Title: Cortical Neuronal and Glial Pathology in TgTauP301L Transgenic Mice
Abstract: Recapitulation of tau pathologies in an animal model has been a long-standing goal in neurodegenerative disease research. We generated transgenic (TgTauP301L) mice expressing a frontotemporal dementia with parkinsonism linked to chromosome 17 (FTPD-17) mutation within the longest form of tau (2N, 4R). TgTauP301L mice developed florid pathology including neuronal pretangles, numerous Gallyas-Braak-positive neurofibrillary tangles, and glial fibrillary tangles in the frontotemporal areas of the cerebrum, in the brainstem, and to a lesser extent in the spinal cord. These features were accompanied by gliosis, neuronal loss, and cerebral atrophy. Accumulated tau was hyperphosphorylated, conformationally changed, ubiquitinated, and sarkosyl-insoluble, with electron microscopy demonstrating wavy filaments. Aged TgTauP301L mice exhibited impairment in hippocampally dependent and independent behavioral paradigms, with impairments closely related to the presence of tau pathologies and levels of insoluble tau protein. We conclude that TgTauP301L mice recreate the substantial phenotypic variation and spectrum of pathologies seen in FTDP-17 patients. Identification of genetic and/or environmental factors modifying the tau phenotype in these mice may shed light on factors modulating human tauopathies. These transgenic mice may aid therapeutic development for FTDP-17 and other diseases featuring accumulations of four-repeat tau, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. Recapitulation of tau pathologies in an animal model has been a long-standing goal in neurodegenerative disease research. We generated transgenic (TgTauP301L) mice expressing a frontotemporal dementia with parkinsonism linked to chromosome 17 (FTPD-17) mutation within the longest form of tau (2N, 4R). TgTauP301L mice developed florid pathology including neuronal pretangles, numerous Gallyas-Braak-positive neurofibrillary tangles, and glial fibrillary tangles in the frontotemporal areas of the cerebrum, in the brainstem, and to a lesser extent in the spinal cord. These features were accompanied by gliosis, neuronal loss, and cerebral atrophy. Accumulated tau was hyperphosphorylated, conformationally changed, ubiquitinated, and sarkosyl-insoluble, with electron microscopy demonstrating wavy filaments. Aged TgTauP301L mice exhibited impairment in hippocampally dependent and independent behavioral paradigms, with impairments closely related to the presence of tau pathologies and levels of insoluble tau protein. We conclude that TgTauP301L mice recreate the substantial phenotypic variation and spectrum of pathologies seen in FTDP-17 patients. Identification of genetic and/or environmental factors modifying the tau phenotype in these mice may shed light on factors modulating human tauopathies. These transgenic mice may aid therapeutic development for FTDP-17 and other diseases featuring accumulations of four-repeat tau, such as Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy. Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is characterized by the insid-ious onset of behavioral and personality changes with dementia and parkinsonism.1Foster NL Wilhelmsen K Sima AA Jones MZ D'Amato CJ Gilman S Frontotemporal dementia and parkinsonism linked to chromosome 17: a consensus conference. 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Tau-associated pathology is prominent in the form of numerous tau-positive pretangles, some neurofibrillary tangles (NFTs), and extensive glial fibrillary tangles (GFTs). In addition, tau protein shifts from the sarkosyl-soluble fraction into sarkosyl-insoluble deposits.9Rizzu P Joosse M Ravid R Hoogeveen A Kamphorst W van Swieten JC Willemsen R Heutink P Mutation-dependent aggregation of tau protein and its selective depletion from the soluble fraction in brain of P301L FTDP-17 patients.Hum Mol Genet. 