Severe In vivo hyper-homocysteinemia is not associatedwith elevation of amyloid-beta peptides in the Tg2576 mice

Abstract

Since hyper-homocysteinemia (HHcy) was recognized as a risk factor for Alzheimer's disease (AD), many studies tried to induce HHcy in animal models to investigate its effect on amyloid-beta protein precursor (AbetaPP) metabolism. Previous reports found that HHcy induced in AD transgenic mouse models, by either feedina a methionine-enriched diet or vitamin Bs deficient diet, is associated with elevation of amyloid-beta (Abeta) levels. However, there is no data available on the effect of dietary intervention which combines both excessive methionine and low levels of vitamin Bs on amyloidogenesis in any of these models. In the current study, we investigated the effect of a combination diet, which was both enriched in methionine and deficient in folate, vitamin B6 and B12, in an AD mouse model, the Tg2576. We found that 7 months treatment of this diet induced severe HHcy in these mice with plasma homocysteine level higher than 150 microM. However, no difference was detected in brain Abeta levels or deposition between the diet-treated and control group. As shown by western blot, severe HHcy did not alter the steady state levels of proteins involved in AbetaPP metabolism, either. These results demonstrate that this combination diet-induced severe HHcy does not influence amyloidogenesis in vivo.

Fig. 1

Plasma homocysteine levels in Tg2576 mice after receiving a combination diet. Mice were fed with a diet which combines excessive methionine and low levels of the folate, vitamin B₆ and B₁₂ (Diet group) or vehicle (Ctrl group) for 7 months. Values represent mean ± S.E.M. ^**P < 0.01.

Fig. 2

Severe HHcy in Tg2576 mice and A β peptide levels. RIPA-soluble (RIPA) and formic acid (FA) extractable Aβ_1-40 (A, B), and Aβ_1-42 (C, D) levels in the cortex (Ctx) and hippocampus (Hippo) from Tg2576 on the special diet (Diet) or vehicle (Ctrl) were measured by sandwich ELISA. Values represent mean ± S.E.M.

Fig. 3

Severe HHcy in Tg2576 mice and Aβ deposition. A) Representative sections of brains of Tg2576 receiving special diet (Diet), or vehicle (Ctrl) immunostained with 4G8 antibody. B) Quantification of the area occupied by Aβ immunoreactivity in hippocampus and somatosensory cortex (SSC) of Tg2576. Values represent mean ± S.E.M.

Fig. 4

AβPP metabolism in Tg2576 mice with severe HHcy A) Representative western blots of AβPP, sAβPPa, sAβPPβ, CTFs (C99 and C83), in brain homogenates from Diet group or Ctrl group. B) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (white bars: Ctrl group; black bars: Diet group). Values represent mean ± S.E.M.

Fig. 5

AβPP proteolytic pathways in Tg2576 mice with severe HHcy. A) Representative western blots of ADAM10, BACE1, PS1, and Nicastrin in brain homogenates from Diet group or Ctrl group. B) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (white bars: Ctrl group; black bars: Diet group). Values represent mean ± S.E.M.

Fig. 6

Aβ catabolic pathways in Tg2576 mice with severe HHcy. A) Representative western blots of NEP, IDE, and APOE in brain homogenates from Diet group or Ctrl group. B) Densitometric analyses of the immunoreactivities to the antibodies shown in panel A (white bars: Ctrl group; black bars: Diet group). Values represent mean ± S.E.M.