Fig. 5

Trem2 deletion promotes tau distribution to endosomal and pre-exosomal compartments, and promotes exosomal tau release. A Trem2 deletion enhances endosomal tau sorting to pre-exosomal compartments. WT or Trem2 KO microglia were incubated with 2.5 μg/ml tau oligomers for 24 h, and stained for endosomal (Rab5, Rab7), lysosomal (LAMP1), and markers for pre-exosomal vesicles (CD63, Tsg101) (red), and co-localization with human tau (green) was visualized by confocal microscopy. Region of overlap between intracellular markers (red) and tau (green) were determined as a proportion of total internalized tau in microglia the graphs shown (mean ± SE, where individual plots represent one cell from 3 independent experiments). Bar = 5 μm. Statistical significance was determined by unpaired Student’s t-test (***p < 0.001). B Experimental schematic: WT or Trem2 KO microglia were loaded with 2.5 μg/ml tau oligomers for 24 h, washed with PBS and treated with 10 μM GW4869 for 3 h. Microglia were then treated with LPS/ATP and conditioned media was collected and subjected to tau quantification (ELISA) or neuronal tau uptake. C Exosomes were purified from conditioned media from tau-loaded WT or Trem2 KO microglia in the presence (+inhib) or absence (con) of GW4869, and subjected to ELISA analysis to quantify exosomal tau released with LPS/ATP in conditioned media. D-F Quantifying tau uptake from microglia conditioned media in primary neurons. Primary WT mouse neurons were incubated for 48 h with conditioned media from tau-loaded WT or Trem2 KO microglia untreated (D) or treated with GW4869 (E), and tau uptake was observed by confocal microscopy in cells stained for tau (T13, green) and tubulin (red); nuclei were visualized by DAPI staining (blue). Bar = 20 μm. F Quantification of tau puncta in neurons (left graph, tau puncta/neuron) or tau staining intensity (right graph, arbitrary units). Graphs represent mean ± SE, statistical significance was determined by One-way ANOVA with Tukey’s multiple comparison (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). G Electron micrograph of exosomes prepared from WT or Trem2 KO microglia. Bar = 100 nm. H, I Trem2 deletion does not affect exosomal size. Exosomes purified from WT or Trem2 KO microglia were plotted for exosomal vesicle size (H), as well as vesicle size distribution (I). Mean and quartiles are shown in violin plots in (H), graph in (I) represents mean ± SE