Unless otherwise noted, reagents were obtained from Sigma (St. Louis, MO). Antibodies were purchased from the following: anti-Drebrin (clone M2F6, MBL, Nagoya, Japan), anti-PSD-95 (Upstate Biotech, Lake Placid, NY), anti-GFAP (Sigma, St. Louis, MO), anti-SNAP-25 (Sternberger Monoclonals, Lutherville, MD), anti-p85α (Stressgen, Victoria, Canada, and BD-Pharmingen, San Diego, CA), anti-neuronal nuclei (NeuN), synaptophysin (MAB368), and anti-actin (Chemicon international, Temecula, CA), anti-phosphoAkt, anti-phospho-BAD (ser136) and anti-total BAD (Cell Signaling, Beverly, MA) and anti-14-3-3 (Santa Cruz Biotechnology, Santa Cruz, CA). Affinity purified anti-fractin rabbit polyclonal antibody was developed and characterized in our laboratory (Yang et al., 1998
Animals and Diets
Seventeen-month-old male and female Tg2576 Tg(+) and Tg(−) mice from twelve litters were randomly assigned among three treatment groups. Mice were fed for 103 ± 5 days with control diet (PMI 5015, PMI International LabDiet, St. Louis, MO), safflower oil-based diet depleted of n-3 polyunsaturated fatty acids (TD 00522, Harlan Teklad, Madison, WI), or this low DHA diet supplemented with 0.6% (w/w) DHA (Martek Bioscience, Columbia, MD) (see ). The three diets were similarly supplemented in minerals and vitamins. Animals were perfused with 0.9% normal saline followed by HEPES buffer (pH 7.2) containing protease inhibitors. Brain regions were dissected from one hemisphere as previously described (Lim et al., 2001
). Unless otherwise noted, biochemical measurements were performed on the residual cortex (cortex region without frontal, entorhinal, or piriform areas). A second set of similarly aged animals fed on the same diets out to 21–22 months were prepared for the behavior study.
Preparation of Tissue Samples
Tissue samples were homogenized in 10 volumes of TBS containing a cocktail of protease inhibitors [20 mg/ml each of pepstatin A, aprotinin, phosphoramidon, and leupeptin; 0.5 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF); 1 mM EGTA; 5 mM fenvalerate; and 5 mM cantharidin]. Samples were sonicated briefly (2 × 10 s) and centrifuged at 100,000 × g for 20 min at 4°C to generate a TBS-soluble fraction (cytosol fraction). The TBS-insoluble pellet was sonicated in 10 volumes of lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, 1% Triton X-100, 0.5% SDS, and 0.5% deoxycholate) containing the same protease inhibitor cocktail. The resulting homogenate was centrifuged at 100,000 × g for 20 min at 4°C to produce a lysis buffer-soluble fraction (membrane fraction).
Adult mutant mice were anesthetized with pentobarbital (60 mg/kg) and perfused with 4% para-formaldehyde (PFA) and 0.1% glutaraldehyde in PBS. After dissection, the brains were kept overnight at 4°C in 4% PFA and then kept in cold PBS. Vibratome sections (100 μm) were cryoprotected, permeabilized by freeze thawing, rinsed in PBS, and immersed for 20 min in 50 mM ammonium chloride and for 30 min in PBS with 0.1% gelatin (PBSg). Sections were incubated for 12 hr (4°C) in the anti-fractin antibody (1:100 in PBSg), and the antibody binding sites were detected using a biotinylated goat anti-rabbit antibody (1:500 in PBSg; Jackson, West Grove, PA) and avidin-biotin-HRP (Vector Laboratories. Inc., Burlingham, CA). After dehydration and osmium staining, the sections were flat embedded. Observations of ultrathin sections (pale yellow) were contrasted with uranyl acetate and lead citrate. Images were collected with a JEOL 100CXII electron microscope.
Samples (30 μg protein) were electrophoresed on 10% acrylamide gels and transferred to PVDF membranes (Immobilon, Millipore, MA) before blocking in 10% nonfat dry milk and 0.1% gelatin in PBS for 1.5 hr. Blots were immunoblotted with the appropriate primary and secondary antibody and chemiluminescence (ECL, Amersham/Pharmacia biotech, Piscataway, NJ, or Supersignal, Pierce, Rockford, IL). Band intensities were scanned and quantified with densitometric software (Molecular Analyst II). Immunoblot data were normalized to total protein load by quantification of all samples in a single assay before loading and confirmation of equal loading by image analysis of scanned Coomassie blue-stained gels after blotting.
Fatty Acid Measurement
Fatty acid analysis in frontal cortex was performed using Folch’s extraction method and gas chromatography with flame ionization detection, as previously described (Moriguchi et al., 2000
). Fatty acid data in diets from Harlan were analyzed by Cornell University, Diagnostic Laboratory, Nutritional and Environmental Analytical Services (Ithaca, NY).
