3-Amino-1-propanesulfonic acid (3-APS) (Ref. A4147), Phenylmethylsulfonyl fluoride (PMSF), EDTA, EGTA, 2-Mercaptoethanol, MES, HEPES, Trizma Base, NaF, Na3VO4 and SDS were purchased from Sigma. Triton X-100, Tween-20, NaCl and MgCl2 were obtained from Merck. Acrylamide/bisacrylamide solution and Bradford reagent was supplied by Bio-Rad. Protran Nitrocellulose Transfer Membrane was from PerkinElmer. The chemiluminiscent detection kit (Western Light) was purchased from Tropix. DAPI (Ref. 268298) was obtained from Calbiochem. Thioflavin S (Th-S) (Ref. T-1892) was purchased from Sigma.
For immunoblot analysis, we used anti-β-tubulin (1/2000; Sigma), anti-β-actin (1/2500; Sigma), and 7.51(anti-tau antibody; a gift from Dr. C. M. Wischik, UK) antibodies followed by relevant secondary antibodies (1/2000; DAKO).
For immunofluorescence, we used anti-β-tubulin (1/500; Sigma), Tau-5 (1/500; Chemicon) and T14(1/500; Zymed Laboratories). The secondary antibodies (Molecular Probes) were used at 1/1000.
Actin staining was done by using phalloidin TRITC (1/300; Sigma). 0,01% Thioflavin S in PBS 1× was used for staining of treated or untreated cell cultures.
Reverse transcription of RNA and polymerase chain reaction (RT-PCR)
Total RNA from HEK 293 and HEK 293 tau expressing cells were prepared using the reagent TRIzol (Invitrogen) and following the supplier's protocol. Reverse transcription was performed using the first cDNA synthesis kit (Roche Applied Science) on 5 μg of RNA with oligo(dT) primers. PCR was performed with the primers for tau R1 (5'-GGCGAATTCGGATCCATGCCAGACCTGAAGAATG-3') and R2 (5'-GGCCTGC AGTTACTCGCGGAAGGTCAGCTTGTGGG-3'). The amplifications were performed basically with the following protocol: 30 cycles of 94°C for 45s, 55°C for 1 min, and 72°C for 1 min. The PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. As a loading control, RT-PCR for actin was performed for each sample. The used primers for murine actin were H1 (5'-GCATGGAGTCCTG TGGCATCCACG-3') and H2 (5'-GGGTGTAACGCAACTAAGTCATAG-3'). Differences among groups were analysed by Student's unpaired t-test to determine significant differences between means.
HEK 293 tau cells
HEK 293 tau expressing cells (expressing tau 3R isoform, a kind gift from Dr. Miguel Medina) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM Glutamine, 1 mM Piruvate, 100 U/ml penicillin, 100 U/mL streptomycin, and 0.2 mg/ml Zeocin in a humified atmosphere of 5% CO2/95% air at 37°C. Proliferating HEK 293 tau expressing cells were plated on glass cover slips coated with 1 mg/ml poly-L-lysine, maintained in the same culture medium during 24 hours and treated with 3-APS for 24,48 and 72 hours , at 100 μM, 500 μM 3-APS.
Human neuroblastoma SH-SY5Y cells [40
] were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM Glutamine, 100 U/ml penicillin, 100 U/mL streptomycin in a humified atmosphere of 5% CO2
/95% air at 37°C. For differentiation, proliferating SH-SY5Y cells were plated and then cultured in Neurobasal-B27 medium (Gibco, Grand Island, NY) supplemented with 2 mM dibutyril cyclic AMP and 1 mM glutamine for 7 days. At this time, about 90% of the cells has extended long neurites and became postmitotic (showing no significant incorporation of tritiated thymidine into DNA).
After 7 days differentiated SH-SY5Y cells were treated during 48 hours with 100 μM, 500 μM 3-APS.
