Chemicals and Reagents
Synthetic human β-amyloid peptide 1-42 were purchased from BACHEM. Dithiothreitol (DTT), protease inhibitors cocktail (P8430) were obtained from Sigma-Aldrich. 4-Hydroxy-2-nonenal (HNE) was obtained from A.G. Scientific, Inc. Mac-R-P-P-G-F-S-A-F-K (Dnp)-OH Fluorogenic Peptide Substrate V, RNAse-free DNase came from R&D and Promega, respectively. GE nitrocellulose membrane was purchased from ISC BioExpress. An enhanced emiLuminescence (ECL) kit and Seize® Classic (G) Immunoprecipitation Kit were obtained from Pierce. Human neprilysin ELISA kit and Aβ1-40 protein ELISA kit are from R&D and Invitrogen, respectively. Other general chemicals and reagents were from Fisher Scientific.
Antibodies were from the following sources: Rabbit anti-HNE and Rabbit anti-NEP antibody (Chemicon); CD10 clonal 56C6 (Thermo Scientific). Anti-human/mouse Rhodamine conjugated affinity purified secondary antibody, goat anti-rabbit fluorescein conjugated secondary antibody, HRP conjugated secondary antibody were purchased from Chemicon.
SH-SY5Y neuroblastoma cells, obtained from the American Type Culture Collection (ATCC), were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Cells were seeded into plates or dishes in DMEM/F12 (1:1) medium, supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin.
Human neuroglioma H4 cell line was obtained from ATCC (item number: HTB-148). H4 cells stably transfected with human βAPP695wt cDNA under control of a CMV promoter (pcDNA3, Invitrogen) was kindly provided by B.C Eckman’s lab (Mayo Clinic, Jacksonville). H4 were grown in Opti-MEM (Gibco). The media were supplemented with 10% FBS and penicillin/streptomycin. The polyclonal H4 APP695wt line was maintained in 500 μg/ml geneticin (Gibco).
Cell treatments with HNE and β-amyloid peptide
SH-SY5Y cells were seeded into 100 mm dishes at a density of 2 × 104 cells per ml. Experiments were carried out 24–48 h after cells were seeded. Different concentrations of HNE (dissolved in 3% DMSO) or Aβ (dissolved in 0.4% DMSO) were added to the cultures 24 h before harvest. Final concentrations of DMSO in medium were <0.003%. Vehicles (with same concentrations of DMSO) were added to the cultures as controls.
Real time RT-PCR
Total RNA from a well of 6-wells plate was isolated using the RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. The concentration of nucleic acids was determined spectrophotometrically at 260 nm and 280 nm, taking into account the dilution factor. RQ1 RNase-free DNase (Promega) was used to remove relict of DNA which might interfere the output of PCR. For the PCR first strand synthesis was performed using the Omiscript® RT Kit (Qiagen Inc.). The resulting cDNA was then assayed by real time PCR.
Real time PCR was performed in 0.2 ml thin wall PCR plates using the iCycler thermal cycler (Bio-rad) and carried out with iQ SYBR Green supermix (Bio-rad) according to the manufacturer’s instructions. The standard reaction mix consisted of iQ SYBR Green supermix, forward and reverse primers at a final concentration of 500 nM each, 10 pg DNA template, DNase free water to give final volume of 20 μl. The mixture was heated to 95°C for 3 min followed by 35 cycles with denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. Human S26 was used as reference gene. Primer sequences for real time PCR: S26:5′-CGC AGC AGT CAG GGA CAT TT-3′ (F), 5′-TTC ACA TAC AGC TTG GGA AGC-3′ (R); NEP: 5′-GCC TCA GCC GAA CCT ACA AG-3′ (F), 5′-AGT TTG CAC AAC GTC CTC AAG TT-3′ (R).
Relative quantification of genes expression was carried out by comparative Ct method according to manufacture’s protocol (User Bulletin #2: ABI PRISM 7700 Sequence Detection System). Briefly, the genes mRNA level was expressed in cycle threshold (Ct) value; the Ct values for each sample were averaged from duplicate. Differences between the mean Ct values of NEP and reference gene were calculated as ΔCtsample= Ct NEP−Ct s26 for different treated groups, and that of the ΔCt for the control groups were set for calibrator (ΔCtcalibrator). Final results, the sample-calibrator ratio, expressed as N-fold differences of NEP expression in the HNE or Aβ1-42 groups compared with control, were determined as 2−(ΔCt sample−ΔCt calibrator).
