Region-specific Dissociation of Neuronal Loss and Neurofibrillary Pathology in a Mouse Model of Tauopathy
2006; Elsevier BV; Volume: 168; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2006.050840
ISSN1525-2191
AutoresTara L. Spires‐Jones, Jennifer D. Orne, Karen S. SantaCruz, Rose Pitstick, George A. Carlson, Karen H. Ashe, Bradley T. Hyman,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoNeurofibrillary tangles form in a specific spatial and temporal pattern in Alzheimer's disease. Although tangle formation correlates with dementia and neuronal loss, it remains unknown whether neurofibrillary pathology causes cell death. Recently, a mouse model of tauopathy was developed that reversibly expresses human tau with the dementia-associated P301L mutation. This model (rTg4510) exhibits progressive behavioral deficits that are ameliorated with transgene suppression. Using quantitative analysis of PHF1 immunostaining and neuronal counts, we estimated neuron number and accumulation of neurofibrillary pathology in five brain regions. Accumulation of PHF1-positive tau in neurons appeared between 2.5 and 7 months of age in a region-specific manner and increased with age. Neuron loss was dramatic and region-specific in these mice, reaching over 80% loss in hippocampal area CA1 and dentate gyrus by 8.5 months. We observed regional dissociation of neuronal loss and accumulation of neurofibrillary pathology, because there was loss of neurons before neurofibrillary lesions appeared in the dentate gyrus and, conversely, neurofibrillary pathology appeared without major cell loss in the striatum. Finally, suppressing the transgene prevented further neuronal loss without removing or preventing additional accumulation of neurofibrillary pathology. Together, these results imply that neurofibrillary tangles do not necessarily lead to neuronal death. Neurofibrillary tangles form in a specific spatial and temporal pattern in Alzheimer's disease. Although tangle formation correlates with dementia and neuronal loss, it remains unknown whether neurofibrillary pathology causes cell death. Recently, a mouse model of tauopathy was developed that reversibly expresses human tau with the dementia-associated P301L mutation. This model (rTg4510) exhibits progressive behavioral deficits that are ameliorated with transgene suppression. Using quantitative analysis of PHF1 immunostaining and neuronal counts, we estimated neuron number and accumulation of neurofibrillary pathology in five brain regions. Accumulation of PHF1-positive tau in neurons appeared between 2.5 and 7 months of age in a region-specific manner and increased with age. Neuron loss was dramatic and region-specific in these mice, reaching over 80% loss in hippocampal area CA1 and dentate gyrus by 8.5 months. We observed regional dissociation of neuronal loss and accumulation of neurofibrillary pathology, because there was loss of neurons before neurofibrillary lesions appeared in the dentate gyrus and, conversely, neurofibrillary pathology appeared without major cell loss in the striatum. Finally, suppressing the transgene prevented further neuronal loss without removing or preventing additional accumulation of neurofibrillary pathology. Together, these results imply that neurofibrillary tangles do not necessarily lead to neuronal death. Neurofibrillary tangles, which are characteristic of Alzheimer's disease (AD) and other tauopathies, consist of paired helical filaments of hyperphosphorylated tau protein.1Kidd M Paired helical filaments in electron microscopy in Alzheimer's disease.Nature. 1963; 197: 192-193Crossref PubMed Scopus (770) Google Scholar, 2Brion JP Couck AM Passareiro E Flament-Durand J Neurofibrillary tangles of Alzheimer's disease: an immunohistochemical study.J Submicrosc Cytol. 1985; 17: 89-96PubMed Google Scholar, 3Grundke-Iqbal I Iqbal K Tung YC Quinlan M Wisniewski HM Binder LI Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology.Proc Natl Acad Sci USA. 1986; 83: 4913-4917Crossref PubMed Scopus (2896) Google Scholar, 4Kosik KS Joachim CL Selkoe DJ Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.Proc Natl Acad Sci USA. 1986; 83: 4044-4048Crossref PubMed Scopus (1148) Google Scholar In AD, these lesions accumulate with well defined spatial and temporal progression and correlate with cognitive decline and neuron loss.5Ball MJ Neuronal loss, neurofibrillary tangles and granulovacuolar degeneration in the hippocampus with ageing and dementia. 