Alzheimer's disease-like tau neuropathology leads to memory deficits and loss of functional synapses in a novel mutated tau transgenic mouse without any motor deficits

Abstract

Tau transgenic mice are valuable models to investigate the role of tau protein in Alzheimer's disease and other tauopathies. However, motor dysfunction and dystonic posture interfering with behavioral testing are the most common undesirable effects of tau transgenic mice. Therefore, we have generated a novel mouse model (THY-Tau22) that expresses human 4-repeat tau mutated at sites G272V and P301S under a Thy1.2-promotor, displaying tau pathology in the absence of any motor dysfunction. THY-Tau22 shows hyperphosphorylation of tau on several Alzheimer's disease-relevant tau epitopes (AT8, AT100, AT180, AT270, 12E8, tau-pSer396, and AP422), neurofibrillary tangle-like inclusions (Gallyas and MC1-positive) with rare ghost tangles and PHF-like filaments, as well as mild astrogliosis. These mice also display deficits in hippocampal synaptic transmission and impaired behavior characterized by increased anxiety, delayed learning from 3 months, and reduced spatial memory at 10 months. There are no signs of motor deficits or changes in motor activity at any age investigated. This mouse model therefore displays the main features of tau pathology and several of the pathophysiological disturbances observed during neurofibrillary degeneration. This model will serve as an experimental tool in future studies to investigate mechanisms underlying cognitive deficits during pathogenic tau aggregation.

Figure 1-6933

Generation of the THY-Tau Tg mouse model. A: The human 412 amino acid double-mutated tauconstruct used for microinjection. Exons 2 and 10 are depicted by black boxes and microtubule binding repeats by gray-shaded boxes. The human 4-repeat tauwas site-directed mutated at G272V and P301S and cloned as XhoI-fragment into a Thy1.2-expression vector. B: THY-Tau mouse lines, transgene DNA copy number, and relative protein levels of total human tau in cortex, hippocampus, and spinal cord compared to endogenous tau in WTs from three adult animals at 3 and 12 months: (+), traces; +, low levels (similar to endogenous tau); ++, medium levels (twofold to threefold greater than endogenous tau); +++, high levels (fourfold to fivefold greater than endogenous tau); ++++, very high levels (fivefold to sixfold greater than endogenous tau; detection with M19G and tau-5). C: Positive grasping reflex in response to tail hang in a 10-month-old tau Tg mouse of line 30 (THY-Tau30) with hind limb paralysis. Mice from line 22 (THY-Tau22) and WTs at the same age do not display this reflex. D: Expression of the transgene in different tissues from THY-Tau22. Immunoreactivity for tau-5 (total tau) is only found in the brain and minor traces in spinal cord. Representative immunoblot from a 6-month-old male THY-Tau22 (Tg) and WT.

Figure 2-6933

Accumulation of pathological tau species in THY-Tau22. Immunohistochemical studies revealed the accumulation of abnormal tau conformation and phosphorylation in 12-month-old THY-Tau22 compared to non-Tg WTs in hippocampus (A and B, note the arrows pointing to the most representative dots in the Tg), CA1 sector (C–J), and spinal cord (K and L, note the arrow). Hyperphosphorylation was identified as brown color and detected with AT8 (A–D and K–L), AT100 (F), AT270 (G), AP422/988 (H), human-specific TP20 tau antibody (I), AT180 (J), and abnormal tau conformation with MC1 (E). No positive labeling was observed after parallel processing of littermate WT tissue (A and C, 12 months old). Representative sections are shown of 10 animals used at the age indicated. Scale bars: 200 μm (A, B); 25 μm (C–L).

Figure 3-6933

Age kinetics of neurofibrillary inclusions and tau accumulation in THY-Tau22 mice. Progressive formation of PHF-1 immunoreactivity (A–C, brown color) and Gallyas silver-positive inclusions (D–F, black color) were age-dependent in CA1 in tau Tg mice showing phospho-tau and NFT-like formation starting from 6 months in the CA1 region. Representative sections of three to five animals used at the age indicated are shown. Scale bar = 25 μm.

Figure 4-6933

Increase of tau hyperphosphorylation and abnormal tau phosphorylation in the aged THY-Tau22 mouse brain. A: Immunoblot analysis of the major AD-relevant tau phosphorylation epitopes. Hyperphosphorylation of tau in cortex at sites Thr231 (AT180), Thr181 (AT270), Ser202/Thr205 (AT8), and Ser396 are detectable from 3 to 6 months and increase with age. Only phosphorylation of murine tau-Ser396 can be detected in aged WT. Abnormal tau phosphorylation at sites Thr212/Ser214 (AT100) and Ser422 (AP422/988) starts at 6 to 10 months. The levels of total human tau protein increase slightly with age in THY-Tau22 mice. Brain homogenate from an AD patient is loaded in the first lane. Triplets of hyperphosphorylated tau isoforms are indicated (69, 64, and 60 kd). Most antibodies detect a duplet of hyperphosphorylated tau in mice, indicated by the black and gray arrowheads. B: Quantification of site-specific tau phosphorylation of three individual blots with at least five animals per age and genotype. Densities of the 64 (bottom bars in dark gray; * = significance) and 69-kd band (top bars in light gray, ^§ = significance) were determined in duplicates. Background noise was subtracted for each lane (unpaired Student’s t-test, */^§P < 0.05, **/^§§P < 0.01 and ***/^§§§P < 0.001). C: Immunoblot of 12E8, an antibody that detects phosphorylation of Ser262 and Ser356, which are microtubule-binding domains 1 and 3. 12E8 allows for the discrimination between AD and PiD. Old tau Tg revealed a phosphorylation profile that is comparable to that found in AD. Note that even endogenous murine tau is detected in the WT and Tg. Representative immunoblots are shown.

