After overnight incubation with primary antibodies, cells were washed with PBS and incubated with secondary antibodies (Alexa Fluor 555Cconjugated goat anti-mouse IgG (1:2,000) and Alexa Fluor 488Cconjugated goat anti-rabbit IgG (1:2,000); Invitrogen) for 2 h at room temperature. 1) internalization and degradation of AOs; 2) release of extracellular vesicles containing active catalase; and 3) selective secretion of interleukin-6, interleukin-10, and vascular endothelial growth factor to the medium. Results support the notion that MSCs may represent a promising alternative for cell-based therapies in AD. AD models (9,C12). For example, Lee (13, 14) showed that transplantation of bone marrow MSCs into the hippocampus of the APP/PS1 mouse model of AD reduced A deposition and Tau hyperphosphorylation and reversed learning and spatial memory deficits. However, the mechanisms underlying those neuroprotective actions of MSCs have not been elucidated. It is generally accepted that MSCs do not exert their beneficial actions through direct differentiation into neural tissue, but rather by acting as NCT-502 trophic mediators releasing immune modulatory, proangiogenic, NCT-502 and/or proneurogenic factors (15). Additional mechanisms involved in paracrine signaling promoted by MSCs include the secretion of specific cytokines (16) and the transfer of extracellular vesicles (EVs) or even of healthy mitochondria to cells with impaired mitochondrial function (17,C19). Here, we aimed to investigate the neuroprotective potential of MSCs in an model of AD, to gain insight into possible mechanisms of cell-to-cell communication (20) that could be exploited in future therapeutic approaches. We demonstrate that MSCs and MSC-derived EVs block oxidative stress and synapse damage induced by AOs in hippocampal neurons and unveil novel neuroprotective mechanisms of action of MSCs, namely the clearance of extracellular AOs, selective secretion of cytokines, and the release of active catalase via EVs. Results MSCs are resistant to AOs We initially evaluated the effects of exposure of MSCs to AOs (500 nm) by investigating cell viability (Fig. 1, and (= 3 independent cultures, with triplicate wells in each experimental condition). and = 3 independent cultures, with triplicate coverslips in each experimental condition). (= 6 independent cultures, with triplicate coverslips in each experimental condition). = 3 independent cultures). In all graphs, data are represented as NCT-502 means S.E. (see Ref. 24), there was no indication of neuronal death under our experimental conditions (500 nm AOs, 6 h of exposure) either in the absence or in the presence of MSCs, as revealed by inspection of bright field images corresponding to each DCF fluorescence image (Fig. 2). Open in a separate window Figure 2. Oxidative stress in hippocampal neurons exposed to AOs in the absence or presence of MSCs. Photomicrographs showing DCF fluorescence (each fluorescence image. show integrated fluorescence for AO-exposed neurons (= 6 NCT-502 independent cultures, with triplicate coverslips in each experimental condition); *, < 0.05; two-way ANOVA followed by Tukey's post hoc test; = 4 independent cultures, with triplicate coverslips in each experimental condition); *, < 0.05; two-way ANOVA followed by Dunnett's post hoc test; to detect Igf1 elevated levels of AOs in AD brain extracts) (26, 27). Results showed a time-dependent reduction in AO immunoreactivity in the culture medium of MSCs, suggesting that AOs were internalized (Fig. 4and and or and and in indicate AO-containing roughly globular cytoplasmic structures in MSCs, suggesting that AOs are located within an intracellular compartment after 3 h of exposure (indicate orthogonal sections of confocal imaged cells. shows that fluorescent HyLite AOs (prepared using HyLite A, as described under Methods) were bound NCT-502 to the surface of MSCs after 20 min of exposure and remained bound 3 h after exchange of the medium containing oligomers for fresh medium (Fig. 4in the representative image shown in Fig. 4and and and = 2 independent cultures, with triplicate wells per experimental condition). *, < 0.05; #, < 0.05; one-way ANOVA followed by Tukey's post hoc test. To establish whether the release of IL-6, IL-10, and VEGF to the culture medium was mechanistically connected to the protection of neurons against oxidative stress induced by AOs, we performed experiments in which the three cytokines were blocked by neutralizing antibodies added to the cultured medium. To this end, we cocultured MSCs with neurons for 24 h.