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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

2006; Elsevier BV; Volume: 169; Issue: 2 Linguagem: Inglês

10.2353/ajpath.2006.060002

1525-2191

Katharina Schindowski, Alexis Bretteville, Karelle Leroy, Séverine Bégard, Jean‐Pierre Brion, Malika Hamdane, Luc Buée,

Cholinesterase and Neurodegenerative Diseases

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. 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. Alzheimer's disease (AD) is the most common form of dementia in the elderly and is characterized neuropathologically by the presence of intracellular neurofibrillary tangles (NFTs) and senile plaques in the brain and by a major loss of synaptic connections. NFTs are neuronal inclusions of the microtubule-associated tau protein and are composed of aggregated phosphorylated tau. In AD, NFTs occur in the hippocampus, the entorhinal and polymodal association cortices, and in the basal forebrain. These brain areas are also severely affected by neuronal and synaptic loss. The loss of neurites, synapses, and neurons represent one of the reasons for the cognitive deficits and dementia of AD.1Arendt T Bigl V Arendt A Tennstedt A Loss of neurons in the nucleus basalis of Meynert in Alzheimer's disease, paralysis agitans and Korsakoff's Disease.Acta Neuropathol (Berl). 1983; 61: 101-108Crossref PubMed Scopus (622) Google Scholar, 2Scott SA DeKosky ST Sparks DL Knox CA Scheff SW Amygdala cell loss and atrophy in Alzheimer's disease.Ann Neurol. 1992; 32: 555-563Crossref PubMed Scopus (81) Google Scholar, 3West MJ Coleman PD Flood DG Troncoso JC Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease.Lancet. 1994; 344: 769-772Abstract PubMed Scopus (991) Google Scholar, 4Gomez-Isla T Price JL McKeel Jr, DW Morris JC Growdon JH Hyman BT Profound loss of layer II entorhinal cortex neurons occurs in very mild Alzheimer's disease.J Neurosci. 1996; 16: 4491-4500PubMed Google ScholarIn addition to the neuropathological hallmarks of AD, there are also a number of other pathophysiological disturbances that are observed in AD brain including the inflammatory reaction of glial cells (gliosis).5Blasko I Stampfer-Kountchev M Robatscher P Veerhuis R Eikelenboom P Grubeck-Loebenstein B How chronic inflammation can affect the brain and support the development of Alzheimer's disease in old age: the role of microglia and astrocytes.Aging Cell. 2004; 3: 169-176Crossref PubMed Scopus (287) Google Scholar It is now increasingly recognized that synaptic dysfunction is a major pathophysiological feature of AD. It has been suggested that disruption of neuronal communication is an early event in the disease progression and could occur independently of neuronal loss per se.6Coleman P Federoff H Kurlan R A focus on the synapse for neuroprotection in Alzheimer disease and other dementias.Neurology. 2004; 63: 1155-1162Crossref PubMed Scopus (275) Google ScholarTau transgenic (Tg) and gene-targeted mice are valuable models that reproduce various aspects of tauopathies and AD tau pathology with associated cognitive changes. Several models with single tau mutations, including P301L,7Lewis J McGowan E Rockwood J Melrose H Nacharaju P Van Slegtenhorst M Gwinn-Hardy K Paul Murphy M Baker M Yu X Duff K Hardy J Corral A Lin WL Yen SH Dickson DW Davies P Hutton M Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.Nat Genet. 2000; 25: 402-405Crossref PubMed Scopus (1123) Google Scholar, 8Gotz J Chen F Barmettler R Nitsch RM Tau filament formation in transgenic mice expressing P301L tau.J Biol Chem. 2001; 276: 529-534Crossref PubMed Scopus (396) Google Scholar, 9Santacruz K Lewis J Spires T Paulson J Kotilinek L Ingelsson M Guimaraes A DeTure M Ramsden M McGowan E Forster C Yue M Orne J Janus C Mariash A Kuskowski M Hyman B Hutton M Ashe KH Tau suppression in a neurodegenerative mouse model improves memory function.Science. 2005; 309: 476-481Crossref PubMed Scopus (1523) Google Scholar P301S,10Allen B Ingram E Takao M Smith MJ Jakes R Virdee K Yoshida H Holzer M Craxton M Emson PC Atzori C Migheli A Crowther RA Ghetti B Spillantini MG Goedert M Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein.J Neurosci. 2002; 22: 9340-9351Crossref PubMed Google Scholar G272V,11Gotz J Tolnay M Barmettler R Chen F Probst A Nitsch RM Oligodendroglial tau filament formation in transgenic mice expressing G272V tau.Eur J Neurosci. 