Figure 5-6933

Ultrastructural aspect of fibrillar inclusions in hippocampal neurons from THY-Tau22. A: A hippocampal neuron of a 15-month-old THY-Tau22 mouse contains several massive fibrillar inclusions (asterisks). B: Higher magnification of the boxed area in A. Fibrillar inclusions are composed of bundles of straight filaments. C: Bundles of abnormal filaments in another hippocampal neuron. D: Higher magnification of the boxed area in C, showing the co-existence of straight filaments and occasional wider filaments with regular constrictions. Representative sections are shown. Scale bars: 2 μm (A); 0.5 μm (B, C); 100 nm (D).

Figure 6-6933

Loss in cell density, neurodegeneration, and ghost tangles in aged THY-Tau22. A–D: DAPI-stained neurons of the CA1 cell layer in a 12-month-old WT (A) and in THY-Tau22 at 6 (B), 12 (C), and 17 (D) months. Note the decrease in DAPI staining intensity and the reduced cell density with increasing age in the tau Tg mice. Scale bar = 20 μm. E: Decrease of cell density in aged THY-Tau22 and semiquantification of Nissl/Cresylviolet-stained cells in the CA1 cell layer (analysis of variance with Bonferroni’s post test **P < 0.01 Tg versus WT at 12 months; n = 3 per age and genotype). Scale bar = 20 μm. F: AT8 immunolabeling combined with G: Gallyas staining and hematoxylin staining. Three cells with different staining patterns are shown by the arrows (from left to right): left: an AT8 and Gallyas-positive cell, with a nucleus; middle: an AT8-positive but Gallyas-negative cell, with a nucleus; right: a Gallyas-positive and AT8-negative cell, without nucleus. Note the loss of AT8 immunoreactivity and the argyrophilic extracellular NFT in the latter, which corresponds to a late ghost tangle.

Figure 7-6933

Gliosis in THY-Tau22 mice. GFAP-immunoreactivity in the hippocampal hilus is increased in THY-Tau22 mice compared to WT animals at 3, 6, and 12 months. Representative sections of 10 mice are shown. Scale bar = 20 μm.

Figure 8-6933

Decreased synaptic transmission in the hippocampus of THY-Tau22 mice. A: Input/output plots of the amplitude of the field excitatory postsynaptic potentials in the CA1 area elicited by stimuli of increasing intensity in hippocampal slices prepared from 6- to 7-month-old WT (open circles) and THY-Tau22 Tg mice (filled squares). Two-way analysis of variance of the EPSP amplitude values throughout the 100- to 300-μA stimulation intensity range indicated no significant effect of genotype (F[1,8] = 0.0931; P > 0.7). B: Input/output plots from 14- to 15-month-old animals, indicating impaired synaptic function by 80% compared to age-matched WT. Analysis of variance indicated a highly significant effect of genotype (F[1,4] = 207.2; ***P < 0.001). At least five animals were used per group. C: Protein levels of synaptotagmin I and synaptophysin, proteins that are involved in synaptic integrity, are not significantly changed between Tg (THY-Tau22) and WT. Thus, a small decrease by 20% was detected in 15-month-old THY-Tau22. However, a synaptic malfunction of basal transmission in old THY-Tau22 appears not to be due only to a minor loss of synaptic proteins. Representative immunoblots are shown.

Figure 9-6933

Altered anxiety in the elevated plus maze in THY-Tau22 mice. Impaired anxiety in THY-Tau22 mice. A: Model of the elevated plus maze (EPM) used with two enclosed arms and two open arms. The mice were placed in the middle facing an open arm and their movements were recorded throughout 5 minutes. Representative locomotion traces for THY-Tau22 (B, D) and WT (C, E) mice in the elevated plus maze. F: THY-Tau22 mice spend more time in the open arms of the elevated plus maze compared to their littermate WTs (157.9 ± 21.23 seconds for male THY-Tau22 versus 39.29 ± 7.334 seconds for male WT, ***P < 0.0001; 101.3 ± 32.62 seconds for female THY-Tau22 versus 27.95 ± 8.078 seconds for female WT, *P < 0.05; n = 8 per group, 6 months old).

Figure 10-6933

Retarded learning and reduced memory in THY-Tau22 mice. Delayed learning in the MWM in THY-Tau22 mice. A: Escape latency, the time to find the hidden platform in the MWM, during the 4-day training period in 10-month-old THY-Tau22 mice (filled squares, n = 8) and WT (open circles, n = 8). Day 1, THY-Tau22 42.95 ± 3.979 seconds versus WT 37.33 ± 3.815 seconds; day 2, THY-Tau22 36.52 ± 3.412 seconds versus WT 27.34 ± 3.894 seconds, *P < 0.05; day 3: THY-Tau22 38.08 ± 3.849 seconds versus WT 27.00 ± 4.912 seconds, *P < 0.05; day 4, THY-Tau22 30.21 ± 3.140 seconds versus WT 26.51 ± 4.901 seconds; Two-way analysis of variance, *P > 0.05 for genotype, *P > 0.05 for learning). B: Escape latency in MWM in 2- to 3-, 10- and 14-month-old THY-Tau22 and WT on training day 3. THY-Tau22 display a delay in learning (two-way analysis of variance **P < 0.01 for genotype, *P < 0.05 for age, n = 8 per group). C: Five days after the last training the platform was removed and a probe test was performed. The time the mice spent in the quadrant where the platform used to be (= goal quadrant) is plotted against age. Aged animals show a mild memory deficit compared to age-matched WTs (n = 8 per group). Representative locomotion traces of THY-Tau22 (D, F) and WTs (E, G) during the learning period (D, E) and the probe test (F, G).