2000; 9: 3075-3082Crossref PubMed Scopus (52) Google Scholar Although FTDP-17 is caused by mutations in the single gene tau, the clinical and pathological features are markedly different among patients carrying distinct mutations and often even among patients within the same family.7Bugiani O Murrell JR Giaccone G Hasegawa M Ghigo G Tabaton M Morbin M Primavera A Carella F Solaro C Grisoli M Savoiardo M Spillantini MG Tagliavini F Goedert M Ghetti B Frontotemporal dementia and corticobasal degeneration in a family with a P301S mutation in tau.J Neuropathol Exp Neurol. 1999; 58: 667-677Crossref PubMed Scopus (358) Google Scholar, 10Bird TD Nochlin D Poorkaj P Cherrier M Kaye J Payami H Peskind E Lampe TH Nemens E Boyer PJ Schellenberg GD A clinical pathological comparison of three families with frontotemporal dementia and identical mutations in the tau gene (P301L).Brain. 1999; 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95: 13103-13107Crossref PubMed Scopus (453) Google Scholar, 18Poorkaj P Grossman M Steinbart E Payami H Sadovnick A Nochlin D Tabira T Trojanowski JQ Borson S Galasko D Reich S Quinn B Schellenberg G Bird TD Frequency of tau gene mutations in familial and sporadic cases of non-Alzheimer dementia.Arch Neurol. 2001; 58: 383-387Crossref PubMed Scopus (147) Google Scholar We report here a line of transgenic mice expressing a P301L mutant version of the longest form of human tau [denoted TgTau(P301L)23027, for brevity TgTauP301L] that developed pretangles and numerous GFTs and NFTs with pathological features seen in FTDP-17. The age-related appearance of NFTs and GFTs was closely related to neuronal and synaptic losses with cerebral atrophy and impaired hippocampal-dependent explicit spatial and working memory, as well as hippocampal-independent implicit associative learning. The longest isoform of human 2N4R wild-type (wt) tau cDNA containing a eukaryotic Kozak initiation sequence19Kozak M An analysis of vertebrate mRNA sequences: intimations of translational control.J Cell Biol. 1991; 115: 887-903Crossref PubMed Scopus (1452) Google Scholar upstream of the start codon was ligated to the SalI-digested site of a linearized cos.Tet expression vector containing the Syrian hamster prion protein gene promoter,20Scott MR Köhler R Foster D Prusiner SB Chimeric prion protein expression in cultured cells and transgenic mice.Protein Sci. 1992; 1: 986-997Crossref PubMed Scopus (220) Google Scholar, 21Citron M Westaway D Xia W Carlson G Diehl T Levesque G Johnson-Wood K Lee M Seubert P Davis A Kholodenko D Motter R Sherrington R Perry B Yao H Strome R Lieberburg I Rommens J Kim S Schenk D Fraser P St. George Hyslop P Selkoe DJ Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice.Nat Med. 1997; 3: 67-72Crossref PubMed Scopus (1175) Google Scholar packaged in vitro, and plated on Escherichia coli DH1 to obtain a bacterial stock containing the recombinant cosmid clone (Figure 1). Similar procedures were adopted for a P301L mutant construct. Transgene inserts were purified, digested, and microinjected into fertilized oocytes of 129SvEv × FVB/N F1 mice, as previously described,21Citron M Westaway D Xia W Carlson G Diehl T Levesque G Johnson-Wood K Lee M Seubert P Davis A Kholodenko D Motter R Sherrington R Perry B Yao H Strome R Lieberburg I Rommens J Kim S Schenk D Fraser P St. George Hyslop P Selkoe DJ Mutant presenilins of Alzheimer's disease increase production of 42-residue amyloid beta-protein in both transfected cells and transgenic mice.Nat Med. 1997; 3: 67-72Crossref PubMed Scopus (1175) Google Scholar and then the resultant mice were backcrossed to 129 mice. A short noncoding region (∼100 bp) was also included in the transgene.22Basler K Oesch B Scott M Westaway D Wälchli M Groth DF McKinley MP Prusiner SB Weissmann C Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene.Cell. 1986; 46: 417-428Abstract Full Text PDF PubMed Scopus (649) Google Scholar