Increased expression of the neuronal glutamate transporter (EAAT3/EAAC1) in hippocampal and neocortical epilepsy

Abstract

Purpose: To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy.

Methods: The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis.

Results: The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with non-dysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue.

Conclusions: Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.

FIG. 1

Top, representative cDNA array probed with aRNA from single dentate granule cells from control, latent period, and epileptic rats and from humans with intractable temporal lobe epilepsy (TLE). Note increase in hybridization intensity to EAAC1 cDNA in latent period and epileptic dentate granule cells with no change in β-actin, EAAT2/GLT-1, or pBS hybridization. Bottom, graphic representation of relative EAAT3/EAAC1 expression (percent of β-actin expression) in dentate granule cells from control, latent, and epileptic rats after pilocarpine treatment, as well as in dentate granule cells from six patients with TLE (n = 36 cells). Significant differences (*) between mean percentage hybridization in control and epileptic rat dentate granule cells are noted (y-axis, ±SE bar; *p < 0.05, **p < 0.01). There is no change in EAAT2/GLT-1 mRNA expression in the latent or epileptic phases. Statistics were not performed comparing rat and human cells.

FIG. 2

Relative EAAT3/EAAC1 abundance is increased in single dysplastic neurons and heterotopic neurons compared with control neurons (n = 30–40 cells in each group). Significant differences (*) between mean percentage hybridization (y-axis, ±SE bar; p < 0.05) are depicted.

FIG. 3

A: Modest staining in the dentate gyrus granule cell layer in control rats (arrows). B: Increased immunolabeling of the dentate gyrus granule cell layer in rats receiving pilocarpine after the onset of spontaneous seizures (arrows).

FIG. 4

A: Modest staining in the dentate gyrus granule cell layer in humans without epilepsy (arrows). B: Increased labeling of the dentate gyrus granule cell layer in patients with medically intractable temporal lobe epilepsy (arrows).

FIG. 5

A: EAAT3/EAAC1 staining in control cortex is modest and enriched in pyramidal cells. B: Increased expression of EAAT3/EAAC1 in focal cortical dysplasia. Note selective enrichment in larger dysplastic neurons.