Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites

doi:10.1016/0304-3940(95)12206-0

Neuroscience Letters

Volume 202, Issues 1–2, 29 December 1995, Pages 81-84

https://doi.org/10.1016/0304-3940(95)12206-0 Get rights and content

Abstract

Previously, we determined sites of tau protein phosphorylation by tau protein kinase (TPK) I/glycogen synthase kinase 3β (GSK-3β) and TPKII/(cyclin-dependent kinase 5 (CDK5) + p23). We prepared antibodies specific for these sites of tau phosphorylated by TPKI and TPKII, using chemically synthesized phosphopeptides as antigens. Each antibody specifically reacts with each phosphorylation site. With these antibodies, it was confirmed that TPKI and TPKII are responsible for these phosphorylation sites, as reported previously, except that Ser404 is also weakly phosphorylated by TPKI alone. It was also observed that TPKII-phosphorylation enhances TPKI-phosphorylation. These results indicate that these antibodies are useful tools for investigation of the phosphorylation of tau by TPKI and TPKII.

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Citation Excerpt :
Sections were incubated at room temperature with 0.01% Triton X-100 in PBS for 30 min and for another hour in 3% bovine serum albumin (BSA) in PBS. For immunolabeling, sections were incubated with antibodies to β-amyloid 1-40/42 (Alpha Diagnostic International), eNOS (Sigma Chemical), poly (ADP-ribose) polymerase (PARP) p85 (Promega, Madison, WI, USA), phospho tau 404 (Ishiguro et al., 1995), and phospho-GSK-3α/β-Ser279/216 (Biosource International) overnight at 4 °C. After washing, sections were incubated with biotinylated anti-rabbit IgG (1:5000) in TNB buffer for 1 h, followed by 2 h incubation with streptavidin–HRP (1:5000) and Alexa 594 anti-mouse IgG (Molecular Probes, Eugene, OR, USA) in TNB buffer (1:400).
To define mechanisms underlying neurovascular injury following brain embolism-induced neurodegeneration, we investigated temporal and spatial pathological changes in brain microvessels up to 12 weeks after microsphere embolism (ME) induction in aged male rats. Mild ME upregulated endothelial nitric oxide synthase (eNOS) and protein tyrosine nitration in brain microvessels. Strong β-amyloid immunoreactivity coincident with increased eNOS immunoreactivity was observed in microvessels. Immunoblotting of purified brain microvessels revealed that β-amyloid accumulation significantly increased 1 week after ME induction and remained elevated for 12 weeks. Importantly, β-amyloid accumulation in brain parenchyma was also observed in areas surrounding injured microvessels at 12 weeks. Levels of Alzheimer's-related hyperphosphorylated tau proteins also concomitantly increased in neurons surrounding regions of β-amyloid accumulation 12 weeks after ME induction, as did glycogen synthase kinase (GSK3β) (Tyr-216) phosphorylation. Taken together, ME-induced aberrant eNOS expression and subsequent protein tyrosine nitration in microvessels preceded β-amyloid accumulation both in microvessels and brain parenchyma, leading to hyperphosphorylation of neuronal tau proteins through GSK3β activation.

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Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites

Abstract

FEBS Lett.

Neuron

Neurosci. Lett.

J. Biol. Chem.

Neurosci. Lett.

Neurosci. Lett.

FEBS Lett.

FEBS Lett.

FEBS Lett.