2001; 13: 2131-2140Crossref PubMed Google Scholar V337M,12Tanemura K Murayama M Akagi T Hashikawa T Tominaga T Ichikawa M Yamaguchi H Takashima A Neurodegeneration with tau accumulation in a transgenic mouse expressing V337M human tau.J Neurosci. 2002; 22: 133-141Crossref PubMed Google Scholar and R406W,13Tatebayashi Y Miyasaka T Chui DH Akagi T Mishima K Iwasaki K Fujiwara M Tanemura K Murayama M Ishiguro K Planel E Sato S Hashikawa T Takashima A Tau filament formation and associative memory deficit in aged mice expressing mutant (R406W) human tau.Proc Natl Acad Sci USA. 2002; 99: 13896-13901Crossref PubMed Scopus (230) Google Scholar, 14Ikeda 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 (94) Google Scholar or overexpression of human tau isoforms15Brion JP Tremp G Octave JN Transgenic expression of the shortest human tau affects its compartmentalization and its phosphorylation as in the pretangle stage of Alzheimer's disease.Am J Pathol. 1999; 154: 255-270Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 16Ishihara T Hong M Zhang B Nakagawa Y Lee MK Trojanowski JQ Lee VM Age-dependent emergence and progression of a tauopathy in transgenic mice overexpressing the shortest human tau isoform.Neuron. 1999; 24: 751-762Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 17Duff 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 over-expressing human genomic and cDNA tau transgenes.Neurobiol Dis. 2000; 7: 87-98Crossref PubMed Scopus (239) Google Scholar have been generated. However, none of these mouse models fully recapitulates the neuropathological spectrum of tangle pathology observed in AD. Moreover, most published models show motor deficits and hind limb paralysis with increasing pathology caused by tau expression in the spinal cord. Motor dysfunction makes it very difficult to study these animals with behavioral experiments or in the late stages of tau pathology because they usually die earlier. Therefore, our aim was to generate a new tau mouse model without APP pathology that does not display any motor dysfunction to investigate the pure pathogenic tau accumulation and its effects in AD tau pathology. In the present study, we describe the construction and characterization of a mouse model expressing double-mutated human tau that displays several of the key features of tau pathology and other pathological changes, such as loss of synaptic function and impaired behavior, observed in AD and other types of tauopathies.Materials and MethodsGeneration of Tg Mice Expressing G272V and P301S Human TauThe cDNA of the 412 amino acid 4-repeat isoform of human tau (htau 46, cloned from human brain cDNA) was a kind gift from M. Goedert (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK).18Lo MM Fieles AW Norris TE Dargis PG Caputo CB Scott CW Lee VM Goedert M Human tau isoforms confer distinct morphological and functional properties to stably transfected fibroblasts.Brain Res Mol Brain Res. 1993; 20: 209-220Crossref PubMed Scopus (19) Google Scholar To introduce a Kozak-sequence and XhoI site, the cDNA was reamplified with the following primers: Tau5′tg 5′-CTCGAGGATGGCTGAGCCCCGCCAGG-3′ and Tau3′tg 5′-CTCGAGTCACAAACCCTGCTTGGCCAGGGAGG-3′ and mutated at G272V and P301S (numbering according to the longest human tau isoform) by polymerase chain reaction (PCR)-based site-directed mutagenesis (Stratagene, Amsterdam, The Netherlands). This cDNA-construct was inserted into the XhoI-sites of a Thy1.2 expression vector.19Vidal M Morris R Grosveld F Spanopoulou E Tissue-specific control elements of the Thy-1 gene.EMBO J. 1990; 9: 833-840Crossref PubMed Scopus (164) Google Scholar, 20Wirths O Multhaup G Czech C Blanchard V Tremp G Pradier L Beyreuther K Bayer TA Reelin in plaques of beta-amyloid precursor protein and presenilin-1 double-transgenic mice.Neurosci Lett. 2001; 316: 145-148Crossref PubMed Scopus (55) Google Scholar The Thy1.2 promotor specifically drives expression in neurons19Vidal M Morris R Grosveld F Spanopoulou E Tissue-specific control elements of the Thy-1 gene.EMBO J. 1990; 9: 833-840Crossref PubMed Scopus (164) Google Scholar that starts at postnatal day 621Hirrlinger PG Scheller A Braun C Quintela-Schneider M Fuss B Hirrlinger J Kirchhoff F Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice.Mol Cell Neurosci. 