In CBD, the characteristic astrocytic pathology (e.g. astrocytic plaques) showed extensive co-localization between TNT1, TOC1 and R1 in the frontal cortex (Fig. 5I–L).
Similarly, the characteristic Pick bodies in the frontal cortex were well labeled by TNT1, TOC1 and R1 in PiD tissue (Fig. 5M–P)
Right angle laser light scattering showed significantly greater scattered light intensity in all 4R tau isoforms when compared to 3R isoforms (one-way ANOVA with Holm-Sidak post-hoc, F(5, 18) = 60.22, p < 0.0001; Fig. 2A)
Similar results were seen in the ThS assay, where the 4R isoforms were significantly higher than 3R isoforms (one-way ANOVA with Holm-Sidak post-hoc, F(5, 18) = 19.99, p < 0.0001; Fig. 2B), and no differences were found in comparisons between the individual 4R isoforms or between the 3R isoforms
In contrast, 3R isoforms were primarily composed of globular oligomers and only very rare long filaments were found (Fig. 2F–H)
The band patterns in the immunoblots showed that the AD cases contained a mixture of isoforms, the PiD cases clearly contained 3R isoforms but also some 4R isoforms, while the vast majority of pathology in CBD cases were comprised of 4R tau isoforms
A mixture of long, intermediate and short filaments, as well as globular oligomers were present in 4R isoform reactions (Fig. 2C–E)
Indeed, early pre-tangle neurons within the hippocampus were labeled with all antibodies in Braak I-II cases (Fig. 5A–D)
Similarly, the soluble fraction from AD contained the greatest level of TOC1 reactivity, followed by CBD and then PiD had the lowest signal (Fig. 6D; one-way ANOVA with Holm-Sidak post-hoc, F(2,9) = 16.57, p = 0.001)
TOC1 detected significantly more oligomeric tau in AD compared to CBD and PiD and more in CBD compared to PiD (Fig. 6G; one-way ANOVA with Holm-Sidak post-hoc, F(2,9) = 35.32, p < 0.0001)
In contrast, AD soluble tau displayed the highest level of TNT1 followed by CBD, with PiD having the lowest levels (Fig. 6C; one-way ANOVA with Holm-Sidak post-hoc, F(2,9) = 24.87, p = 0.0002).
Total tau levels in the soluble fractions were similar for AD, CBD and PiD, as indicated by the Tau5 sandwich ELISA (Fig. 6B; one-way ANOVA, F(2,9) = 3.283, p = 0.085)
Total tau levels in the insoluble fractions, as detected by Tau5, were highest in AD, followed by CBD and PiD contained the least (Fig. 6E; one-way ANOVA with Holm-Sidak post-hoc, F(2,9) = 25.93, p = 0.0002)
TNT1 detected significantly more PAD exposed tau in AD compared to PiD, and more in CBD when compared to PiD, but AD and CBD were not different (Fig. 6F; one-way ANOVA with Holm-Sidak post-hoc, F(2,9) = 12.07, p = 0.0028)
Collectively, these studies indicate that inhibition of anterograde FAT represents a toxic effect common to all tau aggregates, regardless of isoform composition
As expected, monomer and aggregated samples of all six tau isoforms showed equal reactivity for TNT1 and TOC1 when the samples were denatured because this exposes the epitopes making them equally accessible (Student’s t-tests, for all comparisons p > 0.05; Fig. 3C–H)
Comparisons between isoform monomers showed that hT39 monomer signal was significantly higher than hT24 and hT23 monomers (Kruskal-Wallis ANOVA with Dunn’s post-hoc, H = 18.4, p = 0.0025)
Comparisons between isoform monomers showed that hT39 monomer signal was significantly higher than hT24 and hT23 monomers (Kruskal-Wallis ANOVA with Dunn’s post-hoc, H = 18.6, p = 0.0023)
hT40 showed the highest amount of light scattering compared to other 4R isoforms, and there were no differences between the different 3R isoforms
In severe AD cases (i.e. Braak stage V-VI), all markers continue to colocalize in classic NFTs within the hippocampus that characterize AD tau pathology (Fig. 5E–H)
In general, the remarkable co-localization between TNT1, TOC1 and R1 in all tauopathies confirms that PAD exposure and tau oligomerization occur simultaneously in cells displaying tau pathology, irrespective of isoform composition
The hT24 aggregates showed the highest TNT1 signal, which reached significance compared to hT40, hT39, hT37 and hT23 aggregates (one-way ANOVA with Holm-Sidak post-hoc, F(5, 18) = 19.11, p < 0.0001)
Aggregates of all six tau isoforms showed significant increases in TNT1 reactivity when compared to their respective monomer samples (Fig. 3A; Mann-Whitney test, for all comparisons p = 0.029)
The hT24 aggregates showed the highest TOC1 signal, which reached significance compared to hT40, hT39, hT37 and hT23 aggregates, while hT34 aggregates were significantly different from hT39, hT37 and hT23 aggregates, and both hT40 and hT39 aggregates are significantly higher than hT37 and hT23 (one-way ANOVA with Holm-Sidak post-hoc, F(5, 18) = 50.77, p < 0.0001)
Aggregated samples for all six isoforms showed significant increases in TOC1 reactivity when compared to their respective monomer samples (Fig. 3B; Mann-Whitney tests, for all comparisons p = 0.029)
Similarly, perfusion of squid axoplasms with hT39, hT37 and hT23 aggregates significantly impaired anterograde FAT (Fig. 4A) when compared to the respective monomers (all at 2 μM)
hT40, hT34, hT24, hT37 and hT23 aggregates did not significantly impair retrograde FAT when compared to the respective monomers, but hT39 aggregates elicited a mild inhibitory effect on retrograde FAT (Fig. 4B)
Pairwise comparisons within tau species showed that hT24 aggregates produced significantly more inhibition of anterograde FAT when compared to hT34 and hT39 aggregates.
Perfusion of hT40, hT34 and hT24 aggregates into squid axoplasms significantly impaired anterograde transport (Fig. 4A) when compared to the respective monomers (all at 2 μM).
Aggregation-induced increases in PAD exposure and oligomerization are common features among all tau isoforms. The extent of PAD exposure and oligomerization was larger for tau aggregates composed of 4-repeat isoforms compared with those made of 3-repeat isoforms.
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.