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

本项目由SBE博士后研究奖学金（SPRF）计划资助。与年龄相关的认知能力下降，以下简称认知衰老，是生活中的一个事实。为此，即使在健康的个体中，大脑结构和功能的变化也必然导致认知功能的下降。尽管在减少与疾病状态相关的认知缺陷的方法上有大量的工作，但对认知衰老的机制知之甚少。这令人担忧，因为预期寿命在增加，认知老化导致老龄人口总体健康状况恶化。减少认知老化的一种选择是在母体饮食中补充胆碱（MCS），这是一种与维生素B复合物组合在一起的必需营养素。初步研究表明，MCS可改善认知老化。该博士后项目的主要目标是阐明与MCS益处相关的潜在神经和分子机制。此外，PI计划检查与记忆形成相关的大脑结构中的基因目标，以分离因MCS和衰老而不同改变的基因。这可能会揭示各种基因靶点，这将是未来认知衰老研究的重点。由于胆碱是一种在食物中发现的无毒营养物质，可以很容易地口服补充，研究小组认为，对建议的每日摄入量进行任何修改，以减少认知衰老，都将加速。为此，这项应用的结果可能有助于建立新的指导方针，指导如何实施MCS的饮食方案，以期望女性减少其后代的认知衰老。数据将通过在会议和公共论坛上的介绍与科学界和一般社区共享。第2部分：技术描述认知功能的丧失是衰老过程中普遍存在且常常使人衰弱的特征。为此，即使在健康的个体中，大脑结构和功能的变化也必然导致认知功能的下降。最近的研究表明，在母体饮食（MCS）中补充胆碱（一种与维生素B复合物组合在一起的必需营养素）可以减少认知衰老。然而，与MCS益处相关的分子机制仍然难以捉摸。同型半胱氨酸水平升高与认知衰老相关，DNA甲基化降低介导的基因异常表达可能导致认知衰老。胆碱是同型半胱氨酸转化为蛋氨酸和s -腺苷型蛋氨酸（SAM）生产所需甲基的主要饲料来源。SAM是表观遗传机制（如DNA甲基化）的关键底物。因此，我们假设MCS可能通过(1)减少同型半胱氨酸水平的积累，以及(2)在发育过程中改变胎儿表观遗传机制，导致晚年功能改善，从而减少认知衰老缺陷。为此，我们将繁殖2月龄C57Bl/6小鼠。三分之一的繁殖对将被饲养在CTL饮食（胆碱正常饮食，标准胆碱含量为1.1 g/kg氯化胆碱），而其余小鼠将被饲养在母体胆碱补充（MCS）饮食（5 g/kg氯化胆碱），从受孕到出生后第21天。后代将与父母保持相同的胆碱饮食，直到出生后21天断奶。值得注意的是，MCS组的一组小鼠将每隔一天注射一种甜菜碱-同型半胱氨酸S-甲基转移酶阻滞剂S-（ä-carboxybutyl）- dl -同型半胱氨酸（chcy），以防止胆碱介导的同型半胱氨酸水平下降。因此，我们将能够确定MCS的益处是否与同型半胱氨酸水平直接相关。小鼠将在2、8、15和18个月大时采用纵向和横截面策略进行行为测试，以收集多个时间点的数据并控制再次测试的效果。将对组织进行处理，以(1)检查海马内树突棘的数量和形态，(2)检查神经元树突形态相关基因（Dlg4, Rac1, RhoA, Doc2b）启动子区域DNA甲基化的改变。我们将通过使用无偏方法来鉴定MCS差异甲基化的基因来补充这项工作。这些实验将仅在通过激光捕获显微解剖分离的海马CA1神经元中进行。如果成功，我们的结果将剖析潜在的分子机制，即补充胆碱减少认知衰老。了解MCS在行为、神经和分子水平上的益处，可能会导致孕妇所需的胆碱推荐量的改变，以获得最佳的认知功能和预防认知衰老。

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.