2005; 30: 291-303Crossref PubMed Scopus (136) Google Scholar and therefore does not directly affect embryonic development. The vector was then microinjected into a C57BL6/CBA background and backcrossed to C57BL6 for more than five generations. Tg mice were screened for the Tau transgene by PCR analysis of DNA prepared from tail biopsies (DNeasy tissues kit; Qiagen, Courtaboeuf, France). The forward (5′-ATGGCTGAGCCCCGCCAGGAG-3′) and the reverse (5′-TGGAGGTTCACCAGAGCTGGG-3′) primers were used to amplify a 250-bp fragment of Tau DNA. The number of integrated copies was determined by Southern blot analysis.All Tg mice used in the present study were heterozygous. Non-Tg littermates were used as wild-type (WT) controls for all experiments. The chosen line (THY-Tau22) is fertile with normal frequency and size of litters and stably transmits the transgene to its offspring. All experiments on animals were performed in compliance with, and following the approval of, the Centre National de la Recherche Scientifique–Institute of Laboratory Animal Resources Committee, in accordance with standards for the care and use of laboratory animals and with French and European Community rules.Western Blot AnalysisWhole brains were dissected by separating the cortex from the hippocampus and thalamus. Cortical and hippocampal preparations were homogenized in cell-lysis buffer (Cell Signaling Technology, Danvers, MA) using several strokes of sonification and then boiled at 100°C for 10 minutes. For Western analysis, 10 or 25 μg of total protein were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose or polyvinylidene difluoride membranes (all from Invitrogen), incubated with appropriate antibodies (Table 1), and developed using the ECL chemiluminescence kit (Amersham/GE Healthcare, Orsay, France). Protein levels were visualized and quantified using an imaging system (LAS-3000 2.0; Fuji Photo Film Co. Ltd.). Human brain samples were from the Association d'Etudes et de Recherche sur la Maladie d'Alzheimer brain bank.22Mailliot C Sergeant N Bussiere T Caillet-Boudin ML Delacourte A Buee L Phosphorylation of specific sets of tau isoforms reflects different neurofibrillary degeneration processes.FEBS Lett. 1998; 433: 201-204Abstract Full Text PDF PubMed Scopus (100) Google ScholarTable 1Antibodies and Lectin Used in This StudyAntibodySpeciesSpecificityDilutionSourceAT8MouseTau; pSer202/pThr2051:400 (WB); 1:10,000 (ICH/IF)Innogenetics (Gent, Belgium)pSer396RabbitTau; pSer3961:5000Biosource (Camarillo, CA)AT180MouseTau; pThr2311:500InnogeneticsAT270MouseTau; pThr1811:4000 (WB); 1:2000 (IHC)InnogeneticsAP422/988MouseTau; pSer4221:5000Bussiere et al61Bussiere T Hof PR Mailliot C Brown CD Caillet-Boudin ML Perl DP Buee L Delacourte A Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration.Acta Neuropathol (Berl). 1999; 97: 221-230Crossref PubMed Scopus (89) Google ScholarAT100MouseTau; pThr212/pSer2141:1000 (WB); 1:2000 (IHC)InnogeneticsM19GRabbitTau; human-specific, 1 to 19 amino acids1:20,000Bussiere et al61Bussiere T Hof PR Mailliot C Brown CD Caillet-Boudin ML Perl DP Buee L Delacourte A Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration.Acta Neuropathol (Berl). 1999; 97: 221-230Crossref PubMed Scopus (89) Google ScholarMC1MouseTau; conformational epitope, 7 to 9 and 326 to 330 amino acids1:1000P. DaviesPHF-1MouseTau; pSer396/pSer4041:50,000P. DaviesTau-5MouseTau; human/mouse-specific1:10,000BiosourceTP20RabbitTau; human-specific, 33 to 41 amino acids1:2000Brion et al15Brion JP Tremp G Octave JN Transgenic expression of the shortest human tau affects its compartmentalization and its phosphorylation as in the pretangle stage of Alzheimer's disease.Am J Pathol. 1999; 154: 255-270Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar12E8MouseTau; pSer2621:400Seubert et al62Seubert P Mawal-Dewan M Barbour R Jakes R Goedert M Johnson GV Litersky JM Schenk D Lieberburg I Trojanowski JQ Lee VMY Detection of phosphorylated Ser262 in fetal tau, adult tau, and paired helical filament tau.J Biol Chem. 1995; 270: 18917-18922Crossref PubMed Scopus (337) Google ScholarGFAPRabbitGFAP1:20,000 (IF)DAKO (Trappes, France)SynaptophysinRabbitSynaptophysin1:1000Santa Cruz Biotechnology (Santa Cruz, CA)SynaptotagminRabbitSynaptotagmin I1:1000Synaptic System (Göttingen, Germany)LectinSpecificityDilutionSourceBiotinylated tomato lectin (Lycopersicon esculentum)Microglia6 μg/mlSigma-Aldrich L-0651 (Sigma-Aldrich, Lyon, France) Open table in a new tab HistologyImmunohistochemistry and ImmunofluorescenceTg and WT mice from 3 to 17 months