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However, although neurofibrillary tangles correlate with neuronal loss in AD, the amount of loss far exceeds the amount of tangle formation.8Gomez-Isla T Hollister R West H Mui S Growdon JH Petersen RC Parisi JE Hyman BT Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease.Ann Neurol. 1997; 41: 17-24Crossref PubMed Scopus (1139) Google Scholar Thus, whether tangles cause neuronal death remains unclear. The P301L mutation in the tau protein is associated with a hereditary tauopathy characterized by dementia and development of neurofibrillary pathology.10Hutton 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 Che LK Norton J Morris JC Reed LA Trojanowski JQ Basun H Lannfelt L Neystat M Fahn S Dark F Tannenberg T Dodd PR Hayward N Kwok JB Schofield PR Andreadis A Snowden JS Craufurd D Neary D Owen F Oostra BA Hardy J Goate A Van Swieten JC Mann DM Lynch T Heutink P Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17.Nature. 1998; 393: 702-705Crossref PubMed Scopus (2927) Google Scholar A recently developed reversible mouse model of tauopathy, the rTg4510 strain, exhibits progressive memory deficits that can be prevented and even ameliorated by transgene suppression.11SantaCruz 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 (1587) Google Scholar Initial investigations of these mice revealed spatial memory deficits, neurofibrillary lesions, and dramatic neuron loss in the CA1 area of the hippocampus.11SantaCruz 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 (1587) Google Scholar Although the transgene is ubiquitously expressed throughout the cortex, limbic system, and basal ganglia, we noticed that different regions appeared to accumulate neurofibrillary changes and neuronal loss differentially. In tauopathies, formation of neurofibrillary lesions and neuronal loss also occur with regional specificity. For example, in AD the entorhinal cortex and hippocampus are particularly vulnerable12Braak H Braak E Neuropathological staging of Alzheimer-related changes.Acta Neuropathol (Berl). 1991; 82: 239-259Crossref PubMed Scopus (11766) Google Scholar with pathology occurring later in other cortical and subcortical areas, whereas in frontotemporal dementias neuronal loss is most prominent in frontal and temporal neocortex. 13Mirra SS Hyman BT Ageing and dementia/Alzheimer's disease.in: Graham DI Lantos PL Greenfield's Neuropathology. Arnold, New York2002: 200-226Google Scholar Here, we describe characterization of region-specific neuronal loss and neurofibrillary changes in rTg4510 mice using stereological methods and find a region-specific dissociation between neuronal loss and the accumulation of neurofibrillary pathology. In this study, we used a recently developed mouse model of tauopathy (rTg4510) in which expression of human tau containing the frontotemporal dementia-associated P301L mutation can be suppressed with doxycycline (dox).11SantaCruz 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 (1587) Google Scholar The human four-repeat tau gene with the P301L mutation is downstream of a tetracycline-operon responsive element. To express tau, this tau responder gene must be co-expressed with an activator transgene consisting of the tet-off open reading frame,14Gossen M Bujard H Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.Proc Natl Acad Sci USA. 1992; 89: 5547-5551Crossref PubMed Scopus (4268) Google Scholar which is downstream of Ca2+-calmodulin kinase II promoter elements, resulting in P301L tau expression restricted to forebrain structures.15Mayford M Bach ME Huang YY Wang L Hawkins RD Kandel ER Control of memory formation through regulated expression of a CaMKII transgene.Science. 1996; 274: 1678-1683Crossref PubMed Scopus (1110) Google Scholar Tau-expressing mice (transgenic for both a tau responder transgene and an activator transgene) and littermate control mice that do not express tau (lacking either the tau responder transgene or the activator transgene) between 2.5 and 10 months of age were used. No differences were observed between control mice lacking the tau responder transgene and those lacking the activator transgene. dox administration of 200 ppm in the food was used to suppress the tau transgene for 6 weeks. Control mice were also treated with dox. Animals were housed and treated according to institutional and National Institutes of Health standards. Tissue from the temporal and frontal lobes of one human subject was