A validated antibody panel for the characterization of tau post-translational modifications

The peptide array was prepared by Peptides and Elephants GmbH (Berlin, Germany). Synthesized peptides with or without specified modifications were quality controlled by HPLC and MS and were attached C-terminally on trioxatridecanediamine (TOTD) membranes. The membranes were treated 10 min with methanol and washed three times with 1X TBS to activate. After 2 h of blocking with 4% BSA in 1X TBST (1X TBS, 0.05% Tween-20), overnight incubation was performed in blocking solution with 1 μg/ml primary antibody at 4 °C. The next day, the membranes were washed three times with 1X TBST and secondary antibody incubations (1:20,000, IRDye Donkey anti-mouse 800 and IRDye Goat anti-rabbit 680) were done in blocking solution for 2 h at room temperature. The membranes were then washed three times with 1X TBST and once with 1X TBS and developed with a Li-Cor Odyssey CLx Imaging station.

Mice were maintained in a temperature-controlled room (22 °C, 60% air humidity) with a light/dark cycle of 12 h/12 h and had access to food and water ad libitum. Animals were sacrificed at nine months of age by cervical dislocation and brains were snap-frozen in liquid nitrogen and stored frozen at −80 °C until lysis. All procedures were in compliance with German Animal Protection Law and were approved by the competent authorities (Landesamt für Naturschutz und Verbraucherschutz Nordrhein-Westfalen; AZ 87–51.04.2011.A049/01). Anonymized human post-mortem tissue was obtained from the London Neurodegenerative Diseases Brain Bank, a member of the Brains for Dementia Research Network. Tissue donor characteristics were as follows: Donor BBN_18399: male, 77 years, 11 h post-mortem interval, cause of death: metastatic prostate cancer. Donor BBN_24243: female, 69 years, 48 h post-mortem interval, cause of death: advanced pancreatic cancer. Donor BBN_13802: male, 74 years, 22.5 h post-mortem interval, cause of death: respiration failure; cancer.

Lysates from hTg-Tau and Tau-KO mice and human entorhinal cortices were prepared by dounce homogenizer in 350 μl of lysis buffer containing 150 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100 and protease, phosphatase, demethylase (500 μM IOX1, 2 μM Daminozide, 10 μM Paragyline Hydrochloride), deacetylase (10 μM Trichostatin A, 5 mM Nicotinamide), O-GlcNAcase (1 μM Thiamet-G) inhibitors. The protein concentration was measured by BCA assay according to manufacturer’s instructions (BioRad). 20 μg of protein, which was boiled at 95 °C for 5 min in sample buffer, was separated on an SDS-gel and was immunoblotted on PVDF membranes. The membranes were blocked with 5% BSA in 1X TBST and primary antibody (1:1000) incubations were performed in blocking solution overnight at 4 °C. The next day, secondary antibody incubations (1:20,000, IRDye Donkey anti-mouse 800 and IRDye Goat anti-rabbit 680) were done in blocking solution for 2 h at room temperature. The membranes were then washed three times with 1X TBST and one with 1X TBS and developed on a Li-Cor Odyssey CLx imaging station. In the case of no or low signal detection, the immunoblotting was repeated with 50 μg of protein and primary antibodies diluted 1:500. Actin (Sigma-Aldrich, Cat No. A2103) was used as a loading control.

The tau 2N4R isoform (aa1–441) and the truncated tau (aa1–421) was recombinantly expressed in E. coli strain BL21(DE3) (Sigma-Aldrich, Cat. No. CMC0014) using pET19b as expression vector. Protein purification was performed as previously described with modifications described below [9]. Briefly, the frozen E.coli pellets were suspended in sodium-phosphate buffer (50 mM NaPi, 1 mM EGTA, 1 mM DTT, pH 7) and incubated for 30 min at 100 °C. After centrifugation (12 000×g, 4 °C, 15 min) the filtrated supernatant was purified with liquid chromatography using a HiTrap™ IEX HP column (Fisher Scientific, Cat. No. 11748508): The column was washed with sodium-phosphate buffer (50 mM NaPi, 1 mM EGTA, 1 mM DTT, pH 7) and the protein was eluted using a gradient of 0–300 mM NaCl in sodium-phosphate buffer. The column fractions containing pure protein, as determined by gel electrophoresis, were pooled and concentrated with Amicon centrifugal filter units (Merck Millipore, Cat. No. UFC203024).

