Conference Agenda

During development, microglia mediate beneficial synaptic pruning through phagocytosis of aberrant synapses tagged by complement factors. However, how complement factors are delivered to synapses is not completely clear. Our previous evidence shows that extracellular vesicles (EVs) released by microglia, carry multiple signals implicated in synaptic pruning (PS, complement factors) and move at the neuron surface scanning actin protrusions before establishing a stable interaction on mature dendrites, thus representing ideal vehicles to tag synapses with molecules guiding microglia-mediated removal. To assess this hypothesis, we first delivered single microglial EVs through optical manipulation to neurons transfected with the presynaptic marker VGlut and explore whether EVs may adhere to synapses. A fraction of EVs stopped moving at VGlut+ puncta and staining for C1q and synaptic markers confirmed EV association to synapses. We then co-cultured neurons with wild type (wt) or C9orf72 knock out (ko) microglia, which produce a double amount of EVs and complement factors (C1q/C3) compared to wt cells, as indicated by TRPS and WB analysis, and found that wt microglia induced a decrease in the density of Shank-2+ (post-synaptic) but not Bassoon+ (pre-synaptic) puncta while ko microglia reduced the density of both Bassoon+ and Shank2+ puncta and enhanced microglial engulfment of pre-synaptic material. Importantly, pretreatment of ko microglia with GW4869, an inhibitor of EV biogenesis that reduces EV production, restored normal pre-synaptic density. On the other hand, addition of microglial EVs to neurons before microglia co-culturing caused a decrease in Bassoon+ puncta and preincubation of EVs with synaptosomes enhanced their engulfment by microglia. Taken together, our data indicate that microglial EVs promote engulfment of pre-synapses in vitro. Analysis of synaptic density in the hippocampi of P17 ko mice showed a lower level of VGlut in CA1 area compared to wt, linking enhanced microglial EV production to excessive pruning during brain development.

The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown. However, it is believed that the cellular environment of the MN may be crucial to its survival. There is evidence to suggest that vesicles may play a role in ALS. For example, extracellular vesicles may carry toxic substances from astrocytes to MN, and pathological proteins have been found in the circulating extracellular vesicles of ALS patients. Since MN degeneration at the neuromuscular junction is a feature of ALS, and muscle tissue secretes vesicles, we hypothesized that muscle vesicles may also be involved in ALS pathology.

Our recent study found a distinct disease signature in both ALS muscle biopsies and muscle stem cells compared to healthy subjects (n=27 sporadic ALS patients, and n=30 healthy subjects). We noted an accumulation of multivesicular bodies and exosome-like vesicles in the muscle biopsies and muscle stem cells of ALS patients, as well as a disruption of RNA-processing in ALS muscle cells. ALS muscle cells secreted more exosome-like vesicles (MuVs) compared to healthy myotubes. When MuVs from ALS myotubes were administered to healthy MN, it induced shortened and less branched neurites, MN death, and disrupted the localization of RNA and RNA-processing proteins. When ALS MuVs were applied to healthy astrocytes, the proportion of stellate astrocytes was increased.

Interestingly, none of these events was observed when healthy MuVs or when larger extracellular vesicles of ALS or healthy subjects were applied to healthy MN or healthy astrocytes. Furthermore, ALS MuVs were enriched in the RNA-processing protein FUS and its binding partners. The toxicity of the MuVs correlated with the level of FUS expression in the recipient cells.

ALS MuVs can be harmful to MN, suggesting that skeletal muscles may contribute to vesicle-mediated toxicity in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by upper and lower motor neurons (MNs) death, with an important contribution of glial cells.

We previously demonstrated that the intravenous administration of mesenchymal stem cells (MSCs), prolonged survival probability and ameliorated pathological features in SOD1^G93A ALS mice. We postulated that the beneficial effect observed could be mediated by the paracrine activity of extracellular vesicles (EVs) secreted by MSCs.

The aim of the study was to evaluate the in-vitro effect of MSCs-derived EVs on astrocytes and the efficacy of the EVs in-vivo treatment in symptomatic SOD1^G93A_. In spinal cord astrocytes isolated from symptomatic SOD1^G93A mice, 24h in-vitro exposure to MSC-derived EVs significantly reduced the overexpression of glial activation markers (GFAP, vimentin and S100β) and pro-inflammatory factors (TNF-α, IL-1β, IL-6 and CCL2). In human astrocytes (iAstrocytes) differentiated from inducible neural progenitor cells (iNPCs) derived from ALS patients and healthy donors, the exposure to EVs increased the expression of the Nrf2 anti-oxidant factor and reduced the accumulation of reactive oxygen species. We tested the neurotoxicity of ALS astrocytes on MNs survival, that was significantly reduced after EVs exposure. The transfection with synthetic mimics of miRNAs upregulated in MSCs, reverted the reactive phenotype of SOD1^G93A ALS astrocytes and upregulated the Nrf2 antioxidant pathway in iAstrocytes.

Consequently, we tested the chronic intranasal administration of MSC-derived EVs in SOD1^G93A mice starting at 90 days of life (early symptomatic stage). The in-vivo MSC-derived EVs administration significantly slowdown the progression of the disease and increased life span in EV-treated male mice only. Spinal cord MNs survival was significantly preserved at 115 days in male treated mice compared to control.

Our pre-clinical evidence suggests MSCs-derived EVs or even EV-mimicking synthetic particles as a promising pharmacological tool for ALS treatment.

Degeneration of myelinating oligodendrocytes (OLs) and consequent disruption of the myelin sheath enwrapping motoneuronal axons represent major contributing factors to neurodegeneration and disability in ALS. Indeed, our results in the SOD1^G93A mouse model showed an abnormal increase of oligodendrocyte precursor cells (OPCs) expressing the GPR17 receptor, a pivotal regulator of OPC maturation, and a degeneration of mature oligodendrocytes, which are not successfully replaced. Highly relevant, OPCs isolated from the spinal cord of SOD1^G93A mice display defective differentiation compared to control cells. On this basis, fostering endogenous myelin repair sustained by OPCs represents a promising therapeutic approach for ALS. A central role in shaping remyelination is played by microglia, that regulate both myelin damage and repair processes by acquiring different functional states. In this respect, our results show that extracellular vesicles (EVs) released by microglia enhance the differentiation of OPCs surrounding demyelinated lesions in vivo. These findings are corroborated by in vitro experiments showing direct beneficial actions of microglial EVs on OPC migration, differentiation, and myelin deposition. However, the mechanisms underlying EV-induced beneficial effects on OPCs are still obscure and may reveal novel targets to counteract OL dysfunction in ALS. To this aim, we performed a transcriptomic profiling of primary OPCs exposed to inflammatory (i-EVs) or regenerative (IL4-EVs) microglial EVs, to detect early molecular changes which may be responsible for enhanced OL maturation. Results revealed prominent changes induced by IL4-EVs compared to control, while i-EVs were less potent. Moreover, functional enrichment analysis indicated that significant modifications of bioenergetic pathways localized at the inner mitochondrial membrane underlie the pro-differentiating effects of IL4-EVs on recipient OPCs. Globally, EVs emerge as multimodal mediators able to influence OPCs at myelin lesions, which may be exploited to develop novel approaches restoring OPC maturation in ALS. Supported by AriSLA, grant GPR17ALS-1.