Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

Chihiro Sugiyama,Yaichiro Kotake,Masafumi Yamaguchi,Kanae Umeda,Yumi Tsuyama,Seigo Sanoh,Katsuhiro Okuda,Shigeru Ohta

doi:10.1016/j.toxrep.2014.12.014

Chihiro Sugiyama, Yaichiro Kotake + Show 6 more

Open Access

https://doi.org/10.1016/j.toxrep.2014.12.014

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Abstract

In vitro estimating strategies for potential neurotoxicity are required to screen multiple substances. In a previous study, we showed that exposure to low-concentrations of some chemicals, such as organotin, decreased the expression of GluR2 protein, which is a subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors, and led to neuronal vulnerability. This result suggested that GluR2 decreases as an index of neuronal cell sensitivity and vulnerability to various toxic insults. Accordingly, we developed a versatile method that is a large scale determination of GluR2 protein expression in the presence of environmental chemicals by means of AlphaLISA technology. Various analytical conditions were optimized, and then GluR2 protein amount was measured by the method using AlphaLISA. The GluR2 amounts were strongly correlated with that of measured by western blotting, which is currently used to determine GluR2 expression. An ideal standard curve could be written with the authentic GluR2 protein from 0ng to 100ng. Subsequently, twenty environmental chemicals were screened and nitenpyram was identified as a chemical which lead to decrease in GluR2 protein expression. This assay may provide a tool for detecting neurotoxic chemicals according to decreases in GluR2 protein expression.

Highlights

These data demonstrate the formation of essential complexes between the tested anti-GluR2 antibodies and beads, and validate their use in AlphaLISA assays of GluR2 in cell lysates
We previously showed that long-term exposure of rat cortical neurons to environmental chemicals, organotin and lead, led to decreased GluR2 protein expression and greater susceptibility of neurons to glutamate stimulation [6,7]
Small numbers of chemicals have been assessed for neurotoxicity as well-developed methods and markers of neurotoxicity are not available

Summary

Introduction

Present address: Asahikawa Medical University, Asahikawa 0788510, Japan.C. Sugiyama et al / Toxicology Reports 2 (2015) 450–460 barrier allows the passage of neurotoxic environmental chemicals, even at low concentrations [4,5]. We previously showed that long-term exposure of rat cortical neurons to low concentrations of organotin decreases GluR2 protein expression, leading to increased neuronal susceptibility to glutamate stimulation compared with that in control neurons [6]. We showed that long-term lead exposure induces neuronal cell death in association with decreased GluR2 expression [7]. Recent studies show that GluR2 is an essential regulator of the memory phenomena [13]. These studies suggest that decreases in GluR2 may be utilized as an index for conditions under which neuronal cells are sensitive and vulnerable to other stimulants. Western blotting for GluR2 expression is unsuitable for high throughput screening

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