For the caspase digestion, 3 μg of purified tau 2N4R protein (tau 1–441) were mixed with caspase 3 enzyme (Enzo Life Science, Cat. No. BML-AK010–0001) in caspase buffer (100 mM HEPES pH 7,4, 200 mM NaCl, 0,2% CHAPS, 2 mM EDTA, 20% Glycerol, 10 mM DTT). This mixture was incubated for 1 h at 37 °C. The digestion was analyzed by Western blot.

Meso Scale Discovery (MSD) Gold Streptavidin small-spot 96-well plates were blocked with 5% (w/v) Blocker A solution in Tris Wash Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.02% Tween-20). Plates were sealed and allowed to block for 1 h at room temperature (RT) on a plate shaker. The plates were then washed three times with Tris Wash Buffer and coated with 30 μL of biotinylated antibody diluted in 1% Blocker A solution. The biotinylation of the antibodies were performed according to the manufacturer’s instructions (EZ-Link Sulfo-NHS-Biotin, Cat No. 21217, Thermo Scientific). Before biotinylation, BSA was removed with the Melon Gel IgG Purification Kit (Cat. No 45212, Thermo Scientific) if necessary. After incubating the plates for 1 h at RT on a plate shaker, they were washed three times with Tris Wash Buffer. 5 μg of protein lysates (diluted in 50 μl) were added and incubated for 1 h at RT on a plate shaker. Plates were washed three times with Tris Wash Buffer to get rid of unbound lysates and were incubated with 25 μl of 0.5 μg/ml detection antibody (Tau12 labeled with MSD Sulfo-Tag-NHS-Ester, Cat. No: R31AA, Meso Scale Discovery) diluted in 1% Blocker A solution for 1 h at RT on a plate shaker. The plates were then washed three times with Tris Wash Buffer and 150 μl of 2X Read Buffer (Cat. No. R92TC, Meso Scale Discovery) were added 5 min before the signal was measured on a Meso Scale Discovery Quickplex platform.

hiPSCs generated from healthy donor fibroblasts reprogrammed with Sendai Virus were obtained through the StemBANCC consortium. Cells were cultured in mTeSR1 medium (Stemcell Technologies) on Matrigel (Corning). To generate hiPSC – derived neurons, hiPSCs were first differentiated into neural progenitor cells (NPCs) according to [10] with minor modifications. Briefly, NPCs were induced from iPSCs in N2/B27 medium (consisting of Advanced DMEM/F12:Neurobasal (1:1), 1X N2, 1X B27, 1% L-Glutamine, 1% Pen/Strep (all Thermo), 5 μg/mL BSA (Sigma)) supplemented with 10 ng/mL hLIF (Millipore), 4 μM CHIR99021, 3 μM SB431542 (both Tocris), 0.1 μM Compound E (Calbiochem) for 11 days. NPCs were further cultured on Matrigel - coated plates in N2/B27 medium supplemented with hLIF, CHIR99021, SB431542, with additional 10 ng/ml FGF2 (R&D).

For differentiation into neurons, NPCs were seeded at 8 × 10⁵ cells / cm² on laminin (Sigma) coated plates in N2/B27 without supplements and maintained for 11 days with medium change every 2–3 days. For the final maturation, hiPSC – derived neurons were dissociated using Accutase (Thermo) and re-plated at the same density on poly-D-lysine coated pre-coated plates (Greiner), coated with laminin in maturation medium (N2/B27 supplemented with 1 mM dbcAMP, 200 nM ascorbic acid (both Sigma), 5 μg/ml laminin, 2 ng/ml BDNF (Peprotech) and 2 ng/ml GDNF (R&D)). Medium changes were performed twice a week for additional 4 weeks.

hiPSC-derived neurons in 96-well plates were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton-X100 in 1X TBS for 10 min at RT. Cells were then washed three times with 1X TBS and blocked with 5% BSA in 1X TBS for 1 h at RT. Primary antibody incubation (1:50) was performed in 2.5% BSA in 1X TBS overnight at 4 °C. The primary antibody dilution was decreased to 1:25 when no signal was detected. The next day, the cells were washed three times with 1X TBS and secondary antibody (Alexa Fluor goat anti-mouse 488 and Alexa Fluor goat anti-rabbit 488) incubation was performed in 2.5% BSA in 1X TBS for 2 h at RT. Cells were washed two times with 1X TBS and incubated with 1X TBS containing Hoechst (1:20,000) for 10 min at RT and then washed once with 1X TBS and cells were kept in 1X TBS for microscopy. Microscopy was performed with Zeiss inverted fluorescence microscope with 20X objective and images were processed in ImageJ.

