, before focusing on the genetic aetiology of ALS and considering the potential relationship of mutations of different genes to variations in phenotype. For this purpose, a systematic review is conducted collating data from 107 original published clinical studies on monogenic forms of the disease, surveying the age and site of onset, disease duration and motor neuron involvement. The collected data highlight the complexity of the disease’s genotype–phenotype relationship, and thus the need for a nuanced approach to the development of clinical assays and therapeutics.

Skeletal muscle is increasingly considered an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can affect physiological changes with an impact on different pathological conditions, including regenerative processes, aging, and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 × 10 6 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation. Thus, an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml −1), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons. Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.

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,

wordsSkeletal muscle is increasingly considered as an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can effect physiological changes with impact in different pathological conditions, including regenerative processes, aging and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 x 10 6 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation.Thus an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle