A novel triple repeat mutant tau transgenic model that mimics aspects of pick's disease and fronto-temporal tauopathies

Abstract

Tauopathies are a group of disorders leading to cognitive and behavioral impairment in the aging population. While four-repeat (4R) Tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and Alzheimer's disease, three-repeat (3R) Tau is the most abundant splice, in Pick's disease. A number of transgenic models expressing wild-type and mutant forms of the 4R Tau have been developed. However, few models of three-repeat Tau are available. A transgenic mouse model expressing three-repeat Tau was developed bearing the mutations associated with familial forms of Pick's disease (L266V and G272V mutations). Two lines expressing high (Line 13) and low (Line 2) levels of the three-repeat mutant Tau were analyzed. By Western blot, using antibodies specific to three-repeat Tau, Line 13 expressed 5-times more Tau than Line 2. The Tau expressed by these mice was most abundant in the frontal-temporal cortex and limbic system and was phosphorylated at residues detected by the PHF-1, AT8, CP9 and CP13 antibodies. The higher-expressing mice displayed hyperactivity, memory deficits in the water maze and alterations in the round beam. The behavioral deficits started at 6-8 months of age and were associated with a progressive increase in the accumulation of 3R Tau. By immunocytochemistry, mice from Line 13 displayed extensive accumulation of 3R Tau in neuronal cells bodies in the pyramidal neurons of the neocortex, CA1-3 regions, and dentate gyrus of the hippocampus. Aggregates in the granular cells had a globus appearance and mimic Pick's-like inclusions. There were abundant dystrophic neurites, astrogliosis and synapto-dendritic damage in the neocortex and hippocampus of the higher expresser line. The hippocampal lesions were moderately argyrophilic and Thioflavin-S negative. By electron microscopy, discrete straight filament aggregates were detected in some neurons in the hippocampus. This model holds promise for better understanding the natural history and progression of 3R tauopathies and their relationship with mitochondrial alterations and might be suitable for therapeutical testing.

Fig 1. Generation of tg mice expressing the mutant 3R Tau under the mThy-1 promoter.

A. Diagram of the mThy-1 construct with the mutant 3R Tau. B. Levels of human 3R Tau and C murine Tau mRNA expressed as a ratio to the housekeeping gene GAPDH in non-tg and 3R Tau Lines 2, and 13. D. Representative Western blot (SDS) and analysis of the levels of E Total Tau and F 3R Tau with the insoluble (membrane) fractions showing that Line 13 was a higher expresser of total Tau and 3R Tau compared to Line 2 and non-tg mice. Across all lines Tau was detected as double bands between 50–60 kDa. In the Pick’s Disease cases 3R Tau co-migrated at a similar level as in the 3R Tau tg mice. For analysis, N = 6 non-tg and N = 6 mThy-1 3R Tau tg mice (3–4 months old) from each line were utilized. * = P < 0.05 when compared to non-tg control using one way ANOVA with Dunnett’s posthoc test. # = P < 0.05 when comparing Line 2 and 13 using one way ANOVA with Fisher’s posthoc test.

Fig 2. Effects of aging on biochemical alterations in the higher expresser mutant 3R Tau tg mice.

A Representative Western blot (SDS) and analysis of levels of B 3R Tau, C Total Tau, and D PHF-1. Across all antibodies there was a significant increase in the amount of protein at the older time points (12–14 months of age) compared to the earliest time point (3–4 months of age) and non-tg mice. E. Levels of 3R Tau mRNA levels at 3–4, 6–8, and 12–14 months of age were uniformly increased compared to non-tg mice. N = 8 for each age group * = P < 0.05 when compared to non-tg control using one way ANOVA with Dunnett’s posthoc test. # = P < 0.05 when comparing Line 2 and 13 using one way ANOVA with Fisher’s post hoc test.

Fig 3. Memory alterations in low and higher expresser mutant 3R Tau tg mice.

Mice were evaluated for context-dependent learning in an open field area at 8–10 months using a Kinder SmartFrame Cage Rack Station activity monitor system. A. Non-tg controls and 3R Tau tg Line 2 (lower-expresser) mice had reduced levels of activity over time as they became more familiar with the environment in the cage. B and C. Water maze testing presented as distance traveled and time to find the platform, respectively. During the cued portion of the test both the non-tg and mutant 3R Tau tg mice performed as expected. However during the hidden portion of the test, the higher expresser (Line 13) 3R Tau tg mice took a longer time and went a farther distance to find the platform compared to the non-tg. The lower expresser 3R Tau Line 2 mice was comparable to non-tg controls for distance but took longer a time to find the platform during the hidden portion of the test. D. At day 8, during the probe portion of the test (with platform removed) both Lines 2 and 13 displayed impairments in memory retention, E. Probe test with the visual platform confirmed that no visual alterations were detected. N = 12 non-tg, N = 12 3R Tau tg Line 2, N = 12 3R Tau tg Line 13. * = P < 0.05 compared with non-tg by one-way ANOVA with Dunnett’s post hoc analysis. # = P < 0.05 when comparing Line 2 and 13 using one way ANOVA with Fisher’s posthoc test.

Fig 4. Activity and motor alterations in low and higher expresser mutant 3R Tau tg mice.

A-C. Mice were evaluated for motor functioning in a round beam apparatus. Compared to non-tg controls and Line 2 mice, the 3R Tau tg mice Line 13 displayed increased numbers of errors when walking in the round beam. D-F. Mice were evaluated for activity in a Kinder SmartFrame Cage Rack Station activity monitor system. Compared to non-tg controls and Line 2 mice, the 3R Tau tg mice Line 13 displayed increased rearing, total activity and thigmotaxis. (One-way ANOVA with Dunnett’s multiple comparisons post-hoc test). Mice are 8–10 month old. N = 12 non-tg, N = 12 3R Tau tg Line 2, N = 12 3R Tau tg Line 13. * = P < 0.05 compared with non-tg by one-way ANOVA with Dunnett’s post hoc analysis. # = P < 0.05 when comparing Line 2 and 13 using one way ANOVA with Fisher’s posthoc test.

Fig 5. Effects of aging on memory and motor activity in higher expresser 3R Tau tg mice.

A. Context-dependent open field showed no alterations in 3–4 m old 3RTau tg mice, while mice at 6–8 and 12–14 months of age displayed similar level of alteration. B. Motor testing in the round beam showed that at 3–4 months of age the higher expresser 3R Tau Line 13 mice performed at a similar level to non-tg when crossing the beam, while at 6–8 and 12–14 months of age the 3R Tau Line 13 mice showed impairments with a higher rate of errors. C. Testing for spontaneous locomotor activity showed increased total activity in Line 13 mice beginning at 3–4 m of age with a time dependent increase 6–8 and 12–14 m of age. 3–4 (N = 10), 6–8 (N = 8) and 12–14 (N = 8) months of age. * = P < 0.05 when compared to non-tg control using one way ANOVA with Dunnett’s post hoc test.

Fig 6. Comparison of the patterns of 3R and pTau distribution in the brains of the low and higher expresser mutant 3R Tau tg mice.

A. Vibratome sections were immunostained with an antibody against 3R Tau and analyzed by digital bright field microscopy. Panels to the left are low-magnification (20X) photomicrographs of regions of interest, including the neocortex, hippocampus CA1 and dentate gyrus (DG) regions in the non-tg and mutant 3R Tau tg Lines 2 and 13 lines mice. Panels to the right are higher magnification (600X) of the corresponding regions displaying low levels of 3R Tau immunoreactivity in neuronal cells bodies in Line 2 and high levels of immunostaining in Line 13. B. Image analysis of numbers of neuronal cells in the neocortex and C dentate gyrus displaying 3R Tau aggregates. D. Vibratome sections were immunostained with the PHF-1 antibody against pTau (Ser396 and Ser404) and analyzed by digital bright field microscopy. Panels to the left are low-magnification (20X) photomicrographs of regions of interest, including the neocortex, hippocampus CA1 and DG regions in the non-tg and mutant 3R Tau tg Lines 2 and 13. Panels to the right are higher magnification (600X) of the corresponding regions displaying low levels of PHF-1 immunoreactivity in neuronal cells bodies in Line 2 and high levels of immunostaining in Line 13. Image analysis of the overall levels of PHF-1 immunoreactivity expressed as optical density in the E neocortex and F dentate gyrus. Mice were 8–10 month old. N = 12 non-tg, N = 12 3R Tau tg Line 2, N = 12 3R Tau tg Line 13. * = P < 0.05 compared with non-tg by one-way ANOVA with Dunnet post hoc analysis. # = P < 0.05 when comparing Line 2 and 13 using one way ANOVA with Tukey-Kramer posthoc test. Low magnification box scale bar = 250 μm; high power panels scale bar = 25 μm.

Fig 7. Effects of aging on 3R Tau accumulation and dendritic loss in the mutant 3R Tau tg mice.

A. Age-dependent increase in the immunoreactivity for 3R Tau in layers II-III and V pyramidal neurons in the neocortex and quantification in the B neocortex and C hippocampus at ages 3–4, 6–8, and 12–14 months of age in Line 13 and non-tg. D. Visualization of MAP2 immunoreactive dendritic densities in the neocortex at ages 3–4, 6–8, and 12–14 months of age in Line 13 and non-tg. Quantification of MAP2 immunoreactivity in the E neocortex and F hippocampus showed that the dendritic densities were preserved in 3R Tau tg mice (Line 13) 3–4 months of age, but were diminished in the older groups. 3–4 (N = 10), 6–8 (N = 8) and 12–14 (N = 8) months of age. * = P < 0.05 when compared to non-tg control using one way ANOVA with Dunnett’s post hoc test.

Fig 8. Histochemical and immunocytochemical characteristics of the aggregates in the higher expresser mutant 3R Tau tg mice.

