Carol L. Cheatham, Ph.D. - US grants

DESCRIPTION (provided by applicant): The proposed training is designed to provide the candidate with a background in animal cognition and testing as well as training in molecular mechanisms of development. The candidate is a well-trained developmental cognitive neuroscientist with a dedication to at-risk children. However, human research is limiting in that mechanisms cannot be established due to the lack of available tissue. The training proposed in this application will facilitate the candidate's pursuit of her long-term goal of being able to ask scientific questions by moving from the clinic to the bench and back again. Specifically, the candidate will work on the mechanisms involved in the effects of fetal alcohol exposure. This preventable, highly prevalent developmental phenomenon is characterized by a constellation of adverse outcomes including neurodevelopmental abnormalities, growth retardation, and facial structure anomalies. Scientists working with animal models have provided evidence that choline supplementation given concomitant with or after fetal alcohol exposure mediates the effects. The proposed research will establish the mechanisms involved in this mediation by describing any epigenetic modifications and will extend previous research by testing choline as a preventative measure, giving it before and during alcohol exposure in a rat model. In Experiment 1, the candidate will develop a novel choline-supplemented liquid diet for the rats. This diet will be important in the avoidance of the stress of injections that is inherent in cholin administration. The goal of Experiment 1 is to determine if the taste of choline will deter the rat from consuming as much ethanol as the ethanol-only group. If so, subcutaneous injections of choline and vehicle will be necessary. In Experiment 2, pregnant rats will be divided into 3 experimental groups: ethanol-exposed, choline-supplemented, and ethanol-exposed with choline supplementation. Each experimental group will have a matching control group, and an unhandled pellet fed control will be included for a total of 7 diet groups. One male pup from each litter (n=10) will comprise groups for analyses. Hippocampal slices, blood DNA, and brain DNA will be collected on GD 18 (after cell proliferation in hippocampus), PD 45 (prior to Morris water maze training), and PD 54 (after Morris water maze training). Hippocampal slices and blood will be assessed with bisulfite pyrosequencing (epigenetics) and real-time RT-PCR (gene expression) for a selection of genes related to hippocampal development, choline, and alcohol metabolism. Protein levels will be assessed by immunolabeling of frozen brain sections. The research will be completed with a mentoring team comprised of top experts in the field: Steven Zeisel (choline and epigenetics), Jennifer Thomas (animal models of fetal alcohol exposure and mediation of same with choline), and Phillip May (sequelae of fetal alcohol effects in humans). The candidate and two of the mentors are on the faculty of the University of North Carolina at Chapel Hill Nutrition Research Institute (NRI) on the new North Carolina Research Campus in Kannapolis, North Carolina. Having only opened in 2008, this campus is outfitted with state-of-the-art facilities. Laboratories in the NRI are equipped with the newest of DNA sequencing, genotyping, and gene expression equipment. The David H Murdock Research Institute (DHMRI) provides over 110,000 square feet of instrumentation, resident expertise, and well-equipped -omics laboratories. In addition, the DHMRI maintains a 40,000 square foot vivarium with top-of-the-line equipment. All equipment needed for this project is available on this campus; many scientists are housed here and are available to provide expert support to the candidate. The results of these studies will inform an R01 application (candidate's short-term goal) in which the candidate will propose an intervention to determine the effects of choline supplementation in human fetuses who are at risk for fetal alcohol exposure. This work is integral to the understanding of the fetal alcohol exposure phenotype and will be the initial steps toward the use of a nutrient - choline - to mediate or even prevent the effects of fetal alcohol. The candidat has demonstrated her commitment to at-risk children. The proposed training will provide her with a deeper understanding of the mechanisms involved in developmental phenomenon thereby strengthening her ability to form and test hypotheses. As a result of being able to move between the clinic and the bench, the candidate will build a strong, independent research program.

