Strang et al. Acta Neuropathologica Communications (2017) 5:58 DOI 10.1186/s40478-017-0458-0 RESEARCH Open Access Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau Kevin H. Strang1,2, Marshall S. Goodwin1,2, Cara Riffe1,2, Brenda D. Moore1,2, Paramita Chakrabarty1,2,3, Yona Levites1,2,3, Todd E. Golde1,2,3 and Benoit I. Giasson1,2,3,4* Abstract Tauopathies are a group of neurodegenerative disorders, including Alzheimer’s disease, defined by the presence of brain pathological inclusions comprised of abnormally aggregated and highly phosphorylated tau protein. The abundance of brain tau aggregates correlates with disease severity and select phospho-tau epitopes increase at early stages of disease. We generated and characterized a series of novel monoclonal antibodies directed to tau phosphorylated at several of these phospho-epitopes, including Ser396/Ser404, Ser404 and Thr205. We also generated phosphorylation independent antibodies against amino acid residues 193–211. We show that most of these antibodies are highly specific for tau and strongly recognize pathological inclusions in human brains and in a transgenic mouse model of tauopathy. They also reveal epitope-specific differences in the biochemical properties of Alzheimer’s disease sarkosyl-insoluble tau. These new reagents will be useful for investigating the progression of tau pathology and further as tools to target the cellular transmission of tau pathology. Keywords: Alzheimer’s disease, Antibodies, Phosphorylation, Tau, Transgenic mice Introduction The abundance of tau inclusions in the brain correlates well Tau refers to the microtubule-associated protein [10, 47] with disease severity [2]. However, the presence of tau expressed from the MAPT gene located on chromosome pathological inclusions are also a defining feature of many 17q21–22 [1, 39]. In the adult human brain, 6 major tau other types of dementias, such as corticobasal degeneration, isoforms ranging between 352 and 441 amino acids in progressive supranuclear palsy, tangle-only dementia, Pick’s length are produced as a result of alternative RNA disease, and frontotemporal dementia and parkinsonism splicing [20, 21]. Although tau is natively highly soluble linked to chromosome 17 with tau pathology (FTDP-17 t) [11, 43], it can polymerize to form amyloid fibrils [16] [15, 17, 28, 31]. Indeed, the fact that FTDP-17 t is caused that aberrantly coalesce to form intracellular inclusions by more than 50 different mutations in the MAPT gene, that define a large spectrum of neurodegenerative dis- resulting in either tau protein amino acid changes or altered eases termed tauopathies [15, 29, 31, 46]. ratio of tau splicing isoforms, is irrefutable evidence for a Alzheimer’s disease (AD) is the most common form of pathogenic role of tau in neurodegeneration [17, 28]. tauopathy where tau aggregates to form somatodendritic Hyperphosphorylation of tau is a hallmark of tau neurofibrillary tangles (NFTs), parenchymal neuropil pathological inclusions in human brains [24–26, 38] and threads and dystrophic neurites that are intertwined with pathological findings indicate that phosphorylation of extracellular deposits of amyloid-β peptides [15, 29, 31, 46]. tau at specific residues, such as the AT8 and PHF1 epi- topes, occurs early in tau inclusion formation [8, 9, 37]. Given the interest for these epitopes as pathological * Correspondence: [email protected] markers and for immunotherapy [3, 4, 6, 7, 13, 22, 30, 45], 1Department of Neuroscience, College of Medicine University of Florida, Gainesville, FL 32610, USA we have generated and characterized a series of new 2Center for Translational Research in Neurodegenerative Disease, College of monoclonal antibodies targeting these regions of tau with Medicine University of Florida, Gainesville, FL 32610, USA unique phosphorylation specificities. Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Strang et al. Acta Neuropathologica Communications (2017) 5:58 Page 2 of 11 Materials and methods Mouse myeloma (Sp2/O-Ag14; ATCC, Manassas, VA) Mice cells were maintained in high glucose (4.5 g/L) Dulbecco’s Tau null (tau KO) mice [14] and tau transgenic (Tg) mice Modified Eagle Medium (DMEM) with 10% NCTC 135 line PS19 expressing human 1 N/4R tau with the P301S Media (Sigma Aldrich, St. Louis, MO), 20% hybridoma mutation driven by the mouse prion promoter [49] were grade fetal bovine serum (FBS; Hyclone, Logan, UT), obtained from Jackson Laboratory (Bar Habor, ME). Tau 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM L-glu- Tg mice line JNPL3 expressing human 0 N/4R tau with tamine, 0.45 mM pyruvate, 1 mM oxaloacetate, and 0.2 U/ the P301L mutation were previously described [33]. ml insulin at 37 °C and 8% CO2. Spleens were gently ho- mogenized