Early Axonopathy Preceding Neurofibrillary Tangles in Mutant Tau Transgenic Mice
2007; Elsevier BV; Volume: 171; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.070345
ISSN1525-2191
AutoresKarelle Leroy, Alexis Bretteville, Katharina Schindowski, Emmanuel Gilissen, Michèle Authelet, Robert De Decker, Zehra Yilmaz, Luc Buée, Jean‐Pierre Brion,
Tópico(s)Nerve injury and regeneration
ResumoNeurodegenerative diseases characterized by brain and spinal cord involvement often show widespread accumulations of tau aggregates. We have generated a transgenic mouse line (Tg30tau) expressing in the forebrain and the spinal cord a human tau protein bearing two pathogenic mutations (P301S and G272V). These mice developed age-dependent brain and hippocampal atrophy, central and peripheral axonopathy, progressive motor impairment with neurogenic muscle atrophy, and neurofibrillary tangles and had decreased survival. Axonal spheroids and axonal atrophy developed early before neurofibrillary tangles. Neurofibrillary inclusions developed in neurons at 3 months and were of two types, suggestive of a selective vulnerability of neurons to form different types of fibrillary aggregates. A first type of tau-positive neurofibrillary tangles, more abundant in the forebrain, were composed of ribbon-like 19-nm-wide filaments and twisted paired helical filaments. A second type of tau and neurofilament-positive neurofibrillary tangles, more abundant in the spinal cord and the brainstem, were composed of 10-nm-wide neurofilaments and straight 19-nm filaments. Unbiased stereological analysis indicated that total number of pyramidal neurons and density of neurons in the lumbar spinal cord were not reduced up to 12 months in Tg30tau mice. This Tg30tau model thus provides evidence that axonopathy precedes tangle formation and that both lesions can be dissociated from overt neuronal loss in selected brain areas but not from neuronal dysfunction. Neurodegenerative diseases characterized by brain and spinal cord involvement often show widespread accumulations of tau aggregates. We have generated a transgenic mouse line (Tg30tau) expressing in the forebrain and the spinal cord a human tau protein bearing two pathogenic mutations (P301S and G272V). These mice developed age-dependent brain and hippocampal atrophy, central and peripheral axonopathy, progressive motor impairment with neurogenic muscle atrophy, and neurofibrillary tangles and had decreased survival. Axonal spheroids and axonal atrophy developed early before neurofibrillary tangles. Neurofibrillary inclusions developed in neurons at 3 months and were of two types, suggestive of a selective vulnerability of neurons to form different types of fibrillary aggregates. A first type of tau-positive neurofibrillary tangles, more abundant in the forebrain, were composed of ribbon-like 19-nm-wide filaments and twisted paired helical filaments. A second type of tau and neurofilament-positive neurofibrillary tangles, more abundant in the spinal cord and the brainstem, were composed of 10-nm-wide neurofilaments and straight 19-nm filaments. Unbiased stereological analysis indicated that total number of pyramidal neurons and density of neurons in the lumbar spinal cord were not reduced up to 12 months in Tg30tau mice. This Tg30tau model thus provides evidence that axonopathy precedes tangle formation and that both lesions can be dissociated from overt neuronal loss in selected brain areas but not from neuronal dysfunction. Many neurodegenerative diseases are characterized by the presence of filamentous aggregates in neurons and/or in glial cells and composed of abnormally and hyperphosphorylated forms of the microtubule-associated protein tau. These diseases have been referred to as tauopathies1Lee VMY Goedert M Trojanowski JQ Neurodegenerative tauopathies.Annu Rev Neurosci. 2001; 24: 1121-1159Crossref PubMed Scopus (2218) Google Scholar and include some forms of frontotemporal dementia, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and Alzheimer's disease (AD). Several of these tauopathies are characterized by both cognitive and motor dysfunction and widespread tau pathology in brain, brainstem, and spinal cord. For example, cognitive dysfunction and extrapyramidal symptoms are observed in patients with corticobasal degeneration or progressive supranuclear palsy. In the Guam amyotrophic lateral sclerosis-parkinsonism-dementia complex,2Rodgers-Johnson P Garruto RM Yanagihara R Chen KM Gajdusek DC Gibbs Jr, CJ Amyotrophic lateral sclerosis and parkinsonism-dementia on Guam: a 30-year evaluation of clinical and neuropathologic trends.Neurology. 