obtained from the Alzheimer's Disease Research Center at Massachusetts General Hospital. The subject had frontotemporal dementia and carried the P301L tau mutation (70-year-old male, postmortem interval of 12 hours). Tg4510 brains drop-fixed in formalin were embedded in paraffin, and 16-μm parasaggital sections were cut on a Leica RM2155 microtome. Every tenth section (selected on a systematic random basis) was deparaffinized for immunochemistry and blocked in 5% milk for 1 hour. Sections were then immunostained to label tau phosphorylated at serine 396 and 404 with anti-PHF1 primary antibody (1:200 in 5% milk in Tris-buffered saline courtesy of Dr. Peter Davies, Albert Einstein College of Medicine) and horseradish peroxidase-conjugated secondary (1:500 in 5% milk in Tris-buffered saline) developed with diaminobenzidine (0.3 mg/ml in 100 mmol/L Tris plus 0.02% H2O2). Nuclei were counterstained using cresyl violet. Other sections were immunostained with PHF1, CP13 (Peter Davies), or MC1 (Peter Davies) and a secondary anti-mouse IgG conjugated to Cy3 dye (1:200; Jackson ImmunoResearch, West Grove, PA) and then counterstained with Thioflavine S (0.05% in 50% ethanol). After micrographs were obtained, the sections stained with PHF1 and Thioflavine S were restained with Bielchowski silver stain. To confirm that counting neurons based on morphological appearance on cresyl violet-stained sections was accurate as has been previously described,16Gomez-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 Scholar, 17Kordower JH Chu Y Stebbins GT DeKosky ST Cochran EJ Bennett D Mufson EJ Loss and atrophy of layer II entorhinal cortex neurons in elderly people with mild cognitive impairment.Ann Neurol. 2001; 49: 202-213Crossref PubMed Scopus (379) Google Scholar we stained two sections from 9 mice at 4 months of age with NeuN (1:200; Chemicon, Temecula, CA), and horseradish peroxidase secondary antibody binding was developed with diaminobenzidine and counterstained with cresyl violet. Paraffin-embedded human tissues were cut at 8 μm, and two sections were stained with PHF1 and cresyl violet as described above. Negative controls with no primary antibody added showed no positive reaction in any case. Micrographs of stained tissue were obtained on an upright Olympus BX51 (Olympus, Denmark) fluorescence microscope with a DP70 camera using DPController and DPManager software (Olympus). Gross atrophy, neuron loss, and PHF1-positive aggregate formation were assessed on PHF1/cresyl violet-stained sections from mice in five age groups: 2.5, 4, 5.5, 7, and 8.5 to 10 months. At each age, three untreated tau mice and three to six control mice were used. At all ages, three or four tau mice were treated with dox for 6 weeks before sacrificing (to suppress tau transgene expression). Unbiased stereologic counting methods were used to determine neuron density and number, PHF1-positive neuron density and number, and volume in five regions of the brain (hippocampal areas CA1, CA2/3, and dentate gyrus (DG); cortex; and striatum). The optical disector method18West MJ Gundersen HJ Unbiased stereological estimation of the number of neurons in the human hippocampus.J Comp Neurol. 1990; 296: 1-22Crossref PubMed Scopus (1020) Google Scholar was used in a similar fashion to previously described work in transgenic mice.19Irizarry MC McNamara M Fedorchak K Hsiao K Hyman BT APPSw transgenic mice develop age-related A beta deposits and neurophil abnormalities, but no neuronal loss in CA1.J Neuropathol Exp Neurol. 1997; 56: 965-973Crossref PubMed Scopus (575) Google Scholar Briefly, an image analysis system (CAST; Olympus) mounted on an upright BX51 Olympus microscope with an integrated motorized stage (Prior Scientific, Rockland, MA) was used to outline regions, sample, and count neurons. Neurons and PHF1-positive neurons were counted in a 21.8- × 21.8- × 16-μm counting frame placed using a meander sampling paradigm with step lengths determined to sample 100–300 neurons per region in each hemisphere. Typical step lengths were 1250 μm in cortex, 650 μm in striatum, 250 μm in CA2/3, 200 μm in DG, and 350 μm in CA1. Region volumes were determined according to Cavalieri's principle, and the total number of neurons in each region was calculated. Tissues from five rTg4510 hemispheres and three control hemispheres snap frozen in isopentane on dry ice were homogenized using a sonic dismembrator (Model 500; Fisher Scientific, Pittsburgh, PA) in buffer containing 