Epitope: Tau phosphorylated at Ser202 and Thr205
Immunogen: Paired-helical filament tau extracted from AD brain
Clonality: Monoclonal
Isotype: IgG1
Host: Mouse
Reactivity: Human; Mouse; Canine; Rabbit; Rat; Non human primates; Hamster; Chicken/Avian; Drosophila

Overview

Hyperphosphorylated tau polymerizes into paired helical filaments, which aggregate to form neurofibrillary tangles, one of the defining lesions of Alzheimer’s disease.

AT8, which recognizes paired helical filament (PHF) tau, is one of the most widely used anti-tau antibodies. Immunostaining with AT8 can substitute for more difficult silver staining techniques to detect neurofibrillary pathology, although it should be noted that AT8 immunoreactivity reveals “pre-tangles” in addition to the mature neurofibrillary tangles and neuropil threads seen with silver stains (Braak et al., 2006). AT8 is also used to detect PHF tau in western blot and ELISA applications.

While the AT8 epitope is usually described as tau doubly phosphorylated at serine 202 and threonine 205, based on early characterization of the antibody (Goedert et al., 1995), more recent evidence suggests that the epitope on PHFs is actually triply phosphorylated at serine 202, threonine 205, and serine 208 (Malia et al., 2016). This difference in phosphorylation appears to have dramatic consequences for tau’s structure and propensity to aggregate: NMR spectroscopy and molecular dynamic simulations showed that phosphorylation at S202 and T205 induces a turn in the peptide (Gandhi et al., 2015). This turn seems to inhibit tau aggregation, but additional phosphorylation at serine 208 disrupts this turn and promotes aggregation (Despres et al., 2017).

Generation and epitope mapping

Mice were immunized with PHF tau partially purified from the brains of Alzheimer’s patients (Mercken et al., 1992), and their spleen cells were then fused with mouse myeloma cells to create hybridoma cell clones. Antibodies produced by these clones were screened for their ability to recognize tau in ELISAs. Antibodies from clone AT8 detected PHF tau extracted from AD brains, but not normal tau extracted from non-AD brains. Consistent with the ELISA findings, AT8 detected a multiplet of bands in western blots of sarkosyl-insoluble fractions from AD brains—once polymerized into PHFs, tau becomes insoluble in the detergent sarkosyl—but no bands were seen in extracts of normal brains. The antibody also stained flame-shaped structures in neurons in AD brains, very similar in appearance to neurofibrillary tangles revealed by silver stains.

Pre-treatment of brain extracts or tissue sections with phosphatase eliminated AT8 immunoreactivity in ELISAs, western blots, and immunohistochemical applications, indicating that the antibody recognizes a phosphoepitope on tau (Mercken et al., 1992).

Subsequently, site-directed mutagenesis, in which specific serine and threonine residues of recombinant tau were mutated to non-phosphorylatable alanine, followed by phosphorylation in vitro was used to define the phosphoepitopes recognized by AT8. Early studies concluded that phosphorylation at serine 202 and threonine 205 is required for recognition by AT8 (Goedert et al., 1995).

Later studies have shown that additional phosphorylation sites may participate in antibody binding. Weak binding was observed between AT8 and synthetic tau peptides doubly phosphorylated at serines 199 and 202 (Porzig et al., 2007; Malia et al., 2016). In binding studies using synthetic phosphopeptides and a cloned AT8 Fab fragment, AT8 Fab bound 30 times more strongly to tau triply phosphorylated at serine 202, threonine 205, and serine 208 than to tau doubly phosphorylated at residues 202 and 205 (Malia et al., 2016). Co-crystallization of the Fab fragment and synthetic phosphopeptide confirmed that there is a third binding site at serine 208. Notably, the affinity of AT8 Fab for the triply phosphorylated peptide was similar to that for PHF tau extracted from AD brain, suggesting that the natural AT8 epitope in PHFs is tau phosphorylated at serine 202, threonine 205, and serine 208.

Specificity

As noted above, AT8 was generated against tau from Alzheimer’s brains, and the initial characterization of this antibody showed immunoreactivity with AD brains but not normal adult brains (Mercken et al., 1992). These observations subsequently were confirmed by others (see, for example, Koss et al., 2016; Strang et al., 2017).

In the developing brain, AT8 also recognizes MAP2c (Matsuo, et al., 1994), which contains a similar sequence to that around the AT8 epitopes in tau (Sánchez et al., 2000).

AT8 reacts with Alzheimer’s brains, but not normal adult brains. A) Immunoblot. i) Sample dot blot of lysates of temporal cortex from neuropathologically confirmed Alzheimer’s cases (AD) and controls (C). ii) Quantification of dot blot signals. B) Immunohistochemistry. Sections through the hippocampi of a control subject (left) and a subject with AD (right). [A) From Koss et al., 2016, Figure 2a.i-ii; licensed under Creative Commons BY 4.0. B) From Strang et al., 2017, Figure 5; licensed under Creative Commons BY 4.0.]