1986; 36: 7-13Crossref PubMed Google Scholar and in a subset of frontotemporal dementia,3Lomen-Hoerth C Anderson T Miller B The overlap of amyotrophic lateral sclerosis and frontotemporal dementia.Neurology. 2002; 59: 1077-1079Crossref PubMed Scopus (581) Google Scholar patients present with motorneuron disease and dementia. 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Neurofibrillary tangles (NFTs) developed in mice expressing mutant tau, but the exact relationship between their formation and neuronal dysfunction and death has not been firmly established. Recent findings suggested that formation of NFTs could be dissociated from other pathologies in P301L mutant tau mice.16Santacruz 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 (1630) Google Scholar, 22Spires TL Orne JD SantaCruz K Pitstick R Carlson GA Ashe KH Hyman BT Region-specific dissociation of neuronal loss and neurofibrillary pathology in a mouse model of tauopathy.Am J Pathol. 2006; 168: 1598-1607Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar To investigate the relationship between NFT formation and pathological phenotypes in other mutant tau models, we have generated a new transgenic mouse line (Tg30tau) expressing in the forebrain and the spinal cord a human tau protein bearing two pathogenic mutations (P301S and G272V). The P301S tau mutation causes frontotemporal dementia (FTD) or corticobasal degeneration23Bugiani 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 (362) Google Scholar and is associated to widespread tau pathology and an early age of onset. The G272V mutation24Hutton 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 Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2988) Google Scholar was identified in a Dutch family with FTDP-17.25Heutink P Stevens M Rizzu P Bakker E Kros JM Tibben A Niermeijer MF van Duijn CM Oostra BA van Swieten JC Hereditary frontotemporal dementia is linked to chromosome 17q21–q22: a genetic and clinicopathological study of three Dutch families.Ann Neurol. 1997; 41: 150-159Crossref PubMed Scopus (160) Google Scholar We previously characterized another tau transgenic line expressing this mutant tau protein only in the forebrain and developing neurofibrillary lesions in the hippocampus and the cortex: these mice showed delayed learning and reduced spatial memory but no motor deficits.26Schindowski K Bretteville A Leroy K Begard S Brion JP Hamdane M Buee L 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.Am J Pathol. 2006; 169: 599-616Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar The transgenic mice described here developed brain atrophy and a motor neuron disease with axonopathy preceding neurofibrillary pathology and without overt neuronal loss in the hippocampus and the spinal cord, suggesting that these lesions can lead to neuronal dysfunction without cell death in these brain areas. The generation of transgenic line Tg30tau has been previously reported,26Schindowski K Bretteville A Leroy K Begard S Brion JP Hamdane M Buee L 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.Am J Pathol. 2006; 169: 599-616Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar but the pathological analysis of line Tg30tau has not been described before and is presented here. These mice express a mutant tau transgene (1N4R human tau isoform) mutated at positions G272V and P301S, under the control of a modified thy-1 promoter to drive expression in neurons.27Vidal M Morris R Grosveld F Spanopoulou E Tissue-specific control elements of the Thy-1 gene.EMBO J. 1990; 9: 833-840Crossref PubMed Scopus (168) Google Scholar, 28Wirths O Multhaup G Czech C Blanchard V Tremp G Pradier L Beyreuther K Bayer TA Reelin in plaques of β-amyloid precursor protein and presenilin-1 double-transgenic mice.Neurosci Lett. 2001; 316: 145-148Crossref PubMed Scopus (55) Google Scholar Only heterozygous animals were used in the present study; nontransgenic littermates were used as wild-type controls. Transgenic animals were identified by polymerase chain reaction amplification of the tau transgene in genomic DNA as reported.29Boutajangout A Authelet M Blanchard V Touchet N Tremp G Pradier L Brion JP Cytoskeletal abnormalities in mice transgenic for human tau and familial Alzheimer's disease mutants of APP and presenilin-1.Neurobiol Dis. 2004; 15: 47-60Crossref PubMed Scopus (77) Google Scholar All studies on animals were performed in compliance and following approval of the school of medicine ethics committee for animal care and use. Motor