Restored glial glutamate transporter EAAT2 function as a potential therapeutic approach for Alzheimer's disease

Abstract

Glutamatergic systems play a critical role in cognitive functions and are known to be defective in Alzheimer's disease (AD) patients. Previous literature has indicated that glial glutamate transporter EAAT2 plays an essential role in cognitive functions and that loss of EAAT2 protein is a common phenomenon observed in AD patients and animal models. In the current study, we investigated whether restored EAAT2 protein and function could benefit cognitive functions and pathology in APPSw,Ind mice, an animal model of AD. A transgenic mouse approach via crossing EAAT2 transgenic mice with APPSw,Ind. mice and a pharmacological approach using a novel EAAT2 translational activator, LDN/OSU-0212320, were conducted. Findings from both approaches demonstrated that restored EAAT2 protein function significantly improved cognitive functions, restored synaptic integrity, and reduced amyloid plaques. Importantly, the observed benefits were sustained one month after compound treatment cessation, suggesting that EAAT2 is a potential disease modifier with therapeutic potential for AD.

Figure 1.

Increased EAAT2 expression significantly protected against Aβ_25-35-induced neuronal damage in primary cultures. Cortical primary neuron and astrocyte mixed cultures were prepared from EAAT2 transgenic or WT pup littermates. 7-d-old primary cultures were treated with 40 µM Aβ_25-35 for 24 h. (A) Quantitative analysis of EAAT2 protein levels in Aβ_25-35-treated and nontreated cultures by Western blotting. Representative Western blot result is shown. EAAT2 signal intensity was normalized to its actin signal intensity on the same blot. It is notable that data can only be compared between treated and nontreated cultures prepared from the same pup brains (n = 4). (B) Glutamate uptake analysis. Left panel is total [³H]glutamate uptake, and right panel is DHK-sensitive [³H]glutamate uptake (n = 4–5; ***, P < 0.001, Student’s t test). Raw data are presented in Table S1. (C) Representative images of immunocytochemistry staining with MAP2 and Hoechst 33342 showing neurite degeneration and nuclear condensation. The neurons indicated with arrows are magnified in the images on the right. The nuclear staining is represented in the insets. Bar, 50 µm. (D) Quantitative analysis of the number of neurons showing condensed nuclei. Approximately 500 neurons per culture were evaluated as the percentage of neurons showing condensed nuclei, and four to five cultures per group were statistically analyzed (*, P < 0.05, one way ANOVA followed by Holm-Šídák method). (E) Quantitative analysis of MAP2 immunoreactivity. A total of 2.2-mm² area per culture was evaluated, and the mean intensity of no treatment samples was arbitrarily set as 100%. Four to five cultures per group were statistically analyzed (*, P < 0.05, one way ANOVA followed by Holm-Šídák method). All data are from three independent experiments. Mean ± SEM is shown.

Figure 2.

Functional EAAT2 protein was restored in APP/EAAT2 mice. (A) Quantitative analysis of EAAT2 protein levels in the forebrain of the indicated mice. EAAT2 protein levels for each sample were normalized to actin protein levels (n = 6; *, P < 0.05; **, P < 0.01, Student’s t test). Representative Western blot analysis of EAAT2 protein levels at 9 mo of age is shown. (B) Representative images of EAAT2 immunofluorescent staining for 12-mo-old mice in the hippocampal dentate gyrus region (n = 3). Bar, 50 µm. (C) DHK-sensitive [³H]glutamate uptake activity (n = 3; ***, P < 0.001, one way ANOVA followed by Holm-Šídák method). PMVs prepared from mouse forebrains were used for glutamate uptake measurement. (D) EAAT2 mRNA levels in the forebrain of the indicated mice measured by real-time RT-PCR analysis (n = 6). Data are from six (A and D) and three (B and C) independent experiments. Mean ± SEM is shown.

Figure 3.

APP/EAAT2 mice exhibited improved premature death rate, memory, and pathology compared with their APP littermates. (A) Survival curves were generated from 246 mice: 63 APP/EAAT2, 69 APP_Sw,Ind, 59 EAAT2, and 55 WT littermates. (B–D) Behavior tests were conducted in 12–14-mo-old mice (n = 14–17 per group). (B) Y-maze tests assessing short-term memory (***, P < 0.001, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previously visited arm. (C) T-maze tests assessing spatial learning memory (*, P < 0.05, one-way repeated ANOVA followed by Student’s t tests). (D) Novel object recognition tests assessing long-term nonspatial memory (**, P < 0.01; ***, P < 0.001, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previous object. (E, left) Representative images of synaptophysin immunostaining in the CA3 region. The right micrographs show high-magnification images of the boxed areas in the left micrographs. (right) Quantitative analysis of synaptophysin immunoreactivity in the CA3a region indicated by the boxed areas (n = 3–5 mice per group; *, P < 0.05, Student’s t test). (F, left) Quantitative analysis of Aβ immunostaining (n = 4–5 mice per group; *, P < 0.05; **, P < 0.01, Student’s t test). (right) Representative images of Aβ immunostaining in the dentate gyrus region are shown. Nuclei were stained with Hoechst 33342 (blue). (E and F) Bars, 50 µm. Data are from six (A), four (B–D), and three (E and F) independent experiments. Mean ± SEM is shown.