PROGRAM SUMMARY The main aim of the NIH Nutrition Obesity Research Center (NORC) is to assist researchers in advancing their research in the field of obesity and nutrition. The Precision Nutrition core, directed by Dr. Voruganti is one of the UNC NORC cores that provides an all-encompassing support system providing investigators with the design and implementation of nutrigenetic, nutrigenomics and microbiome research components of obesity studies. In this administrative supplement, we propose to a pilot study in which we will generate a model that can predict the risk for cognitive decline based on their genetic, dietary, metabolic and demographic variables. Cognitive decline is a key clinical feature of the neurodegenerative disease, Alzheimer?s disease (AD), an extremely debilitating condition. The prevalence of AD is expected to increase to 14 million by 2050 in the US and 130 million worldwide. Considering that there is no cure for AD, it is imperative that we take measures to predict its onset and design interventions which can either prevent and/or slow down the progression of its symptoms, particularly cognitive decline. Cognitive decline is influenced by multiple genetic and environmental factors and it has proven difficult to predict. Previous models predicting the risk for cognitive decline have either been based on invasive markers or focused on only a few sets of genotypes or phenotypes. In this project we will use an established cohort of elderly adults for developing a risk prediction model and then use data from the Baltomore Longitudinal Study of Aging (BLSA) data repository to test, validate and confirm this model. We expect that the resultant model will facilitate efficient and cost- effective nutritional interventions to prevent or slow the rate of progression of cognitive decline in middle- aged and elderly adults.

7. PROJECT SUMMARY Despite prolific marketing campaigns explaining the dangers of consuming alcohol during pregnancy, the prevalence of Fetal Alcohol Spectrum Disorders (FASD) in the United States is 2-5%, which is higher than the rate of Autism Spectrum Disorders. Whether it is because a woman was unaware of the pregnancy, ignorant of the risk, dismissing of the risk, or unable to abstain, women do drink during pregnancy, and therefore, it is imperative that we find a way to prevent FASD. Optimal nutrition is essential for the developing fetal brain and is generally lacking in active drinkers. One nutrient, choline, has been shown to ameliorate the effects of fetal alcohol exposure in animal models. Translation to the clinic shows promise, but scientists report mixed or limited results. Importantly, with each successive pregnancy, DHA stores become depleted, and with each successive pregnancy, the risk of FASD increases. Thus, maternal DHA deficiency may be implicated in FASD. Indeed, DHA and choline work together to deliver DHA to the brain where it supports brain function. This synergistic activity is further enhanced by the addition of uridine. To explore this synergy hypothesis, we will test a natural therapeutic prenatal prophylactic that contains DHA, choline, and uridine and is easily translatable to the clinic - the chicken egg - in a preclinical study. Our central hypothesis is that the synergistic activity of DHA, choline, and uridine in egg will mitigate the effects of fetal alcohol exposure on the developing hippocampus and frontal lobes. Importantly, these experiments will be the first in a program of research designed to establish the mechanisms through which synergistic nutrients ameliorate the effect of prenatal alcohol exposure on fetal brain development. In the proposed work, pregnant dams will be fed experimental diets from gestational day GD5 to GD20 in a 2 (Prenatal Exposure: Ethanol, Pair-fed Control) X 5 (Diet: Egg, Choline, DHA, Uridine, Unsupplemented) design. Subsequent pups will be observed for early physical (ear unfurling, eye opening, appearance of fur) and behavioral (e.g., self-righting, cliff avoidance) development in a series of tests. Groups will be compared on day of success in a Chi-square analysis for discrete variables. Starting on PD45, random subsets (one male and one female per litter) will be trained and tested on a classic Morris Water Maze spatial and working memory tests. Data will be subjected to analyses of variance between groups. Brain tissue will be assayed for phospholipid and uridine content and synaptic proteins will be quantified with immunoblots. We predict that the ethanol effects will be reduced in the egg-supplemented groups compared to the non-egg groups. The results of this experiment will inform interventions for pregnant women who are unable to abstain from consuming alcohol during pregnancy and their at-risk fetuses. If our preclinical work shows promise, we will translate the work to the clinic, working with support programs in the US and Canada to provide the food to their clients and thereby, improve the outcomes and the subsequent lives of their unborn children.