were anesthetized and transcardially perfused sequentially with 0.9% NaCl and 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS) (pH 7.4) or 10% formalin and 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4). Brains and spinal cords were removed and stored in 4% paraformaldehyde. Some samples were embedded in paraffin and cut on a sliding microtome at a thickness of 10 μm. Cryosections (14 μm) were cut on a cryostat and mounted on chrome alum-coated slides. Endogenous peroxidase was quenched by treating the section with methanol containing 0.3% H2O2 for 30 minutes. Sections were blocked in 10% horse serum. Primary antibodies were used according to Table 1 and incubated overnight at 4°C in the presence of 1% horse serum. All secondary biotinylated or fluorescein-, Texas Red-, and AMCA-coupled antibodies, fluorochromes, ABC-kit, and 3,3′-diaminobenzidine as chromogen for peroxidase activity were from Vector Laboratories, Burlingame, CA. Incubation with the secondary antibody was done at room temperature for 1 hour. All washing steps (3 × 10 minutes) and antibody dilution were done using phosphate-buffered saline (0.1 mol/L PBS, pH 7.4) or Tris-buffered saline (0.01 mol/L Tris, 0.15 mol/L NaCl, pH 7.4). Incubation with the ABC complex and detection with 3,3′-diaminobenzidine was done according to the manufacturer's manual. Hematoxylin counterstaining was performed according to standard procedures. A minimum of three mice per genotype, age, and sex was used for each determination. Data were analyzed by analysis of variance with Bonferroni's post test.Gallyas Silver ImpregnationFloating cryosections (14 μm) were silver stained according to Braak and Braak.23Braak H Braak E Demonstration of amyloid deposits and neurofibrillary changes in whole brain sections.Brain Pathol. 1991; 1: 213-216Crossref PubMed Scopus (458) Google Scholar4′,6-Diamidino-2-Phenylindole (DAPI) StainingCryosections were mounted in VectaShield containing DAPI (Vector Laboratories).Nissl/Cresylviolet StainingMounted cryosections (14 μm) were rinsed 5 minutes in distilled water and transferred to a 1.5% cresyl-violet solution. Sections were then incubated in a solution containing 1% glacial acetic acid and 16% ethanol in distilled water. Sections were then transferred to an ascending alcohol series (70%, 90%, 96%, and 99% ethanol), and finally to toluene, before they were coverslipped using VectaMount (Vector Laboratories).Semiquantitative Estimation of Cell DensityImages were made using a software-controlled (Leica FW 4000; Leica, Rueil-Malmaison, France) digital camera (Leica DC 300FX) attached to a microscope (Leica DM RB), stored onto hard disk, and analyzed offline. For analysis, images were taken using a ×40 objective. In each image, stained cells were counted in a counting frame.Transmission Electron MicroscopyTg and WT mice were anesthetized with chloral hydrate and perfused intracardially with a solution of 2% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 mol/L phosphate buffer at pH 7.4.15Brion JP Tremp G Octave JN Transgenic expression of the shortest human tau affects its compartmentalization and its phosphorylation as in the pretangle stage of Alzheimer's disease.Am J Pathol. 1999; 154: 255-270Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar Tissue blocks were quickly dissected and further fixed by immersion with 4% (w/v) glutaraldehyde in 0.1 mol/L phosphate buffer at pH 7.4 for 90 minutes. After washing in Millonig's buffer with 0.5% (w/v) sucrose for 24 hours, the tissue sections were postfixed in 2% (w/v) OsO4 for 30 minutes, dehydrated, and embedded in Epon. Semithin sections were stained with toluidine blue. Ultrathin sections were counterstained with uranyl acetate and lead citrate and observed with a Zeiss EM 809 transmission electron microscope at 80 kV. Measurements of the diameter of filaments were performed on digitalized images using the public domain NIH ImageJ program and given in mean ± SD.Electrophysiological RecordingsHippocampal slices (400 μm thick) were prepared from male tau Tgs and WT controls at 6 to 7 and 14 to 15 months of age. Slices were cut transversally on a McIlwain tissue chopper (Campden Instruments, Loughborough, UK) and placed in a submersion-type recording chamber through which artificial cerebrospinal fluid (ACSF: 124 mmol/L NaCl, 3 mmol/L KCl, 1.25 mmol/L NaH2PO4, 1.3 mmol/L MgSO4, 2 mmol/L CaCl2, 26 mmol/L NaHCO3, and 