Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau

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.

Fig. 1

Specificity of novel tau antibodies raised against tau peptide p391–409 as determined by immunoblotting with recombinant tau proteins phosphorylated in vitro with p42 MAPK or GSK3β. WT, S396A and S404A 2 N/4R tau were incubated with p42 MAPK or GSK3β or without any kinase as described in “Material and Methods.” The proteins were resolved onto 10% polyacrylamide gels and analyzed by immunoblotting with novel tau antibodies (as indicated for each blot) PHF2, PHF15, PHF17, PHF20 or PHF22. Similar blots were performed with previously characterized antibody PHF1 and total tau antibody H150. The mobilities of molecular mass markers are shown on the left

Fig. 2

Characterization of the specificity of novel tau antibodies by immunoblotting analyses using total brain lysates from nTg, tau KO and PS19 tau Tg mice. Brains from tau KO, nTg, and PS19 tau Tg mice were harvested and lysed in 2% SDS/50 mM Tris, pH 7.5 as described in “Material and Methods.” Equal amounts of proteins (40 μg) from each sample was resolved onto 10% polyacrylamide gels and analyzed by immunoblotting with each antibody indicated above. Arrows depict human tau expressed in PS19 mice. The mobilities of molecular mass markers are shown on the left

Fig. 3

Specificity of novel tau antibodies raised against tau peptide p193–211 as determined by immunoblotting with recombinant tau proteins phosphorylated in vitro with p42 MAPK or GSK3β. WT, S199A, S202A and T205A 0 N/3R tau (mutants numbered according to 2 N/4R human tau) were incubated with p42 MAPK or GSK3β or without any kinase as described in “Material and Methods.” The proteins were resolved onto 10% polyacrylamide gels and analyzed by immunoblotting with novel tau antibodies (as indicated for each blot) 1H5, 2D1, 3C9, 4A10, 5F2, 6G12, 7F2, 8G5, and 10G12 and total tau antibody H150. The mobilities of molecular mass markers are shown on the left

Fig. 4

Immunocytochemistry of representative tau pathology in human AD brain and JNPL3 Tg mice with new antibodies PHF17 and PHF20. Immuno-reactivity of previously characterized phospho-tau antibodies PHF1 and new tau antibodies PHF17 or PHF20 in the hippocampus of a control individual or a subject with AD, and in the spinal cord of 12 month old nTg and JNPL3 Tg mice. Arrows indicating NFTs in human brain or NFT-like inclusion pathology in JNPL3 mice. Asterisk depicting dystrophic neurites within senile plaques. Bar = 100 μm, and 200 μm for insets

Fig. 5

Immunocytochemistry of representative tau pathology in human AD brain and JNPL3 Tg mice with new antibodies 2D1 and 7F2. Immuno-reactivity of previously characterized phospho-tau antibodies AT8 and new tau antibodies 2D1 or 7F2 in the hippocampus of a control individual or a subject with AD, and in the spinal cord of 12 month old nTg and JNPL3 Tg mice. Arrows indicating NFTs in human brain or NFT-like inclusion pathology in JNPL3 mice. Asterisk depicting dystrophic neurites within senile plaques. Bar = 100 μm, and 200 μm for insets

Fig. 6

Characterization of the novel tau antibodies in detecting biochemically sarkosyl-insoluble tau in human brain lysates from AD patients. Immunoblotting analysis of the sarkosyl-insoluble fraction from the temporal cortex of human AD cases (n = 3) and control cases (CTR; n = 2). Samples were biochemically fractionated as described in “Material and Methods.” Equal amounts of proteins (10 μg) from each sample was resolved onto 10% polyacrylamide gels and analyzed by immunoblotting with each antibody indicated above blot, including total tau antibody 3026. The mobilities of molecular mass markers are shown on the left