Humanization of the entire murine Mapt gene provides a murine model of pathological human tau propagation

Characterization of human MAPT KI mice

Normal adult human brains express six distinct isoforms of tau generated by alternative splicing (14). They are classified into 3R-tau and 4R-tau, depending on the number of repeated microtubule-binding domains (3 repeats in 3R and 4 in 4R). Importantly, NFTs in human AD comprise an equal mixture of all 3R and 4R tau isoforms (14). In contrast, 4R-tau aggregates may cause corticobasal degeneration or progressive supranuclear palsy, whereas deposition of 3R-tau aggregates characterize Pick's disease (14, 16).

The major difference between tau isoforms present in adult mouse and human brains is that the former possesses 4R-tau only, although they express 3R-tau in development like humans, whereas normal adult human brains also possess 3R-tau (17). In contrast, almost all existing mouse models of tauopathy overexpress one of the six possible tau isoforms (18). These mice, with or without FTDP-17 mutations, do not fully recapitulate the pathophysiology of tauopathy. In addition, because the presence of murine tau may hamper human tau aggregation, models in which endogenous murine tau is deleted are preferred (19). Ideally, the mouse model of tauopathy should be based on the KI strategy because overexpression of tau may disturb the normal physiological functions of neurons, such as microtubule assembly and synaptic functions (20).

We therefore generated mutant mice, in which the entire murine Mapt gene has been humanized (MAPT KI mice) by homologous recombination as described (21). The tau protein in MAPT KI mice localized to axons (e.g. mossy fibers) in a manner similar to endogenous tau in WT mice (10) (Fig. 1, A and B). In clear contrast, P301S-tau transgenic mice, which express human tau protein at 5-fold higher levels than endogenous mouse tau (22) (Fig. 1B), exhibit an abnormal distribution of tau, such as dendritic mislocalization (23) (Fig. 1C). Moreover, the humanization of murine tau did not affect Aβ levels in the brain (Fig. 1D). These observations suggest that the human tau proteins in MAPT KI mice are functionally, physiologically, and structurally normal and that MAPT KI incorporates correctly into murine neuronal systems.

Humanization of Mapt gene in App KI mice

We next cross-bred MAPT KI mice with App^NL-F and App^NL-G-F mice (5). 6-Month-old App^NL-G-F/MAPT dKI mice exhibited a significantly higher tau phosphorylation status than single MAPT KI mice at Ser-202/Thr-205, Ser-396/Ser-404, and Ser-422, as recognized by AT8, PHF-1, and anti-pS422 antibodies, respectively (Fig. 2, A and B). Similarly, 6-month-old single App^NL-G-F KI mice also showed greater tau phosphorylation than WT mice in a similar manner (Fig. 2, C and D). Immunohistochemistry allowed us to detect AT8 signals in dystrophic neurites around Aβ plaques, but we observed no NFTs in single App^NL-G-F KI mice or the dKI mice, where we used P301S-tau Tg and AD brains as positive controls (Fig. 2, E and F). Humanization of the Mapt gene did not affect Aβ-amyloid deposition, neuroinflammation (Fig. 3), or memory in App^NL-G-F mice (Fig. S1).

At 24 months of age, App^NL-G-F/MAPT dKI mice exhibited significantly higher tau phosphorylation than single MAPT KI mice at Ser-202/Thr-205, Ser-396/Ser-404, and Ser-422 (Fig. 4, A and B). Aβ-amyloidosis accelerated phosphorylation of murine and human tau proteins to a similar extent in the App KI and App/MAPT dKI mice, respectively (Fig. 4, C and D), indicating that murine and human tau proteins behave comparably in terms of Aβ-amyloidosis–evoked phosphorylation. We also observed that Aβ-amyloidosis induced more prominent neuroinflammation in these mutant mice (21). Consistently, the single App^NL-G-F KI and App^NL-G-F/MAPT dKI mice exhibited more dystrophic neurites than the WT and single MAPT KI mice without NFT formation (Fig. 4, E and F). App^NL-F mice exhibited a much less aggressive pathology than the App^NL-G-F mice (5), but App^NL-F/MAPT dKI mice exhibited higher tau phosphorylation than single MAPT KI mice (Fig. 5). Thus, both in App^NL-F and App^NL-G-F mice, Aβ-amyloidosis augmented tau phosphorylation.

