Identification of biomarkers to identify ALS subjects among patients affected by motor neuron disorders

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disorder characterized by heterogeneous clinical symptoms and disease course, making both diagnosis and prognosis difficult. Biomarkers are urgently needed to minimise diagnostic delay and improve prognostic tracking, as well as to allow for a better understanding of the disease towards future therapeutic discoveries. Extracellular-vesicles (EVs), carrying diverse bioactive materials from cell-to-cell, have been found to transport neurotoxic contents in ALS. Their ability to circulate to distant cells, from their original secreting cell, and their abundance in body fluids, makes them interesting as a novel source of biomarkers. Potential contributions to pathology from multiple tissues are increasingly explored in ALS, and we have shown that EVs released by the skeletal muscle cells of ALS patients are toxic towards motor neurons.

The aim of this thesis was to (1) investigate the role of two categories of EVs released by muscle cells, exosomes and microvesicles, on their surrounding microenvironment and in ALS pathology; (2) determine the potential of EVs as prognostic and diagnostic biomarkers.

Exosomes and microvesicles isolated from the culture medium of healthy patient muscle cells were distinct from each other in terms of their surface markers and buoyant properties. Exosomes from healthy subjects exhibited an enrichment in protein content, increased secretion, as well as preferential uptake by all cell types in in vitro studies, compared to microvesicles. Moreover, unlike microvesicles, skeletal muscle-derived exosomes (MuVs) were preferentially absorbed by motor neurons compared to both astrocyte and muscle cell types, with specific toxicity of ALS MuVs towards motor neurons followed by the muscle as a secondary target. These results highlight the specific toxicity of MuVs and more generally the potential contribution of muscle EVs to ALS pathology.

In vitro characterization of the MuV content revealed that ALS MuVs were enriched in sphingomyelins, and proteins including muscle specific markers, that could be used as potential biomarkers. In ALS plasma samples, the level of total lipid in circulating EVs decreased over the course of ALS progression and correlated with the motor function decline, revealing a potential source of prognostic biomarker. Muscle protein markers were detected in plasma derived EVs of patients, suggesting the presence of muscle EVs in circulation. Among the protein cargoes distinguishing ALS and healthy MuVs in vitro, an exosomal marker heat-shock cognate 71 kDa protein (HSPA8), was correlated with the muscle function in vivo, suggesting it as a prognostic biomarker of ALS severity. More importantly, EVs positive for HSPA8 and a muscle marker dystroglycan (DAG), were increased in ALS compared to Parkinson’s and Healthy controls, so these represent promising candidate diagnostic biomarkers.

These findings suggest that the EV content could reflect the muscle health at a distance and be used as a source of biomarkers.

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