Expression profile of mutant tau in Tg mice. (A) Western blot analysis of tau expression in Tg mice (5 months). Equal amount of proteins of TBS-soluble fractions from the cortex (Co), olfactory bulb (Ob), hippocampus (Hp), striatum (St), thalamus and midbrain (Tm), medulla oblongata (Mo), cerebellum (Ce), and spinal cord (Sc) were dephosphorylated, subjected to SDS/PAGE, and probed with Tau-1. An arrowhead indicates the position of the mutant tau. (B) A fluorescent photomicrograph of a sagittally sectioned Tg brain immunostained with the anti-human tau antibody (H-150). Note that mutant tau is robustly expressed in the forebrain regions, but weakly expressed in the cerebellum and brainstem.

Tau filament formation in aged Tg mice. (A) Western blot analysis of Sarkosyl-insoluble tau. TBS-soluble (a), Sarkosyl-soluble (b), and Sarkosyl-insoluble (c) fractions from 22-month-old control (−) and Tg (+) brains were subjected to SDS/PAGE and stained with pan-tau antibody JM. (B) An immunoelectron photomicrograph of Sarkosyl-insoluble tau. Abnormal thin filaments intensely labeled by JM were recovered from Sarkosyl-insoluble fractions of the Tg brain but never from those of the control brain. (Scale bar = 50 μm.)

Impaired associative memory in aged Tg mice. (a) Tg mice (19 months old) showed reduced levels of freezing during cued testing (*, P = 0.0359) conducted 48 h after conditioning. (b) Tg mice showed significantly reduced levels of freezing during contextual testing conducted 15 days after conditioning (15 d; **, P = 0.0072). There was no significant difference in freezing between genotypes during contextual testing conducted 24 h after conditioning (24 h; P = 0.6291).

Formation of tau inclusions in aged Tg mice. (a–h) Immunostaining of the hippocampal region (CA1) of a 19-month-old Tg mouse (a, c, e, and g) and a non-Tg littermate (b, d, f, and h) with monoclonal anti-myc antibody (a and b), PS199 (c and d), AT180 (e and f), and PS404 (g and h). (i) Immunostaining with Alz50. An arrowhead indicates a tangle-like inclusion in a large neuron of the striatum. (j–l) Double immunostaining of the hilar neurons of the Tg hippocampus with JM (k) and monoclonal anti-ubiquitin antibody (j). A merged image is shown in l. (m and n) Photomicrographs of Congo red-stained sections. The images were photographed without (m) or with (n) a polarizing filter set. Arrowheads indicate Congo red birefringent fibrils. (o and p) A neuron of the Tg hippocampus stained with polyclonal anti-tau antibody (JM) (o) was further stained with thioflavin S (p). (q–t) Photomicrographs showing Gallyas silver-stained sections of the hippocampus (q), neocortex (s), and amygdala (t) from the Tg mouse and the hippocampus from the non-Tg littermate (r). (Scale bars = 10 μm.)

Characterization of tau inclusions in aged Tg mice. (A) Western blots comparing the specificity of polyclonal antibodies AR406 and AW406 (19) with the anti-tau antibody JM. AR406 selectively stained mouse wild-type tau, whereas AW406 stained only mutant human tau. (B) Photomicrographs of two serial sections stained with AR406 and AW406. Note that both antibodies stained the same irregularly shaped neurons in the CA1 region of the aged Tg hippocampus. Arrows and asterisks indicate blood vessels. (C–F) Double immunostaining with AR406 and anti-α-tubulin antibodies. Note that irregularly shaped neurons (arrowheads in C; phase contrast) in the aged Tg hippocampus are stained with AR406 (D) but lack tubulin immunoreactivity (E). A merged image is shown in F. (Scale bars in B and C = 10 μm.) (G–I) An ultrastructural analysis of an irregularly shaped neuron in the CA2 of aged Tg hippocampus. Arrowheads in G indicate the somatodendritic part of an irregularly shaped neuron. N, nucleus. (Scale bar = 20 μm.) Boxed images in G are enlarged in H (large box) and I (small box). Note that the dendrite of a degenerating neuron was electron-dense, filled with organelles (H) and sometimes with bundles of thin filaments (white arrowheads in I). Negatively stained tau filaments (black arrowheads) from the Sarkosyl-insoluble fraction used in Fig. 3 were scale-adjusted and shown in the inset in I. Ly, lysosome; Rb, ribosome; Mito, mitochondria; MT, microtubules. (Scale bars in H and I =100 nm.)