Date Published: January 19, 2017
Publisher: Public Library of Science
Author(s): Tiffany J. Mellott, Olivia M. Huleatt, Bethany N. Shade, Sarah M. Pender, Yi B. Liu, Barbara E. Slack, Jan K. Blusztajn, Masuo Ohno.
Prevention of Alzheimer’s disease (AD) is a major goal of biomedical sciences. In previous studies we showed that high intake of the essential nutrient, choline, during gestation prevented age-related memory decline in a rat model. In this study we investigated the effects of a similar treatment on AD-related phenotypes in a mouse model of AD. We crossed wild type (WT) female mice with hemizygous APPswe/PS1dE9 (APP.PS1) AD model male mice and maintained the pregnant and lactating dams on a control AIN76A diet containing 1.1 g/kg of choline or a choline-supplemented (5 g/kg) diet. After weaning all offspring consumed the control diet. As compared to APP.PS1 mice reared on the control diet, the hippocampus of the perinatally choline-supplemented APP.PS1 mice exhibited: 1) altered levels of amyloid precursor protein (APP) metabolites–specifically elevated amounts of β-C-terminal fragment (β-CTF) and reduced levels of solubilized amyloid Aβ40 and Aβ42 peptides; 2) reduced number and total area of amyloid plaques; 3) preserved levels of choline acetyltransferase protein (CHAT) and insulin-like growth factor II (IGF2) and 4) absence of astrogliosis. The data suggest that dietary supplementation of choline during fetal development and early postnatal life may constitute a preventive strategy for AD.
The development of a treatment for AD constitutes a major goal for biomedical sciences. A vast amount of resources have been devoted to this challenge, consistent with its enormous societal need. Very little thought has been given however to the possibility that AD might be preventable or that its onset might be delayed by the use of a prevention strategy. We have previously shown that high choline intake during gestation and perinatal period in rodent models prevents age-related memory decline  and in the current study we test the idea that this preventive strategy will be effective in a model of AD. Choline was classified as an essential nutrient, by the Food and Nutrition Board (FNB) of the Institute of Medicine of the National Academy of Sciences, relatively recently. It was only in 1998 that the FNB issued dietary reference intake values for this nutrient . Significantly, the FNB recognized that the requirements for choline are increased during pregnancy and nursing (Adequate Intake values for women (mg/day): non-pregnant 425; pregnant 450; lactating 550). Because of this short history, our understanding of the significance of choline nutrition in human health and disease remains inadequate. The 2007 National Health and Nutrition Examination Survey (NHANES) study reported that in the US fewer than 15% of pregnant women consume the recommended amount . Moreover, at least 25% of women in a California cohort consumed so little choline that they were at 4-fold increased risk of having babies with neural tube defects [4, 5]. Several additional studies confirm that Americans consume far less choline than recommended with only approximately 25% of adults meeting the AI values [6–10]. These data indicate that increased intake of choline by our population is a desirable public health goal.
To assess the effects of choline supplementation on the progression of amyloidosis, we measured the amount of solubilized Aβ40 and Aβ42 by ELISA and soluble Aβ by Western blot analysis in hippocampal tissue, as well as the number of plaques and total plaque area in both anterior and posterior hippocampal sections of wild-type and APP.PS1 mice. First, solubilized Aβ40 and Aβ42 were measured in hippocampal lysates in females (Fig 1A and 1C) and males (Fig 1B and 1D) at 6-, 9-, and 12-months of age. Females from the control diet group had more solubilized Aβ40 than control males at both 9- (P270) and 12- months (P360). Choline supplementation significantly reduced the levels of Aβ40 and Aβ42 in APP.PS1 female mice at the 9-months of age but not at 12-months (Fig 1A and 1C). In contrast, there were no significant differences in Aβ40 and Aβ42 levels in 9-month-old males, but at 12-months choline-supplemented males had dramatically less solubilized Aβ40 and Aβ42 than controls (approximately 13% and 39% of controls, respectively) (Fig 1B and 1D). Western blot analysis with an anti-APP antibody was used to visualize full-length APP and Aβ levels in the hippocampus of control and choline-supplemented APP.PS1 mice. There were no significant differences in the levels of full-length human APP between dietary groups at either 9- or 12-months of age, regardless of sex (Fig 1E and 1F). In both dietary groups and in both sexes, however, there were significant reductions in the amounts of full length APP at 12-months of age compared to those at 9-months. The analysis of total soluble Aβ levels in the hippocampus via immunoblot produced similar results to those obtained using ELISA, such that choline-supplemented females had significantly less soluble Aβ than control mice at 9-months-old (Fig 1E) and choline-supplemented males had less Aβ at 12-months (Fig 1F). In both sexes, the amount of soluble Aβ significantly increased from 9-months to 12-months, regardless of diet. In the hippocampus of 12-month-old APP.PS1 mice, we also measured the levels of the products of APP cleavage catalyzed by the α and β secretase enzymes, i.e. the α- and β-C-terminal fragments (CTFs) of APP. β-CTF is the substrate of γ secretase that produces the Aβ peptides. Using Western blot analysis with an antibody raised against the C-terminal end of APP (Fig 2), we found that, while choline supplementation had no effect on the α-CTF levels, it increased the levels of the β-CTF by approximately 30% as compared to controls in both females and males (Fig 2A and 2B).
These data show that high dietary choline consumption by mouse mothers throughout pregnancy and nursing ameliorates two central pathophysiologic features of their AD model APP.PS1 offspring: accumulation of amyloid plaques and reductions in CHAT protein levels in the hippocampus. The characteristic age-dependent progressive amyloidosis in brain of the APP.PS1 mice [45, 62] was dramatically attenuated by an increased supply of choline during fetal and early postnatal development.