Figure 2.

Neuroprotection mediated by exosomes. (a) pCN exposed to pOL exosomes in Boyden chamber co-cultures and subjected to OGD compared to control conditions (pCN ctrl, pOL-conditioned medium depleted of exosomes). After OGD followed by reoxygenation, neuronal metabolic activity was determined by MTT assay. The relative metabolic activity correlates OGD co-cultures to respective cultures grown under normoxic conditions. Error bars, s.e.m. (***p < 0.001; n = 5, Students t-test). (b–d) Western blot analysis of pCN lysates after co-culture with Oli-neu cells (ON) or pOL. (b) Presence of hSOD1-EGFP in isolated exosomes (left) and transfer of hSOD1-EGFP from transfected ON cells to pCN (right). Control pCN were co-cultured with untransfected ON. Alix and Hsp70 identify exosomes. (c) Western blotting of isolated pOL-derived exosomes demonstrates presence of catalase (Cat, left). PLP/DM20 serves as an oligodendrocyte-specific exosome marker and calnexin (CNX) as a contamination marker. Catalase levels determined after co-culture of pCN with pOL (right). Detection of PLP/DM20 in pCN indicates exosome transfer. (d) Quantification of catalase normalized to tubulin (Tub) as derived from Western blot signals in (c) (right). Error bars, s.e.m. (**p < 0.01; Wilcoxon test, n = 10). (e) Images of pCN co-cultured with pOL (right panel), which were stained with PKH67 to label pOL-derived exosomes (green). Control pCN were cultured in absence of pOL (left panel). pCN were immuno-stained for catalase (red) and the neuronal marker Tuj1 (blue). Scale bar, 20 µm.