deficits in Tg30tau mice were assessed by testing on a Rotarod apparatus (Ugo Basile, Comerio, Italy) at 8, 10, and 12 months of age. Animals were first submitted to training sessions (three trials per day during 3 consecutive days) during which they were placed on the rod rotating at constant speed (4 rpm). Animals were individually separated the day before the test and randomly evaluated on two consecutive test sessions, using an experimental setting of progressive acceleration from 4 to 40 rpm throughout 300 seconds. The latency to fall off the Rotarod was recorded. Animals staying up to 300 seconds were removed from the Rotarod, and their latency to fall recorded as 300 seconds. The brains and spinal cords of wild-type and Tg30tau transgenic mice were dissected at 1, 3, 6, and 12 months. After wet weight estimation, tissues were fixed in 10% formalin or in 4% (w/v) paraformaldehyde and embedded in paraffin. Formalin-fixed sections (10-μm thick) were stained with hematoxylin and eosin, with the Nissl method, with the Gallyas silver-staining method, or with Congo Red and thioflavin. Tissue sections were examined with a Zeiss Axioplan microscope (Carl Zeiss GmbH, Jena, Germany) and digital images acquired using an Axiocam HRc camera (Carl Zeiss). The density of Gallyas-positive cell profiles in the cortex, in the hippocampus, and in the spinal cord was expressed as the total number of Gallyas-positive cell profiles visible on whole sagittal (cortex and hippocampus) or coronal (lumbar spinal cord) tissue section of 3-, 6-, and 12-month-old mice (n = 3 different animals at each age). The B19 antibody is a rabbit polyclonal antibody raised to adult bovine tau proteins, reacting with all adult and fetal tau isoforms in bovine, rat, mouse, and human nervous tissue in a phosphorylation-independent manner.30Brion JP Hanger DP Couck AM Anderton BH A68 proteins in Alzheimer's disease are composed of several tau isoforms in a phosphorylated state which affects their electrophoretic mobilities.Biochem J. 1991; 279: 831-836Crossref PubMed Scopus (108) Google Scholar The TP20 and M19G rabbit polyclonal antibodies were raised against a synthetic peptide of human tau and react with human tau and not with mouse tau.8Brion 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 disease.Am J Pathol. 1999; 154: 255-270Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 31Buée-Scherrer V Condamines O Mourton-Gilles C Jakes R Goedert M Pau B Delacourte A AD2, a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer's disease.Mol Brain Res. 1996; 39: 79-88Crossref PubMed Scopus (148) Google Scholar The TP007 and TP70 rabbit polyclonal antibodies were raised against synthetic peptides mapping at the extreme N and C termini of human tau, respectively.32Davis DR Brion J-P Couck A-M Gallo J-M Hanger DP Ladhani K Lewis C Miller CCJ Rupniak T Smith C Anderton BH The phosphorylation state of the microtubule-associated protein tau as affected by glutamate, colchicine and b-amyloid in primary rat cortical neuronal cultures.Biochem J. 1995; 309: 941-949Crossref PubMed Scopus (78) Google Scholar, 33Brion JP Couck AM Robertson J Loviny TLF Anderton BH Neurofilament monoclonal antibodies RT97 and 8D8 recognize different modified epitopes in PHF-tau in Alzheimer's disease.J Neurochem. 1993; 60: 1372-1382Crossref PubMed Scopus (65) Google Scholar The anti-cleaved tau (Asp421) mouse monoclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The mouse monoclonal antibody AD2 is specific for tau phosphorylated at Ser396.31Buée-Scherrer V Condamines O Mourton-Gilles C Jakes R Goedert M Pau B Delacourte A AD2, a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer's disease.Mol Brain Res. 1996; 39: 79-88Crossref PubMed Scopus (148) Google Scholar The AT8, AT180, AT270, and AT100 mouse monoclonal antibodies (purchased from Innogenetics, Ghent, Belgium) are specific for tau phosphorylated at Ser202 and Thr205 (AT8),34Goedert M Jakes R Vanmechelen E Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205.Neurosci Lett. 1995; 189: 167-170Crossref PubMed Scopus (495) Google Scholar at Thr231 (AT180), at Thr181 (AT270),35Goedert M Jakes R Crowther RA Cohen P Vanmechelen E Vandermeeren M Cras P Epitope mapping of monoclonal antibodies to the paired helical filaments of Alzheimer's disease: identification of phosphorylation sites in tau protein.Biochem J. 1994; 301: 871-877Crossref PubMed Scopus (354) Google Scholar and a specific PHF-tau epitope involving phosphorylation at Thr212 and Ser214 (AT100).36Hoffmann R Lee VMY Leight S Varga I Otvos Jr, L Unique Alzheimer's disease paired helical filament specific epitopes involve double phosphorylation at specific sites.Biochemistry. 