Investigating mechanisms of neurometabolic disease using zebrafish and cellular models

Background:
Disruption of neuronal function can lead to epilepsy, developmental delay and behavioural abnormalities. These are all features of the severe neurometabolic disease, Non-Ketotic Hyperglycinemia (NKH), an inherited disorder caused by mutation of GLDC, encoding the glycine cleavage system. There is no cure for NKH and current treatments are ineffective, meaning that there is a need to better understand the function of the glycine cleavage system in nervous system development and function and to develop models for testing possible novel treatments. Analysis of metabolite levels in Gldc-deficient mice, Gldc knock-out zebrafish and in other experimental models shows highly conserved biochemical effects, including accumulation of glycine and related molecules. Similarly, these models show neurological abnormalities and/or motor deficits. An important next step is to understand the mechanisms linking metabolic abnormalities to neuronal dysfunction. The rationale for the programme of work in the host laboratories is that parallel use of mouse, human iPSC-derived cells and zebrafish models allows a multi-faceted approach taking advantage of the strengths of each system.

Aims/Objectives:
The overall aim of the project is to investigate the effect of Gldc loss of function on neuronal and astrocytic development and function. The objectives are to:

• Generate novel zebrafish model(s) carrying missense mutations in Gldc, and characterise effects on survival, motor function and behaviour. Induction of partial loss of function will allow comparison of phenotypes with Gldc-null fish.
• Evaluate neuronal and astrocyte generation, morphology and activity using transgenic reporters and calcium-imaging in fish larva. We hypothesise that this will reveal specific subsets of cells with abnormal function.
• Read-outs of cell activity and fish phenotypes will be used to test the effect of treatments to alter metabolic function and/or screen potential therapeutic agents.
• Applicability of findings to mammalian systems and the NKH disease will be explored, making us of availability of the NKH mouse model and human cellular models.

Methods:
The project will benefit from the combined expertise of the Greene and Alexandre groups in working with experimental models of Gldc dysfunction, developmental neurobiology, zebrafish technology, advanced imaging, gene targeting and metabolomics (e.g. Refs 1-3).

• CRISPR/Cas9 technology will be used for generating targeted genetic modification
• Confocal imaging (single and multi-photon) will allow analysis of cell numbers and morphology
• Transgenic reporters will allow image analysis of calcium flux in vivo
• Behavioural assays will make use of video tracking with computational analysis
• Validation of cellular abnormalities in mammalian models will provide experience in use of mouse genetic models and cell culture

Data will be analysed in the context of transcriptomic and metabolomic datasets generated in mouse and cellular models of NKH.

Timeline:
Year1 - generation of fish lines and initiate the behavioural essays.
Year2 - calcium and cellular imaging.
Year3 - finalise calcium and cellular imaging and apply to mammalian models.

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.            Riche R, Liao M, Pena IA, Leung K-Y, Lepage N, Greene ND, Sarafoglou K, Schimmenti LA, Drapeau P, Samarut E. Glycine decarboxylase deficiency impairs motor behaviour in zebrafish rescued by counterbalancing glycine at synaptic level. JCI Insight. 3:e124642  (2018).
3.            Hadjivasiliou Z, Moore RE, McIntosh R, Galea GL, Clarke JDW, Alexandre P. Basal protrusions mediate spatiotemporal patterns of spinal neuron differentiation. Dev. Cell. 49: 906-917 (2019).