Several studies have shown an aberrant increase of exon 10–containing 4R-tau isoforms in AD and tauopathy cases (24, 25). Exon 10 encodes one of the microtubule-binding domains, and its inclusion gives rise to the production of 4R-tau. However, it has been difficult to analyze tau splicing using existing mouse models because of the lack of a suitable model expressing all human isoforms without overexpression of tau. To examine the contribution of Aβ pathology to tau splicing, we determined the level of each tau isoform in MAPT KI and App^NL-G-F/MAPT dKI mice. The relative expression ratio of mRNA for 4R-tau/3R-tau was almost the same between aged MAPT KI mice and App^NL-G-F/MAPT dKI mice, with values of 0.71 ± 0.08 and 0.69 ± 0.11 (n = 4 each), respectively, obtained for each strain (Fig. 4, G and H). The isoform profile of tau protein remained unaltered in the aged dKI mice (Fig. 4, I and J), suggesting that Aβ-amyloidosis does not directly affect alternative splicing of the tau gene.

We previously observed no atrophy in these regions in aged App^NL-G-F/MAPT dKI mice from 18 to 24 months and detected only a low level of caspase-3 activation in App^NL-G-F/MAPT dKI mice as well as in App^NL-G-F KI mice (21). These findings indicate that humanization of the Mapt gene does not affect neurodegenerative processes in these mouse models of extensive Aβ-amyloidosis.

Propagation of AD brain-derived pathological tau proteins in WT, App KI, MAPT KI, and App^NL-G-F/MAPT dKI mice

Propagation of pathological proteins associated with neurodegeneration, such as tau, Aβ, α-synuclein, and TDP-43, has been shown to represent the primary mechanism that accounts for progressive spreading of pathogenic agents (26). Based on the experimental paradigm described previously (13), we analyzed whether humanization of murine tau protein affects pathological tau propagation in WT and App KI mouse brains (Fig. 6).

The first notable observation is that humanization of murine tau in WT mice boosted propagation of AD brain-derived pathological tau (Fig. 6A, left panels), indicating that humanization of the host animal tau affects the transmission of the pathogenic agents. In addition, the presence of Aβ plaques augmented tau pathology seeded by the AD brain–derived pathological tau proteins in single App^NL-G-F KI mice (Fig. 6, A and B) in agreement with the previous report (13). Of particular note, App^NL-G-F/MAPT dKI mice exhibited greater pathological propagation than App^NL-G-F KI mice (Fig. 6, A and B). In addition, we observed many more AT8-positive tau aggregates in dystrophic neurites surrounding Aβ plaques in App^NL-G-F/MAPT dKI than single App^NL-G-F KI mice (Fig. 6, C and D), but we did not detect thioflavin S– or Gallyas silver–positive NFT formation (data not shown). These differences between WT and MAPT KI mice or App KI and App KI/MAPT dKI mice were undetectable unless the AD brain–derived pathological tau was introduced. Our findings probably imply that there exists a species preference (i.e. mice versus humans) in the dynamic interactions between the pathological and normal tau proteins. This may possibly apply to some other pathogenic proteins, such as α-synuclein, associated with neurodegenerative diseases.

