Accumulation of Filamentous Tau in the Cerebral Cortex of Human Tau R406W Transgenic Mice
2005; Elsevier BV; Volume: 166; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)62274-2
ISSN1525-2191
AutoresMasaki Ikeda, Toshitaka Kawarai, Takeshi Kawarabayashi, Etsuro Matsubara, Tetsuro Murakami, Atsushi Sasaki, Yasushi Tomidokoro, Yasushi Ikarashi, Hisashi Kuribara, Koichi Ishiguro, Masato Hasegawa, Shu‐Hui Yen, M. Azhar Chishti, Yasuo Harigaya, Kōji Abe, Koichi Okamoto, Peter St George‐Hyslop, David Westaway, Mikio Shoji,
Tópico(s)Nuclear Receptors and Signaling
ResumoMissense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3β and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD). Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3β and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD). Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a familial neurodegenerative disease characterized by autosomal dominant inheritance, personality change, progressive dementia, and parkinsonism. Extensive tau accumulation with neurofibrillary tangles (NFT) and loss of neurons are characteristic pathological changes and are associated with frontotemporal lobe atrophy.1Foster NL Wilhelmsen K Sima AA Jones MZ D'Amato CJ Gilman S Frontotemporal dementia and parkinsonism linked to chromosome 17: a consensus conference.Ann Neurol. 1997; 41: 706-715Crossref PubMed Scopus (606) Google Scholar Following the initial discovery of missense mutations in the tau gene,2Poorkaj P Bird TD Wijsman E Nemens E Garruto RM Anderson L Andreadis A Wiederholt WC Raskind M Schellenberg GD Tau is a candidate gene for chromosome 17 frontotemporal dementia.Ann Neurol. 1998; 43: 815-825Crossref PubMed Scopus (1250) Google Scholar, 3Hutton M Lendon CL Rizzu P Baker M Froelich S Houlden H Pickering-Brown S Chakraverty S Isaacs A Grover A Hackett J Adamson J Lincoln S Dickson D Davies P Petersen RC Stevens M de Graaff E Wauters E van Baren J Hillebrand M Joosse M Kwon JM Nowotny P Heutink P et al.Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2973) Google Scholar, 4Spillantini MG Murrell JR Goedert M Farlow MR Klug A Ghetti B Mutation in the tau gene in familial multiple system tauopathy with presenile dementia.Proc Natl Acad Sci USA. 1998; 95: 7737-7741Crossref PubMed Scopus (1335) Google Scholar numerous exonic and intronic mutations have been reported.5Reed LA Wszolek ZK Hutton M Phenotypic correlations in FTDP-17.Neurobiol Aging. 2001; 22: 89-107Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar The majority of mutations are clustered within or close to the microtubule (MT)-binding domains, or in the 5′-splice site of exon 10.5Reed LA Wszolek ZK Hutton M Phenotypic correlations in FTDP-17.Neurobiol Aging. 2001; 22: 89-107Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar Most of the exonic mutations lead to tau proteins with a decreased ability to promote MT assembly and an increase in self-aggregation.6Hasegawa M Smith MJ Goedert M Tau proteins with FTDP-17 mutations have a reduced ability to promote microtubule assembly.FEBS Lett. 1998; 437: 207-210Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar Some of the exonic, and all of the intronic, mutations cause an increase in the level of four-repeat tau.3Hutton M Lendon CL Rizzu P Baker M Froelich S Houlden H Pickering-Brown S Chakraverty S Isaacs A Grover A Hackett J Adamson J Lincoln S Dickson D Davies P Petersen RC Stevens M de Graaff E Wauters E van Baren J Hillebrand M Joosse M Kwon JM Nowotny P Heutink P et al.Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2973) Google Scholar, 4Spillantini MG Murrell JR Goedert M Farlow MR Klug A Ghetti B Mutation in the tau gene in familial multiple system tauopathy with presenile dementia.Proc Natl Acad Sci USA. 1998; 95: 7737-7741Crossref PubMed Scopus (1335) Google Scholar An increase in four-repeat tau is hypothesized to promote tau self-aggregation and decrease MT assembly.7Spillantini MG Goedert M Crowther RA Murrell JR Farlow MR Ghetti B Familial multiple system tauopathy with presenile dementia: a disease with abundant neuronal and glial tau filaments.Proc Natl Acad Sci USA. 1997; 94: 4113-4118Crossref PubMed Scopus (311) Google Scholar These gain-of-function effects have been suggested to cause tau accumulation leading to NFT formation and neuronal cell death. Several neurodegenerative diseases that display tau accumulation, such as Alzheimer's disease, frontotemporal dementia, Pick's disease, progressive supranuclear palsy, and corticobasal degeneration are now classified as tauopathies.8Spillantini MG Goedert M Tau protein pathology in neurodegenerative diseases.Trends Neurosci. 1998; 21: 428-433Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar It is therefore important to clarify the mechanism by which mutant tau accumulates and leads to NFT formation and to determine whether or not there is a single common pathological pathway of tauopathies. Although pedigrees segregating the tau R406W mutation have a variety of clinical and pathological characteristics, consistent pathological features include frontotemporal atrophy, abundant tau accumulation, and neurofibrillary tangles containing both paired helical filaments and straight filaments.9Reed LA Grabowski TJ Schmidt ML Morris JC Goate A Solodkin A Van Hoesen GW Schelper RL Talbot CJ Wragg MA Trojanowski JQ Autosomal dominant dementia with widespread neurofibrillary tangles.Ann Neurol. 1997; 42: 564-572Crossref PubMed Scopus (164) Google Scholar, 10van Swieten JC Stevens M Rosso SM Rizzu P Joosse M de Koning I Kamphorst W Ravid R Spillantini MG Niermeijer MF Heutink P Phenotypic variation in hereditary frontotemporal dementia with tau mutations.Ann Neurol. 1999; 46: 617-626Crossref PubMed Scopus (205) Google Scholar, 11Saito Y Geyer A Sasaki R Kuzuhara S Nanba E Miyasaka T Suzuki K Murayama S Early-onset, rapidly progressive familial tauopathy with R406W mutation.Neurology. 2002; 58: 811-813Crossref PubMed Scopus (48) Google Scholar The tau R406W mutation has far a weaker effect on MT assembly-promoting activity than that of tau P301L and tau V337M mutations,6Hasegawa M Smith MJ Goedert M Tau proteins with FTDP-17 mutations have a reduced ability to promote microtubule assembly.FEBS Lett. 1998; 437: 207-210Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar and stable or transient transfection of tau R406W in non-neuronal cell lines showed that tau R406W was less phosphorylated than wild-type tau.12Matsumura N Yamazaki T Ihara Y Stable expression in Chinese hamster ovary cells of mutated tau genes causing frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).Am J Pathol. 1999; 154: 1649-1656Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar While the sarcosyl-soluble tau R406W was less phosphorylated than sarcosyl-soluble wild-type tau, sarcosyl-insoluble tau R406W was as highly phosphorylated as the insoluble wild-type tau.13Miyasaka T Morishima-Kawashima M Ravid R Heutink P van Swieten JC Nagashima K Ihara Y Molecular analysis of mutant and wild-type tau deposited in the brain affected by the FTDP-17 R406W mutation.Am J Pathol. 2001; 158: 373-379Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar These results suggested unique molecular effects of tau R406W on NFT formation. For this reason, we generated transgenic (Tg) mice expressing tau R406W (TgTauR406W). These mice were then followed using behavioral, neuropathological, and neurochemical methods. While the majority of these mice did not develop an overt behavioral or neuropathological phenotype, a small proportion of these mice (<20%) developed a behavioral, neuropathological, and neurochemical phenotype that displayed several features reminiscent of human tauopathies. The cause of this variation in expressivity/penetrance is being explored and remains unknown (possibly the effects of genetic background modifiers although environmental effects cannot yet be excluded). Nevertheless, in its most highly expressed form, the TgTauR406W mice developed an illness characterized by extensive