Fig. 2

Hippocampal tau hyperphosphorylation is greatly increased by concurrent beta-amyloid pathology. Western blot and immunofluorescent analysis was used to examine the effects of concurrent Aβ and tau pathology on tau accumulation, phosphorylation, and solubility. Examination of the soluble fraction of microdissected hippocampi (a), revealed an electrophoretic shift in total human tau (HT7) to produce a second band at 65 kDa, likely representing hyperphosphorylation of tau (p = 0.0007), but no significant change in the unphosphorylated band at 60 kDa (p = 0.71). Western blotting for several tau phosphoepitopes revealed similar 60 and 60 kDa bands. In this case, as both bands represent phosphoryalation of that epitope, quantification was performed on the combined bands. While phosphorylation at Ser199 and Ser202 was unchanged between Thy-Tau22 and T5x mice, several other pathological phospho-epitopes including AT100 (p = 0.0002), AT270 (p = 0.0002), and PHF1 (p = 0.003) exhibited robust 2–4 fold increases in T5x hippocampal fractions versus Thy-Tau22 lysates (b). Whereas total levels of insoluble human tau (HT7) and Ser199 and Ser202 epitopes were unchanged between T5x and Thy-Tau22 mice, T5x mice exhibited dramatic 7–10 fold increases of insoluble AT100, AT270, and PHF1 phosphorylated tau (p < 0.0001). Representative immunohistochemical labeling of AT8 (c) and PHF1 (d) immunoreactive tangles in the hippocampus further illustrate the enhancement of tau hyperphosphorylation induced by Aβ pathology in T5x mice. Labeling for the tau conformational epitope MC1 at both low (e) and high (f) power magnification likewise reveals a large increase in MC-1 immunoreactive CA1 neurons (quantification shown in Additional file 2: Figure S2). Data are represented as mean ± SEM, normalized to % of WT group, n ≥ 8 mice/group. * Indicates p < 0.05 for both ANOVA and Fisher’s protected least-significant difference (PLSD) post hoc tests with significance versus all other groups, whereas *over a bar indicates significance between 2 or 3 particular groups. Scale Bar = 100 μm in (c-e) and 50 μm in (f)