Mesenchymal stem cell-derived exosomes and exosome-shuttled miRNAs ameliorate the reactive and neurotoxic phenotype of mouse SOD1G93A astrocytes and human-derived SOD1A4V astrocytes

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting primarily motor neurons (MNs) but involving also non-neuronal cells. Nowadays, it is well recognised that astrocytes, microglia and oligodendrocytes play a central role in disease onset and progression. In particular, astrocytes acquire a toxic phenotype characterized by an abnormal proliferation and by the release of neurotoxic factors, including pro-inflammatory cytokines (Lee et al., 2016). We have previously shown that the intravenous administration of mesenchymal stem cells (MSCs) in SOD1G93A mice prolonged survival, ameliorated motor skills and reduced gliosis and inflammation in spinal cord. These beneficial effects were not associated with MSC differentiation, being possibly mediated through paracrine mechanisms. We hypothesized that MSC-derived exosomes and exosome-shuttled miRNAs could mediate these positive effects. To verify our hypothesis we tested here the effects of exosomes derived from INF-activated MSCs on cultured astrocytes prepared from the spinal cord of 120 day-old late-symptomatic SOD1G93A mice. The phenotype of SOD1G93A astrocytes and the efficacy of the exosome treatment were characterized by Western blotting, confocal microscopy and ELISA immunoassay. Vimentin, GFAP and S100β, astrogliosis markers, were increased in astrocytes from 120 days-old SOD1G93A mice vs. age-matched WT astrocytes and their expression was reduced after exposure to exosomes. Nrf2, a booster of the response to oxidative stress, was decreased in SOD1G93A astrocytes vs. age-matched WT astrocytes. Exosome treatment normalized Nrf2 down-regulation both in the cytoplasm and nucleus. The quantification of TNF-α,IL-1β, IL-6 and CCL2 expression and release showed that these four pro-inflammatory factors were more expressed in and more efficiently released from SOD1G93A astrocytes and that the exposure to exosomes resulted in a significant decrease of their over-expression and release. Conversely, the anti-inflammatory cytokine IL-10 was decreased in SOD1G93A astrocytes and its expression was normalized after exposure to exosomes. Also NLRP3 expression, a marker of the multiprotein oligomer inflammasome, was increased in SOD1G93A astrocytes and the increase was reversed by exosomes. The amelioration of SOD1G93A astrocyte phenotype had a positive impact on MN viability in astrocyte-MN co-cultures. We observed a constant decrease of MN survival during time, both in control and exosome-treated co-cultures; however, the viability of MNs seeded on exosome-treated SOD1G93A astrocytes was always significantly higher when compared to co-cultures with untreated astrocytes. Exosome cargo was analysed for miRNAs and potential mediators of exosome activity were identified. The selected miRNAs showed a significant efficacy to reduce GFAP, IL-1β and TNF-α expression. Computational analysis highlighted their possible involvement in the modulation of NFκB and MAPK pathway activation, affecting numerous kinases and transcription factors involved in the regulation of these inflammatory signalling pathways. qPCR analysis confirmed the ability of these four miRNAs to reduce MAPK11 expression, regulating TNF-α synthesis. Unfortunately, the other selected targets were not affect by mimic transfection. Finally, we translated this study to human astrocytes derived from healthy donors and ALS patients carrying A4V-SOD1 mutation. Human ALS astrocytes were treated with exosomes derived from human MSCs, activated with INF. We observed only a slight amelioration of ALS astrocyte phenotype after the exosome treatment. Remarkably, analysis of MN viability showed an increased MN number in co-cultures with exosome-treated astrocytes compared to those co-cultured with untreated astrocytes. These results indicate that exosomes and exosome-shuttled miRNAs can reduce astrocyte reactivity and that this effect has a positive impact on MN viability. The in-vitro exosome activity, both in mouse and human models, paves the way to translational preclinical in-vivo treatments in SOD1G93A mice.