Antibodies, Reagents and Plasmids
The following antibodies were used for immunoblotting and/or immunofluorescence: Tau-5 antibody (1:1000, Biosource), which recognizes phosphorylated and non-phosphorylated forms of Tau. Phosphorylation-dependent Tau antibodies included PHF-1 monoclonal antibody, which recognizes Ser-396 and Ser-404 residues (1:500, provided by P. Davies, Albert Einstein College of Medicine); T181 monoclonal antibody (1:1000, Sigma); S199/202 rabbit monoclonal antibody (1:1000, Sigma). Also used were Flag antibody (1:1000, Sigma); rabbit polyclonal BAG2 antibody (1:500, Abcam, clone ab58682); mouse anti-alpha tubulin (1:50, Sigma), mouse mono- and poly ubiquitinylated proteins, (1:20, clone FK2, BIOMOL), mouse β-actin monoclonal (1:10000, Sigma), rabbit anti-CHIP (N-terminal) (Sigma-Aldrich, C9118), and mouse anti-Hsp70/Hsc70 mAb (Stressgen, BB70). Lactacystin, a proteasome inhibitor (Fenteany and Schreiber, 1998
), was used at 10 μM (Calbiochem). Benzyloxycarbonyl-valinyl-alaninyl-aspartyl fluoromethyl ketone (Z-VAD.FMK), an interleukin-1β-converting enzyme (ICE)-like protease inhibitor was used at 20 μM.
Human 4R Tau and mouse BAG2-Flag cDNAs were cloned into pEYFP-C1, pDsRed2-C1 and pECFP-C1 vectors (Clontech). The RNAi sequences were obtained by running an algorithm for picking siRNA sites (Heale et al., 2005
) and cloned into pSilencer™
4.1-CMV puro vector (Ambion). The negative control construct was altered so that the sequence was no longer complementary to BAG2 mRNA. The BAG2 shRNAi sequences synthesized were: Sense strand GCCGGACCCUCACGGUUGAgg and antisense strand UCAACCGUGAGGGUCCGGCcc (overhang in lower case). The wild type human ubiquitin UBC expression plasmid (plamid # 11928) and the Ub-KO plasmid with all seven lysines of ubiquitin mutated to arginines (plasmid # 11934) were purchased from Addgene (Dantuma et al., 2006
; Bergink et al., 2006
). The K48R ubiquitin mutant was prepared by site-directed mutagenesis on a plasmid expressing mVenus-UBB (Quick-Change II site-directed mutagenesis Kit, Stratagene). Forward primer 5′-gctcatctttgcaggccggcagctggaagatggc, and reverse primer 5′-gccatcttccagctgccggcctgcaaagatgagc were used to introduce a lysine codon (aag) for an arginine codon (cgg) at position 48 of the ubiquitin protein. The mutagenesis was confirmed by sequencing using the primer 5′-cttaccggcaagaccatc.
Monkey kidney COS-7 cells were grown in Dulbeco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen) in a 5% CO2 humidified incubator at 37 °C. Cells were transfected with Lipofectamine (Invitrogen) and lysed with Ripa Buffer (1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 50 mM TrisHCl pH 7.4). Protein concentration was estimated by the BCA protein assay kit (Pierce) and was adjusted to 1 μg/μl.
Pregnant embryonic day 18 (E18) Sprague Dawley rats were sacrificed by CO2 incubation, and embryos were removed immediately by Cesarean section. Hippocampi were removed in dissection media without Calcium and Magnesium (HEPES Buffered Hanks’ Balanced salt solution (HBSS), HEPES, 10 mM, pH 7.3, and Pen/Strep) and digested in 0.25% trypsin with the same dissection media at 37°C for 15 min. The tissue was then washed 2X with HBSS and manually dissociated with a fire-bored Pasteur pipette. Cells were plated at 250,000 cells per six well plate for immunoblot analyses and 90,000 cells per six well plate for immunofluorescence. The plates were previously coated overnight with poly-L-Lysine and incubated with Glial medium (MEM, 20% glucose, pyruvate, Pen/Strep and 10% Horse serum) until plating. 3 h after plating, the medium was changed to Neurobasal medium containing B27 supplement and 0.5 mM glutamine. Very few glial cells were observed in these cultures.
