Understanding the function of mitochondrial one-carbon metabolism in brain development

Background:
The formation of the brain during embryonic development depends on regulation of gene expression, cellular properties and coordinated tissue movements. Abnormalities in these processes can lead to structural malformations and this project focuses on two such conditions, neural tube defects (NTDs) and obstructive hydrocephalus, which remain common birth defects worldwide.  Mutation of Gldc, encoding glycine decarboxylase, can cause NTDs and hydrocephalus in humans and mice (1-3). Gldc is a component of mitochondrial one-carbon folate metabolism and loss-of-function models therefore provide a paradigm for understanding the requirement for this metabolic network in development, and for design and testing of novel therapies. Abnormal function of Gldc has been implicated in a range of cellular abnormalities including suppression of one-carbon dependent reactions, oxidative stress, diminished proliferation and impaired stem cell pluripotency. Whether and how these abnormalities contribute to defects of brain development is not well known. Defining the cell- and stage-specific requirement for Gldc function is a key next step in understanding its requirement in brain development.

Aims/Objectives:
The aim of this project is to understand the molecular and cellular mechanisms by which alteration in Gldc function leads to NTDs, hydrocephalus and other brain malformations. We have developed mouse models in which Gldc expression can be selectively switched on and off in specific tissues and stages of development.  Using these models, the objectives are to:

• Use transgenic labelling of Gldc-expressing cells and their descendants to identify the Gldc-lineage and its contribution to different brain regions during development.
• Perform tissue- and stage-dependent knockout and rescue of Gldc expression to identify the specific cells/tissues that are essential for prevention of NTDs, hydrocephalus and other defects. 
• Analyse selected cell populations for alterations in gene expression, metabolite profile and mitochondrial function and test for normalisation by small molecule or genetic manipulation. Potential therapeutic approaches will taken forward for testing in vivo for rescue of birth defects.
• Assess conservation of effects of Gldc dysfunction by analysing human neural progenitors derived from iPS cells carrying GLDC mutations.

Methods:
The project will provide training in a range of technologies, with a flexible approach allowing focus on the student’s interests. Core methods will be use of mouse genetic models, including conditional knockout/rescue by cre-lox technology and lineage tracing with transgene reporters. Imaging will use light and fluorescent confocal microscopy with image analysis tools. Functional studies will include RNA-seq and bioinformatic analysis, metabolomic screening tools and cellular assays (e.g. proliferation, mitochondrial function). In order to extend the project to validate findings in human cells, iPS cell lines carrying mutations of GLDC will be cultured and differentiated to cell populations of interest.

References:
1.            Pai YJ, Leung K-Y, Savery D, Hutchin T, Prunty H, Heales S, Brosnan ME, Brosnan JT, Copp AJ, Greene NDE. Glycine decarboxylase deficiency causes neural tube defects and features of non-ketotic hyperglycinemia in mice. Nat. Commun. 6: 6388 (2015).
2.            Leung, K-Y, Pai YJ, Chen Q, Santos C, Calvani E, Sudiwala S, Savery D, Ralser M, Gross SG, Copp AJ, Greene NDE. Partitioning of one-carbon units in folate and methionine metabolism is essential for neural tube closure. Cell Reports, 21: 1795-1808 (2017).
3.            Santos C, Pai YJ, Mahmood MR, Leung KY, Savery D, Waddington SN, Copp AJ, Greene NDE. Impaired folate metabolism causes formate-preventable hydrocephalus in Gldc-deficient mice. J. Clin. Invest. Doi: 10.1172/JCI132360 (2020).