Enantiomers of 2-methylglutamate and 2-methylglutamine selectively impact mouse brain metabolism and behavior

Abstract

Imbalance of excitatory and inhibitory neurotransmission is implicated in a wide range of psychiatric and neurologic disorders. Here we tested the hypothesis that insertion of a methyl group on the stereogenic alpha carbon of l-Glu or l-Gln would impact the γ-aminobutyric acid (GABA) shunt and the glutamate-glutamine cycle. (S)-2-methylglutamate, or (S)-2MeGlu, was efficiently transported into brain and synaptosomes where it was released by membrane depolarization in a manner equivalent to endogenous l-Glu. (R)-2MeGlu was transported less efficiently into brain and synaptosomes but was not released by membrane depolarization. Each enantiomer of 2MeGlu had limited activity across a panel of over 30 glutamate and GABA receptors. While neither enantiomer of 2MeGlu was metabolized along the GABA shunt, (S)-2MeGlu was selectively converted to (S)-2-methylglutamine, or (S)-2MeGln, which was subsequently slowly hydrolyzed back to (S)-2MeGlu in brain. rac-2MeGln was also transported into brain, with similar efficiency as (S)-2MeGlu. A battery of behavioral tests in young adult wild type mice showed safety with up to single 900 mg/kg dose of (R)-2MeGlu, (S)-2MeGlu, or rac-2MeGln, suppressed locomotor activity with single ≥ 100 mg/kg dose of (R)-2MeGlu or (S)-2MeGlu. No effect on anxiety or hippocampus-dependent learning was evident. Enantiomers of 2MeGlu and 2MeGln show promise as potential pharmacologic agents and imaging probes for cells that produce or transport l-Gln.

Enantiomers of 4‐aminopentanoic acid act as false GABAergic neurotransmitters and impact mouse behavior

“…Mouse cerebral synaptosomes were prepared exactly according to the published methods (Gajera et al, 2019;Wawro et al, 2021).…”

“…Synaptosome uptake and high potassium-induced release assays were performed as described (Wawro et al, 2021). Uptake assays used 250μl aliquots of the synaptosome suspension transferred to a pre-chilled 0.8 ml 96-well plate.…”

“…Our previous work showed that insertion of a methyl group on the stereogenic alpha carbon of l-glutamate reduced substantially the susceptibility of the product, 2-methylglutamate (2MeGlu), towards enzymatic decarboxylation in mouse brain synaptosomes or in vivo (Wawro et al, 2021), consistent with previous studies using bacterial GAD (Rumping et al, 2019). Although metabolically isolated from the GABA shunt, 2MeGlu along with its metabolite 2-methylglutamine (2MeGln) are enantiomer-specific modulators of the glutamate-glutamine cycle, and the S enantiomer of 2MeGlu, (S)-2MeGlu, is a false glutamatergic neurotransmitter (Wawro et al, 2021). Exposure to high-dose (≥100 mg/kg IP) (S)-2MeGlu or (R)-2MeGlu acutely causes marked reduction in response to novelty and locomotion, and chronic low dose (10 mg/kg IP) of (S)-2MeGlu tended to improve cue-dependent learning in young healthy mice (Wawro et al, 2021).…”

“…Although metabolically isolated from the GABA shunt, 2MeGlu along with its metabolite 2-methylglutamine (2MeGln) are enantiomer-specific modulators of the glutamate-glutamine cycle, and the S enantiomer of 2MeGlu, (S)-2MeGlu, is a false glutamatergic neurotransmitter (Wawro et al, 2021). Exposure to high-dose (≥100 mg/kg IP) (S)-2MeGlu or (R)-2MeGlu acutely causes marked reduction in response to novelty and locomotion, and chronic low dose (10 mg/kg IP) of (S)-2MeGlu tended to improve cue-dependent learning in young healthy mice (Wawro et al, 2021).…”

“…As part of these studies, we also investigated the would-be decarboxylated metabolite of 2MeGlu, 4-aminopentanoic acid (4APA) (Wawro et al, 2021). We showed that racemic (rac) 4APA is taken up into mouse cerebral synaptosomes in a time-and temperaturedependent manner, that rac-4APA is released from synaptosomes upon depolarization with high concentration of potassium ion, and that rac-4APA is not detectably carboxylated to 2MeGlu, oxidized 4-oxopentanoic acid (methyl analogue of succinic semialdehyde), or reduced to 4-hydroxypentanoic acid (methyl analogue of γ-hydroxybutyrate) (Wawro et al, 2021). Here, we have investigated the enantiomer-specific neurochemical and pharmacological properties of 4APA in vitro in mouse cerebral synaptosomes and in vivo in young adult mice (Figure 1).…”