COS7 cells were cotransfected with TAU and BAG2 or transfected with TAU in the absence of BAG2. 16 hours postransfection, cells were incubated for 30 min in DMEM methionine/cysteine Free, supplemented with dyalized FBS and L-Glutamine. Cell were incubated in the same media with Expre35S35S protein labelling mix (200uCi/mL; PerkinElmer, MA) for one hour. After this pulse period, media was removed and cells were washed twice with pre-warmed PBS and incubated up to 28 hs in the same media with unlabelled L-cysteine-HCl (500 ug/mL; Sigma-Aldrich, MO) and L-methionine (100 ug/mL; Sigma-Aldrich). Cells were harvested at 0, 4, 8, 24, and 28 hrs after the pulse period. For harvesting, cells were washed once in pre-warmed PBS, scraped from the well, centrifuged at 2300 rpm/5 min/4 C. The pellet of cells were resuspended in lysis buffer (150 mM Kcl, 25 mM trisHCl, 2 mM EDTA, 0.5 mM DTT, 0.5% NP-40 and 1X Protease Inhibitor Coctail-Sigma Aldrich), and incubated at 4C for 30 min, in a head-to-tail mixer. Lysates were centrifuged at 13200 rpm/1 min/4 C and the supernatant were transferred to a microcentrifugue tube. The lysates were immunoprecipitated with PHF-1 antibody, using protein-G sepharose. Spin Column (Sigma-Aldrich) were used to minimize sepharose beads lost during washing times. Immunoprecipitated p-Tau was eluted using 1X Laemmli sample buffer and separated in an SDS-PAGE 10% gel (1.0 mm). Acrylamide gels were Coomassie-blue stained and then incubated for 30 minutes with EN3HANCE enhancer solution (PerkinElmer), washed on cold water with 1% glycerol and dried. A Phosphorimager screen was exposed for 3 days at −80C and scanned images were quantified with quantity-one software (Bio-Rad Laboratories, CA)
Transfections, RNA Isolation, qPCR
To over-express Tau and BAG2 protein, COS-7 cells were transfected with 4 μg of BAG2 RNAi or a non-silencing RNAi vector in a 6 well plate with Lipofectamine (Invitrogen). 48 h later, the cells were co-transfected with 2 μg of 4R Tau (pDsRed2-C1) and 2 μg of BAG2-Flag pEYFP-C1. 24 h later cells were lysed for immunoblotting. All experiments in primary neurons were initiated five days after plating, and the amount of transfected plasmid was half of the amount used in COS-7 cells. To over-express mir-128a we transfected 250,000 DIV 5 neurons with 75 nM or 250 nM pre-mir-128a or pre-scrambled (Ambion) using Lipofectamine. For transfections, lysates were harvested 48 h later. For RNA isolation and qPCR, precipitates were added to cells and medium was renewed after 5 h. After the indicated time, RNA was extracted by using the miRVana Isolation Kit (Ambion) and DNase treated (DNA-free, Ambion). Reverse transcription was performed by using Superscript III (Invitrogen). Quantitative real-time PCR was performed in an Applied Biosystems PRISM 7900HT Fast Real-Time PCR System with SYBR green PCR master mix (Applied Biosystems). BAG2 Ct was normalized to that of GAPDH.
Dual Luciferase Assay
Downstream of the Firefly luciferase reporter vector pMIR-Report (Ambion) we inserted a 60 bp sequence of the BAG2 3′UTR that contained the miR-128a predicted target site. Our negative control construct was altered so that three bases at the seed region were no longer complementary to miR-128a. We seeded 50,000–60,000 HeLa cells 24 h prior to transfection in 24-well tissue culture plates. The next day 200 ng of the pMIR-Report vector, 20 ng of the transfection control Renilla vector phRLTK (Promega) and 30 pmol pre-mir-128a or pre-srambled were transfected using 3 μl of lipofectamine. Lysates were harvested 24 h after transfection and reporter activity was measured using the Dual Luciferase Assay (Promega). The Firefly luciferase units from every sample were normalized to the units of the transfection control Renilla.
Preparation of Sarkosyl Insoluble Tau
Preparations of sarkosyl insoluble Tau were previously decribed (Cho and Johnson, 2004
; DeTure et al., 2002
). Briefly, cells were prepared in lysis buffer containing 50 mM TrisHCl pH 7.4, 0.15 M NaCl, and 1% Sarkosyl and scraped off the plate after 30 min incubation at 4 °C. The samples were vortexed, incubated for 30 min at RT, centrifuged 20 min at 3000 g. The supernatants were recovered and centrifuged at 170,000 g for 2 h at 4°C. Recovered supernatants were mixed with electrophoresis sample buffer. Pellets containing sarkosyl insoluble material were mixed with 2X sample buffer. All the samples were boiled at 100 °C for 5 min. Protein was separated on a 4–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a Protran membrane, incubated with the relevant antibodies and detected with horseradish peroxidase conjugated antibodies. Bands were visualized with chemiluminescence (Pierce).
