Secretion of toxic exosomes by muscle cells of ALS patients: role in ALS pathogenesis

Background: The causes of ALS remain unknown. For sporadic and familial cases, several studies show an abnormal accumulation of lysosomally-directed protein aggregates in the cytosol. Mutations in genes involved in the autophagy pathways and multivesicular body biogenesis are now known to occur among familial cases. These data suggest a potential disruption of the endosome and lysosome pathways, thus affecting exosome genesis. Work in our lab and others has shown that the skeletal muscle has a functional secretory activity. The muscle secretome contains exosomes - vesicles that carry out intercellular transport of functional proteins, mRNA, and miRNA. Aims: We hypothesize that exosome secretion is disrupted in ALS muscle cells, and that this affects the intercellular communication between muscles and motor neurons. Materials & Methods: Muscle stem cells were purified from ALS, SBMA, SMAIII-IV and age-matched healthy subjects (n=18, n=12, n=12, n=21 respectively). Exosomal toxicity was tested by adding them to the culture medium of healthy human muscle cells, human IPS-derived motor neurons or primary murine motor neurons. Results: Whereas clinically overlapping pathologies SBMA and SMA-IV have gene expression profiles similar to healthy controls, myotubes of sporadic ALS patients have a strongly specific gene expression signature. The 30 genes most strongly contributing to this ALS-specific signature encode proteins localized to a form of secreted vesicle known as the exosome. We confirmed by RT-qPCR, immunostaining and electron microscopy that the exosomal content is significantly upregulated in both ALS myotubes and muscle biospises. In electron microscopy of sporadic ALS muscles cells we observed multi-vesicular bodies that are significantly more filled with exosomes (1.40 ± 0.14 exosomes/mm2 in ALS, 0.9 ± 0.07 exosomes /mm2 in control), and ALS myotubes released 2-fold more exosomes than healthy controls. ALS exosomes added to the culture medium of healthy muscle cells or of healthy motor neurons induced: (1) muscle fiber atrophy, (2) cellular stress by stimulating membrane blebbing, (3) cell death of muscle cells and motor neurons. Conclusion: The secretion of toxic exosomes occurs independently of muscle denervation and could be a potential mechanism to explain the progressive spread of the disease across muscles and motor groups.