Understanding the role of the Glycine Cleavage System in Neural Tube Defects

Neural tube defects (NTDs) are common birth defects that arise in early pregnancy caused by incomplete formation of the neural tube, which will later develop into the brain and spinal cord. As a result, the brain and/or spinal cord of the fetus become irreversibly damaged, resulting in death around birth, or long term disability in surviving children. The most common forms of NTDs are anencephaly, affecting the brain, and spina bifida, which affects the lower spinal cord. They occur in approximately 1-2 per 1,000 pregnancies and total at least 170,000 new cases per year worldwide.
The risk of NTDs depends on both inherited factors and environmental influences such as maternal diet, diabetes or exposure to certain chemicals. Because of the many possible contributory factors, the exact causes of NTDs in any affected individual are usually unknown. 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 NTDs are prevented by folic acid - perhaps up to 50% of all cases fail to respond - and so additional therapies are needed. In order to make further progress towards prevention of all NTDs we need a better understanding of their causes, in particular the genes that increase a person's risk of NTD. Moreover, it will be important to identify new preventive therapies for NTDs which may be used individually or in combination with folic acid. In families where genetic risk factors have been identified this also means that family-specific therapies may be offered.
All cells require efficient handling of small molecules called folates, which are related to folic acid, for many different functions. It appears that some NTDs are caused by an inherited abnormality in the way cells in the embryo handle folates. We studied a group of proteins called the "glycine cleavage system" (GCS), that are involved in folate handling. Some patients with NTDs had defects in these proteins, whereas unaffected people did not. This finding suggests that problems with the GCS may directly cause NTDs. In support of this idea, mouse embryos that have GCS defects also develop NTDs.
This project will make use of mouse models lacking function of glycine decarboxylase (Gldc), part of the GCS. The mouse models provide an opportunity to study the role of these folate handling proteins in the embryo and how the associated NTDs may be prevented. We have found that handling of folates is altered in Gldc-deficient embryos and we will now use detailed metabolic studies to work out exactly how these changes come about. We will use genetic approaches to turn off Gldc function or to restore function only in the neural tube. This will tell us which tissues in the developing embryo need Gldc function, to ensure normal development.
The next step is to understand which of the outputs of folate metabolism are disrupted in Gldc-deficient embryos and to test which of the changes are responsible for NTDs. This will be achieved by detailed biochemical analysis and embryo imaging using markers of particular cellular changes, followed by use of supplements to correct these defects. Folate metabolism is essential for synthesis of DNA, which is needed for cells to divide, and methylation reactions that modify gene expression and other functions. Folate metabolism may also be needed for regulation of the level of reactive oxygen species (free radicals) which can be damaging within cells. An imbalance in production and removal of these molecules, termed oxidative stress, is important in a number of diseases, including diabetes. We will examine whether Gldc-deficiency causes oxidative stress in developing embryos, that may contribute to NTDs.
Understanding the molecular and cellular causes of NTDs in Gldc-deficient embryos is an important step towards developing new therapies to prevent more NTDs in humans than is currently possible using folic acid alone.

This project aims to understand how abnormal folate one-carbon metabolism (FOCM) causes neural tube defects (NTDs). We focus on a mitochondrial-specific component of FOCM, the glycine cleavage system (GCS), which has been implicated in human NTDs. Mice lacking expression of the GCS component glycine decarboxylase (Gldc) exhibit abnormal FOCM, elevated glycine levels and NTDs. Main goals are:
1. To understand the metabolic basis of NTDs in Gldc-deficient embryos, we will adopt complementary approaches: (i) metabolic labelling to track one-carbon units; (ii) mass spectrometry-based quantification of folate profiles in differing subcellular compartments and cell types; (iii) gene expression/pathway analysis.
2. Genetic approaches, involving conditional knockout and rescue of Gldc expression, will be used to determine whether there is tissue heterogeneity in the requirement for GCS activity in FOCM and to test whether Gldc function in the neuroepithelium is necessary and sufficient for neural tube closure.
3. Outputs of FOCM include provision of one-carbon units for nucleotide biosynthesis and/or methylation reactions. We will determine which of these outputs are compromised in Gldc-deficient embryos and whether they mechanistically contribute to NTDs. Downstream effects on DNA copy number/integrity and DNA/protein methylation will be evaluated. The relative importance of diminished methylation and nucleotide biosynthesis in Gldc-mediated NTDs will be tested by supplementation strategies and genetic approaches that channel one-carbon units towards either output.
4. Mitochondrial FOCM contributes to production of NADPH, required for redox balance. We will ask whether Gldc-deficiency results in redox imbalance and oxidative stress using mass spectrometry based read-outs of reactive oxygen species, as well as imaging and reporter assays in embryos. The functional contribution of oxidative stress to NTDs will be assessed using cellular markers and rescue experiments.

A key long-term aim of this research will be to develop therapeutic approaches for NTDs that will complement folic acid to allow prevention of more NTDs than is currently possible. The major beneficiaries will therefore be (a) children who would otherwise have been born with a birth defect, (b) families who have encountered NTDs and are motivated towards progress on prevention; (c) healthcare professionals whose aim is to improve health status of mother and fetus during pregnancy; (d) the pharmaceutical industry, whose nutritional supplement sector has a growing interest in prevention of common birth defects such as NTDs.

Neural tube defects are clinically devastating to affected individuals: anencephaly is lethal at birth and spina bifida may result in lifelong disabilities. There are major implications for health care provision - the estimated cost of life time medical care for a spina bifida patient in the USA is estimated at in excess of $500,000. If failure of closure occurs, the only treatments available are palliative; eg. in utero surgery may prevent further degeneration but does not result in recovery of damaged tissue. The optimum approach is primary prevention as exemplified by population-wide use of folic acid supplements when planning pregnancy.

At the present time the recurrence risk for NTDs is more than 10-fold higher than for a first affected pregnancy. Prospective mothers are advised to take folic acid and, while this has proven a very successful strategy, risk is only reduced by up to 70%. In fact, following fortification of the food supply with folic acid in the USA, only a 26% reduction in NTD prevalence has been detected. The applicants previously identified inositol as a preventive therapy for genetically-determined mouse NTDs that are unresponsive to folic acid. This finding has been taken forward to clinical testing and a pilot randomised, double blind trial, to test the efficacy of inositol for prevention of NTDs , has been completed. The experience of the applicants in taking a therapy from the laboratory to the clinic will facilitate the exploitation of findings from the current project towards clinical impact. This could be achieved on a timescale of 5-10 years.

In the longer term, genetic analysis could be offered to allow identification of parental risk factors, which would then indicate the most effective preventive therapy. This strategy would also address the possibility that therapies which are effective in some individuals may be inactive or even harmful in others. Such a 'pharmacogenomics' type of model is currently unrealistic for NTDs. However, in an era when personalised exome sequencing will become a reality, it is time to begin development of therapies that may be appropriate for a subset of NTDs with a particular underlying pathology.

Identification of genetic risk factors is the aim of large-scale studies in the NTD research community. The next step will involve development of preventive strategies based on measurable metabolic and/or cellular defects in NTD models/patients. In the current project this approach will be used to address NTDs associated with a specific group of genes (encoding the glycine cleavage system) that appear to play a key role in neural tube development in both mice and humans. This offers the possibility of: (a) refining the likely range of influence of existing treatments, particularly folic acid (i.e. which NTDs are likely to respond to folic acid and which are not?), and (b) identifying promising novel therapies which may be more effective than folic acid in NTDs with particular patterns of molecular pathology.