accumulation of tau and subsequent alterations in the neocortex, hippocampus, and amygdala associated with motor and memory disturbances. The longest isoform of wild-type human four-repeat tau cDNA containing a eukaryotic Kozak initiation sequence (GCCGCCACC)14Kozak M Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2386) Google Scholar, 15Kozak 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 into the SalI restriction site of the cos.tet expression vector containing the Syrian hamster prion protein promoter gene,16Scott MR Kohler 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 packaged in vitro and plated on E. coli DH1 to obtain a bacterial stock containing the recombinant cosmid clone. To generate the tau R406W mutation, wild-type human four-repeat tau cDNA was mutated by an oligonucleotide-mediated method with a proofreading DNA polymerase ("Quick change", Stratagene, La Jolla, CA). Following confirmation of the site-directed mutagenesis by direct sequencing, the mutated tau R406W cDNA was reintroduced into the cos.tet expression vector. The transgenes were purified and microinjected into fertilized oocytes of FVB/N mice as previously described.17Citron 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, 18Chishti MA Yang DS Janus C Phinney AL Horne P Pearson J Strome R Zuker N Loukides J French J Turner S Lozza G Grilli M Kunicki S Morissette C Paquette J Gervais F Bergeron C Fraser PE Carlson GA George-Hyslop PS Westaway D Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695.J Biol Chem. 2001; 276: 21562-21570Crossref PubMed Scopus (795) Google Scholar Positive founders were subsequently bred with FVB wild-type mice and offspring were genotyped using a human tau cDNA fragment radiolabeled by the random-primer method. To analyze gene expression of human tau, RT-PCR was performed using 2 μl of mRNA isolated using the QuickPrep Micro mRNA purification kit (Amersham Biosciences, UK) from brains of Tg mice (21807) and non-Tg mice brains at 6 and 10 months old (n = 3, respectively) in the reaction tube of Ready-To-Go RT-PCR Beads (Amersham Biosciences, UK) with PCR primers sets as follows: mouse tau exon 9F (5′-CACCAAAATCCGGAGAACGA-3′) and mouse exon 11R (5′-CTTTGCTCAGGTCCACCGGC-3′): human tau exon 9F (5′-CTCCAAAATCAGGGGATCGC-3′) and human tau exon 11R (5′-CCTTGCTCAGGTCAACTGGT-3′). PCR conditions included 30 cycles of 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 45 seconds with a final 72°C extension phase for 10 minutes, as in a previous report.19Duff K Knight H Refolo LM Sanders S Yu X Picciano M Malester B Hutton M Adamson J Goedert M Burki K Davies P Characterization of pathology in transgenic mice overexpressing human genomic and cDNA tau transgenes.Neurobiol Dis. 2000; 7: 87-98Crossref PubMed Scopus (248) Google Scholar For semi-quantification, RT-PCR of β-actin was performed as internal control.20Elliott JL Cytokine upregulation in a murine model of familial amyotrophic lateral sclerosis.Brain Res Mol Brain Res. 2001; 95: 172-178Crossref PubMed Scopus (165) Google Scholar Ten μl of PCR products were analyzed by 2.5% agarose gel elecrophoresis. The intensity of ethidium-stained bands was analyzed by Scion Image (Scion Corporation, USA). Transgenic founders were used to create Tg mice lines bearing the tau R406W mutation: TgTauR406W 21807, TgTauR406W 21783, and TgTauR406W 21768. Twenty-one positive Tg progenies, 16 F1 Tg and 98 F2 Tg were analyzed. The TgTauR406W 21807 line (n = 11) was used for the rotarod and passive avoidance tests. All animal experiments were performed according to guidelines established in the "Guide for the Care and Use of Laboratory Animals." The following antibodies were used: two human tau-specific antibodies, anti-tau154 (1:200; antibody to synthetic amino acids of 154–168 of human tau 441), E1 (1:1000)21Crowe A Ksiezak-Reding H Liu WK Dickson DW Yen SH The N terminal region of human tau is present in Alzheimer's disease protein A68 and is incorporated into paired helical filaments.Am J Pathol. 