Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal

Abstract

Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a GABAA receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of GABAA receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated GABAA receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing GABAA receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders.

Figure 1. Glo1 copy number regulates Glo1 expression.

(A) The BAC (RP23-247F19) contained Glo1 and partial copies of Dnahc8 and Btbd9. The first exons of Dnahc8 and Btbd9 were ablated by inserting ampicillin (AMP) and kanamycin (KAN) cassettes, respectively. (B) BAC copy number was estimated by qPCR on genomic DNA (n = 8 WT and 3–5 Tg per line). P < 10^–10, 1-way ANOVA. (C) Glo1 mRNA was estimated in whole brain by qPCR (n = 12 WT and 4–7 Tg per line). P < 10^–14, 1-way ANOVA. (D and E) GLO1 protein was estimated in whole brain by immunoblot (n = 16 WT and 3–6 Tg per line). P < 10^–11, 1-way ANOVA. Data are mean ± SEM. *P < 0.05; **P < 0.0001.

Figure 2. Glo1 overexpression increases anxiety-like behavior.

WT and Tg littermates were tested in the OF test (n = 74 WT [pooled across all lines] and 14–21 Tg per line). (A) Center time. P < 0.001, 1-way ANOVA. (B) Total distance. P > 0.1, 1-way ANOVA. Data were pooled from multiple individual experiments. Data are mean ± SEM. *P < 0.05.

Figure 3. MG regulates anxiety-like behavior.

(A) GLO1 enzymatic activity in whole brain (n = 3 WT and 3 Tg). (B and C) HPLC measurement of MG concentration in whole brain. (B) Representative chromatograms from WT (solid) and Tg (dashed) mice. An enlarged view of the relevant peak is shown in the inset. (C) Average MG concentration (n = 9 WT and 8 Tg). (D) HPLC measurement of MG concentration in whole brain after i.p. treatment with MG. Assay order had a significant effect on MG concentration and was used as a covariate in a 1-way analysis of covariance (ANCOVA) for the factor treatment (n = 4–7 per group). P = 0.009. (E and F) MG decreased anxiety-like behavior in the OF test. MG (50 mg/kg) (E) increased time in the center of the OF but (F) did not change total distance traveled (n = 18 per group). mAU, milli absorbance units. Data are mean ± SEM. *P < 0.05, **P < 0.0005.

Figure 4. MG has GABAergic effects in vivo.

(A) 100 mg/kg MG caused locomotor depression (n = 8 per group). (B) 300 mg/kg MG increased foot slips on the balance beam (n = 14 per group). Mann-Whitney U test, U = 26; P = 0.0002. (C) 300 mg/kg MG caused hypothermia (n = 10 per group). Data are mean ± SEM. *P < 0.05, **P < 0.0005.

Figure 5. MG is a GABA_A receptor agonist in CGNs.

(A) CGNs are depolarized by MG (white circles) or GABA (black circles) (EC₅₀, 10.5 ± 0.5 μM MG; Hill coefficient, 1.17). The relative amplitude of depolarization is shown normalized to the response of each cell to 100 μM MG. (B) MG evokes inward currents in a concentration-dependent manner (EC₅₀, 12 ± 0.7 μM; Hill coefficient, 1.13). The amplitude of currents is shown normalized to the peak response of each cell (I/Imax). (C) Depolarization evoked by 10 μM MG (left) or GABA (middle) was blocked by 10 μM SR. Mean data are plotted as a histogram (right). (D) Inward currents evoked by 10 μM MG (left) or GABA (middle) were also blocked by 10 μM SR. Mean data are plotted as a histogram (right). (E) Currents evoked by 100 μM GABA were reduced by coapplication of MG (left). Scale bars: 200 pA/pF, 25 s. Mean data are normalized to the current evoked by 100 μM GABA in each cell (right). (F) Currents observed approximately 40 seconds after the application of 10 μM MG to the inside of macropatches excised from CGNs were blocked when 10 μM SR was included in the pipette. Application of 10 μM GABA to the inside of macropatches did not evoke a current. Mean data are plotted as a histogram. The bar above each trace shows the duration of drug application. Data are mean ± SEM. n = 6–12 cells or macropatches per condition.

Figure 6. MG is a GABA_A receptor agonist in HNs.

(A) Both GABA and MG evoked concentration-dependent inward currents in HNs. The EC₅₀ of currents evoked by MG was 9.5 ± 0.9 μM, with a Hill coefficient of 1.05. The relative amplitude of the currents was normalized to that cell’s response to 100 μM MG. Currents evoked by 100 μM GABA were reduced by coapplication of MG in a concentration-dependent manner. Scale bars: 1 nA, 10 s. Mean data for competition experiments are shown normalized to the current evoked by 100 μM GABA in each cell. (B) Currents evoked by 10 μM MG or GABA were augmented by coapplication of 500 nM diazepam, midazolam, or zolpidem. Scale bars: 1 nA, 10 s. Mean data are plotted as histograms. The bar above each trace shows the duration of drug application. Data are mean ± SEM for 8–10 cells per condition.

Figure 7. Pharmacological inhibition of GLO1 reduces anxiety-like behavior.

(A) Enzymatic activity of 10 ng purified GLO1 protein treated with vehicle or 100 μM BrBzGCp2 (n = 3 vehicle and 4 BrBzGCp2). (B) HPLC measurement of MG concentration in whole brain 2 hours after i.p. treatment with vehicle or BrBzGSHCp2. Assay order had a significant effect on MG concentration and was used as a covariate in a 1-way ANCOVA for the factor treatment (n = 8 per group). P < 0.0001. (C and D) B6 mice were treated with i.p. vehicle or 30 mg/kg BrBzGCp2 and then tested in the OF test 2 hours after injection (n = 25 vehicle and 24 BrBzGCp2). (C) Center time. (D) Total distance. mU, milliunits (μmol/min). Data are mean ± sem. *P < 0.05, **P < 0.0001.