Hippocampal neurons were cultured as described by Banker and Cowan [41
]. Pregnant wild type females were sacrificed at gestional day 18, and the embryos were removed in sterility. Dissociated hippocampal neurons were plated on glass cover slips coated with 1 mg/ml poly-L-lysine and then 20 μg/ml laminin. After incubating in medium containing 10% horse serum (Gibco-BRL, Gaithersburg, MD) for 24 hours, the medium was changed and supplemented with N2 and B27 (Gibco-BRL). At this point, neurons were treated with 3-APS for 24, 48 and 72 hours, at 100 μM, 500 μM 3-APS.
Cell Lysis and Western Blot Analysis
HEK 293 and HEK 293 tau expressing cells were washed once with phosphate-buffered saline (PBS), placed on ice, and then homogenized in a buffer containing: 20 mM HEPES, pH 7.4; 100 mM sodium chloride (NaCl); 100 mM sodium fluoride (NaF); 1% Triton X-100; 1 mM sodium orthovanadate (Na3VO4); 5 mM EDTA; and the Complete protease inhibitor cocktail (Roche Diagnostics, Barcelona, Spain). After determination of the protein content via Bradford assay, samples containing the same amount of protein were mixed with electrophoresis buffer containing sodium dodecyl sulfate (SDS), boiled for 5 min, and separated by gel electrophoresis in the presence of SDS on 10% acrylamide gels. The proteins were then transferred to nitrocellulose membranes by following standard procedures, and the membranes were blocked with 10% nonfat dried milk in PBS, 0.2% Tween-20 (PBST). The blocked membranes were incubated overnight with primary antibodies diluted in blocking solution at 4°C. The membranes were then rinsed three times in PBST and incubated with the corresponding peroxidase-conjugated secondary antibody for 1 hr at room temperature. The immunoreactive proteins were visualized by using an enhanced chemiluminescence detection system (Amersham), and subsequent densitometric analysis was performed with an imaging densitometer (GS-710 model; Bio-Rad, Hercules, CA). Western blot analysis were also done using an antibody raised aginst actin (Sigma) as loading control. Protein extracts prepared from differetiated SH-SY5Y cells were processed and analysed by Western Blot using tau antibody 7.51 (1/100) as previously indicated.
Immunoblot analysis of aggregated Tau
Treated HEK 293 tau expressing cells, or SH-SY5Y neuroblastoma cells, were homogenized in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1% SDS, 5 mM 4-(2-aminoethyl) benzene-sulfonyl fluoride hydrochloride, 1 μg/ml proteases inhibitors (Complete protease inhibitor cocktail (Roche Diagnostics, Barcelona, Spain)), 1 mM NaF, 1 mM Na3VO4, and 1 mM β-glycerophosphate) without thawing by using a polytron homogenizer (Kinematica, Kriens, Switzerland) at its highest speed for 30 s. Before centrifugation to fractionate the detergent soluble from detergent insoluble material, an aliquot from each sample was taken to determine if each sample contains, or not, a similar amount of actin (loading control). To do that we have used an antibody raised against actin. Tau protein aggregates from homogenates were isolated by centrifugation (4°C) for 20 min at 20000–30000 g (table-top centrifuge at 15000 rpm). One fourth of the supernatant volume, after centrifugation, was taken to characterize the protein present in that fraction. The whole aggregated protein present in the insoluble fraction was then diluted in O+ buffer (62.5 mM Tris-HCl pH 7.0, 10% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, 2.3 % (w/v) SDS, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM Na3VO4 1 mM PMSF (phenylmethyl-sulfonyl fluoride), 1 μg/ml proteases inhibitors (Complete protease inhibitor cocktail (Roche Diagnostics, Barcelona, Spain)), boiled for 5 min, and separated by gel electrophoresis in the presence of SDS on 10% acrylamide gels. Then, the protein content was analized by western blot as previously described.