10 mmol/L glucose) was continuously superfused at 2.5 to 3 ml/minute. The ACSF was bubbled with a mixture of 95% O2 and 5% CO2 and maintained at 30°C. Test stimulations (0.1 ms in duration) were delivered at constant intensity every 30 seconds through bipolar stainless steel electrodes placed in the stratum radiatum of the CA1 area. Extracellular field excitatory postsynaptic potentials (EPSPs) were recorded with a monopolar tungsten electrode implanted in the same region. To analyze synaptic transmission efficiency, input/output plots were constructed individually for each slice by applying single stimuli in increments of 20 μA from 100 μA (threshold) to 300 μA. To evaluate synaptic plasticity, a 30-minute baseline was recorded with the stimulation intensity set at half-maximal strength. Long-term potentiation (LTP) was induced by applying a single train of tetanic stimulation at a frequency of 100 Hz for 1 second, after which the size of the EPSP was monitored for 90 minutes.Behavioral TestsModified Irwin ExaminationA comprehensive screen, modified from Irwin,24Irwin S Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse.Psychopharmacologia. 1968; 13: 222-257Crossref PubMed Scopus (1134) Google Scholar was used to determine whether any of the mice exhibited physiological, behavioral, or sensorimotor impairments related to their genotype. To explore motor skills, coordination, and muscle strength, the mice were placed on a wire that was tightened between two 30-cm-high columns and their ability to balance on the wire was assessed. In addition, their ability to grasp and hang on the wire with all four paws for at least 5 seconds and to climb back on the wire was determined.Rotarod TestMotor coordination and balance were tested using an accelerating rotarod (Rota-ROD LE 8200, Bioseb, Chaville, France). The mice were allowed to explore the rotarod for 2 minutes without rotation and then the drum was slowly accelerated to 14 rotations per minute (rpm). The mice were subjected to 2-minute training trials twice a day for 2 consecutive days. On day 3 the rotarod test was performed by placing the mice on the rotating drum (25 rpm) for 2 minutes and the time each animal was able to maintain its balance walking on top of the rod was measured. Data were analyzed by Student's t-test.Elevated Plus MazeThe elevated plus maze is a test of anxiety and motor behavior. The maze was set at a height of 65 cm and consisted of four white Plexiglas arms, each 6 cm wide and 28 cm long with 10-cm-high gray walls surrounding the two enclosed arms. Individual mice were placed in the center of the maze facing an open arm and the total distance and time spent in each arm was measured by a video camera connected to a computer and analyzed with the Videotrack Software (View Point S.A., Champagne au Mont d'Or, France). The total time spent in open and closed arms, the number of arm entries, and the inactive time were calculated. Data were analyzed by unpaired Student's t-tests.Spatial Learning and Memory in the Morris Water Maze (MWM)This experiment was performed in a circular pool, 90 cm in diameter, made of white plastic and filled with milky colored water. An escape platform, 8 cm in diameter, made of clear plastic was submerged 0.5 cm under the water level. Visual clues were provided by different geometrical forms printed in A4-sized letters and placed on the four surrounding walls (distance from the pool was ∼50 to 70 cm). Each mouse was given four trials daily (5- to 7-minute interval between trials, a total of 16 trials) for 4 days. Each trial was performed from one of four different starting points. The movement of the mice was monitored using Videotrack Software (View Point). The time taken to locate the escape platform (escape latency; up to 60 seconds) was determined. After locating the platform the mouse was allowed to sit on it for 15 seconds. Mice who failed to find the platform within 60 seconds were guided to it and allowed to stay on it for 15 seconds. A latency of 60 seconds was entered into the record for such an occurrence. All four trials per day were averaged for statistical analysis, except for the first trial on day 1. On day 9 (5 days after the last training) mice were subjected to a 60-second probe trial in which the platform was removed and the mice were allowed to search for it. The time that each animal spent in each quadrant was recorded (quadrant search