Generation and initial characterization of PS1M146L/APP751sl (PS1/APP) tg mice has been reported previously [10, 58, 59]. Heterozygous PS1/APP double tg mice (C57BL:6 background) were generated by crossing homozygous PS1 tg mice with heterozygous Thy1-APP751SL mice. Only male mice were used in this work. Age-matched non-transgenic male mice (WT) of the same genetic background (C57BL:6) were used as controls.
Mice were first anesthetized with sodium pentobarbital (60 mg/kg), the hippocampi were dissected and immediately frozen and stored at −80°C until use. For immunohistochemistry, anesthetized mice were perfused transcardially with a paraformaldehyde-based solution (see details below). All animal experiments were performed in accordance with the guidelines of the Committee of Animal Research of the University of Seville (Spain) and the European Union Regulations.
RNA and total protein extraction
Total RNA from mice hippocampi or cultured cells was extracted using Tripure Isolation Reagent (Roche) as described previously [10, 11, 27, 58, 59]. After isolation, RNA integrity was assessed by agarose gel electrophoresis. The yield of total RNA was determined by measuring the absorbance (260:280 nm) of isopropanol-precipitated aliquots of the samples. The recovery of RNA was comparable in all studied groups (1.2-1.5 μg/mg of tissue).
The protein pellets, obtained using the Tripure Isolation Reagent and isopropanol-mediated precipitation, were resuspended in 4% SDS and 8M urea in 40 mM Tris–HCl, pH 7.4 and rotated overnight at room temperature to get complete protein solubilization.
Reverse transcription and real-time RT-PCR
Retrotranscription (RT) was performed using random hexamers, 4 μg of total RNA as template and High-Capacity cDNA Archive Kit (Applied Biosystems) following the manufacturer recommendations [10, 27]. For real time RT-PCR, commercial Taqman™ probes (Applied Biosystems) were used for amplification. Alternatively, SYBRgreen dye and designed specific primers were used for amplification of human APP751 (forward: 5´-GGATATGAAGTTCATCATCA-3´; reverse: 5´-TCACTGTCGCTATGACAACA-3´), and human PS1 (forward: 5´-TGGCTCATCTTGGCTGTG-3´; reverse: 5´-ACCAGCATACGAAGTGG-3´). PCR reactions were carried out using either ABI Prism 7000 or 7900HT sequence detector systems (Applied Biosystems). A standard curve was first constructed for every assay, using increasing amounts of cDNA. In all cases, the slope of the curves indicated optimal PCR conditions (slope 3.2-3.4). The cDNA levels of the different mice were determined using GAPDH as housekeeper. Therefore, GAPDH amplification was done in parallel with the gene to be analyzed, and this dada used to normalize target gene results.
Independent of the analyzed gene, results were always expressed using the comparative Ct method, following the Bulletin number 2 from Applied Biosystems. As a control condition, we selected 6-month-old WT mice. In consequence, the expression of all tested genes, for all ages and mouse strains, was referenced to the expression levels observed in 6-month-old WT mice.
Western blots were performed as described . Briefly, 5–20 μg of proteins from the different samples were loaded on 16%-SDS-tris-tricine-PAGE or 12%-SDS-tris-glycine-PAGE and transferred to nitrocellulose (Hybond-C Extra; Amersham).
After blocking, using 5% non-fat milk, membranes were incubated overnight, at 4°C, with the appropriate antibody: phospho-neurofilament (clone SMI-31; 1:1,000; Abcam), total-neurofilament (clone SMI-32; 1:1000; Abcam), phospho-Ser199:202-Thr205-PHF-tau (clone AT8; 1:1000; Pierce), phospho-Thr212-Ser214-PHF-tau (clone AT100; 1:1,000; Innogenetics), kinesin-1 heavy chain (1:1,000; Abcam), dynein-1 intermediate chain (1:1000; Millipore), ATP-synthetase-Beta (1:1000; BD Transduction Laboratories), Abeta peptide (clone 6E10; 1:5000; Signet), APP C-terminal (1:6000; Calbiochem), PS1 C-terminal (1:2000; Millipore), BACE-1 (1:1000; Abcam), V0a1-proton-pump subunit (1:1000; Synaptic Systems), LC3B (1:1000; Cell Signaling), cathepsin B (1:1000, Santa Cruz); cathepsin-D (1:5000; ABFrontier Co. Ltd), Lamp-1 (1:1000; Developmental Studies Hybridoma Bank; University of Iowa), ubiquitin (1:1000; Sigma-Aldrich) and Beta-actin (1:5000; Sigma-Aldrich). Membranes were then incubated with the corresponding horseradish-peroxidase-conjugated secondary antibody (Dako, Denmark) at a dilution of 1:8000. Each blot was developed using the ECL-plus detection method (Amersham).
For quantification, the scanned (Epson 3200) images were analyzed using PCBAS program. For normalization purposes, proteins were first estimated by Lowry and protein loading corrected by beta-actin. The intensity of bands from 6-month-old WT or PS1/APP (Figures 4A, E and F; Figures 5C and D) mice were averaged and considered as 1 relative unit. All other data were then normalized by the specific signal observed in 6-month-old WT or PS1/APP group or negative control for “in vitro” experiments.
Enzymatic activity determination
BACE-1, Cathepsin B and D activities were determined using commercial kits (R&D Systems, Germany; Calbiochem, Germany and Sigma-Aldrich, respectively) following the manufacturer instructions. Briefly, fresh hippocampal samples were homogenized in the buffer supplied by the manufacturer or in PBS (for proteasome activity), centrifuged at 10,000xg (15 min at 4°C) and the supernatant (100–200 μg of protein per assay) was used. BACE-1 and cathepsin B and D activities were determined using substrates provided by the manufacturer
Proteasome chymotrypsin-like activity was determined as described . Briefly, soluble fractions (30–50 μg of protein per assay) were diluted in 50 μM reaction substrate Suc-Leu-Leu-Val-Tyr-aminomethylcoumarin (AMC) (Sigma-aldrich), 0.1mM EDTA, 5mM DTT, 0.01% (w:v) CHAPS; 100mM NaCl; 1% (v:v) Glycerol, 50mM HEPES-KOH pH 7.5. Duplicated reactions were placed in a 96-well black polystyrene microplate (BD Transduction Labs.) and incubated at 37°C. Fluorescence was determined at excitation 360-380nm and emission 460-480 nm.
For each enzymatic assay, the fluorescence intensity was determined every 15 minutes (starting by the addition of the substrate) for a 1–2 hours final incubation time, using a Synergy HT Multi-mode microplate reader (Biotek). The activities were calculated from the maximal slope of the fluorescence intensity vs time curves and corrected by the amount of protein added. The results were then normalized by the activity observed in 6 months WT mice or 6 months PS1/APP mice (for BACE-1).
Also for each enzymatic activity, a reaction without substrate, a reaction without sample, and reaction with an inhibitor were used as negative controls. The inhibitors used were: Cathepsin B, Inhibitor Ref. 219385 (Callbiochem); Cathepsin D, pepstatin A Solution Ref. P3749 (Sigma-Aldrich); Proteasome 10 μM MG-132 (Sigma-Aldrich). Independently of the enzymatic activity assay, each experiment was repeated, at least, three times for each age and genotype.
Gamma-secretase activity was determined following previously described protocols with some modifications . Briefly, membrane pellets from PS1/APP animals (n=6 per age) were thawed and resuspended (at 3 mg of protein per ml) in 150 mM Citrate Buffer, pH 6.4, containing protease inhibitors (Roche). Aliquots (150 μg of proteins) were used for each assay. As negative control, 100 μM L-685-458 gamma-secretase inhibitor (Calbiochem) was added prior assay. Samples were then incubated, at 37°C with orbital shaking at 400 rpm, for 2 hours. After incubation, membranes were sonicated (at 80 W for 30 seconds) and centrifuged at 30,000xg (30 minutes, 4°C). Supernatants were used to determine AICD production by western blot using anti-APP C-terminal as primary antibody.
Tissue preparation for immunohistochemistry
After anesthesia with sodium pentobarbital (60 mg/kg), 6, 12, and 18-month-old control (WT) and PS1/APP tg male mice (n=4/age/genotype) were perfused transcardially with 0.1 M phosphate-buffered saline (PBS), pH 7.4 followed by 4% paraformaldehyde, 75 mM lysine, 10 mM sodium metaperiodate in 0.1 M phosphate buffer (PB), pH 7.4. Brains were then removed, post-fixed overnight in the same fixative at 4°C, cryoprotected in 30% sucrose, sectioned at 40 μm thickness in the coronal plane on a freezing microtome and serially collected in wells containing cold PBS and 0.02% sodium azide. All animal experiments were approved by the Committee of Animal Use for Research of the Malaga University (Spain) and the European Union Regulations.
Coronal free-floating brain sections (40 μm thick) from 6 and 12–18 month-old control (WT) and PS1/APP mice were processed simultaneously in the same solutions and conditions to prevent processing variables. Sections were first treated with 3% H2O2/3% methanol in PBS and with avidin-biotin Blocking Kit (Vector Labs, Burlingame, CA, USA), and then incubated overnight at room temperature with one of the following antibodies: anti-APP-C-terminal rabbit polyclonal (1:20,000; Sigma-Aldrich), anti-phospho-Ser199:202:Thr205-PHF-tau mouse monoclonal (clone AT8; 1:500; Pierce) anti-ubiquitin rabbit polyclonal (1:2,000, Dako), anti-LC3 goat polyclonal (1:1,000, Santa Cruz Biotechnology), anti-BACE-1 rabbit polyclonal (1:1,000, Abcam) or anti-Lamp2 rabbit polyclonal (1:500, Abcam).
