Elucidating the molecular mechanisms linking maternal choline supplementation to healthy cognitive aging

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Part 1: Non-technical DescriptionThis project is supported under the SBE Postdoctoral Research Fellowships (SPRF) program. Age-related cognitive decline, hereafter referred to as cognitive aging, is a fact of life. To this end, structural and functional brain changes invariably lead to decreased cognitive functions even in otherwise healthy individuals. Although there is a large body of work on ways to reduce cognitive deficits associated with disease states, little is known about the mechanisms underlying cognitive aging. This is concerning given that life expectancy is increasing and cognitive aging leads to a deterioration of general health for the aging population. One option to reduce cognitive aging is the supplementation of the maternal diet with choline (MCS), an essential nutrient grouped with the vitamin B complex. Preliminary work has demonstrated that MCS leads to amelioration of cognitive aging. The primary goal of this postdoctoral project is to elucidate the underlying neural and molecular mechanisms linked to MCS benefits. Additionally, the PI plans to examine genetic targets within brain structures associated with memory formation to isolate genes that are differentially altered by MCS and aging. This will likely reveal various gene targets that will be the focus of future cognitive aging studies. Because choline is a non-toxic nutrient found in food and can be easily supplemented orally, the research team believes that any modifications to the recommended daily intake amount to reduce cognitive aging will be expedited. To this end, the results of this application may help establish new guidelines on how a diet regimen of MCS should be implemented in expecting women to reduce cognitive aging in their offspring. Data will be shared with both the scientific and general community through presentations at conferences and public forums. Part 2: Technical DescriptionThe loss of cognitive function is a pervasive and often debilitating feature of the aging process. To this end, structural and functional brain changes invariably lead to decreased cognitive functions even in otherwise healthy individuals. Recent work has shown that supplementation of choline, an essential nutrient grouped with the vitamin B complex, in the maternal diet (MCS) reduces cognitive aging. However, the molecular mechanisms linked to MCS benefits remains elusive. Elevated homocysteine levels correlate with cognitive aging, and aberrant gene expression mediated by reduced DNA methylation may contribute to cognitive aging. Choline is the major dietary source of methyl groups for the conversion of homocysteine to methionine, and for the production of S-Adenosyl methionine (SAM). SAM is a key substrate for epigenetic mechanisms, such as DNA methylation. Therefore, we hypothesize that MCS may reduce cognitive aging deficits by (1) reducing the buildup of homocysteine levels, and (2) by altering fetal epigenetic mechanisms during development leading to functional improvements in late life. Herein, we will breed 2-month-old C57Bl/6 mice. One-third of the breeding pairs will be kept on a CTL diet (choline normal diet, with standard choline content of 1.1 g/kg choline chloride), while the remaining mice will be kept on a maternal choline supplemented (MCS) diet (5 g/kg choline chloride), from conception through postnatal day 21. The offspring will be kept on the same choline diet as the parents until weaning at postnatal day 21. Notably, a group of dams from the MCS groups will be injected every other day with a betaine-homocysteine S-methyltransferase blocker S-(ä-carboxybutyl)-DL-homocysteine (CBHcy) that prevents the choline-mediated decrease in homocysteine levels. Thus, we will be able to determine whether the benefits of MCS are directly linked to homocysteine levels. Mice will be tested behaviorally using a longitudinal and a cross sectional strategy at 2, 8, 15 and 18 months of age to collect data at multiple time points and control for re-test effects. Tissue will be processed to (1) examine dendritic spine number and morphology within the hippocampus and (2) to examine alterations of DNA methylation in the promoter region of neuronal dendritic morphology-related genes (Dlg4, Rac1, RhoA, Doc2b). We will complement this work by using an unbiased approach to identify genes that are differentially methylated by MCS. These experiments will be done exclusively in hippocampal CA1 neurons isolated by laser-capture microdissection. If successful, our results would dissect the underlying molecular mechanisms whereby choline supplementation reduces cognitive aging. Understanding MCS benefits at the behavioral, neural and molecular level may lead to a modification in the recommended amounts of choline required for pregnant mothers for optimal cognitive functioning and prevention of cognitive aging.