In the case of the P301L construct, one Tg line was obtained, and first generation offspring were used to start two breeding colonies. All animal experiments were performed according to guidelines established in the Guide for the Care and Use of Laboratory Animals (Japan) and Canadian Council on Animal Care guidelines (Canada). After mice were sacrificed under anesthesia, the brains were removed and cut sagittally on the midline. One hemisphere was fixed in 0.1 mol/L phosphate buffer (pH 7.6) containing 4% paraformaldehyde and embedded in paraffin. For immunostaining, sections were treated with 99% formic acid for 3 minutes. After blocking with 5% normal goat or horse serum in 50 mmol/L phosphate-buffered saline (PBS) containing 0.05% Tween-20 and 4% Block-Ace (Snow Brand, Sapporo, Japan), sections were incubated with primary antibodies for 6 hours. The specific labeling was visualized using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). Immunostained tissue sections were counterstained with hematoxylin. Five-μm coronal hemisphere sections were used for hematoxylin and eosin (H&E), Gallyas-Braak, Bielschowsky, Bodian, thioflavin-S, and Nissl staining. The following antibodies were used for immunostaining: Tau154 (a polyclonal antibody against amino acids 154 to 168 of human tau 441, 1:200), tau-C (a polyclonal antibody against C-terminal amino acids 422 to 438 of both human and mouse tau, 1:200), and Alz50 (an antibody against paired-helical-filament tau, 1:100).23Ikeda M Shoji M Kawarai T Kawarabayashi T Matsubara E Murakami T Sasaki A Tomidokoro Y Ikarashi Y Kuribara H Ishiguro K Hasegawa M Yen SH Chishti MA Harigaya Y Abe K Okamoto K St. George-Hyslop P Westaway D Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice.Am J Pathol. 2005; 166: 521-531Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar CP27 (all forms of human tau, 1:500), MC1 (early epitope of conformationally changed tau, 1:500), and Tg3 (an Alzheimer-specific conformation of pThr231 tau, 1:50) were also used.24Weaver CL Espinoza M Kress Y Davies P Conformational change as one of the earliest alterations of tau in Alzheimer's disease.Neurobiol Aging. 2000; 21: 719-727Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar For the detection of phosphorylated tau, PS199 (serine-199, 1:500), AT8 (serine-202/threonine-205, 1:200; Innogenetics, Ghent, Belgium), and PHF1 (serine-396/serine-404, 1:100) were used.23Ikeda M Shoji M Kawarai T Kawarabayashi T Matsubara E Murakami T Sasaki A Tomidokoro Y Ikarashi Y Kuribara H Ishiguro K Hasegawa M Yen SH Chishti MA Harigaya Y Abe K Okamoto K St. George-Hyslop P Westaway D Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice.Am J Pathol. 2005; 166: 521-531Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 24Weaver CL Espinoza M Kress Y Davies P Conformational change as one of the earliest alterations of tau in Alzheimer's disease.Neurobiol Aging. 2000; 21: 719-727Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar Antibodies against glial fibrillary acidic protein (GFAP) (1:2000; DAKO, Glostrup, Denmark), synaptophysin (1:400, DAKO) and ubiquitin (Ubi-Q, 1:500)25Murakami T Shoji M Imai Y Inoue H Kawarabayashi T Matsubara E Harigaya Y Sasaki A Takahashi R Abe K Pael-R is accumulated in Lewy bodies of Parkinson's disease.Ann Neurol. 2004; 55: 439-442Crossref PubMed Scopus (125) Google Scholar were also used. Immunofluorescence study was performed with antibodies for tau and glial markers. The samples were incubated with AT8 and either anti-GFAP antibody or Olig2 (a marker for oligodendrocytes, 1:500; IBL, Fujioka, Japan) overnight at room temperature. Then the samples were incubated with fluorescein isothiocyanate-labeled (1:2000; Vector Laboratories) and rhodamine-labeled (1:2000; Chemicon, Temecula, CA) secondary antibody for 3 hours. The sections were examined by immunofluorescence microscopy (BX-51; Olympus, Tokyo, Japan). For electron