50 mmol/L Tris/HCl (pH 7.4), 175 mmol/L NaCl, 5 mmol/L EDTA (pH 8.0), and proteinase inhibitor mixture (complete mini; Roche Applied Science, Manheim, Germany). Homogenates were diluted in 2× Tris/glycine sample buffer and run on 10 to 20% polyacrylamide gels using the BioRad mini system at 100 V for approximately 2 hours. Proteins were transferred from the gel to a polyvinylidene difluoride membrane overnight at 20 V and 4°C. Blots were probed with primary antibodies against tau (CP27, mouse monoclonal, 1:2000; Dr. Peter Davies,) and actin (rabbit polyclonal, 1:1000; Sigma-Aldrich, St. Louis, MO), followed by secondary anti-mouse IgG conjugated to Alexa 680 (1:1000; Molecular Probes, Carlsbad, CA) and anti-rabbit IgG conjugated to IR800 (1:1000; Molecular Probes). Blots were imaged using the Odyssey Infrared Imager (Licor BioSciences, Lincoln, NB), and the optical density of the lanes was quantified using Image J (National Institutes of Health, Bethesda, MD). Data are presented as mean ± SD from the mean. Statistical analyses were performed using StatView software (SAS Institute, Cary, NC). Three experimental conditions were used: nontransgenic (control) animals, untreated tau animals (tau), and tau animals treated with dox for 6 weeks to suppress transgene suppression (tau plus dox). To assess the effects of age and the expression of the tau transgene on neuron number and neurofibrillary pathology, two-way analysis of variances were run with age and condition as independent variables. Bonferroni-Dunn posthoc comparisons between different conditions were performed to assess differences between two conditions, for example, whether the tau group was statistically different from the tau plus dox group. To compare the conditions at individual ages, one-way analysis of variances were used with condition as the independent variable and the posthoc Bonferroni-Dunn tests were split by age. Student's t-test (two-tailed equal variance not assumed) were performed to analyze the difference between one dox-treated age and the age that treatment began in non-dox-treated animals. In a previous study, marked atrophy of the rTg4510 brain was noted along with neuronal loss in the hippocampal CA1 region.11SantaCruz 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 (1587) Google Scholar Here we characterize neuronal loss in five different regions: hippocampal areas CA1, CA2/3, and dentate gyrus, neocortex, and neostriatum using stereological estimations of cresyl violet-labeled neuronal nuclei (Figure 1). Neurons were identified by nuclear morphology. Comparisons of previous studies that used cresyl violet or NeuN labeling of neurons show almost identical neuron counts in human entorhinal cortex.16Gomez-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 Scholar, 17Kordower JH Chu Y Stebbins GT DeKosky ST Cochran EJ Bennett D Mufson EJ Loss and atrophy of layer II entorhinal cortex neurons in elderly people with mild cognitive impairment.Ann Neurol. 2001; 49: 202-213Crossref PubMed Scopus (379) Google Scholar All regions examined exhibited significant neuronal loss (two-way analysis of variance, Bonferroni-Dunn post hoc test control versus tau, P < 0.0167). The extent and time course of neuronal loss in rTg4510 mice varied substantially by region, with the hippocampal formation exhibiting the most dramatic neuron loss compared to the average control neuron number (Figure 2 and Table 1). By 8.5 months, rTg4510 neuronal loss amounted to 85% in DG, 82% in CA1, 69% in CA2/3, and 52% in cortex. Although significant, the neuronal loss in striatum was minimal (Figure 2). In all areas except striatum, neuronal loss was age related. Bonferroni-Dunn posthoc analyses of one-way analysis of variances split by age were used as a conservative measure of significantly different numbers of neurons at each age (control versus tau), showing that neuron loss begins in CA1 and CA2/3 by 5.5 months, very early in DG (2.5 months), later in cortex (8.5 months), and no significant loss at any specific age in striatum (although as mentioned above, an analysis of variance that considers all age groups together indicates some neuronal loss in this area).Table 1Onset AgesRegionAge-related neuronal lossOnset of neuronal loss (months)Onset of PHF1 accumulation (months)Prevention of loss with tau suppressionPrevention of PHF1 accumulation with tau suppressionCA1Yes5.52.5YesOnly before 2.5 monthsCA2/3Yes5.52.5YesOnly before 5.5 monthsDGYes2.57YesNoCortexYes8.52.5YesOnly