Inositol-preventable neural tube defects: understanding the molecular causes and mechanisms of prevention

During early pregnancy, a crucial event in the developing embryo is the formation of the neural tube , which will later develop into the brain and spinal cord. Failure of the neural tube to form correctly leads to a group of birth defects called neural tube defects (NTDs), in which the brain and/or spinal cord of the fetus become irreversibly damaged, resulting in death before or shortly after birth, or handicap in surviving babies. Overall, NTDs occur in around 1 per 1,000 pregnancies although the rate varies and is significantly higher in some regions (e.g. Northern Ireland and Scotland). Worldwide, approximately 130,000 cases occur every year. The risk of NTDs depends on both inherited genetic factors and non-genetic factors such as diet, but the exact causes are not well understood. However, the risk of an affected pregnancy can be substantially reduced if the mother takes folic acid supplements before and during early pregnancy. Unfortunately, not all cases of NTDs are preventable by folic acid and for this reason, we are seeking additional protective approaches. Using a mouse model that naturally develops folate-resistant NTDs we found that another vitamin, inositol, can prevent NTDs, and we are now carrying out a clinical trial to test whether inositol prevents NTDs in humans. There are two main aims of the present research proposal. First, we will investigate the cause of the inositol-preventable NTDs in the mouse. We have used a technique that lets us separate many proteins and compare which ones are present in normal embryos and those that develop NTDs. We have detected several proteins that seem to be present at lower abundance or in abnormally modified forms in embryos developing NTDs. We will build on these studies by analysing the proteins that seem to be abnormal in the embryos that develop spina bifida. Understanding the role of specific proteins in the mouse may indicate risk factor genes in humans. The second aim of the research is to work out exactly how inositol prevents NTDs, as this may allow improvements to be made in the therapeutic approach. Knowledge of the causes of NTDs in humans may then allow more accurate counselling for affected families who are considering a further pregnancy, and may allow identification of women who will benefit from inositol supplementation.

Neural tube defects (NTDs), such as spina bifida and anencephaly, are severe congenital malformations caused by failure of closure of the embryonic neural tube. Folic acid supplementation can prevent many cases of NTDs but a significant proportion (at least 30%) are unresponsive, and these defects remain a major health issue in the UK and worldwide. As a system to investigate the mechanisms underlying development of ?folate-resistant? NTDs, and to evaluate potential therapies, the curly tail mutant mouse provides a well-studied model. The incidence of defects in curly tail is influenced by both multiple genetic and environmental factors, as in humans.
We found previously that NTDs in curly tail can be prevented by inositol, acting through a protein kinase C (PKC)-dependent mechanism. This finding suggests that inositol may represent a possible adjunct therapy to folic acid for prevention of NTDs. Indeed, we recently initiated a clinical trial to test this possibility. Our present goals are: (1) to understand the molecular basis of NTDs in the curly tail mouse and (2) to further define the mechanism by which NTDs can be prevented by inositol. In a recent proteomic screen, we compared curly tail and genetically matched wild type embryos, using 2D protein gels and mass spectrometry. We identified a number of protein differences involving changes in protein abundance and post-translational protein modification. Our working hypothesis is that these changes may be associated with predisposition to NTDs.
Here, we will characterise the specific changes in individual candidate proteins by detailed examination of the abundance and spectrum of post-translational variants. We will then evaluate the potential role of the most promising candidate proteins in neural tube closure. Correction of mutant protein profiles by inositol treatment will support such a role, particularly if this is dependent on the PKC isoforms we previously found to be necessary for prevention of NTDs. Tissue expression and subcellular localization will be examined as these may indicate disruption of protein function. Since the curly tail mutation is thought to represent a hypomorphic allele of the transcription factor, Grhl3, we will also examine the protein profile of Grhl3 null mutants, which also exhibit NTDs, in parallel with curly tail. These studies will allow us to prioritise candidate proteins for functional analysis to directly test their requirement in neural tube closure and/or the prevention of NTDs by inositol.