Tau PTMs have been studied extensively in the context of AD as well as other neurodegenerative diseases [6]. Many studies using different experimental approaches and model systems show that PTMs affect the function of tau by modulating its binding to microtubules, its degradation, subcellular localization and secretion [6]. However, the excessive information coming from human samples, different model systems and experimental approaches makes it difficult to summarize current knowledge about tau modifications in human brain. Additionally, researchers are becoming increasingly aware of interplay between different PTMs, in particular on heavily modified proteins such as tau. PTMs can be mutually exclusive (targeting the same amino acid), or prevent or promote subsequent modifications through conformational changes [11]. In order to provide an overview of the current state of knowledge of the tau PTM landscape and also facilitate the visualization of potential or proven interactions between PTMs, we designed an interactive tool, which comprises all modifications that have been shown in human brain so far. Initially, tau PTMs were retrieved from the dbPTM database, which combines experimentally validated PTM sites from a number of resources including the Human Protein Reference Database (HPRD 9.0), the Phospho.ELM database (PhosphoELM.10011), PhosphoSitePlus (Phosphositeplus.1010730), UniProtKB/Swiss-Prot (Phosphositeplus.1010730), and SysPTM (SysPTM 1.1). We used the Uniprot ID: P10636 to query dbPTM for microtubule-associated protein tau (MAPT) entries, and mapped the modified residue positions to the PNS-tau isoform and then to the respective residue in the 2N4R tau isoform. We then verified the entries and added recent information by manual literature search. While curating the data we applied these rules: 1) The modification has been shown in human brain samples with mass spectrometry and/or immunohistochemistry. 2) The modifications shown in other model systems such as mouse or cultured cells were only added as supporting information in the literature section. These stringent criteria prevent the inclusion of PTMs that may have arisen as artifacts of the model system with no relevance for human brain. The current version of TauPTM contains 53 phosphorylation, 8 acetylation, 2 deamidation, 10 methylation, 5 dimethylation, 4 nitration, 4 ubiquitination and 6 proteolytic cleavage sites present on human tau (www.​tauptm.​org). In addition to the single PTMs of tau, we entered known cross-reactions between modifications such as the finding that acetylation at K280 prevents the phosphorylation at T212 and S214, whereas it promotes the phosphorylation at S262. These cross-reactions are visualized by color-codes on the respective amino acids as well as in a table format, allowing for the fast visual inspection of potential known interactions for single PTMs.

We next used our TauPTM tool to search for commercially available antibodies raised against the single modifications. We found several antibodies covering 26 different modification sites (Fig. 1), approximately one third of all known human tau PTMs are therefore covered by commercially available antibodies. To test antibody specificities for PTM-tau over non-modified tau, we synthesized 15 amino acid-long peptides derived from the human tau sequence (2N4R isoform) with either no, single or multiple PTMs on a membrane. Since antibody epitopes may include amino acids N- or C-terminal of the detected PTM, we designed at least two peptides for each antibody, with the modified amino acid moved by five positions.

We tested a total of 36 nitration-, acetylation-, dimethylation- and phosphorylation-specific antibodies, focusing only on antibodies detecting specific single modifications rather than conformation specific antibodies which require more than one modification and a specific conformation/aggregation state of tau oligomers, as these conformational epitopes cannot be mimicked with synthesized peptides.

Among the antibodies detecting nitrated, acetylated and dimethylated tau (Fig. 2), those raised against nitration on Y18 and Y29 (Fig. 2a, b), acetylated K280 (Fig. 2c) and dimethylated K311 (Fig. 2d) showed high specificity for their respective modifications with no detection of non-modified peptides. Furthermore, their binding was not affected by PTMs in the surrounding tau sequence, an important advantage when analyzing heavily modified protein samples.

Next, we tested commercially available phosphorylation-specific tau antibodies. As phosphorylation is a major PTM on tau and hyperphosphorylation is linked to tauopathies, a plethora of antibodies is available. We tested 32 phospho-tau antibodies raised against 22 phosphorylation sites that have been reported in human brain. All antibodies shown in Figs. 3 and 4 were specific for their respective phosphorylation site, however, for 7 out of 19 their binding was impaired by phosphorylation events at neighboring sites. For instance, binding of the antibody against phosphorylated S198, while highly specific for the modification, was hindered by other phosphorylation events in close vicinity (Fig. 3b).

