Fig. 3

Representative confocal microscopy images of 21DIV primary hippocampal neurons inoculated with AD-tau samples at 7DIV for 14 days. A All six AD-tau inoculates (AD1-6) trigger misfolding/aggregation of endogenous mouse tau to various degrees with different fluorescent intensities and sizes of the fluorescence signal (red), MAP2 (somatodendritic marker, cyan) is present to some extend in mouse tau-positive aggregates. The pattern of tau aggregation is different in an inoculum-to-inoculum manner. Squares of 4 × 4 µm were applied as regions of interest in areas of profoundly aggregated tau and the plot profiles for mouse tau (red) and MAP2 (cyan) are displayed. B, C Violin plots of maximum grey values from plot profiles of 4 × 4 µm areas for aggregated mouse tau (B) and MAP2-positive signal in areas with aggregated tau (C), mean ± SEM. One-way ANOVA showed statistical significance in the maximum intensity of mouse tau aggregates and MAP2 fluorescence present in these aggregates’ areas across all six inoculum-treated neurons (*** p < 0.0001). Bonferroni test in the graph (B) confirmed significant variability among all six inocula to trigger tau aggregation (*** p < 0.0001, except AD1 vs AD6: *p = 0.0163, AD2 vs AD6: *p = 0.0083). The occurrence of MAP2 in tau aggregates greatly varied among most of the AD inocula (p < 0.0001, Bonferroni test), except among samples AD1 vs. AD3 (p = 0.768), AD1 vs. AD4 (p = 0.361), AD3 vs. AD4 (p = 1), and AD2 vs. AD6 (p = 1). The max intensity values of plot profiles of 4 × 4 µm ROIs were averaged from two independent experiments (ROIs, n = 36 per inoculum). D Images of neuronal cultures inoculated for 14 days with AD1-tau sample of three different concentrations (5, 15, and 45 ng/well) show that the degree of mouse tau aggregation is inoculum concentration-dependent. Graph representing x-fold increase of mouse tau aggregates as number of particles (0.1-infinite µm size) to untreated cells (medium only, 0 ng of AD1-tau). One-way ANOVA did not show statistical significance (p = 0.343) due to high variability, mean ± SEM. Values were collected from confocal images of two independent experiments (n = 15). All images are presented as maximum intensities of 35 z-stacks (0.35 µm each), scale bars: 50 µm of large images, and 20 µm of cropped areas. The staining for aggregated tau was performed after ice-cold methanol fixation