Western Blot AnalysisAnimals were sacrificed by decapitation, the brains were extracted, and either the brain or hippocampi and EC were dissected. Tissue was homogenized in radio-immunoprecipitation assay buffer (Sigma) supplemented with a cocktail of protease and phosphatase inhibitors (Roche). Samples were homogenized using a Polytron and stored at −80°C. The materials for SDS-PAGE were obtained from Invitrogen (NuPAGE system). Protein lysates were boiled in sample buffer consisting of lithium dodecyl sulfate sample buffer and reducing agent and resolved on 4%–12% Bis-Tris polyacrylamide precast gels in a 3-(n-morpholino)propanesulfonic acid-SDS running buffer containing antioxidant. For most analyses, 30 μg/lane were loaded, unless indicated otherwise. Gels were transferred onto Nitrocellulose Membranes Protran (Whatman) in transfer buffer containing 20% methanol. Blots were blocked in Odyssey blocking buffer (Li-Cor biosciences), followed by incubation with primary antibodies (β -actin [Sigma; 1:10,000]; Total Tau [Dako; 1:10,000], HT7 [1:5,000], TauY9 [1;1,1000], mTau [Naruhiko Sahara; 1:5,000], AT180 [pT231, Thermo Scientific; 1:1,000], PHF1 [courtesy of Peter Davies; 1:5,000], CP13 [courtesy of Peter Davies, 1:1,000], and DA9 [courtesy of Peter Davies; 1: 10,000]) and detected with anti-mouse or anti-rabbit IgG conjugated to IRDye 680 or 800 (Li-Cor Biosciences; 1:10,000). Densitometric and MW analyses were performed using ImageJ software (National Institutes of Health). Band density values were normalized to β-actin or total tau levels when tau phosphorylation levels were analyzed. Mean band densities for samples from rTgTauEC mice were normalized to corresponding samples from control mice.

Each of the eight age groups studied (3, 6, 9, 12, 15, 18, 21, 24 months) contained transgenic and control animals. A total of 183 animals were used for this study, including 97 transgenic and 86 control animals.

Age-matched littermates expressing only the activator transgene and the responder transgene were used as controls.

We generated the line of transgenic animals (called rTgTauEC [reversible tau restricted to EC]) by crossing FVB-Tg(tetO-TauP301L)4510 mice (Santacruz et al., 2005xTau suppression in a neurodegenerative mouse model improves memory function. Santacruz, K., Lewis, J., Spires, T., Paulson, J., Kotilinek, L., Ingelsson, M., Guimaraes, A., DeTure, M., Ramsden, M., McGowan, E. et al. Science. 2005; 309: 476–481Crossref | PubMed | Scopus (1091) | Google ScholarSee all ReferencesSantacruz et al., 2005) with a transgenic mouse line on a C57BL/6 genetic background expressing tet transactivator under the control of the neuropsin promoter (developed at The Scripps Research Institute [Yasuda and Mayford, 2006xCaMKII activation in the entorhinal cortex disrupts previously encoded spatial memory. Yasuda, M. and Mayford, M.R. Neuron. 2006; 50: 309–318Abstract | Full Text | Full Text PDF | PubMed | Scopus (59) | Google ScholarSee all ReferencesYasuda and Mayford, 2006]). Only F1 offspring were used as experimental animals, ensuring a uniform 50:50 mix of FVB and C57BL/6 genetic background. The human Tau gene with the P301L mutation in rTg4510 mice is downstream of a tetracycline-operon responsive element and requires the presence of the tet transactivator (Gossen and Bujard, 1992xTight control of gene expression in mammalian cells by tetracycline-responsive promoters. Gossen, M. and Bujard, H. Proc. Natl. Acad. Sci. USA. 1992; 89: 5547–5551Crossref | PubMed | Scopus (3808) | Google ScholarSee all ReferencesGossen and Bujard, 1992). Since the tet transactivator is downstream of the promoter of neuropsin, human tauP301L expression is restricted to EC-II. Mice with both the activator and responder transgenes are abbreviated rTgTauEC.

