Functional retrieval of exosomal cargo by neurons.

<p>(A–G) Boyden chamber co-culture of pOL transduced by recombinant AAV/MBP-Cre (pOL-MBP-Cre) and CN transduced by AAV/CBA-floxstop-hrGFP reporter vector. hrGFP expression indicates Cre-mediated recombination. Tuj1 staining (red) of CN exposed to (A) nontransduced pOL, (B) pOL-MBP-Cre, or (C) pOL-MBP-Cre treated with 5 µM GW4869. Scale bar, 100 µm. (D–G) Reporter gene expression (hrGFP) in target neurons detected by Western blotting of neuronal lysates. CN were exposed to (D) pOL-MBP-Cre treated or not with 100 µM glutamate, (E) transfected with Rab35 or control siRNA, or (F) treated or not with 5 µM GW4869. (F, right lane) Exposure of CN to exosome-depleted culture supernatant from pOL-MBP-Cre does not lead to reporter gene expression in neurons. (G) pOL, Oli-neu cells, or HEK cells transduced with AAV/MBP-Cre co-cultured with CN. (H) Cre RT-PCR and (I) Western blot of exosomes derived from pOL-MBP-Cre or control cells. Pgk1 (mRNA), PLP/DM20, and Alix (protein) are shown as standards. (J–L) Stereotactic injection of exosomes derived from pOL-MBP-Cre into the (J, K) hippocampus (HC) and (L) cerebellum of ROSA26-lacZ reporter mice. β-galactosidase positive cells were stained using X-gal and neurons were stained for (K) neuron-specific enolase (NSE) or for (L) GABA-R_α6. Mice (<i>n</i> = 5) were analyzed 14 d after injection.