AlzAntibodies

Tau (PHF-1)

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Epitope: Tau phosphorylated at Ser396 and Ser404
Immunogen: Paired-helical filament tau extracted from brains of individuals with neurofibrillary pathology
Clonality: Monoclonal
Isotype: IgG1
Host: Mouse
Reactivity: Human; Mouse; Rat; Non human primates

RRID: AB_2315150
Availability: Available through the Feinstein Institutes for Medical Research (“Feinstein”), on behalf of Albert Einstein College of Medicine (“Einstein”), under UBMTA. Contact MTA@einsteinmed.edu to initiate the request. Separate agreements with both Einstein and Feinstein are required. Fees may apply. Please note: The PHF-1 antibody generated against paired helical filaments of tau, which recognizes tau phosphorylated at serines 396 and 404, should not be confused with antibodies directed against PHD finger protein 1.

Application Reference(s)

ELISA

Otvos et al., 1994

Sandwich ELISA

Acker et al., 2013

Immunohistochemistry

Rye et al., 1993; Augustinack et al., 2002; Andorfer et al., 2003; Hoover et al., 2010; Oh et al., 2010; Rockenstein et al., 2015; Strang et al., 2017; Goodwin et al., 2021

Immunoelectron microscopy

Ksiezak-Reding et al., 1994; Andorfer et al., 2005; Campbell et al., 2015; Reilly et al., 2017; Torres et al., 2021

Immunoblot

Greenberg et al., 1992; Andorfer et al., 2003; Petry et al., 2014; Rockenstein et al. 2015; Koss et al., 2016; Strang et al., 2017; Huynh et al., 2022

Blocking/Neutralizing

Boutajangout et al., 2011; Chai et al., 2011

Overview

This monoclonal antibody, generated against paired helical filaments isolated from the brains of Alzheimer’s patients, recognizes tau doubly phosphorylated at serines 396 and 404. Immuno-electron microscopy showed that the antibody binds to tau filaments (Ksiezak-Reding et al., 1994; Takahashi et al., 2002; Reilly et al., 2017).

  • Recognizes tau doubly phosphorylated at serines 396 and 404
  • Immunoreactivity seen in Alzheimer's disease and other tauopathies but not controls
  • Used to assess the progression of tau pathology in human disease and in the brains of animal models of tauopathy
  • Validated in knockout and cell-based assays

PHF-1 is often used to assess the progression of tau pathology in human disease and in the brains of animal models of tauopathy. The antibody recognizes tau in the brains of individuals with Alzheimer’s disease and other tauopathies, with little, if any, immunoreactivity towards control brains. It also recognizes tau in transgenic mouse models of tauopathy. Some PHF-1 immunoreactivity has been seen in the brains of presumably healthy non-transgenic animals, but at lower levels than in disease models.

Targeting the PHF-1 epitope provided therapeutic benefits in preclinical studies. Systemic administration of PHF-1 was reported to reduce tau pathology and provide functional benefits in transgenic mice expressing tau with mutations linked to frontotemporal dementia (Boutajangout et al., 2011; Chai et al., 2011), as did PHF-1-derived single-chain variable fragments or intrabodies expressed from recombinant adenoviral vectors injected into the central nervous system (Goodwin et al., 2020). In another study, adenoviral expression of recombinant PHF-1 in the hippocampus of MAPT-P301S transgenic mice prevented hippocampal atrophy in addition to reducing tau pathology (Liu et al., 2016).

The PHF-1 epitope is sensitive to the post-mortem interval prior to fixation (Gärtner et al., 1998).

PHF-1 recognizes tau filaments. Immunoelectron microscopy of fibrils isolated from the brains of individuals with Alzheimer’s disease and corticobasal degeneration, stained with PHF-1. Scale bar, 100 nm. [Adapted from Ksiezak-Reding et al., 1994, American Journal of Pathology. © 1994 American Society for Investigative Pathology.]

Generation and epitope mapping

Monoclonal antibody PHF-1 was generated against “relatively soluble” paired helical filaments (PHFs) isolated from Alzheimers’ brains. The PHF-1 epitope on PHF tau was shown to be sensitive to treatment with alkaline phosphatase or hydrofluoric acid, indicating that the antibody recognizes a phosphoepitope on tau (Greenberg et al., 1992). When tested in ELISAs against synthetic phosphopeptides representing tau fragments, PHF-1 was shown to react with tau phosphorylated at serine-396 or serine-404, with approximately 10-fold increased affinity when both sites were phosphorylated, compared with either site alone (Otvos et al., 1994). Site-directed mutagenesis of recombinant tau reinforced this finding: Replacement of either serine-396 or serine-404 with an alanine residue, which cannot be phosphorylated, largely eliminated PHF-1 reactivity with tau on western blots (Otvos et al., 1994; Strang et al., 2017).