The experimental inoculation of the pathological tau protein derived from AD brain, however, did not evoke NFT formation or neurodegeneration in App^NL-G-F/MAPT dKI mice. These findings suggest that tau propagation in dystrophic neurites is likely an initial stage of tau pathology formation and neurodegeneration, which proceeds over decades in human brains (12). Because many of the pathogenic mutations in the MAPT gene that cause FTDP-17 are located in the genomic regions corresponding to microtubule-binding domains (27) and because some mutations in the exon 10–intron 10 interface are also pathogenic by affecting alternative splicing (27), introduction of these mutations into the human MAPT KI mice may lead to a generation of relevant tauopathy models by accelerating the pathological processes without depending on the overexpression paradigm.

Mutant mice

App^NL-G-F mice, which include the Swedish, Arctic, and Iberian mutations, and MAPT KI mice were maintained as described previously (5, 21). P301S-tau transgenic mice (PS19) were described previously (22) and then backcrossed to C57BL/6J mice. Tau-deficient mice were obtained from the Jackson Laboratory (stock no. 007251). All mice used in the experiments were on the C57BL/6J background. We used the male mice for the 6-month-old analysis and the female mice for 24-month-old analysis in the present study. All animal experiments were carried out according to the RIKEN Brain Science Institute's guidelines for animal experimentation.

ELISA of Aβ

Soluble materials from mouse cortical hemispheres were dissolved in Tris-HCl–buffered saline (TS fraction) and insoluble materials in guanidine HCl solution (GuHCl fraction) as described previously (38). Aβ_X-40 and Aβ_X-42 levels in brain extracts were quantified using an Aβ ELISA kit (Wako, Tokyo, Japan) according to the manufacturer's instructions. Aβ_X-40 and Aβ_X-42 carrying the Arctic mutation were quantified based on standard curves using synthetic human Arctic Aβ peptides (Peptide Institute, Osaka, Japan) as described previously (5).

Western blot analysis of tau

The cortical brain tissue samples were homogenized in lysis buffer (50 mm Tris-HCl (pH 8.0) containing 150 mm NaCl, 50 mm EDTA, 1% Triton X-100, and protease/phosphatase inhibitor mixture) and centrifuged at 20,000 × g for 20 min at 4 °C, as described previously (39). Brain extracts as the total fraction (15 μg of protein/lane) were subjected to Western blotting using anti-Tau antibodies (both mouse and human tau-specific antibody, Tau5 (catalog no. AHB0042, Thermo Fisher Scientific; epitope: amino acid residues 210–214 on the longest isoform of human tau); human tau-specific antibody, Tau13 (catalog no. sc-21796, Santa Cruz Biotechnology, Inc.; epitope: amino acid residues 2–18 on the longest isoform of human tau); 3R-tau–specific antibody, RD3 (catalog no. 05-803, Merck Millipore; epitope: a junction-flanking sequence coded by adjacent exons 9 and 11 in the absence of exon 10); 4R-tau–specific antibody, RD4 (catalog no. MBS604301, MyBiosource, San Diego, CA; epitope: a junction-flanking sequence coded by adjacent exons 9 and 10 of Tau)), anti-phospho-Tau antibodies (AT8 (catalog no. 90206, Innogenetics, Ghent, Belgium; epitope: phosphorylated serine 202 and threonine 205); PHF-1 (kindly provided from Peter Davies, Albert Einstein College of Medicine; epitope: phosphorylated serine 396 and 404); pS422 (catalog no. ab62639, Abcam, Cambridge, UK; epitope: phosphorylated serine 422)), and internal controls (anti-β-tubulin antibody (catalog no. ab11312, Abcam) and anti-GAPDH antibody (catalog no. 2275-PC-100, TREVIGEN, Gaithersburg, MD)). To show the specificity of antibodies, brain extract derived from Tau-KO mice was used as a negative control. For analysis of the isoform pattern of tau, brain extracts were dephosphorylated with λ-phosphatase (catalog no. sc-200312A, Santa Cruz Biotechnology) according to the manufacturer's instructions. All six isoforms of human Tau ladder were purchased from rPeptide. Each set of experiments was repeated at least three times to confirm the results. Band intensity was determined with a LAS4000 densitometer (Fujifilm, Tokyo, Japan).

