Diet-Induced Hyperhomocysteinemia Increases Amyloid-β Formation and Deposition in a Mouse Model of Alzheimers Disease

Abstract

Hyperhomocysteinemia (HHcy) has been recognized as a risk factor for developing Alzheimer’s disease (AD). However, its underlying molecular mechanisms are still elusive. Here we show that HHcy induces an elevation of amyloid beta (Aβ) levels and deposition, as well as behavioral impairments, in a mouse model of AD-like amyloidosis, the Tg2576 mice. This elevation is not associated with significant change of the steady state levels of the Aβ precursor protein (APP), β - or α-secretase pathways, nor with the Aβ catabolic pathways. By contrast, HHcy significantly reduces glycogen synthase kinase 3 (GSK3) Ser21/9 phosphorylation, but not total GSK3 protein levels. Similar results are obtained in brains homogenates from a genetic mouse model of HHcy. In vitro studies show that homocysteine increases Aβ formation, reduces phosphorylated GSK3 levels, without changes in total APP and its metabolism, and these effects are prevented by selective GSK3 inhibition. Overall, these data support a potential link between GSK3 and the pro-amyloidotic effect of HHcy in vivo and in vitro.

Homocysteine exacerbates β‐amyloid pathology, tau pathology, and cognitive deficit in a mouse model of Alzheimer disease with plaques and tangles

“…Additionally, immunoblot analyses showed that these cells had a significant increase in tau phosphorylation at the same epitopes as for the brain tissues from the diet mice and a selective activation of the cdk5 pathway. Because recent data from transgenic mice support the hypothesis that A β can modulate cellular metabolic events leading to phosphorylation-specific changes in tau, 20 and considering that Hcy can act on A β metabolism per se, 10–12 it was possible that in our study the effect on tau was secondary to that on Ab. However, based on our results we conclude that the effect of Hcy on tau phosphorylation is independent from Ab, because suppression of A β formation did not alter the Hcydependent tau phosphorylation.…”

“…11 Our group and others have reported that a diet low in folate, vitamin B6, and vitamin B12 produces high circulating Hcy levels and results also in an exacerbation of AD-like brain amyloidosis. 10,12 However, due to the limitations of the APP mice, which develop only 1 of the 2 major hallmark lesions of AD (ie, amyloidosis), no data are available on whether high Hcy levels also influence the development of tau neuropathology, and importantly, whether these effects are independent from each other. With this knowledge in mind, for this study we used the 3xTg mice, which develop both amyloid plaques and tau fibrillary tangles, and fed them a folate-, B6-, and B12-deficient diet, which is a well-established model to induce high levels of Hcy.…”

“…The cells were cultured to 80 to 90% confluence in 6-well plates and then changed to fresh medium containing 50 μ M DL-Hcy (Sigma, St Louis, MO) with 40 μ M adenosine (Sigma) and 10 μ M erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride (Sigma), as previously described. 10,17 Cell toxicity was monitored by measuring the amount of the lactate dehydrogenase enzyme released in the supernatant at the end of the incubation time by a colorimetric assay kit (Cell Biolabs, San Diego, CA). After 24 hours of treatment, media were collected for A β 1–40 measurement, and cell lysates were harvested after treatment with radioimmunoprecipitation assay (RIPA) buffer for Western blotting analyses.…”

“…10, 15, 16 Briefly, samples were elec trophoresed on 10% Bis–Tris gels or 3 to 8% Tris–acetate gel (Bio-Rad, Richmond, CA), transferred onto nitrocellulose membranes (Bio-Rad), and then incubated overnight with the appropriate primary antibodies as indicated in the Table. After 3 washings with T-TBS (pH 7.4), membranes were incubated with IRDye 800CW-labeled secondary antibodies (LI-COR Bioscience, Lincoln, NE) at room temperature for 1 hour.…”

“…Thus, we and others have shown that genetic and diet-induced chronic high Hcy both result in a significant increase in brain b-amyloid (Ab) levels and deposition in transgenic mouse models of AD-like amyloidosis. 10–12 However, because these mice mainly model only 1 of the 2 major pathological features of the AD phenotype, it would be of great scientific interest to investigate the role that Hcy may have on the other hallmark lesion, tau neuropathology, and whether the 2 effects are linked.…”