PHF-1 reacts with tau phosphorylated at serine-396 and serine-404. Western blot of recombinant human tau phosphorylated in vitro with GSK3β, probed with PHF-1. WT, wild-type tau; S396A, S404A, tau in which serine-396 and serine-404, respectively, were mutated to non-phosphorylatable alanines. [Strang et al., 2017, Figure 1, cropped; licensed under Creative Commons BY 4.0]

Specificity

In immunohistochemical (Rye et al., 1993; Augustinak et al., 2002; Strang et al., 2017) and immunoblotting (Greenberg et al., 1992; Koss et al., 2016; Strang et al., 2017) applications, the PHF-1 antibody recognizes tau from Alzheimer’s brains, with very little, if any, immunoreactivity towards control brains.

PHF-1 antibody recognizes tau from Alzheimer’s brains but not control brains. A) Western blot. B) Dot blot. C) Immunohistochemistry. [(A) and (C) from Strang et al., 2017, Figures 6 and 4 (selected panels), respectively; licensed under Creative Commons BY 4.0. (B) Koss et al., 2016, Figure 2.b.i-ii, licensed under Creative Commons BY 4.0.]

PHF-1 immunoreactivity has also been observed in the brains of individuals with other tauopathies, including argyrophilic grain disease (Tolnay, et al., 1997), progressive supranuclear palsy (Takahashi et al., 2002), Pick’s disease (Rockenstein et al., 2015), and pallido-ponto-nigral degeneration—a subtype of frontotemporal dementia (Reed et al., 1998).

PHF-1 reactivity has been used as a marker of pathology in transgenic rodent models of tauopathy, although some reactivity also has been reported in the brains of non-transgenic animals. PHF-1 immunoreactivity was detected by ELISA (Acker et al., 2013), western blot (Lin et al., 2011; Petry et al., 2014; Rockenstein et al., 2015; Strang et al., 2017), and immunohistochemistry (Hoover et al., 2010; Oh et al., 2010; Rockenstein et al., 2015; Strang et al., 2017) in the brains of mice expressing human tau with mutations linked to frontotemporal dementia or Pick’s disease. The antibody also reacted with tau in the brains of htau mice (Andorfer et al., 2003; Acker et al., 2013), which over express wild-type human tau in the absence of mouse tau and eventually develop neurofibrillary tangles. Little or no immunoreactivity was detected by immunohistochemistry (Hoover et al., 2010; Rockenstein et al., 2015; Strang et al., 2017), western blot (Lin et al., 2011; Rockenstein et al., 2015) or ELISA (Acker et al., 2013) in the brains of non-transgenic mice, compared with the brains of transgenic mice assessed in the same experiments. However, in at least two studies, PHF-1 detected bands on western blots of brain extracts from non-transgenic mice that were absent from extracts of tau knockouts run on the same blots (Petry et al., 2014; Strang et al., 2017). Additionally, the antibody stained neurons in the brains of wild-type rats (Gärtner et al., 1998). Most recently, PHF-1 immunoreactivity was observed in mitochondria located at synapses in aged wild-type mice (Torres et al., 2021). These findings suggest that some PHF-1-immunoreactive tau is present in the brains of presumably healthy animals, albeit at lower levels than in disease models.

PHF-1 reactivity is increased in transgenic mouse models of tauopathy. A) ELISA of brain extracts from wild-type mice, transgenic mice that over express wild-type human tau in the absence of mouse tau (htau), transgenic mice that express human tau with the P301L mutation linked to frontotemporal dementia (P301L), and Mapt knockout mice. B) Western blot of brain extracts from Mapt knockout mice (KO), non-transgenic mice (nTg), and transgenic mice that express human tau with the P301L mutation (PS19). C) Immunohistochemistry on brain sections from non-transgenic mice (TgNeg), transgenic mice that over express wild-type human tau (rTgWT), or transgenic mice that express human tau with the P301L mutation (rTgP301L). [A) From Acker et al., 2013, Neurobiology of Aging © 2012 Elsevier Inc. B) Adapted from Strang et al., 2017, Figure 2, selected panel; licensed under Creative Commons BY 4.0.  C) From Hoover et al., 2010, Neuron © 2010 Elsevier Inc.]