time). Four groups of male mice were used at 2 to 3, 7, 10, and 14 months. The 7-month-old Tg and WT mice showed severe freezing behavior (eg, lying motionless in the water and refusing to swim) that strongly interfered with the test and were excluded from the data analysis. All behavioral tests were conducted under a quiet and light-reduced environment. Data were analyzed by Student's t-tests.ResultsGeneration and Selection of THY-Tau Tg MiceThe tau mutations G272V and P301S were generated by site-directed mutagenesis PCR into the human 4-repeat tau cDNA and subcloned into the Thy1.2-expression vector (Figure 1A). After microinjection and implantation, eight viable THY-Tau mouse lines were obtained. A variable number of DNA copies were detected in each of the lines from 1 copy up to 10 copies (Figure 1B). Human tau protein expression in the heterozygous animals from all lines was analyzed in the cortex, hippocampus-enriched fractions, and spinal cord. Five lines expressed low levels of human tau in mouse brain and were excluded. One important aim of this study was to generate a tau Tg mouse line with tau pathology similar to that observed in AD in the absence of any motor dysfunction. Therefore, expression of tau protein in the spinal cord, accompanied by motor deficits and paralysis, was an exclusion criterion for each line. Because mice from line 30 show rather high levels of human tau in spinal cord and starting by the age of 7 to 8 months they display dystonic posture and paralyzed hind limbs (Figure 1, B and C), the line was excluded. Nevertheless, the tau profile in the brain of line 30 is still quite interesting with high levels in hippocampus and rather low levels in the cortex. The tau pathology of line 30 is currently under investigation. Line 52 expresses low levels of the tau construct in hippocampus and cortex and develops tau accumulation at more than 18 months of age. Homozygous animals of line 52 are viable and their characterization is also currently being investigated.By contrast, line 22 showed high levels of human tau protein in brain homogenates, only minor traces in the spinal cord (Figure 1D), and no dystonic hind limbs (Figure 1C) in all ages investigated (up to 18 months) and was therefore chosen as the line for further characterization in this study. Several tissues from line 22 were analyzed for expression of human tau ∼65 kd by immunoblotting. No Tg tau was found in other organs (Figure 1D), even after long overexposure (data not shown). All animals characterized in this study derived from line 22 are heterozygous and are named THY-Tau22. During this study two females (5 to 7 months old) and three males (17 months old) died, representing an overall mortality of 3% in THY-Tau22 mice. No mortality was observed in the littermate WT controls. The Tg mice had a reduced bodyweight compared to their WT littermates by ∼15% (THY-Tau22, 22.87 ± 0.47 g; WT, 26.95 ± 0.44 g, n = 8 males per group, 2 months old, ***P < 0.001; THY-Tau22, 28.25 ± 0.53 g; WT, 34.76 ± 1.25 g, n = 8 males per group, 7 months old, ***P < 0.001; THY-Tau22, 27.42 ± 1.43 g; WT, 32.47 ± 0.76 g, n = 8 males per group, 14 months old, **P < 0.01). This effect was similar in females.AD-Relevant Pathological Tau Hyperphosphorylation and Conformation in THY-Tau22 MiceUnder normal physiological conditions tau is a highly soluble protein that becomes insoluble by conformational changes and pathological phosphorylation in AD and in frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). The result is a translocation of the insoluble tau species from the axon, accumulation in the cell bodies, and formation of NFT.Sections of THY-Tau22 mouse brain and homogenates from dissected cortices were screened with a battery of phospho-specific antibodies (Figure 2, Figure 4 and Supplementary Figure S1 at http://ajp.amjpathol.org). Phosphorylation of tau was present from the age of 3 months (AT8 and AT270) and 6 months (PHF-1, AT100, AT180, and AP422/988; Figure 4A) and abnormal conformational changes of tau using MC1 antibody by 3 months (data not shown). Abnormal tau species were detected in neocortex, the hippocampus—starting early and being very prominent in the CA1 pyramidal layer (Figure 2, C–J) and spreads later to the dentate gyrus (DG) and CA3 subfield —the striatum, the olfactory bulb, the occipital cortex, the amygdala, the ventral thalamic nuclei, and deep layers of the entorhin