Figure 1.
Comparison between single transgenic mice, double transgenic mice, and littermate controls.
Representative photographs of 12 months old wild-type (A), Tg-Tau (B), Tg-FDD-Tau (C), and Tg-FDD (D) showing clasping of the hindlimb and bilaterally pulling of the hind paws toward the abdomen when suspended by the tail. Performance of wild-type (WT, n = 7) and Tg-FDD-Tau (2x-Tg, n = 9) animals on an accelerating rotating rod apparatus at 6 months of age (E). No significant differences in performance were observed between Tg-FDD-Tau mice compared with age-matched WT animals. No differences in daily performances were observed in females (f) (F) and males (m) (G) WT and Tg-FDD-Tau animals.
Figure 2.
BRI2 expression and processing in transgenic mice.
Schematic diagram of the Danish amyloid precursor protein (ADanPP) (A). BRI2 is a type-II single trans-membrane (TM) domain protein. Processing of ADanPP by pro-protein convertases (PCs) generates the 34 amino acid peptide (ADan) and a mature form of BRI2 (m-BRI2). Processing by ADAM10 in the ectodomain of BRI2 releases the BRICHOS domain and an N-terminal fragment (NTF). The NTF is also the subject of additional proteolysis by SPPL2, releasing an intracellular domain (ICD) and a C-terminal peptide fragment (BRI2 C-peptide) [18]. Disulfide bonded loops in the BRICHOS domain and in the carboxy-terminus of BRI2 (amino acids 5 and 22 of the ADan peptide) are indicated. The figure shows the localization of the Abs used. Western blot analysis of PNS from neocortex of Tg-FDD-Tau (2x, n = 6) mice, Tg-FDD (FDD, n-6) mice, and wild-type (WT, n = 6) control mice using the BRI2-amino-terminal antibody 14307 (B). The ectodomain processing of the FDD mutant form of BRI2 by PCs seems to be compromised in FDD. In Tg-FDD mice, two bands can be observed corresponding to full-length ADanPP and m-BRI2, while in WT mice most of the detectable BRI2 protein can be seen as m-BRI2. Samples were run in triplicates. Representative samples are shown. The densitometric values of the bands representing ADanPP and m-BRI2 immunoreactivity were normalized to the values of the corresponding actin band using ImageJ software. No significant differences were observed between Tg-FDD-Tau and Tg-FDD mice (independent t test).
Figure 3.
Amyloid deposition and BRI2 accumulation in transgenic mice.
Amyloid deposition is seen throughout all cortical layers (A), the hippocampal formation (B), and in leptomeningeal vessels of the cerebellum (C) of Tg-FDD-Tau mice. Antibodies against ADan immunolabeled cortical blood vessels (D) and amyloid deposits in the hippocampus (E). Amyloid plaques in Tg-FDD mice are surrounded by globular dystrophic neurites (DNs) labeled by the BRI2-amino-terminal Ab 14307 in the neocortex (F) and hippocampus (G, H). The Ab also labeled intracellular deposits and swollen neurites. Arrows indicate the presence of ADan amyloid plaques. Sections were from a 12 month old (A–E) Tg-FDD-Tau mice and a 21 month old Tg-FDD mouse (F–H). Thioflavine S (A–C). Immunohistochemistry using Abs 1699/1700 (D, E) and Ab 14307 (F–H). Scale bars: A–C, F, 100 µm; G, 50 µm; H, 25 µm.
Figure 4.
Tau deposition and inflammatory changes in double transgenic mice.
Reactive astrocytes in the hippocampus (A) and neocortex (B), and keratan sulfate-positive-activated microglia in the hippocampus (C). The phosphorylation-dependent anti-tau Ab AT8 immunolabeled tau deposits in the hippocampus (D, E), and the neocortex (F). Sections were from 6 (F), 10 (B), and 12 month old (A, C, D, E) Tg-FDD-Tau mice. Immunohistochemistry using anti-GFAP (A, B), anti-keratan sulfate (C), and Ab AT8 (D–F). Scale bars: A–F, 50 µm.
Figure 5.
Enhanced AT8 immunopositive neuronal perikarya in Tg-FDD-Tau mice.
Staining of a section of the neocortex of a 6 month old Tg-Tau mouse (A) and a Tg-FDD-Tau (B) mouse. A statistically significant difference in the number of AT8-positive neuronal perikarya is observed between Tg-FDD-Tau (2x) mice and single Tg-Tau (Tau) mice (C) (****P<0.0001, two tail t test). Immunohistochemistry using Ab AT8 (A, B). Scale bars: A, B, 50 µm.
Figure 6.
Enhanced Tau-C3-positive neuronal perikarya in Tg-FDD-Tau mice.
Analysis of caspase-truncated tau at D421. Serial sections of the neocortex of Tg-FDD-Tau (A, B) and single Tg-Tau (C, D) mice were analyzed at 6 (A, C) and 11 (B, D) months of age. A statistically significant difference in the number of Tau-C3-positive neuronal perikarya is observed between Tg-FDD-Tau (2x) mice and single Tg-Tau (Tau) mice at 6 (E) and 11 (F) months of age. The number of Tau-C3-positive neuronal perikarya in Tg-FDD-Tau mice increased with statistical significance between 6 and 11 months of age (G). (****P<0.0001, two tail t test). Representative immunoblots showing pro-caspase-3 levels in PNS from 11 months old wild-type (WT, n = 6), Tg-Tau (Tau, n = 6), Tg-FDD-Tau (2x, n = 6), and Tg-FDD (FDD, n = 6) mice (H). Samples were run in triplicates. No significant differences in the levels of pro-caspase-3 was observed between the four groups. β-actin was used to normalize protein loading. Optical densities of the individual bands were quantified using NIH ImageJ. Statistical analyses were performed with GraphPad Prism 5.04. Immunohistochemistry using Ab Tau-C3 (A–D). Scale bars: A–D, 50 µm.
Figure 7.
Biochemical analysis of tau in transgenic mice.
Representative immunoblots of tris-soluble and sarkosyl-insoluble fractions from 9 month old Tg-FDD-Tau (2x) and Tg-Tau (Tau) (four to five mice per group were analyzed). Samples were run on SDS-PAGE and immunoblotted with anti-tau Abs d29, AT8, AT100 and Tau-C3. β-actin was used to normalize protein loading.
Figure 8.
Biochemical analysis of synaptophysin levels in transgenic mice.
Representative immunoblots and densitometry analysis showing synaptophysin and PSD-95 levels in PNS from wild-type (WT, n = 6), Tg-FDD (FDD, n = 6), Tg-Tau (Tau, n = 6), and Tg-FDD-Tau (2x, n = 6) mice. Samples were run in triplicates. No significant changes were observed at 3 months of age in synaptophysin levels between Tg-FDD-Tau and WT mice (A). A significant decrease in synaptophysin levels in Tg-FDD-Tau mice is observed at 6 months of age (B). At 9 months of age, synaptophysin levels are also decreased in single transgenic mice, particularly in Tg-Tau mice (C). A short and an extended (long) exposure of the film are shown. β-actin was used to normalize protein loading. Optical densities of the individual bands were quantified using NIH ImageJ. Statistical analyses were performed with GraphPad Prism 5.04. (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001, two tail t test).