Immunoprecipitation, Immunoblotting and Protein Quantification
After incubation with HNE (5 and 10 μM) or Aβ (1 and 2 μM) for 24 h, cells were harvested, lysed in Passive Lysis Buffer containing protease inhibitors cocktail (Sigma P8430). Lysates were incubated for 30 min on ice, and then centrifuged at 10,000 × g for 10 min at 4 °C. Protein concentration in the supernatant was determined by Coomassie blue protein binding method using protein quantification Kit-rapid (Sigma/Fluka) with bovine serum albumin as standard. The same amount of protein from HNE-treated, Aβ1-42 and vehicle-treated cells were used for western blotting assay and immunoprecipitation.
For NEP protein levels detection, samples containing equal amounts of protein were denatured in protein sample buffer (100 mM Tris-Cl pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 20% H2O, 200 mM DTT) at 100°C for 10 min and loaded, separated on 10% SDS–polyacrylamide gels and transferred to nitrocellulose membranes in a Bio-rad electrophoresis system. After blocking with TBST containing 5% non-fat milk, the membranes were kept at 4 °C overnight with primary antibodies (1:5000 for β-actin, 1:1000 for NEP, respectively), followed by HRP-conjugated secondary antibodies (1:5000 to 1:10000 dilution) at room temperature for 2 h. The target protein bands were detected using the ECL Western blotting detection system (Pierce) and autoradiography film (Fisher).
Immunoprecipitation with anti-NEP was performed with Seize® Classic (G) Immunoprecipitation Kit according to the manufacturer’s protocol. For total cell extract preparation, SH-SY5Y cells were lysed in Passive Lysis Buffer containing protease inhibitors cocktail (P8430, Sigma). Lysates were incubated for 30 min on ice, and then centrifuged at 10,000 × g for 15 min at 4°C. Supernatants with same amount of protein were incubated with rabbit anti-NEP polyclonal antibody (Chemicon, 1 μg/ml) at 4°C overnight. After washing Immobilized Protein G by adding 0.4 ml of BupH™ Modified Dulbecco’s PBS twice, the immune complexes were added to the spin cup containing the equilibrated immobilized protein G and incubated for more than 1 hour at 4°C. The samples were centrifuged briefly to remove the flow-through solution. The beads were extensively washed with BupH™ Modified Dulbecco’s PBS. The immunoprecipitated proteins were eluted by adding equal volume of ImmunoPure® Elution Buffer and then analyzed by Western blotting with anti-HNE and anti-NEP antibodies.
The density of the NEP and HNE blotting bands were quantified by ImageJ software (1.37v, Wayne Rasband, National Institutes of Health, USA) as described (Farrer et al. 2004
; Wang et al. 2007
Double immunofluorescence staining
Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 20 min and permeabilized with 0.2% Triton X-100 in PBS. After blocking for 1 h at RT with 1% goat serum/PBS, cells were incubated overnight at 4°C in a humidity chamber with primary anti-NEP (1:200). At the end of the incubation, the cells were rinsed three times with PBS-Tween-20 (0.05%) and incubated with the FITC conjugated goat anti-rat (1:50) for 60 min at room temperature. Then the cells were fixed with 4% paraformaldehyde at RT for 15 min. After rinsing with PBS for 3 times, second primary rabbit anti-HNE (1:200) were added and incubated for 2 h at RT or overnight at 4°C. Secondary, TRITC conjugated anti-rabbit IgG (1:50) were incubated for another 60 min at RT. All primary and secondary antibodies were diluted in PBS with 1% normal goat serum. After a rinse with PBS, the cells were photographed and analyzed with a fluorescent microscope (Nikon, E600, Japan). The immunofluorescence staining was done at least three times to assess the reproducibility of the results. In order to confirm the specificity of immunostaining for the second cycle, the primary or secondary antibodies were omitted or replaced with preimmue IgG.