microscopy, tissue blocks from the brains of 14-month-old TgTauP301L mice were immersed in 2.5% glutaraldehyde, 0.1 mol/L phosphate buffer, pH 7.4, for 4 hours and washed in 0.1 mol/L phosphate buffer containing 7% sucrose. The blocks were then postfixed in 2% osmium tetroxide, dehydrated in ethanol and propylene oxide, and embedded in Quetol 812 (Nisshin EM, Tokyo, Japan). Ultrathin sections were stained with uranyl acetate and lead acetate before observation. For immunoelectron microscopy, pre-embedding was performed. In brief, samples were fixed in periodate-lysine-paraformaldehyde at 4°C, washed in PBS containing graded concentrations of sucrose, and rapidly frozen in liquid nitrogen. The sections (6 to 8 μm) were incubated with AT8 antibody followed by anti-mouse IgG F(ab′)2 conjugated with peroxidase (Vector Laboratories). After immunostaining, the sections were embedded in Quetol 812, and ultrathin sections were prepared. They were observed without additional staining by electron microscopy. Half of each brain was homogenized in 9 vol of Tris-saline buffer with the protease inhibitors [50 mmol/L Tris-HCl and 150 mmol/L NaCl, pH 7.6, 0.5 mmol/L diisopropyl fluorophosphate (DIFP), 0.5 mmol/L phenylmethyl sulfonyl fluoride, 1 μg/ml N-tosyl-l-lysine chloromethyl ketone (TLCK), 1 μg/ml antipain, 1 μg/ml leupeptin, 0.1 μg/ml pepstatin, and 1 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA)] and centrifuged at 55,000 rpm for 60 minutes at 4°C. The supernatant was analyzed as the Tris saline buffer-soluble fraction. The pellet was homogenized again in 4 vol of 1% sarkosyl in Tris saline inhibitors, incubated on ice for 30 minutes, and centrifuged at 55,000 rpm for 60 minutes at 4°C. The pellet was analyzed as the sarkosyl-insoluble fraction. The samples were boiled at 70°C in 4 vol of sodium dodecyl sulfate sample buffer and separated on 4 to 12% NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, CA), and the blots were probed with antibodies tau-C, Tau154, CP27, Alz50, MC1, AT8, or Tg3. Signals were visualized with an enhanced chemiluminescence detection system (Amersham, Buckinghamshire, UK) and quantified using a luminoimage analyzer (LAS 1000-mini; Fuji Film, Tokyo, Japan). TgTauP301L and non-Tg littermates in Okayama (TgTauP301L(O)) were enrolled in behavioral experiments. In this cohort, the Morris water-maze (MWM) test was performed at ages 9 and 12 months for reference and for the working memory tasks.26Janus C Pearson J McLaurin J Mathews PM Jiang Y Schmidt SD Chishti MA Horne P Heslin D French J Mount HT Nixon RA Mercken M Bergeron C Fraser PE St. George-Hyslop P Westaway D A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer's disease.Nature. 2000; 408: 979-982Crossref PubMed Scopus (1382) Google Scholar The eight-arm radial maze (8-ARM) test was performed also in the same cohort at 9 and 13 months.27Egashira N Tanoue A Higashihara F Mishima K Fukue Y Takano Y Tsujimoto G Iwasaki K Fujiwara M V1a receptor knockout mice exhibit impairment of spatial memory in an eight-arm radial maze.Neurosci Lett. 2004; 356: 195-198Crossref PubMed Scopus (63) Google Scholar The number of correct choices in the initial eight chosen arms and the number of errors (defined as choosing arms already visited) were assessed. After all behavior examinations, TgTauP301L(O) and non-Tg littermates were examined histologically. Among TgTauP301L(O) mice, ∼37% had pretangles; 42% had pretangles and GFTs; and 21% had pretangles, GFTs, and NFTs. Two cohorts of old TgTauP301L mice and their non-Tg littermates were studied in Toronto (TgTauP301L(T)). The first cohort (16 Tg and 10 non-Tg littermates) was 16 to 18 months of age, and the second cohort (13 Tg and six non-Tg littermates) was 16 months of age. Each cohort of mice was administered a battery of behavioral tests in the following sequence: an open-field test, a reference memory, and a cued (visible platform) version of the MWM test, followed by a conditioned taste aversion (CTA) test.28Pennanen L Welzl H D'Adamo P Nitsch RM Götz J Accelerated extinction of conditioned taste aversion in P301L tau transgenic mice.Neurobiol Dis. 2004; 15: 500-509Crossref PubMed Scopus (76) Google Scholar, 29Janus C Welzl H Hanna A Lovasic L Lane N St. George-Hyslop P Westaway D Impaired conditioned taste aversion learning in APP transgenic mice.Neurobiol Aging. 