Reactivity with AT8 has also been reported in transgenic mice that express human tau with mutations linked to frontotemporal dementia (see, for example, Petry et al., 2014; Strang et al., 2017).

AT8 stains neurons in the brains of transgenic mice that express human tau with a mutation linked to frontotemporal dementia. Sections through the spinal cords of non-transgenic (left) and JNPL3 (right) mice. [From Strang et al., 2017, Figure 5; licensed under Creative Commons BY 4.0.]

Validation

AT8 has been tested against tau knockouts in western blot and immunohistochemical applications to assess the selectivity of AT8 for tau versus other proteins. Two studies (Petry et al., 2014; Wobst et al., 2017) reported that AT8 failed to recognize protein species in brain lysates from tau knockout mice, when used as a detection antibody for western blots, while a third study found non-specific bands in lysates from knockouts, with multiple lots of antibody (Strang et al., 2017). AT8-positive cells were found by immunohistochemistry in the brains (Wobst et al., 2017) and by immunofluorescence in the retinae (Rodriguez et al., 2018) of transgenic mice that express human tau, but the antibody did not stain the corresponding tissues of tau knockout mice.

Validation of AT8 for Western blotting. A) AT8 does not bind any protein in the brains of tau knockout (TKO) mice and displays elevated signals in the brains of mice expected to have increased levels of hyperphosphorylated tau—transgenic mice that express human amyloid precursor protein, presenilin-1, and tau with disease-linked mutations (3xTg) and hypothermic wild-type mice (Hypo). Blot probed with AT8 followed by a secondary antibody that recognizes non-denatured mouse IgG. B) AT8 recognizes a protein in brain lysates from wild-type mice (WT) but not tau knockout mice (KO). C) Non-specific high molecular weight bands recognized by AT8 in western blots of mouse brain extracts. These bands were detected in tau knockouts (KO), as well as in wild-type (WT) mice and a transgenic mouse that expresses human tau with a mutation linked to FTD (PS19). Authors noted that these bands were seen using three different lots of AT8. (Arrow points to presumed phospho-tau band.) [A) From Petry et al., 2014; Figure 2.A2.AT8-TB, labels added; licensed under Creative Commons BY 4.0. B) Adapted from Wobst et al., 2017, Supplementary Figure 2, selected lanes and labels added; licensed under Creative Commons BY 4.0. C) From Strang et al., 2017, Figure 2; licensed under Creative Commons BY 4.0.]

Validation of AT8 for immunohistochemistry. A) AT8 stains neurons in the cortex and hippocampus of transgenic mice that over express human tau with the P301S mutation linked to frontotemporal dementia (hTau.P301S), but staining is absent from the brains of tau knockout (KO) mice. Sections are counterstained with hematoxylin. B) A strong signal is seen in the retina of hTau mice, which express human tau in the absence of mouse tau, compared with tau knockout (mTKO) mice. The nerve fiber layer is at the bottom and the outer nuclear layer at the top in these images. [A) Adapted from Wobst et al., 2017, Figure 3c, selected panels and relabeled; licensed under Creative Commons BY 4.0. B) From Rodriguez et al., 2018, Figure 1B, selected panels; licensed under Creative Commons BY 4.0.]

AT8 was found to be highly specific for tau phosphorylated at serine-202 in a cell-based assay designed to quantify the degree of non-specific binding of antibodies directed against defined phosphorylation sites on tau (Li and Cho, 2020).

Specificity of AT8 evaluated by flow-cytometric cell-based assay. Top: HEK293FT cells were co-transfected with GSK-3β (glycogen synthase kinase 3β) and either EGFP-tagged wild-type tau or IRFP-tagged tau with serine-202 mutated to alanine to prevent phosphorylation at this site. Cells were fixed, permeabilized and incubated with AT8 followed by a fluorescently tagged secondary antibody. Antibody binding to the cells transfected with tau-S202A is considered non-specific. Specificity was quantified by the value Φ, defined as 1-(fraction of antibody binding that is non-specific); 1 indicates no non-specific binding and 0 indicates that all binding is non-specific. Here Φ = 0.95 ± 0.07 (mean ± standard deviation from two experiments). Bottom: HEK293FT cells were co-transfected with GSK-3β and either EGFP-tagged wild-type tau or IRFP-tagged wild-type tau. Prior to antibody incubation, the IRFP-tau-transfected cells were treated with lambda phosphatase. Antibody binding to the phosphatase-treated cells is considered non-specific. Here Φ = 0.95 ± 0.05. X axis, tau expression; Y axis, antibody binding. [Adapted from Li and Cho, 2020, Journal of Neurochemistry © 2019 International Society for Neurochemistry.]

