The main focus of the Loeken laboratory is to understand how maternal diabetes causes congenital malformations. Using a mouse model of diabetic pregnancy, Loeken and colleagues have shown that increased glucose metabolism by embryos inhibits expression of Pax3, a gene required for neural tube closure, and that inhibition of Pax3 expression leads to neural tube defects (NTDs), one of the most common malformations that occur in human diabetic pregnancy. The NTDs resulting from PAX3 insufficiency appear to be caused by derepression of the p53 tumor suppressor protein, leading to neuroepithelial apoptosis.The Loeken lab has derived mouse embryonic stem cells (ESC) in low (physiological) glucose media that they call LG-ESC. These cells respond to changes from normal to high glucose concentrations like embryos respond to changes from a nondiabetic to a diabetic pregnancy. These cell lines allow the study of how a diabetic environment causes molecular and biochemical disturbances that is not possible using the mouse embryo model. In addition to wild type LG-ESC lines, they have derived lines that encode either wild type or mutant Pax3 alleles and carry a plasmid that can inducibly knock down expression of the mRNA encoding p53. Because p53 can promote cell cycle withdrawal, differentiation, and increased oxidative fuel metabolism, PAX3 may be required during establishment of the neural tube to repress p53 and its effects on cell cycling, differentiation, and fuel metabolism. We propose to grow these cells under conditions to induce neuronal precursors in normal and high glucose, with and without p53 knock down, and study expression and function of genes regulating neural tube differentiation and glucose metabolism. I will test the hypotheses that inhibition of PAX3 expression, by high glucose culture or Pax3 gene mutation: 1. Inhibits glycolytic metabolism and stimulates oxidative metabolism. 2. Inhibits expression of the pluripotency markers, Nanog, Oct4, and Sox2, and increases expression of neural differentiation markers such as Sox1 and Nanog.3. Inhibits cell cycling.4. And that all of these effects of PAX3 are mediated by p53.These studies will provide novel insight into how maternal diabetes regulates neural tube defects through gene expression that affects fuel metabolism.
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