2004; 25: 1213-1219Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar After all behavioral tests, the ratio of insoluble to soluble human tau in the brains of TgTauP301L(T) and non-Tg littermates was examined by Western blotting. For statistical analysis, two-way repeated measure analysis of variance with post hoc tests or the unpaired Student's t-test was performed (SPSS version 12 and GraphPad Prism version 4) as indicated in the text or as described in detail in the Supplementary Material (see http://ajp.amjpathol.org). TgTauP301L mice were derived from a series of microinjections also involving wt and TauR406W constructs. All constructs were based on a cDNA encoding the longest from of human tau (2N, 4R) inserted into the cos.Tet hamster PrP cosmid expression vector (Figure 1), with pathologies in some resultant TgTauR406W lines being described previously.23Ikeda M Shoji M Kawarai T Kawarabayashi T Matsubara E Murakami T Sasaki A Tomidokoro Y Ikarashi Y Kuribara H Ishiguro K Hasegawa M Yen SH Chishti MA Harigaya Y Abe K Okamoto K St. George-Hyslop P Westaway D Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice.Am J Pathol. 2005; 166: 521-531Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar From 3 months of age onwards, TgTauP301L mice showed prominent dot-like tau immunoreactivity in the neurons of the pyramidal cell layers of the hippocampus (Figure 2a), amygdala, and cerebral cortex. Thereafter, tau immunoreactivity in TgTauP301L mice spread and progressed with age. By 10 months of age, tau-positive pretangles were observed in numerous neurons. By 12 months of age, perineural or flame-shaped NFTs were observed in the cerebral cortex (Figure 2c), hippocampus, amygdala, basal forebrain nucleus, locus ceruleus, and substantia nigra. NFTs were labeled by Gallyas-Braak (Figure 2d), Bielschowsky (Figure 2e), and thioflavin-S (Figure 2f) staining procedures. The antibodies PHF1, AT8, Alz50, and Ubi-Q-labeled NFTs and numerous neuropil threads (Figure 2, g–j). Staining with MC1 (Figure 2k), CP27 (Figure 2l), and Tg3 (Figure 2m) also detected NFTs in the cerebral cortex. We have not yet found any abnormalities associated with expression of normal human tau in aged mice of a comparable Tg wt line denoted TgTau(wt)25355, implying a crucial pathogenic role for the P301L mutation (not shown). In the spinal cord of TgTauP301L mice, a small number of glial tangles were observed, but NFT formation was not apparent (Figure 2n). Interestingly, florid glial tau pathology occurred independently of (and typically preceded) neuronal tau cytopathology. At 8 months of age, tufted astrocytes and plaque-like deposits were detected in the cerebral cortex (Figure 2o), hippocampus, striatum, brainstem, and spinal cord. The glial plaques were observed in the cerebral cortex of ∼52% of the TgTauP301L older than 12 months of age and closely resembled those seen in CBD or PSP. In some affected animals, fields of glial pathology merged to yield almost confluent immunostaining (Figure 2p). Immunofluorescence study revealed that most of the plaque-like deposits were stained with both AT8 and GFAP (Figure 2, q–s), suggesting that these tau deposits were mainly in astrocytes. The cytoplasm of some oligodendrocytes also showed immunoreactivity for AT8, although the appearance was different from that of the astrocytic plaques (not shown). In homozygous Tg mice, NFTs started to appear at 4 months of age and increased in severity with age, but the rate of appearance of NFTs and GFTs was ∼50% in the homozygous TgTauP301L mice even in the late phase of the disease course. NFTs were extensively observed in the frontotemporal cortex and amygdala at 18 to 24 months of age (Figure 2t). Classic flame-type NFTs in the cortex and globose-type NFTs in the brainstem were also observed. Approximately 27% of TgTauP301L mice older than 12 months of age showed NFTs. Although spinal cord and brainstem pathological features were prominent in previous reports of Tg Tau mice, the severity of cumulative neuropathology in the cortex of aged TgTauP301L mice was remarkable. A low-power view of a 17-month-old heterozygote immunostained with AT8 antibody showed extensive immunostaining corresponding to the appearance of NFTs and GFTs in the hippocampus, frontal cortex, caudate, and accumbens nucleus (Figure 2, u and v). In electron microscopy analyses, massive wavy tubules in the perikarya and proximal dendrites were seen in both the cortical and hippocampal neurons (Figure 3a, arrows). Most tubules were composed of filaments 12 to 22 nm wide and had a wavy appearance. Because the distance between the crossovers of the twist was variable, periodicity was not determined. These wavy tubules were in longitudinally parallel arrays, although they showed branching and crossover (Figure 3, b and c). Immunoelectron microscopy showed that antibody AT8 labeled these filaments (Figure 3d). Aggregates of criss-crossing straight filaments (herringbone-like structure)30Lewis J Dickson DW Lin WL Chisholm L Corral A Jones G Yen SH Sahara N Skipper L Yager D Eckman C Hardy J Hutton M McGowan E Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP.Science. 2001; 293: 1487-1491Crossref PubMed Scopus (1296) Google Scholar were observed (Figure 3e). Astrocytic end-feet around the vessels were packed with wavy filaments (Figure 3f). TgTauP301L showed brain atrophy by 18 months (Figure 4a), whereas non-Tg littermates did not (Figure 4b). Atrophy was especially prominent in the temporal lobe [Figure 4, a (arrow) and c] and hippocampus (Figure 4a, arrowhead). Nissl staining in the hippocampus, amygdala, and the entorhinal cortex showed neurons with degenerated cytoplasm and condensed nuclei. The severity of degeneration paralleled the frequency of NFTs, neuropil threads, and GFTs. Synaptic density was reduced throughout the hippocampus (Figure 4d). In some TgTauP301L mice, neuronal degeneration was so severe that neurons in the pyramidal cell layer in CA1 and CA2 almost disappeared (Figure 4, e and f). Severe reactive astrocytosis (Figure 4h) was also detected. The net expression level of tau in TgTauP301L mice detected by tau-C was 1.7 times that of endogenous mouse tau at 4 months of age (Figure 5a). Western blotting using Tau154 showed the specific expression of a 68-kd band, the longest spliced form of human tau, in the Tris saline-soluble fractions, with the levels being similar in the cortical areas, hippocampus, basal ganglia, cerebellum, brainstem, and spinal cord. With aging, sarkosyl-insoluble forms of human tau became apparent and were phosphorylated in many regions of the central nervous system in Tg mice but were especially increased in the cortical areas, hippocampus, and basal ganglia (Figure 5b). However, more marked phenotypic variation was seen in the accumulation of sarkosyl-insoluble tau compared with soluble tau. The amount of insoluble tau was correlated with the histopathological severity detected by AT8 (Figure 5c). Pair-wise analysis of two TgTauP301L mice with either low or high histopathology showed prominent enhancement of NFT-specific conformational changes (Tg3, Alz50, MC1) and phosphorylation (AT8, PHF1) in the accumulated insoluble tau (Figure 5d). Extensive behavioral testing showed no gross motor deficits in TgTauP301L(O) or TgTauP301L(T) mice. Spatial reference memory, as measured by the MWM test, was normal in TgTauP301L(O) mice at 9 or 12 months of age [TgTauP301L(O) a