Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons (MNs) and currently lacks effective therapies. It has been shown that astrocytes carrying the hSOD1G93A mutation reduce MN survival, although the underlying molecular mechanisms remain unknown. These astrocytes release extracellular vesicles (EVs) containing differential microRNAs that decrease neuronal viability, but their transcriptomic impact is still unclear. Our project investigates the gene expression of control MNs and MNs treated with EVs derived from non-Tg and hSOD1G93A astrocytes through high-throughput sequencing. We have cultured non-Tg and hSOD1G93A astrocytes, isolated and quantified their EVs, and cultured MNs to assess their viability in the presence or absence of EVs. We will perform RNA-seq to identify transcriptomic changes and determine whether differentially expressed mRNAs are targeted by microRNAs present in SOD1G93A EVs, evaluating their post-transcriptional regulation using Western blot or immunofluorescence, with EVs, mimics, and antagomiRs. In addition, we treated astrocytes with antimycin A (AA) to analyze changes in EV quantity and protein levels. This study contributes to understanding astrocyte–MN interactions in ALS, the mechanisms of neurodegeneration, and potential microRNA-based therapeutic strategies, with possible health and social impact.