Fig 2: Anti-HA immunolabelling in His6-HA-SUMO1 KI neurons permeabilised with 20 µg/ml digitonin is mainly nuclear.Hippocampal neurons from either His6-HA-SUMO1 KI mice or SUMO1 KO mice were fixed in 4% PFA and permeabilised using 20 µg/ml digitonin for 20 min, then blocked with 10% horse serum for 20 min. Primary antibodies were 1:1000 mouse anti-HA (green), 1:2000 rabbit anti-synapsin 1/2 (red), and 1:500 chicken anti-Map2 (blue). In His6-HA-SUMO1 KI cells, anti-HA labelling was primarily observed in nuclei. Faint anti-HA labelling and occasional puncta were also observed, which did not generally correspond with synapsin-positive puncta (arrowheads). Data representative of 2 independent experiments. Scale bars, 10 µm.DOI: http://dx.doi.org/10.7554/eLife.26338.011

Fig 3: Astrocytic hApoEe4 potentiates MC1 pathology produced by tau oligomers. Scale bar of representative images represents 50 µM. High-content imaging and associated quantification represents data from one independent experiment. (a) Immunocytochemistry of co-cultures five days after addition with 100 nM of tau monomers or tau oligomers. Oligomers were added five days after transduction with the PGFAP construct of interest. Cells were fixed and stained with MC1 to measure tau pathology and MAP2 to label neurons. Hoechst stain was used to label nuclei. Arrowheads highlight examples of MC1-positive cell bodies and neurites. Top panel depicts images without MAP2 imaging overlay to aid in visualization. (b) High-content imaging to quantify MAP2-positive Hoechst-labeled nuclei (n = 4 wells/group). (c,d) Quantification of MC1 intensity (c) and MC1 area (d) in MAP2-labeled cell bodies (n = 4 wells/group). Data were normalized to the oligomer-treated PGFAP::EV group (n = 4 wells/group). NS not significant, *p < 0.05, **p < 0.01, Tukey multiple comparisons test following one-way ANOVA.

Fig 4: Measuring synaptic content from excitatory and inhibitory synapses using CellProfiler. A, Multiplexed images of PRISM stained DIV21 hippocampal neurons are used as input for the CellProfiler pipeline. Raw images (top) are aligned using MAP2 signal. A white-top filter is applied to enhance puncta (middle). Puncta are thresholded and separated into individual puncta using peak intensity (bottom). B, Synapses are labeled excitatory (E) or inhibitory (I) using the presence or absence of excitatory-specific (vGlut1) or inhibitory-specific (vGAT) markers. Synapses with both markers are labeled as dual (D). Clusters that do not contain either marker are labeled as unknown (U). C, Representative image (top) and enlarged dendrite (bottom) from a MAP2-stained DIV21 hippocampal neuron with excitatory (red) and inhibitory (green) synapses labeled as colored circles. The size of the colored circle represents the relative synapsin1 area. Scale bar: 1 µm.

Fig 5: Dendritic alterations correlate with anxiety-like behaviors 52 weeks following blast exposure. Pearson’s correlation analysis between area fraction of MAP2 in the hippocampus and time spent in the enclosed arm of the EPM arena (A–D). (A) No significant correlation was found between area fraction of MAP2 in the DG and time spent in the enclosed arm (p = 0.79, r = -0.06). (B) A significant negative correlation between area fraction of MAP2 in the CA1 and duration in the enclosed arm (p = 0.007, r = -0.6). (C) No significant correlation was found between area fraction of MAP2 in the CA2 and duration in the enclosed arm (p = 0.55, r = -0.14). (D) Correlation between area fraction of MAP2 in the CA3 and time spent in the enclosed arm was found to be significant (p = 0.04 r = -0.5). Overall regression lines are black with 95% confidence intervals denoted with black dotted lines.