Interestingly, for three antibodies (pT205, pT212 and pS409), only phosphorylation events C-terminal of the specific PTM site interfered with detection (Fig. 3f and g, Fig. 4g). Overall, however, this finding is concerning, as tau is most often modified at multiple sites at the same time and phosphorylation events may thus block antibody detection. Moreover, the antibodies raised against pS202, pS199/202, pT205, pS416 and pS422 detected other neighboring phosphorylation sites in addition to their specific residues (Figs. 3d-f, Fig. 4h and Fig. 5f).

Furthermore, we found a number of antibodies that, despite being sold as specific for phospho-tau at particular sites, did not distinguish between modified and non-modified versions of tau-derived peptides. Five different antibodies against phosphorylation at site Ser262 (pS262) gave very strong signals with non-modified peptides (Fig. 5a), and we were unable to identify one with sufficient specificity. Similarly, the only commercially available antibody against pT50 we are aware of did not meet our specificity criteria (Fig. 5b). For sites pS400, pS404 and pS422, we had to test several antibodies to identify one that performed well. Three out of four antibodies tested for pS404, and one out of two for each pS400 and pS422 showed strong binding to bare peptides or peptides modified at different sites (Fig. 5d-f).

Interestingly, only the antibody against the pY18 site showed no signal on the array (Fig. 5c). As the antibody was raised against a tau peptide spanning aa12–24, which is covered by our array, it is unlikely that its epitope is outside of the region covered in our experiment. However, it is possible that conformational changes caused by the PTM, which are not recapitulated on the array, are necessary for detection.

Having determined the specificity of antibodies for different PTM-modified versions of tau, we went on to assess non-specific signals from brain lysates by immunoblotting. In this assay, we included a panel of widely used, commercially available pan-tau antibodies (Tau1, Tau5, Tau12, BT2, HT7, Dako-Tau), which have various epitopes spanning the entire tau sequence (Fig. 6a), isoform-specific antibodies (0 N, 1 N, 2 N, 3R, 4R) and an antibody against Caspase 3-cleaved tau at D421 (Fig. 1, C3-D421). In order to show the specificity of the antibodies for tau, we used cerebral lysates from nine-month-old hTg-Tau mice expressing all six human tau isoforms on a mouse tau knockout background [8], and the corresponding Tau-KO mouse [12]. Amongst all pan-tau antibodies tested, only BT2 and HT7 showed nonspecific signals around 35 kD and 75 kD, respectively, as these bands were detected in Tau-KO samples (Fig. 6d, e).

In order to test the specificity of the C3-D421 antibody, we used recombinant tau truncated at D421 (tau 1–421) as well as full length tau cleaved with caspase 3 in vitro (tau 1–441 + Casp 3). While all tau forms (full length, truncated and cleaved) were detected with the Dako antibody against total tau (Fig. 7a, left), we observed that C3-D421 antibody specifically detects tau 1–421, both expressed as a truncated protein and generated through caspase 3 cleavage in vitro (tau 1–441 + Casp3) (Fig. 7a, right).

To test the specificity of isoform specific antibodies, we used a commercially available tau ladder, which includes all six isoforms expressed in human brain. 1 N and one of the tested 4R antibodies were found to be nonspecific (Fig. 7b+c), whereas 0 N, 2 N, 3R and another 4R antibody only detected their corresponding isoforms. However, the 2 N antibody had an additional nonspecific signal around 50 kD as detected in Tau-KO samples (Fig. 6i). Most of the post-translational modification specific antibodies, which were validated by peptide arrays, showed specific binding to tau except pT181, pT199, pS356, pS396 and pS416. These antibodies bind to other proteins in Tau-KO samples (Fig. 6). The meK311, pS217, pS238, and C3-D421 specific antibodies did not give any signal (Fig. 7d-g) although the experiment was repeated multiple times with higher amounts of lysates and higher concentrations of the antibodies. Taken together, these results suggest that the meK311, pS217, pS238 and C3-D421 modifications either do not exist or only exist at nondetectable levels in the mouse brains.

We summarized the peptide array and immunoblotting results in Table 1, which shows the source of the antibodies and how they performed in both assays. When we classified the antibodies according to peptide array results we categorized them as very good, good, and average. If the antibody detected an additional modification site, it was classified ‘average’, and if its detection was hindered by other neighboring modifications as ‘good’. The antibodies classified as ‘very good’ showed no non-specific binding and their detection was not hindered by other modifications. The antibodies, which gave strong signals with non-modified peptides (Fig. 5), were discarded.