before 4 monthsStriatumNo5.5NoOnset ages were determined by the first age at which posthoc analysis showed a difference between tau and control (for neuronal loss and PHF1 accumulation) and between tau and dox-treated tau (for prevention of PHF1 accumulation). Open table in a new tab Onset ages were determined by the first age at which posthoc analysis showed a difference between tau and control (for neuronal loss and PHF1 accumulation) and between tau and dox-treated tau (for prevention of PHF1 accumulation). The more dramatic loss of neurons in hippocampus was not caused by higher levels of tau expression. Western blots from five rTg4510 mice and three control mice at 2.5 months show equal amounts of tau protein expressed in the hippocampus, cortex, and striatum of rTg4510 mice (Figure 3, A and B). These data also indicate that the early loss of neurons in the DG (beginning between 2.5 and 4 months) was not due to higher levels of tau in the hippocampal formation. Further, recent work from Ramsden et al showed that, in the forebrain, levels of soluble tau protein remain stable throughout the life of the transgenic animals, although Sarkosyl-insoluble tau increases with age.20Ramsden M Kotilinek L Forster C Paulson J McGowan E SantaCruz K Guimaraes A Yue M Lewis J Carlson G Hutton M Ashe KH Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L).J Neurosci. 2005; 25: 10637-10647Crossref PubMed Scopus (496) Google Scholar rTg4510 mice developed age-related PHF1-positive inclusions that closely resemble those from human tauopathy (Figure 3, C and D).11SantaCruz 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 (1587) Google Scholar, 12Braak H Braak E Neuropathological staging of Alzheimer-related changes.Acta Neuropathol (Berl). 1991; 82: 239-259Crossref PubMed Scopus (11766) Google Scholar, 21Ishizawa K Ksiezak-Reding H Davies P Delacourte A Tiseo P Yen SH Dickson DW A double-labeling immunohistochemical study of tau exon 10 in Alzheimer's disease, progressive supranuclear palsy and Pick's disease.Acta Neuropathol (Berl). 2000; 100: 235-244Crossref PubMed Scopus (33) Google Scholar In the temporal lobe of a 70-year-old patient with dementia associated with the P301L mutation, 4.4% of neurons were PHF1-positive, similar to the 10% of cortical neurons that were positive in P301L mice at an advanced age. In all of the regions examined in rTg4510 mice, intraneuronal PHF1-positive tau aggregates accumulated in an age-dependent manner (analysis of variances F[1,16] P < 0.0001 in all regions). Surprisingly, however, PHF1-positive tau staining did not always correlate with the pattern of neuronal loss. Although in CA1 there was both substantial accumulation of PHF1-positive cells and neuronal loss, other regions showed a clear dissociation. In DG, 53% of neurons were lost by 4 months of age, before PHF1-positive neurons appeared, and by 8.5 months, only 18% of the remaining neurons (ie, ∼3% of the original DG cells) were PHF1 immunoreactive (Figure 2). By contrast, in striatum, 11% of neurons at 7 months and 25% at 8.5 months of age exhibited PHF1 immunoreactivity, but there was no statistically significant loss of neurons at these time points (Figure 2). Because PHF1 is a relatively late marker of tau pathology, we also used CP13 (an early marker for tau phosphorylated at serine 20222Jicha GA Weaver C Lane E Vianna C Kress Y Rockwood J Davies P cAMP-dependent protein kinase phosphorylations on tau in Alzheimer's disease.J Neurosci. 1999; 19: 7486-7494Crossref PubMed Google Scholar) and MC1 (an early marker of conformational change23Jicha 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 (400) Google Scholar) on 2.5-month sections to determine whether the early loss of neurons in DG is preceded by these earlier markers. We found almost no cells positive for these markers in DG (three DG cells labeled with CP13 and one with MC1 in two sections each from four animals among thousands of unstained DG neurons), although there were some positive cells in CA1 and cortex at this age, as has been recently reported.20Ramsden M Kotilinek L Forster C Paulson J McGowan E SantaCruz K Guimaraes A Yue M Lewis J Carlson G Hutton M Ashe KH Age-dependent neurofibrillary tangle formation, neuron loss, and memory impairment in a mouse model of human tauopathy (P301L).J Neurosci. 