Original Research: Influence of okadaic acid on hyperphosphorylation of tau and nicotinic acetylcholine receptors in primary neurons

Abstract

The aim of the study was to investigate the influence of hyperphosphorylation of tau induced by okadaic acid on the expression of nicotinic acetylcholine receptors and the neurotoxicity of β-amyloid peptide. Primary cultures of neurons isolated from the hippocampus of the brains of neonatal rats were exposed to okadaic acid or/and Aβ1-42 Tau phosphorylated at Ser404 and Ser202, and the protein expressions of α7, α4 and α3 nAChR subunits were quantified by Western blotting, and their corresponding mRNAs by real-time PCR. Superoxide dismutase activity was assayed biochemically and malondialdehyde by thiobarbituric acid-reactive substance. As compared to controls, phosphorylations of tau at Ser404 and Ser202 in the neurons were elevated by exposure to 20 nM okadaic acid for 48 h but not by 1 or 2 µM Aβ1-42 Treatment with 20 nM okadaic acid or 1 µM Aβ1-42 for 48 h resulted in the reduced α7, α4 and α3 proteins, and α4 and α3 mRNAs, as well as the decreased activity of superoxide dismutase and the increased malondialdehyde. Okadaic acid and Aβ1-42 together caused more pronounced changes in the expressions of α7 and α4, superoxide dismutase activity and lipid peroxidation than either alone. When pre-treatment with vitamin E or lovastatin, the neurotoxicity induced by okadaic acid was significantly attenuated. These findings indicate that hyperphosphorylation of tau induced by okadaic acid inhibits the expression of nicotinic acetylcholine receptors at both the protein and mRNA levels, as well as enhances the neurotoxicity of β-amyloid peptide.

Keywords: Okadaic acid; neurotoxicity; nicotinic acetylcholine receptor; primary neurons; tau protein; β-amyloid peptide.

Figure 1

Immunostaining of primary cultures of neurons originating from the hippocampus of the brains of neonatal rats. The mouse antibody against the NeuN and rabbit antibody against the GFAP were used. NeuN-positive neurons (a) and GFAP-positive astrocytes (b) were shown. (c) The proportions of neurons and astrocytes was shown as bar graph.(A color version of this figure is available in the online journal.)

Figure 2

MTT levels in the primary neurons treated with OA orAβ_1–42. MTT reduction was detected after the primary neurons were treated with 1–40 nM OA (a) or 1–2 µM Aβ_1–42 (b) for 48 h. The results represent means ± SD (n = 20). *P < 0.05 and **P < 0.01 in comparison to untreated cells, as determined by analysis of variance (ANOVA), followed by the Student–Newman–Keul's test

Figure 3

The level of phosphorylation of tau in primary-cultured neurons exposed to OA or Aβ_1–42. Tau protein was detected with rabbit polyclonal anti-tau phosphorylated antibody at Ser404 (a and b) or Ser202 (c and d) to OA (10 or 20 nM) and Aβ_1–42 (1 or 2 µM), respectively, for 48 h. The results are expressed as means ± SD (n = 8). *P < 0.05 in comparison to untreated cells

Figure 4

The levels of α7 (a), α4 (b) and α3 (c) nAChR proteins in primary-cultured neurons exposed to OA or/and Aβ. Immunoblotting was carried out with rabbit monoclonal anti-α7, and goat polyclonal anti-α4 and -α3 antibodies after primary-cultured neurons were exposed to 20 nM OA or/and 1 µM Aβ_1–42 for 48 h. The results are expressed as means ± SD (n = 6). *P < 0.05 in comparison to controls; and ^#P < 0.05 in comparison to the treatment of OA or Aβ alone. Representative Western blots are depicted beneath the bar graphs

Figure 5

The levels of α7 (a), α4 (b) and α3 (c) nAChR mRNA in primary-cultured neurons exposed to OA (20 nM) or/and Aβ_1–42 (1 µM) for 48 h. The mRNA levels were determined by real-time PCR. The results are expressed as means ± SD (n = 6). *P < 0.05 in comparison to controls; and ^#P < 0.05 in comparison to the treatment of OA or Aβ alone