Marie Salvador

Mentor: Dr. Xinyin Jiang

Title: The effect of postnatal choline supplementation on DNA methylation and gene

expression in mouse progeny exposed to gestational choline-deficiency.

Proposal: Choline is a nutrient critical for fetal development, because it is required for cellular

growth and proliferation. The availability of choline during gestation and early postnatal periods affects

neurogenesis and synaptogenesis, thereby influencing early brain development with lasting impacts on

lifelong memory and learning 1. Choline deficiency during early development increases neuronal cell

death, decreasing proliferation and differentiation of cells in the hippocampus and cortex of the brain1.

However, it remains unknown whether the deleterious influence of prenatal and early postnatal choline

deficiency on brain development can be overcome by choline supplementation later in life.

The prevalence of choline insufficiency during pregnancy has not been explored due to the lack of a

sensitive and reliable marker of choline status in the body. However, the NHANS data suggests that less

than 10% of pregnant women in the U.S. meet the current recommended intake (in the form of Adequate

Intake) of 450 mg choline per day 2. Genetic polymorphisms, obesity, pregnancy and a high-fat dietary

style may lead to higher demand of choline, and therefore higher risk of suboptimal intake of choline in

certain populations. Therefore, research on whether post-weaning choline intake can reverse the effects of

low choline exposure during early development can not only reveal the temporal-specific interaction

between choline and brain functioning, but also provide a potential solution to improve long-term

cognitive outcomes of offspring affected by prenatal choline deficiency.

Choline is involved in brain development via three major pathways. It acts as a precursor to the

neurotransmitter acetylcholine (Ach) 3,4,5. Choline is an essential component of the myelin sheath, as well

as cellular and lipoprotein membranes, and thus participates in neural cell structure and essential lipid

delivery to the developing brain. Choline is also oxidized to betaine by which it donates labile methyl

groups for methylation reactions including DNA methylation. DNA methylation is a major epigenetic

modification which alters gene expression without altering the genomic sequence. An array of genes

related to memory and neuroendocrine functioning such as the brain derived neurotrophic factor (BDNF)
Marie Salvador
Mentor: Dr. Xinyin Jiang

and corticotropin-releasing hormone (CRH) are regulated by DNA methylation. The epigenome of the

developing brain demonstrates exceptional plasticity to exposures such as maternal methyl group

availability. The epigenetic marks established in early brain developmental stages are often maintained

into later life.

The objective of the proposed study is to determine whether in a mice model post-weaning

choline supplementation can rescue the changes in DNA methylation and gene expression in the

offspring’s brain resulted from choline deficiency during gestation and early postnatal development. The

hypothesis is that post-natal choline supplementation will normalize the DNA methylation and gene

expression levels of mice exposed to prenatal and early postnatal choline deficiency.

Specific Aim 1. Determine whether gene expression changes in the hippocampus of mouse brains

resulted from early choline deficiency can be rectified by post-weaning choline supplementation.

C57BL/6J female mice will be fed with either a choline deficient diet (GCD) with 1 g/kg of choline or a

control diet (GCG) with 2 g/ kg of choline, 6 weeks prior to and during gestation and during lactation.

After weaning, choline will be provided for the mice offspring, in two levels: 8 g/kg for the supplemented

choline group (CS), 2g/kg for the control group (CG) and 2g/kg for the sufficient choline group (CE).

Treatment will continue for 6 weeks. Expression of genes regulating neuronal development (e.g. Bdnf),

cellular growth (e.g. Igf2), and apoptosis (e.g. Casp3) will be measured in the hippocampus of the brain.

We anticipate that GCD group would result in lower expression of Bdnf and Igf2, and higher expression

of Casp3 than GCG mice, while post-weaning CS would normalize these alterations.

Specific Aim 2. Determine whether epigenetic changes in the mouse brain due to early choline

deficiency can be rescued by post-weaning choline supplementation.

The global DNA methylation of mouse hippocampi will be assessed by a DNA methylation kit (Cayman

Chemicals). We anticipate that GCD would decrease global DNA methylation in the hippocampus,

whereas post-weaning CS would normalize DNA methylation.

Marie Salvador
Mentor: Dr. Xinyin Jiang

Timeline

Date: Task

03/26/18 Acclimation of mice for two weeks. We provided regular diet of 2 g/kg choline provided
to all mice.

04/10/18 The mice were assigned to either a choline deficient group receiving 1g/kg of choline or
to a control group receiving 2g/kg of choline. The mating process was also started, as
we placed 2 females with one male per cage. We checked daily for vaginal plugs that
indicated gestation.

04/17/18 We weighted female mice, for gestation confirmation. We separated mice once
gestation was confirmed.

04/18/18 Check daily for birth of pups. We planned to wean pups after they reached their 3-
weeks of birth.

05/21/18 We weaned the offspring mice, all pups were acclimated, on a control diet of 2 g/ kg
choline.

05/29/18 We assigned all pups to their postnatal diet to either 8g/kg or 2g/kg supplemental
choline. We weighted mice offspring and food in and out, weekly.

07/10/18 -07/11/18 We euthanized animals to dissect the brain and the liver for analysis.

07/17/18 Start RNA extraction of some samples.

08/08/18 Start of reverse transcription cDNA of some samples.

08/22/18 Start of DNA extraction of some samples.

08/27/18- 12/16/2018 Continue experiments and continue working on writing of thesis.

References

1. Sanders, L. M., & Zeisel, S. H. (2007). Choline: Dietary Requirements and Role in Brain
Development. Nutrition Today, 42(4), 181–186.
http://doi.org/10.1097/01.NT.0000286155.55343.fa
Marie Salvador
Mentor: Dr. Xinyin Jiang

2. NIH. (2018) “Office of Dietary Supplements - Choline.” NIH Office of Dietary Supplements, U.S.
Department of Health and Human Services, ods.od.nih.gov/factsheets/Choline-
HealthProfessional/.
3. Caudill, Marie A. “Pre- and Postnatal Health: Evidence of Increased Choline Needs.” Journal of
the American Dietetic Association, vol. 110, no. 8, 2010, pp. 1198–1206.,
doi:10.1016/j.jada.2010.05.009
4. Zeisel, S. H. (2010). Choline: Clinical Nutrigenetic/Nutrigenomic Approaches for Identification
of Functions and Dietary Requirements. World review of nutrition and dietetics, 101, 73-83.
doi:10.1159/000314512
5. Biasi, Elisabetta. “The Effects of Dietary Choline.” Neuroscience Bulletin, vol. 27, no. 5, 2011,
pp. 330–342., doi:10.1007/s12264-011-1523-5.
6. Monk, Bradley R., et al. (2012) “The Effects of Perinatal Choline Supplementation on
Hippocampal Cholinergic Development in Rats Exposed to Alcohol during the Brain Growth
Spurt.” Hippocampus, vol. 22, no. 8, 2012, pp. 1750–1757., doi:10.1002/hipo.22009.