Figure 4.

EAAT2 protein levels were restored by LDN/OSU-0212320 in 12-mo-old APP_Sw,Ind mice. (A) Structure of LDN/OSU-0212320. (B) 12-mo-old APP_Sw,Ind mice were treated with the indicated dosages of compound by i.p. injection for 3 d. PMVs were prepared from forebrains to determine EAAT2 protein levels by Western blot analysis. EAAT2 signal intensity was normalized to its actin signal intensity on the same blot (n = 3; *, P < 0.05; **, P < 0.01, one way ANOVA followed by Holm-Šídák method). Representative Western blots are presented. (C) Mice were treated with 30 mg/kg LDN/OSU-0212320 i.p. for 2 mo starting at 12 mo of age and were subjected to behavior tests (results shown in Fig. 6). After all behavior tests, PMVs were prepared from forebrains to determine EAAT2 protein levels by Western blot analysis. EAAT2 signal intensity was normalized to its actin signal intensity on the same blot (n = 3–4; *, P < 0.05, Student’s t test). Representative Western blots are presented. All data are from three independent experiments. Mean ± SEM is shown.

Figure 5.

LDN/OSU-0212320–ameliorated memory deficits and pathology in 7-mo-old APP_Sw,Ind mice. Mice were treated with 30 mg/kg LDN/OSU-0212320 i.p. starting at 7 mo of age. After 10 d of treatment, mice were subjected to Y-maze test, which was repeated after 30 d of treatment. After 60 d of treatment, mice underwent three cognitive tests (n = 18–24 per group) and then pathological experiments (n = 4 per group). (A) Y-maze tests assessing short-term memory (*, P < 0.05; **, P < 0.01; ***, P < 0.001, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previously visited arm. (B) T-maze tests assessing spatial learning memory (*, P < 0.05, one-way repeated ANOVA followed by Student’s t tests). (C) Novel object recognition tests assessing long-term nonspatial memory (**, P < 0.01; ***, P < 0.001, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previous object. (D, left) Representative images of synaptophysin immunostaining in the CA3 region. The right micrographs show high-magnification images of the boxed areas in the left micrographs. (right) Quantitative analysis of synaptophysin immunoreactivity in the CA3a region indicated by the boxed areas (*, P < 0.05, Student’s t test). (E, left) Quantitative analysis of Aβ immunostaining (*, P < 0.05, Student’s t test). (right) Representative images of Aβ immunostaining in the dentate gyrus region are shown. Nuclei were stained with Hoechst 33342 (blue). (D and E) Bars, 50 µm. Data are from six (A–C) and three (D and E) independent experiments. Mean ± SEM is shown.

Figure 6.

LDN/OSU-0212320–ameliorated memory deficits and pathology in 12-mo-old APP_Sw,Ind mice. Mice were treated with 30 mg/kg LDN/OSU-0212320 i.p. starting at 12 mo of age. After 10 d of treatment, mice were subjected to Y-maze test, which was repeated after 30 d of treatment. After 60 d of treatment, mice underwent three cognitive tests (n = 18–21 per group) and then pathological experiments (n = 4 per group). (A) Y-maze tests assessing short-term memory (**, P < 0.01, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previously visited arm. (B) T-maze tests assessing spatial learning memory (*, P < 0.05; **, P < 0.01, one-way repeated ANOVA followed by Student’s t tests). (C) Novel object recognition tests assessing long-term nonspatial memory (***, P < 0.001, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previous object. (D) Quantitative analysis of synaptophysin immunostaining in the CA3a region (**, P < 0.01, Student’s t test). (E) Quantitative analysis of Aβ immunostaining (**, P < 0.01, Student’s t test). Data are from four (A–C) and three (D and E) independent experiments. Mean ± SEM is shown.

Figure 7.

The beneficial effects of LDN/OSU-0212320 were sustained 1 mo after treatment was ceased. 12-mo-old mice were treated with 30 mg/kg LDN/OSU-0212320 i.p. for 2 mo and subjected to behavior tests (n = 10–11 mice per group) and pathological experiments (n = 4 mice per group) 1 mo after treatment cessation. (A) Y-maze assessment was measured 10, 20, and 30 d after treatment cessation (*, P < 0.05; **, P < 0.01, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previously visited arm. (B) T-maze tests (*, P < 0.05; **, P < 0.01, one-way repeated ANOVA followed by Student’s t tests). (C) Novel object recognition tests (*, P < 0.05; ***, P < 0.001, one way ANOVA followed by Holm-Šídák method). The dotted line represents the point at which mice did not remember the previous object. (D) Quantitative analysis of synaptophysin immunostaining in the CA3a region (*, P < 0.05, Student’s t test). (E) Quantitative analysis of Aβ immunostaining. Data are from four (A–C) and three (D and E) independent experiments. Mean ± SEM is shown.