Glutamine Supplementation Preserves Glutamatergic Neuronal Activity in the Infralimbic Cortex, Which Delays the Onset of Mild Cognitive Impairment in 3×Tg-AD Female Mice

Abstract

It was recently found that glutamine (Gln) supplementation activates glutamatergic neurotransmission and prevents chronic-stress-induced mild cognitive impairment (MCI). In this study, we evaluated the effects of Gln on glutamatergic activity in the medial prefrontal cortex and the onset of cognitive impairment in a triple-transgenic Alzheimer's disease mouse model (3×Tg-AD). Female 3×Tg-AD mice were fed a normal diet (3×Tg) or a Gln-supplemented diet (3×Tg+Gln) from 2 to 6 months of age. Glutamatergic neuronal activity was analyzed at 6 months, and cognitive function was examined at 2, 4, and 6 months. 3×Tg mice exhibited a decrease in glutamatergic neurotransmission in the infralimbic cortex, but 3×Tg+Gln mice did not. The 3×Tg group showed MCI at 6 months of age, but the 3×Tg+Gln group did not. The expressions of amyloid peptide, inducible nitric oxide synthase, and IBA-1 were not elevated in the infralimbic cortex in the 3×Tg+Gln group. Therefore, a Gln-supplemented diet could delay the onset of MCI even in a mouse model predisposed to cognitive impairment and dementia through genetic modification.

Keywords: 3×Tg-AD; glutamatergic neuronal activity; glutamine; mild cognitive impairment; oxidative stress.

Figure 1

Glutamine (Gln) supplementation protected glutamatergic neurotransmission activity in 3×Tg-AD mice. (A) Dietary groups and experimental schedule. (B) Microscopic images of glutamatergic neurons in the infralimbic cortex utilized for measuring spontaneous excitatory postsynaptic current (sEPSC) and representative sEPSC signals. (C) sEPSC frequency. (D) Cumulative amplitude. Vglut2-ires-Cre::tdTomato (CTL; n = 7 cells; 2 mice), 3×Tg-Vglut2-ires-Cre::tdTomato (3×Tg; n = 13 cells; 3 mice), and Gln-supplemented 3×Tg (3×Tg+Gln; n = 11 cells; 3 mice). (E) Representative superimposed sEPSC traces. More than 30 peaks in each group were superimposed. Bars represent the means ± SEM. ** p < 0.01 vs. CTL, and ## p < 0.01 vs. 3×Tg in 1-way ANOVA with Tukey’s multiple comparison tests. *** p < 0.001 in 2-way ANOVA with Tukey’s multiple comparison tests.

Figure 2

Gln supplementation in 6-month-old 3×Tg-AD mice exhibited inhibitory effects on mild cognitive impairment (MCI) and the induction of oxidative stress and amyloid beta (Aβ) accumulation in the plasma. (A) Dietary groups and experimental schedule. (B) Body weights and (C) food intake were measured weekly. (D) Discrimination index (DI) in the object recognition test (ORT) in wild-type (WT, n = 5), 3×Tg-AD (3×Tg, n = 3), and Gln-diet 3×Tg (3×Tg+Gln, n = 4) mice at 2, 4, and 6 months of age. (E) Change in DI values in the ORT in mice between the age of 2 and 6 months. (F) Reactive oxygen/nitrogen species (ROS/RNS) analysis in plasma. (G) Aβ_1–42 content in neuron-derived exosomes (NDEs) isolated from plasma. ELISA results of Aβ_1–42 were normalized to those for CD81. Bars represent the means ± SEM. * p < 0.05, *** p < 0.001 vs. WT, and # p < 0.05 vs. 3×Tg in 1-way ANOVA with Tukey’s multiple comparison tests.

Figure 3

Gln supplementation prevented increases in amyloid content and expression of proteins related to ROS/RNS production in the infralimbic cortex of 6-month-old 3×Tg mice. Representative immunohistochemistry images and signal intensity quantification for amyloid (A), IBA-1 (B), and iNOS (C). The white signal corresponds to DAPI. Red or green signals represent target proteins. Scale bar sizes are indicated in the images. Bars represent the means ± SEM. n = 4/group. * p < 0.05, *** p < 0.001 vs. WT, and # p < 0.05 vs. 3×Tg in 1-way ANOVA with Tukey’s multiple comparison tests.