COS-7 cells were transfected with 4 μg of each plasmid (BAG2 and Tau). 24 h later, cells were washed in cold PBS and harvested in immunoprecipitation buffer (150 mM NaCl, 0.5% TritonX100, 50 mM TrisHCl pH 7.5, 5 mM EDTA, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail). The lysate was pre-cleared for 1 h at 4 °C with 25 μl of protein G (Sigma) and centrifuged at 14,000 rpm. The supernatant was incubated with 5 μg of antibody against TAU-5 and 60 μl of protein G and rocked at 4°C overnight. The protein G beads were pelleted and washed with immunoprecipitation buffer. The precipitates were resolved on SDS-PAGE gel and immunoblotted. Beta-actin was used to normalize Tau loading. Statistical analysis were carried out with PRISM Software (GraphPad software) using the Dunnetts’s multiple comparison test after one-way analysis of variance (ANOVA).
Primary rat hippocampal neuronal cultures were infected at DIV 1 with BAG2-FLAG lentivirus or a GFP control lentivirus and grown for one week. The cells were then washed twice with pre-warmed PBS and incubated for 30 min in lysis buffer (150 mM KCl, 25 mM TrisHCl, 2 mM EDTA, 0.5 mM DTT, 0.5% NP-40 and 1X Protease Inhibitor Cocktail (Sigma Aldrich)) at 4 C. They were scraped from the well and the lysates were centrifuged at 13,200 rpm/10 min/4 C, and the supernatant was recovered. Approximately, 600 uL of lysate were immunoprecipitated with PHF-1 antibody or with anti-FLAG M2 agarose affinity gel (Sigma-Aldrich, MO). Immunoprecipitates were eluted with 1X loading sample buffer and western blot was performed with anti-FLAG, PHF-1, anti-CHIP, anti-HSP70.
For immunofluorescent labeling, neurons or COS-7 cells were grown on glass coverslips and gently washed (3×1 min) in warmed MEM-H (1.423% of MEM with HEPES, 0.22% NaHCO3), pH 7.2. The cells were fixed in 4% (wt/vol) paraformaldehyde (15min, 37°C), washed (3×1 min) with PBS, permeabilized with 0.1% triton X-100 (15 min), washed 2X in PBS and blocked for 20min with blocking medium (MEM-H, 1% BSA, 10% Horse serum). Cells were incubated with primary antibodies in blocking medium for 2 h at room temperature, washed (3×5 min) with PBS and incubated for an additional 1 h with secondary antibodies. Finally, cells were washed in PBS and visualized by fluorescence microscopy (Nikon eclipse TE300).
To analyze the effect of BAG2 over-expression, BAG2 RNAi over-expression or BAG2 RNAi non-silencing on Tau levels, BAG2-pEYFP-C1 vector, BAG2RNAi + pEYFP-C1 vector and BAG2 RNAi non-silencing-pEYFP-C1 vector were separately transfected into 5 day old neurons plated on glass coverslips. After three days neurons were fixed and labeled with antibody. In each transfection experiment, 100 BAG2 positive and 100 BAG2 negative neurons were randomly chosen in the same plate. Optical Density (OD) values of PHF-1 stained neurons were compared among the transfected and non-transfected neurons in the same plate. Quantification of pixel intensity was performed with Metamorph software and normalized to background. The number of cells in each group with OD >200 and OD <200 were binned.
For co-localization studies, COS-7 cells were transfected with BAG2 and Tau for 24 h, fixed in 4% paraformaldehyde, and stained with mouse monoclonal anti-α-tubulin and counterstained with Alexa 594 antibody. Data was acquired on a spinning disc microscope (Olympus) and then post-processed by ImageJ software (http://rsb.info.nih.gov/ij/
). Images were processed using successive median and Wiener filtering to reduce noise.
BAG2 mobility assay
COS-7 cells were transfected with BAG2-pEYFP-C1 plasmid and/or with Tau-pECFP-C1 plasmid in 1:4 ratio. 6 hr after transfection, 100 μM 3,4-Methylenedioxy-γ-benzylidine-γ-butyrolactam (KNK437) was added. After 20 h, the cells were imaged on an Olympus IX50 inverted microscope with an Olympus UPLSAPO 60× NA=1.2 water immersion objective and a DVC-1310 CCD camera (DVC Company). With the 8-bit camera used for this study, gain was set to avoid saturated pixels in the image. Images were analyzed with custom-written Matlab (Mathworks) programs in order to extract puncta trajectories. Images were first segmented to extract the puncta, their center was determined by adjusting the background- and intensity-corrected intensity profile to a Gaussian curve. Puncta trajectories were then determined by matching puncta positions between frames using ascending pair wise distances and using puncta diameter and intensity as criteria to confirm the match. See Supplemental Data
for additional details.