Neurotoxicity of skeletal muscle extracellular vesicles in Amyotrophic Lateral Sclerosis (ALS)

: vesicle sub-type specificity, target cell type specificity, and mechanisms of uptake

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative and multi-system disease characterised by the gradual degeneration and loss of upper and lower motor neurons with associated muscle weakness, atrophy, and paralysis. Numerous mechanisms such as dysfunctional mitochondria and oxidative stress; axonal transport; glutamate excitotoxity; protein homeostasis; and dysregulated RNA processing are suggested to be responsible for the prevailing pathology,including extracellular vesicle-mediatedpropagation involving the transport of toxic misfolded and/or aggregated proteins.Extracellular vesicles, especially small extracellular vesicles (exosomes), are secreted byprincipal cell types implicated in ALS, including motor neurons, astrocytes, microglia and,recently reported, skeletal muscles. To investigate the contribution of the skeletal musclesecretome to pathology, this thesis focuses on the functionality and uptake mechanismsof skeletal muscle-derived small extracellular vesicles (or MuVs).

In this thesis, a protocol for the efficient isolation of MuVs from conditioned media of myotubes was developed and validated, yielding MuVs with canonical markers characteristic of small extracellular vesicles. Secondly, vesicles secreted by differentiated myotubes exhibited selective toxicity based on functional studies in the principal cells affected in ALS (motor neurons, astrocytes, and skeletal muscle). MuVs derived from ALS myotubes were specifically and selectively toxic to motor neurons and myotubes, unlike larger vesicles or ectosomes derived from ALS myotubes that exhibited no toxicity. Thirdly, MuVs uptake by myotubes requires actin polymerization and the involvement of endocytic pathways such as macropinocytosis and clathrin-mediated endocytosis and lipid rafts. In addition, interactions of the EV surface with the recipient cell surface appear necessary for docking and subsequent internalisation of MuVs.

Altogether, this work refines our understanding of the potential role of MuVs in vesicle-mediated propagation and the spread of toxicity in ALS.

Date of Award	May 2022
Original language	English
Supervisor	William Duddy (Supervisor) & Stephanie Marie Duguez (Supervisor)