Fig. 4: Increased synaptic engulfment by astrocytes in PSP SN and frontal cortex demonstrated using array tomography and immunogold electron microscopy.

a–d, A single 70 nm segmented array tomography section with orthogonal views of the image stack (a,b), and 3D reconstructions (c,d) of PSP SN (a,c) and frontal cortex (b,d) immunostained for presynapses (SYO, cyan), tau (AT8, magenta) and astrocytes (GFAP, gray). Arrows and insets show colocalization between synapses (SYO) and astrocytes (GFAP). Large boxes = 50 μm × 50 μm, scale bar = 10 μm. Small boxes = 5 μm × 5 μm. e,f, Array tomography image stacks were taken from PSP (n = 5 donors SN, 7 frontal cortex) and control (n = 6 SN, 7 frontal cortex). Two to three image stacks per region per case were taken as technical replicates. Boxplots show the first quartile to third quartile (box) with the median marked, and whiskers show maximum and minimum values calculated from each image stack, excluding outliers. Data points show case means (biological replicates). Data were analyzed with LMEMs (~diagnosis × brain region + 1 | case) with post hoc pairwise Tukey-corrected comparisons. Quantification reveals that in PSP SN and frontal cortex, there is astrogliosis (e, SN estimate = 0.334, 95% CI (0.039, 0.629), t(16.6) = 2.393, P = 0.0289; frontal cortex estimate = 0, 95% CI (0.0316, 0.601), t(14.1) = 2.382, P = 0.0319, square root transformed data LMEM ~ diagnosis × brain_region + 1 | case). Further in PSP, there is increased colocalization between presynapses (SYO) and astrocytes (GFAP; f, model run on Tukey transformed data, SN estimate = 0.360, 95% CI (0.116, 0.60), t(26.8) = 3.029, P = 0.0054, frontal cortex estimate = 0.243, 95% CI (0.026, 0.459), t(23.2) = 2.320, P = 0.0295). g–i, Immunogold electron microscopy of human postmortem PSP frontal cortex with anti-SYO antibody labeled with 10 nm gold particles. Astrocytes were identified by ultrastructure (g) and often included lysosomes (ly). A higher magnification image of the astrocytic cytoplasm in the human brain (h) shows phagocytosed debris, including membranous structures abutting lysosomes. Higher magnification image (i) shows synaptic vesicle-containing structures (arrows) labeled for SYO with 10 nm gold in the astrocytic cytoplasm and gold particles in the lysosome (arrowheads). Scale bars = 3 µm (g), 500 nm (h) and 200 nm (i).