Hyperhomocysteinemia increases beta-amyloid by enhancing expression of gamma-secretase and phosphorylation of amyloid precursor protein in rat brain

Abstract

Hyperhomocysteinemia and beta-amyloid (Abeta) overproduction are critical etiological and pathological factors in Alzheimer disease, respectively; however, the intrinsic link between them is still missing. Here, we found that Abeta levels increased and amyloid precursor protein (APP) levels simultaneously decreased in hyperhomocysteinemic rats after a 2-week induction by vena caudalis injection of homocysteine. Concurrently, both the mRNA and protein levels of presenilin-1, a component of gamma-secretase, were elevated, whereas the expression levels of beta-secretase and presenilin-2 were not altered. We also observed that levels of phosphorylated APP at threonine-668, a crucial site facilitating the amyloidogenic cleavage of APP, increased in rats with hyperhomocysteinemia, although the phosphorylation per se did not increase the binding capacity of pT668-APP to the secretases. The enhanced phosphorylation of APP in these rats was not relevant to either c-Jun N-terminal kinase or cyclin-dependent kinase-5. A prominent spatial memory deficit was detected in rats with hyperhomocysteinemia. Simultaneous supplementation of folate and vitamin-B12 attenuated the hyperhomocysteinemia-induced abnormal processing of APP and improved memory. Our data revealed that hyperhomocysteinemia could increase Abeta production through the enhanced expression of gamma-secretase and APP phosphorylation, causing memory deficits that could be rescued by folate and vitamin-B12 treatment in these rats. It is suggested that hyperhomocysteinemia may serve as an upstream factor for increased Abeta production as seen in patients with Alzheimer disease.

Figure 1

Hyperhomocysteinemia promotes amyloidogenic cleavage of APP. Homocysteine (Hcy, 400 μg/kg/day) was injected through vena caudalis into the rats for different time periods (n = 6 to 7 for each group) (A, B). The correlation between decrease of APP and increase of Aβ was analyzed by Pearson (C, D). Alternatively, Hcy was injected in different concentrations for 2 weeks (Hcy1 = 400 μg/kg/day, Hcy2 = 1600 μg/kg/day) with or without supplement of folate and Vit-B12 (FB, 4 mg/kg/day or 250 μg/kg/day) (n = 10 for each group) (E, F). The same volume of saline was injected as control (Con). The hippocampal extract was used for Western blotting (A, E) and quantitative analysis (B, F). The relative Aβ level was accessed by 4G8 and 6E10 and the full length APP was measured by 22C11 and 369, all of which were normalized against β-actin. All bands shown in the blots were collected for the calculation of APP and Aβ, and all data were expressed as mean ± SD. *P < 0.05, **P < 0.01 vs control; ^#P < 0.05, ^##P < 0.01 vs Hcy1; ^ΔP < 0.05, vs Hcy2.

Figure 2

Hyperhomocysteinemia increases Aβ40 in cortex and hippocampus. The rats received injection of Hcy (Hcy1 = 400 μg/kg/day, Hcy2 = 1600 μg/kg/day) with or without supplement of folate and Vit-B12 (FB, 4 mg/kg/day or 250 μg/kg/day) for 2 weeks, and the same volume of saline was injected as controls (Con). The rats were then sacrificed and the levels of Aβ40 in the hippocampus and cortex were measured by ELISA (A, B) and immunocytochemistry staining (E, 400 μg/kg/day of Hcy and 250 μg/kg/day of FB) by using an Aβ40-specific antibody. The correlation between decrease of APP (see Figure 1, E and F) and increase of Aβ40 measured by ELISA was analyzed by Pearson (C, D). All data expressed as mean ± SD; n = 7 for ELISA; n = 3 for immunocytochemistry, scale bar = 100 μm. *P < 0.05, **P < 0.01 vs control; ^#P < 0.05, ^##P < 0.01 vs Hcy1; ^ΔP < 0.05, ^ΔΔP < 0.01 vs Hcy2. CA1-CA4 denote the Cornu Ammonis areas and DG represents the denate gyrus.

Figure 3

Hyperhomocysteinemia selectively increases the mRNA and protein levels of PS-1. Hcy (400 μg/kg/day) was injected into the rats for 2 weeks with or without supplement of folate and Vit-B12 (FB, 250 μg/kg/day). Then the hippocampus extracts were prepared and the mRNA and protein levels of APP and its secretases, including PS-1, PS-2 and BACE, were measured and quantitatively analyzed by reverse transcription-PCR (A, B) and Western blotting (C, D), respectively. The β-actin and GAPDH were used as internal controls. All data were expressed as mean ± SD; n = 4 for reverse transcription-PCR; n = 6 for Western blotting. *P < 0.05, **P < 0.01 vs control; ^#P < 0.05 vs Hcy.

Figure 4

Hyperhomocysteinemia increases the phosphorylation of APP at Thr668. Hcy (400 μg/kg/day) was injected into the rats for 2 weeks with or without supplement of folate and Vit-B12 (FB, 250 μg/kg/day). Then the hippocampal extracts were prepared and the phosphorylation level of APP at Thr668 (pT668-APP) was measured by Western blotting (A) and quantitative analysis (B), and the expression of pT668-APP was also detected by immunohistochemistry staining in hippocampus (Hip) and cortex (Cor) (C). All data expressed as mean ± SD (n = 5). *P < 0.05, **P < 0.01 vs control; ^#P < 0.05 vs Hcy. Scale bar = 100 μm.

Figure 5

Phosphorylation of APP increases the total binding level to the secretases without affecting the binding capacity. Hcy (400 μg/kg/day) was injected into the rats for 2 weeks with or without supplement of folate and Vit-B12 (FB, 250 μg/kg/day). The hippocampal extracts were immunoprecipitated (IP) with pT668-APP antibody and the precipitates were analyzed by immunoblotting (IB) using the antibodies against pT668-APP, BACE1, PS1 and PS2 as labeled (A). The relative levels of the pT668-APP and the bound secretases in the precipitates were quantitatively analyzed as indicated (B), and the binding capacity of the secretases to the pT668-APP was calculated by normalized against the level of pT668-APP in each group and expressed by setting the vehicle control group as 1 (C). All data expressed as mean ± SD (n = 5). *P < 0.05, **P < 0.01 vs control; ^#P < 0.05, ^##P < 0.01 vs Hcy.

Figure 6

Hyperhomocysteinemia inactivates JNK and Cdk-5. Hcy (400 μg/kg/day) was injected into the rats for 2 weeks with or without supplement of folate and Vit-B12 (FB, 250 μg/kg/day). The levels of total JNK (t-JNK) and the phosphorylated (p-JNK, the activated form), and Cdk-5, p35, and p25 were measured by Western blotting (A and C) and quantitatively analyzed (B and D). All data expressed as mean ± SD (n = 5). *P < 0.05. **P < 0.01 vs control; ^##P < 0.01 vs Hcy.

Figure 7

Hyperhomocysteinemia impairs the spatial memory of the rats. Hcy (400 μg/kg/day) was injected for 2 weeks with or without supplement of folate and Vit-B12 (FB, 250 μg/kg/day) after a consecutive training for 6 days in Morris water maze. Then, the spatial memory retention of the rats was measured at day 15 after the injection. The path to find the platform before (pre-) and after (post-) injection (A), the escape latency to find the hidden platform (B), the path swimming in the maze for one minute after removed the platform (C), and the distance swimming in the third quadrant in one minute (D) of the rats were shown. All data expressed as mean ± SD (n = 12). * P < 0.05, ** P < 0.01 vs control.