A. Representative photomicrographs (frontal cortex) of brain sections from the higher expresser Line 13 mice stained with Bielchowsky and Gallyas silver impregnation technique and Thioflavin-S and immunostained with the antibodies against 3R Tau, pTau (AT8, CP9, MC-1, PHF-1) and ubiquitin. B. Summary table with semiquantitative assessment of the abundance of pathological Tau lesions in the brains of the mutant 3R Tau tg mice. C. Representative photomicrographs of frontal cortex from a Pick’s disease case immunostained for 3R Tau, AT8, and ubiquitin. Scale bar = 10 μm. Mice were 8–10 months old from each line.

Fig 9. Ultrastructural and immunogold analyses of the neuronal alterations in the hippocampal dentate gyrus in the higher expresser mutant 3R Tau tg mice.

Vibratome sections were post-fixed with glutaraldehyde and embedded in epon-araldyte, and ultra-thin sections from the hippocampus were prepared for transmitted electron microscopy (TEM) and immunogold analysis. A. Representative electron micrographs from the neuropil of non-tg mice displaying normal characteristics for dentate granular (DG) cells, mitochondria, axons, synapses and dendrites. B. In the neuropil of the higher mutant 3R Tau tg Line 13 the mitochondria were enlarged and irregular, there were extensive axonal dystrophy and accumulation of electrodense bodies in dendrites and synapses accompanied by filamentous aggregates. C. In the non-tg no immunogold labeling was observed. D. With an antibody against 3R Tau, the intra-neuronal filamentous aggregates were decorated by gold particles in the tg mice. Mice were aged 8–10 months. Bar for A and B = 1 μm, for C and D = 100 nm.

Fig 10. Analysis of neurodegeneration and synaptic-dendritic distribution of 3R Tau in the higher expresser mutant 3RTau tg mice.

A and B. Vibratome sections were immunostained with antibodies against the neuronal marker NeuN (visualized with DAB, bright field microscopy) and the dendritic marker MAP2 (visualized with FITC, confocal microscopy). Compared to the non-tg and lower expresser Line 2, Line 13 mice had decreased immunoreactivity in the neocortex (arrows) and hippocampus (arrowheads). C and D. Image analysis for the numbers of NeuN positive cells and area of the neuropil covered by MAP2 immunoreactive dendrites in the hippocampus showing a significant reduction in the mutant 3RTau tg Line 13 compared to Line 2 and non-tg controls. E. Sections were double-labeled with antibodies against human 3R Tau (red channel) and synaptophysin (SY38, green channel) and imaged with the laser scanning confocal microscope. Split panels show the somatic and synaptic localization of 3RTau and co-localization with synaptophysin in the hippocampus. The box to the right represents a zoomed area for the dashed box to the left showing details about the co-localization. F. Image analysis for the % of synaptophysin terminals showing 3R Tau immunoreactivity. G. Sections were double-labeled with antibodies against human 3R Tau (red channel) and MAP2 (green channel) and imaged with the laser scanning confocal microscope. Split panels show the somatic and dendritic localization of 3R Tau in the hippocampus. The box to the right represents a zoomed area for the dashed box to the left showing details about the co-localization. H. Image analysis for the percent of MAP2 dendrites showing 3R Tau immunoreactivity between the two synaptic markers. Scale bars in the single channel images = 25 μm and 5 μm in the enlarged merged images. Mice were 8–10 month old. N = 12 non-tg, N = 12 3R Tau tg Line 2, N = 12 3R Tau tg Line 13. * = P < 0.05 and ** = P < 0.01, respectively, compared with non-tg by one-way ANOVA with Dunnet post hoc analysis.

Fig 11. Analysis of astroglial cells in the higher expresser mutant 3R Tau tg mice.

A. Sections were immunolabeled with antibodies against GFAP and imaged with a digital bright field microscope. Representative low power (20X) photomicrographs of the neocortex and hippocampus from non-tg and mutant 3R Tau tg mice displaying increased GFAP immunoreactivity in the tg mice. Higher magnification (600X) images of the hippocampus corresponding to the area in the box showing the astroglial cells in the hippocampus. Bottom panel represents cells immunostained with antibody against 3R Tau displaying a glial-like appearance. B. Image analysis for levels of GFAP immunoreactivity in the hippocampus expressed as optical density showing increased levels in tg mice compared to non-tg controls. C. Sections double-labeled with antibodies against GFAP (green channel) and human 3R Tau (red channel) and imaged with the laser scanning confocal microscope. Split panels showing processes of astroglial cells displaying localization of 3R Tau (arrows). D. Image analysis for the percent of GFAP positive cells showing 3R Tau immunoreactivity. Mice were 8–10 month old. N = 12 non-tg, N = 12 3R Tau tg Line 2, N = 12 3R Tau tg Line 13. * = P < 0.05 compared with non-tg by one-way ANOVA with Dunnet post hoc analysis. Scale bar for upper panel in A = 100 μm and 50 μm for the lower panel. Scale bar for panel in C = 10 μm.