in 5% FBS/Hank’s balanced salt solution (HBSS; Lonza, Walkersville, MD) and centrifuged to pellet Antibodies cells. The cell pellet was resuspended in red blood cell AT8 (Thermo-fisher) is a mouse monoclonal antibody lysis buffer for one minute (Sigma Aldrich, St. Louis, MO) specific towards phosphorylation sites S202 and T205 in and diluted with HBSS. The cells were then washed twice tau [19] that can also be influenced by phosphorylation by centrifuging at 100 x g for 10 min and resuspended in at S199 or S208 [34, 42]. PHF1 (generously provided by HBSS. Sp2/O-Ag14 cells were also washed twice with Dr. Peter Davies, Albert Einstein University, NY, NY) is HBSS. Five million Sp2/O-Ag14 cells were added to 50 a mouse monoclonal antibody specific towards phos- million spleen cells, and after centrifuging at 100 x g for phorylation sites S396 and S404 in tau [40]. Rabbit poly- 10 min onto a culture dish, fusion was induced with 50% clonal antibody (H-150) raised against the first 150 polyethylene glycol 1450 (Sigma Aldrich, St. Louis, MO). amino acids of human tau was obtained from Santa After washing with HBSS, cells were incubated in Sp2/O- Cruz Biotechnologies (Dallax, TX), and rabbit polyclonal Ag14 media at 37 °C with 8% CO overnight. The next antibodies 3026 and 3029 raised against recombinant 2 day, the cells were gently detached from the plate and dis- full-length 0 N/3R tau was generated as a service by tributed into 96 well plates with Sp2/O-Ag14 media/0.5% GenScript USA Inc. (Piscataway, NJ). hybridoma enhancing supplement (Sigma Aldrich, St. Louis, MO)/HAT selection supplement (Sigma Aldrich, Generation of new mouse tau monoclonal antibodies St.Louis,MO). All procedures were performed according to the NIH Guide for the Care and Use of Experimental Animals Hybridoma screening and were approved by the University of Florida Institutional All hybridoma clones were screened for reactivity to the Animal Care and Use Committee. Phosphopeptides respective unconjugated peptide that was used for EIVYKpSPVVSGDTpSPRHLSC (p391–409) and DRSGYS immunization by enzyme-linked immunosorbent assay pSPGpSPGpTPGSRSRC (p193–211) corresponding, re- (ELISA). MaxiSorp plates (Thermo Scientific, Waltham, spectively, to residues 391–409 and 193–211 in the 2 N/4R MA) were coated with 1 μg/ml peptide in PBS and human tau isoform with a C residue added at the carboxy- blocked with 5% FBS/PBS. Media from the hybridomas termini for chemical conjugation were synthesized and was applied to plates, which were then incubated at purified by GenScript USA Inc. (Piscataway, NJ). Lyophi- room temperature. Next, the plates were washed with lized peptides were reconstituted in phosphate buffered sa- PBS and incubated with goat anti-mouse secondary line (PBS) and conjugated to Imject maleimide-activated antibody conjugated to horseradish peroxidase (HRP; mariculture keyhole limpet hemocyanin (mcKLH; Thermo Jackson Immuno Research Labs, West Grove, PA) at Scientific, Waltham, MA). 2–3 month old female BALB/c room temperature. Then, plates were washed and TMB mice (Jackson Laboratory, Bar Harbor, ME) were used for substrates (Pierce, Rockford, IL) were applied until color immunization. Injection solutions emulsified by vortexing changes were observed. Reactions were then quenched for 15 min were prepared by combining 100 μgKLH- with 1 M HCl and absorbance was measured at 450 nm. conjugated peptide in 200 μlPBSwith100μlofFreund’s Clones that were positive by ELISA were transferred to complete adjuvant (Sigma Aldrich, St. Louis, MO) for the larger culture plates as needed. The positive clones were initial subcutaneous injection. Three weeks following the next screened by immunohistochemistry of a human AD initial injection, mice were boosted with an intraperitoneal autopsy case with abundant tau pathology. (IP) injection of 50 μg KLH-conjugated peptide in 200 μl Antibody clones were isotyped with the mouse mono- PBS emulsified with 100 μl of Freunds incomplete ad- clonal antibody isotyping kit purchased from Sigma- juvant (Sigma Aldrich, St. Louis, MO). Three weeks Aldrich (St. Louis, MO). later, mice were boosted with an IP injection of 50 μg KLH-conjugated peptide in PBS. Three days later, Immunohistochemistry analyses mice were euthanized and spleens were harvested Paraffin embedded, formalin fixed human brain tissue was using aseptic technique. obtained through the University of Florida Neuromedicine Strang et al. Acta Neuropathologica Communications (2017) 5:58 Page 3 of 11 Human Brain Tissue Bank. Sequential tissue sections were Preparation of total mouse brain protein lysates deparaffinized with xylenes and sequentially rehydrated Tau KO, PS19 Tg or non-transgenic (nTg) mice were hu- with graded ethanol solutions (100–70%).