Effects of a Dehydroevodiamine-Derivative on Synaptic Destabilization and Memory Impairment in the 5xFAD, Alzheimer's Disease Mouse Model

Abstract

Carboxy-dehydroevodiamine·HCl (cx-DHED) is a derivative of DHED, which improves memory impairment. Carboxyl modification increases solubility in water, indicating that its bioavailability is higher than that of DHED. Cx-DHED is expected to have better therapeutic effects on Alzheimer's disease (AD) than DHED. In this study, we investigated the therapeutic effects of cx-DHED and the underlying mechanism in 5xFAD mice, transgenic (Tg) mouse model of AD model mice. In several behavioral tests, such as Y-maze, passive avoidance, and Morris water maze test, memory deficits improved significantly in cx-DHED-treated transgenic (Tg) mice compared with vehicle-treated Tg mice. We also found that AD-related pathologies, including amyloid plaque deposition and tau phosphorylation, were reduced after the treatment of Tg mice with cx-DHED. We determined the levels of synaptic proteins, such as GluN1, GluN2A, GluN2B, PSD-95 and Rabphilin3A, and Rab3A in the brains of mice of each group and found that GluN2A and PSD-95 were significantly increased in the brains of cx-DHED-treated Tg mice when compared with the brains of Tg-vehicle mice. These results suggest that cx-DHED has therapeutic effects on 5xFAD, AD model mice through the improvement of synaptic stabilization.

Keywords: 5xFAD; Alzheimer's disease; carboxy-dehydroevodiamine; memory impairment; synaptic destabilization.

Figure 1

Schematic timeline of drug treatment and order of behavioral tests in 5xFAD mice and wild-type (WT) control mice and the effects of cx-DHED on learning and memory deficits in 5xFAD mice during behavioral training trials. (A) The 4-months 5xFAD transgenic male mice were tested after 2 months of five times weekly cx-DHED (cx-DD), donepezil (DP), or vehicle (v) injections after 1 week of habituation in the animal room. At the end of the tests, all mice were decapitated, and their brains were collected. (B) The percentage of spontaneous change increased significantly in response to cx-DD and DP compared with that in Tg mice (left panel). No difference in the number of arm entries was detected between the experimental groups (right panel). (C) When mice entered the dark chamber, an electrical foot shock (0.2 mA) was delivered for 2 s. Latency increased significantly in the mice treated with cx-DD and DP compared with that in the untreated Tg mice. (D) The training trial was carried out four times daily for five consecutive days. The treatments with Cx-DD and DP significantly decreased latency in Tg mice. All data were given as means ± standard error of the mean (SEM) (N = 10 mice per group). **P<0.01, ***P<0.001 compared with WT-v mice, ^#P<0.05, ^##P<0.01, ^###P<0.001 compared with Tg-v mice by ANOVA. Vehicle-treated wild type mice (WT-v); cx-DHED-treated wild type mice(WT-cx-DD); vehicle-treated 5xFAD Tg mice (Tg-v); cx-DHED-treated 5xFAD Tg mice(Tg-cx-DD); DP-treated 5xFAD Tg mice (Tg-DP).

Figure 2

Effects of cx-DHED on amyloid beta (Aβ) plaque deposition. (A) Immunostaining of brain tissues with the 6E10 antibody from WT-v, WT-cx-DD, Tg-v, Tg-cx-DD, and Tg-DP groups. Scale bars, 40 × . a–d: the cortex (a,c) and dentate gyrus (b,d) of the hippocampal region are enlarged images of square box in Tg-v and Tg-cx-DD, scale bars, 100 × . (B,C) Plaque counts in the cortex (CX; B) and dentate gyrus (DG; C) of the hippocampal region decreased in Tg-cx-DD mice. All data were given as means ± standard error of the mean (SEM) (N = 7 mice per group). ^##P<0.01, ^###P<0.001 compared with Tg-v mice.

Figure 3

Effects of cx-DHED on phosphorylated tau deposits. (A) Immunostained brain tissues with the AT-8 antibody in WT-v, WT-cx-DD, Tg-v, Tg-cx-DD, and Tg-DP. Scale bars, 40 × . a–d: the cortex (a,c) and dentate gyrus (b,d) of the hippocampal region are enlarged images of square box in Tg-v and Tg-cx-DD, scale bars, 100 × . (B,C) Phosphorylated tau counts in CX (B) and DG (C) of the hippocampal region decreased in Tg-cx-DD mice. Scale bars, 100 × . All data were given as means ± standard error of the mean (SEM) (N = 4 mice per group). *P<0.05, **P<0.01 compared with WT-v mice, ^#P<0.05, ^##P<0.01 compared with Tg-v mice.

Figure 4

Effects of cx-DHED on synaptic proteins. Synaptosome fractions of cortical lysates were used to detect the loss of post-synaptic receptor immunoreactivity in mice synaptic proteins. (A) Western blot analysis of GluN2A, PSD-95, Rabphilin-3A, and RAB3A from cortex tissue lysate. (B–C) The bar shows the percentage of beta-actin normalized to the density of GluN2A (B) and PSD-95 (C), on Western blot bands from cortex tissue lysate. All data were given as means ± standard error of the mean (SEM) (N = 4 mice per group). ***P<0.001 compared with WT-v mice, ^#P<0.05, ^##P<0.01, ^###P<0.001 compared with Tg-v mice.