1991; 139: 1463-1470PubMed Google Scholar and two human and mouse tau antibodies, PHF-1 (1:100, kindly provided by Dr. P. Davies, Albert Einstein College of Medicine),22Greenberg SG Davies P Schein JD Binder LI Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau.J Biol Chem. 1992; 267: 564-569Abstract Full Text PDF PubMed Google Scholar and anti-tau-C against C-terminal tau (1:200, 422–438 amino acid of human tau 441),23Yamaguchi H Ishiguro K Uchida T Takashima A Lemere CA Imahori K Preferential labeling of Alzheimer's neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3β and cyclin-dependent kinase 5, a component of TPK II.Acta Neuropathol (Berl). 1996; 92: 232-241Crossref PubMed Scopus (238) Google Scholar a conformation-dependent tau antibody Alz-50 (1:100, kindly provided by Dr. P Davies);24Wolozin BL Pruchnicki A Dickson DW Davies P A neuronal antigen in the brains of Alzheimer patients.Science. 1986; 232: 648-650Crossref PubMed Scopus (514) Google Scholar an anti-glial fibrillary acidic protein antibody (GFAP, 1:20,000, DAKO, Denmark), an anti-microglia antibody (F4/80, 1:20, BMA Biomedicals, Switzerland), an anti-ubiquitin antibody (1:500, kindly provided by Dr. D. Dickson, Mayo Clinic Jacksonville); phosphorylation-site specific tau antibodies: anti-PS199 (phosphorylated serine 199, 1:500),25Tomidokoro Y Ishiguro K Harigaya Y Matsubara E Ikeda M Park J-M Yasutake K Kawarabayashi T Okamoto K Shoji M Aβ amyloidosis induces the initial stage of tau accumulation in APPsw mice.Neurosci Lett. 2001; 299: 169-172Crossref PubMed Scopus (92) Google Scholar, 26Ishiguro K Sato K Takamatsu M Park J Uchida T Imahori K Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.Neurosci Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar CP13 (phosphorylated at serine 202, 1:100, kindly provided by Dr. P. 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299: 169-172Crossref PubMed Scopus (92) Google Scholar, 26Ishiguro K Sato K Takamatsu M Park J Uchida T Imahori K Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.Neurosci Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar anti-PY216 (anti-activated GSK-3β, 1:250),25Tomidokoro Y Ishiguro K Harigaya Y Matsubara E Ikeda M Park J-M Yasutake K Kawarabayashi T Okamoto K Shoji M Aβ amyloidosis induces the initial stage of tau accumulation in APPsw mice.Neurosci Lett. 2001; 299: 169-172Crossref PubMed Scopus (92) Google Scholar, 26Ishiguro K Sato K Takamatsu M Park J Uchida T Imahori K Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.Neurosci Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar, 29Planel E Yasutake K Fujita SC Ishiguro K Inhibition of protein phosphatase 2A overrides tau protein kinase I/glycogen synthase kinase 3 beta and cyclin-dependent kinase 5 inhibition and results in tau hyperphosphorylation in the hippocampus of starved mouse.J Biol Chem. 2001; 276: 34298-34306Crossref PubMed Scopus (180) Google Scholar anti-PS9 (anti-inactive GSK-3β, 1:25),25Tomidokoro Y Ishiguro K Harigaya Y Matsubara E Ikeda M Park J-M Yasutake K Kawarabayashi T Okamoto K Shoji M Aβ amyloidosis induces the initial stage of tau accumulation in APPsw mice.Neurosci Lett. 2001; 299: 169-172Crossref PubMed Scopus (92) Google Scholar, 26Ishiguro K Sato K Takamatsu M Park J Uchida T Imahori K Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.Neurosci Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar anti-Cdk5 antibody (anti-cyclin-dependent kinase 5, 1:100)25Tomidokoro Y Ishiguro K Harigaya Y Matsubara E Ikeda M Park J-M Yasutake K Kawarabayashi T Okamoto K Shoji M Aβ amyloidosis induces the initial stage of tau accumulation in APPsw mice.Neurosci Lett. 2001; 299: 169-172Crossref PubMed Scopus (92) Google Scholar, 26Ishiguro K Sato K Takamatsu M Park J Uchida T Imahori K Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.Neurosci Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar and anti-MAPK (anti-mitogen-activated protein kinase, 1:250).25Tomidokoro Y Ishiguro K Harigaya Y Matsubara E Ikeda M Park J-M Yasutake K