Deciphering the role of O-GlcNAcylation in neuronal development and functioning

Abstract

O-GlcNAcylation is an essential intracellular protein modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). This modification occurs on serine and threonine residues of thousands of proteins, exerting diverse effect on their function. Since its discovery, the role of the modification in various disease states has become increasingly appreciated. As a key integrator of metabolic state with signalling and transcription the modification is crucially involved in the pathogenesis of diabetes, cancer, neurodegenerative conditions, and intellectual disability. The role of OGT in intellectual disability has only recently been discovered and remains poorly understood.

The work presented in this thesis furthers the understanding of intellectual disability caused by mutations in OGT, termed OGT-CDG, through modelling disease mutations in Drosophila. The results presented here demonstrate the crucial role of O-GlcNAcylation in synaptogenesis and normal sleep. Additionally, while alternate hypotheses aim to explain the pathogenesis of OGT-CDG, the rescue of phenotypes associated with a patient mutation modelled in Drosophila by increasing global O-GlcNAcylation suggests reduced levels of this modification are central to this disorder. Furthermore, the behavioural phenotypes assayed were amenable to rescue in adulthood, raising the possibility that beyond affecting developmental processes, the aetiology of OGT-CDG involves impaired functioning of the adult nervous system.

The aim of identifying OGT substrates causal in OGT-CDG remains elusive. The second chapter of this thesis describes bioinformatic approaches at narrowing the pool of potential conveyors of this disorder. Through screening a recently compiled database of all known human O-GlcNAc sites and missense mutations segregating with disease, the results of this chapter provide several candidates for future empirical validation. Several of these candidate conveyors are conserved in Drosophila and are associated with phenotypes found in flies modelling OGT-CDG patient mutations, further supporting their relevance to the pathophysiology of the disorder.

The final chapter of this thesis focuses on the first in vivo implementation of a novel methodology to site-specifically elevate O-GlcNAc stoichiometry, through cysteine mutagenesis of modified serine or threonine residues. This approach relies on the ability of OGT to modify cysteine residues, while the resulting S-GlcNAcylation is resistant to OGA hydrolysis. The protein chosen to test this approach is the circadian rhythm protein Period. Recent research has demonstrated the profound impact of O-GlcNAcylation of Period on circadian rhythm in flies, and the fluctuating levels of the modification on this protein render it suitable for this approach. While conclusive identification of S-GlcNAcylation of Period remained elusive, the phenotypic characterisation of this line suggests its presence.

Date of Award	2023
Original language	English
Awarding Institution	University of Dundee
Supervisor	Daan van Aalten (Supervisor) & Leeanne McGurk (Supervisor)