Species

PHF-1 was shown to react with human (see examples cited above), mouse (Petry et al., 2014; Strang et al., 2017), rat (Gärtner et al., 1998), and baboon (Schultz et al., 2000) tau.

Validation

PHF-1 has been tested against tau knockouts in western blot (Petry et al., 2014; Strang et al., 2017; Rodriguez et al., 2018) and ELISA (Acker et al., 2013) applications to assess the selectivity of PHF-1 for tau versus other proteins. PHF-1 immunoreactivity was absent in the tau knockouts.

PHF-1 was found to be highly specific for tau phosphorylated at serines 396 and 404 in a cell-based assay designed to quantify the specificity of antibodies directed against defined phosphorylation sites on tau (Li and Cho, 2020).

Specificity of PHF-1 evaluated with a 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 serines 396 and 404 mutated to alanines to prevent phosphorylation at these sites.  Cells were fixed, permeabilized and incubated with PHF-1 followed by a fluorescently tagged secondary antibody.  Antibody binding to the cells transfected with tau-S396A,S404A 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 = 0.98 ± 0.00 (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 = 0.98 ± 0.03. X axis, tau expression; Y axis, antibody binding. [Adapted from Li and Cho, 2020, Journal of Neurochemistry © 2019 International Society for Neurochemistry.]