Kawarabayashi T Okamoto K Shoji M Aβ amyloidosis induces the initial stage of tau accumulation in APPsw mice.Neurosci Lett. 2001; 299: 169-172Crossref PubMed Scopus (92) Google Scholar, 26Ishiguro K Sato K Takamatsu M Park J Uchida T Imahori K Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.Neurosci Lett. 1995; 202: 81-84Crossref PubMed Scopus (82) Google Scholar After mice were sacrificed under ether anesthesia, brains were removed and cut sagittally at the midline. One hemisphere was fixed in 4% paraformaldehyde with 0.1 mol/L phosphate buffer (PB, pH 7.6) for 1 week and embedded in paraffin. Five μm-thick sections were prepared for staining. Sections were immersed in 0.5% periodic acid and treated with 99% formic acid for 3 minutes for tau immunostaining. After blocking with 5% normal goat serum in 50 mmol/L phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 4% Block Ace (Snow Brand, Japan), sections were incubated for 6 hours with the primary antibodies. The specific labeling was visualized using a Vectastain Elite ABC kit (Vector Labs, Burlingame, CA). These tissue sections were counterstained with hematoxylin. Conventional Gallyas-Braak silver staining for NFT and Nissl staining for neuronal cell counts were performed. We adopted the terminology defined based on an atlas of the mouse brain.30Paxinos G Franklin KB G Paxinos KB Franklin The Mouse Brain in Stereotaxic Coordinates. ed 2. Academic Press, San Diego2001Google Scholar The number of neurons/mm2 was counted in three Nissl-stained 5-mm sections from the piriform cortex and amygdala of 11 Tg mice, including no. 8859 and no. 9731, and 11 age-matched 10-month-old non-Tg mice. The brain tissues were immersed in a fixative solution (2.5% glutaraldehyde, 0.1 mol/L phosphate buffer, pH 7.4) for 4 hours and washed several times in 0.1 mol/L PB containing 7% sucrose. Blocks were then post-fixed in 2% osmium tetroxide, dehydrated in ethanol and propylene oxide, and embedded in Quetol 812 (Nisshin EM, Japan). Ultra-thin sections were stained with uranyl acetate and lead acetate before observation with an electron microscope. For immuno-electron microscopic study, PBS-washed pellets, after sarcosyl extraction of Tg mice brains, were applied to carbon-coated 400 mesh EM grids (VECO, Holland). The samples were incubated with an anti-tau154 antibody at room temperature for 3 hours. Each specimen was washed with PBS and incubated with 12-nm colloidal gold conjugated anti-rabbit IgG (Jackson Immunoresearch Labs, PA) at room temperature for 1 hour, then examined by electron microscopy (EM). Half of the brains from Tg or non-Tg mice at 5 and 13 months of age were weighed and homogenized using a Teflon-homogenizer in nine volumes of Tris-saline buffer (TS) with protease inhibitors (TS inhibitors: 50 mmol/L Tris-HCl and 150 mmol/L NaCl, pH 7.6, 0.5 mmol/L DIFP, 0.5 mmol/L PMSF, 1 μg/ml TLCK, 1 μg/ml antipain, 1 μg/ml leupeptin, 0.1 μg/ml pepstatin, 1 mmol/L EGTA). The homogenate was centrifuged at 55,000 rpm for 60 minutes at 4°C and the supernatant was analyzed as the TS-soluble fraction. Then, the pellets were homogenized again in four volumes of 1% sarcosyl in TS inhibitors, incubated on ice for 30 minutes, and centrifuged at 55,000 rpm for 60 minutes at 4°C. The supernatant and pellet were analyzed as sarcosyl-soluble or sarcosyl-insoluble fractions, respectively. Each 2 μl of sample was boiled at 70°C in four volumes of sodium dodecylsulfate (SDS) sample buffer and separated on 4 to 12% NuPAGE Bis-Tris Gel (Invitrogen Corp., Carlsbad, CA). The signal intensity was detected using the ECL-Plus system (Amersham Bioscience Corp., NJ) and a luminoimage analyzer (LAS 1000-Mini, Fuji Film, Tokyo). Tg mice (n = 11) and age-matched non-Tg control mice (n = 11) at 10 and 12 months old were assessed for how long they could stay on a rotating rod treadmill apparatus (Ugo Basile, Biological Research Apparatus, Milan, Italy). Mice were placed on the rod rotating at a speed of 16 rpm for 30 seconds, and the time they stayed