After treatments, HEK 293 and HEK 293 tau expressing cells were fixed with either cooled methanol (-20°C) (tubulin immunofluorescence) or 4% paraformaldehyde (actin and tau immunofluorescence) for 20 min at 4°C or 37°C, respectively, and then washed with buffer A (0.1 M MES; 2 mM EGTA; 0.5 mM MgCl2) or with phosphate buffered saline (PBS), respectively. Fixed cells were incubated with 1 M glycine 30 min then permeabilized with 0.2% Triton X-100 in PBS or buffer A for 5 min at room temperature. The cover slips were blocked with 1% BSA/PBS or Buffer A for 1 h at room temperature and incubated in primary antibodies in 1% BSA, in PBS, or in buffer A, for 1 h at room temperature. After washing three times with PBS or with buffer A, the secondary antibodies were incubated for 1 h, at room temperature. DAPI (1 μg/ml) staining was performed 10 minutes before finishing secondary antibody incubation. Finally, the covers lips were washed three times with PBS or with buffer A and once with H2O, and mounted with FluorSave Reagent (Calbiochem).
Fluorescence microscopy was used to measure fluorescence intensity. After staining with 0.01 % Th-S and T14 antibody cell were observed on a Zeiss Axiovert200 fluorescent microscope. T14 antibody and thioflavin-S fluorescence images were captured through a 100× objetive on a high resolution CCD camera (SPOT RT Slider, Diagnostic). The images were saved for later analysis and quantitation. Camera exposure and light settings were keot constant during each experiment. The fluorescence intensity measurements were carried out using the image analysis software Metamorph 6.1 r6 (Universal Imaging).
A similar protocol was followed for SH-SY5Y neuroblastoma cells and primary cultures of hippocampal neurons.
Binding of tau to microtubules
For in vitro
assembly of tau protein with microtubules, purified tubulin was assembled in the presence of taxol [42
] and mixed with tubulin, after incubation of the mixture, the polymerized protein was isolated by centrifugation in Airfuge (Beckman) at room temperature, for 15 min at 100000 g. The protein present in supernatant and pellet was analyzed by gel electrophoresis followed by western blot using anti-tau and anti-tubulin antibodies.
Determination of Cell Viability
Cell viability was assessed by calcein-propidium iodide uptake [43
]. Calcein/acetoxymethyl ester is taken up and cleaved by esterases present in living cells, yielding yellowish-green fluorescence. In contrast, propidium iodide is taken up only by dead cells, which then exhibit orange-red fluorescence. Briefly, cells were incubated for 30 min with 8 μM propidium iodide (Sigma) and 1 μM calcein/acetoxymethyl ester (Molecular Probes). The cultures were then rinsed once with Hanks balanced salt solution containing 2 mM CaCl2
, and the cells were visualized by fluorescence microscopy with a Zeiss Axiovert 135 microscope. Three randomly selected fields were analyzed per well (100–200 cells/field) in at least three independent experiments. Cell viability was expressed as the percentage of calcein-positive cells with respect to the total number of cells.
Protein and peptide preparation
Recombinant human tau (whole molecule), N-terminal tau (residues 1–251), peptide 3RC (containing three tubulin binding motifs and the carboxyl-terminal region), peptide 3R (containing three tubulin binding motifs), have been synthesized and purified as previously reported [4
]. Tau peptide containing residues 317–335 was obtained as previously indicated (meter referencia 33).
Assembly of Tau Peptides into Filaments
Filaments were grown by vapor diffusion in hanging drops in the standard way used for protein crystallization as previously indicated [44
]. In a typical experiment, 0.5–2 μg of peptide was resuspended in 10–15 μL of buffer A (0.1 M MES (pH 6.4), 0.5 mM MgCl2
, and 2 mM EGTA) containing 50 mM NaCl and 3-APS at concentrations ranging from 1 to 4 mM. In other assays, a similar amount of tau (0.5–2 μg) but in the presence of 0.5 mg/mL heparin was resuspended in buffer A [4
]. The reservoir in this case contained 0.2 M NaCl in buffer A. Filaments were obtained after incubation for 4 days at 4°C. The samples we revisualized by electron microscopy as described [4
]. Electron micrographs were obtained at a magnification of 80000 on Kodak SO-163 film. Micrographs were digitized using an Eikonix IEEE-488 camera with a pixel size equivalent to 7 Å in the specimen plane. Processing and measurements were performed using the Digital micrograph 2.1 software from Gatan. Several standards were used for the control of the measurements. Alternatively, tau polymers were stained with Th-S and visualized by immunofluorescence [27