Fig. 4

Activation of microglia treated with neuron extracellular vesicles. Extracellular vesicles (EVs) were isolated from primary cortical neurons by centrifugation. To determine EV counts, samples were diluted and analyzed for size and concentration using NanoSight imaging. A Representative still frame image from NanoSight acquisition is shown on left. Intensity-size scatter plot and size histogram are shown on right. B, C Mouse cortical neurons were infected at plating with AAV1.Syn.shISG15 to silence neuronal ISG15 expression or AAV1.Syn.EGFP control and at 12 DIV treated with IFNγ for 72 h prior to EV isolation. Micro-RNA was isolated from EVs, and small RNA libraries were prepared and acquired by paired end 100 bp RNA sequencing. Differentially expressed miRNA species were determined using a 5% FDR cut off. MicroRNAs upregulated (B) and downregulated (C) upon ISG15 knockdown are shown. D Primary microglial cultures were prepared from p1 mouse pups. E Primary astrocyte cultures were prepared from p3–4 mouse pups. Astrocytes (DIV29) and microglia (DIV7) were treated with EVs from neurons that were infected at plating with AAV1.Syn.ISG15 or AAV1.Syn.EGFP. RNA was isolated from astrocytes and microglia 24 h after EV treatment and expression of the indicated transcripts determined by RT-PCR relative to cells treated for 24 h with control EVs. **P < 0.01 by unpaired Student’s t-test. Mean ± SEM are shown