Fluorometric assay of NEP activity
NEP activity was determined by Fluorescence Resonance Energy Transfer (FRET). After extraction with a final concentration of 0.1% Triton X-100 in phosphate-buffered saline (PBS; pH 7.4) for 30 minutes on ice, NEP activity in cell lysates was analyzed using a synthetic NEP fluorogenic peptide substrate (Mca-RPPGFSAFK[Dnp]-OH; R&D Systems, Inc., Minneapolis, MN) (Johnson and Ahn 2000
) at room temperature in the presence or absence of the NEP inhibitor, thiorphan. Samples dissolved in 50 mM HEPES buffer [pH 7.5] were preincubated with 10 μM thiorphan or PBS for 10 minutes prior to adding fluorogenic peptide substrate (dissolved in HEPES). Fluorescence was read after excitation at 320 nm and emission at 405 nm on a fluorescent ELISA plate reader (Spectra Max Gemini; Molecular Devices, Sunnyvale, CA). For kinetic analysis the cell lysates were incubated with increasing concentrations of substrate (4–20 μM) at room temperature. Fluorescence over the time was measured for 1 hour. The specific NEP activity was determined as the fluorescence difference occurring in the presence or absence of 10 μM thiorphan. Kinetic isotherms (Vmax
values) for NEP activity were determined by means of non-linear least squares fitting to the Michaelis-Menten equation using GraphPad Prim software (version 3.0 for Windows, ISI Software San Diego. CA, USA).
To measure the activity of membrane-bound enzyme, cell suspensions were prepared, then washed with PBS. 5 × 104 cells were resuspended in 50 μl of PBS contained in each well of black microtiter plates. The 50 μl substrate (10 μM in 50 mM HEPES) was added, and incubated at room temperature for 60 min. As above, thiorphan was used to determine the specific NEP activity in intact cells.
Sandwich ELISA measurement of NEP protein level and Immunocaptured specific NEP activity assay
Cells treated by vehicles, HNE or Aβ1-42 were lysated with Passive Lysis Buffer containing protease inhibitors cocktail (Sigma P8430). After 30 min incubation on ice, the lysates were centrifuged at 1300 rpm for 15 min at 4°C. Every single sample was assigned to the assays of NEP level (ELISA) and NEP activity (immunocapture-based).
NEP protein level and specific activity were measured by using DuoSet® ELISA kit (R&D). Sandwich ELISA assay of NEP was performed according to the protocol provided by manufacturer. Standard curves were produced from serial dilutions of recombinant human NEP.
For the immunocapure-based NEP activity (Miners et al, 2008), 96-well high binding ELISA plates (BD) were coated with 100 μl capture NEP (goat anti-human, 1.6 μg/mL) antibody diluted in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2) and left for 18 h at room temperature. The plates were washed with PBS containing 0.05% tween-20 for 3 times. After 60 min blocking with PBS containing 1% BSA fraction V, 100 μL of standards or supernatants were added and incubated at 4°C overnight. After 3 washes, the fluorogenic peptide (10 μM) diluted in 100 mM Tris-HCl pH 7.5, 50 mM NaCl and 10 μM ZnCl2 was added and incubated at 37°C in dark. Fluorescent readings were taken after 60 min. Control wells included in each plate contained PBS and fluoregenic peptide..
Sandwich ELISA measurement of Aβ
3 ml of SH-SY5Y and H4 APP695wt cells suspensions were seeded into 6-well plates. The cells were then allowed to grow to confluence (resting phase). The medium in each well was replaced with 1 ml of the appropriate fresh media and treated with 5, 10 and 20 μM HNE for 24 h after which the media were collected. Sandwich ELISAs for the detection of Aβ was performed according to the protocol provided by manufacturer (Biosource). In brief, 50 μl of Aβ peptide standards, controls and dilutions of samples were added into wells pre-coated with primary antibody specific for the NH2-terminus of Human Aβ (capture antibody) and allowed to incubate overnight at 4°C. 50 μl/well of secondary (detection) antibody was allowed to bind 4 h at room temperature. After 4 washes with buffer, 100 μl/well anti-rabbit Ig’s-HRP solution was added and continuously incubated for 30 min at room temperature. Developing was performed using stabilized chromogen (TMB) and the reaction stopped by the addition of 100 μl of stop solution. Plates were read at 450 nm in a SpectraMax Plus spectrophotometer (Molecular Devices) and analyzed by SOFTmax® PRO software. Aβ values in the unknowns were calculated by comparison to the values obtained for the synthetic Aβ standards analyzed on the same plate.
All results are given as mean ± SEM. Statistical analyses were performed with One-way ANOVA followed by least significant difference post hoc analysis (multiple comparisons) and t-test with threshold of P < 0.05.