Elsevier

Neuroscience Research

Volume 31, Issue 4, August 1998, Pages 317-323
Neuroscience Research

Activation of tau protein kinase I/glycogen synthase kinase-3β by amyloid β peptide (25–35) enhances phosphorylation of tau in hippocampal neurons

https://doi.org/10.1016/S0168-0102(98)00061-3Get rights and content

Abstract

According to the amyloid hypothesis for the pathogenesis of Alzheimer’s disease (AD), amyloid β peptide (Aβ) directly affects neurons, leading to neurodegeneration and tau phosphorylation, followed by the production of paired helical filaments (PHF) in neurofibrillary tangles (NFT). To analyze the relationship between the phosphorylation sites of tau and the activation of kinases in response to Aβ, we treated cultured rat hippocampal neurons with a peptide fragment of Aβ, Aβ(25–35). Aβ(25–35) treatment activated tau protein kinase I/glycogen synthase kinase-3β (TPK I/GSK-3β) but not glycogen synthase kinase-3α (GSK-3α) or mitogen activated protein kinase (MAP kinase) in primary culture of hippocampal neurons. Using antibodies that recognize phosphorylated sites of tau, we showed that tau phosphorylation was enhanced in at least five sites (Ser199, Ser202, Ser396, Ser404, and Ser413 numbered according to the human tau isoform containing 441 amino acid residues), to an extent that depended on the level of TPK I/GSK-3β. Treatment with TPK I/GSK-3β antisense oligonucleotide inhibited the enhancement of tau phosphorylation induced by Aβ(25–35) exposure. Thus, TPK I/GSK-3β activation by Aβ(25–35) may lead to extensive tau phosphorylation.

Introduction

Alzheimer’s disease (AD) is histochemically characterized by the accumulation of amyloid β protein (Aβ) in senile plaques (Selkoe, 1991), and paired helical filaments (PHFs) composed of hyperphosphorylated tau in neurofibrillary tangles (NFTs) (Grundke-Iqbal et al., 1986a, Grundke-Iqbal et al., 1986b, Ihara et al., 1986, Lee et al., 1991, Selkoe, 1991), neuropil threads and plaque-associated neurites. Tau is a group of microtubule-associated proteins expressed predominantly in axons (Binder et al., 1985, Kosik and Finch, 1987) that promote microtubule assembly in vitro (Kanai et al., 1989), and that stabilize microtubules in vivo (Drubin and Kirschner, 1986). Hyperphosphorylated tau is observed in the developing fetal brain and in the AD brain (Goedert et al., 1989, Brion et al., 1993, Kanemaru et al., 1992, Kenessey and Yen, 1993, Watanabe et al., 1993). In both cases, some neurons are degenerating, implying that the phosphorylation of tau may directly or indirectly play a part in neuronal death through promoting the instability of microtubules. The phosphorylation of tau can be explained by the up-regulation of protein kinase and/or down-regulation of phosphatase activity. In vitro studies have shown that tau can be a substrate for various protein kinases (Ishiguro et al., 1991, Ishiguro et al., 1992, Ishiguro et al., 1993, Correas et al., 1992, Drewes et al., 1992, Vulliet et al., 1992, Biernat et al., 1993, Scott et al., 1993, Yang et al., 1993, Alonso et al., 1994) including tau protein kinase I/glycogen synthase kinase-3β (TPK I/GSK-3β), GSK-3α and MAP kinase, and that highly phosphorylated tau, which carries the epitope of PHF-tau, loses its ability to support microtubule formation.

According to the amyloid hypothesis for the pathogenesis of AD (Hardy and Higgins, 1992), Aβ directly affects neurons, leading to neurodegeneration and formation of PHF in NFT. Consistent with this idea, primary cultures of embryonic rat hippocampal neurons undergo progressive degeneration as well as expression of an epitope for phosphorylated tau after exposure to Aβ(1–40) or its fragment peptide Aβ(25–35) (Yankner et al., 1990, Mattson et al., 1992, Takashima et al., 1993, Busciglio et al., 1995). Using an antisense oligonucleotide, we obtained evidence that TPK I/GSK-3β is involved in tau phosphorylation and neuronal death in Aβ-treated hippocampal cultures (Takashima et al., 1993). However, the involvement of other kinases cannot be ruled out, and it is unclear which site of tau is phosphorylated in Aβ-treated hippocampal neurons.

In this study, we analyzed tau phosphorylation in rat hippocampal cultures treated with Aβ(25–35) using polyclonal peptide antibodies against each phosphorylation site of PHF-tau, and the involvement of kinases in tau phosphorylation was examined.

Section snippets

Cell culture

Cultures of embryonic rat hippocampal cells were prepared as described (Takashima et al., 1993). Cells were seeded at a density of 2×105 cells/cm2 onto multi-well, poly-l-lysine-coated tissue culture plates. In most experiments, cells were grown in Dulbecco’s modified Eagle’s medium supplemented with heat-inactivated 5% fetal bovine serum (HyClone) and 5% horse serum (JRH) for the first 3 days, and then the culture medium was changed to neurobasal medium (Gibco) supplemented with B-27 (Gibco)

Tau phosphorylation by exposure of rat hippocampal culture to Aβ(25–35)

To assess tau phosphorylation after Aβ(25–35) (20 μM) exposure of cells for various periods, the soluble cytoplasmic fraction (supernatant) and insoluble materials (pellet) of the cell homogenate were subjected to immunoblotting analysis using the phosphorylation-dependent antibodies described above. In the cytoplasmic soluble fraction (sup) (Fig. 1A), the immunoreactivity of PS396 and PS413 reached 6 times the control level within 6 h after Aβ exposure. The immunoreactivity of PS404 and PS202

Discussion

The accumulation of Aβ in senile plaques and of PHF composed of hyperphosphorylated tau in NFT can be observed in the AD brain. According to the amyloid hypothesis for the pathogenesis of AD (Hardy and Higgins, 1992), Aβ directly affects neurons, leading to neurodegeneration and formation of PHF in NFT. We have shown here that primary cultures of embryonic rat hippocampal neurons undergo progressive degeneration as well as the enhancement of tau phosphorylation after exposure to Aβ(25–35),

Acknowledgements

The authors would like to thank Dr James G. Kenimer for his valuable discussion and critical review of our manuscript. 5E2 was a kind gift from Dr K.S. Kosik (Harvard Medical School, Boston, USA). This work was partly supported by the Takeda Medical Foundation.

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Present address: Laboratory for Alzheimer’s Disease, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 350-01, Japan.

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