Immunohistochemical studies

Paraffin-embedded sections were immunostained using antibodies specific to the N termini of Aβ (Aβ_1-X) (40) and 82E1 (catalog no. 10323, IBL, Gunma, Japan), to Iba1 (Wako), to GFAP (catalog no. MAB3402, Merck Millipore, Darmstadt, Germany), to tau (pan-tau–specific antibody, Tau1 (catalog no. MAB3420, Merck Millipore, Darmstadt, Germany); murine tau-specific antibody, T49 (catalog no. MABN827, Merck Millipore), Tau5, Tau13), to phosphorylated tau (AT8), and to MAP2 as a dendritic marker (catalog no. M121, Leinco Technologies, St. Louis, MO). To show the specificity of antibodies, brain sections derived from Tau-KO mice were used as a negative control. When necessary, tyramide signal amplification (PerkinElmer Life Sciences) was used as described previously (41). We performed antigen retrieval by autoclave (121 °C for 5 min).

We quantified the immunoreactive areas using MetaMorph imaging software (Universal Imaging Corp., West Chester, PA) as described previously (41). To reduce the variation of results across tissue sections, we used the average of data from at least four sections per mouse.

RNA extraction and semiquantitative RT-PCR

Total RNA was extracted from brain samples using RNAiso Plus (Takara, Shiga, Japan) according to the manufacturer's instructions. We performed semiquantitative RT-PCR for detection of both 3R- and 4R-tau mRNA as described previously (42) with minor modifications. The primer pair used for tau was 5′-AAGTCGCCGTCTTCCGCCAAG-3′ and 5′-GTCCAGGGACCCAATCTTCGA-3′, and that for GAPDH was 5′-CCATGGCACCGTCAAGGCTGA-3′ and 5′-GCCAGTAGAGGCAGGGATGAT-3′. PCR products of 288 and 381 bp correspond to 3R-tau and 4R-tau, respectively. RT-PCR products were detected by 24, 26, 28, 30, 32, and 34 PCR cycles to mortgage the linearity of the products, and the result was decided by 26 cycles. Band intensity was calibrated using ImageJ software.

Tau propagation analysis

AD-tau extracted from an AD patient's brain, as described previously (43), was prepared in the laboratory of V. M. Y. L. and J. Q. T. In this study, AD-tau was provided from the same batch of extracts (43). We injected AD brain–derived tau into the mouse brain by stereotaxic injection as described previously (13, 44). The mice (19-month-old males) were deeply anesthetized and immobilized in a stereotaxic frame (Muromachi). AD brain–derived tau was injected in dorsal hippocampus and overlying cortex in the right hemisphere (bregma: −2.5 mm; lateral −2 mm; depth −1.7 mm and −0.5 mm from brain surface) with a total amount of 2 μg of AD-tau using a Hamilton syringe as described (44). After 3 months, we collected the brains, which were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4-μm thickness, as described previously (13), and performed immunohistochemical analysis. Protocols involving the use of human tissue were approved by the Institutional Review Board of the RIKEN Brain Science Institute and University of Pennsylvania, respectively. All of the human materials were taken in accordance with the Declaration of Helsinki.

AD brain sections

AD brain sections were used as a positive control of tauopathy in all studies. Paraffin-embedded brain (neocortical and hippocampal region) sections from normal humans and AD patients were purchased from Wako. Protocols involving the use of human tissue were approved by the institutional review board of the RIKEN Brain Science Institute.

Statistics

All data are shown as the mean ± S.E. For comparison between two groups, statistical analysis was performed by Student's t test. For comparisons among three or more groups, two-way analysis of variance (ANOVA) or repeated measures ANOVA followed by a post hoc test (Scheffe's F test) were used. Normality was tested using Statcel 3 (add-in software for Microsoft Excel). Data were collected and processed in a randomized and blinded manner. No statistical methods were used to predetermine sample size, but our sample sizes are similar to those generally employed in the field.