Fig. 5.
Exosomal PRR7 is sufficient for the removal of excitatory synapses. a Representative immunofluorescent images of PSD-95/vGLUT and vGAT/GABAARγ2 in hippocampal neurons treated with CS harvested from GFP control-transfected or HA-PRR7-transfected neurons. b Quantitation of the CS effect on excitatory and inhibitory synapses. c, d Quantitation of the CS effects on PSD-95 harvested from GFP-transfected, HA-PRR7-transfected or PRR7-GFP-transfected neurons. Cluster density (c) and intensity (d). n = 12 per condition. e Exosomal secretion of various PRR7 mutants. CB Coomassie blue staining. f Reversal of PSD-95 clusters by replacing PRR7-overexpressing CS with normal conditioned medium at 12 h. g Time course of the effect of HA-PRR7-exosomes on PSD-95. Top panels show representative images and quantified data are shown bottom. n = 15 each. h–j Uptake of exosomal PRR7 by neurons. Representative images of hippocampal neurons treated with exosomes purified from either GFP-transfected (control) or HA-PRR7-transfected neuron culture. Total staining of HA and PSD-95 in recipient neurons after the spot application of exosomes (h). Total and surface staining of HA and MAP2 after the application of evenly mixed HA-PRR7-containing exosomes (i). Western blotting analyses of TCLs prepared from Txf and exosome-treated recipient (Rec.) neurons (j). Scale bars, 5 (a), 10 (g), and 20 μm (h, i). All data are mean ± SEM. Two-way (b) or one-way ANOVA (c, d) Post hoc Tukey’s test, F1,56 = 30.71 (b), F2,33 = 78.78 (c), and F2,33 = 14.66 (d). ***P < 0.0001. Mr relative molecular weight