Enhancement of Glutamate Uptake as Novel Antiseizure Approach: Preclinical Proof of Concept

Abstract

Objective: Excitotoxicity is a common hallmark of epilepsy and other neurological diseases associated with elevated extracellular glutamate levels. Thus, here, we studied the protective effects of (R)-AS-1, a positive allosteric modulator (PAM) of glutamate uptake in epilepsy models.

Methods: (R)-AS-1 was evaluated in a range of acute and chronic seizure models, while its adverse effect profile was assessed in a panel of standard tests in rodents. The effect of (R)-AS-1 on glutamate uptake was assessed in COS-7 cells expressing the transporter. WAY 213613, a selective competitive EAAT2 inhibitor, was used to probe the reversal of the enhanced glutamate uptake in the same transporter expression system. Confocal microscopy and Western blotting analyses were used to study a potential influence of (R)-AS-1 on GLT-1 expression in mice.

Results: (R)-AS-1 showed robust protection in a panel of animal models of seizures and epilepsy, including the maximal electroshock- and 6 Hz-induced seizures, corneal kindling, mesial temporal lobe epilepsy, lamotrigine-resistant amygdala kindling, as well as seizures induced by pilocarpine or Theiler's murine encephalomyelitis virus. Importantly, (R)-AS-1 displayed a favorable adverse effect profile in the rotarod, the minimal motor impairment, and the Irwin tests. (R)-AS-1 enhanced glutamate uptake in vitro and this effect was abolished by WAY 213613, while no influence on GLT-1 expression in vivo was observed after repeated treatment.

Interpretation: Collectively, our results show that (R)-AS-1 has favorable tolerability and provides robust preclinical efficacy against seizures. Thus, allosteric enhancement of EAAT2 function could offer a novel therapeutic strategy for treatment of epilepsy and potentially other neurological disorders associated with glutamate excitotoxicity. ANN NEUROL 2025;97:344-357.

FIGURE 1

Chemical structure of (R)‐AS‐1 together with a summary of the previously published data (*Abram et al. ²³ ) and studies reported in the current manuscript.

FIGURE 2

Efficacy of (R)‐AS‐1 in the IHKM and TMEV models in mice. (A) Mean number of spontaneous HPDs after single i.p. administration of (R)‐AS‐1 at dose of 50 mg/kg in the IHKM model. Data are presented as the mean ± SEM. Statistical analysis: paired t‐test; *p < 0.05 versus baseline. (B) Cumulative seizure burden analysis following inoculation with TMEV and treatment with (R)‐AS‐1. ( R)‐AS‐1 was administered at dose of 100 mg/kg i.p. BID for 5 days, starting 3 days post‐inoculation. Data are presented as the mean ± SEM. Statistical analysis: Multiple Mann–Whitney test; ****p < 0.001 versus control (vehicle).

FIGURE 3

Efficacy of (R)‐AS‐1 in the PILO SE in mice. (A) Effect of (R)‐AS‐1 (15 mg/kg, i.p., 30 min before PILO) on PILO‐induced seizures or lethality. Statistical analysis: Fisher's exact probability test; *p < 0.05 versus the vehicle + PILO (300 mg/kg) group (B) Fluoro Jade B (FJB, green) and 4′,6‐diamidino‐2‐phenylindole (DAPI, blue) staining for neurodegeneration in the hippocampus after PILO SE. (a) vehicle + PILO (300 mg/kg) treated mice, (b–f) (R)‐AS‐1 + PILO (300 mg/kg) ‐treated mice. DG = dentate gyrus; CA1, CA2, CA3 = cornu ammonis areas 1, 2, 3.

FIGURE 4

Evaluation of the effect of (R)‐AS‐1 on glutamate uptake mediated by EAAT2 in the presence or absence of the specific competitive EAAT2 inhibitor WAY 213613. Uptake assays conducted on COS‐7 cells transiently transfected with CMV or EAAT2 were pre‐incubated for 10 min with either a vehicle, (R)‐AS‐1 alone at concentrations ranging from 10⁻⁴ M to 10⁻¹² M or a combination of (R)‐AS‐1 (at concentrations 10⁻⁴ M to 10⁻¹² M) with 0.1 μM WAY 213613. (A) Results were normalized to a percentage of control (EAAT2) and expressed as mean ± SEM from 3 to 6 independent experiments performed in technical triplicate. Statistical analysis: Kruskal‐Wallis test with post‐hoc Dunn's multiple comparisons test; ^# p < 0.05, (R)‐AS‐1 versus (R)‐AS‐1 + WAY 213613 (0.1 μM), ^#### p < 0.0001 (R)‐AS‐1 versus WAY213613; A one‐way ANOVA with post‐hoc Dunnett's multiple comparison test was used to analyze differences between various concentrations of (R)‐AS‐1 versus. control; *p < 0.05 (0.1 nM), ****p < 0.0001 (1 nM–100 μM). (B) Sample results from a single experiment expressed in DPM (disintegrations per minute) expressed as mean ± SEM for selected concentration of (R)‐AS‐1 (10 nM) alone and combination of (R)‐AS‐1 (10 nM) with WAY 213613 (0.1 μM). Statistical analysis: Kruskal‐Wallis test with post‐hoc Dunn's multiple comparisons test; **p < 0.01, EAAT2 versus (R)‐AS‐1 (10 nM) or t‐test; ^### p < 0.001, (R)‐AS‐1 (10 nM) versus (R)‐AS‐1 (10 nM) + WAY 213613 (0.1 μM); p > 0.05 (n.s. = not significant), EAAT2 versus WAY 213613.

FIGURE 5

Effect of (R)‐AS‐1 on GLT‐1/EAAT2 expression. (A) Representative immunoblot of 14 DIV neuron–glia cultures stained with EAAT2 antibody (green) and β‐actin‐control (red), after 72 h treatments with vehicle (control), 100 nM and 100 μM of (R)‐AS‐1 and 500 μM and 1 mM of ceftriaxone (CEF) in neuron–glia cultures. Molecular weight ladder (kDa) on far‐left side. (B) Quantification of blot intensities of the EAAT2 monomers represented in A. Graphs are replicates of 3–4 independent groups of neuron–glia cultures combined. Data are presented as the mean ± SEM. Statistical analysis: One‐way ANOVA with Dunnett's post hoc test; **p < 0.01. (C) Qualitative assessment of GLT‐1 expression in the mice hippocampus: Control (vehicle); (R)‐AS‐1 (100 mg/kg, i.p., BID for 7 days); amitriptyline (AMI, 10 mg/kg, i.p., QD for 7 days), a positive control for GLT‐1 expression. Positive GLT‐1 signals are stained red; blue – cell nuclei. DG = dentate gyrus; CA1‐CA3 = cornu ammonis areas. (D) Western blotting for GLT‐1 expression in the mice cerebrum lysates (n = 6/group). Representative immunoblot illustrating EAAT2 (green bands) and β‐actin (red bands) expression in mice cortical samples after 7‐day BID (i.p.) administration of either vehicle, (R)‐AS‐1 (100 mg/kg, i.p.) or AMI (10 mg/kg, i.p.). (E) Quantification of immunoblots shows no significant difference in EAAT2 expression between vehicle and (R)‐AS‐1‐treated mice, and a significant difference between vehicle and AMI‐treated animals. Data are presented as the mean ± SEM. Statistical analysis: One‐way ANOVA with Dunnett's post hoc test; *p < 0.05. EAAT2 was normalized to β‐actin, and (R)‐AS‐1 and AMI were normalized to vehicle.