The tissue-bound primary antibody was detected by incubating with the corresponding biotinylated secondary antibody (1:500 dilution, Vector Laboratories), and then followed by streptavidin-conjugated peroxidase (Sigma Aldrich) diluted 1:2000. The reaction was visualized with 0.05% 3-3’-diaminobenzidine tetrahydrochloride (DAB, Sigma Aldrich), 0.03% nickel ammonium sulphate and 0.01% hydrogen peroxide in PBS. When required, immunolabeled sections were then incubated for 3 minutes in a solution of 20% Congo red. Sections were then mounted on gelatin-coated slides, air dried, dehydrated in graded ethanols, cleared in xylene and coverslipped with DPX (BDH) mounting medium. Specificity of the immune reactions was controlled by omitting the primary antiserum.
Quantitative analysis of Abeta plaques-associated dystrophic neurites
The number of APP-immunopositive dystrophic neurites per plaque was quantified over Congo red stained Abeta deposits in sections from PS1/APP animals at young (4 and 6 month-old group) and old (12 and 18 months-old group) ages (n= 6/group; 5 sections per animal through the antero-posterior extent of the hippocampus). Quantification was done in CA1 subfield which was defined using a 10x objective and the number of dystrophic neurites was counted using a 100x objective in an Olympus BX61 microscope equiped with NewCAST software package (Olympus, Glostrup, Denmark). All plaques present in the CA1 region of each section were quantified. The number of dystrophic neurites per plaque was normalized to the mean plaque area to allow comparisons between groups.
Synaptosome and microsome fractions isolation
Synaptosomal fractions were obtained basically as described previously [17, 63]) with some modifications. Briefly, one mouse hemicortex was gently homogenized with a glass Dounce homogenizer in cold Buffer A (0.32 M sucrose, 1 mM EDTA, 1mM EGTA in 20 mM Tris–HCl pH 7.5, plus 1mM sodium orthovanadate, 50 mM sodium floride and a complete protease inhibitor cocktail (Roche) at a ratio of 40-50 mg of tissue per ml of buffer. This homogenate was first centrifuged at 1500 g and the post-nuclear supernatant was again centrifuged at 12,600 g for 20 minutes at 4°C to get the crude synaptosomes fraction. This pellet was resuspended in 13% (w:v) (final concentration) Ficoll PM400 (in buffer A) and layered on the bottom of a discontinuous gradient, composed by buffer A and 7% Ficoll (in buffer A). The gradient was centrifuged at 100,000g (45 minutes, 4°C) in a TLS-55 swimming bucket rotor (Beckman-Coulter), and synaptosomes were isolated at the 7.5–13% interface. After washing (twice with buffer A), the pellet of synaptosomes was resuspended in Buffer A. The possible contamination with vacuolated postsynapses was evaluated by testing the presence of tau (presynaptic) and MAP2 (postsynaptic) proteins. Results (not shown) indicated the existence of a minimal contamination with vacuolated postsynapses.
On the other hand, the microsomal fraction was obtained after additional centrifugation of the 12,600 g supernatant at 100,000 g (1 hour, 4°C) in a TLA-110 rotor (Beckman-Coulter). The pellet of microsomes was also resuspended in Buffer A. The protein content of the both synaptosomal and microsomal fractions was determined by Lowry.
APPswe-expressing N2a cultures
APPswe-stably transfected Neuroblastoma cells were generously donated by Dr. Gopal Thinakaran (University of Chicago) . N2aAPPswe cells were cultured in high glucose DMEM-Optimem (50%-50%) supplemented with 2 mM glutamine and 5% fetal bovine serum (PPA Company), in presence of Penicillin and Streptomycin (100 units/ml and 0.01 mg/ml respectively) and G418 (PPA Company) as clonal selection antibiotic (0.2 μg/ml) . For cell drug treatments, a 0.2μm filtered stock solution of Chloroquine diphosphate salt (Sigma-Aldrich) or MG132 (Sigma-Aldrich) were diluted in the same media at a final concentration of 10 or 5 μM, respectively. This media was kept for 6 or 24 hours before collecting the cells and isolating RNA and protein as described above.
Data were expressed as mean ± SD. The comparison between two mice groups (WT and PS1/APP mice) was done by t test. For comparison between several age groups, we used one-way ANOVA followed by Tukey post hoc multiple comparisons test (Statgraphics plus 3.1). As stated above, for most experiments, the different xgroups were compared with 6-month-old WT mice. In some cases (Figures 4A, E and F; Figure 5C and D) 6-month-old PS1/APP mice were used as reference. The significance was set at 95% of confidence.