1997; 36: 8114-8124Crossref PubMed Scopus (151) Google Scholar The mouse monoclonal antibodies PHF-1 (kindly provided by Drs. P. Davies and S. Greenberg, Albert Einstein College of Medicine, Bronx, NY) and AP422 (kindly provided by Drs. M. Hasegawa and M. Goedert, Medical Research Council-Laboratory of Molecular Biology, Cambridge, UK) are specific for tau phosphorylated at Ser396/404 and Ser422, respectively.37Hasegawa M Jakes R Crowther RA Lee VMY Ihara Y Goedert M Characterization of mAb AP422, a novel phosphorylation-dependent monoclonal antibody against tau protein.FEBS Lett. 1996; 384: 25-30Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 38Otvos Jr, L Feiner L Lang E Szendrei GI Goedert M Lee VM-Y Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404.J Neurosci Res. 1994; 39: 669-673Crossref PubMed Scopus (415) Google Scholar The tau monoclonal antibody MC1 (kindly provided by Dr. P. Davies) recognizes a conformational epitope requiring both an N-terminal fragment and a C-terminal fragment.39Jicha GA Bowser R Kazam IG Davies P Alz-50 and MC-1, a new monoclonal antibody raised to paired helical filaments, recognize conformational epitopes on recombinant tau.J Neurosci Res. 1997; 48: 128-132Crossref PubMed Scopus (422) Google Scholar BioSource (Nivelles, Belgium) provided the phosphospecific rabbit polyclonal tau antibodies to pThr212, pSer214, pSer262, pThr403, and pSer409. The mouse monoclonal antibody to ubiquitin (clone MAB1510) was from Chemicon (Temecula, CA). The mouse monoclonal antibodies to α-tubulin (clone DM1-A), to β-actin (clone AC15), and to GFAP (clone GA5) were from Sigma (Bornem, Belgium). The polyclonal rabbit antibodies to neurofilament L (NA1214) and M (NA1216), to phosphorylated neurofilament H (NA 1211) and the mouse monoclonal antibody to neurofilament H (clone SMI32) and phosphorylated neurofilament H (SMI31) were from Affiniti (Exeter, UK). The anti-phosphotyrosine antibodies 4G10 and PT-66 were purchased from Upstate Biotechnology and Sigma. For detection of Aβ, we used a polyclonal antibody raised to amino acids 12 to 28 of the Aβ peptide.40Brion JP Couck AM Bruce M Anderton BH Flament-Durand J Synaptophysin and chromogranin A immunoreactivities in senile plaques of Alzheimer's disease.Brain Res. 1991; 539: 143-150Crossref PubMed Scopus (58) Google Scholar Several antibodies to protein kinases or phosphatases were also used. The mouse monoclonal antibodies to cdc2 (clone 17) and cdk5 (clone DC17) and polyclonal antibodies to cdk5 (C-8 and H291), p35, ERK1 (C-16), and casein kinase Id (R-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse monoclonal antibody to ERK1 was from Affiniti and to Jun kinase (phospho Thr183/Tyr185) from Cell Signaling (Suffolk, UK). The anti-GSK-3β (TPK1) antibody was from Transduction Laboratories (San Jose, CA). The antibodies to GSK-3 phosphorylated on Tyr216 were from BioSource and from Upstate Biotechnology. The antibody to active caspase3 was from Promega (Madison, WI). Brain and spinal cord of Tg30tau and wild-type mice were quickly dissected after lethal anesthesia and separately homogenized in a buffer containing proteases and phosphatase inhibitors (50 mmol/L Tris, 10 mmol/L ethylenediamine tetraacetic acid, pH 7.4, 1 mmol/L phenylmethyl sulfonyl fluoride, 25 μg/ml leupeptin, 25 μg/ml pepstatin, 1 mmol/L Na3VO4, 10 mmol/L Na4P2O7.10 H2O, and 20 mmol/L NaF). Samples were mixed with sodium dodecyl sulfate sample buffer containing a reducing agent and then boiled at 100°C for 10 minutes (all done as recommended by Invitrogen). For Western blot analysis, 10 μg of total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NuPAGE; Invitrogen, Carlsbad, CA), blotted onto nitrocellulose or polyvinylidene difluoride membranes (Hybond and Hybond Phosphate from Amersham/GE Health Care, Orsay, France). The membranes were blocked and incubated with the appropriate primary antibody and then incubated with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit A4914 from Sigma-Aldrich, Lyon, France; and horse anti-mouse from Vector Laboratories, Burlingame, CA). Finally, the peroxidase activity was detected with the ECL detection kit and visualized with Hyperfilm ECL (Amersham/GE Health Care). The immunohistochemical labeling was performed using the ABC method. In brief, tissue sections were treated with H2O2 to inhibit endogenous peroxidase and incubated with the blocking solution [10% (v/v) normal horse serum in Tris-buffered