Neural tube defects (NTDs) are the second most common birth defect worldwide and can occur do to environmental factors like micronutrition. Deficiencies in folic acid concentrations have been heavily associated with human NTDs due to neurulation’s dependency on the methionine cycle and folate cycle. Neurulation is the formation of the nervous system from the ectoderm that involves the fusion of the neural folds in the middle of the embryo that moves cranially and caudally. The cells of the neural plate fold back onto themselves and create the neural tube. NTDs can occur in any region on the neuronal axis. An example of an NTD in the Cranial region is anencephaly, where the brain is severely underdeveloped. An example of an NTD in the caudal region would be spina bifida, which is the incomplete closure of the spinal cord and column. NTDs can also vary in severity, where you have the least severe forms show near complete closure of the neural tube. Along with folate, zinc is also proposed to influence the incidence of NTDs. In mice, zinc-deficient diets have been associated with NTDs and in humans, lower zinc levels in maternal and fetal tissues are associated with NTDs and cultured cells from NTD patients show characteristics of decreased zinc uptake. However, the mechanism by which zinc affects neural tube closure is unclear.
To investigate whether zinc deficiency leads to failure of neural tube closure, researchers cultured mouse embryos under zinc-deficient conditions. They introduced zinc deficiency in the embryos by treating the tissue with TPEN, which is a zinc chelator. In high dosages of TPEN (15 micromolar), neural tube closure failed in all embryos and a reduction in size of the cranial region and dead tissue were seen. In low dosages of TPEN (10 micromolar), neural tube closure occurred, but small amounts of dead tissue were seen. In high dosages of TPEN plus a zinc supplement, neural tube closure occurred, just much later (-5hrs late). They concluded here that zinc deficiency does cause a failure in neural tube closure. To clue them into how zinc deficiency lead to the failure of neural tube closure, they analyzed time lapsed movies to see how folding is disrupted. Cranial neural tube closure requires inflection and zippering of the neural folds. They found weak to no effects of zinc deficiency on zippering, but they found that zinc deficiency disrupts inflection of neural folds. The question of why dead tissue was seen in zinc deficient conditions remained and researchers used the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method and a Cleaved Casp3 assay to see how many cells were undergoing apoptosis in control, zinc deficient, and zinc deficient + zinc supplement treated embryos. TUNEL detects DNA fragmentation generated during apoptosis and a cleaved Casp3 propagates an apoptotic signal. They found significantly higher TUNEL and cleaved Casp3 signals in zinc deficient tissue. They also saw that cleaved Casp3-positive cells and TUNEL positive cells in the cranial region are lessened by adding zinc, so apoptosis can be suppressed with more zinc. Since p53 is a major player in apoptosis, researchers investigated p53 in zinc deficient tissue, and through fluorescent imaging found significant levels of p53 in zinc deficient tissue as compared to little to no p53 in control and zinc supplemented tissue. They discovered through protein gels, that Zinc deficiency attenuates p53 ubiquitylation by interfering with its binding to E3 ubiquitin ligase, Mdm2.
Li H, Zhang J, Niswander L. Zinc deficiency causes neural tube defects through attenuation of p53 ubiquitylation. Development. 2018;145(24):dev169797. Published 2018 Dec 13. doi:10.1242/dev.169797