The link between protein O-GlcNAcylation and intellectual disability

Supervisors: Daan van Aalten, Marios Stavridis

Project Description

Understanding and controlling the balance between pluripotent self-renewal and differentiation is the major aim of the field of stem cell biology and it is the limiting factor for successful, safe, widespread use of embryonic stem cells (ESCs) in a clinical setting. In recent years it has become clear that these ESC functions are regulated by environmental cues (growth factors, nutrient supply, other cells) via signalling pathways operating on several proteins. One such pathway involves the addition of a single sugar molecule of N-acetylglucosamine (GlcNAc) on certain proteins by a single enzyme called Ogt. It appears that Ogt is essential for life, as attempts to delete this X-linked gene in ESCs or animals result in cell death. Ogt responds to nutrient availability and is thought to act as a sensor of "feast" or "famine" states in cells. The modification it carries out on proteins is found deregulated in diseases such as cancer, diabetes and Alzheimer's, highlighting the importance of correct Ogt function in the maintenance of a healthy state. Recently, mutations in Ogt have also been identified in patients with X-linked intellectual disability (XLID) [e.g. 1], further linking the function of this enzyme to neuronal function.

Recent work by us and others has identified a key role for O-GlcNAc in the differentiation of embryonic stem cells [2], in particular towards neural and neuronal lineages. Nevertheless, studies on the function of this modification are limited due to the apparently essential role for Ogt function for cell survival which hampers the dissection of links between reduced O-GlcNAcylation on the proteome and cellular function. This project is aimed at understanding how this gene regulates ESC and neuronal fate (including cell survival, pluripotency, differentiation and neuronal maturation) by altering the function of Ogt by a series of bespoke ("knock-in") mutations, using the CRISPR/Cas9 approach that we have recently established in our laboratory. We will then determine the minimum activity of Ogt that is required for survival, pluripotency, neural differentiation and maturation and then study what proteins are modified by it at each activity level, giving us starting points for mechanistic biology.

These experiments will enable us to determine the minimal set of modified proteins that are essential for life (by mass spectrometry), the effects of reducing levels of Ogt activity on gene expression and protein modification, on ESC pluripotency and neuronal function. We will carry out experiments to compare the behaviour and function of ESC as well as ESC-derived neurons in conditions of varying Ogt catalytic activity. We will engineer mutations mimicking those found in XLID patients as well as other mutations we have recently demonstrated [3] to reduce Ogt function to the bare minimum required for development and compare the gene expression of cells in each of these conditions as Ogt is known to play important roles in maintaining controlled gene expression (epigenetic regulation) as well as potentially other roles linked to XLID. This programme will expand our understanding of the role of the modification in normal cells and elucidate the critical functions of Ogt in gene expression and protein modification. These findings will inform future studies on ESC biology as well as for the understanding of Ogt and O-GlcNAc in disease.

References:

1 T. S. Niranjan et al., PLoS One. 2015 Feb 13;10(2):e0116454.

2 C. M. Speakman et al., Stem Cells. 2014 Oct;32(10):2605-15.

3 D. Mariappa et al., Open Biol. 2015 Dec;5(12):150234.

 

Other: