Tissue from control and AD brains was selected from the South West Dementia Brain Bank. The brains had been divided mid-sagittally at autopsy: the left half had been sliced and frozen at −80°C for biochemical studies and the right half fixed in formalin for detailed neuropathological assessment. Diagnosis of AD was made according to Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) criteria (18
). According to National Institute on Aging-Reagan criteria (19
), 17/19 of these cases had a high likelihood that the dementia was due to AD. The remaining 2 cases (ID numbers 23 and 29, ) had an intermediate likelihood that dementia was due to AD. Aβ load and cerebral amyloid angiopathy (CAA) scores were determined as previously described (20
) (). CAA was graded in arteriolar vessels in the cortex and in the leptomeninges according to the Olichney method (21
To determine the specificity of the picalm antibody, brain tissue homogenates were prepared in sodium-dodecyl sulphate buffer as previously described (22
) and were run alongside GST-tagged recombinant picalm protein (AbNova, Taipei, Taiwan) on a 4% to 20% Tris-HCl precast gel (Bio-Rad, Hertfordshire, UK. 150V, 1 hour). Protein was transferred to nitrocellulose membrane (Anachem, Luton, UK. 20V, overnight [ON] at 4°C), and detected using the Millipore SNAP i.d. protein detection system (Millipore, Billerica, MA). The membrane was blocked in 0.5% milk powder in Tris-buffered saline/0.05% Tween-20, incubated with picalm (1:50, 10 minutes; Calm C-18, Santa Cruz Biotechnology, Heidelberg, Germany) and with peroxidise anti-goat (1:10,000, 10 minutes; Vector Laboratories, Peterborough, UK). Visualization of the target protein was by enhanced chemiluminescence using the Amersham ECL detection reagents (GE Healthcare, Buckinghamshire, UK).
Seven-μm-thick sections from formalin-fixed, paraffin-embedded blocks of frontal and temporal lobe from 7 control and 7 AD brains were immunostained for picalm. The sections of frontal lobe included the superior and middle frontal gyri (Brodmann area 6), cingulate cortex (area 24), paracentral lobule (area 32), and underlying white matter. Those of temporal lobe included the parahippocampal gyrus (area 36), and inferior (area 20), middle (area 21) and superior temporal (area 22) gyri and the hippocampus. Dewaxed and hydrated sections were immersed in methanol containing 3% H2O2 (30 minutes), boiled in sodium citrate buffer (pH 6), blocked in 10% normal rabbit serum (20 minutes), and incubated with anti-picalm antibody (1:800, ON; Calm C-18, Santa Cruz Biotechnology). This was followed by incubations with biotinylated anti-goat secondary antibody (1:100, 20 minutes; Vector Laboratories), with avidin-biotin horseradish peroxidase complex (20 minutes; VectaElite ABC; Vector Laboratories) and with 3,3′-diaminobenzidine (DAB, 10 minutes; Vector Laboratories). All incubations were conducted at room temperature. Sections were counterstained with hematoxylin, dehydrated, cleared and mounted.
Double Immunofluorescent Labeling of Brain Tissue
Fluorescein tyramide-labeled factor VIII-related antigen/von Willebrand factor (FVIIIRA/VWF) was combined with streptavidin Alexa Fluor 555 labeling of biotinylated picalm. Sections were initially treated as above. Following treatment with sodium citrate buffer, sections were incubated in 0.12% potassium permanganate in phosphate-buffered saline (PBS; 25 minutes) and in 1% oxalic acid and 1% potassium pyrosulphate in PBS (15 minutes) to reduce autofluorescence. Sections were washed and labeled for FVIIIRA/VWF (VWF; Abcam, Cambridge, UK) using the tyramide signal amplification (TSA) fluorescence system (Perkin Elmer, Milano, Italy). This involved initial immersion in Tris-sodium chloride buffer containing 0.5% TSA blocking reagent for 30 minutes and subsequent incubation in primary antibody (1:800; ON, room temperature), peroxidise anti-rabbit antibody (1:200; 30 min) and fluorescein-labeled tyramide (1:50; 10 minutes). This was followed by ON incubation with anti-picalm (1:100), 20-minute incubation with biotinylated anti-goat (1:100) and 1-hour incubation with streptavidin Alexa Fluor 555 (Invitrogen, Paisley, UK). Sections were mounted in Vectashield mounting medium (Vector Laboratories) and images acquired using a confocal laser-scanning microscope (Nikon Instruments, The Netherlands).
The TSA biotin system (Perkin Elmer) was used for double immunofluorescent labeling of picalm and Aβ (4G8; Covance Cambridge Bioscience, Cambridge, UK). Following treatment with sodium citrate buffer as above, sections were blocked in TSA blocking buffer (30 minutes), incubated with anti-picalm (1:800, ON), biotinylated anti-goat (30 minutes), streptavidin-HRP (30 minutes), biotinyl tyramide (10 minutes) and streptavidin Alexa Fluor 555 (1 hour). Sections were washed and incubated ON with 4G8 (1:2000) and with donkey anti-mouse Alexa Fluor 488 (1 hour).
Double Immunofluorescent Labeling of Cerebrovascular Endothelial Cells
Human brain microvascular endothelial cells (TCS Cellworks, Buckingham, UK) were grown on fibronectin-coated coverslips in endothelial cell medium supplemented with 5% fetal bovine serum and 1% growth supplement (TCS Cellworks) at 37°C in 5% CO2. Cells were fixed in 4% paraformaldehyde (10 minutes; Alfa Aesar, Lancashire, UK), blocked and permeabilized in a solution containing 5% normal donkey serum and 0.1% triton X-100 in PBS (20 minutes) then incubated ON at 4°C with FVIIIRA/VWF (1:100) and picalm (1:50) antibodies. Cells were subsequently incubated with donkey anti-rabbit Alexa Fluor 488 (1 hour), with biotinylated anti-goat antibody (1:100, 20 minutes) and with streptavidin Alexa Fluor 555 (1 hour). Cells were mounted in Vectashield mounting medium and images acquired using a confocal laser-scanning microscope.
RNA Extraction and Reverse Transcription
RNA extraction from the frontal and temporal cortex and from the thalamus of 20 controls and 19 AD cases was performed as previously described (23
). Profiles of the cases are listed in . There were no significant differences in postmortem delay (p = 0.832) or age (p = 0.433) between the 2 groups. RNA concentration was determined using a Ribogreen RNA quantification kit (Invitrogen). RNA was reverse transcribed using the High Capacity c-DNA Archive Kit (Applied Biosystems, Foster City, CA), and the concentration of the resulting cDNA determined using the Picogreen DNA quantification kit (Invitrogen). These kits were used according to the manufacturers’ instructions.
Real-Time Polymerase Chain Reaction
The expression of PICALM and the calibrator genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FVIIIRA/VWF and CD31/platelet endothelial cell adhesion molecule were measured using the ABI 7000 sequence detection system (Applied Biosystems, Hs00200318_m1, Hs99999905_m1, Hs01109438_m1 and Hs01065279_m1 respectively). The 20-μl reaction mixture comprised SensiMix dT (Quantace, London, UK), TaqMan gene expression assay (Applied Biosystems) and 10 ng cDNA. Each sample was analyzed in triplicate using the following program settings: 50°C (2 minutes), 95°C (10 minutes), and 40 cycles of 95°C (15 seconds) and 60°C (1 minute). PICALM expression in relation to each calibrator gene was calculated (ΔCt) and used to determine the fold change in PICALM expression (2−ΔΔCt) in each case relative to the mean expression in controls.
The Mann-Whitney U test was used to assess the significance of changes in PICALM expression between AD and control groups. P values < 0.05 were regarded as significant.