on the rotating rod was measured. The trial was performed three times and then repeated three additional times after 10 minutes of rest for every mouse. Statistical analysis was conducted by the Mann-Whitney test. Tg mice (n = 11) and age-matched non-Tg control mice (n = 11) at 10 months of age were examined. The apparatus (AP model, O'Hara Co., Tokyo, Japan) for the step-through passive avoidance test consisted of two compartments; one was illuminated [light at the top of the compartment (27 watt, 3000 lux)] and the other was a dark compartment. After the mouse was placed in the illuminated safe compartment, the compartment was lit, and the mouse stepped through an open guillotine door into the dark compartment. The time spent in the illuminated compartment was defined as the latency time. Three seconds after the mouse entered the dark compartment, a foot-shock (0.3 mA, 50 V, 50 Hz AC, for 3 seconds) was given. The retention of avoidance memory trials was carried out once a week for 9 weeks after 5 days of serial acquisition trials. The retention latency time was measured for up to 300 seconds without delivering a foot-shock.31Ikarashi Y Kuribara H Shiobara T Takahashi A Ishimaru H Maruyama Y Learning and memory in mice treated with choline oxidase, a hydrolytic enzyme for choline.Pharmacol Biochem Behav. 2000; 65: 519-522Crossref PubMed Scopus (7) Google Scholar, 32Ikarashi Y Harigaya Y Tomidokoro Y Kanai M Ikeda M Matsubara E Kawarabayashi T Kuribara H Younkin SG Maruyama Y Shoji M Decreased level of brain acetylcholine and memory disturbance in APPsw mice.Neurobiol Aging. 2004; 25: 483-490Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Statistical analysis was performed by a two-way repeated measure analysis of variance (SPSS Version 11). At 3 months of age (n = 5), there was no detectable accumulation of human tau by immunocytochemistry. At 6 months of age (n = 5), tau 154 labeled some neurons and processes in the dentate gyrus of the hippocampus (Figure 1a, arrowhead) and neurons in the amygdala (Figure 1a, arrow). At 8 months of age (n = 5), tau deposition was observed in the cell bodies and processes of neurons of the neocortex, hippocampus, and amygdala (Figure 1b). At 14 months old (n = 4), accumulation of tau protein progressively extended to the caudate putamen, white matter, and cerebellar cortex (Figure 1c). This tau immunoreactivity was enhanced by formic acid pretreatment. Human tau was not detected in the brains of non-Tg control mice at 14 months of age (Figure 1d). Accumulation of human tau was intensely observed in the neocortex and the piriform cortex, amygdala, and hippocampus in 10-month-old Tg mice brains (n = 13) (Figure 1, i to k). In the neocortex, accumulation of tau was prominent in layer II and IV-VI (Figure 1k). Moderate accumulation of tau was observed in the entorhinal cortex, caudate putamen, anterior and posterior horn of the spinal cord, brainstem and cortex of the cerebellum (Figure 1, l to o). The staining of tau in the olfactory bulb, thalamus, and hypothalamus was weak (Figure 1, i to l). Tau was also observed in subcortical areas, such as the corpus callosum, internal capsule, white matter of the cerebellum, and white matter of the spinal cord. No atrophy was observed in skeletal muscles. The accumulated tau localized predominantly in the cell bodies and processes of the neocortical neurons (Figure 1e). Granular dot-like stainings were frequently observed all around the neurons (Figure 1e). No human tau was observed in non-Tg control mice (Figure 1f). Tau accumulated in the cell bodies and dendrites of neurons in the anterior horn of the spinal cord (Figure 1g) and in neuronal processes in the neocortex (Figure 1h). In 10-month-old Tg mice, GFAP immunostaining demonstrated marked astrogliosis in the neocortex, amygdala (Figure 2a, arrow), and hippocampus (Figure 2a, arrowhead) compared with non-Tg mice (Figure 2b). Numerous large GFAP-positive bizarre astrocyte
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