Genetics: Worm Genes Confirmed and Cloned for Proteomic Tool Kit Developmental Biology: Diabetes-boosted Oxygen Radicals Block Neural Tube Closure Epidemiology: Link Strengthened Between MS and Epstein-Barr Diversity: Set an Example for Others, U.S. Surgeon General Advises eAddendum: Senator Kennedy Holds SARS Briefing at HMS   Study Writes 'Guidebook' to Chromosome 7 Alzheimer's Marker Revealed in Eye Informatics System Simplifies Complex Image Analysis   HMS Faculty Council Report Students Take On Big Questions at Soma Weiss U.S. News and World Report Ranks HMS Top Med School Junior Science Awards Dana-Farber to Host Antibody Library In Memoriam: Brian McGovern   'Humor' Belittles Pregnant Student   Labs Look for Write Stuff   Front Page

DEVELOPMENTAL BIOLOGY Diabetes-boosted Oxygen Radicals Block Neural Tube Closure Oxidative Stress Shown to Disrupt Gene Expression in Mouse Model Oxidative stress may be to blame for higher rates of neural tube defects in diabetic pregnancies, according to research led by Mary Loeken, HMS assistant professor of medicine at Joslin Diabetes Center. Diabetic women who are pregnant are warned to keep their glucose levels under control or risk birth defects like spina bifida. But how glucose thwarts the development of certain tissues in the embryo has been unclear. This latest study, published in the April Diabetologia, shows that the oxidative stress caused by diabetes can disrupt expression of a gene needed for neural tube development in a mouse model. Mary Loeken's latest study shows that even mild oxidation from excess glucose can disturb developmental genes in mouse embryos. (Photo by Steve Gilbert) Neural tube defects arise in the first weeks of pregnancy, when the tiny folded disc of tissue destined to become the brain and spinal cord fails to zip up completely into an enclosed tube. Babies of diabetic mothers have a higher rate of birth defects than babies of nondiabetic women, and neural tube defects are among the most common. The risk of these defects rises significantly if glucose is not kept under strict control. Cell Suicide Prevention Loeken and her colleagues previously found that expression of the gene encoding Pax-3, a transcription factor, is expressed at lower levels in mouse embryos with diabetic mothers. Since Pax-3 is required for neural tube closure, and mice lacking Pax-3 have severe neural tube defects, this may explain the increased neural tube defects associated with diabetic pregnancy. Last year, they reported that the protein's action is to keep p53 from sending death signals to the cells of the neural tube (see Focus, March 22, 2002). Without Pax-3, there is nothing to save the cells from suicide. "Pax-3 is not required to regulate neural tube closure--other genes do that--but you do need to inhibit p53-dependent apoptosis in order to get neural tube closure," Loeken said. "There may be something about the timing of the induction of the neural tube that is very sensitive to whether or not oxidants are being produced." --Mary Loeken But how a mother's high glucose levels dampen Pax-3 was still unknown. "The concentration of glucose in the embryo's cells seems to be at equilibrium with maternal glucose levels," Loeken said. In an embryo, most of this glucose is metabolized through the glycolysis pathway. But some of it is also metabolized oxidatively, which produces oxygen free radicals that are known to wreak havoc on cells. "It was known from other work that you could prevent malformations induced by diabetic pregnancy in rat embryos if you administered antioxidants," Loeken added. She speculated that oxidation might affect expression of Pax-3. Loeken's team found that by feeding a large dose of vitamin E to pregnant diabetic mice, they could normalize Pax-3 expression in the embryos and prevent neural tube defects. Levels of lipid peroxidation in embryonic tissues were lower in these embryos than in those whose mothers were not fed vitamin E. In mice and in cultured embryonic tissue, they found that inducing oxidative stress with the chemical antimycin A inhibits Pax-3 expression as well. Oxidative stress was thought to harm an embryo, typically by causing direct damage to DNA and triggering apoptosis, but Loeken's team found no evidence of DNA damage when they replicated the events in cultured cells. Instead, the finding suggests that even relatively low levels of oxidative stress can cause damage in an embryo by perturbing the expression of specific genes. Michael Brownlee, a professor of diabetes research at Albert Einstein College of Medicine, who was not an author on the paper, said that the study helps connect Pax-3 to the known link between oxidation and neural tube defects in animals. But it also points the way to a larger paradigm, that a perturbation in gene expression at vulnerable times during development may have long-term effects, including disease. Loeken noted that many proteins, including transcription factors, depend on their redox state for activation. "It could be that cells have a way of sensing the redox potential and determining whether they're ready to switch on a control gene," she said. "There may be something about the timing of the induction of the neural tube that is very sensitive to whether or not oxidants are being produced." Bruce Demple, professor of toxicology at HSPH, said that several examples of oxidative stress regulating gene expression have been established in recent years, so "this is likely a very widespread mechanism." Furthermore, Loeken said, "There are a lot of ways an embryo could be exposed to oxidative stress in addition to the increase in glucose metabolism associated with diabetes." Folic acid deficiency, for instance, can lead to mild oxidative stress through the accumulation of homocysteine, an oxidant. So this mechanism may explain some of the benefits of taking folic acid supplements during pregnancy. Easing Oxidative Stress Ultimately, it is apoptosis through p53 that causes the neural tube defect. Since p53 responds to oxidative stress as well, hyperglycemia may raise levels of p53 while putting a damper on its inhibitor Pax-3. Although the dietary levels of vitamin E given to mice were very high--twentyfold over normal--the study suggests that antioxidants may have the potential to prevent congenital defects in the babies of diabetic mothers. Diabetic women of childbearing age would need to be proactive about prevention, however, since neural tube closure occurs in an embryo around the same time many women find out they are pregnant. The link between PAX-3 and neural tube development has still not been made in humans. Loeken said that it would be difficult to determine whether expression of the human gene was disturbed at a critical time, but the structural similarity of the human PAX-3 gene to those of other vertebrates, and the similar mechanisms of neural tube closure among higher vertebrates, argue strongly for similar developmental roles. --Courtney Humphries