Identification of translational activators of glial glutamate transporter EAAT2 through cell-based high-throughput screening: an approach to prevent excitotoxicity

Abstract

Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.

FIG. 1

(A) The EAAT2 transcripts with long 5′ UTR are not constitutively translated but require extracellular factors to induce translational read-through. The 5′ UTR may form secondary structure, or stem-loops, which negatively affect translation by impeding the binding or migration of 40S ribosomal subunits. (B) A schematic illustration of the enzyme-linked immunosorbent assay procedures.

FIG. 2

Validation of the enzyme-linked immunosorbent assay (ELISA). PA-EAAT2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and treated with retinoic acid (1.5 μg/mL) for 72 h and then subjected to (A) ELISA, (B) Western blot analysis, or (C) [³H]glutamate uptake assay. The rate of retinoic acid–induced EAAT2 protein expression as measured by ELISA (n = 192 for each; *p < 0.0001) was correlated with that measured by Western blotting or glutamate uptake (n = 12 for each; *p < 0.0001). For glutamate uptake data, the fold increase of the retinoic acid–treated cells was normalized to the DMSO-treated cells.

FIG. 3

Screen results. (A) Percent activation for 14,000 compounds screened at 2.4 μM concentration. Data are representative of the ~140,000 compounds screened. Note: DMSO and retinoic acid control data appear after the break. (B) Percent coefficients of variation (%CVs) for DMSO vehicle control and retinoic acid–treated wells for each plate screened.

FIG. 4

The enzyme-linked immunosorbent assay (ELISA) 12-point dose responses for 3 compounds. Compounds for this assay were applied to quadruplicate wells at concentrations ranging from 0.1 nM to 30 μM. Error bars represent the standard deviation. EC₅₀s are provided on the inset of each graph.

FIG. 5

Confirmation of the hits. PA-EAAT2 cells were treated with indicated concentrations of compound 9 in Dulbecco’s modified Eagle’s medium (DMEM) for 72 h and then harvested (A) for determining EAAT2 protein levels by Western blotting (equal protein loading was confirmed by Ponceau S staining and also by reprobing the blots with antiactin antibodies), (B) for measuring glutamate transport function by [³H]-glutamate uptake assay, (C) for determining induced EAAT2 protein cellular localization by immunofluorescent staining using anti-EAAT2 antibodies (scale bar, 50 μm), and (D) for measuring EAAT2 mRNA levels by real-time RT-PCR analysis. For glutamate uptake and RT-PCR data, the fold increase of the compound treated cells was normalized to the DMSO-treated cells.