Modeling neurodevelopmental risk of imbalanced micronutrient supply in cerebral organoids

In 1998, the Food and Drug Administration mandated the fortification of grain products with folic acid (FA) to reduce the incidence of neural tube defects. While this mandate has been highly successful, in combination with rising supplementation it has led to a substantial increase in FA consumption in the U.S. At the same time a critical gap exists in our understanding of potentially detrimental effects of high FA intake on neurodevelopment of the fetus. Indeed, a series of epidemiological and experimental observations let us hypothesize that excessive intake of FA during pregnancy poses risks to brain development of the fetus that can predispose to neurodevelopmental disorders. These observations are supported by our own work in mice, which has confirmed that excess prenatal FA exposure can modify developmental neurogenesis and, in the process, alter cortical cytoarchitectural integrity in the offspring. Intriguingly, deviations observed with FA excess closely mimicked those seen in folate deficiency, a recognized cause of neurodevelopmental disorders. A distinct micronutrient, vitamin B12 (B12), has seen population level decreases in intake during the same period FA intake has increased. B12 is critically required for folate cycle progression and regeneration of tetrahydrofolate, which leads us to further hypothesize that neurodevelopmental risk of FA excess is heightened in the face of B12 deficiency. To bridge the gap between preclinical observations in animal models and human neurodevelopment, the research objective of this interdisciplinary proposal is to experimentally test the developmental and biochemical consequences of excessive FA exposure in engineered human cerebral organoids. Such organoids provide an excellent model in the study of early neurodevelopmental events, including those that may be critically disturbed in neurodevelopmental disorders as other studies have suggested. Simultaneously, we will test the effects of B12 deficiency. We will culture cerebral organoids under nine different conditions of folate and B12 supply in the media to measure in Aim 1 their effects on differentiation and growth, rates of neuron generation, and cellular composition at different timepoints. In Aim 2, we propose detailed biochemical investigations of folate pathway dysregulations. In addition, we will test the effects of folate/B12 supply on global and site-specific DNA methylation levels by whole-genome bisulfite sequencing and examine transcriptomic and proteomic dysregulations. In Aim 3, we will measure the effects of folate/B12 imbalance on electrophysiological function of organoid neural networks using high-density microelectrode arrays. Project Number: 1R01HD122185-01A1 | Fiscal Year: 2026 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Konstantinos Zarbalis (+1 co-PI) | Institution: UNIVERSITY OF CALIFORNIA AT DAVIS, DAVIS, CA | Award Amount: $702,556 | Activity Code: R01 | Study Section: Nutrition and Metabolism in Health and Disease Study Section[NMHD] View on NIH RePORTER: https://reporter.nih.gov/project-details/11367770