5A6 (generated by G.V. Johnson, 1997; 1:1,000)

Figure 2Biochemical Characterization of Tau Pathology Reveals Age-Dependent Increase in Tau Hyperphosphorylation and Presence of Sarkosyl-Insoluble Tau in rTgTauEC(A–F) Representative immunoblots from western blot analysis of mouse brain extracts from 12-, 18-, and 24-month-old rTgTauEC and control mice. Antibodies specific to phosphorylated tau (AT180 [pT231], PHF1 [pS396/404], and CP13 [pS202]), total tau, and human tau (htau) were used. Quantification of immunoblots shows that rTgTauEC mice present an age-dependent increase in total tau (B), human tau (results are expressed as percent of total tau expression at 24 months, the highest expression point defined as 100%) (C), and phosphorylated epitopes (D–F) (n = 4 mice per group). A series of ultracentrifugation and extraction steps were used to obtain sarkosyl-insoluble fractions of tau from whole brain of 24-month-old rTgTauEC, control (25 μg of protein loaded), and 18-month-old rTg4510 mice (5 μg of protein loaded) (positive control).(G) Representative western blots of sarkosyl fractions are shown. Sarkosyl soluble (S) and insoluble fractions (I) were immunoblotted with total tau antibody (phosphorylation-independent, Dako). Control mice did not show sarkosyl-insoluble tau. In contrast, immunoblotting of fractions from rTgTauEC mice revealed sarkosyl-insoluble complexes of tau, of the same type as found in the brains of rTg4510 mice. A 64 kDa insoluble hyperphosphorylated tau species was detected by immunoblotting in both rTg4510 and rTgTauEC brains but was absent in age-matched control brains when analyzed using total tau antibody. In the soluble fraction, the 55 kDa species of tau was also present, similar to the species present in rTg4510 mice.(H) Quantification of samples of sarkosyl fractions (n = 3 mice per group). Results are expressed as the mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 1MEC-Restricted Expression of Human P301L-Mutated Tau Causes Progressive Tauopathy in EC Neurons(A) rTgTauEC transgenic mice express human tauP301L under a tetracycline-responsive promoter (tetO) and the tetracycline transactivator (tTA) under the neuropsin promoter.(B and C) Low-magnification view of a medial horizontal section of rTgTauEC mouse brain stained with DAPI (B) and FISH showing human tau mRNA (C) demonstrates the restriction of expression of human tauP301L to layer II of the MEC and the pre- and parasubiculum (Pre, Para). The EC and MEC are outlined with a red dotted line. Note the lack of expression in the LEC, the subiculum (Sub), and the hippocampus including the DG, CA3, and CA1. There was no transgene expression in age-matched controls (C, right panel; scale bar, 1 mm). Higher-magnification inset in (C) shows the white outline EC area (scale bar, 100 μm).(D) Immunohistolabeling with phosphorylation-independent tau antibody 5A6 shows that tau protein is intensely expressed from 3 months of age compared to an age-matched control brain. Scale bar, 1 mm left and right panels, 200 μm in higher-magnification insets (center panels).(E) Human tau-expressing neurons in the MEC develop progressive tau pathology beginning at 3 months of age, with Alz50-positive tau misfolding in the axon terminal zone (molecular layer of the DG, left panel; scale bar, 200 μm). By 6 months of age, MEC neurons have Alz50-positive tau staining in their soma, and the soma in this region increasingly accumulate pathological forms of tau with age, shown here by markers of later tau pathology including PHF1 (tau phosphorylated at Ser396 and Ser404) starting from 12 months, Gallyas silver stain-positive neurons from 18 months, and Thioflavin S-positive neurofibrillary tangles by 24 months of age. Scale bar, 50 μm for all soma images.

We also invite 500 word blog post style responses to "I can't use the Web to publish my research because..." which we will use to seed discussion during the open session.

pipeline

in shorter-term, less in-depth projects [1, 2].

The