Last Updated: 05 Feb 2024

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References

Research Models Citations

  1. htau
  2. JNPL3(P301L)
  3. Tau P301S (Line PS19)
  4. rTg(tauP301L)4510

Paper Citations

  1. Ksiezak-Reding H, Morgan K, Mattiace LA, Davies P, Liu WK, Yen SH, Weidenheim K, Dickson DW. Ultrastructure and biochemical composition of paired helical filaments in corticobasal degeneration. Am J Pathol. 1994 Dec;145(6):1496-508. PubMed.
  2. Takahashi M, Weidenheim KM, Dickson DW, Ksiezak-Reding H. Morphological and biochemical correlations of abnormal tau filaments in progressive supranuclear palsy. J Neuropathol Exp Neurol. 2002 Jan;61(1):33-45. PubMed.
  3. Reilly P, Winston CN, Baron KR, Trejo M, Rockenstein EM, Akers JC, Kfoury N, Diamond M, Masliah E, Rissman RA, Yuan SH. Novel human neuronal tau model exhibiting neurofibrillary tangles and transcellular propagation. Neurobiol Dis. 2017 Oct;106:222-234. Epub 2017 Jun 10 PubMed.
  4. Boutajangout A, Ingadottir J, Davies P, Sigurdsson EM. Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain. J Neurochem. 2011 Aug;118(4):658-67. PubMed.
  5. Chai X, Wu S, Murray TK, Kinley R, Cella CV, Sims H, Buckner N, Hanmer J, Davies P, O'Neill MJ, Hutton ML, Citron M. Passive immunization with anti-Tau antibodies in two transgenic models: reduction of Tau pathology and delay of disease progression. J Biol Chem. 2011 Sep 30;286(39):34457-67. PubMed.
  6. Goodwin MS, Sinyavskaya O, Burg F, O'Neal V, Ceballos-Diaz C, Cruz PE, Lewis J, Giasson BI, Davies P, Golde TE, Levites Y. Anti-tau scFvs Targeted to the Cytoplasm or Secretory Pathway Variably Modify Pathology and Neurodegenerative Phenotypes. Mol Ther. 2021 Feb 3;29(2):859-872. Epub 2020 Oct 14 PubMed.
  7. Liu W, Zhao L, Blackman B, Parmar M, Wong MY, Woo T, Yu F, Chiuchiolo MJ, Sondhi D, Kaminsky SM, Crystal RG, Paul SM. Vectored Intracerebral Immunization with the Anti-Tau Monoclonal Antibody PHF1 Markedly Reduces Tau Pathology in Mutant Tau Transgenic Mice. J Neurosci. 2016 Dec 7;36(49):12425-12435. PubMed.
  8. Gärtner U, Janke C, Holzer M, Vanmechelen E, Arendt T. Postmortem changes in the phosphorylation state of tau-protein in the rat brain. Neurobiol Aging. 1998 Nov-Dec;19(6):535-43. PubMed.
  9. Greenberg SG, Davies P, Schein JD, Binder LI. Hydrofluoric acid-treated tau PHF proteins display the same biochemical properties as normal tau. J Biol Chem. 1992 Jan 5;267(1):564-9. PubMed.
  10. Otvos L Jr, Feiner L, Lang E, Szendrei GI, Goedert M, Lee VM. Monoclonal antibody PHF-1 recognizes tau protein phosphorylated at serine residues 396 and 404. J Neurosci Res. 1994 Dec 15;39(6):669-73. PubMed.
  11. 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.
  12. Rye DB, Leverenz J, Greenberg SG, Davies P, Saper CB. The distribution of Alz-50 immunoreactivity in the normal human brain. Neuroscience. 1993 Sep;56(1):109-27. PubMed.
  13. Augustinack JC, Schneider A, Mandelkow EM, Hyman BT. Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer's disease. Acta Neuropathol. 2002 Jan;103(1):26-35. PubMed.
  14. 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.
  15. Tolnay M, Spillantini MG, Goedert M, Ulrich J, Langui D, Probst A. Argyrophilic grain disease: widespread hyperphosphorylation of tau protein in limbic neurons. Acta Neuropathol. 1997 May;93(5):477-84. PubMed.
  16. Rockenstein E, Overk CR, Ubhi K, Mante M, Patrick C, Adame A, Bisquert A, Trejo-Morales M, Spencer B, Masliah E. A novel triple repeat mutant tau transgenic model that mimics aspects of pick's disease and fronto-temporal tauopathies. PLoS One. 2015;10(3):e0121570. Epub 2015 Mar 24 PubMed.
  17. Reed LA, Schmidt ML, Wszolek ZK, Balin BJ, Soontornniyomkij V, Lee VM, Trojanowski JQ, Schelper RL. The neuropathology of a chromosome 17-linked autosomal dominant parkinsonism and dementia ("pallido-ponto-nigral degeneration"). J Neuropathol Exp Neurol. 1998 Jun;57(6):588-601. PubMed.
  18. Acker CM, Forest SK, Zinkowski R, Davies P, d'Abramo C. Sensitive quantitative assays for tau and phospho-tau in transgenic mouse models. Neurobiol Aging. 2013 Jan;34(1):338-50. Epub 2012 Jun 21 PubMed.
  19. Lin WL, Dickson DW, Sahara N. Immunoelectron microscopic and biochemical studies of caspase-cleaved tau in a mouse model of tauopathy. J Neuropathol Exp Neurol. 2011 Sep;70(9):779-87. PubMed.
  20. 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.
  21. Hoover BR, Reed MN, Su J, Penrod RD, Kotilinek LA, Grant MK, Pitstick R, Carlson GA, Lanier LM, Yuan LL, Ashe KH, Liao D. Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration. Neuron. 2010 Dec 22;68(6):1067-81. PubMed.
  22. Oh KJ, Perez SE, Lagalwar S, Vana L, Binder L, Mufson EJ. Staging of Alzheimer's pathology in triple transgenic mice: a light and electron microscopic analysis. Int J Alzheimers Dis. 2010;2010 PubMed.
  23. Andorfer C, Kress Y, Espinoza M, de Silva R, Tucker KL, Barde YA, Duff K, Davies P. Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms. J Neurochem. 2003 Aug;86(3):582-90. PubMed.
  24. Torres AK, Jara C, Olesen MA, Tapia-Rojas C. Pathologically phosphorylated tau at S396/404 (PHF-1) is accumulated inside of hippocampal synaptic mitochondria of aged Wild-type mice. Sci Rep. 2021 Feb 24;11(1):4448. PubMed.
  25. Schultz C, Dehghani F, Hubbard GB, Thal DR, Struckhoff G, Braak E, Braak H. Filamentous tau pathology in nerve cells, astrocytes, and oligodendrocytes of aged baboons. J Neuropathol Exp Neurol. 2000 Jan;59(1):39-52. PubMed.
  26. 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.
  27. 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. American Journal of Pathology
  2. licensed
  3. Creative Commons BY 4.0
  4. licensed
  5. Creative Commons BY 4.0
  6. licensed
  7. htau
  8. Neurobiology of Aging
  9. Neuron
  10. Journal of Neurochemistry

Further Reading

No Available Further Reading