CHARACTERIZATION OF AXONAL TRANSPORT AND EXTRACELLULAR VESICLE PROFILE IN ALS IPSC-DERIVED MOTOR NEURONS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of motor neurons, leading to muscle weakness and reduced survival. Disease-modifying treatments are lacking, and sensitive biomarkers to monitor disease progression and therapeutic response are needed. Extracellular vesicles (EVs) have emerged as promising biomarkers of disease-related cellular alterations. Additionally, impaired axonal transport is an early and key pathogenic feature of ALS, as efficient trafficking of organelles and vesicles is essential for motor neuron function. This study investigated whether EVs released by motor neurons can serve as indicators of neuronal dysfunction in ALS, assessed by alterations in axonal transport. To this end, we employed induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the TARDBP A382T mutation and a CRISPR-corrected isogenic control. Axonal transport was assessed by using mitotracker and lysotracker to monitor mitochondrial and lysosomal movement, together with two fluorescent peptidic probes synthesized in-house that bind kinesin or dynein. EVs were isolated from conditioned media by differential ultracentrifugation and characterized by nanoparticle tracking analysis to determine their concentration and size distribution. Our results showed that ALS motor neurons exhibited alterations in axonal transport, evidenced by reduced lysotracker, mitotracker and dynein transport speeds. In parallel, conditioned media from ALS motor neurons displayed a decreased concentration of large EVs compared to the isogenic control. These findings indicate concomitant defects in axonal transport and EV release in ALS motor neurons and support the use of EVs as readouts of neuronal alterations in ALS.