2005; 25: 10637-10647Crossref PubMed Scopus (496) Google Scholar Thus, the early loss of neurons in DG does appear to precede any detectable changes in tau confirmation, phosphorylation, or solubility. Suppression of tau transgene expression for 6 weeks using dox halted the progressive neuronal loss. In all five regions examined, the number of neurons per hemisphere was not significantly different between the age when suppression began and after 6 weeks of treatment (Figure 2, dotted lines, and Table 1), even when significant loss would be expected over this time period as was the case in CA1 and cortex between 4 and 5.5 months and in DG before 2.5 months. Figure 4 shows examples in CA1 and DG where transgene suppression with 6 weeks of dox treatment produced a qualitatively discernable, dramatic prevention of neuronal loss. In DG, the early drastic neuronal loss in rTg4510 mice was completely prevented by transgene suppression from 0 to 2.5 months, indicating that neuronal loss is degenerative and not due to developmental abnormalities. In contrast, transgene suppression did not remove existing neurofibrillary pathology (eg, the number of PHF1-positive cells did not decrease from the levels at the beginning of treatment in any region in any age group tested (Figure 2)). In fact, in a few cases, the amount of PHF1-positive cells increased over 6 weeks of treatment despite transgene suppression. This was the case in CA1 and CA2/3 with suppression from 4 to 5.5 months and in CA2/3, DG, and cortex with treatment from 5.5 to 7 months (Figure 2). In CA2/3, suppression from 4 to 5.5 months prevented as many new PHF1-positive cells from appearing as would occur without treatment, indicating a selective differential fate of PHF1-immunoreactive tau deposition in different regions. Suppression beginning before pathology started to accumulate did prevent PHF1 accumulation. Animals that had been treated from birth to 2.5 months did not have any PHF1-positive cells in any region, even though in CA1, CA2/3, and cortex there are significant numbers of PHF1-positive cells at 2.5 months without treatment. Counterstaining fluorescent immunostaining for PHF1 with Thioflavine S and subsequent staining with Bielchowski silver stain show that, after transgene suppression, Thioflavine S-positive and argyrophilic neurofibrillary lesions also persisted (Figure 5). This suggests that the persistence of neurofibrillary pathology after 6 weeks of transgene suppression is not merely conservation of the hyperphosphorylated state. Over this same time period of suppression, previous data showed a reduction of ∼85% in transgene mRNA and 70% in soluble protein levels.11SantaCruz 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 (1587) Google Scholar This shows that phosphorylated tau aggregates are relatively long-lived despite drastic reductions in tau production, but they do not necessarily lead to neuronal death. In AD and other tauopathies, phospho-tau-immunoreactive inclusions and cell death occur differentially in different brain regions. Regional vulnerability has been extensively characterized in AD over the past decades. In AD layer II of the entorhinal cortex, CA1, and subiculum are particularly vulnerable to both neurofibrillary lesions and neuronal death, whereas CA2/3, DG, and presubiculum are far less affected.24Hyman BT Van Horsen GW Damasio AR Barnes CL Alzheimer's disease: cell-specific pathology isolates the hippocampal formation.Science. 1984; 225: 1168-1170Crossref PubMed Scopus (1788) Google Scholar, 25Hof PR Morrison JH The cellular basis of cortical disconnection in Alzheimer disease and related dementing conditions.in: Terry R Katzman R Bick KL Sisoda SS Alzheimer disease. Lippincott Williams & Wilkins, Philadelphia1999: 207-232Google Scholar In the AD cortex, primary sensory and motor areas are less vulnerable than association areas.26Pearson RC Esiri MM Hiorns RW Wilcock GK Powell TP Anatomical correlates of the distribution of the pathological changes in the neocortex in Alzheimer disease.Proc Natl Acad Sci USA. 1985; 82: 4531-4534Crossref PubMed Scopus (910) Google Scholar, 27Lewis DA Campbell MJ Terry RD Morrison JH Laminar and regional distributions of neurofibrillary tangles and neuritic plaques in Alzheimer's disease: a quantitative study of visual and auditory cortices.J Neurosci. 1987; 7: 1799-1808PubMed Google Scholar, 28Arnold SE Hyman BT Flory J Damasio AR Van Hoesen GW The topographical and neuroanatomical distribution of neurofibrillary tangles and neuritic plaques in the cerebral cortex of
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