saline (0.01 mol/L Tris and 0.15 mol/L NaCl, pH 7.4)]. After an overnight incubation with the diluted primary antibody, the sections were sequentially incubated with horse anti-mouse antibodies conjugated to biotin (Vector Laboratories, Burlingame, CA) followed by the ABC complex (Vector). The peroxidase activity was revealed using diaminobenzidine as chromogen. For immunolabeling with the Aβ antibodies, rehydrated tissue sections were pretreated with 100% formic acid for 10 minutes before incubation with the blocking solution. Double immunolabeling was performed using fluorescent markers. The first antibody was detected using an anti-rabbit or an anti-mouse antibody conjugated to fluorescein isothiocyanate (Jackson, Boston, MA), and the second antibody was detected using an anti-rabbit or an anti-mouse antibody conjugated to biotin, followed by streptavidin conjugated to Alexa 594 (Molecular Probes, Carlsbad, CA). Double immunolabeling was followed by nuclear 4,6-diamidino-2-phenylindole staining. Selected areas in tissue sections were photographed after double-fluorescent immunolabeling and stained with the Gallyas silver staining method to identify neurons containing fibrillary material. The number of phosphotau-positive cell profiles in the cortex, in the hippocampus, and in the gray matter of the spinal cord was determined in 3-, 6-, and 12-month-old mice as described above for Gallyas staining. TUNEL staining was performed on tissue sections to detect DNA cleavage associated with apoptotic cell death using digoxigenin-dUTP, as previously reported.41Boutajangout A Leroy K Touchet N Authelet M Blanchard V Tremp G Pradier L Brion JP Increased tau phosphorylation but absence of formation of neurofibrillary tangles in mice double transgenic for human tau and Alzheimer mutant (M146L) presenilin-1.Neurosci Lett. 2002; 318: 29-33Crossref PubMed Scopus (35) Google Scholar For stereological analysis, wild-type and transgenic animals were intracardially perfused with 10% (v/v) formalin, and their brain and spinal cord were dissected and embedded in paraffin. Brains and lumbar spinal cord were serially cut in coronal sections (15-μm thickness) that were then stained with Cresyl violet. The total numbers of hippocampal pyramidal cells was estimated in the left hemispheres of 12-month-old transgenic mice (n = 3) and wild-type mice (n = 3). For each animal, 10 coronal sections at 75-μm intervals throughout the hippocampal pyramidal cell layer were used. The pyramidal cell layer was delineated as depicted in Figure 3C. Counting of total numbers of neurons was performed with a ×100/1.30 oil objective by using a random-sampling stereological counting tool, the optical fractionator,42West MJ Slomianka L Gundersen HJ Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator.Anat Rec. 1991; 231: 482-497Crossref PubMed Scopus (2654) Google Scholar, 43Williams RW Rakic P Three-dimensional counting: an accurate and direct method to estimate numbers of cells in sectioned material.J Comp Neurol. 1988; 278: 344-352Crossref PubMed Scopus (388) Google Scholar which is implemented within a stereology workstation consisting of a modified light microscope (Leica DMR, Wetzlar, Germany), Leica PL Fluotar objectives (×10/0.30; ×100/1.30 oil), a motorized specimen stage for automatic sampling (BioPoint 2; Ludl Electronic Products, Hawthorne, NY), an electronic microcator (Heidenhain, Traunreut, Germany), a charge-coupled device color video camera (HV-C20A; Hitachi, Tokyo, Japan), and a personnel computer with stereology software (Stereoinvestigator version 5.05.4; MicroBrightField, Inc., Colchester, VT). Optical disectors were automatically randomly distributed throughout the delineated area, and each pyramidal cell whose nuclear top came into focus within an optical disector was counted. Estimated total numbers of pyramidal cells per specimen were hence calculated from the number of counted neurons, the volume of the pyramidal cell layer, and the sampling probability.44Schmitz C Hof PR Recommendations for straightforward and rigorous methods of counting neurons based on a computer simulation approach.J Chem Neuroanat. 2000; 20: 93-114Crossref PubMed Scopus (222) Google Scholar Supplemental Table 1 (see http://ajp.amjpathol.org) summarizes the details of the counting procedure. For measuring the volume of the hippocampal pyramidal cell layer, the projection area of this cell layer was delineated on the sections used for cell counting, and its projection area was measured on all ana
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