Characterization of the extracellular vesicles released by astrocytes cultured from the spinal cord of late symptomatic SOD1G93A mice, a model of amyotrophic lateral sclerosis

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects upper and lower motor neurons (MNs), leading to muscle atrophy, paralysis, and death within 3-5 years from symptom onset. ALS is a non-cell-autonomous disease in which astrocytes play a crucial role in MN damage and death, possibly by secretion of soluble factors or extracellular vesicles (EVs). This study addressed the part played by EVs produced by astrocytes cultured from adult late symptomatic SOD1G93A mice, an ALS animal model, in the astrocyte and MN interplay in ALS. Methods Astrocytes were prepared from 120-day-old spinal cord SOD1G93A and SOD1WT mice. The tissue was mechanically dissociated in DMEM medium + N2 factor, and astrocytes were cultured at 37°C and 5% CO2 for 20 DIV before experiments. EVs were isolated by nickel-based isolation (NBI) from the supernatant of astrocytes by using positively charged agarose beads and eluted by EDTA-NaCl elution buffer. MNs were isolated by gradient from the spinal cord of E13,5 WT embryos, cultured in neurobasal medium and counted for viability from day 8 to day 14 after seeding. EVs-RNA was extracted from NBI-isolated EVs using the Single Cell RNA extraction kit (Norgen Biotek) and analyzed by RNA sequencing. Results The number of cells obtained from SOD1G93A and WT mice was 6.37x105 and 1.61x105, respectively. EVs recovered per cell were 6,73x105 from SOD1G93A and 1,78x106 from WT astrocytes. We did not detect size or Z potential differences between EVs from the two mouse genotypes. We have completed the RNA sequencing to define the microRNA repertoire of SOD1G93A and WT astrocyte-derived EVs. Bioinformatic analysis is ongoing. Exposure to EVs from SOD1G93A astrocytes significantly reduced WT-MN viability compared to controls. We observed the same when treating SOD1G93A MNs with EVs from SOD1G93A astrocytes. Conclusions Our results indicate that SOD1G93A mouse-derived astrocytes negatively influence MN viability by secreting EVs that can carry unique information which defines their neurotoxic activity. Data from RNA sequencing should shape EV characteristics and decipher the microRNAs with modified expression for further in-vitro studies.