For immunoblotting, antibodies that gave strong or weak but specific signal to tau were classified as ‘very good’. Antibodies detecting weak non-specific bands in tau-KO samples were classified as ‘good’. If the antibody was not specific to their intended target, such as 1 N and one of the 4R antibodies, they were classified as ‘bad’. If we could not detect any signal, we classified the antibody as ‘no signal’. Importantly, this last category does not reflect on the antibody quality, but rather on the absence of the analyte in hTg-Tau brain samples.

Next, we tested our validated antibodies on human entorhinal cortex lysates to confirm their suitability for the investigation of tau PTMs in patient samples (Fig. 8). We tested a total of 20 antibodies consisting of five pan-tau and 15 PTM-specific tau antibodies. Total tau antibodies with epitopes at the N-terminus or in the central region detected full-length tau isoforms, ranging from approximately 45–70 kDa on the gel, as well as shorter, presumably truncated forms (Fig. 8a-d). The Dako antibody on the other hand was raised against a C-terminal epitope (aa 243–441) and does not detect these fragments, suggesting they are C-terminally truncated (Fig. 8e). The isoform-specific antibodies raised against the repeat region further show that this epitope is still intact in some tau fragments ranging from approximately 35-40 kDa (Fig. 8h and i).

Western blotting with PTM-specific antibodies revealed that meK311, pY18, pT231, pS356, pS400 and pS404 are present in our samples (Fig. 8l–s), while nY18, acK280, pT212, pS238, and pS422 were not detectable, even when the amount of protein loaded on the gel was increased (Fig. 8j–t). This suggests that either these tau modifications do not exist or are below detection level in our human brain samples.

In addition to the immunoblotting, we tested the specificity of the antibodies for tau by electrochemiluminescence ELISA using the same mouse brain lysates as for immunoblotting (Fig. 9). We chose this technique to assess the specificity of the antibodies to native tau, since we used denaturing conditions for immunoblotting. Moreover, ELISA can be performed in a sandwich format using a pair of antibodies, which may increase the specificity for tau. The electrochemiluminescence detection furthermore provides a quantitative and sensitive readout with a large linear range [13, 14].

We first biotinylated the antibodies in our panel and captured them on streptavidin-coated plates, then incubated the plates with cerebral lysates from hTg-Tau and Tau-KO mice followed by detection of the captured tau species with the pan-tau antibody Tau12. The capture antibody was linked to the sulfo-tag necessary to generate the electrochemiluminescent signal. We used biotinylated GAPDH antibody as a control for the background binding of tau on empty spots on the well and detected by sulfo-Tau12 antibody, and then subtracted this background signal from all other experimental data.

Amongst the pan-tau capture antibodies, HT7, BT2, Tau5, and Tau1 gave high signal in a similar range, which was 10⁵-fold higher in hTg-Tau compared to Tau-KO brain lysates. Dako-Tau, 1 N- and 2 N–isoform specific antibodies still showed a 10⁴-fold higher signal in hTg-Tau lysates compared to Tau-KO controls. Interestingly, when Tau12 was used both as capture and as detection antibody, we only detected background signal, suggesting that the Tau12 epitope is fully occupied in the capture step, preventing the binding of the sulfo-Tau12 detection antibody (Fig. 9a).

We then tested all other validated PTM-specific antibodies. The signal strength of the antibodies ranged between 10⁴ and 10⁶, whereas the background signal for all was around 10² (Figs. 9b and c). These data suggest that all tau modifications tested are present in hTg-Tau mouse brain.

We next decided to use our panel of validated antibodies to determine whether PTMs alter the subcellular localization of tau in iPSC-derived neurons. While all tested pan-tau antibodies except Tau5 showed signal by immunofluorescence, the isoform specific antibodies 3R and 4R did also not work with this technique (Fig. 10). Furthermore, we could detect tau with all PTM-specific antibodies except C3-D421 (Figs. 11, 12 and 13). Interestingly, we observed differences in the localization of tau dependent on its modification. For example, tau phosphorylated at S198 localized both to cell body and neurites, whereas tau phosphorylated at T181 and acetylated at K280 mainly localized only to neurites or to cell body, respectively. In Table 2, we summarized the differential localization of tau species according to their presence in the cell body, in neurites or in both compartments.