Amounts of oxidized proteins containing carbonyl groups were measured in the membrane fraction of cortex samples using an Oxyblot kit (Intergen, Purchase, NY) as previously described (Lim et al., 2001
Hemi-brains were fixed with 4% PFA (4°C overnight), cryoprotected with 10% and 20% sucrose-PBS, snap frozen at −70°C, and cryostat sectioned into coronal (12 μm) sections. For fractin/drebrin double labeling and NeuN labeling, slides were soaked in 75% ethanol (2 min at room temperature) and dH2O resin. Sections were processed in antigen unmasking buffer (Vector Labs, Burlingame, CA), steamed 15 min, and rinsed in 0.3%Triton X-100 in Tris-buffered saline (10 min at room temperature). Incubation with fractin (1:20) and drebrin (1:30) (or NeuN antibody [1:2000]) was performed in 0.1% Tween-20 in 3% BSA Tris-buffered saline for 1 hr at 37°C. Secondary antibodies (1:1000, 1 hr incubation at room temperature) were goat anti-rabbit-FITC and goat anti-mouse-Rhodamine (Molecular Probes, OR). For phospho-BAD, sections were exposed to three successive 3 min acetone (50%–100%–50%) washes, rinsed with 5 μg/ml Proteinase K (5 min at room temperature), 0.3% Triton X-100 (5 min at room temperature), and H2O2 in methanol (5 min at room temperature). Sections were blocked with 5% normal goat serum for 30 min at 37°C and incubated with phospho-BAD antibody (1:15) for 60 min at 37°C. After incubation with secondary antibody and ABC reagent (Vector Labs, CA), sections were developed using metal enhanced DAB kits (Pierce, Rockford, IL). Image analysis on sections (at Bregma −1.5, −2, and −3) was performed with an Optronix Engineering LX-450A CCD video system using NIH Image software. At sacrifice, there was no treatment-dependent change in brain weights, nor in Bregma-matched brain region areas, indicating the absence of major treatment-related shrinkage of brain regions. For NeuN, quantitative analysis for neuronal density was performed in all cortical layers (entorhinal II, entorhinal III/IV, parietal II, parietal III/IV, parietal V/VI, frontal II, frontal III/IV, frontal V/VI) and hippocampal pyramidal cell layers (CA1, CA2, CA3). We measured neuronal nuclei density by image analysis in these neuronal layers in four consecutive sections for each of four different Bregmas (1.0 mm, 1.7mm, −2.7 mm, and −3 mm).
Handling of Human Tissue
Postmortem tissue from temporal lobe was obtained from the USC AD Center and the UCLA AD Research Center. Nine controls were compared to ten AD patients with moderate disease. Postmortem interval, age of death, and gender were comparable between both groups. The tissue was processed as for mouse tissue except that the concentration of SDS in the lysis buffer was raised to 2% and Triton X-100 and deoxycholate were removed.
Quantitative Real-Time RT-PCR of p85α PI3-K mRNA
Total RNA was isolated from brain using the RNAqueous kit (Ambion) as per the manufacturer’s instructions and was treated with DNase. RNA (0.7 μg) was reverse transcribed using dT primers using the Retroscript kit (Ambion) and was then aliquoted. Analysis of RNA levels was performed using QPCR with the SYBR Green PCR Master Mix (Applied Biosystems) and an SDS7700 (Applied Biosystems), followed by dissociation curve analysis that verified the amplification of a single PCR species. Thermocycling parameters were standard for the SDS7700 (Applied Biosystems) instrument: 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Each sample was tested in triplicate. A standard curve was run to allow relative comparisons of sample values. The standard curve was made using cDNA (using dT primers) from brain tissue RNA reverse transcribed at two times the concentration of RNA (1.4 μg) compared to that used for reverse transcription of the sample RNA; standard curves typically had an r2 > 0.99. The primers for p85α were designed using Primer Express software (Applied Biosystems) using the mouse p85α sequence NCBI# U50413. The primer sequences were forward, ACCTGTGAACTGAGCTGCAGAA; reverse, TAGAAACGTCTGGTCATCCAACA. GAPDH mRNA levels were also measured for each cDNA sample as an internal housekeeping gene control using the primers from the TaqMan Rodent GAPDH Control Reagents (Applied Biosystems). Levels of p85α mRNA were normalized to levels of GAPDH mRNA for each sample.
We followed Morris water maze protocols based on extensive characterization of age-dependent cognitive deficits in an APP mouse (Westerman et al., 2002
). Mice were trained for six blocks (four consecutive swim trials per block, two blocks per day, one block a day in aged animals) to find a visible platform in our mouse pool (5 foot diameter swimming pool), maintained at 26°C to prevent hypothermia. There is a video camera on the ceiling, attached to a laptop with HVS image tracking capabilities, so calculation of mouse swim paths and latencies was streamlined and facilitated. Besides training mice, the visible testing phase was used to exclude mice that have visual or motor deficits (Westerman et al., 2002
). If mice did not find the platform within 60 s, they were led to the platform with a fish net. If mice sank, they were picked up and prevented from drowning. After the sixth block, mice started the hidden platform trials, which consisted of 12 blocks of training. Probe trials for retention deficits (60 s swim without platform to determine percent path in target quadrant) were then performed. During swims, the staff handling the mice moved out of sight of the mice so that only distal stationary cues were present. Learning was assessed by significant regressions of block on latency, and despite weekend breaks, the number of blocks of testing was limited by excessive fatigue and their imminent mortality. At the end of each block (four consecutive swim trials), mice were towel dried and then placed back in their cages.
Statistical comparisons of data were performed using an ANOVA followed by post hoc pairwise comparisons with Fisher’s probability of least significant difference test (PLSD). Square root transformation to establish homogeneity of variance was used for p85 data in mice. Coefficients of correlation and significance of the degree of linear relationship between parameters were determined with a simple regression model. For visible and hidden platform tests, swim latencies, paths, and swim speeds were analyzed. Regressions of latencies on blocks were calculated to determine whether learning was occurring. 2 × 2 ANOVA (Combined Group Blocks × treatment) and repeated measures ANOVA were performed to determine treatment and transgene differences in latencies. Percentage path in target and opposite quadrants in probe trial was also assessed using 2 × 2 ANOVA (transgene × treatment).