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References

Research Models Citations

1. JNPL3(P301L)
2. 3xTg
3. Tau P301S (Line PS19)
4. hTau.P301S
5. htau

Mutations Citations

1. MAPT P301S

Paper Citations

1. Braak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici K. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry. Acta Neuropathol. 2006 Oct;112(4):389-404. PubMed.
2. Goedert M, Jakes R, Vanmechelen E. Monoclonal antibody AT8 recognises tau protein phosphorylated at both serine 202 and threonine 205. Neurosci Lett. 1995 Apr 21;189(3):167-9. PubMed.
3. Malia TJ, Teplyakov A, Ernst R, Wu SJ, Lacy ER, Liu X, Vandermeeren M, Mercken M, Luo J, Sweet RW, Gilliland GL. Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti-tau antibody AT8. Proteins. 2016 Apr;84(4):427-34. Epub 2016 Feb 5 PubMed.
4. Gandhi NS, Landrieu I, Byrne C, Kukic P, Amniai L, Cantrelle FX, Wieruszeski JM, Mancera RL, Jacquot Y, Lippens G. A Phosphorylation-Induced Turn Defines the Alzheimer's Disease AT8 Antibody Epitope on the Tau Protein. Angew Chem Int Ed Engl. 2015 Jun 1;54(23):6819-23. Epub 2015 Apr 16 PubMed.
5. Despres C, Byrne C, Qi H, Cantrelle FX, Huvent I, Chambraud B, Baulieu EE, Jacquot Y, Landrieu I, Lippens G, Smet-Nocca C. Identification of the Tau phosphorylation pattern that drives its aggregation. Proc Natl Acad Sci U S A. 2017 Aug 22;114(34):9080-9085. Epub 2017 Aug 7 PubMed.
6. Mercken M, Vandermeeren M, Lübke U, Six J, Boons J, Van de Voorde A, Martin JJ, Gheuens J. Monoclonal antibodies with selective specificity for Alzheimer Tau are directed against phosphatase-sensitive epitopes. Acta Neuropathol. 1992;84(3):265-72. PubMed.
7. Porzig R, Singer D, Hoffmann R. Epitope mapping of mAbs AT8 and Tau5 directed against hyperphosphorylated regions of the human tau protein. Biochem Biophys Res Commun. 2007 Jun 29;358(2):644-9. PubMed.
8. Koss DJ, Jones G, Cranston A, Gardner H, Kanaan NM, Platt B. Soluble pre-fibrillar tau and β-amyloid species emerge in early human Alzheimer's disease and track disease progression and cognitive decline. Acta Neuropathol. 2016 Dec;132(6):875-895. Epub 2016 Oct 21 PubMed.
9. Strang KH, Goodwin MS, Riffe C, Moore BD, Chakrabarty P, Levites Y, Golde TE, Giasson BI. Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau. Acta Neuropathol Commun. 2017 Jul 31;5(1):58. PubMed.
10. Matsuo ES, Shin RW, Billingsley ML, Van deVoorde A, O'Connor M, Trojanowski JQ, Lee VM. Biopsy-derived adult human brain tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau. Neuron. 1994 Oct;13(4):989-1002. PubMed.
11. Sánchez C, Pérez M, Avila J. GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling. Eur J Cell Biol. 2000 Apr;79(4):252-60. PubMed.
12. Petry FR, Pelletier J, Bretteville A, Morin F, Calon F, Hébert SS, Whittington RA, Planel E. Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions. PLoS One. 2014;9(5):e94251. Epub 2014 May 2 PubMed.
13. Wobst HJ, Denk F, Oliver PL, Livieratos A, Taylor TN, Knudsen MH, Bengoa-Vergniory N, Bannerman D, Wade-Martins R. Increased 4R tau expression and behavioural changes in a novel MAPT-N296H genomic mouse model of tauopathy. Sci Rep. 2017 Feb 24;7:43198. PubMed.
14. Rodriguez L, Mdzomba JB, Joly S, Boudreau-Laprise M, Planel E, Pernet V. Human Tau Expression Does Not Induce Mouse Retina Neurodegeneration, Suggesting Differential Toxicity of Tau in Brain vs. Retinal Neurons. Front Mol Neurosci. 2018;11:293. Epub 2018 Aug 24 PubMed.
15. Li D, Cho YK. High specificity of widely used phospho-tau antibodies validated using a quantitative whole-cell based assay. J Neurochem. 2020 Jan;152(1):122-135. Epub 2019 Sep 4 PubMed.

External Citations

1. Mercken et al., 1992
2. licensed
3. Creative Commons BY 4.0
4. licensed
5. licensed
6. Creative Commons BY 4.0
7. licensed
8. licensed
9. licensed
10. Journal of Neurochemistry

Further Reading